New Biotechnology · Volume 31S · July 2014 SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION) Orals Sunday 13 July Industrial biotechnology from fundamentals to practice (ACIB Session) ACIB-1 Two promising biocatalytic tools: regioselective carboxylation of aromatics and asymmetric hydration of alkenes Silvia M. Glueck 1,∗ , Christiane Wuensch 1 , Tamara Reiter 1 , Johannes Gross 1 , Georg Steinkellner 1 , Andrzej Lyskowski 1 , Karl Gruber 2 , Kurt Faber 2 1 2 ACIB GmbH c/o University of Graz, Department of Chemistry, Austria University of Graz, Austria Due to the growing demand for alternative carbon sources, the development of CO2 -fixation strategies for the production of valuable chemicals is a current challenge in synthetic organic chemistry [1]. The enzymatic carboxylation of aromatic compounds [2,3] catalyzed by various decarboxylases represents a promising ‘green’ alternative to the classic Kolbe-Schmitt reaction [4] which requires harsh reaction conditions. Depending on the type of enzyme, the carboxylation proceeded in a highly regio-complementary fashion: Benzoic acid decarboxylases selectively catalyzed the ortho-carboxylation of phenolic substrates (conv. up to 80%) whereas phenolic acid decarboxylases exclusively catalyzed the ␤-carboxylation of styrenes (conv. up to 40%) [5]. During these studies a promiscuous ‘hydratase-activity’ of phenolic acid decarboxylases was discovered, which leads to the stereoselective hydration of hydroxystyrenes to yield the corresponding sec-alcohols with high conversion (up to 82%) and e.e.’s of up to 71% [6]. Acknowledgements: This work has been supported by the Austrian BMWFJ, BMVIT, SFG, Standortagentur Tirol and ZIT through the Austrian FFG-COMET-Funding Program. 1871-6784/$ — see front matter References [1].Tsuji Y, Fujihara T. Chem Commun 2012;48:2365. [2].Glueck SM, Gümüs S, Fabian WMF, Faber K. Chem Soc Rev 2010;39:313. [3].Matsui T, Yoshida T, Yoshimura T, Nagasawa T. Appl Microbiol Biotechnol 2006;73:95. [4].Lindsey AS, Jeskey H. Chem Rev 1957;57:583. [5].Wuensch C, Gross J, Steinkellner G, Lyskowski A, Gruber K, Glueck SM, Faber K. RSC Adv 2014;4:9673. [6].Wuensch C, Gross J, Steinkellner G, Gruber K, Glueck SM, Faber K. Angew Chem Int Ed 2013;52:2293. http://dx.doi.org/10.1016/j.nbt.2014.05.1615 ACIB-2 Unconventional substrates for enzymatic reduction: carboxylates and nitriles M. Winkler 1,∗ , K. Napora-Wijata 1 , B. Wilding 1 , N. Klempier 2 1 ACIB GmbH, Petersgasse 14/III, 8010 Graz, Austria Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, 8010 Graz, Austria 2 Keywords: Biocatalysis; Nitrile Reductase; Carboxylate reductase; Reduction; Enzyme The reduction of carbonyls by enzymes has been extensively studied and is now widely applied on industrial scale. In comparison, enzyme catalyzed carboxylic acid/carboxylate and nitrile reduction are relatively young fields. Their full potential is currently being explored. The chemical reduction of nitriles and carboxylic acids requires strong reducing agents due the low reactivity of these moieties. Consequently, mild alternatives would be a valuable addition to the current toolbox of biocatalysis. The products of nitrile reduction are primary amines and we have recently expressed nitrile reductase enzymes (NReds), in order to apply these enzymes as biocatalysts. The substrate scopes of wild type and mutant NReds as well as their biochemical characteristics were studied [1]. The reaction mechanism of NReds involves the www.elsevier.com/locate/nbt S1 SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION) New Biotechnology · Volume 31S · July 2014 Acknowledgement: This project is financed by the European Union’s Seventh Framework Programme FP7/2007-2013 under grant agreement no. 289646 (Kyrobio). References Figure 1 Whole cell biotransformation approach towards 3hydroxytyrosol – an antioxidant from olives. reaction of the substrate with an active site thiol and subsequent NADPH mediated reduction of the intermediate [2], which is similar to the mechanism proposed for carboxylate reductase enzymes (CARs) [3]. The latter enzyme was in focus of the development of a whole cell system (Figure 1) for the production of the antioxidant 3-hydroxytyrosol [4]. [1].Winkler M, et al. Comprehensive chirality. Synthetic methods VI, vol. 7. Amsterdam: Elsevier; 2012. p. 350–71. [2].Hajnal I, et al. FEBS J 2013;280:5815–28. http://dx.doi.org/10.1016/j.nbt.2014.05.1617 ACIB-4 Enzyme responsive polymers Alexandra Rollett 1,∗ , Andrea Heinzle 2 , Konstantin Schneider 2 , Doris Schiffer 2 , Gregor Tegl 3 , Eva Sigl 2 , Georg Guebitz 1 1 BOKU-Vienna/ACIB GmbH, Austria ACIB-Austrian Centre of Industrial Biotechnology GmbH, Austria 3 BOKU-Vienna, Austria 2 References [1].(a) Wilding B, et al. Adv Syn Catal 2012;354(11–12):2191–8; (b) Wilding B, et al. Chem Eur J 2013;19(22):7007–12. [2].Chikwana VM, et al. J Biol Chem 2012;387:30560–70. [3].Venkitasubramanian P, et al. J Biol Chem 2007;282:478–85. [4].Napora-Wijata K, et al. ChemCatChem, Patent application pending [in print]. http://dx.doi.org/10.1016/j.nbt.2014.05.1616 ACIB-3 Engineering of cupin hydroxynitrile lyases Kerstin Steiner 1,∗ , Romana Wiedner 1 , Bettina Kothbauer 1 , Mandana Gruber-Khadjawi 1 , Helmut Schwab 2 1 2 ACIB GmbH, Austria TU Graz, Austria Enantiopure cyanohydrins serve as versatile building blocks for a broad range of chemical and enzymatic reactions resulting in highly valuable products for many applications. Hydroxynitrile lyases comprise a diverse group of enzymes which catalyze the reversible cleavage of cyanohydrins to carbonyl compounds and hydrogen cyanide [1]. For a long time HNLs were only known to exist in plants, but recently (R)-selective HNL activity has been identified in several bacterial proteins [2]. The structure of one target protein from Granulicella tundricola (GtHNL) was solved and showed high similarity to proteins of the cupin superfamily, a fold, which has not been reported for HNLs before. Cupins are ubiquitous small beta-barrel proteins and ideal candidates for application in industrial processes and as scaffold for enzyme engineering as they are easily expressed as soluble proteins in exceptionally high yield (> 50% of total protein) in E. coli. A detailed biochemical characterisation of GtHNL revealed that the enzyme is metal-dependent. Several amino acids were identified, which are important for activity. The initial specific activity of 1.74 U/mg at pH 6 with (R)-mandelonitrile, and the 80% conversion of benzaldehyde to (R)-mandelonitrile with 90% enantiomeric excess were significantly improved by applying random and rational protein engineering approaches. S2 www.elsevier.com/locate/nbt Smart polymers can change their properties depending on certain stimuli like pH, temperature, light or electrical fields. Especially in biomedical materials, but also in technical applications, like the packaging industry, enzymes are useful biomarkers for sensoring purposes. Here we present different strategies towards enzyme responsive polymer systems for the detection of microbial contaminations in distinctive fields. Enzyme responsive hydrogel based systems were constructed using methacrylated biopolymers like polygalacturonic acid [1], alginic acid [2], carboxymethylcellulose [3], peptidoglycan, gelatin, collagen, alginate and agarose [4–6] while both stability and sensitivity were tuned via the degree of UV crosslinking. Theses systems were successfully used to detect contaminating microorganisms based on their extracellular enzymes, and infection of wounds based on enzymes from the human immune system. The latter included myeloperoxidase, cathepsin G, elastase and lysozyme, which were found to show an elevated activity in infected wounds. References [1].Schneider, et al. N Biotechnol 2012;29:502–9. [2].Schneider, et al. Enzyme Microbial Technol 2011;48:312–8. [3].Schneider, et al. Process Biochem 2012;47:305–11. [4].Hasmann, et al. Exp Dermatol 2011;20:508–13. [5].Heinzle, et al. Wound Repair Regen 2013;21:482–9. [6].Hasmann, et al. Diag Microbiol Infect Dis 2011;71:12–23. http://dx.doi.org/10.1016/j.nbt.2014.05.1618 New Biotechnology · Volume 31S · July 2014 SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION) ACIB-5 ACIB-6 Esterases from Clostridium are involved in anaerobic degradation of synthetic polyester Designing robust Saccharomyces cerevisiae strains against stresses encountered during bioethanol fermentations from lignocellulosic biomass Veronika Perz 1,∗ , Veronika Perz 2 , Armin Baumschlager 2 , Klaus Bleymaier 2 , Andrzej Łyskowski 2 , Altijana Hromic 2 , Karl Gruber 3 , Carsten Sinkel 4 , Ulf Küper 4 , Melanie Bonnekessel 4 , Doris Ribitsch 2 , Georg Guebitz 5 Vinod Kumar 1,∗ , Darren Greetham 1 , Tithira Wimalasena 2 1 2 The University of Nottingham, United Kingdom Kingston Research Limited (BP and DuPont JV), United Kingdom 1 ACIB GmbH, Austria ACIB GmbH, Petersgasse 14, 8010 Graz, Austria 3 Institute of Molecular Biosciences, University of Graz, Austria 4 BASF SE, Carl-Bosch-Straße 38, 67056 Ludwigshafen, Germany 5 Institute of Environmental Biotechnology, University of Natural Resources and Life Sciences, Austria 2 The anaerobic degradation of synthetic aliphatic–aromatic polyesters used in food packaging is of great importance during anaerobic digestion. Several studies have proven the biodegradability of PBAT (poly(butylene adipate-co-butylene terephthalate)) under aerobic conditions while there is only little information on anaerobic degradation. In this study, imaging analysis (CLSM, SEM) and quantification of degradation products indicated anaerobic hydrolysis of PBAT in biogas sludge. However, the detected hydrolysis rates are still too low for efficient PBAT degradation in industrial biogas plants. Consequently, hydrolysis of PBAT by enzymes from different anaerobic organisms (Clostridium species) was investigated. Therefore, various hydrolases from these organisms were successfully heterologously expressed in E. coli BL21-Gold(DE3). The kinetic parameters on the standard substrate p-nitrophenyl acetate were determined and revealed high activity of up to 700 U/mg (vmax ). Analysis of the crystal structure of one esterase from C. botulinum disclosed the presence of a Zn2+ metal ion that lies deep beneath the protein surface. The degradation of synthesized oligomeric and polymeric model substrates was studied in order to get a deeper insight into the reaction mechanisms of these new hydrolases. Esterases from C. hathewayi and C. botulinum were indeed able to hydrolyze aliphatic-aromatic polyesters like PBAT and different model substrates as indicated by HPLC/MS quantification of the hydrolysis products terephthalic acid, adipic acid, mono(4-hydroyxbutyl)terephthalate and bis(4hydroxybutyl)terephthalate. We could demonstrate that the esterases show activity under mild conditions as well as in biogas sludge. http://dx.doi.org/10.1016/j.nbt.2014.05.1619 During the pre-treatment of lignocellulosic biomass inhibitory compounds are released which can exert adverse effects on cellular growth, metabolism and ethanol production. In addition, osmotic stress caused by the high concentrations of available sugars and end-stage ethanol toxicity reduce overall rates of bioethanol fermentation. In previous studies, F1 hybrid segregants derived from clean lineage Saccharomyces cerevisiae strains were assessed for tolerance to a range of stresses encountered during industrial bioethanol fermentations. Using a systems biology approach (QTLQuantitative Trait Locus) chromosomal loci conferring resistance against weak acid and osmotic stress were identified, and within those loci genes COX20 (acetic acid) and RCK2 (osmotic) were determined as important for stress response. In the present study, strains in which COX20 or RCK2 have either been deleted or inserted into on tetracycline induced vectors. Phenotypic microarray assessment (Biolog, US) of these strains under acetic acid, formic acid, furfural, HMF, vanillin and sorbitol stress have been determined and compared against appropriate controls. Results have highlighted that presence of COX20 improves tolerance to weak acids while RCK2 gene conferred resistance to osmotic stress. The presence of either gene also enhanced the tolerance against furanic compounds such as furfural and HMF. http://dx.doi.org/10.1016/j.nbt.2014.05.1620 ACIB-7 Systems biology of Pichia pastoris Brigitte Gasser Dep. of Biotechnology, BOKU University of Natural Resources and Life Sciences Vienna, Austria Pichia pastoris is the most frequently used yeast system for heterologous protein production today, however, the toolbox of available genetic elements is rather limited. To enable more robust and cost-effective production processes for biopharmaceutical proteins and for industrial enzymes, it is crucial to understand the molecular physiology of the host, and the specific limitations that the product may exert on expression. Instead of classical genetic approaches, we applied systems biology tools to improve several aspects of the P. pastoris production platform. Combined transcriptomics, proteomics, metabolomics and flux data were used to investigate the interplay between protein production and cell metabolism. Thereby we gained insight in key regulatory effects exerted during heterologous protein production processes. These genome scale data were then further exploited for the identification of novel regulatory elements and www.elsevier.com/locate/nbt S3 SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION) New Biotechnology · Volume 31S · July 2014 the prediction of cell engineering targets for improved productivity and robustness. Additionally, the endowment of genes involved in the protein secretory pathway was analysed in a comparative genomics approach, as productivities are often limited at the level of protein folding and secretion. ACIB-9 http://dx.doi.org/10.1016/j.nbt.2014.05.1621 Martina Baumann 1,∗ , Elisabeth Gludovacz 2 , Sabine Vcelar 1 , Nicole Borth 3 Utilization of recombinase mediated cassette exchange (RMCE) for the generation of recombinant CHO cell lines with defined expression properties 1 ACIB-8 Cross-species comparison of recombinant protein secretion in CHO cells and Pichia pastoris Nils Landes 1,∗ , Andreas Maccani 2 , Christian Leitner 3 , Michael Maurer 4 , Alexandra B. Graf 4 , Minoska Valli 2 , Clemens Gruber 5 , Gerda Modarres 4 , Friedrich Altmann 5 , Brigitte Gasser 3 , Wolfgang Ernst 3 , Renate Kunert 3 , Diethard Mattanovich 3 1 ACIB, Austria Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria 3 Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria 4 School of Bioengineering, University of Applied Sciences FH Campus Wien, Vienna, Austria 5 Department of Chemistry, University of Natural Resources and Life Sciences Vienna, Austria 2 Chinese hamster ovary (CHO) cells are currently the workhorses in biopharmaceutical industry. However, yeasts such as Pichia pastoris are about to enter this field. Although a vast number of research studies has focused on characterization of each of the individual expression system, information on direct cross-species comparative studies are limited. Here we present a comprehensive comparison of recombinant P. pastoris strains and CHO cell lines, including bioprocess engineering aspects as well as systems biology approaches. Two model proteins of different complexity were chosen: monomeric and non-glycosylated human serum albumin and a more complex 3D6 single-chain Fv-Fc fusion antibody (3D6scFvFc), which is secreted as a homodimer and contains the Fc-specific glycosylation sites. High and low producing strains or cell lines of the two model proteins were established and characterized in lab-scale bioreactor cultivations. To evaluate the performance of each expression system, fed batch cultivations were performed. The production processes were characterized by monitoring biomass and product formation as well as product quality. The two host systems were then compared regarding their biomass specific secretion rates and space-time yields of the processes. Obtained results show that P. pastoris is the preferred host system for production of HSA, whereas CHO cells are more suited for the production of a complex molecule like the 3D6 scFv-Fc fusion protein. For transcriptomics and proteomics analysis steady state samples from chemostat cultures were taken. Similarities and differences between the investigated host systems as well as protein specific effects will be presented. http://dx.doi.org/10.1016/j.nbt.2014.05.1622 S4 www.elsevier.com/locate/nbt ACIB, Austria BOKU, Austria 3 BOKU/ACIB, Austria 2 For diverse applications, including generation of recombinant production cell lines, stable integration of transgenes into the genome of the host cell is a pre-requisite. The precise position of the integrated transgene is a major determining factor, both for gene expression level and long-term stability, which makes the screening for a suitable cell clone very time-and labour intensive. The utilization of site-specific Recombinase Mediated Cassette Exchange (RMCE) opened up the possibility to transfer the gene of interest (GOI) into pre-selected genomic locations with defined expression properties. In this study we developed a strategy supporting the identification of recombinant Chinese Hamster Ovary (CHO) cells with integration sites favourable to persistent high transgene expression and good gene-exchangeability. A sortable reporter gene (CD4) with a leaky start codon, flanked by heterospecific FRT (Flippase recognition targets) sequences was stably transfected into CHO cells. The leaky start codon reduces translation efficiency and allows sorting for the highest transcription rates, while the FRT sites mediate subsequent exchange of the expression cassette. Two rounds of RMCE followed by FACS sorting for top producers were performed to select for sites that allow reliable cassette exchange besides high transgene expression. The resulting master cell line can be used repeatedly for insertion of the GOI without major genetic alterations. A combination of chemical selection using alternative selection markers and FACS sorting for absence of CD4 expression as criterion for successful gene exchange enables fast establishment of recombinant cell lines with predictable expression properties. http://dx.doi.org/10.1016/j.nbt.2014.05.1623 ACIB-10 Integrated continuous refolding and precipitation of proteins in a tubular reactor Siqi Pan ∗ , Monika Zelger, Rainer Hahn Austrian Centre of Industrial Biotechnology, Austria Large amounts of therapeutic proteins expressed in Escherichia coli as inclusion bodies have created a bottleneck in downstream processing. Process integration of continuous processes is one way to alleviate this bottleneck. In view of this consideration, laboratory scale tubular reactors for the continuous processing of recombinant proteins were developed. Benefits include faster mixing, high design flexibility and efficient heat transfer. With these advantages in mind, refolding strategies like pulsed refolding and New Biotechnology · Volume 31S · July 2014 SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION) temperature leap refolding in tubular reactors were successfully performed. Additionally, the tubular reactor offer better integration between processing steps while the ability to reach steady state ensured consistent product quality. In an exemplary example, a fully continuous process for an autoprotease fusion protein was established. This includes inclusion body dissolution, refolding and autocleavage, followed by purification by precipitating out impurities. Process runs were robust over extended periods while reaction rates, product quality and yields were equal to batch processing. In an advanced setup, oxidative refolding by aeration in loop configuration, equipped with static mixers, sparging elements and air-traps was successfully performed. The integration of inline sensors also allowed the monitoring of critical process parameters. Process runs confirmed that productivity with the tubular reactor were more than twice of the batch reactor. With a growing interest in integrated continuous biomanufacturing, the clear advantages of the tubular reactor would be essential to this field. cells? Such a problem is called a (minimal) hit-ting set [(M)HS] problem–a classical problem in combinatorial optimization. The problem of calculating MHSs is often encountered in computational biology with various applications in shotgun proteomics, gene expression analysis, multi-genome alignment and metabolic engineering. Here we present MHScalculator, which allows for fast calculation of MHSs in large systems. MHScalculator was developed at acib and is freely available. We illustrate the power of MHScalculator for genomics, systems pharmacology, and systems biotechnology by aligning multiple genomes, by predicting minimal drug cocktails, and by finding minimal metabolic intervention strategies to turn microorganism into optimal cell-factories for bio-production of chemical commodities. MHScalculator is able to process large input data sets typically within a minute and allows for a fast and comprehensive analysis on standard computer infrastructure outperforming alternative solvers. http://dx.doi.org/10.1016/j.nbt.2014.05.1625 http://dx.doi.org/10.1016/j.nbt.2014.05.1624 ACIB-11 From multi-sequence alignment to metabolic engineering to minimal drug cocktails: a new hitting set calculator Juergen Zanghellini ∗ , Christian Jungreuthmayer University of Natural Resources and Life Sciences, Austria Suppose you are given a set of drugs for the treatment of malignant cell lines. Typically a single drug does not treat all malignant cells. So what is the (smallest) set of drugs killing all malignant www.elsevier.com/locate/nbt S5 BIOMOLECULAR TECHNOLOGY OF PROTEINS Biomolecular technology of proteins BIOTOP-1 Design and engineering of next generation mammalian cell factories David James The University of Sheffield, United Kingdom The majority of new biopharmaceuticals are made in Chinese hamster ovary cells, a transformed cell type originally isolated in the 1950s. The adaptability and utility of CHO cell factories derives from our exploitation of their acquired genetic/functional variation using high-throughput functional screening and selection processes which enable industry to isolate and maintain cell factories with unusual and desirable properties. However, we still have a limited understanding of enabling cellular mechanisms that underpin the ideal manufacturing phenotype. This knowledge would permit design and construction of intrinsically better cell factories using the new concepts and tools of systems and synthetic biology. We now have the potential to provide disruptive new solutions for cell and process engineering, where we will be able to create bespoke cell factories with a predictable ability to manufacture complex biopharmaceutical proteins. http://dx.doi.org/10.1016/j.nbt.2014.05.1626 New Biotechnology · Volume 31S · July 2014 penetratin. Flow cytometry measurements and confocal imaging show that human Oct4-PTD internalizes into live cells slightly more efficiently than penetratin. However, the cellular distribution differs greatly, with Oct4-PTD showing diffuse cytosolic and nuclear staining, whereas penetratin is strictly localized to a punctuate pattern in the cytoplasm. By using a Cre/loxP-based reporter system, we further show that this peptide also drives translocation of a functionally active Oct4-PTD-Cre-fusion protein into reporter cells. Finally, recombinant full length Oct4 is shown to translocate into human and mouse fibroblasts even without addition of any kind of cationic fusion tag. Our data therefore support the idea that transcription factors might be part of an alternative signalling pathway, where the proteins are transferred to neighbouring cells to actively change the behaviour of the recipient cell. http://dx.doi.org/10.1016/j.nbt.2014.05.1627 BIOTOP-3 From slow to fast: effects of growth rate on global gene expression and recombinant protein secretion in Pichia pastoris Corinna Rebnegger 1,∗ , Alexandra B. Graf 2 , Minoska Valli 3 , Brigitte Gasser 3 , Michael Maurer 2 , Diethard Mattanovich 1 1 Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria 2 School of Bioengineering, University of Applied Sciences FH-Campus Vienna, Vienna, Austria 3 Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria BIOTOP-2 Characterization of a novel cell penetrating peptide derived from human Oct4 Eva Harreither 1,∗ , Hanna Rydberg 2 , Helene Amand 2 , Vaibhav Jadhav 1 , Lukas Fliedl 3 , Christina Benda 4 , Miguel Esteban 5 , Duanqing Pei 5 , Nicole Borth 1 , Regina Grillari-Voglauer 1 , Oliver Hommerding 6 , Frank Edenhofer 7 , Bengt Nordén 2 , Johannes 1 Grillari 1 Department of Biotechnology, BOKU University Vienna, Austria Department of Chemical and Biological Engineering/Physical Chemistry, Chalmers University of Technology, Gothenburg, Sweden 3 ACIB GmbH, Austrian Center of Industrial Biotechnology, Austria 4 Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, Guangzhou, China 5 Chinese Academy of Sciences, Guangzhou, China 6 Stem Cell Engineering Group, Institute of Reconstructive Neurobiology, University of Bonn, Germany 7 Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell Biology, Julius-Maximilians-University Würzburg, Germany 2 Oct4 is a transcription factor that plays a major role for the preservation of the pluripotent state in embryonic stem cells as well as for efficient reprogramming of somatic cells to induced pluripotent stem cells (iPSC) or other progenitors. We here report that a 16 amino acid peptide representing the third helix of the human Oct4 homeodomain can internalize in mammalian cells upon conjugation to a fluorescence moiety thereby acting as a cell penetrating peptide (CPP). This Oct4-protein transduction domain (PTD) was characterized by comparison to the well studied CPP S6 www.elsevier.com/locate/nbt In Pichia pastoris recombinant protein production is related to the specific growth rate ␮. To investigate the impact of varying ␮ on productivity in more detail, we studied secretion rates and the transcriptome of a recombinant P. pastoris strain producing human serum albumin (HSA) at ␮ ranging from 0.015 to 0.15 h−1 in glucose-limited chemostat cultures. Production rates of HSA correlated positively with ␮ while product quality remained unaffected. Analysis of global gene expression showed that ribosomal genes and other genes involved in gene expression and translation as well as mitochondrial genes were upregulated with increasing growth rate, while many transcriptional regulators, carbon source responsive genes, autophagy and other proteolytic processes were downregulated at higher ␮. Expression of mating and sporulation genes peaked at intermediate ␮ of 0.05 and 0.075 h−1 , and at very slow growth (␮ = 0.015 h−1 ) many transporter genes were differentially expressed. Analysis of a subset of genes related to protein folding and secretion revealed that unfolded protein response targets such as translocation, endoplasmic reticulum genes, and cytosolic chaperones were upregulated with increasing ␮ while proteolytic degradation of secretory proteins was downregulated. We therefore conclude that a high ␮ positively affects specific protein secretion rates by acting on multiple cellular processes. http://dx.doi.org/10.1016/j.nbt.2014.05.1628 BIOMOLECULAR TECHNOLOGY OF PROTEINS New Biotechnology · Volume 31S · July 2014 BIOTOP-4 The human anti-HIV antibodies 2F5, PG9 and 2G12 differ in their proteolytic susceptibility Melanie Niemer 1,∗ , Melanie Niemer 2 , Ulrich Mehofer 2 , Juan Antonio Torres Acosta 2 , Maria Verdianz 2 , Theresa Henkel 2 , Andreas Loos 2 , Richard Strasser 2 , Daniel Maresch 2 , Thomas Rademacher 3 , Herta Steinkellner 2 , Lukas Mach 2 1 University of Natural Resources and Life Sciences Vienna, Austria University of Natural Resources and Life Sciences, Austria 3 Institute of Molecular Biotechnology, RWTH Aachen University, Germany 2 The tobacco-related species Nicotiana benthamiana has recently emerged as a promising host for the manufacturing of protein therapeutics. However, the production of recombinant proteins in N. benthamiana is frequently hampered by undesired proteolysis. Here we show that the expression of the human anti-HIV antibodies 2F5, PG9 and 2G12 in N. benthamiana leaves leads to the accumulation of discrete heavy-chain derived degradation products of 30–40 kDa. Incubation of purified 2F5 with N. benthamiana intercellular fluid resulted in rapid conversion into the 40-kDa fragment, whereas 2G12 proved largely resistant to degradation. Such a differential susceptibility to proteolytic attack was also observed when these two antibodies were exposed to various types of proteinases in vitro. While serine and cysteine proteinases are both capable of generating the 40-kDa 2F5 fragment, the 30-kDa polypeptide is most readily obtained by treatment with the latter class of enzymes. The principal cleavage sites reside within the antigen-binding domain, the VH -CH 1 linker segment and the hinge region of the antibodies. Collectively, these results indicate that down-regulation of endogenous serine and cysteine proteinase activities could be used to improve the performance of plant-based expression platforms destined for the production of biopharmaceuticals. http://dx.doi.org/10.1016/j.nbt.2014.05.1629 BIOTOP-5 Chlorite dismutases and dye-decolorizing peroxidases – similarities and differences within a structural superfamily of heme proteins Irene Schaffner ∗ , Stefan Obinger Hofbauer, Paul Furtmüller, Christian BOKU Vienna, Austria Chlorite dismutases (Clds) are heme b containing enzymes which were discovered in chlorate- and perchlorate-reducing bacteria (PCRBs) [1] but are found in many other bacterial and archaeal phyla [2]. Cld protects PCRBs from the accumulation of the harmful metabolic intermediate chlorite by degrading it to chloride and dioxygen [1]. This catalytic function turns Cld into a highly interesting enzyme for bioremediation as the environmental pollutants chlorate and chlorite are used as bleaching agents, as disinfectants, in pesticides etc. Additionally, Cld is extremely interesting from a biochemical point of view as it is the only known enzyme system which efficiently catalyzes O-O bond formation beside photosystem II. Moreover, Clds are phylogenetically related with a recently discovered new heme peroxidase family called dye-decolorizing peroxidases (Dyps). They were shown to have a unique tertiary structure with a distal heme region that is different from conventional peroxidases from plants and animals [3] but similar to Cld. Here, we show an up-to-date phylogenetic analysis of Clds and Dyps and point out their structural relationship as well as their functional differences [4]. References [1].van Ginkel CG, Rikken GB, Kroon AG, Kengen SW. Arch Microbiol 1996;166:321–6. [2].Maixner F, Wagner M, Lücker S, Pelletier E, Schmitz-Esser S, Hace K, et al. Environ Microbiol 2008;10:3043–56. [3].Sugano Y, Muramatsu R, Ichiyanagi A, Sato T, Shoda M. J Biol Chem 2007;282:36652–8. [4].Hofbauer S, Schaffner I, Furtmüller PG, Obinger C. Biotechnol J 2014 [in press]. http://dx.doi.org/10.1016/j.nbt.2014.05.1630 BIOTOP-6 Enzymatic oxidation of plant polysaccharides adsorbed to cellulose surfaces Filip Mollerup 1,∗ , Matti Häärä 2 , Kirsti Parikka 3 , Chunlin Xu 2 , Maija Tenkanen 3 , Emma Master 4 1 Department of Biotechnology and Chemical Technology, Aalto University, Finland 2 Laboratory of Wood and Paper Chemistry, Åbo Akademi University, Finland 3 Department of Food and Environmental Science, University of Helsinki, Finland 4 Department of Chemical Engineering and Applied Chemistry, University of Toronto, Canada The development of renewable energy and materials depends on intelligent utilization of abundant plant resources. While biotechnology for biofuels has received considerable attention, the potential of enzymes to tailor plant polymers for novel biopolymer synthesis is comparatively unexplored. In particular, enzymes that catalyze selective oxidation of specific hydroxyls to carbonyl and carboxyl groups can facilitate targeted chemical derivatizations of polysaccharides to create renewable polymers with preferred physical and chemical properties [1]. In this regard, galactose oxidase (GaOx) continues to be a promising catalyst for specific delivery of new functionality to plant polysaccharides. Current chemo-enzymatic protocols with GaOx begin by oxidizing galactose substituents in polysaccharides (e.g. galactoglucomannan, xyloglucan) that are suspended in solution. Oxidized polysaccharides can then be adsorbed to cellulose surfaces to modify the surface reactivity and/or barrier properties of cellulose-based materials. Although this is an elegant approach to expanding the range of cellulosic bioproducts, oxidation and derivatization of poly and oligo-saccharides can reduce their affinity to cellulose surfaces. www.elsevier.com/locate/nbt S7 BIOMOLECULAR TECHNOLOGY OF PROTEINS Accordingly, the aim of this study was to investigate the potential to oxidize xyloglucan and galactoglucomannan following adsorption of these polysaccharides to cellulose (Whatmann filter paper no. 1). Wild-type GaOx along with GaOx fused to a family 29 CBM from Piromyce equi were used in the analysis. Recent characterization of the GaOx RQW mutant fused to a cellulose-bind CBM3 will also be presented. S8 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 Reference [1].Xu C, Spadiut O, Araújo AC, Nakhai A, Brumer H. Chemo-enzymatic assembly of clickable cellulose surfaces via multivalent polysaccharides. ChemSusChem 2012;5:661–5. http://dx.doi.org/10.1016/j.nbt.2014.05.1631 New Biotechnology · Volume 31S · July 2014 OPENING CEREMONY Synthetic biology for synthetic chemistry atively high reliability), synthetic biologists will soon design and build engineered biological systems. We have used synthetic biology to create inexpensive, effective, anti-malarial drugs. Currently, malaria infects 300–500 million people and causes 1-2 million deaths each year, primarily children in Africa and Asia. One of the principal obstacles to addressing this global health threat is a lack of effective, affordable drugs. The chloroquine-based drugs that were used widely in the past have lost effectiveness because the Plasmodium parasite which causes malaria has become resistant to them. The faster-acting, more effective artemisinin-based drugs – as currently produced from plant sources – are too expensive for large-scale use in the countries where they are needed most. The development of this technology will eventually reduce the cost of artemisinin-based combination therapies significantly below their current price. To reduce the cost of these drugs and make them more widely available, we have used synthetic biology to engineer microorganisms to produce artemisinin from renewable resources. Having successfully completed the artemisinin work, we are now engineering the metabolism of the same microorganisms (Escherichia coli and Saccharomyces cerevisiae) for production of advanced biofuels and chemicals that might otherwise be produced from petroleum. Unlike ethanol, the advanced biofuels have the full fuel value of petroleum-based biofuels, will be transportable using existing infrastructure, and can be used in existing automobiles and airplanes. Similarly, the microbially sourced chemicals can be dropped into existing processes used to produce existing materials. These chemicals will be produced from natural biosynthetic pathways that exist in plants and a variety of microorganisms as well as from pathways that have no representation in the natural world. Large-scale production of these chemicals and fuels will reduce our dependence on petroleum and reduce the amount of carbon dioxide released into the atmosphere, while allowing us to take advantage of our current transportation infrastructure and products supply chains. Jay Keasling a,b,c http://dx.doi.org/10.1016/j.nbt.2014.05.1633 Opening Ceremony PL1 – 1 How to get biotechnology to work for us? Anne Glover (CBE) CSA to President Barroso, European Commission One thing scientists are not short of is imagination and our endeavours in biotechnology are an example of that. At this conference we will hear about synthetic polymers, plants as bio-factories, stem cell therapies, climate change mitigation and biofuels to mention just some of the topics. This harnessing of cellular and biomolecular processes to develop new products and processes and safeguard our planet requires more than just scientists to make sure it is the success that we hope for. We need communicative scientists who can talk about the opportunities as well as the threats of new technologies; we need citizens to engage and question what we do; we need honest and transparent businesses to translate knowledge into the economy and offer new possibilities; we need imaginative policymakers to develop smart options to allow exploitation of the knowledge that we produce and we need effective and transparent politicians who make sure that citizens realise the maximum benefit of the research that they have funded. We are living in an era dominated by biology and the bioeconomy has never held out so many possibilities. Only if we all work together will we be able to achieve the best for citizens, our lifestyle and our planet. http://dx.doi.org/10.1016/j.nbt.2014.05.1632 PL1 – 2 a Departments of Chemical Engineering and Bioengineering, University of California, Berkeley, CA 94720, United States b Synthetic Biology Department, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, United States c Joint BioEnergy Institute, Emeryville, CA 94608, United States Synthetic biology is the design and construction of new biological entities such as enzymes, genetic circuits, and cells or the redesign of existing biological systems. Synthetic biology builds on the advances in molecular, cell, and systems biology and seeks to transform biology in the same way that synthesis transformed chemistry and integrated circuit design transformed computing. The element that distinguishes synthetic biology from traditional molecular and cellular biology is the focus on the design and construction of core components (parts of enzymes, genetic circuits, metabolic pathways, etc.) that can be modeled, understood, and tuned to meet specific performance criteria, and the assembly of these smaller parts and devices into larger integrated systems that solve specific problems. Just as engineers now design integrated circuits based on the known physical properties of materials and then fabricate functioning circuits and entire processors (with rel- www.elsevier.com/locate/nbt S9 MONDAY 14 JULY BIOCHEMICAL ENGINEERING JOURNAL YOUNG INVESTIGATOR AWARD LECTURE Monday 14 July Biochemical Engineering Journal Investigator Award Lecture Young YI – 1 A glimpse into the future of mammalian cell culture process development: innovative approaches to impact time to clinic, product quality, and cost of process development and commercial manufacturing Chetan Goudar Amgen Inc., United States There has been remarkable progress in cell line generation and cell culture processes over the past 2 decades. Expression levels have increased by orders of magnitude and a broad spectrum of complex biotherapeutics have been successfully manufactured at commercial scale. Despite these advances, it is important to recognize that the future poses fundamentally different challenges. While development costs and speed to in vivo validation of biology will face increasing pressure, decentralized manufacturing is increasingly becoming a necessity for global reach of a S10 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 biotherapeutic, both from regulatory and economic standpoints, which has implications for commercial manufacturing. Increased regulatory clarity around biosimilars has intensified efforts in this space and matching product quality of molecules developed decades ago with current processes is most effectively done with robust mechanistic understanding of cellular processes rather than by purely empirical approaches. In this changing landscape, recent advances in systems biology and a renewed interest in continuous perfusion cultivation offer avenues to bring about paradigm shifts in cell culture process development and commercial manufacturing. An approach for streamlined cell line and early-stage cell culture process development will be presented which can accelerate time to human clinical testing by as much as 8 months. Application of a poly-omics systems biology approach for identifying modulators for a critical product quality attribute will be presented followed by validation of these targets using cell line engineering. Recent dramatic productivity enhancements from continuous perfusion cultures will be presented and the implications of these advances on next-generation commercial manufacturing facilities will be discussed. http://dx.doi.org/10.1016/j.nbt.2014.05.1634 New Biotechnology · Volume 31S · July 2014 Symposium 1: Stem cell applications and gene therapies: where are we? O1-1 Engineering synthetic stem cell niches Matthias P. Lutolf Laboratory of Stem Cell Bioengineering and Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland In vivo, the behavior of stem cells in a tissue is governed by the three-dimensional (3D) microenvironment, or ‘niche’, which involves a dynamic interplay between biochemical and mechanical signals provided by the extra-cellular matrix (ECM), cell-cell interactions and soluble factors. The complexity of niches and the context-dependent cell-responses that arise from these interactions have posed a major challenge to understanding the underlying regulatory mechanisms and to identify artificial niches controlling the behaviour of specific stem cell types. To systematically dissect the role of the various interacting factors that determine stem cell fate in a 3D context, we have developed experimental paradigms to simultaneously generate hundreds to thousands of unique microenvironments and probe their effects on cell fate. In this talk I will discuss our approach by way of several experiments in which we have measured the combined effects of microenvironment stiffness, proteolytic degradation, and three classes of signaling proteins on pluripotent stem cell fate, unveiling a comprehensive map of the interactive involvement of these parameters in regulating self-renewal and early differentiation. Our approach is broadly applicable to gain a systems-level understanding of multifactorial 3D cell-matrix interactions and opens the door for discovering unique artificial niches that control the behavior of difficult-to-culture mammalian stem cells. http://dx.doi.org/10.1016/j.nbt.2014.05.1635 O1-2 A quick potency assay for osteogenic and chondrogenic differentiation of adipose derived stem cells Eleni Oberbauer 1,∗ , Florian Hildner 2 , Ara Hacobian 1 , Susanne Wolbank 1 , Carolin Steffenhagen 1 , Georg Feichtinger 1 , Anja Peterbauer 2 , Christian Gabriel 2 , Heinz Redl 1 1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Austria 2 Red Cross Blood Transfusion Service of Upper Austria, Austria The field of bone- and cartilage regeneration has evoked strong interest in tissue engineering. Osteogenic and chondrogenic differentiation potential of adipose derived stem cells (ASC) are challenging and promising for bone and cartilage repair. Current in vitro methods to analyze the differentiation capacity are time consuming and thus we designed and established novel enhancer and tissue-specific promoter for osteogenic and chondrogenic differentiation of ASC together with a quick potency bioassay. For this, osteocalcin or collagen type II promoter was coupled SYMPOSIUM 1: STEM CELL APPLICATIONS AND GENE THERAPIES: WHERE ARE WE? to a strong viral enhancer for signal amplification. Human primary ASC were co-transfected with luciferase based reportergenes pCMVE/mOCP-MetLuc (osteocalcin) or pCMVE/mACDC-MetLuc (collagen type II) together with renilla control plasmid. For transfection, a mild lipofection was applied and a green fluorescence protein (GFP) control was included to assess transfection efficiency. Secreted metridia luciferase allows to measure promoter activation in the supernatant without need of cell lysing. The activity of the reportergenes was enhanced in transfected ASC treated with osteogenic or chondrogenic differentiation media compared to control condition. Osteogenic differentiation was confirmed with Alizarin red quantitative and histological stainings whereas chondrogenic differentiation was confirmed with collagen type II staining. These findings were further verified by qRT-PCR. Although osteocalcin and collagen type II are late markers, the activation of the signal-amplified and sensitive reportergenes can already be determined after 3 days of differentiation in vitro. http://dx.doi.org/10.1016/j.nbt.2014.05.1636 O1-3 Repeated systemic administration of human adiposederived stem cells attenuate diabetic nephropathy in the rats Xue-Yuan Bai ∗ , Yuxiang Ma, Xiangmei Chen Department of Nephrology, Chinese PLA General Hospital, China Diabetic nephropathy (DN) is a major complication of diabetes and represents the leading cause of end-stage renal disease worldwide. Unfortunately, once the patients develop overt proteinuria, there is no cure for DN. Therefore, the availability of a strategy aimed to delay or revert DN would be highly desirable. To investigate the role of human adipose-derived stem cells (ASCs) in treatment of diabetic nephropathy, Sparague-Dawley rats were made diabetes by intraperitoneal injection of streptozotocin after uninephrectomy. After 12 weeks proteinuria was well-established. The rats received injection of human ASCs via tail vein at 5 × 106 every 4 weeks for five times. Reduction of proteinuria was not observed in diabetic rats until 24 weeks after three doses of ASCs. However, since 28 weeks, urinary protein excretion was significantly suppressed, and persisted up to 32 weeks after streptozotocin. Hypoalbuminemia and hyperlipidemia were also improved in ASCs treated group. ASCs significantly attenuated glomerulus hypertrophy and renal tubular interstitial injury as well as downregulation of podocyte markers, WT-1 and snaptopodin. Hoechst labeled ASCs were injected into DN rat via tail vein. Cells were detected in lung and spleen, and peritubular regions, but rarely in glomeruli and pancreas within 48 hours after injection. Gene expression of human Alu could be detected in rat lung and spleen till 4 weeks after injection. The ASCs did not improve hyperglycemia and pancreatic damage. These findings indicate that repeated intravenous ASCs can reduce diabetic kidney damage in rats even at the progressive stage. http://dx.doi.org/10.1016/j.nbt.2014.05.1637 www.elsevier.com/locate/nbt S11 New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 1: STEM CELL APPLICATIONS AND GENE THERAPIES: WHERE ARE WE? O1-4 Producing and harvesting culture-derived platelets with functional activity from blood stem cells William Miller 1,∗ , Alaina Schlinker 1 , Katherine Christopher Wegener 2 , K. Augustine Min 2 1 2 Radwanski 2 , Northwestern University, United States Fresenius Kabi, USA Platelet production in culture by megakaryocytic cells (Mks) derived from hematopoietic stem and progenitor cells (HSPCs) would supplement donations, but it is very challenging to purify the platelets. As an alternative to multi-step centrifugation, we used a polycarbonate spinning membrane with 4-␮m cylindrical pores. Because platelet release is asynchronous, we explored whether we could harvest platelets generated early in culture and reseed immature Mks to generate platelets later in culture. Experiments with immortalized Mks and apheresis platelets showed efficient platelet recovery and the exclusion of Mks. Recovered platelets had little pre-activation and the Mks retained viability and proplatelet formation. Next, primary CD34+ HSPCS were differentiated to Mks that released platelets. As for the Mk line, primary Mks were excluded from the platelet fraction and the viability and ploidy distribution of recovered Mks were similar to those of input Mks. Recovered platelets expressed surface markers and spread after activation by thrombin in a similar manner to unprocessed platelets. However, culture-derived platelet recovery was lower than for apheresis platelets (70 vs. 90% at 3000 rpm). This is likely due to the larger size of culture-derived platelets and preplatelets (5.6 vs. 3.3 ␮m). After screening experiments with even larger red blood cells (7.3 ␮m), we increased culturederived platelet recovery with essentially no Mk contamination by decreasing the rotation rate to 2000 rpm. Recovered platelets showed minimal pre-activation and were activated by thrombin. Importantly, Mks in the cell fraction that were returned to culture released platelets that were harvested two days later. http://dx.doi.org/10.1016/j.nbt.2014.05.1638 O1-5 Adipose derived stem cells respond to in vitro extracorporeal shockwave treatment with increased stemness and multipotency Christina Schuh 1,∗ , Philipp Heher 1 , Anna Weihs 2 , Asmita Banerjee 1 , Susanne Wolbank 1 , Rainer Mittermayr 1 , Heinz Redl 1 , Dominik Rünzler 2 , Andreas Teuschl 2 1 LBI for Experimental and Clinical Traumatology, Austria University of Applied Sciences Technikum Wien – Department of Biochemical Engineering, Austria 2 Tissue resident adipose derived progenitor/stem cells (ASCs) are a promising tool for tissue engineering, addressing the problem of tissue and organ shortage. Limiting factors for the use of ASCs are donor variation and senescence, loss of differentiation capacity as a consequence to loss of multipotency. For several decades extracorporeal shockwave treatment (ESWT) has been proven to be a S12 www.elsevier.com/locate/nbt suitable clinical tool to improve regeneration of a variety of tissues, however, the mechanisms underlying these beneficial effects still remain widely unknown. In this study we address the effects of ESWT onto ASCs and their stemness as well as differentiation potential in high passages. In our study we show that human and rat ASCs respond strongly to repetitive shockwave treatment in vitro, resulting in maintenance and significant elevation of mesenchymal markers (flow cytometry: CD73, CD90, CD105), while cell viability and proliferation remain at a comparable level to control group. Another effect observed was a significant increase in differentiation capacity into osteogenic (von Kossa staining; PCR: osteocalcin, biglycan) and adipogenic lineage (Oilred O staining) as well as into Schwann like cells (flow cytometry: P75, S100, P0) in high passages. Our results indicate that ESWT preserves stemness and multipotency of ASCs even in higher passages after extensive expansion. Hence, ESWT might be a promising tool to improve the quality and consistency of ASCs for cell therapy in tissue engineering and regenerative medicine. Acknowledgement: Financial support from FFG (#818412) and the “City of Vienna Competence Team Tissue Engineering Bioreactors” is gratefully acknowledged. http://dx.doi.org/10.1016/j.nbt.2014.05.1639 O1-6 Engineering bacteria for the discovery of potential therapeutic compounds against protein misfolding diseases Georgios Skretas 1,∗ , Dafni Delivoria 2 , Ilias Papaevgeniou 2 , Niki Chondrogianni 2 Matis 2 , Nikoletta 1 National Hellenic Research Foundation, Greece Institute of Biology, Medicinal Chemistry & Biotechnology – National Hellenic Research Foundation, Greece 2 It has now been widely recognized that many incurable diseases with enormous socioeconomic impact, such as Alzheimer’s disease, Parkinson’s disease, type 2 diabetes, etc., are initiated by a common mechanism: the misfolding of specific proteins. Here, we describe the use of engineered bacterial cells as a platform for the discovery of potential therapeutics against such protein misfolding diseases (PMDs). The topic of the described research is the application of molecular evolution approaches for the discovery of compounds that rescue the misfolding of PMD-associated proteins. To achieve this, Escherichia coli cells are first engineered to biosynthesize large libraries of test compounds with high structural diversity. Then, the same cells are modified further so that they allow the identification of the rare molecules that correct the folding of particular misfolding-prone and PMD-associated proteins (MisPs) with the use of a genetic screen. Lead compounds identified by this initial screen, are then subjected to more detailed evaluation by biochemical and biophysical methods of protein analysis, and their ability to inhibit MisP-induced pathogenicity is tested using appropriate human cell assays or in vivo models of the disease of interest. The molecules capable of rescuing the misfolding of the target MisP and of antagonizing its associated New Biotechnology · Volume 31S · July 2014 pathogenicity become drug candidates against the specific disease. We will describe our efforts to identify such “pharmacological chaperones” against the misfolding of the amyloid ␤ (A␤) peptide and of certain carcinogenic misfolded variants of human p53, with the aim of developing potentially therapeutic compounds against Alzheimer’s disease and cancer, respectively. http://dx.doi.org/10.1016/j.nbt.2014.05.1640 O1-7 Computational prediction of associations between psoriasis, rheumatoid arthritis and osteoarthritis Tuba Sevimoglu ∗ , Kazim Yalcin Arga Marmara University, Turkey Microarray technologies have enabled rapid and efficient expression profiling of thousands of genes simultaneously. Microarray studies on disease datasets have already revealed genes that could be implicated in the pathogenesis of that disease thus opening the path to accurate diagnosis and potential biomarkers. This study aims at comparative analysis of three common diseases with important genetic origin: Osteoarthritis (OA), rheumatoid arthritis (RA) and psoriasis. SYMPOSIUM 1: STEM CELL APPLICATIONS AND GENE THERAPIES: WHERE ARE WE? A comparison of gene expression data from five independent studies, obtained from Gene Expression Omnibus has been performed. Each dataset was statistically analyzed in order to identify differentially expressed genes (DEGs). Proteins encoded by DEGs were determined and integrated with protein-protein interaction (ppi) data for further analyses and to identify hub proteins. Enrichment analyses were performed to map the interconnectivities between diseases and biological pathways. Comparative analyses indicated that six DEGs were common between all three diseases, which are immune related and have associations to cardiovascular diseases. Enrichment analyses lead to a significant association with PPAR signaling pathway. There are 34 Transcriptional Factors regulating these DEGs. The hub proteins in the disease ppi network are: CEBPD and MMP1. This study suggests a genetic cause common between psoriasis, OA and RA. The commonality between psoriasis and RA comes from the fact that they are both autoimmune diseases. In the case of OA and psoriasis, Psoriatic Arthritis might be the link that connects psoriasis to OA. This study will elucidate molecular and cellular mechanisms which will lead to a better understanding and better drug design for these diseases. http://dx.doi.org/10.1016/j.nbt.2014.05.1641 www.elsevier.com/locate/nbt S13 New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 2: PLANTS FOR THE PRODUCTION OF HIGH VALUE CHEMICALS Symposium 2: Plants for the production of high value chemicals O2-1 Cultured plant cambial meristematic cells as a chassis for the production of pharmaceuticals Susan Howat 1 , Marisol Ochoa Villarrea 1 , Rabia Amir 1 , Eunjung Kwon 1 , Zejun Yan 1 , Young-Woo Jin 2 , Eun-Kyong Lee 2 , Gary J. Loake 1,∗ 1 Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh, King’s Buildings, Edinburgh EH9 3JR, UK 2 Unhwa Corp., 874-1, 2Ga, Wooah-Dong, Dukjin-gu, Jeonju, South Korea selection of four markers had significant genetic effect. In the two groups of ++/+ genotype, the average fiber strength of individuals were 31.21-32.62 cN/tex, and 30.77-32.50 cN/tex for the +/− genotype, selection effects of single marker were 0.80-1.51cN/tex; In the groups for QTL-1 × QTL-3 and QTL-2 × QTL-3Combinations, the average fiber strength of individuals while pyramiding two QTLs were 33.40-34.08 cN/tex, the selection effects were 2.73-3.56 cN/tex which compared to the plants without the two QTL, and the selection effects of 1.12-3.02 cN/tex compared to the plants with one of the two QTL. In addition, QTL-1 and QTL-2 might be the same QTL, QTL-2 and QTL-3 had obvious epistasis effect. So, This shows it is feasibility to use the molecular marker for assisting the pyramiding selection of fiber strength. http://dx.doi.org/10.1016/j.nbt.2014.05.1643 A plethora of important, chemically diverse natural products are derived from plants. In principle, plant cell culture offers an attractive production platform for some natural products but often is not a commercially viable strategy because of difficulties associated with culturing dedifferentiated plant cells (DDCs) on an industrial scale. To address this issue we have isolated and cultured innately undifferentiated cambial meristematic cells (CMCs). Utilizing a combination of deep sequencing technologies, we identified marker genes and transcriptional programs consistent with a stem cell identity. CMCs derived from Taxus cuspidata and Panax ginseng, sources of the key anticancer drug, paclitaxel and neuroprotective ginsenosides, respectively, circumvented obstacles routinely associated with the commercial growth of DDCs. Subsequent molecular strategies uncovered a network of regulators that control the expression levels of genes integral to the biosynthesis of these key natural products. We envisage both natural and where appropriate, engineered CMCs, will provide a cost-effective and environmentally friendly platform for the sustainable production of a wide variety of important plant-derived pharmaceuticals. http://dx.doi.org/10.1016/j.nbt.2014.05.1642 O2-2 Molecular marker-assisted selection and pyramiding effect of major QTLs for cotton fiber strength Youlu Yuan ∗ , Tiankang Wang, Yuzhen Shi, Haihong Shang, Aiying Liu, Junwen Li, Juwu Gong, Tao Wang, Wan-kui Gong, Tingting Chen, Botao Li Institute of Cotton Research of Chinese Academy of Agricultural Sciences/State Key Laboratory of Cotton Biology/Key Laboratory of Biological and Genetic Breeding of Cotton, The Ministry of Agriculture, China Two elite fiber quality materials 0-153 and Xinluzao 24, and two commercial cotton cultivars (lines) Lumianyan 28 and Jimian 516 were used as parents to develop two double-cross combinations (Jimian 516 × 0-153) × (Jimian 516 × Xinluzao24)Pop1, (Lumianyan 28 × 0-153) × (Lumianyan 28 × Xinluzao 24)Pop2, Four SSR markers, which were linked to three QTLs of fiber strength and derived from 0-153 or Xinluzao 24, were used respectively to study the efficiency of molecular marker-assisted selection and pyramiding effect of fiber strength. The result showed that assisted S14 www.elsevier.com/locate/nbt O2-3 The CO2 microalgae biorefinery: high value products and biofuels using halophilic microalgae in the “D-Factory” Patricia Harvey 1,∗ , David Bailey 1 , Ami Ben-Amotz 2 , Vitor Verdelho 3 , Guy Harris 4 , David Rooke 4 , Herre Hoekstra 5 , Paul Goacher 6 , Joao Crespi 7 , Guido Reinhardt 8 , Laura Martinelli 9 , Declan Schroder 10 , Richard Pipe 10 , Nadine Igl-Schmid 11 , Antonis Kokossis 12 , Karin Perrson 13 1 University of Greenwich, United Kingdom Nature Beta Technologies Ltd., (NBT), Israel 3 A4F AlgaFuel, Portugal 4 Dynamic Extractions, United Kingdom 5 Evodos, Germany 6 Hafren Investments, United Kingdom 7 Instituto de Biologia Experimental e Tecnológica (IBET), Portugal 8 IFEU – Institute for Energy and Environmental Research, United States 9 IN SRL Italy, Italy 10 Marine Biological Association of the UK, United Kingdom 11 NATECO2 GmbH & Co. KG, Germany 12 National Technical University of Athens, Greece 13 SP Technical Research Institute of Sweden AB, Sweden 2 Fuel-only algal systems are not economically feasible because yields are too low and costs too high for producing microalgal biomass compared to using agricultural residues e.g. straw. Biorefineries which integrate biomass conversion processes and equipment to produce fuels, power and chemicals from biomass, offer a solution. The CO2 microalgae biorefinery (D-Factory) is a 10 million Euro FP7-funded project which will cultivate the microalga Dunaliella in highly saline non-potable waters in photobioreactors and open raceways and apply biorefinery concepts and European innovations in biomass processing technologies to develop a basket of compounds from Dunaliella biomass, including the high value nutraceutical, ␤-carotene, and glycerol. Glycerol now finds markets both as a green chemical intermediate and as a biofuel in CHP applications as a result of novel combustion technology. Driving down costs by recovering the entire biomass of Dunaliella cells from saline cultivation water poses one of the many challenges for the D-Factory because Dunaliella cells are both motile, and do not possess an external cell wall, making them highly susceptible to cell rupture. Controlling expression of desired metabolic pathways to deliver the desired portfolio of compounds flexibly and New Biotechnology · Volume 31S · July 2014 sustainably to meet market demand is another. The first prototype D-Factory in Europe will be operational in 48 months, and will serve as a robust manifestation of the business case for global investment in algae biorefineries and in large-scale production of microalgae. http://dx.doi.org/10.1016/j.nbt.2014.05.1644 O2-4 Exploiting nature’s chemists: high value bioactive compounds from algae Christine Edwards ∗ , Linda Lawton IDEAS/Robert Gordon University, United Kingdom Algae are an essential component of global ecosystems using light and carbon dioxide to produce organic carbon and oxygen. Consequently they are found in diverse habitats including terrestrial and aquatic habitats, hot springs to Antarctic mats. The ecological diversity has resulted in chemical diversity and they increasingly investigated as sources of new chemical entities in drug discovery programs, in particular anti-cancer. In order to exploit the chemical diversity in target algae, 400–500 L algae per month, are grown in parallel batch cultures. Cells are harvested and bioactive compounds are extracted using methanol, cleaned up by automated flash chromatography with final polish by preparative high performance liquid chromatography (HPLC). Throughout the process quality and quantity of compounds in extracts/fractions are tracked by ultra performance liquid chromatography–photodiode array-mass spectrometry (UPLC-PDA-MS) to ensure high purity is achieved. Due to the highly toxic nature of the compounds, extreme precautions must be exercised throughout processing, with frequent review. In addition, shipping is a challenge as they are categorised as extremely dangerous according to transport regulations so they usual couriers such as FedEx will not take them. In addition, as they are considered as potential weapons of mass destruction, export licence and control is essential. Despite all the challenges 14 products are globally available via a fine biochemical company, generating in excess of £120 K per annum. http://dx.doi.org/10.1016/j.nbt.2014.05.1645 O2-5 Use of synthetic biology in creating high-value metal nanoparticles from phytoremediated waste Matthew Edmundson ∗ , Michael Capeness, Louise Horsfall Edinburgh University, United Kingdom Metal and metalloid contamination of land is a major problem on former industrial sites in the UK and in the wider world. Phytoremediation has been used as a method to clean up this contamination in the past but disposal of the contaminated plant waste is still a problem. The Cleaning Land for Wealth (CL4 W) SYMPOSIUM 2: PLANTS FOR THE PRODUCTION OF HIGH VALUE CHEMICALS consortium is looking into financially incentivising land decontamination by producing high-value products from the waste, with our initial case studies focusing on arsenic, platinum and palladium. CL4 W is following a number of research tracks, including investigating the optimal plant species for phytoremediating a number of different metals and developing cost-effective methods of processing the phytoremediation waste. We are also using synthetic biology tools to employ micro-organisms in harvesting heavy metals from the waste, engineering bacteria to convert these metals into high-value nanoparticles for use in medicine and industry, and looking into extracting energy from the left-over plant material. The data presented here will detail the progress made so far on the CL4 W project, highlighting our work in evaluating contaminated industrial sites and our current model of assessing the costs and benefits of the project. As part of the nanoparticle production research track we are working with bacteria able to reduce platinum and palladium, and have identified a number of the genes involved in these pathways. We have also engineered a strain of E. coli able to tolerate high levels of arsenic, and which at the same time produces nano-scale structures when in the presence of arsenic. http://dx.doi.org/10.1016/j.nbt.2014.05.1646 O2-6 Gene isolation and its identification of salinity stress on G. hirsutum L. Wuwei Ye Institute of Cotton Research, CAAS, China Soil salinization has become a serious global problem affecting the agricultural development and the ecological environment. Salinityas one of the most important abiotic stresses in the world, severely limits the production of crop. Saline-alkali land in our country is widely distributed with the character of multi types and serious salt-deposition. In order to carry out the utilization of saline-alkali land efficiently, it is necessary to develop the agriculture on the saline-alkali land. Cotton, the major cash crop in China, is playing a crucial role in national economic development. China, with less cultivated lands and more people, faces the contradiction between food and cotton, which seriously affects the cultivation and production of cotton. Therefore, it is an effective way to farm saline land and to enhance the sustainable agricultural development by develop the salinity-tolerant varieties of cotton. Identification of salinity-tolerance also plays a vital role on cotton breeding. 11 salt-tolerance related genes, H+ -pyrophosphatase gene and S-adenosylmethionine synthetase gene and others, were cloned from the salt-tolerance material on Gossypium hirsutum, which were named GhVP and GhSAMS, respectively. The bioinformatics analysis and their transformed accessions were tested and identified. http://dx.doi.org/10.1016/j.nbt.2014.05.1647 www.elsevier.com/locate/nbt S15 New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 3: GLYCOBIOTECHNOLOGY Symposium 3: Glycobiotechnology O3-1 Development of new synthetic and analytical tools in glycobiotechnology Sabine L. Flitsch The University of Manchester, United Kingdom Carbohydrates provide the largest biomass on Earth and are central to many aspects of biotechnology with applications in biofuels, biomaterials, food and medicine. Carbohydrates are complex biomolecules and there is an urgent need to develop robust synthetic and analytical methodologies to fully exploit opportunities presented by glycobiotechnology. We have developed a toolbox for the synthesis and analysis of complex carbohydrates and their function with a focus on (i) chemoenzymatic synthesis of glycoconjugates such as glycopeptides, (ii) glycoarrays to study glycoenzyme activity and discover carbohydrate-protein interactions and (iii) ion mobility mass spectrometry for high resolution structural analysis of carbohydrates and (iv) mass spectrometry for the label free identification of carbohydrate-binding proteins [1–5]. References [1].Both, et al. Nat Chem 2014;6(1):65. [2].Noble, et al. J Am Chem Soc 2012;134(31):13010–7. [3].Šardzík, et al. J Am Chem Soc 2012;134(10):4521–4. [4].Castangia, et al. Angew Chem 2012;51(52):13016–8. [5].Rannes, et al. J Am Chem Soc 2011;133(22):8436–9. http://dx.doi.org/10.1016/j.nbt.2014.05.1648 O3-2 Pichia pastoris GlycoDelete: the way out when N-glycans are a burden Bram Laukens 1,2,∗ , Katrien Claes 1,2 , Charlot De Wachter 1,2 , Nico Callewaert 1,2 1 Unit for Medical Biotechnology, Inflammation Research Centre (IRC), VIBUGent, Technologiepark 927, B-9052 Ghent-Zwijnaarde, Belgium 2 Department of Biochemistry and Microbiology, Laboratory for Protein Biochemistry and Biomolecular Engineering, UGent, K.L.-Ledeganckstraat 35, B-9000 Ghent, Belgium In order to simplify bio-production of glycoproteins produced in Pichia pastoris, new approaches need to be developed to deal with the heterogeneity that arises from the fungal-type Nglycosylation. Here, we present a method that enables the in-vivo removal of N-glycans during production of recombinant glycoproteins in Fungi. The methylotrophic yeast P. pastoris is an excellent host for recombinant protein production thanks to the strong, tightly controlled methanol-inducible promoter system and the wellestablished upscaling processes. Moreover, Pichia does not secrete a lot of endogenous proteins which facilitates downstream processing. However, the sometimes very extensive oligomannosetype N-glycosylation pattern can be of concern during downstream processing and characterization of the recombinant product. S16 www.elsevier.com/locate/nbt Although some tools are available to produce proteins with tailored N-glycans in P. pastoris (GlycoSwitch® ), such engineering process is costly and time-consuming. We developed a method that enables the in vivo removal of N-glycans by inducible co-expression of a fungal endoglycosidase, removing N-glycosylation associated heterogeneity without impairing strain viability and growth. This approach, called Pichia GlycoDelete, drastically reduces sample heterogeneity, facilitates protein purification and alleviates many of the problems that are associated with yeast-type N-glycosylation. http://dx.doi.org/10.1016/j.nbt.2014.05.1649 O3-3 Enzymatic remodelling of chitin for agrochemical applications Rémi Chambon 1,∗ , Guillaume Despras 2 , Dominique Urban 2 , Boris Vauzeilles 3 , Jean-Marie Beau 3 , Sébastien Fort 4 , Sylvie Armand 4 , Sylvain Cottaz 4 1 CERMAV-CNRS, Grenoble Université Paris Sud and CNRS – Institut de Chimie Moléculaire et des Matériaux d’Orsay, France 3 Université Paris Sud and CNRS – Institut de Chimie Moléculaire et des Matériaux d’Orsay – Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, France 4 Centre de Recherches sur les Macromolécules Végétales – CNRS, France 2 Lipo-chitinoligosaccharides (LCOs) are produced by bacteria (genus Rhizobium) and arbuscular mycorrhizal fungi, and are involved in the establishment of symbiosis with plants. They promote the assimilation of atmospheric nitrogen in legumes (Nod factors [1]) and intake of water and nutrients in 80% of plant species (Myc factors [2]). These Nod and Myc factors consist of a backbone of four or five N-acetylglucosamine residues modified by a fatty acid on the non-reducing unit and have various decorations on some hydroxyl groups. The LCOs, which are active at subnanomolar concentration, are produced naturally in extremely small quantities. The detailed analysis of their recognition mechanisms as well as their agrochemicals applications requires the development of efficient synthetic methods. In this context we developed a chemo-enzymatic chitin remodeling approach to access LCOs via combined use of the chitin-N-deacetylase NodB (Sinorhizobium meliloti) and chitin oligomers chemically functionalized on the reducing unit [3]. References [1].Lerouge P, Roche P, Faucher C, Maillet F, Truchet G, Prome JC, et al. Nature 1990;344(6268):781–4. [2].Maillet F, Poinsot V, Andre O, Puech-Pages V, Haouy A, Gueunier M, et al. Nature 2011;469(7328):58–63. [3].This project is supported by the ANR (ANR-10-CD2I-0008, BioCOS 2010-2014). http://dx.doi.org/10.1016/j.nbt.2014.05.1650 New Biotechnology · Volume 31S · July 2014 O3-4 Polysaccharides production by autotrophic cultures of microalgae Giuseppe Olivieri 1 , Renato S. Coellho 2 , Telma T. Franco 2 , Antonino Pollio 3 , Antonio Marzocchella 1,∗ 1 DICMaPI – Università degli Studi di Napoli Federico II, Italy Faculdade de Engenharia Química – University of Campinas, Brazil 3 Biological Science Department – Università degli Studi di Napoli “Federico II”, Italy 2 Light energy and CO2 may be considered as the most abundant and cheap feedstock for bioprocesses aimed to produce energy vectors as well as green chemical building blocks. Microalgae can be cultivated on waste streams of gas (CO2 polluted) and liquids (industrial effluents and salt supplements) for producing biofuels and carbohydrates, a building block for bioplastics production. The aim of this contribute is to report about recent joint progress of a research activity carried out in Campinas (BR) and in Napoli (IT). The study regards autotrophic cultures of microalgae selected to produce a significant amount of carbohydrates coupled, if possible, with lipids as biofuel resource. Selected strains were autochthon in Brazil and from the collection available at the University of Napoli ACUF (http://www.biologiavegetale.unina.it/acuf.html). Microalgae were grown in flasks and then inoculated in the photobioreactors. Both the broth and the cells have been characterized for cultures carried out under a wide interval of operating conditions. Main measured data were cell concentration, total organic carbon (TOC) in the liquid phase, polysaccharide concentration and protein concentration. The attention was paid on both the extracellular and intracellular polysaccharides. http://dx.doi.org/10.1016/j.nbt.2014.05.1651 O3-5 Synthesis of potential prebiotics using Pseudomonas syringae DC3000 levansucrase Lsc3 Triinu Visnapuu 1,∗ , Anneli Aasamets 1 , Karin Mardo 1 , Eerik Jõgi 1 , Heiki Vija 2 , Tiina Alamäe 1 1 2 University of Tartu, Estonia National Institute of Chemical Physics and Biophysics, Estonia Plant-derived inulin-type oligofructans are considered to be effective prebiotics that function as specific growth substrates for beneficial gut bacteria. The effects of levan-type FOS (fructooligosaccharides) containing ␤-2,6 linkages are poorly studied as they are not commercially available. Some studies prove their enhanced prebiotic effect [1]. Polymeric levan has potential applications as anti-cancer, anti-inflammatory and immune stimulating agent. Levansucrases (EC 2.4.1.10) are bacterial enzymes belonging to GH68 family of glycoside hydrolases. Pseudomonas syringae DC3000 encodes three levansucrases (Lsc1, Lsc2, Lsc3). Recombinant Lsc3 splits sucrose and synthesizes ␤-2,6-linked FOS, polymeric levan and also heterooligofructans when transfructosy- SYMPOSIUM 3: GLYCOBIOTECHNOLOGY lating alternative acceptors [2]. Lsc3 has very high catalytic activity (kcat ∼500 1/s), stability and therefore a high biotechnological potential. In this study, we optimized levan and FOS production by Lsc3 protein. High-performance liquid chromatography system coupled with ELS detector was used to detect and quantify saccharides, levan was quantified spectrophotometrically. We showed that Lsc3 produced up to 15.4 g of FOS per mg of protein under optimized conditions. Product yield and spectrum depended on reaction conditions. Also, permeabilized bacterial cells expressing levansucrase were shown to serve as effective catalyst for FOS production. We developed a method for enzymatic production of levan-type FOS from sucrose and a simple yeast-based method for the removal of monosaccharides that form as by-products of the reaction. Acknowledgements: This work was supported by ERF grant 3.2.0701.12-0041 (SLOMR12215T) managed by Archimedes Foundation and an ETF grant GLOMR9072. References [1].Marx SP, et al. FEMS Microbiol Lett 2000;182:163–9. [2].Visnapuu T, et al. J Biotechnol 2011;155:338–49. http://dx.doi.org/10.1016/j.nbt.2014.05.1652 O3-6 7-Hydroxydehydronuciferine induces human melanoma A375.S2 autophagy and apoptosis and inhibits metastasis in vitro and in vivo Hui Min Wang Kaohsiung Medical University, Taiwan Melanoma is the deadliest cancer. We identified 7hydroxydehydronuciferine (7-HDNF) isolated from the leaves of Nelumbo nucifera Gaertn cv. Rosa-plena to be a bioactive agent against human melanoma A375.S2 cells. 7hydroxydehydronuciferine (7-HDNF) was known to induce autophagy and apoptosis response mechanisms, and antimigratory activity of melanoma in vitro and in vivo. Cell proliferation assay was used to test cell viability. Acridine orange (AO) staining and flow analysis were applied to observe cell morphology. The apoptotic cell death ratio was measured via two-dimensional flow cytometry by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double stained. Western blot was applied to examine protein expressions whereas wound healing assay was to examine cell activity. Strong anticancer effects of 7-HDNF exhibited in a dose-dependent manner, and displayed minor cytotoxicities on normal human skin cells.7-HDNF induced the formation of intracellular vacuoles and the augmentation of acidic vesicular organelles (AVO). 7-HDNF increased the cellular arrest in cell cycle at G2/M phase. Cellular membrane asymmetry lost was confirmed. Protein expressions were discovered to verify autophagy and apoptosis response mechanisms sharing the associated pathways. 7-HDNFpresented the high-quality anti-migratory activity. 7-HDNF inhibited melanoma tumor growth in mice xenograft model, accompanied with a decrease www.elsevier.com/locate/nbt S17 SYMPOSIUM 3: GLYCOBIOTECHNOLOGY of phosphorylation of AKT. We demonstrated the mechanism of this compound starting with the formation and accumulation of AVO leading to autophagy. 7-HDNF caused the cellular membrane asymmetry lost, triggering the G2/M cell cycle arrest in caspase-dependent apoptosis. 7-HDNF presented high-quality S18 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 anti-migratory bio-functions and inhibited melanoma tumor growth in mice xenograft model. http://dx.doi.org/10.1016/j.nbt.2014.05.1653 New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 4: ROBUST BIOCATALYSTS FOR THE PRODUCTION OF NOVEL BIO-BASED PRODUCTS Symposium 4: Robust biocatalysts for the production of novel bio-based products O4-1 Molecular design of transglucosidases for polysaccharide and oligosaccharide synthesis P. Monsan a,b,c,d,e,f a Université de Toulouse; INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France b CNRS, UMR 5504, F-31400 Toulouse, France c INRA, UMR 792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France d Toulouse White Biotechnology, 3 rue des Satellites, F-31400 Toulouse, France e INRA, UMS 1337TWB, F31400 Toulouse, France f CNRS 3582, F-31400 Toulouse, France Among the diversity of transglucosidase enzymes, a specific interest is given to the glucansucrases of the GH70 family, which are produced extracellularly by different lactic acid bacteria: Leuconostoc sp., Lactococcus sp., Streptococcus sp., Weissella sp. These enzymes are able to use the energy of the osidic linkage of the sucrose molecule (27.6 kJ/mol) to catalyse the efficient transfer of its ␣-D-glucopyranosyl unit. This results in the synthesis of a wide variety of products, polysaccharides, oligosaccharides and glucoconjugates, which contain a diversity of linkages: ␣-1,2; ␣-1,3; ␣-1,4; ␣-1,6. It is possible to combine the biochemical and structural characterization of these transglucosidases with gene sequence data and alignement analysis, to develop structure-function relationship studies and molecular engineering based on both rational in silico and combinatorial in vitro approaches. The resulting variants present new and improved catalytic properties, resulting in the synthesis of new products. In addition, original transglucosidases have been obtained from sequencing the genome of several lactic acid bacteria and isolation of the corresponding genes. They are able to decorate dextran polysaccharides (␣-1,6 main backbone linkage) with ␣-1,2 and ␣1,3 osidic branching and generate a new series of polysaccharides and oligosaccharides. We have solved the structures of TAs of the Pfam classes, III, IV and V in order to further understand their mechanism and substrate specificities. The class III ␻-amino acid TAs catalyse transamination of ␻amino acids such as ␤-alanine or ␥-aminobutyric acid where the transferred amino group is not adjacent to the carboxyl group. The crystal structures and inhibitor complexes of two ␻-TAs from Pseudomonas aeruginosa and Chromobacterium violaceumhave been determined to understand differences in their substrate specificity. The class V TA enzymes include the serine:pyruvate transaminases having broad substrate specificity including a reaction with a ␤-hydroxyl substrate. The structure of the thermophilic archaeal Sulfolobus solfataricus serine TA has been determined to 1.8 Å resolution and in complex with the inhibitor, gabaculine. These structures have shown the conformational changes in the enzyme active site during the course of the catalytic reaction. The structure of the class IV Nectria haematococca transaminase enzyme has been determined in the holo and inhibitor bound form which offers a detailed insight into the structural basis for substrate specificity and enantioselectivity of (R)-selective amine:pyruvate transaminases [1–4]. References [1].Sayer S, et al. Acta Cryst 2007;F63:117–9. [2].Sayer S, et al. Acta Cryst 2012;D68:763–72. [3].Sayer S, et al. Acta Cryst 2013;D69:564–76. [4].Sayer S, et al. FEBS J 2014 [in press]. http://dx.doi.org/10.1016/j.nbt.2014.05.1655 O4-3 Current developments on the engineering of Escherichia coli biofilms for enzymatic biosynthesis of halotryptophans Isaac Vizcaino-Caston 1,∗ , James Thomas Leech 1 , Tania Triscari Barberi 2 , Rebeca J.M. Goss 2 , Mark J.H. Simmons 1 , Tim W. Overton 1 1 2 The University of Birmingham, United Kingdom University of St. Andrews, United Kingdom http://dx.doi.org/10.1016/j.nbt.2014.05.1654 O4-2 Structural studies on transaminase enzymes and applications in biocatalysis Jenny Littlechild 1,∗ , C. Sayer 1 , M. Isupov 1 , J. Ward 2 , J. Littlechild 1 1 2 University of Exeter, United Kingdom University College London, United Kingdom The transaminases (TAs) catalyse the transfer of an amino group from an amino acid to a keto acid using the cofactor pyridoxal 5’phosphate (PLP) and are important for the production of optically pure amines and amino alcohols used in the synthesis of many important drugs. Halogenated molecules are being utilized on a daily basis by the fine chemistry and pharmaceutical industries in diverse reactions for the production of enantiomerically pure compounds. The chemical synthesis of such molecules requires harsh solvents and conditions which result in expensive and environmentally unfriendly processes. We propose an alternative to such methods utilizing recombinant bacterial biofilms to synthesise 5-halotryptophans. The plasmid pSTB7 expressing recombinant tryptophan synthase TrpBA was transformed into diverse strains of E. coli and used to generate engineered biofilms [1,2]. These biofilms were used as biocatalysts for the bioconversion of 5haloindoles into 5-halotryptophans. Biofilm cultures showed higher yield than planktonic bacteria, bacterial lysates or immobilised TrpBA enzyme [3]. Biofilm topology and viability were assessed using confocal microscopy before and after reactions. In order to enable scale-up, a novel method to adhere bacteria to supports was devised. We will discuss the ability of different cell www.elsevier.com/locate/nbt S19 SYMPOSIUM 4: ROBUST BIOCATALYSTS FOR THE PRODUCTION OF NOVEL BIO-BASED PRODUCTS immobilization methods to develop biofilm communities as well as their ability to perform biotransformations. References [1].Tsoligkas AN, Winn M, Bowen J, Overton TW, Simmons MJH, Goss RJM. Engineering biofilms for biocatalysis. ChemBioChem 2011;12:1391–5. [2].Tsoligkas AN, Bowen J, Winn M, Goss RJM, Overton TW, Simmons MJH. Characterisation of spin coated engineered Escherichia coli biofilms using atomic force microscopy. Colloids and Surfaces B: Biointerfaces 2012;89:152–60. [3].Perni S, Hackett L, Goss R, Simmons M, Overton T. Optimisation of engineered Escherichia coli biofilms for enzymatic biosynthesis of L-halotryptophans. AMB Express 2013;3:66. http://dx.doi.org/10.1016/j.nbt.2014.05.1656 O4-4 Structural and biochemical characterization of two novel enzymes with promiscuous ene-reductase activity Tea Pavkov-Keller 1,∗ , Alexandra Binter 2 , Steinkellner Georg 1 , Christian C. Gruber 1 , Kerstin Steiner 2 , Christoph Winkler 3 , Helmut Schwab 4 , Kurt Faber 3 , Peter Macheroux 5 , Karl Gruber 6 1 ACIB GmbH, C/o ZMB, Austria 2 ACIB GmbH, Austria 3 Department of Chemistry, University of Graz, Austria 4 Institute of Molecular Biotechnology, Graz University of Technology, Austria 5 Institute of Biochemistry, Graz University of Technology, Austria 6 Institute of Molecular Biosciences, University of Graz, Austria An approach using three-dimensional motifs reflecting specific active site arrangements (catalophore) was developed in our group. It does not depend on overall protein similarity and therefore enables the search across enzyme families and the detection of potential catalytic promiscuity. This catalophore approach led to the discovery of two novel enzymes with ene-reductase activity. Enzymes of this family have recently been shown to possess a great potential for (industrial) biotransformations. Neither the amino acid sequence of these two enzymes nor their overall structure is related to those of the well-known old yellow enzymes (OYE). These two flavoproteins contain FMN as a cofactor and exist as homodimers. We cloned, expressed and purified both enzymes and subjected them to crystallization trials. Obtained crystals were soaked with putative substrates/inhibitors. The enzymatic characterization was pursued by stopped-flow and difference titration experiments. Additionally, several typical OYE substrates (i.e. alkenes bearing an electron-withdrawing activating group) were tested to assess the biocatalytic performance. The analysis showed some salient features of typical OYEs as well as some striking differences, i.e. a stereocomplementary behaviour. In conclusion, the two novel enzymes can be described as NADPH-dependent quinone reductases with significant OYE-like side activities. http://dx.doi.org/10.1016/j.nbt.2014.05.1657 S20 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 O4-5 Lessons on directed evolution of hydrolases and glucose oxidase Ulrich Schwaneberg RWTH – Aachen University, Chair of Biotechnology, Germany Protein engineering by directed evolution and semi-rational design has become a standard method to tailor enzyme properties to industrial demands. Improving thermal stability and activity simultaneously is often challenging since high activity often requires flexibility whereas thermal resistance relies and ‘strong’ interactions within a protein. On the example of proteases (BgAP [1,2], S41 [3]) and a phytase [4,5], lessons learned from improving both properties individually and simultaneously will be presented. Subsequently, lessons on improving detergent and salt (ionic liquid) resistance of a protease (subtilisin E [6,7]) and a cellulase (CelA2 [8,9]) will conclude the hydrolase reengineering examples. As a highlight, the generation of oxygen independent and highly active glucose oxidase variants (GOx from A. niger) [10,11] will conclude the presentation. References [1]. Martinez R, et al. Biotechnol Bioeng 2013;110:711–20. [2]. Jakob F, et al. Appl Microbiol Biotechnol 2013;52:2359–63. [3]. Martinez R, et al. Protein Eng Des Sel 2011;24:533–44. [4]. Shivange A, et al. J Biotechnol 2014;170:68–72. [5]. Shivange AV, et al. Appl Microbiol Biotechnol 2012;95:405–18. [6]. Li Z, et al. ChemBioChem 2012;13:691–9. [7]. Li Z, et al. J Biotechnol 2014;169:87–94. [8]. Lehmann C, et al. Green Chem 2012;14:2719–3272. [9]. Pottkämper J, et al. Green Chem 2009;11:691–7. [10].Arango Gutierreza E, et al. Biosens Bioelectron 2013;50:84–90. [11].Prodanovic R, et al. Anal Bional Chem 2012;404:1439–47. http://dx.doi.org/10.1016/j.nbt.2014.05.1658 O4-6 Immobilization of carbonic anhydrase for biomimetic CO2 capture in slurry absorber Sara Peirce 1,∗ , Maria Elena Russo 2 , Viviana De Luca 3 , Clemente Capasso 3 , Mosè Rossi 3 , Giuseppe Olivieri 4 , Piero Salatino 4 , Antonio Marzocchella 4 1 Dipartimento di Ingegneria Chimica dei Materiali e della Produzione Industriale – Università degli Studi di Napoli Federico II, Italy 2 Consiglio Nazionale delle Ricerche – Istituto di Ricerche sulla Combustione, Italy 3 Consiglio Nazionale delle Ricerche – Istituto di Bioscienze e Biorisorse, Italy 4 Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale – Università degli Studi di Napoli Federico II, Italy Novel post-combustion treatments include biomimetic Carbon Capture and Storage (CCS) processes based on CO2 absorption into aqueous solution assisted by enzyme catalysis. Carbonic anhydrase (EC 4.2.1.1) catalyzes CO2 hydration and it has been proposed as industrial biocatalyst for biomimetic CCS processes [Lacroix and Larachi, 2008, Recent Patent Chem Eng; Russo et al., 2013, Sep Pur Technol]. New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 4: ROBUST BIOCATALYSTS FOR THE PRODUCTION OF NOVEL BIO-BASED PRODUCTS The present contribution reports results of immobilization of carbonic anhydrase (CA) on fine solids. The aim of the study is twofold: the improvement of the biocatalyst stability at the operating conditions typical of CCS processes; the confinement of the biocatalyst in the CO2 absorption reaction volume during continuous operations. Studies reported in the literature suggest the use of slurry reactors as an effective technology for the maximization of the absorption rate enhancement in the presence of heterogeneous catalysis [Alperet al., 1980, Chem Eng Sci; Penders-van Elk et al., 2012–2013, Int J Greenhouse Gas Control; Russo et al., 2013]. In agreement with observation reported in the literature, a covalent CA immobilization technique has been investigated. The carriers have been fine paramagnetic, non-porous and polyglutaraldehyde functionalized particles. Bovine CA was used as model enzyme to optimize immobilization procedure and solid biocatalyst activity assay. Results have pointed out that the activity of the immobilized enzymes is satisfactory. The immobilized enzymes may be effectively used in biomimetic CCS processes. Further investigation will be focused on the immobilization of thermostable recombinant carbonic anhydrase [Capasso et al., 2012, Chem Eng Trans]. ages to the enzyme itself, the surrounding environment and a more effective electron transfer to the redox mediator or electrode surface. In this study we aimed to identify amino acid residues involved in oxygen reactivity. We applied a semi-rational protein engineering approach. Eleven amino acids around the active site were chosen as target positions for site-saturation mutagenesis. Using a screening assay in 96-well plates with ABTS and 2,6-Dichlorophenolindophenol (DCPIP) [4] five variants with decreased oxidase activity (0.1 to 39%) and maintained dehydrogenase activity were identified. Our results show that the exchange of one amino acid can reduce the unfavourable production of H2 O2 of POx without losing activity with alternative e− acceptors. References [1].Leitner C, Haltrich D, Nidetzky B, et al. Appl Biochem Biotechnol 1998;70–72:237–48. [2].Spadiut O, Brugger D, Vasile C, et al. Electroanalysis 2010;22:813–20. [3].Leitner C, Volc J, Haltrich D. Appl Environ Microbiol 2001;67:3636–44. [4].Brugger D, Krondorfer I, Zahma K, et al. Biotech J 2013, http://dx.doi.org/10.1002/biot.201300336. http://dx.doi.org/10.1016/j.nbt.2014.05.1660 http://dx.doi.org/10.1016/j.nbt.2014.05.1659 O4-7 Engineering of pyranose 2-oxidase for modified oxygen reactivity Dagmar Brugger 1,∗ , Dietmar Haltrich 2 , Clemens Karl Peterbauer 2 1 2 Food Biotechnology, BOKU Vienna, Austria BOKU Vienna, Austria The flavoprotein pyranose 2-oxidase (POx) [1] attracts interest as potential component in electrochemical devices [2]. POx catalyses the oxidation of aldopyranoses, preferentially D-glucose, using molecular oxygen, benzoquinone, radicals or chelated metal ions [3] as e− acceptors. Reduced oxygen reactivity is a desirable property for applications in biosensors/biofuel cells because of reduced H2 O2 production and consequently reduced dam- www.elsevier.com/locate/nbt S21 New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 5: SYNTHETIC BIOLOGY Symposium 5: Synthetic biology O5-1 Design and production of new-to-nature antimicrobials by synthetic biology Oscar P. Kuipers ∗ , Auke van Heel, Dongdong Mu, Liang Zhou, Andrius Buyvidas, Manuel Montalbán-López Department of Molecular Genetics, University of Groningen, Germany Antibiotic resistance in human pathogens is on the rise and this rise is not met with new approved antibiotics to combat these resistant bacteria. To solve this growing problem of resistance, alternative sources of antibiotics should be explored. One of these sources could be the class of ribosomally synthesized, posttranslationally modified peptides called lantibiotics. We’ll describe four different approaches to develop novel antimicrobials: (1) Developing a synthetic biology approach to efficiently use the large amount of sequenced genomes (>4000) as a resource for novel lantibiotics. Here we present the results of the first 25 candidates. (2) Use of heterologously expressed post-translational modification enzymes to hypermodify lantibiotics. Various documented posttranslational modifications have been introduced into lantibiotic peptides broadening the antimicrobial spectrum (3) Design and production of novel lantibiotics by ring moduleand hinge-variation. Specific lantibiotic modules have been randomly fitted in a defined architecture, and the nisin induction and modification machinery are exploited for the required modifications. The screening of more than 10,000 chimeric molecules for biological activity has already rendered several more active antimicrobials. (4) Biomodules for introducing various types of circular and heterocyclic modifications in lantibiotics. Purpose is to design three unique biomodules that introduce heterocyclic, cyclic and circular (head-to-tail) modifications in lantibiotics (already containing lanthionine rings http://dx.doi.org/10.1016/j.nbt.2014.05.1661 O5-2 Synthetic transcription factors allow regulon wide control and shifting the nitrogen/carbon balance in bacteria Jorg Schumacher 1,∗ , Baojun Wang 2 , Ana Claudia Bonatto 3 , Martin Buck 1 1 Imperial College London, UK University of Edinburgh, UK 3 Universidade Federal do Paraná, Curitiba, PR, Brazil 2 Synthetic approaches to enhance the production of a desired product commonly involve altering the DNA control sequences regulating expression of the enzymes required. However, many metabolic pathways involve many core and functionally associ- S22 www.elsevier.com/locate/nbt ated genes, so that optimising and testing each control element step by step to achieve desired outcomes is laborious. We have engineered chimaeric activators of the ␴54 RNA polymerase to control transcription of the bacterial ntr regulon, comprising over 100 genes involved in the regulation, metabolism, scavenging and transport of nitrogen compounds. We functionally combined the signal input and functional output domains from various ␴54 RNA polymerase activators with the dimerisation/promoter recognition domains of the master Ntr regulator NtrC and established orthogonal signal input/transcription output transfer functions. Such transcription activators allow to activate transcription ‘against the grain’ of the cell physiology. By force driving transcription of dozens of genes and measuring systems changes (transcriptomic, metabolomic and proteomic) we could observe which aspects of the cell system resisted or gave way to the synthetic perturbation and at which level along the central dogma of molecular biology. This revealed inter-network connections and control hierarchies between these levels and how they contribute to cellular robustness. We found that in the synthetic cell system, the intracellular carbon/nitrogen levels can be shifted towards higher nitrogen assimilation compared to the wild type E. coli. http://dx.doi.org/10.1016/j.nbt.2014.05.1662 O5-3 Cofactor uptake in 1,2-propanediol metabolising microcompartments Matthias Mayer 1,∗ , Stefanie Frank 2 , Evelyne Lawrence 2 , Mark Smales 2 , Martin Warren 2 1 2 Deery 2 , Andrew University of Kent/Warren Group, United Kingdom University of Kent, United Kingdom Propanediol utilising microcompartments are proteinaceous structures consisting of a shell that incorporates enzymes necessary for 1,2-propanediol degradation. The shell is formed by 7 different types of proteins that form hexamers (assemble to form the faces of the shell) or pentamers (vertices of the shell) with central pores and is thought to protect the cell from the toxic propionaldehyde intermediate as well as to transport substrate and cofactors across the protein shell. The dehydration of 1,2-propanediol to propionaldehyde, which is catalysed by the protein complex PduCDE, is B12 (adenosylcobalamin) dependent. Previously it was shown that the pdu microcompartment houses enzymes to regenerate B12 . Uptake of B12 into enteric cells and reactivation are well described, but it still remains unclear if B12 is transported or accumulated inside Pdu microcompartments. We investigated if Pdu microcompartments take up or accumulate B12 . Recombinantly expressed and purified empty microcompartments (shell proteins) and full Pdu microcompartments (fully expressed pdu operon) showed a higher content of B12 than the crude cell lysate, suggesting that Pdu microcompartments have an inherent ability to bind and gather B12 . Different cobalamins were found to be co-purified with both the empty and the fully expressed microcompartments. This finding was confirmed by in vitro co-localisation studies of purified microcompartments New Biotechnology · Volume 31S · July 2014 with fluorescently labelled B12 . The uptake mechanism remains unclear and is part of our current research. An understanding of transport along the protein shell and its selectivity is an important landmark in our efforts to generate engineered compartments capable of sequestering complex synthetic metabolic pathways. http://dx.doi.org/10.1016/j.nbt.2014.05.1663 O5-4 Use of transporter plug-ins in building effective microbial cell factories for chemical and fuel production Christopher Grant ∗ , Phattaraporn Morris, Frank Baganz University College London, United Kingdom Synthetic biology hopes to predictably build cellular factories for the production of chemicals, pharmaceuticals and fuels from cheap renewable feedstocks. A critical, yet understudied, element of this is control of compound transport both into and out of the cell as a means of avoiding issues such as substrate access limitations, substrate and product inhibition and aiding product recovery. We report here a successful strategy for the contextual evaluation of this topic using a library of transport modifying plug-ins combined with multifactorial characterisation. Auxiliary plasmids (pUMP) were used to express a library of transporters as plug-ins alongside two biosynthesis plasmids pGEC41, which oxidises hydrocarbons into fatty alcohols and pADAR7942 which synthesizes bioalkanes from metabolic fatty acid precursors. We demonstrate here benefits of the transporter plug-in approach for: (i) Facilitated delivery hydrophobic substrates to improve whole-cell biocatalysis rates by up to 70 fold (ii) Industrially relevant product yields of over 40 g/Lorganic phase (8 g/Ltotal ) (iii) Reducing byproduct formation in whole-cell bioconversion of alkanes to alkanols (iv) Improving bioalkane synthesis yields from glycerol by >5 fold (v) Reducing alkanol and aldehyde intermediate formation in biosynthesis of bioalkanes by > 10 fold (vi) The integration with in situ product removal strategies to improve bioalkane yields by 10 fold compared to the starting process. This library and plug-in approach is of broad appeal for biological production of hydrophobic compounds and could be a key enabling technology for biological routes for producing a wider range of hydrophobic compounds such as biofuels, fine and specialty chemicals and pharmaceutical intermediates. http://dx.doi.org/10.1016/j.nbt.2014.05.1664 SYMPOSIUM 5: SYNTHETIC BIOLOGY O5-5 Novel tuneable gene expression systems based on orthogonal riboswitches Neil Dixon MIB – Manchester, United Kingdom Strategies that permit precisely controlled, differential, and simultaneous expression of multiple genes would be extremely useful for a broad range of metabolic engineering, protein expression and synthetic biology applications. A new paradigm in genetic regulation emerged with the discovery of novel genetic regulatory elements within the 5′ UTR of bacterial mRNA. Upon binding to a specific metabolite, these so-called ‘riboswitches’ change conformation, permitting differential gene regulation to occur. As these switches operate via a small molecule-dependent, protein-free mechanism, they present themselves as attractive targets for use as novel genetic control elements. Previously, we showed that it is possible to re-engineer riboswitches so that they no longer bind to their original cognate ligands, but are instead activated by synthetic ligands, and further that these ‘orthogonal’ riboswitches can be used to control heterologous gene expression in vivo [Dixon et al., PNAS 2010]. We have further developed these into multi-component systems that permit fine-tuning over co-expression output stoichiometry, with wide ranging potential applications in functional and structural analysis [Dixon et al., Angew Chem, 2012]. Finally, I will discuss the development of these cellular systems and molecular devices from simply proof-of-principle studies, into the tuneable modular expression system RiboTite, and demonstrate the application of this expression technology for the production of proteins of biotechnological interest. http://dx.doi.org/10.1016/j.nbt.2014.05.1665 O5-6 Signal transduction engineering: a powerful platform technology for enhancing secondary metabolite production Jian-Jiang Zhong ∗ , Yi-Ning Xu, Gao-Yi Tan, Linquan Bai Shanghai Jiao Tong University, China Streptomycetes and higher fungi produce many bioactive secondary metabolites. Ganoderic acids (GAs) produced by Ganoderma lucidum, a higher fungus, have significant anti-tumor and anti-metastasis activities. Validamycin, an anti-fungal antibiotic produced by Streptomyces hygroscopicus 5008, is an efficient rice sheath blight controller, and can be used as the source for chemical synthesis of voglibos, an antidiabetic drug. Engineering of regulatory mechanism was done to enhance the production of target secondary metabolites. The effects of metal ions on the GAs biosynthesis in liquid cultures of G. lucidum were investigated, and the increased enzyme activities and up-regulation of transcriptional levels of genes in the triterpene biosynthesis were www.elsevier.com/locate/nbt S23 SYMPOSIUM 5: SYNTHETIC BIOLOGY observed. The regulation mechanism of Mn2+ on the GA biosynthesis was found to be via calcineurin signaling transduction. For the validamycin biosynthesis, our previous study indicated the involvement of A-factor-like cascade. A recent genome-wide analysis reveals three pairs of afsA-arpA in S. hygroscopicus 5008. This work aims to decipher the regulatory role of the multiple afsA-arpA homologs in the A-factor-like cascade, and then to improve the validamycin production by engineering the regulatory cascade. By double deletion of shbR1/R3, the transcriptions of adpA-H and the validamycin biosynthetic genes were up-regulated, and the validamycin production and productivity were enhanced significantly for both the wide-type and a high-producing industrial strain. The transcriptomic analysis revealed that the engineering of A-factorlike signaling cascade caused a shift from primary to secondary metabolism. The signal transduction engineering proposed here is very useful for efficient production of those secondary metabolites in cultivation processes. http://dx.doi.org/10.1016/j.nbt.2014.05.1666 New Biotechnology · Volume 31S · July 2014 so as to improve the engineering efficiency, we devised two novel methods termed as “Genome Replication Engineering Assisted Continuous Evolution (GREACE)” and “Stress-InducedMutagenesis Based Adaptive Evolution (SIMBAE)”. Both methods implant controllable in vivo mutagenesis machineries into the cell. Starting the mutagenesis machineries under stressful conditions will trigger continuous mutation generation andsynchronous selection process, and then result a continuous and efficient evolution process which enabled “Mutagenesis coupled-with Selection”. Specifically, GREACE uses a library of activity-compromised proofreading elements of the main DNA polymerase during genome replication as the mutagenesis machinery. In SIMBAE, the mutagenic state is generated by constructing a SIM module which re-produces complex cellular stress-responses implemented by upregulation and down-regulation of various genes. Either method is capable of increasing genomic mutation rate up to 3000-fold. Significantly improved chemical tolerance (n-butanol, acetate, kanamycin), themotolerance, and osmotic tolerance of E. coli were achieved within 9–90 days using the two methods. http://dx.doi.org/10.1016/j.nbt.2014.05.1667 O5-7 Development of two continuous genome engineering strategies for efficient microbial evolution Zhen Cai ∗ , Guodong Luan, Linjiang Zhu, Yin Li Institute of Microbiology, Chinese Academy of Sciences, China Engineering complex phenotypes of microbes, for instance stress tolerance, remains to be a big challenge in this field. Current evolutionary engineering methods including physical and chemical mutagenesis, global transcription machinery engineering, and artificial transcription factors engineering, use “Mutagenesis followed-by Selection” as the core principle. That is, mutations or genetic perturbations are firstly introduced by exogenous mutagens or genetic manipulations, followed by selection of desired phenotypes. Thus iterative rounds of mutagenesis-selection and frequent manual interventions are often required, resulting in discontinuous and inefficient strain improvements. To address the discontinuity of the existing evolutionary engineering approaches S24 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 Symposium 6: Assimilation of CO2 , CO and CH4 into biobased products O6-1 Microbial fixation of CO2 in water bodies and in drylands to combat climate change, soil loss and desertification Roberto De Philippis Department of Agrifood Production and Environmental Sciences, University of Florence, Florence, Italy The growing concern for the increase of the global warming effects raises the challenge of finding novel technological approaches to stabilize CO2 emissions in the atmosphere. Biological-CO2 mitigation, triggered through biological fixation, is considered a promising and eco-sustainable method. Microorganisms such as cyanobacteria, green algae and some autotrophic bacteria could potentially fix CO2 more efficiently than higher plants, due to their faster growth. Some examples of the potential of Biological-CO2 mitigation will be reported and discussed in this lecture. In arid and semiarid environments, soil carbon sequestration (CO2 fixation) by cyanobacteria and Biological Soil Crusts is considered an eco-friendly and natural process to increase soil C content and a viable pathway to soil restoration after one disturbance event. Another way for Biological-CO2 mitigation intensively studied in the last few years is related to the possibility to perform carbon dioxide sequestration using microalgae, obtaining at the same time bioproducts of industrial interest. Another possibility under study is the exploitation of specific chemotrophic bacteria for CO2 fixation coupled with the production chemicals such as polyhydroxyalkanoates. In spite of the potential of these processes, multiple factors still have to be optimized in order to achieve a cost-effective CO2 sequestration. http://dx.doi.org/10.1016/j.nbt.2014.05.1668 O6-2 Microalgal biofuels from native biological resource of Pearl River Delta Maurycy Daroch ∗ , Zongchao Jia, Cong Shao, Hui Guo, Ying Liu, Jay J. Cheng Peking University School of Environment and Energy, China Algal biofuels are seen as promising solutions of global energy crisis and climate change for the years to come. Major advantages of algae are potentially high yield and no competition with food crops for arable land and fresh water. Although the coastal areas of Pearl River Delta have long been known for huge diversity of aquatic life little work has been done to assess the possibility of using local microalgae resources for biofuel production. Our study fills this niche and tests the feasibility of producing renewable fuels ethanol and biodiesel from local algal strains. A total of 89 unique algal strains from Peking University Algae Collection were isolated and screened as feedstocks for biofuel SYMPOSIUM 6: ASSIMILATION OF CO2 , CO AND CH4 INTO BIOBASED PRODUCTS production. Microalgal strains M. afer PKUAC 9 and S. abundans PKUAC 12 isolated from coastal waters of Pearl River Delta region were used for saccharification and subsequent fermentative bioethanol production where as Hindakia sp. PKUAC 169 and Chlorella sp. PKU AC 102 were optimal for biodiesel production. Hindakia sp. PKUAC 169 isolate was successfully cultivated in two derivatives of BG11 medium: nitrogen starved and salt induced. Both methods yielded improved lipid accumulation, but only salt induction resulted in increased overall lipid productivity. Derivatisation of algal lipids to FAMEs showed different lipid profiles under selected growth conditions and suggest that salt induced medium is better suited for biodiesel production than nitrogen starved medium. Optimisation of heterotrophic cultivation of algal strain Chlorella sp. PKUAC 102 resulted in tenfold higher lipid productivities than autotrophically grown strains. http://dx.doi.org/10.1016/j.nbt.2014.05.1669 O6-3 Exploring the potential of microalgae for bioenergy production Frank Baganz ∗ , Yanan Xu, Paul Hellier, Nicos Ladommatos, Saul Purton University College London, United Kingdom Intensive research is being applied to biofuel production from algal biomass owing to their fast growth rates and high lipid content. This work explores key steps in algal bioprocessing focusing on algal biomass harvesting through flocculation with chitosan; and the use of algal biomass for engine combustion. Initially flocculation with chitosan to harvest algal biomass of the green alga Chlorella sorokiniana was explored. Chitosan proved to be highly efficient in the induction of flocculation with the clarification efficiency reaching >99% below pH 7 at optimal dosage. Influencing factors of flocculation efficiency and its effect on the subsequent dewatering process were also evaluated. It was shown to reduce the volume to be processed by 20–50 folds, and significantly reduce energy input and material costs of centrifugation or filtration operations. In order to reduce the complicated and costly downstream processes of algal biofuel production, this work explores an alternative way of utilizing energy from algal cells by blending algal slurry into fossil diesel using specific combinations of surfactants. We will show that this approach provides benefits by reducing the consumption of fossil fuel and also reducing exhaust emissions particularly NOx thus benefiting the environment. http://dx.doi.org/10.1016/j.nbt.2014.05.1670 www.elsevier.com/locate/nbt S25 New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 6: ASSIMILATION OF CO2 , CO AND CH4 INTO BIOBASED PRODUCTS O6-4 Development of luminescent photobioreactors improved microalgae cultivation for Seyedeh Fatemeh Mohsenpour ∗ , Nik Willoughby Heriot-Watt University, United Kingdom Effect of light conditions on the growth, photosynthetic pigments and lipid production of green algae Chlorella vulgaris and cyanobacteria Gloeothece membranacea was investigated. The microalgae were cultivated in luminescent acrylic bubble column photobioreactors (PBR) under varying conditions. Luminescent acrylic PBR in blue, green, yellow, orange, and red ranges, capable of spectral conversion of simulated full-spectrum daylight were used. The results showed significant variations in culture growth and photosynthetic pigmentation dependent on culture conditions and illumination. The highest biomass productivity of 184 mg L−1 day−1 was obtained in red luminescent PBR. Increasing the initial culture density reduced the rate of productivity although lipid accumulation was significantly induced. Blue and yellow PBR doubled lipid accumulation up to 11% in high density cultures. However, blue PBR provided the least efficient light condition for bio-pigment production. Red PBR was the most effective portion of PAR ranges to enhance chlorophyll production up to 0.75% in G. membranacea whilst green PBR induced pigmentation in C. vulgaris. Phycobiliproteins were the dominant pigments in G. membranacea and red and green light favoured synthesis of these pigments. The average production of phycoerythrin and phycocyanin was slightly improved in high density cultures. The proposed illumination strategy offers improved microalgae growth without resorting to artificial light sources, reducing energy use and costs of cultivation. http://dx.doi.org/10.1016/j.nbt.2014.05.1671 O6-5 Identification of new auxiliary enzymes for the hydrolysis of lignocellulose Oriana Salazar ∗ , Gonzalo Carvajal, Javier Devia, Tania Quiroz University of Chile, Chile Despite important technological advances, the cost of lignocellulosic bioethanol as an energy source is still high compared to fossil fuels. Cellulose hydrolysis is carried out by the action of cellulases and, given the recalcitrance of the biomass high loading of enzymes is required, impacting significantly the total cost of the process. Efficient hydrolysis of lignocellulose depends on the concerted action of cellulases and auxiliary proteins, which can help to reduce cellulase loading in the conversion to monomer sugars. In order to identify auxiliary proteins from rot fungi and improve the action of cellulases on pretreated wheat straw, extracellular proteins from Gloeophyllum trabeum, Fusarium oxysporum and Trametes versicolor cultured in wheat straw were fractioned by S26 www.elsevier.com/locate/nbt exclusion chromatography. Individual fractions were analyzed for the capability to increase the activity of a commercial cellulase mix. Fractions showing the highest cellulolytic activity were analyzed by LC GC/MS. Two new polysaccharide monooxygenases (PMO, former GH61) and one xylanase were identified, amongst other hydrolytic and oxidative enzymes. Corresponding genes were cloned by reverse transcription and PCR, and sequenced. Proteins GtLPMO9A, FoLPMO9A and GtXyl1 were produced in the heterologous host Pichia pastoris and purified. Different combinations of these enzymes with cellulases, in reactions with wheat straw as substrate, were tested for production of glucose and xylose. Results indicate that GtLPMO9A, FoLPMO9A and GtXyl1 increase the production of monomer sugars, suggesting that they could be useful tools for improvement of lignocellulose degradation. Acknowledgement This work was financed by Fondecyt, Project 1121088. http://dx.doi.org/10.1016/j.nbt.2014.05.1672 O6-6 Engineering biofuel producing microbes for efficient hemicellulose utilisation using synthetic biology Gavin Thomas 1,∗ , Rosanna C. Hennessy 1 , Henrique I. Neves 1 , Preben Krabben 2 , Elizabeth Jenkinson 2 1 2 University of York, United Kingdom Green Biologics Limited, United Kingdom Rapid depletion of fossil fuels and oil reserves is driving the development of biofuels to replace reliance on petroleum usage. Hemicellulose is the second most abundant renewable polymer on Earth, and thus an attractive resource for the production of bulk chemicals and fuels (e.g. biobutanol). Development of biomass-derived biofuels is however challenged by the recalcitrance of lignocellulosic materials to degradation. Harsh and costly pretreatment methods are required to break down structurally robust plant biomass to fermentable sugars, which can release inhibitors and retard subsequent fermentations. An alternative method is microbial-based enzymatic degradation. While diverse hemicellulolytic bacteria have been identified, many are poorly characterised with limited genetic manipulation tools and importantly lack industrially relevant pathways. A synthetic biology approach is therefore being used to engineer hemicellolytic ability into butanol producing bacteria. Synthetic gene clusters comprising a minimal enzyme system required to catalyse the common hemicellulose xylan will enable such bacteria to transport and metabolise hemicellulose-derived sugars more efficiently to butanol. Synthetic gene clusters will include endoxylanases and beta-xylosidases to hydrolyse xylan into xylose monomers but also enzymes that hydrolyse side chains or modifications. As the rate of substrate uptake is a key factor in determining the rate of product formation, transporters will be an important design consideration. At the bioprocess level, milder pretreatment procedures will enable deployment of a cost-competitive technology with reduced envi- New Biotechnology · Volume 31S · July 2014 ronmental impact for the production of biofuels from renewable resources. http://dx.doi.org/10.1016/j.nbt.2014.05.1673 O6-7 Economic assessment of microbial lipids for biodiesel production: Competitiveness with microalgae and agricultural plant oils Ho Nam Chang ∗ , Gwon Woo Park KAIST, Republic of Korea SYMPOSIUM 6: ASSIMILATION OF CO2 , CO AND CH4 INTO BIOBASED PRODUCTS cell mass yield of glucose and wood hydrolyzates were 0.5 g/g. Lipid contents from 10 to 90% of cell mass, bioreactor productivity of 0.5–64 g/L/h and plant capacity of 20,000–1,000,000 tons/year were assumed. A $0.902/kg-lipid was predicted with $0.2/kg wood hydrolyzates and $0.1/kg VFAs with productivity, 12 g/L/h and capacity, 100,000 tonnes/year and 75% lipids content. The raw materials accounted for 55.7% of total operating costs followed by direct fixed cost dependent 17.4%. A 50% Chemical oxygen demand (COD) recycle of waste empty cell mass to VFAs further reduced the cost to $0.886.A biodiesel from microbial lipid has a potential to become competitive with diesels from other sources. http://dx.doi.org/10.1016/j.nbt.2014.05.1674 For economic assessment of microbial lipids volatile fatty acids (VFAs) derived from waste organics was used as low cost carbon source and multi-stage continuous high cell density culture (MSCHCDC) process for high bioreactor productivity and titer. The experimental lipid yield was 0.27 g/g-VFA, and the experimental www.elsevier.com/locate/nbt S27 New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 7: APPLICATIONS OF METABOLIC MODELLING Symposium 7: Applications of metabolic modelling [8].Driouch H, Melzer G, Wittmann C. Metab Eng 2012;14:47. [9].Becker J, Zelder O, Häfner S, Schröder H, Wittmann C. Metab Eng 2011;13:159. O7-1 http://dx.doi.org/10.1016/j.nbt.2014.05.1675 Making use of metabolic models – in silico driven design and engineering of industrial microorganisms O7-2 Christoph Wittmann Institute of Systems Biotechnology, Saarland University, 66123 Saarbrücken, Germany Superior microorganisms that produce chemicals, materials and fuels from renewables are major drivers of the developing bio-based economy. Firstly, they hold the key to the optimized production of traditional bio-based products regarding key performance indicators such as titer, yield and productivity [1]. Secondly, they enable future production of important petrochemicals from green resources rather than from fossil fuels [2]. Without doubt, systems metabolic engineering is changing the way to design and optimize such microbial cell factories for industrial production: the integration of systems biology and systems biotechnology with new concepts from synthetic biology enables the global analysis and engineering of microorganisms–and bioprocesses at efficiency and versatility otherwise not accessible. In this regard, the lecture will highlight the integration of model-based prediction and design into industrial strain engineering: driven by massively increasing genomic information, stoichiometric models provide valuable insights into the properties of metabolic networks and give access to theoretical maximum yields, optimal pathways and preferred production hosts [3]. Most importantly, metabolic models can even guide smart and straightforward engineering of cells and processes [4]. The use of models for decision-making and strategic developments appears particularly useful for industrial application. Model-based approaches are fast, feasible at limited level of knowledge in early project stages and allow the investigation of different scenarios, all relevant for short development time and high performance required. Different examples from industrial biotechnology will demonstrate the power of using metabolic models: bio-based production of chemicals [5,6], polymers [7] and recombinant proteins [8]. Regarding industrial lysine production model-based design has provided synthetic strains, which reach the high performance of classical producers derived over the past fifty years [9]. References [1].Becker J, Wittmann C. Curr Opin Biotechnol 2012;23:718. [2].Becker J, Wittmann C. Curr Opin Biotechnol 2012;23:631. [3].Krömer JO, Wittmann C, Schröder H, Heinzle E. Metab Eng 2006;8:353. [4].Melzer G, Eslahpazir M, Franco-Lara E, Wittmann C. BMC Syst Biol 2009;3:12. [5].Buschke N, Becker J, Schäfer R, Kiefer P, Biedendieck R, Wittmann C. Biotechnol J 2013;8:557. [6].Kind S, Neubauer S, Becker J, Yamamoto M, Völkert M, Weong WK, et al. Metab Eng 2014 [in press]. [7].Poblete-Castro I, Binger D, Rodrigues AL, Becker J, Dos Santos V, Wittmann C. Metab Eng 2013;15:113. S28 www.elsevier.com/locate/nbt Design of optimally constructed metabolic networks of minimal functionality David Ruckerbauer ∗ , Christian Jungreuthmayer, Jürgen Zanghellini ACIB GmbH, Austria Background: Metabolic engineering aims to design microorganisms that will generate a product of interest at high yield. Thus, a variety of in silico modeling strategies has been applied successfully, including the concepts of elementary flux modes (EFMs) and constrained minimal cut sets (cMCSs). The EFMs (minimal, steady state pathways through the system) can be calculated given a metabolic model. cMCSs are sets of reaction deletions in such a network that will allow desired pathways to survive and disable undesired ones (e.g., those with low product secretion or low growth rates). Grouping the modes into desired and undesired categories had to be done manually until now. Results: Although the optimal solution for a given set of pathways will always be found with the currently available tools, manual selection may lead to a sub-optimal solution with respect to a metabolic engineering target. A small change in the selection of modes can reduce the number of necessary deletions while only slightly reducing production. Based on our recently introduced formulation of cut set calculations using binary linear programming, we suggest an algorithm that does not require manual selection of the desired pathways. Conclusions: We demonstrated the principle of our algorithm with the help of a small toy network and applied it to a model of E. coli using different design objectives. Furthermore we validated our method by reproducing previously obtained results without requiring manual grouping of modes. http://dx.doi.org/10.1016/j.nbt.2014.05.1676 O7-3 What is the relationship between intracellular and extracellular metabolites? The theory of “metabolic overflow” put into test Silas Villas-Boas 1,∗ , Ting-Li Han 1 , Tim Liu 1 , Sang Kim 1 , Sonia Carneiro 2 , Eugenio Ferreira 2 , Isabel Rocha 2 1 2 The University of Auckland, New Zealand University of Minho, Portugal Compared to our knowledge on metabolic pathways and the establishment of tools to manipulate these pathways, we know very little about the mechanisms behind the secretion of metabolic intermediates. Microorganisms secrete a wide range of metabolic intermediates, and many of them are of great industrial interest. New Biotechnology · Volume 31S · July 2014 Despite the cellular process of metabolite efflux being ubiquitous to all microbial cells, we still do not know clearly how this process works and how it is regulated. It is believed that small metabolites, mainly those end products of fermentation are excreted through the plasma membrane passively, or they are secreted through specialised mechanisms such as vectorial reaction in response to hypo-osmotic stress, or uniport and synport transport systems. However, all of these mechanisms are based on the concept of “metabolic overflow”, which under specific metabolic conditions it is observed a massive excretion of some metabolic intermediates due to their intracellular accumulation. Although this concept seems appropriate to explain the secretion of some intracellular metabolites, it does not apply to many cases studied during continuous culture. Our metabolomics data obtained during different time-series studies of microbial growth under continuous and batch cultures confirm that the concept of “metabolic overflow” cannot be applied to explain the efflux of several intracellular metabolites found in the extracellular medium. Most of our studies indicate that microbial cells very often get rid of some intracellular metabolites in response to an environmental stimulus, even if these metabolites are key intermediates of central carbon metabolism. http://dx.doi.org/10.1016/j.nbt.2014.05.1677 O7-4 Towards genome-scale metabolic pathway analysis: metabolome integration allows efficient enumeration of elementary flux modes in metabolic networks Jürgen Zanghellini ∗ , Matthias P. Gerstl, David Ruckerbauer, Christian Jungreuthmayer Austrian Centre of Industrial Biotechnology, Austria Background: Elementary flux mode (EFM) analysis is used to identify all non-decomposable steady state pathways in metabolic networks. EFMs represent minimal functional building blocks. They can be used to characterize metabolic phenotypes and cellular robustness or identify targets in metabolic engineering. Despite its potential EFM analysis is currently restricted to medium scale models as the number of EFMs in a network explodes with the network’s size. Method: We present a novel computational method that integrates the cellular metabolome into an EFM analysis. As the metabolome directly maps to the Gibbs free energy surface of the reaction network, we are able to identify and remove thermodynamically infeasible EFMs during the runtime of an analysis without impacting any biologically relevant EFMs. Thermodynamic infeasibility is determined in the framework of network embedded thermodynamics. In this way we curb the explosion of the number of EFMs in large networks. Results: We present details of our implementation and demonstrate that our new approach successfully tackles the major bottleneck strongly reduce the memory consumption. Thus an unbiased characterization of large-scale metabolic models is made possible. In fact, we present the first thermodynamically consistent EFM analysis of iJE660a, a genome-scale metabolic model of SYMPOSIUM 7: APPLICATIONS OF METABOLIC MODELLING E. coli. For instance, our analysis confirms the inactivity of glutamate dehydrogenase in E. coli grown on glucose. Conclusion: Thermodynamic EFM analysis successfully integrates the metabolome into an EFM analysis. It is solely based on first principles, and allows for an unbiased characterization of the metabolic capabilities in genome-scale metabolic networks. http://dx.doi.org/10.1016/j.nbt.2014.05.1678 O7-5 Genome scale metabolic modeling of recombinant protein producing yeasts: prediction of process parameters and metabolic engineering targets for efficient production Diethard Mattanovich University of Natural Resources and Life Sciences Vienna, Austria Genome scale metabolic models have been successfully applied to predict genetic interventions to redirect metabolic fluxes towards desired products of the primary and secondary metabolism. Complex polymeric products like heterologous proteins equally demand redistributions of primary metabolic fluxes, their rational design however is far less obvious. Therefore cell engineering for protein overproduction has concentrated mainly to transcription, codon usage, protein folding and secretion. We have developed the first genome scale metabolic model for the yeast Pichia pastoris (PipMBEL1254), and integrated the synthesis of heterologous protein. The model could successfully predict the increase of protein production in oxygen limited conditions, as well as changes of central metabolic fluxes in production strains, as measured by 13 C flux analysis. Metabolic engineering targets for enhanced protein productivity were predicted with FSEOF for gene overexpression and MOMA for gene deletion. After deleting or overexpressing the respective genes as predicted more than 50% of the interventions led to an enhanced production of cytosolic human superoxide dismutase (hSOD), and similarly of other proteins. Beneficial mutations were mainly related to reduction of the NADP/H pool and the deletion of fermentative pathways. We demonstrate that genome scale metabolic modeling is suitable to describe metabolic changes in recombinant strains and can be successfully applied to design genetic interventions to the primary metabolism to increase recombinant protein production. http://dx.doi.org/10.1016/j.nbt.2014.05.1679 O7-6 Use of a novel combinatorial genetics platform to rapidly clone, express and select target biocatalytic activities for multigenic metabolic pathway optimization Ian Fotherigham Ingenza, Ltd., United Kingdom Replacement of petroleum-based products and manufacturing processes with competitive bio-based alternatives is attracting www.elsevier.com/locate/nbt S29 New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 7: APPLICATIONS OF METABOLIC MODELLING increased attention due to environmental concerns surrounding petroleum sustainability and supply. Replacement of conventional processes for manufacturing valuable industrial products and the selection of optimal biosynthetic routes requires the construction, and in most cases subsequent context-dependent evaluation, and optimization of multicomponent biosynthetic pathways to generate intermediates and end products. This talk will present the use of Ingenza’s proprietary combinatorial genetics platform (inABLE® ) to rapidly clone, express, select and optimize target activities for many separate enzymatic reactions, from thousands of independent genes derived from metagenomic and phylogenetic discovery approaches. Multiple gene variants comprising of up to ten individual genetic elements are combined in single reactions, generating expression libraries with hundreds or thousands of members in diverse heterologous configurations for HTP interrogation. Obvious synergy exists between this approach and versatile, solid phase screening and selection methods using growth-based, crossfeeding or colorimetric methods to identify colonies of interest. This is illustrated through the rapid identification of critical pathway enzymes, optimal gene coding sequences and enzyme variants from inABLE® -derived high quality variant libraries for bio-based polymer production. The technology aims to bring increasing predictability and overcome limitations associated with iterative and empirical processes for microbial strain improvement. The successful realization of optimal target reactions enables rapid pathway definition and progression to process optimization and scale-up. http://dx.doi.org/10.1016/j.nbt.2014.05.1680 O7-7 Methanol – a potential carbon source for Corynebacterium glutamicum Sabrina Witthoff ∗ , Jan Marienhagen, Michael Bott Forschungszentrum Jülich, Germany Methanol represents an interesting carbon source for microbial production processes, but cannot be assimilated by Corynebacterium glutamicum, an industrial producer of >4 million tons of amino acids and a model organism in white biotechnology. We therefore attempted to introduce the ribulose monophosphate (RuMP) pathway for methanol assimilation in this species. S30 www.elsevier.com/locate/nbt For implementation of the RuMP pathway, three heterologous genes encoding methanol dehydrogenase, 3-hexulose-6phosphate synthase, and 6-phosphate-3-hexuloisomerase were expressed in C. glutamicum wild type. All other enzymes required for conversion of fructose-6-phosphate and regeneration of ribulose-5-phosphate as C1-acceptor can be recruited from endogenous metabolism. The recombinant strain metabolized methanol at a rate of 500 ␮mol h−1 (g CDW)−1 during growth on glucose/methanol mixtures. Further studies revealed, however, that C. glutamicum wild type is able to oxidize methanol via formaldehyde and formate to CO2 with a rate of 200 ␮mol h−1 (g CDW)−1 . The alcohol dehydrogenase AdhA is mainly responsible for the oxidation of methanol to formaldehyde, whereas the oxidation of formaldehyde is predominantly catalyzed by two enzymes, the acetaldehyde dehydrogenase Ald and the mycothiol-dependent formaldehyde dehydrogenase AdhE. The resulting formate is oxidized via formate dehydrogenase FdhF to CO2 [1,2]. A C. glutamicum mutant lacking Ald and AdhE activity is severely impaired in its ability to oxidize formaldehyde to CO2 and thus, represents a promising strain to enforce methanol assimilation and prevent dissimilation. References [1].Witthoff S, Eggeling L, Bott M, Polen T. Microbiology 2012;158:2428–39. [2].Witthoff S, Mühlroth A, Marienhagen J, Bott M. Appl Environ Microbiol 2013;79:6974–83. http://dx.doi.org/10.1016/j.nbt.2014.05.1681 New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 8: INDUSTRIAL BIOTECHNOLOGY OF NATURAL AND SYNTHETIC POLYMERS Symposium 8: Industrial biotechnology of natural and synthetic polymers O8-1 Green polymer processing with enzymes Enrique Herrero Acero a , Doris Ribitsch a , Veronika Perz a , Alessandro Pellis b , Antonino Biundo b , Katrin J. Greimel a,b , Gibson S. Nyanhongo b , Georg M. Guebitz a,b,∗ by open mixed microbial cultures. Lab-scale bioreactors, inoculated with soil as a source of microorganisms, were operated under anaerobic conditions and fed with either glucose or cellulose (two different particle sizes) as only carbon sources. Ethanol yield up to approx. 20% of the theoretical value was obtained with glucose. In the cellulose-fed reactors, cellulose hydrolysis occurred and it was found to occur faster with the smaller particle size. http://dx.doi.org/10.1016/j.nbt.2014.05.1683 a Austrian Centre of Industrial Biotechnology, Konrad Lorenz Strasse 20, 3430 Tulln, Austria b University of Natural Resources and Applied Life Sciences, Vienna, Konrad Lorenz Strasse 20, 3430 Tulln, Austria Environmentally friendly reaction conditions and the highly specific mode of action are among the advantages of enzyme based processing of (bio)synthetic polymers when compared to chemical processes. In coatings, enzymes can replace toxic metal based catalysts or improve dispersing properties of lignins. Their surface specific action is exploited in functionalization of synthetic materials to impart e.g. antimicrobial properties. On the other hand, enzymes have a potential for specific recovery of polymer building blocks from composite materials or mixed plastics waste. To improve the efficiency of enzymes on non-natural polymeric substrates, engineering enzyme surface properties and attachment of binding modules are useful strategies together with engineering of the active site architecture [1–4]. References [1].Herrero Acero E, et al. Biotechnol Bioeng 2013;110(10):2581–90. [2].Ribitsch D, et al. Biomacromolecules 2013;14(6):1769–76. [3].Greimel KJ, et al. Green Chem 2013;15(2):381–438. [4].Nugroho Prasetyo E, et al. Bioresour Technol 2010;101(14):5054–62. http://dx.doi.org/10.1016/j.nbt.2014.05.1682 O8-2 Open mixed cultures for bioethanol production from lignocellulosic materials Davide Dionisi ∗ , Tom Adams, Craig Dempster University of Aberdeen, United Kingdom This study investigates the feasibility of a new process for the production of bioethanol from lignocellulosic wastes. In the investigated process, conversion of lignocellulosic materials to ethanol is obtained by using microorganisms for all the required process stages, i.e. lignin and cellulose hydrolysis and carbohydrates conversion to ethanol. The aim is therefore to avoid the use of expensive chemical-physical pretreatments for lignin and cellulose hydrolysis, replacing them with cheaper and more environmentally sustainable microbial processes. A key aspect of the investigated process is the use of open mixed microbial cultures, instead of pure cultures of selected microorganisms. This presentation will discuss the main challenges to be overcome for the development of the investigated process and will present experimental data on glucose and cellulose fermentation O8-3 Strategies for enzymatic functionalization of synthetic polymers Enrique Herrero Acero 1,∗ , Caroline Gamerith 1 , Andreas Ortner 1 , Steinkellner 1 , Karl Gruber 2 , Helmut Doris Ribitsch 1 , Georg Schwab 3 , Georg M. Guebitz 4 1 ACIB GmbH, Austria University of Graz, Austria 3 Graz University of Technology, Austria 4 University of Natural Resources and Applied Life Sciences, Vienna, Austria 2 Polymers have a wide range of uses because of their outstanding bulk properties, but their surfaces are lacking of desired reactivity or properties. Current activation processes rely in the use of concentrated acids/alkali solution or plasma approaches. While harsh chemicals typically decrease the polymer properties, plasma treatment are very energy demanding and unable to functionalize complex geometries like tubes. Enzymes have revealed as powerful catalyst able to overcome all these limitations. Enzymes are able to partially hydrolase the upper most layers of polymers in a controlled manner, introducing anchor groups for subsequent grafting. Following this approach it was possible to graft Human Serum Albumin (HSA) to polylactic acid (PLA) membranes in which the polymer was activated in a first step with a cutinase [1]. The functionalized material had a higher hydrophilicity, antioxidant capacity, which improved its biocompatibility. Using a more elaborated double enzymatic reaction it was possible to covalently graft ferulic acid on polyamide (PA) fibers [2]. In order to improve the enzymatic process we have improved the polymerenzyme interaction via rationale design of the enzyme surface [3] and by fusion of binding modules leading to increased adsorption as measured via Quartz Crystal Microbalance [4]. References [1].Nyanhongo GS, et al. React Funct Polym 2013;73(10):1399–404. [2].Herrero Acero EJ. Mol Catal B: Enzym 2012;79:54–60. [3].Herrero Acero E. Biotechnol Bioeng 2013;110(10):2581–90. [4].Ribitsch D, et al. Biomacromolecules 2013;14(6):1769–76. http://dx.doi.org/10.1016/j.nbt.2014.05.1684 www.elsevier.com/locate/nbt S31 SYMPOSIUM 8: INDUSTRIAL BIOTECHNOLOGY OF NATURAL AND SYNTHETIC POLYMERS O8-4 Influence of nutritional and physicochemical variables in PHB production from raw glycerol by a wild Bacillus megaterium strain Carolina Guzmán Luna 1,∗ , Paalo Andrea Moreno Yañez 1 , Camilo Jose Yañez Diaz 1 , Nilo Sérgio Medeiros Cardozo 2 , Humberto Escalante 1 , Marianny Y. Combariza 1 1 2 Universidad Industrial de Santander, Brazil Federal University of Rio Grande do Sul, Brazil Reducing production costs and increasing strain productivity in biosynthetic processes are decisive factors linked to biodegradable polymers worldwide production and usage. Agro-industrial byproducts as alternative carbon sources and optimization of culture media composition are some of the strategies used to address these issues. Amongst many available biopolymers polyhydroxybutyrate (PHB), intracellularly produced and accumulated by abundant bacteria species, could become a substitute for synthetic plastics due to its thermoplastic and mechanical properties. In this contribution we report on the influence of five nutritional and two physicochemical variables in PHB production by Bacillus megaterium B2, a recently isolated and characterized bacterium with PHB accumulating ability using raw glycerol, from the biodiesel industry, as carbon source [1]. According to Plackett Burman and central composite designs temperature, glycerol and Na2 HPO4 concentrations are the most significant variables on PHB production by B2, with optimal values of 34 ◦ C, 7.6 g/L and 3 g/L, respectively. After 14 hours of fermentation, in shake flasks with optimized medium, B2 produced 0.37 g/L of PHB with an 18% w/w accumulation. This corresponds to an 85% increase in PHB production when compared to initial culture conditions. These results suggest the potential of B. megaterium B2 as PHB producer using raw glycerol an inexpensive, abundant and readily available carbon source. Reference [1].Tarazona N, Moreno P, Yañez C, Combariza MY, Guzmán C. Microbial production of polyhydroxybutyrate by native Bacillus strains using a biodiesel by-product as carbon source. Lisbon, Portugal: Presented at the European Symposium on Biopolymers; October 7–9, 2013. PS 2.1. http://dx.doi.org/10.1016/j.nbt.2014.05.1685 O8-5 Value-added carotenoid production in the pennate diatom Phaeodactylum tricornutum with light emitting diode based photobioreactors Weiqi Fu 1,∗ , Sigurður Brynjólfsson 1 , Bernhard Palsson 2 1 2 University of Iceland, Iceland University of California, San Diego, United States Diatoms are one of the most promising feedstocks for producing feed supplements, bioactive pharmaceuticals and biofuels in a bio-based economy. The marine pennate diatom PhaeodactyS32 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 lum tricornutum is able to accumulate large amounts of specialty carotenoids. Among the valuable carotenoids present in diatoms, fucoxanthin has health promoting effects in humans, including anti-cancer, anti-obesity, and anti-diabetic effects as well as antimalarial activity. Fucoxanthin has also been observed to be more potent than ␤-carotene and astaxanthin in terms of anti-obesity and anti-proliferative effects on adult T-cell leukemia cells. The demand for fucoxanthin in the global market has been increasing dramatically. In this study, we characterized the biomass productivity and biomass yield of P. tricornutum with light emitting diode (LED)-based photobioreactors with different light intensities and wavelengths. Under red LED illumination, increasing the photon flux from 85 to 255 ␮E/m2 /s caused photoinhibition of Phaeodactylum cells when there was either 3.0 mM or 0.3 mM of metasilicate in the medium. In contrast, combined red and blue (50:50) LED illumination produced a higher biomass yield and growth rate. Further, the presence of blue light enhanced the accumulation of fucoxanthin in comparison to illumination with red light only. These results demonstrated the feasibility of fucoxanthin production in P. tricornutum, and a method combining LED technology and synthetic biology approaches was proposed to facilitate its development. http://dx.doi.org/10.1016/j.nbt.2014.05.1686 O8-6 Antibacterial and antifungal activity of charcoal materials and microwave radiation Hee Jin Yang 1,∗ , Yun Jeong Cha 2 , Hern Kim 2 , Shin Sik Choi 2,3 1 Myoungji University, Republic of Korea Department of Energy and Biotechnology, Myongji University, Republic of Korea 3 Department of Food and Nutrition, Myongji University, Republic of Korea 2 Suppression of microbial contamination or growth using antibacterial and antifungal materials has been required in various commercial products including foods and cosmetics. In this study, we have investigated the inhibitory effect of charcoal polymers and microwave radiation on bacterial and fungal cell growth or survival rate. When bacterial and fungal cells were cultured with charcoal polymers, the growth of some microbes was significantly inhibited by nano and micro sized charcoal particles protruded from the surface of polymers. Four species of fungi were also dramatically killed by microwave radiation for less than 5 min. These results suggest that the use of charcoal plastics and microwave has a great potential for reducing microbial contamination in multiple areas. Acknowledgements: This work was supported by NGV of Hyundai Motors Company and BK21 Plus of Ministry of Education (22A20130012051). http://dx.doi.org/10.1016/j.nbt.2014.05.1687 New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 8: INDUSTRIAL BIOTECHNOLOGY OF NATURAL AND SYNTHETIC POLYMERS O8-7 Polyhydroxyalkanoates production by aerobic mixed microbial cultures using crude glycerol Paulo Costa Lemos ∗ , Rita Moita, André Freches, Rita Pontes REQUIMTE/CQFB, Portugal Due to the prospective partial replacement of fossil fuels by biodiesel, its production has continuously grown in the last decade. In order to biodiesel production to be carried out under sustainable conditions, new application to their wastes/byproducts need to be investigated, especially concerning their major by-product – crude glycerol. In this study the feasibility of producing polyhydroxyalkanoates (PHA) by mixed microbial community using crude glycerol as feedstock was investigated. The microbial population selected under aerobic dynamic feeding conditions had the ability to consume both major carbon fractions present in the crude, glycerol and methanol. Two biopolymers were stored, poly-3-hydroxybutyrate (PHB) and glucose biopolymer (GP), apparently using glycerol as the only carbon source for their production. The microbial enrichment obtained has able to accumulate up to 47% PHB of cell dry weight with a productivity of 0.24 g HA/L d. The overall PHA yield on total substrate consumed (0.32 g COD HB/g COD crude glycerol) was in the middle range of those reported in literature (0.08–0.58 g COD PHA/g COD real waste). Molecular biology methods (FISH, DGGE) were used to characterize the microbial population. As opposed to many other reports with real wastes, crude glycerol can be directly used to produce PHA. Its use avoids an additional pre-fermentation step, making the overall production process economically more competitive, reflected in a reduction of the polymer final cost. This was the first study that demonstrates the valorisation of the glycerol fraction present in the crude glycerol into PHA using an aerobic mixed microbial consortium. http://dx.doi.org/10.1016/j.nbt.2014.05.1688 www.elsevier.com/locate/nbt S33 BIOECONOMY Bioeconomy SP3-1 Asian bioeconomy and biobusiness: current scenario and future prospects Satyahari Dey 1,2,∗ 1 2 Indian Institute of Technology Kharagpur, India Deputy Secretary General, Asian Federation of Biotechnology, India The 20th century witnessed exciting inventions in biology that has resulted in innovations today. The recombinant DNA technology, animal cell culture, bioprocess engineering of recombinant cells and the deciphering of human genome are notable. A plethora of ‘omics’ related platform technologies have enabled precise and rapid probing of cellular processes facilitating huge applications in 5 Fs (food, fuel, f(ph)armaceuticals, fabric and fodder). The revenue from bio-industries in some countries, including a few in Asian regions, was as high as 2.5% of GDP. The developing regions in Asia could not experience such a growth uniformly. The sluggish global market over the last 5 years was also a bottleneck to this aspiration. The world trade trends now to recover from slowdown. It is important to set a realistic goal for biobusiness in Asia for 2025. Asia in one hand is blessed with the highest number of biotechnologists of the world in the younger age group, and the largest population of underprivileged people (having lower human development indices) on the other. The bountiful bioresources in Asia if exploited in bioentrepreneurship could eliminate the disparities. Inspiring young biotechnologists towards this goal is essential. A strong academia-industry partnership could accelerate the success. The European Federation of Biotechnology and Asian Federation of Biotechnology together can mobilize their members, and interface with respective Governments facilitating favourable regulatory and policy frameworks. http://dx.doi.org/10.1016/j.nbt.2014.05.1689 SP3-2 Biotech products on the market as a main driver for knowledge based bio-economy Aleksandra Małyska ∗ , Tomasz Twardowski Institute of Bioorganic Chemistry Polish Academy of Sciences, Poland As biotechnology applications are present in broad variety of sectors it has been the key technology for realising the KBBE providing a more cost-effective option and better quality products. At the same time some of its products arouse great public controversy, in particular, the products of genetic engineering in the agri-food sector. Poland is no exception and international studies revealed that attitudes of Poles towards biotech products reflect the views of other Europeans. It is understandable because people are not aware in how many everyday products biotechnology is already applied. S34 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 The supply chain of biotech products involves many stakeholders, organizations, activities and resources, some of which have a direct and often significant influence on consumers purchase behaviors. One of them is the retailers, who are a key players in the supply chain by providing link between the end-consumers and the manufacturers as well as suppliers. Our study took place in 2012 and 2013 and involved more than 250 visits in various retail outlets in Poland and conducting standardized interviews with merchants concerning their views and knowledge of products referring to innovative biotechnology. Within this study we were able to identify goods (manufactured in different EU countries) that were either direct applications of innovative biotechnology or unduly usurped to be such products The study revealed a positive correlation between the level of knowledge and the opinion on biotech products. The results helped to gain an understanding of reasons and motivations underlying retailers’ attitudes towards biotech products. http://dx.doi.org/10.1016/j.nbt.2014.05.1690 New Biotechnology · Volume 31S · July 2014 TUESDAY 15 JULY PLENARY LECTURE: PROSTHETIC GENE NETWORKS FOR BIOMEDICAL APPLICATIONS Tuesday 15 July Plenary lecture: Prosthetic gene networks for biomedical applications PL2-1 Prosthetic networks – synthetic biology-inspired treatment strategies for metabolic disorders Martin Fussenegger ETH Zurich, Department of Biosystems Science and Engineering, Basel, Switzerland Since Paracelsus’ (1493–1541) definition that the dosing makes the drug the basic treatment strategies have largely remained unchanged. We continue to use a precise prescribed dose of a small-molecule drug, a protein therapeutic or a therapeutic transgene to constitutively modulate or complement the activity of a disease-relevant target. However, this treatment concept does neither consider the metabolic dynamics nor the interdependence of the most important pathophysiology’s of the 21st century such as obesity, diabetes and cardiovascular disorders. Synthetic biologyinspired prosthetic networks may act as metabolic prostheses that provide the dynamic interventions, the immediate pre-disease action and the multi-target capacity required to meet with the treatment challenges of the future. Prosthetic networks consist of synthetic sensor-effector gene circuits that (i) seamlessly operate in implanted designer cells, (ii) constantly sense, monitor and score metabolic disturbances in peripheral circulation, (iii) process OFF-level concentrations of pathologic metabolites, and (iv) coordinate an adjusted therapeutic response in an (v) automatic and self-sufficient manner. We will present our latest generation of synthetic mammalian gene circuits and provide a few examples of prosthetic networks operating in animal models of prominent human diseases to highlight the challenges and impact of synthetic biology on future biomedical applications. http://dx.doi.org/10.1016/j.nbt.2014.05.1691 www.elsevier.com/locate/nbt S35 SYMPOSIUM 9: BIOMARKERS AND DIAGNOSTIC TOOLS New Biotechnology · Volume 31S · July 2014 Symposium 9: Biomarkers and diagnostic tools recognize sequence epitopes that are difficult to target using conventional antibodies, and that these novel antibodies are potent inhibitors of protein aggregation. O9-1 http://dx.doi.org/10.1016/j.nbt.2014.05.1693 Designing nanomaterials for ultrasensitive biosensing Molly Stevens Imperial College London, UK Bio-responsive nanomaterials are of growing importance with potential applications including drug delivery, diagnostics and tissue engineering. This talk will provide an overview of our recent developments in the design of materials for ultrasensitive biosensing. Our recent simple conceptually novel approaches to real-time monitoring of protease, lipase and kinase enzyme action using modular peptide functionalized gold nanoparticles and quantum dots will be presented. Furthermore we have recently developed a new approach to ultrasensitive biosensing through plasmonic nanosensors with inverse sensitivity by means of enzyme-guided crystal growth as well as a “Plasmonic ELISA” for the ultrasensitive detection of disease biomarkers with the naked eye. We are applying these biosensing approaches both in high throughput drug screening and to diagnose diseases ranging from cancer to global health applications. http://dx.doi.org/10.1016/j.nbt.2014.05.1692 O9-2 Antibodies by design Peter Tessier Rensselaer Polytechnic Institute, United States The ability of antibodies to recognize target molecules (antigens) with high affinity and specificity is central to their widespread use in diagnostic and therapeutic applications. The binding activity of antibodies is encoded in up to six of their solvent-exposed peptide loops that directly contact antigens. Antibodies are generated by randomly varying the sequences of their antigen-binding loops and selecting rare variants that are complementary to target antigens. Due to the daunting number of possible antibody sequences with variation only within their antigen-binding loops (>103̂0 variants), it seems unlikely that the needles (antibodies with desired binding activity) in the haystack (all possible antibody variants) can be predicted instead of being selected. We have challenged this conventional wisdom by reducing the seemingly intractable problem of designing multiple antibody loops to cooperatively bind antigens to a tractable one in which we design individual antibody loops with binding activity. Using this simplified design strategy that is inspired by natural biological interactions, we find that antibody fragments can be readily engineered to recognize diverse aggregated proteins linked to neurodegenerative disorders (e.g., Alzheimer’s disease) by targeting unique structural features within such proteins. Our innovative approach generates single- and multidomain antibodies that recognize misfolded proteins not only based on their sequence, but also based on their conformation. We also find that our antibodies S36 www.elsevier.com/locate/nbt O9-3 Engineering cofactor specificity of methyltransferases Martin Tengg 1,∗ , Yu Zheng 2 , Mandana Gruber-Khadjawi 3 , Elmar Weinhold 1 1 Institute of Organic Chemistry, RWTH Aachen University, Germany New England Biolabs Inc., Ipswich, MA, USA 3 ACIB GmbH, Austrian Centre for Industrial Biotechnology, Graz, Austria 2 Biological methylations of various substrates occur in every living cell and are essential for cell survival. These transformations are catalyzed by methyltransferases (MTases) which generally transfer the activated methyl group from S-adenosyl-l-methionine (AdoMet) to DNA, RNA, proteins and small biomolecules. Recent studies demonstrated that many MTases can accept AdoMet analogues for transfer of extended carbon chains to their substrates [1,2]. The ability to accept a broad range of cofactor analogues is of great interest both in terms of DNA diagnostics and biocatalytic synthesis. In order to increase transfer rates of extended groups from AdoMet analogues protein engineering approaches are performed. High-throughput directed evolution of the DNA MTase M.SssI is performed by in vitro compartmentalization. This method links genotype and phenotype for an effective selection of active variants. The bacterial enzyme M.SssI is of particular interest because it performs the same reaction as mammalian DNA MTases by targeting 5′ -CG-3′ (CpG) DNA sequences. CpG modifications are key epigenetic signatures in transcriptional regulation and genome imprinting. The transfer of functional groups will provide new tools for DNA labelling as well as DNA modification detection. M.SssI evolution will also guide rational protein engineering of the homologous small molecule MTase NovO, for the synthesis of new fine chemicals as well as bioactive intermediates and products. References [1].Dalhoff C, Lukinavicius G, Klimasăuskas S, Weinhold E. Nat Chem Biol 2006;2:31–2. [2].Stecher H, Tengg M, Ueberbacher BJ, Remler P, Schwab H, Griengl H, Gruber-Khadjawi M. Angew Chem Int Ed 2009;48:9546–8. http://dx.doi.org/10.1016/j.nbt.2014.05.1694 O9-4 Synthetic bioreporters for detection of environmental pollutants Jan Roelof van der Meer ∗ , Davide Merulla, Siham Beggah University of Lausanne, Switzerland One of the main immediate application areas for synthetic biology are bioreporters, living cells with simple designed genetic SYMPOSIUM 9: BIOMARKERS AND DIAGNOSTIC TOOLS New Biotechnology · Volume 31S · July 2014 circuits that permit detection of a specific chemical or group of chemicals, under the concomitant production of an easily but accurately quantifiable reporter signal. Bioreporters have attracted considerable interest because they offer cheap alternatives for chemical analysis in remote areas where high-end instruments are unavailable. Such demands on bioreporters, however, require an almost fail-proof and robust technology that goes much beyond what traditional research “proof-of-principles” have been able to demonstrate. We will show on the example of a bioreporter for arsenic, how important improvements can be made in the circuit design and in the optimization of the assay technology. Arsenic is a recurring noxious contaminant of drinking water in large areas of our planet, and bioreporter technology can provide the means to rapidly quantify its presence in the concentration range of 1–10 ␮g/L. First, we demonstrate how designing a feedback or an uncoupled circuit affects the signal output and detection sensitivity for arsenic. Secondly, we show a new system to better control residual background expression in the circuit while maintaining optimal induction, and we also provide experimental evidence to improve the circuit behaviour by avoiding cross-interference from the host. Finally, we present the use of micro-engineered structures that would permit continuous remote operation of a bioreporter sensor. http://dx.doi.org/10.1016/j.nbt.2014.05.1695 O9-5 A homogeneous quenching resonance energy transfer assay for H-Ras activation cycle monitoring and inhibitor screening Kari Kopra 1,∗ , Arjan van Aldrichem 2 , Markku Syrjänpää 1 , Stefan Veltel 3 , Pekka Hänninen 1 , Daniel Abankwa 4 , Urpo Lamminmäki 5 , Harri Härmä 1 1 Laboratory of Biophysics, University of Turku, Finland Institute for Molecular Medicine Finland, University of Helsinki, Finland 3 University Hospital Hamburg-Eppendorf, Finland 4 Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Finland 5 Department of Biotechnology, University of Turku, Finland 2 Background: Recently, we have developed a homogeneous single-label signaling technique, the Quenching Resonance Energy Transfer (QRET). In this study, the homogeneous QRET technology was applied to monitor GTPase activation cycle and activation cycle inhibition. Methods: The QRET system is based on the protection of target protein bound Eu3+ -GTP from a soluble quencher molecule. (1) The small GTPase cycle (nucleotide exchange and hydrolysis) can be monitored in the competitive assay by tracking GTP hydrolysis in the presence of nanomolar concentrations of H-RasWt , SOScat , and p120GAP. (2) The nucleotide exchange can be monitored using a second assay with H-RasWt and SOScat . The increased timeresolved luminescence (TRL) signal is monitored when either GTP hydrolysis (1) or Eu3+ -GTP association (2) occurs. Results: We have proven the suitability of the QRET system to monitor GTP hydrolysis (1) and nucleotide exchange (2). The GTP hydrolysis assay was used to screen 1280 compound small molecule library whereof twelve inhibitors were found (average Zfactor 0.78). The GTP hydrolysis assay can simultaneously detect inhibitors affecting either nucleotide exchange or GTP hydrolysis. Therefore, same screening was performed by monitoring Eu3+ -GTP association to H-RasWt (average Z-factor 0.78). From the screening assays, seven same inhibitor hits were found. Additionally, five inhibitors were found only in GTP hydrolysis assay. Conclusions: These novel QRET assays for GTPase research can be performed using nanomolar protein concentrations. The presented QRET assays enable the study of whole small GTPase cycle by monitoring the guanine-nucleotide exchange factor (GEF) induced nucleotide exchange and/or GTPase-activating proteins (GAP) catalyzed GTP hydrolysis. http://dx.doi.org/10.1016/j.nbt.2014.05.1696 O9-6 Comparative large scale microRNA expression profiles of cynomolgus monkeys, rat and human reveal miR-182 associated with Type 2 diabetes Hongli Du ∗ , Jinghui Zhou, Yuhuan Meng, Xiaoning Wang South China University of Technology, China Type 2 diabetes (T2D) is a prevalent disease that is present throughout the world, and is usually associated with insulin resistance. MicroRNAs (miRNAs) play important role in the suppression of gene expression and have been shown to be implicated in human diseases. We used a novel animal model, cynomolgus monkey fed with normal and high fatty diet (HFD) respectively, to analyze the miRNA expression profile in whole blood by deepsequencing. Finally in total 24 miRNAs with differential expression were filtered. Among them, mir-182 and mir-183, related to insulin resistance by modulating FOXO1 and PI3 K/AKT cascade, had the greatest copy number in the whole blood. Decrease of mir-182 in T2D cynomolgus individuals is completely consistent with the previous studies in human and rat. Integrating mir-182 tissue expression profile, target genes and copy number in blood revealed that mir-182 plays a key role in FOXO1 modulation that leads to potential hyperglycemia and modulates the insulin secretion. In addition, the possible miRNA regulation system of T2D under the influence of different diet conditions was also interpreted in the present study. The cholesterol content influences mir-182 and potentially other miRNAs expression level that causes the insulin resistance and the various miRNA regulation systems between normal diet and HFD. http://dx.doi.org/10.1016/j.nbt.2014.05.1697 www.elsevier.com/locate/nbt S37 SYMPOSIUM 9: BIOMARKERS AND DIAGNOSTIC TOOLS O9-7 Association of apolipoprotein E gene polymorphism with serum lipid levels Sehrish Fatima ∗ , Syed Shahid, Obaid Khan, Abid Azhar University of Karachi, Pakistan Dyslipidemia has been marked to play an integral role in the development of cardiovascular diseases. Various environmental and genetic factors may influence dyslipidemia. Polymorphism of the apolipoprotein E (apo E) gene may influence lipid metabolism by modulating lipid levels. This study was designed to investigate the role of genetic variants of apo E in dyslipidemic patients. The study was carried out in equal number (n = 110) of normal subjects and dyslipidemic patients. Genotyping was performed by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR- RFLP) at Single Nucleotide Polymorphisms (SNPs) S38 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 rs429358 (ApoE4) and rs7412 (ApoE2). The genotype frequencies were: E3/E3 (75%), E2/E3 (4.1%), E3/E4 (20%), E4/E4 (0%), E2/E4 (0%), and E2/E2 (0.45%). The allele frequencies were 0.025, 0.875 and 0.1 for ␧2, ␧3 and ␧4, respectively. In control group allele frequencies for ␧2, ␧3 and ␧4 were 0.045, 0.83 and 0.127, respectively, whereas in dyslipidemic patients group allele frequencies for ␧2, ␧3 and ␧4 were 0.045, 0.682 and 0.273 respectively. Total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) levels were significantly high in ␧4 allele carriers (p < 0.001) and high density lipoprotein cholesterol (HDL-C) level was high in ␧2 allele carriers (p < 0.05) indicating protective effect of ␧2 allele. Apo E polymorphism has significant influences on lipid profile in dyslipidemic patients group as frequency of ␧4 allele was found to be prevalent in patients group which indicates a relationship of ␧4 allele in apo E gene with dyslipidemia. http://dx.doi.org/10.1016/j.nbt.2014.05.1698 SYMPOSIUM 10: BIOWASTE BIOREFINERY FOR A MORE SUSTAINABLE BIOECONOMY New Biotechnology · Volume 31S · July 2014 Symposium 10: Biowaste biorefinery for a more sustainable bioeconomy O10-1 Biowaste biorefinery for biobased food industry a more sustainable and Fabio Fava DICAM, School of Engineering, Alma Mater Studiorum-University of Bologna, Bologna, Italy Modern biobased industry is giving its close attention on organic waste as a new bioresource. Specific biowaste valorization pathways are focusing on food processing waste, being food sector the first manufacture in Europe. Anyway they need to become integrated, combining biomass pretreatments and recovery of biogenic chemicals with bioconversion processes in order to obtain a large class of chemicals. This will help to (a) use the whole biowaste, by avoiding producing residues and providing to the approach the required environmental sustainability, and (b) producing different biobased products that enter different markets, to get the possible economical sustainability of the whole biorefinery. However, the costs of the developed integrated processes might be high, mostly for the fact that the industry dealing with such issues is still underdeveloped and therefore dominated by high processing costs. Such costs can be significantly reduced by intensifying research & innovation on process integration and downstream processing. The low or no cost of starting material along with the environmental benefits coming from the concomitant biowaste disposal would offset the high capital costs for initiating such a biorefinery. http://dx.doi.org/10.1016/j.nbt.2014.05.1699 O10-2 Development of an advanced biorefinery concept based on valorization of pulp and paper industry waste streams Apostolis Koutinas , Mary Alexandri ∗ , Chrysanthi Pateraki, Anestis Vlysides, Harris Papapostolou Agricultural University of Athens, Greece The biorefinery concept is based on the efficient fractionation of a renewable resource followed by the conversion of individual fractions into marketable products in a way that exploits the full potential of the resource. Research at the Agricultural University of Athens focuses on restructuring conventional industrial plants into integrated biorefineries through the valorization of waste or by-product streams. This study evaluates the potential restructuring of a pulp and paper process employing the sulfite process through fractionation of spent sulfite liquor (SSL) for the production of phenolic compounds as antioxidants, lignosulfonates and succinic acid. SSL is a complex waste stream generated via wood chip digestion with calcium or magnesium sulfite where delignification occurs under high pressure and low pH conditions. SSL digestion leads to hemicellulose degradation to C6 and C5 sugars and lignin depolymerisation by sulfonation and hydrolysis. SSL was processed via ultrafiltration to produce a sugar-rich permeate that is suitable as carbon source for fermentative production of succinic acid. The retentate contained a high concentration of lignosulfonates. An antioxidant-rich fraction was extracted from the permeate stream via solvent extraction with ethyl acetate. The total phenolic content, the antioxidant activity and specific phenolic compounds were analysed in this stream. Production of succinic acid was evaluated with the bacterial strains Actinobacillus succinogenes and Basfia succiniciproducens in batch and fed-batch bioreactor cultures using either free or immobilized cells. SSL valorisation could lead to a sustainable biorefinery concept. Acknowledgements: The authors gratefully acknowledge the financial support by the FP7 research project BRIGIT (KBBE2012-6-311935, www.brigit-project.eu). http://dx.doi.org/10.1016/j.nbt.2014.05.1700 O10-3 Biotechnological conversion of spent coffee grounds into polyhydroxyalkanoates Stanislav Obruca ∗ , Pavla Benesova, Sinisa Petrik, Dan Kucera, Ivana Marova Faculty of Chemistry, Brno University of Technology, Czech Republic Coffee is one of the world’s most popular beverages and has been growing steadily in commercial importance. Nowadays, coffee is, after petroleum, the second largest traded commodity in the world. Hence, coffee industry is responsible for the generation of large amounts of waste, above all, spent coffee grounds (SCG). The aim of this work was to study conversion of SCG into valuable product–polyhydroxyalkanoates (PHAs). These polyesters are accumulated by various bacteria as carbon and energy storing materials. Due to their mechanical properties, they are considered being an alternative to petrochemical plastics. At first, oil extracted from SCG (approx. 15 wt% oil in SCG) was efficiently (YP/S = 0.82) converted into PHA employing Cupriavidus necator H16. Further, the solid residues after oil extraction were hydrolysed (combination of chemical and enzymatic hydrolysis) yielding fermentable sugars, which were further used as a substrate for production of PHAs employing Bacillus megaterium and Burkholderia cepacia. B. cepacia revealed significantly higher PHAs yields and; moreover, it was capable of direct accumulation of copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate without addition of precursors. Finally, solids after SCG hydrolysis possess high calorific value and can be used as a fuel to at least partially cover energetic demands of the process. Hence, entire biomass of SCG can be used for sustainable production of PHAs employing bio-refinery approach. Acknowledgement: This work was supported by projects “Centre for Materials Research at FCH BUT” No. www.elsevier.com/locate/nbt S39 SYMPOSIUM 10: BIOWASTE BIOREFINERY FOR A MORE SUSTAINABLE BIOECONOMY CZ.1.05/2.1.00/01.0012 from ERFD and “Excellent researcher at BUT” No. CZ.1.07./2.3.00/30.0039. young http://dx.doi.org/10.1016/j.nbt.2014.05.1701 O10-4 Extraction of the protein fraction of dry distillers grains with solubles, implementing biocatalytic and chemical methods Maria Villegas Torres ∗ , Gary Lye, John Ward New Biotechnology · Volume 31S · July 2014 obtain extracts rich in sugars, containing around 12.5 g/L sucrose, 7 g/L glucose and 2.5 g/L fructose. All sugars were consumed and SA yield of 0.4–0.5 g SA/g sugar could be obtained, depending on extract concentration. These results show that carob pulp water extracts or glycerol are promising substrates for SA production, demonstrating the high versatility of A. succinogenes. References [1].McKinlay, et al. Appl Microbiol Technol 2007;76:727–40. [2].Carvalho, et al. Nat Biotechnol 2014;31:133–9. http://dx.doi.org/10.1016/j.nbt.2014.05.1703 UCL, United Kingdom O10-6 Distillers dried grain and solubles (DDGS) is a by-product from distilleries and breweries, currently used as animal feed. It contains mainly protein, fats and residual carbohydrates, making it an ideal feedstock for biocatalytic processes. In addition, due to the current expansion of the biofuel industry in the UK, a dramatic increase in DDGS production is expected. Therefore, it is of industrial interest to develop manufacturing processes of industrially relevant compounds implementing DDGS as feedstock. In this work, we focused on the extraction of the protein fraction (up to 40%), which is expected to have a high frequency of specific amino acids, as wheat grain is mostly gluten -a highly elastic protein mainly composed of glutamine and proline. Glutamine has been extensively used as injury treatment, and muscle growth, and because of its physical characteristics can be an ideal source for biomaterials manufacture. We optimized a protein extraction process involving both chemical steps combined with enzyme hydrolysis. The optimized extraction method is a novel approach that can be applied at industrial scale. http://dx.doi.org/10.1016/j.nbt.2014.05.1702 O10-5 Succinic acid production from raw materials by Actinobacillus succinogenes Christophe Roca 1 , Margarida Carvalho 2,∗ , Maria A.M. Reis 2 1 2 REQUIMTE, Portugal REQUIMTE/Universidade Nova de Lisboa, Portugal Succinic acid (SA) is currently considered a key platform chemical as it is used in the production of a wide range of products, from pharmaceuticals to green solvents, fibers and bioplastics [1]. The use of renewable feedstocks as carbon source for the production of SA via fermentation has been suggested as a solution to reduce production costs and develop a sustainable SA production process [2]. In this work, Actinobacillus succinogenes was used as biocatalyst for the conversion of two raw materials into SA: i) glycerol, byproduct of biodiesel industry, is usually poorly metabolized to SA because of a redox imbalance but here, it could be efficiently converted to SA, reaching concentration as high as 49 g/L SA using DMSO as electron acceptor; ii) extracts from carob pods, a by-product of carob locust bean gum industry were used as carbon source in small scale batch cultivations. A simple aqueous extraction was used to S40 www.elsevier.com/locate/nbt Enhanced welan gum production using cane molasses as substrate by Alcaligenes sp. ATCC31555 Jufang Wang ∗ , Hongxia Ai, Min Liu South China University of Technology, China Welan gum is a new high molecular microbial exopolysaccharide with a wide range of potential industrial applications in food, concrete, petroleum, ink and for enhancing oil recovery. Welan gum is more effective than any other polysaccharides in enhancing oil recovery and expected to become a novel oil recovery agent for its excellent stability at high temperature. For new polymers to be a commodity product in the near future, it is crucial to enhance the productivity and lower their production costs. In this work, cane molasses was selected for welan gum production by Alcaligenes sp. ATCC31555 in 5L fermentor. The pre-treatment of cane molasses, agitation and the additives for improving dissolved oxygen were investigated for process optimization. Sulfuric acid hydrolysis was the optimal molasses pretreatment for welan gum production with a maximum welan gum concentration (33.5 g/L), yield (0.62 g/g), productivity (0.28 g/L/h), and broth viscosity (3.375 Pa s), which are 50%, 138%, 47%, and 67% higher, respectively, compare to those without treatment. The process was subsequently optimized by agitation and adding additives for improving the dissolved oxygen at special time point during the fermentation. Optimal welan gum production (41.0 ± 1.41 g/L) was found at 600 rpm under 30 ◦ C with addition of 10% n-dodecane at 36 h in 5L fermentor, which is the highest welan gum concentration so far. Moreover, welan gum from molasses displayed similar rheological properties and thermal stability than that from glucose. It indicated that cane molasses may be an economical industrial substrate for welan gum fermentation. http://dx.doi.org/10.1016/j.nbt.2014.05.1704 New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 10: BIOWASTE BIOREFINERY FOR A MORE SUSTAINABLE BIOECONOMY O10-7 Investigating the biomass modifying and degrading enzymatic toolbox of Aspergillus japonicus var aculeatus FEC 156 with quantitative proteomics and New Generation Sequencing tools George Anasontzis 1,∗ , Thuy Nguyen Thanh 2 , Thanh Vu Nguyen 2 , Lisbeth Olsson 1 1 2 Chalmers University of Technology, Sweden Food Industries Research Institute, Sweden In the bio-based economy concept, the current hydrocarbon fuels and non-biodegradable plastics will be replaced by new products which will derive from natural and renewable resources. The synthesis of such biofuels and biochemicals is still challenged by the difficulties to cost efficiently degrade lignocellulosic materials to fermentable sugars or to isolate the intact polymers. Biomass degrading and modifying enzymes play an integral role both in the separation of the polymers from the wood network, as well as in subsequent modifications, prior to further product development. The type of application usually defines the conditions where the reactions should take place. Thus, novel enzymes with variable combined properties, such as different thermotolerance, pH range of activity, substrate specificity and solvent tolerance, still need to be discovered and developed to achieve the highest possible efficiency in each case. We isolated an Aspergillus japonicus var aculeatus strain and evaluated its cellulase and hemicellulases activities. It was then cultivated in bioreactors with different carbon sources, such as wheat bran, spruce and Avicel and its biomass degrading capacity was determined and semiquantified with TMT (Tandem Mass Tags), through cross species protein identification of its secretome. Information on the genes involved in the different stages of the fermentation and carbon sources have been acquired with next generation sequencing of its total transcriptome. Interesting transcripts are being heterologously cloned and expressed in order to identify their role and potential use in the biorefinery concept. http://dx.doi.org/10.1016/j.nbt.2014.05.1705 www.elsevier.com/locate/nbt S41 SYMPOSIUM 11: NANOTECHNOLOGY: NEW BIOLOGICAL APPLICATIONS Symposium 11: Nanotechnology: new biological applications O11-1 Design, structure and assembly of superparamagnetic core–shell nanoparticles Erik Reimhult Institute for Biologically inspired materials, Department of Nanobiotechnology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria Nanoparticles with carefully controlled core–shell structures can be used in biomedical applications, for example, for separation, as contrast agents, for hyperthermia and in drug delivery [1,2]. Exquisite control allows further applications through assembly into biomimetic membrane and vesicular structures for which permeability externally can be controlled by applied magnetic fields. I will briefly describe a new synthetic toolkit based on nitrocatechol dispersants, monodisperse synthesis of Fe3 O4 cores using capping agents and new approaches to ligand grafting into biocompatible shells on such nanoparticles; these developments allow the synthesis of a range of new magnetic nanoparticles for defined biotechnological applications and for fundamental research. The emphasis is on the purification and characterization of such nanoparticles and further on the assembly of nanoparticles into membrane superstructures that can be magnetically controlled. The relationship between nanoparticle structure, the assembled membrane structure and the release of a model drug from nanoparticle actuated vesicles will be highlighted. References [1].Amstad E, Reimhult E. Nanomedicine 2012;7:145–64. [2].Amstad E, Textor M, et al. Nanoscale 2011;3:2819–43. http://dx.doi.org/10.1016/j.nbt.2014.05.1706 O11-2 Production and characterization of HIV-1 virus-like particles using transient gene expression in mammalian cells Sonia Gutiérrez-Granados ∗ , Laura Cervera, Segura Maria de las Mercedes, Francesc Gòdia Universitat Autònoma de Barcelona, Spain New vaccine strategies based on virus-like particles (VLPs) are rapidly evolving. Upon expression in a number of heterologous host systems, Gag polyprotein of HIV-1 spontaneously assembles in the vicinity of the plasma membrane and it is released by budding producing enveloped particles that resemble immature HIV-1 virions. These particulate immunogens have proven to be potent stimulators of both cellular and humoral immune responses in animal models. Besides, Gag-based VLPs do not contain the viral genome, minimizing biosafety concerns. Thus, VLPs offer great promise as HIV-1 vaccines. Mammalian cells are the preferred system as a cell factory for this kind of vaccines due to the complexity S42 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 of VLP assembly and release from cells. Transient gene expression (TGE) offers the flexibility to rapidly screen a number of product variants with sufficient quantity and quality for preclinical proof of concept studies. In this work, mammalian cells are used for the production of a fluorescent variant Gag-based VLP using a TGE approach. A method based on GFP fluorescence has been developed to quantify both the transfection efficiency and the Gag-GFP VLP production. Additionally, the properties and purity of the obtained VLPs are further characterized by other techniques such as transmission electron microscopy, nanoparticle tracking analysis and size-exclusion chromatography. Discussion will be focused on the characterization of the process as well as the obtained product. http://dx.doi.org/10.1016/j.nbt.2014.05.1707 O11-3 Bacterial microcompartments moving into the world of biotechnology Stefanie Frank 1,∗ , Andrew Lawrence 1 , Allan Pang 2 1 2 University of Kent, United Kingdom University of California, United States Bacterial microcompartments (BMCs) are cytosolic protein structures that are composed of a shell housing sequentially acting enzymes associated with particular metabolic pathways.BMCs permit the enhancement of metabolic processes, which is why they are expected to have great potential for biotechnological applications. One of the most complex BMCs is associated with 1,2-propanediol utilisation (pdu) and the focus of our efforts for microcompartment engineering. The shell of the native Pdu microcompartment is composed of seven proteins (PduA, PduB, PduJ, PduK, PduN, PduT and PduU); only five are required for the self-assembly of empty heterologous microcompartments in E. coli. PduA is one of the most abundant shell proteins and has the ability to self-assemble into higher-order structures which we found can be manipulated by strategic intervention at the molecular level. Targeting of enzymes to the shell is mediated by short peptide sequences. We have solved the structure for the N-terminal targeting peptide (P18) of PduP, an enzyme associated with the Pdu microcompartment and identified a main interaction partner PduK, a protein of the outer shell. We have also fused two heterologous enzymes required for ethanol production (pyruvate decarboxylase and alcohol dehydrogenase) with targeting peptides and found it has been possible to direct these enzymes to empty BMCs in vivo and to generate a functional ethanol bioreactor. Ongoing research is striving to further understand proteinprotein interactions and assembly of microcompartments and related higher order protein structures in molecular detail in order to control and manipulate these structures for the development of ‘optimized’ bioreactors. http://dx.doi.org/10.1016/j.nbt.2014.05.1708 New Biotechnology · Volume 31S · July 2014 O11-4 Protein supramolecular engineering – applications in biotechnology Patrick Shahgaldian 1,∗ , Maria Rita Correro 2 , Alessandro Cumbo 3 , Philippe Corvini 2 1 University of Applied Sciences and Arts Northwestern Switzerland, Switzerland 2 University of Applied Sciences Northwestern Switzerland, Switzerland 3 INOFEA AG, Switzerland A large number of living organisms possess the capability to produce intricately patterned and hierarchically structured biogenic silica. Imitating natural systems’ ability to produce hierarchical silica structures may provide the possibility to design functional (nano)materials with the same degree of complexity. In this lecture, two novel design strategies of nanomaterials capable of either molecular recognition or biocatalysis will be discussed; both approaches are based on the self-assembly of organo-silica precursors using protein templates [1]. The first part of this presentation will be dedicated to the development of a novel class of nanomaterials possessing selective molecular recognition properties of viruses. The synthetic strategy to produce those nanoparticles is based on the formation of a chemical imprint of the template virion at the surface of silica nanoparticles. It is demonstrated that the so-produced particles possess enhanced molecular recognition properties for their target, even in complex media (e.g. human serum). The second part of this lecture will be dedicated to the development of a chemical strategy to produce nanobiocatalysts with enhanced biochemical, physical and chemical stabilities. It is based on the formation of a protective shell at the surface of enzyme proteins that provides a comfortable medium that allows for enzyme stabilization. It will be demonstrated that those systems can find a number of biotech applications. Reference [1].Cumbo A, Lorber B, Corvini PF-X, Meier W, Shahgaldian P. Nat Commun 2013;4:1503. http://dx.doi.org/10.1016/j.nbt.2014.05.1709 O11-5 Biological production of stable copper nanoparticles Nikolaos Pantidos ∗ , Louise Horsfall University of Edinburgh, United Kingdom Many nonferrous industries such as mining and surface treatment plants produce co-products that are high in heavy metals and therefore toxic to the environment. A less obvious producer of heavy metal containing co-products is the whisky industry. Current methods of copper removal from such co-products include electrolysis and membrane filtration which are reported to be impractical and costly. Alternatively, when copper is found as a salt, current methods of removal include settlement, filtration and SYMPOSIUM 11: NANOTECHNOLOGY: NEW BIOLOGICAL APPLICATIONS precipitation. Biological copper ion removal from effluents has been shown to be quite effective. There are two biological methods to remove copper from effluent which involve biosorption and reduction. Biosorption involves bacteria binding to copper via the cysteine-rich transport proteins that are associated with the cell membrane to precipitate it. Some bacteria are also able to reduce higher valency insoluble copper ions into zero valency insoluble forms of the metal. Here we present a metal-reducing bacterium Morganella psychrotolerans, which is able to reduce Cu2+ to insoluble Cu0 nanoparticles. The copper nanoparticles are produced as part of the bacterium’s defence mechanism against the toxic effects of metal ions. M. psychrotolerans grows at low a temperature which makes it ideal for industrial applications as energy for heating is not required; hence it can be potentially cheaper than current methods of copper removal. The copper nanoparticles produced during the distillery co-product retreatment can be isolated and subsequently used for purposes such as optics, catalysts, antimicrobials or recycled in order to make new copper stills for whisky distilleries. http://dx.doi.org/10.1016/j.nbt.2014.05.1710 O11-6 Insight into the physiological role of a compartmentalised ferritin like protein in Rhodospirillum rubrum Jon Marles-Wright ∗ , Didi He, Atanas Georgiev, David Clarke University of Edinburgh, United Kingdom All domains of life have evolved systems for the compartmentalisation of enzymes and metabolic pathways. These include the lipid-bounded organelles in eukaryotes and extend to protein-shelled microcompartments, such as carboxysomes, in prokaryotes. Rhodospirillum rubrum, which is a model organism for the study of bacterial nitrogen fixation, possesses two distinct metabolic compartments. We have characterised the simplest of these compartments, which is encoded in a two-gene locus and comprises a linocin-family shell protein and a ferritin-like protein. Using a combination of structural biology techniques, including X-ray crystallography, native mass-spectrometry, and electron microscopy, in combination within vivostudies we have determined the structure and function of this intriguing metabolic compartment. The compartment shell is build from 60 subunits of the linocin protein and forms a 25 nm icosahedron, much like the capsid of a virus. The ferritin is encapsulated co-translationally through the interaction of a short signal sequence at its terminus with the linocin. It forms a donut shaped structure and presents a unique inverted ferroxidase site, which is formed through the interaction of two protein monomers. The activity of this ferritin is mediated by encapsulation within the linocin shell and is distinct to the ferritins, bacterioferritins and DPS proteins that make up the rest of the family of ferritin-like proteins. This metabolic compartment is widely distributed across bacterial groups and is highly conserved, indicating a common www.elsevier.com/locate/nbt S43 SYMPOSIUM 11: NANOTECHNOLOGY: NEW BIOLOGICAL APPLICATIONS physiological purpose. The promiscuous metal ion binding sites in the ferritin and localisation mechanism give hints into the potential for engineering these compartments. http://dx.doi.org/10.1016/j.nbt.2014.05.1711 O11-7 Catalytic properties improvements of Alcaligenes faecalis nitrilase by self-assembly induced aggregation Shuang Li ∗ , Xiaofeng Yang School of Bioscience and Bioengineering, South China University of Technology, China A large number of studies have been devoted to improve catalytic properties of enzymes by protein engineering. However, these conventional methods, i.e. site directed, random and saturated mutagenesis, always rely on the correlation between structure and function or high-throughput screening approaches, which make it hard to obtain positive mutants in a short time. Here S44 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 we fused the nitrilase from Alcaligenes faecalis with an amphipathic peptides. Results showed that approximately 90% of nitrilase were produced as active inclusion bodies (aggregrates) in Escherichia coli cells. The thermal stability of the self-assembly induced nitrilase was increased by 6.8 and 4.3 folds at 45◦ C and 50◦ C, respectively. When the nitrilase aggregrates are purified by differential centrifugation with sucrose density gradient and negatively stained, a highly ordered amyloid fibrils structure is observed under TEM. After two steps of washing with buffers, the nitrilase aggregates were purified and further immobilized with sodium alginate as the carrier, then designated as iSEA. Thermal stability was further increased by 1.5-fold compared to the aggregrates. Also the iSEA showed good progress in the tolerance of mandelonitril from 30 mM to 200 mM. High expression yields, simple recovery steps of aggregates from the host cells and the favorable catalytic characteristics are attractive features industrially. http://dx.doi.org/10.1016/j.nbt.2014.05.1712 New Biotechnology · Volume 31S · July 2014 Symposium 12: Stress responses in microbial bioprocessing O12-1 Mechanisms of protein folding and quality control in bacteria Bernd Bukau Center for Molecular Biology of the University of Heidelberg, German Cancer Research Center, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany SYMPOSIUM 12: STRESS RESPONSES IN MICROBIAL BIOPROCESSING References [1].Wang B, Kitney R, Joly N, Buck M. Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nat Commun 2011;2:508. [2].Wang B, Buck M. Customizing cell signalling using engineered genetic logic circuits. Trends Microbiol 2012;20(8):376–84. [3].Wang B, Barahona M, Buck M. A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals. Biosens Bioelectron 2013;40:368–76. http://dx.doi.org/10.1016/j.nbt.2014.05.1714 O12-3 Protein homeostasis is established by a complex cellular machinery which assists and regulates regular folding pathways and counteracts protein misfolding and aggregation. A particularly critical process in the life of a protein is the native folding of newly synthesized proteins, which therefore is tightly controlled. Already during ongoing synthesis by the ribosome nascent polypeptides are subject to enzymatic processing, chaperone-assisted folding to the native state or targeting to translocation pores at membranes. The ribosome itself plays a key role in these different tasks by serving as platform for the regulated association of enzymes, targeting factors and chaperones that act upon the nascent polypeptides emerging from the exit tunnel. The molecular mechanisms integrating the different co-translational processes leading to the maturation and native folding of nascent chains will be described. http://dx.doi.org/10.1016/j.nbt.2014.05.1713 O12-2 Engineering customised cell signalling circuits and their biotechnological applications Baojun Wang University of Edinburgh, United Kingdom Cells live in an ever-changing environment and continuously sense, process and react to environmental signals using their inherent signalling and gene regulatory networks. Here I will present the construction of synthetic gene circuits to customise cellular information processing and responses by harnessing the inherent modularity of signalling networks [1–3]. In particular, a set of modular and orthogonal genetic logic gates, e.g. AND and NAND, and analogue circuits such as a gain-tunable genetic amplifier were engineered to modulate multiplein vivo transcriptional signals in either digital-like or bespoke analogue manner. I will then show that how these gene circuits can be used to enhance the specificity and sensitivity of synthetic cell-based biosensors for detecting heavy metal ions and bacterial signalling molecules in an aqueous environment, and to realise robust gene expression control and sensing in single cells over a range of abiotic conditions. Furthermore, we are engineering modular genetic controllers that can act as dynamic stress sensor-regulators to achieve adaptive control of cellular pathway gene expression flows for optimised biomolecule production. Stochastic activation of the GlpR-controlled glp gene cluster in Pseudomonas putida KT2440 results in a bistable growth pattern on glycerol Pablo Ivan Nikel ∗ , Victor de Lorenzo Centro Nacional de Biotecnologia (CNB-CSIC), Spain Phenotypic variation is a widespread trait among prokaryotes, and the molecular mechanisms underlying the phenomenon include genetic changes (e.g., genomic inversions and strandslippage processes), epigenetic variations (e.g., distinct patterns of DNA methylation), as well as feed-back-based multi-stability. All these mechanisms are known to ultimately lead to the appearance of at least two distinct phenotypes within an otherwise isogenic population. However, this complex trait has been scarcely explored in microorganisms that have environmental and industrial interest. The soil bacterium Pseudomonas putida exhibits promising biotechnological potential, together with its generally regardedas-safe certificates, resistance to endogenous and exogenous stress, amenability to genetic manipulation, and suitability as a host for heterologous gene expression. Growth of P. putida KT2440 on glycerol is characterized by an unexpectedly long lag phase (Nikel PI, Kim J, de Lorenzo V. Metabolic and regulatory rearrangements underlying glycerol metabolism in Pseudomonas putida KT2440. Environ Microbiol 2014;16:239-54). In the present contribution, the growth of individual P. putida KT2440 cells was assessed on glycerol to further explore this phenotypic behavior. It was found that the physiological properties of cells growing on this carbon source resulted in the appearance of two distinct cell sub-populations which significantly differed in their metabolic activity. These macroscopic properties are wired to the dual logic of the GlpR regulator, repressing the transcription of the cognate glp genes, encoding the enzymes needed for glycerol catabolism. Analyzed in perspective, these results suggest that P. putida is subjected to a carbon-source-dependent bet-hedging strategy that might be relevant in the natural niches it inhabits. http://dx.doi.org/10.1016/j.nbt.2014.05.1715 www.elsevier.com/locate/nbt S45 SYMPOSIUM 12: STRESS RESPONSES IN MICROBIAL BIOPROCESSING O12-4 The adaptation of the intestinal sulphate reducing bacterium, Desulfovibrio desulfuricans, to nitrosative stress induced by nitric oxide Matthew Faulkner ∗ , Jeff Cole The University of Birmingham, United Kingdom Desulfovibrio desulfuricansis an environmentally important organism that causes widespread bacterial corrosion to metal structures. It is useful for the bioremediation of heavy metals in the environment. The presence of D. desulfuricans populations in the human gut has been correlated with gastro-intestinal disease: its metabolism is thought to form inflammatory products. Unlike Desulfovibrio vulgaris, D. desulfuricans reduces both sulphate and nitrate as terminal electron acceptors. Nitrate is reduced via nitrite to ammonia, a process that generates nitric oxide as a side product. A strong adaptive nitrosative stress response is essential for organisms living in nitrate-rich, micro-oxic environments. Nitric oxide is generated during nitrate reduction in this, and other, organisms. Nitrosative stress, caused by NO production, is induced by macrophages to kill pathogens during human infection. It is therefore important to understand how D. desulfuricans responds to nitrosative stress. The tolerance of D. desulfuricans ATCC 27774 to nitric oxide was defined for bacteria with various histories of exposure. Cultures grown with nitrate as terminal electron acceptor showed a 10-fold increase in tolerance to NO compared to sulphate grown cultures. Nitrate grown cells also reduced nitric oxide at a 5-fold higher rate than sulphate grown cells. These phenotypic changes were investigated in the transcriptome of cells from stressed and unstressed conditions by qPCR. The data reveal that exposure to nitrosative stress induced a global response, that includes the induction of genes encoding proteins of previously unknown function. http://dx.doi.org/10.1016/j.nbt.2014.05.1716 O12-5 Origins of Escherichia coli growth rate and cell shape changes at high external osmolality Teuta Pilizota 1,∗ , Joshua Shaevitz 2 1 2 University of Edinburgh, United Kingdom Princeton University, United States In line with the challenge of designing robust synthetic systems, there has been an increased interest in understanding bacterial growth rates. Growth can be modulated in different ways, including temperature, antibiotics, toxins, nutrients and osmolarity. While the effects of nutrient availability have been extensively characterized, an almost equally common modulation of bacterial growth occurs in response to changes in external osmolarities. In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope, followed by an active recovery. Post recovery E. coli cells have been shown to grow slower, smaller and at a reduced turgor pressure. Despite the S46 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 fact that the active recovery is a key stress response, the nature of these changes is not understood. The biotechnology industry is heavily involved in engineering bacteria for production of useful compounds. High-level excretion of substrate feed during biotechnological applications constantly increases medium concentration. As a result, limited cell growth and low volumetric productivity has been reported. Here, we use fluorescence imaging of single cells during hyperosmotic shocks, combined with custom made microfluidic devices, to show that cells fully recover their volume to the initial value and continue to grow slower immediately after the recovery. We also show that turgor pressure recovers to the initial value along with the cell volume and, unlike previously thought, does not cause the reduction in growth rates. We present results that point to changes in cellular energetics as the main cause of growth slow down at high external osmolarities. http://dx.doi.org/10.1016/j.nbt.2014.05.1717 O12-6 Engineering global regulator cAMP receptor protein (CRP) of E. coli for improved biobutanol tolerance Rongrong Jiang ∗ , Hefang Geng, Huiqing Chong Nanyang Technological University, Singapore The toxicity of biofuel, including isobutanol and 1-butanol, to microbial host is a major drawback during fermentative biofuel production. Previously, UV/chemical mutagens and metabolic engineering have been used for strain engineering. However, these approaches are either labor intensive or time consuming, and require comprehensive metabolic information. In this work, we aimed to improve E. coli tolerance towards isobutanol and 1butanol by rewiring its global transcription factor cAMP receptor protein (CRP). Error-prone PCR was performed to mutate crp, and the random mutagenesis libraries were subjected to isobutanol or 1-butanol stress for screening. After 3–5 subcultures with increased stressor concentration, the mutants with improved tolerance were enriched in cell culture. Mutant IB2 (S179P, H199R) and MT5 (G71D, T127N, D138V, T208N) exhibited enhanced isobutanol/1butanol tolerance respectively. In the presence of 1% (v/v, 9.6 g/L) isobutanol, IB2 had the growth rate of 0.18 hour−1 , 3.6-times faster than the control (0.05 hour−1 ). Similarly, when challenged with 1.2% (v/v, 9.6 g/L) 1-butanol the growth rate of MT5 was 0.18 hour−1 , double that of the control (0.09 hour−1 ). Genome wide DNA microarray revealed that IB2 had 366 differentially expressed genes (>2 fold, p-value < 0.05) under isobutanol stress, including acid resistance genes (gadABCE, hdeABD) and transporters (proVWX, manXYZ). Quantitative real-time PCR showed that 1-butanol stress response genes (rpoH, ompF, sodA, manX, marA) in MT5 demonstrated differential expression compared to the control. Therefore, we believe that engineering CRP can serve as an effective alternative for E. coli engineering. http://dx.doi.org/10.1016/j.nbt.2014.05.1718 SYMPOSIUM 12: STRESS RESPONSES IN MICROBIAL BIOPROCESSING New Biotechnology · Volume 31S · July 2014 O12-7 Heat shock at higher cell densities improves the translocation of measles virus hemagglutinin into the yeast endoplasmic reticulum Bak¯unaitė, R¯uta Rimantas Slibinskas ∗ , Edita Raimundas Ražanskas, Evaldas Čiplys Zinkeviči¯utė, Vilnius University Institute of Biotechnology, Lithuania The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of heterologous proteins. However, secretory expression of the heterologous proteins in yeast is often subject to several bottlenecks that limit yield. Translocation of newly synthesized proteins into endoplasmic reticulum (ER) is the first stage of the secretion pathway. It was earlier shown that synthesis of measles virus hemagglutinin (MeH) is inefficient mostly due to bottleneck in the translocation of precursors of viral protein into the ER of the yeast cells. The aim of this study was to improve translocation of MeH in S. cerevisiae by manipulating cell culture conditions. Induction of MeH expression under various conditions was tested to establish factors that influence the efficiency of MeH translocation. We found that heat shock with subsequent induction of MeH expression at 37◦ C improved translocation of MeH precursors at the higher cell densities. The amount of MeH glycoprotein increased about three-fold after the heat shock in the late-log phases of both glucose and ethanol growth. Our results suggest that the heat shock may be employed for the improvement of expression of the heterologous proteins in the S. cerevisiae secretory pathway. http://dx.doi.org/10.1016/j.nbt.2014.05.1719 www.elsevier.com/locate/nbt S47 FUTURE COLLABORATIONS OF AFOB-EFB AND MOU SIGNING CEREMONY New Biotechnology · Volume 31S · July 2014 Future collaborations of AFOB-EFB and MOU signing ceremony (3) Biopharmaceutical and Medical Biotechnology, (4) Biocatalysis and Protein Engineering, (5) Bioprocess and Bioseparation Engineering, (6) Bioenergy and Biorefinery, (7) Environmental Biotechnology, (8) Marine Biotechnology, (9) Nanobiotechnology, Biosensors and Biochips, (10) Systems and Synthetic Biotechnology, (11) Tissue Engineering and Biomaterials, and (12) Asia Bioeconomy and Biobusiness. Currently, some AFOB Divisions have started to work. For example, the Bioenergy and Biorefinery Division will have the annul division meeting and summit in coming August, in which the leader of EFB Environmental Biotechnology Section has been invited to join as an invited speaker. The division meeting of Bioprocess and Bioseparation Engineering Division is to be held in this September. AFOB and its Divisions have strong interest in (co-)hosting various seminars or symposia with EFB and other societies. In addition to collaborations on congresses of AFOB and EFB, the collaborations between similar divisions from two parties will also be beneficial. For collaboration in Division/Section level, we are expecting academic Divisions of AFOB and their counterpart Sections of EFB to jointly organize academic activities and establish close collaborations. SP6-1 Asian Federation of Biotechnology (AFOB) as a driving force for collaborative growth of biotechnology in Asia and beyond Ho Nam Chang , Jian-Jiang Zhong ∗ , Tai Hyun Park, Hyun Jung Kim Asian Federation of Biotechnology, 508 Meet-You-All Tower Annex B, 12 Gaetbeol-ro, Yeonsu-gu, Incheon, Republic of Korea Asian Federation of Biotechnology (AFOB, homepage: www.afob.org) is a non-profitable organization established in 2008. Since then, 13 Asian regions have joined AFOB as member regions and currently 3000 individual members from Asia are actively involved in AFOB activities. Of course, the number of both member regions and individual members is still increasing. Following the mission to promote co-operation and to expand network between academia and industry of biotechnology, AFOB holds various events including congresses and symposia for scientists and engineers in all areas of biotechnology. Especially, AFOB has established a tradition in Asian Congress on Biotechnology (ACB) since 1990 when APBioChEC was its predecessor, Young Asian Biochemical Engineers’ Community (YABEC) since 1995, AFOB Regional Symposium (ARS) and AFOB International Symposium. AFOB also organizes Annual Meetings and Summits for each division to promote their activities and collaboration with related divisions in partner organizations. In addition, Biotechnology Journal publishes AFOB special issues twice a year as an official partnership between AFOB and the Wiley publisher, enhancing the international status of Asian regions in biotechnology and exhibiting top biotechnology achievements from member institutions and organizations to the world beyond the Asia. All the members of AFOB are working hard for innovative and practical biotechnology for the benefits of our human beings for better life, clean environment and sustainable development of the society. http://dx.doi.org/10.1016/j.nbt.2014.05.1720 SP6-2 Academic Division of AFOB and Possible Collaboration with EFB Wen-Chien Lee ∗ , Hyung Joon Cha, Jian-Jiang Zhong, Ho Nam Chang Asian Federation of Biotechnology, 508 Meet-You-All Tower Annex B, 12 Gaetbeol-ro, Yeonsu-gu, Incheon, Korea Asian Federation of Biotechnology (AFOB) has set up an academic committee to coordinate and support its academic Divisions. Totally twelve academic Divisions are established to cover all areas of applied biotechnology. These are Divisions of (1) Agricultural and Food Biotechnology, (2) Applied Microbiology, S48 www.elsevier.com/locate/nbt http://dx.doi.org/10.1016/j.nbt.2014.05.1721 SP6-3 Advances in Biotechnology in Asia and Future Collaboration with EFB Tai Hyun Park ∗ , Teruyuki Nagamune, Jung-Keug Park, Jian-Jiang Zhong, Ho Nam Chang Asian Federation of Biotechnology, 508 Meet-You-All Tower Annex B, 12 Gaetbeol-ro, Yeonsu-gu, Incheon, Korea The fast advancement of biotechnology around the world provides us with new products, ideas, methods, and tools. Biotechnology in Asia is also growing very fast, and abundant biodiversities and biomass resources are located in Asian region. AFOB and EFB are able to develop a partnership to provide their members with the opportunities for the collaborative research. In this sense, academic societies can play an important role. As an example, during last three years the American Institute of Chemical Engineers (AIChE) has been organizing an International Forum: Biotechnology in Asian Countries. The topics of the International Forum at AIChE annual meetings were as follows: Biotechnology in China (2011), Biotechnology in Taiwan and Southeastern Asia (2012), Biotechnology in South Korea and Japan (2013). In this presentation, we are going to introduce some activities of AFOB and the biotech researches in Asian countries. We would also like to suggest some possible strategies for strengthening the collaboration between AFOB and EFB, and any feedback from EFB colleagues is well expected. http://dx.doi.org/10.1016/j.nbt.2014.05.1722 New Biotechnology · Volume 31S · July 2014 Advances in omic technologies SP4-1 Integrating omics to study human biology and disease Mathias Uhlen Science for Life Laboratory, Sweden The human proteins constitute the major building blocks for the function of the various processes necessary for human life. The mapping of the human genome has allowed us to predict that each human has approximately 20 000 genes encoding for proteins. In the field of proteomics, these proteins are studied using various tools such as mass spectrometry, antibody-based profiling, chromatography, bioimaging, crystallography and spectroscopy. In addition, the new tools in genomics based on next generation sequencing have open up the possibility to study human variation and expression levels in a quantitative manner not possible only a few years ago. We have classified all the protein coding genes in humans using a combination of genomics, transcriptomics, proteomics and antibody-based profiling. We have used this data to study the global protein expression patterns in human cells, tissues and organs as well as a discovery tool to find potential biomarkers for disease, such as cancer [1–9]. ADVANCES IN OMIC TECHNOLOGIES Syst Biol 10,721). iAdipocytes1809 was employed for gaining further insights from transcriptomics data obtained from Swedish Obese Subjects (SOS) Sib Pair study whereas iHepatocytes2322 was employed for the analysis of transcriptomics and metabolomics data obtained from NAFLD patients, and thereby we identified both putative biomarkers as well as therapeutic targets. Personalized GEMs for HCC patients were employed for identifying anticancer drugs that are specific to individual patients using the concept of antimetabolites i.e. drugs that are structural analogs to metabolites. The toxicity of each antimetabolite was predicted by assessing the in silico functionality of 83 healthy cell type specific GEMs. Finally, I will present the integration of data from RNASeq and antibody-based immunohistochemistry across all major human tissues and organs to explore the human tissue proteome with enriched expression (Mol Cell Proteomics 13, 397;Faseb J, doi:10.1096/fj.14-250555) and its use for the validation and improvement of the GEMs. http://dx.doi.org/10.1016/j.nbt.2014.05.1724 References [1].Mardinoglu, et al. Nat Commun 2014;5:3083. [2].Agren, et al. Mol Syst Biol 2014;10:721. [3].Stadler, et al. Nat Meth 2013;10(4):315–23. [4].Danielsson, et al. Proc Natl Acad Sci U S A 2013;110(17):6853–8. [5].Mardinoglu, et al. Mol Syst Biol 2013;9:649. [6].Paik, et al. Nat Biotechnol 2012;30(3):221–3. [7].Uhlen, et al. Nat Biotechnol 2010;28(12):1248–50. [8].Bourbeillon, et al. Nat Biotechnol 2010;28(7):650–3. [9].Vashisht, et al. Science 2009;326:718–21. http://dx.doi.org/10.1016/j.nbt.2014.05.1723 SP4-2 The use of Genome-scale metabolic models for drug target and biomarker identification Adil Mardinoglu 1,∗ , Mathias Uhlen 2 , Jens Nielsen 1 1 2 Chalmers University of Technology Royal Institute of Technology The elucidation of diverse disease mechanisms for identification of novel drug targets and discovery of biomarkers has been a major focus in medicine, and further research efforts are still needed for developing efficient diagnostic and treatment strategies. Genome-scale metabolic models (GEMs) can aid in this by providing a scaffold for integration of omics data and revealing the molecular mechanisms involved in the occurrence of the disease (J Intern Med 271, 142;Biotechnol J 8,985). My presentation will cover the reconstruction and the use of functional GEMs for adipocytes (iAdipocytes1809) (Mol Syst Biol 9, 649), hepatocytes (iHepatocytes 2322) (Nat Commun 5,3083) and personalized GEMs for Hepatocellular carcinoma (HCC) patients (Mol www.elsevier.com/locate/nbt S49 SPONSORED SYMPOSIUM Sponsored Symposium SS-1 Recent developments in scaling down and using single use probes for measuring the live cell concentration by dielectric spectroscopy John Carvell , Matt Lee, Pardip Sandhar ∗ Aber Instruments Ltd, United Kingdom Real-time bioprocess monitoring is fundamental for maximizing yield, improving efficiency and process reproducibility, minimizing costs, optimizing product quality, and full understanding of how a system works. Bioreactors that are monitored continuously and in real-time offer the advantage of meeting current and future supply demands with biological product of the utmost quality and safety, achieved at the lowest overall cost and with least risk. This paper will focus on the latest developments in dielectric spectroscopy for live cell concentration measurement and how the technology has been scaled down allowing bioreactors with less than 100 ml working volume to be monitored in real time. The presentation will also focus on how dielectric spectroscopy can also be applied to single use bioreactors in a cGMP environment and on samples down to 100 ␮l volume. http://dx.doi.org/10.1016/j.nbt.2014.05.1725 New Biotechnology · Volume 31S · July 2014 strain C. glutamicum DM1933. Within oxygen-limited plug-flowcompartment coupled to aerobic stirred-tank-reactor, primary (i.e. deliberately induced) and secondary (resulting from primary) oscillations of substrate and dissolved oxygen concentration as well as pH and metabolic side product concentrations are studied. With anaerobic residence times from 40 s up to several minutes, robustness and performance of microbial metabolism is challenged at continuously oscillating microenvironments. Carbon flux, respiratory activity and side product concentrations show fundamental differences under oscillating conditions. Most strikingly, Omics-data from the metabolome, proteome and transcriptome show only marginal effects with respect to oscillation. This indicates that the regulon of C. glutamicum is very robust against oscillatory conditions, which constitutes an important mechanism for robustness in industrial processes. http://dx.doi.org/10.1016/j.nbt.2014.05.1726 SS-3 Automated development of recombinant bioprocesses–from vision to mission Florian Glauche 1,∗ , Andreas Knepper 1 , Michael Heiser 1 , Lorenz Theuer 1 , Fabian Wollny 1 , Susan Bigesse 1 , Antje Neubauer 2 , Sarina Arain 3 , Gernot Thomas John 3 , Joachim Aschoff 4 , Birgit Stehlik 4 , Ingrid Schmidt 4 , Rudibert King 5 , Norman Violet 5 , Detlef Goelling 6 , Andreas Raab 6 , Gregor Kiesewetter 6 , Rick Nolte 6 , Peter Neubauer 1 1 SS-2 Scale-down study of oscillations in oxygen and substrate supply for Corynebacterium glutamicum Marco Oldiges 1,∗ , Friedrich Käß 1 , Ioanna Hariskos 1 , Andrea Michel 1 , Robert Spann 2 , Peter Neubauer 2 , Stefan Junne 2 , Wolfgang Wiechert 1 1 Forschungszentrum Jülich/Institute of Bio- and Geosciences – IBG-1: Biotechnology, Germany 2 Chair of Bioprocess Engineering/TU Berlin, Germany The platform organism Corynebacterium glutamicum has a successful history of biotechnological application and prevailing significance in industrial biotechnology with applications at bioreactor scales of several hundred cubic meters. Studies in metabolic and bioprocess engineering, however, are mostly limited to cultivation at laboratory scale. An often neglected upscaling effect is that physical transport and metabolic uptake processes of, for example oxygen and substrate lead to stronger concentration gradients with increasing bioreactor working volume. This causes oscillating microenvironments for individual cells. The metabolic impact of such oscillations can be monitored in specialized scaledown bioreactor setups, where large scale effects are simulated in laboratory-scale experiments. In this work, two-compartment reactor systems (stirred-tankreactor connected to plug-flow-reactor) have been used to generate defined oscillations in oxygen and substrate supply during cultivation of C. glutamicum ATCC13032 and lysine producing S50 Chair of Bioprocess Engineering, TU Berlin BioSilta Europe GmbH 3 PreSens Precision Sensing GmbH 4 Infoteam Software AG 5 Chair of Measurement and Control, TU Berlin 6 Organobalance GmbH 2 www.elsevier.com/locate/nbt The development of recombinant protein production processes is a time and labor-intensive task. Implementing Quality by Design (QbD) and Design of Experiments (DoE) principles, already at the screening stage can significantly reduce development risk, time and costs. With the recent advances in laboratory automation, most steps of up- and downstream bioprocess development can be automated with implementation of DoE and QbD. At TU Berlin, in close collaboration with industrial partners, a reference laboratory, which focuses on the automation of recombinant protein production process development, was established. Key features of the system are (i) development of software solutions in order to apply DoE for screening and optimization procedures, (ii) automated atline and on-line analyses by the use of analytical procedures and sensors, (iii) software-supported model creation and development of process models for advanced process control at the bioreactor scale. Microbial cultivations are performed in 96 and 24 well plates as well as in a 10 mL 48-bioreactor system. The application of EnBase® technology to maintain controlled growth is one of the key technologies in this concept. Growing cells in fed-batch cultures over the whole developmental line ensures consistency in operation procedures from microliter and milliliter scales to pilot plant scale. The overall strength of the automation concept SPONSORED SYMPOSIUM New Biotechnology · Volume 31S · July 2014 is demonstrated on a number of difficult to produce proteins in Escherichia coli and Saccharomyces cerevisiae. http://dx.doi.org/10.1016/j.nbt.2014.05.1727 SS-5 Massive protein superfamily data integration applied to smart library design Henk-Jan Joosten SS-4 Scale up of chito-oligomer production via bacterial fermentation Hendrik Waegeman 1,∗ , Ko Wisse 2 , Saskia Vander Meeren 2 , An Vermeulen 2 , Brecht Vanlerberghe 2 1 2 Bio Base Europe Pilot Plant vzw, Belgium Bio Base Europe Pilot Plant, Belgium Chito-oligomers constitute an interesting class of specialty carbohydrates, among other applications used in plant protection and wound healing products. Today’s commercially available chitosans are produced chemically from chitin isolated from shrimp shell wastes. They can be well defined concerning their degree of polymerisation and degree of acetylation, but they are invariably characterised by a random pattern of acetylation (PA), despite this influences the activity greatly. Together with other partners within the ERA-IB ChitoBioEngineering and FP7 Nano3Bio project, Bio Base Europe Pilot Plant aims at establishing, through genetic, metabolic and enzyme engineering, biotechnological ways of producing fully defined, partially acetylated chitosans, of which no production methods are available to date. Our specific goal, is to employ the strains tested at laboratory scale, to develop an industrially viable fermentation and downstream purification process for every specific chito-oligomer. Processes, meeting defined requirements in terms of titer and production rate are scaled up in our facilities. This presentation will show the results of the successful scale up of the first product developed by BBEPP and its partners. Bio-Prodict, Nijmegen, Netherlands Nature offers a wide variety of enzymes that can be utilized in many different processes, but often these enzymes need to be optimized by introducing mutations to meet the requirements of these (biotechnological) processes. In many cases a combination of multiple mutations are needed to reach these goals, but finding the right combination of mutations still is problematic. More and more protein engineers use a strategy referred to as “smart library design”. Smart mutant libraries contain only a small number of mutants and are designed such that they contain a high number of active clones with mutations at positions (hotspots) that are likely to show the desired effect. The quality of a smart library depends on: 1. The selection of hotspots and 2. The prediction of the best amino acid changes at these hotspots. Recently it was shown that the vast amounts of data nowadays available for protein superfamilies can be used for the prediction of both these steps and therefore for the design of high quality smart mutant libraries. Here we present how 3DM, a protein superfamily analysis platform that integrates many different data types for complete protein superfamilies, can be used to design smart libraries. Using this strategy different enzymes features, such as enantioselectivity, activity and thermostability have been optimized. Comparisons with random designed libraries show that, by using 3DM when designing a smart library, libraries are of high quality, which reduces not only the number of clones that need to be screened but it also increases the chance of finding an enzyme with the desired properties. http://dx.doi.org/10.1016/j.nbt.2014.05.1729 http://dx.doi.org/10.1016/j.nbt.2014.05.1728 www.elsevier.com/locate/nbt S51 SYMPOSIUM 13: BIOMEDICAL RESEARCH New Biotechnology · Volume 31S · July 2014 Symposium 13: Biomedical research O13-1 Bacteria fabricate 3D scaffolds for organ regeneration Paul Gatenholm ∗ , Hector Johannesson, Daniel Hägg Martinez, Johan Sundberg, Sara BBV Laboratory and WWSC, Department of Chemical and Biological Engineering, Biopolymer Technology, Chalmers University of Technology, Göteborg, Sweden In recent years, stem cells and biomaterials have emerged as tools to regenerate damaged or diseased tissue. Typically, a biocompatible material that can be engineered to match the shape of the defect and can function as a vehicle to deliver cells and/or biologically active factors that promotes tissue regeneration is used. Bacterial nanocellulose is emerging biomaterial which combines the nanofibril network with hydrogel like behavior which makes it ideal for Tissue Engineering applications. We have previously used bacteria to fabricate artificial blood vessels and menisci substitutes, and excellent biocompatibility has been shown in rat, hamster, pig and sheep. In the present study we describe a novel biofabrication method, which uses the Gluconoacetobacter Xylinus machinery to engineer 3D scaffolds with features at different length scales – ranging from the nano, subcellular to the macroscale. Examples of 3D Bacterial nanocellulose scaffolds prepared in our laboratory include multichannel scaffolds for preparation of microvascular structures, highly porous 3D structures for growth of cartilage tissue and combination of 3D multichannel scaffold with highly porous architecture for growth of vascularized tissue such as bone and adipose tissue. 3D Bacterial nanocellulose scaffolds have shown to support neural network formation and enable stem cell differentiation. Human tissues grown on 3D Bacterial nanocellulose scaffolds show great potential of this new biomaterial-cell constructs for applications in reconstructive surgery and as in vitro model of diseases such as Alzheimer and Osteoarthritis. http://dx.doi.org/10.1016/j.nbt.2014.05.1730 O13-2 Enzyme immobilized polymeric biomaterials Mukesh Doble ∗ , Prabhawathi Veluchamy IIT Madras, India Polymers are widely used in the biomedical technology for the fabrication of medical implants and devices. Implant based infection is highly prevalent in biomaterial which leads to its early rejection. Several physical, chemical and biological methods are practiced to overcome the bacterial attachment and the subsequent formation of biofim. Antibiotics are used as main form of therapy, but emergence of antibiotic resistance bacteria is forcing the researches to search for other methods. Biomaterials are hydrophobic, with no labile chemical groups that might be used with conventional immobilization strategies. Papain, an antimicrobial and biocompatible enzyme is immobilized on two S52 www.elsevier.com/locate/nbt polymers namely, poly urethane and polycaprolactam, which are approved by FDA for medical applications. The enzyme is immobilised using glutaraldehye as the fixing agent. Presence of the enzyme is confirmed using FTIR. The conditions namely, temperature, pH and time of reaction for immobilization are optimized to achieve maximum enzyme activity. The storage stability of the surfaces is tested successfully. The formation of Staphylococcus aureus and Escherichia coli biofilm on these surfaces are tested. These organisms are widely found in implant associated infection. Both the surfaces reduced the number of live colonies (by about 30 times), amounts of biomass, protein and carbohydrate content (by 6 times) in the bacterial biofilm. The composition of the biofilm was probed using FT-Raman spectroscopy. This study indicates that stable antimicrobial surfaces could be prepared using this enzyme. Such surfaces could find applications in the design of implantable or topical biomaterials which require prevention of biofilm. http://dx.doi.org/10.1016/j.nbt.2014.05.1731 O13-3 Osteoblast cell proliferation on magnesium-substituted hydroxyapatite (Mg-HA) coatings Fatma Nese Kok 1,∗ , Sakip Onder 2 , Ayse Ceren Calikoglu 3 , Kursat Kazmanli 4 , Mustafa Urgen 4 , Gamze Torun Kose 3 1 Istanbul Technical University, Turkey Istanbul Technical University, Molecular Biology Genetics and Biotechnology Programme, Turkey 3 Yeditepe University, Genetics and Bioengineering Department, Turkey 4 Istanbul Technical University, Department of Metallurgical and Materials Engineering, Turkey 2 Hydroxyapatite (HA; Ca10 (PO4 )6 (OH)2 ) is a well-known biocompatible material commonly preferred in surface modification of hard tissue implants to ensure better osteointegration with the surrounding bone tissue. Properties of HA may also be improved via doping different ions into HA structure for better osteointegration and cell proliferation properties. In our study, cell proliferation studies on magnesium substituted HA coatings (MgHA) that were deposited on different Mg2+ containing (Ti,Mg)N thin film coatings (0, 4.24 and 10.42% at) were studied to determine the effect of Mg2+ on cell proliferation. Mg-HA coatings and characterization studies were conducted as our previous study [1]. Then, osteoblast cells were seeded on HA and Mg-HA coatings and MTS studies were conducted for 1, 4 and 7 days. Results showed that cell proliferation was better on Mg-HA coatings that were deposited on low Mg2+ (4.24 at%) containing surfaces compared with the HA coatings that were deposited on Mg2+ free surfaces. Cell proliferation slowed down on Mg-HA coatings that were deposited on high Mg2+ (10.42 at%) containing surfaces. High Mg content also accelerated the corrosion so this was taught to be the major reason for the decrease in cell survival. Studies on the biocompatibility of (Ti,Mg)N thin film coatings prepared in different conditions are being tested to better understand the effect of Mg presence in the coatings. The grant from TUBITAK (112M339) is gratefully acknowledged. SYMPOSIUM 13: BIOMEDICAL RESEARCH New Biotechnology · Volume 31S · July 2014 Reference [1].Onder S, et al. Mater Sci Eng C 2013;33(7):37–42. http://dx.doi.org/10.1016/j.nbt.2014.05.1732 O13-4 Bone regeneration through facile binding of bone graft substitute particles using mussel adhesive protein Hyung Joon Cha 1,∗ , Bong-Hyuk Choi 1 , Hogyun Cheong 1 , Yun Kee Jo 1 , Jin-Soo Ahn 2 , Sang-ho Jun 3 1 Pohang University of Science and Technology, Republic of Korea Seoul National University, Republic of Korea 3 Korea University Anam Hospital, Republic of Korea 2 To date, hydroxyapatite and related calcium phosphates have been intensively investigated as the bone substitutes for bone tissue engineering. Among these, inorganic bovine bone is the most commonly used bone graft material. However, lack of cell recognition motifs and/or biochemical factors has been considered a limitation. Mussel adhesive proteins (MAPs) are one of most remarkable and powerful adhesive materials in nature. Previously, recombinant MAPs were successfully demonstrated to be functional cell adhesion materials on various surfaces due to their peculiar adhesive properties. Herein, MAPs were applied as surface coating and functionalization biomaterials to xenograft materials. We successfully coated MAPs onto xenograft surfaces by simply mixing xenografts with the MAP solution. Through in vitro study using mouse osteoblast cell line MC3T3-E1, significant enhancement of cellular activities such as attachment, proliferation, spreading, and osteogenic differentiation was observed on MAP-coated xenografts. In addition, we found that in vivo implantation of MAP-coated xenografts enhanced bone regeneration in a rat calvarial defect model. These results collectively demonstrate that facile coating of xenografts using biofunctional MAP would be a promising strategy for successful bone tissue engineering applications. http://dx.doi.org/10.1016/j.nbt.2014.05.1733 O13-5 The stabilisation of red blood cells in a powdered form Krishnaa Mahbubani ∗ , Nigel K.H. Slater Department of Chemical Engineering and Biotechnology, University of Cambridge, United Kingdom In recent years apprehension surrounding blood transfusions have significantly reduced. However, fears around the supply of blood have become a concern. In metropolitan areas, managing the blood inventory is critical to ensure emergency and elective surgeries can take place without shortages while military blood banking faces a slightly different challenge, in obtaining the required amount and type of blood products at specific times and often in remote areas. Current practice is to refrigerate blood (from donor centres), with an anticoagulant, where it is retained for a mere 42 days. To ensure a readily available safe blood supply, preservation techniques and methods for long-term storage need to be investigated. Cryopreservation in glycerol has shown some success in extending the shelf life of blood. However, prior to use, the glycerol must be removed. This is a demanding, labour intensive and costly process resulting in a limited 24-hour shelf life of deglycerolised sample making it an unfeasible procedure. We present the use of freeze-drying with intracellular sugars as a method for stabilisation. A two part process; freeze-drying involves a freezing step followed by a sublimation process that takes place with the sample held in a vacuum. The ideal product would be a dried powder capable of room temperature storage. With the appropriate additives, the hydrated blood would be suitable for direct transfusion. While the process is still in its infancy; freezedrying has the potential to change the future of blood banking. http://dx.doi.org/10.1016/j.nbt.2014.05.1734 O13-6 Stable aqueous solutions of keratin polypeptides: their properties in solution and at interfaces Fang Pan ∗ , Jian Lu The University of Manchester, United Kingdom Keratins are important structural fibrous proteins and are the main constituting components in skin, hair, wool, feathers, nails, horns and connecting tissues. Their physical and biological studies have direct relevance to health and disease and also bear important implications to hair and skin care. In addition to personal care, there are many other reasons that require us to understand how keratins behave physically and how they interact with other molecules. Wool is predominantly composed of keratin proteins that provide desirable properties. The purpose of this work is to develop water-soluble keratin polypeptides from sheep wool, which can be used to form smooth molecular layers and optically flat thin films. These model biointerfaces can facilitate various physical and biological measurements that would otherwise be too difficult to contemplate. Successful preparation of keratin samples was demonstrated by identification of molecular weights of the key components from gel electrophoresis and measurements of their surface tension and basic solution properties. Zeta potential measurements from keratin samples prepared demonstrated almost identical pH dependent surface charge distributions with isoelectric points around pH 3.5, showing that during purification by dialysis has completed removal of SDS used during wool fibre dissolution. http://dx.doi.org/10.1016/j.nbt.2014.05.1735 www.elsevier.com/locate/nbt S53 SYMPOSIUM 13: BIOMEDICAL RESEARCH New Biotechnology · Volume 31S · July 2014 O13-7 Trehalolipid biosurfactants from Rhodococcus ruber with anti-adhesive and immunomodulatory activities Maria Kuyukina 1,∗ , Irena 1 Kochina , Sergey Gein 2 1 2 Ivshina 1 , Tatiana Baeva 1 , Olesia Institute of Ecology and Genetics of Microorganisms, Russia Perm State University, Russia In recent years, glycolipid biosurfactants traditionally considered as emulsifying and solubilizing agents are attracting an increasing attention as possible biomedical agents with expressed biological activities [1]. Trehalolipids (TL) produced by members of closely related actinobacterial genera Rhodococcus, Nocardia, Corynebacterium, Gordonia, Mycobacterium, Tsukamurella, and Arthrobacter include ␣,␣′ -D-trehalose, a nonreducing disaccharide, which is linked by an ester bond to long-chain fatty acids [2]. Well-known TL of pathogenic Mycobacterium tuberculosis, Corynebacterium diphtheriae play a key role in the infections caused by these actinobacteria, and they are characterized by high immunomodulatory activity [3]. However the pathogeni- S54 www.elsevier.com/locate/nbt city of producers and high cytotoxicity of produced TL limit their biomedical applications. Therefore, the search for TL producers among nonpathogenic actinobacteria is essential. In in vitro experiments, TL biosurfactants from Rhodococcus ruber IEGM 231 stimulated both proinflammatory (interleukin (IL)-1b, IL-6, tumor necrosis factor-a (TNF-␣) and anti-inflammatory (IL-12, IL-18) cytokine production of human monocytes, depending on cell culture composition and induction. Also, diverse anti-adhesive effects of TL towards human monocytes and bacterial species were revealed. Since TL from R. ruber displayed no cytotoxicity against human lymphocytes or bacterial cells, they could be proposed as potential immunomodulatory, antitumor and anti-adhesive agents. Research was funded by the RAS MCB and RF President Leading Science School programs. References [1].Kitamoto, et al. J Biosci Bioeng 2002;94:187–201. [2].Kuyukina, Ivshina. Microbiol Monographs 2010;16:292–313. [3].Ryll, et al. Microbiol Immunol 2001;45:801–11. http://dx.doi.org/10.1016/j.nbt.2014.05.1736 SYMPOSIUM 14: PLANT GENETIC ENGINEERING New Biotechnology · Volume 31S · July 2014 Symposium 14: Plant genetic engineering O14-1 Genetic engineering of secondary metabolism for plant protection J.A. Pickett Rothamsted Research, Harpenden AL5 2JQ, United Kingdom Only recently has secondary plant metabolism become a target for genetic modification, initially to investigate the role of metabolites and now in crop plants for pest management. By generation in the plant, chemically unstable and even highly volatile metabolites can be exploited beyond the toxicants traditionally used to control pests. Expression of the aphid alarm pheromone, (E)-␤farnesene, in wheat will be described leading on to the need for induced or primed expression. Examples of other semiochemicals, in addition to pheromones, such as stress related homoterpenes, that are already exploited in crop protection, demonstrate also induction or priming as a consequence of stress related plant signalling involving secondary metabolism. Such signals themselves then provide alternatives to constitutive expression for use in GM crops and already provide evidence of likely success as well as offering new types of sentinel plants and potentially solutions for problems of perennialisation of arable crops. Secondary metabolites, exploited in these ways, also show promise for not only disease and weed management, but in dealing with other requirements of more sustainable agriculture relating to non-CO2 greenhouse gas production. http://dx.doi.org/10.1016/j.nbt.2014.05.1737 O14-2 Transcriptome and small RNA sequencing analysis of a new dwarf mutant in Gossypium hirsutum L Xiongming Du 1,∗ , Wen-yan An 2 , Jun-ling Sun 1 , Wen-fang Gong 1 , Shou-pu He 1 , Zhao-e Pan 1 1 Institute of Cotton Research of Chinese Academy of Agricultural Sciences (ICR, CAAS), China 2 Institute of Cotton Research of Chinese Academy of Agricultural Sciences (ICR, CAAS)/College of Life Science and Technology, Huazhong Agricultural University, China Plant height is an important trait in upland cotton. Ari1327 was a dwarfed mutant derived from an American upland cotton varieties-Ari971 by 60 Co ␥-ray irradiation. Ari1327 exhibited dwarf trait during germination and cotyledon periods. Using the next generation high-throughput sequencing technology, three cDNA and small RNA libraries of the dwarfed mutant Ari1327, higher plant mutant Ari3697 and their wild type Ari971 were constructed and sequenced with Illumina HiSeqTM 2000 system. Through comprehensive analysis with transcriptome and miRNA regulation level for the plant high mutant, it will be beneficial to reveal the molecular mechanism dwarf mechanism and clone the related genes for dwarf plant breeding in cotton. After comparing the transcriptome data of Ari1327 with Ari971 and Ari3697, 13919 differentially expressed unigenes (DEGs) were identified, of which5406 up-regulated while 8513 down-regulated in Ari1327. The 16 genes in plant hormone signal transduction pathway was validated and analysed using quantitative real time PCR (qRT-PCR). After analysis of the differential expression of miRNA families in each sample, 16 miRNA families were found to up-regulate in Ari1327 and down-regulated in Ari3697. In three miRNAs libraries, we found 25 miRNAs only expressed in Ari1327 and 13 miRNAs only expressed in Ari3697. These 16 miRNA families and 38 specific expressed miRNAs may play significant role in regulating plant height of Ari1327. Some miRNAs were selected to validate the reality of small RNA sequencing by using stem-loop RT-PCR. The target genes of these differentially expression miRNAs were also annotated in A and D genomes in cotton. http://dx.doi.org/10.1016/j.nbt.2014.05.1738 O14-3 Can plant still be a major and cost-effective source for the supply of artemisinin, the most potent anti-malaria drug Kexuan Tang ∗ , Qian Shen, Fangyuan Zhang Shanghai Jiao Tong University, China Artemisinin isolated from Artemisia annua L., is most potent in treating malaria. Since the discovery of artemisinin in 1970s, A. annua has been the only source for artemisinin supply. Recent breakthrough in transgenic yeast has broken the traditional supply way of artemisinin. According to the report, transgenic yeast system can supply 50–60 tons of artemisinin annually at the sale price of $350–400/kg, which is lower than the current artemisinin production cost by using traditional plant varieties. Data showed in this year A. annua plantation has been reduced by over 60%. It brings concerns if farmers are not willing to grow A. annua anymore, is the yeast system ready for providing all the amount of artemisinin the world needs and when? Will plant system to produce artemisinin be out of the stage? Here, we report the development of three strategies, including spraying fertilizer technology, developing high artemisinin content varieties, and transgenic plant technology to increase artemisinin content in A. annua, which dramatically reduces production cost, and could supply artemisinin in large quantity at the sale price of $250 in 3 years. Transgenic lines have been approved for environmental release, the first GM medicinal plants to be approved in the world. Environmental test and animal test demonstrate transgenic lines are safe and artemisinin extracted from transgenic lines has same function with that extracted from non-transgenic varieties. We believe these strategies, together with transgenic yeast system, will secure artemisinin supply enough at affordable price to combat malaria. http://dx.doi.org/10.1016/j.nbt.2014.05.1739 www.elsevier.com/locate/nbt S55 SYMPOSIUM 14: PLANT GENETIC ENGINEERING O14-4 Engineering barley for increased drought resistance Ivo Frébort ∗ , Hana Pospíšilová, Petr Galuszka Palacky University in Olomouc, Czech Republic Barley is an agriculturally important crop and the production of plants with enhanced stress tolerance is one of the important goals in barley breeding. New techniques of molecular cloning and plant transformation accelerate classical breeding techniques helping to produce barley plants with required enhanced traits. Morphology and development of the barley plants have been altered by genetic manipulation with genes coding for cytokinin dehydrogenase (EC 1.5.99.12; CKX), a principal enzyme controlling cytokinin levels in plants. Three unique transgenic barley lines (Hordeum vulgare cv. Golden Promise) transformed with an expression cassette consisting of ␤-glucosidase root specific promoter, modified CKX gene from Arabidopsis thaliana with engineered protein targeting to cytoplasm, vacuoles or apoplast, and NOS terminator were prepared and T2 generations of homozygous plants were analyzed. Selected transgenic lines, distinctive with altered morphology of the root system, showed higher resistance to drought conditions than wild type plants, both when grown in soil or in hydroponic culture. This method of conveying drought resistance may be further exploited in order to create a usable trait that can be transferred to commercial cultivars of barley. http://dx.doi.org/10.1016/j.nbt.2014.05.1740 O14-5 Recombinant protein expression in the chloroplast of the green microalga Chlamydomonas reinhardtii: A case study using a novel green fluorescent protein as a reporter Stephanie Braun Galleani ∗ , Frank Baganz, Saul Purton UCL, United Kinglom Unicellular algae such as Chlamydomonas reinhardtii are attracting increasing interest as low cost, GRAS platforms for the synthesis of high-value heterologous proteins. Foreign genes introduced into its chloroplast genome can be targeted to a precise locus and expressed without suffering gene silencing issues. Fluorescent proteins such as green fluorescent protein (GFP) provide a simple tool for monitoring protein synthesis in vivo. However, the level of fluorescence from GFP expressed in the C. reinhardtii chloroplast has proved disappointingly low. A newly discovered protein called Verde Fluorescent Protein (VFP) has showed superior fluorescence in other organisms, so we are currently investigating its utility as reporter. A codon-optimised gene encoding VFP was synthesised and successfully introduced into the chloroplast genome under the control of the atpA promoter/5′ UTR. Protein expression was confirmed by western blotting, and fluorescence was demonstrated by confocal microscopy and flow cytometry. We explored whether S56 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 protein expression and fluorescence levels could be improved by: (i) expressing VFP under the control of the psaA promoter/5′ UTR; (ii) fusing the VFP coding sequence to that of an endogenous gene (rbcL); (iii) co-expressing a bacterial chaperone in the chloroplast, obtaining increased protein expression for (i) and (iii). These constructs have been grown using different media and temperatures, in order to evaluate growth rate together with level of protein expression and fluorescence. This approach merges both cell engineering and bioprocess optimisation of a strain of algae widely used in research and has the potential to be applied to other recombinant proteins expressed in C. reinhardtii. http://dx.doi.org/10.1016/j.nbt.2014.05.1741 O14-6 Comparative evaluation of bacterial diversity from gm and Non-GM maize rhizosphere Naseer Ahmad 1,∗ , Naseer Ahmad 2 , Zabta Khan Shinwari 3 1 Department of Environmental Sciences, COMSATS Institute of Information Technology (CIIT), Pakistan 2 COMSATS Institute of Information Technology (CIIT), Pakistan 3 Quaid-i-Azam University, Islamabad, Pakistan The rhizosphere is a critical interface supporting the exchange of resources between plants and their associated soil environment. Rhizosphere microbial diversity is influenced by the physical and chemical properties of the rhizosphere, some of which are determined by the genetics of the host plant. However, within a plant species, the impact of genetic variation in the composition of the bacterial biota of GM and Non-GM maize rhizosphere is poorly understood. Here, we studied the bacterial diversity and population dynamics in the rhizosphere of one GM and two Non-GM maize cultivars (IG and IW) grown under field conditions, by traditional cultivation techniques and 16S rRNA gene-based molecular analysis of DNA directly extracted from pre cultivated soil and rhizosphere samples. Rhizosphere and pre cultivated soil samples were taken at three different plant growth stages. The isolated bacterial strains were further screened for different functional characterization. From this study, we find that the transgenic crop has no positional impact on the rhizospheric bacterial community. http://dx.doi.org/10.1016/j.nbt.2014.05.1742 New Biotechnology · Volume 31S · July 2014 O14-7 Understanding the interactions between plant biotic and abiotic stress through characterization of microRNA effectors in jute (Corchorus spp.) – Macrophomina phaseolina interaction system Lalit Kharbikar ∗ , Pran Gobinda Karmakar Central Research Institute for Jute and Allied Fibres (Indian Council of Agricultural Research), India Jute (Corchorus spp.) is an important fibre crop in India. This smallholder’s crop is vulnerable to most devastating pathogen, Macrophomina phaseolina. It causes damping off, root rot and collar rot collectively known as stem rot, which results in up to 30% yield loss and low fibre quality. The pathogen first causes root rot upon drought stress and then colonizes physiologically altered stem causing stem rot at maturity, under favorable weather. However, the molecular mechanisms underlying are unknown. Plant–pathogen–weather interface is regulated by a number of genes. Existing, probe-dependent gene regulation analysis meth- SYMPOSIUM 14: PLANT GENETIC ENGINEERING ods require plant and pathogen cells to be physically separated. However, probe-independent RNA sequencing method allows plant and pathogen transcripts to be analyzed simultaneously. MicroRNA effectors serve the conserved gene regulatory mechanisms which have been reported to control plant–pathogen interactions. Most works on miRNAs have focused only on initial pathogen infection processes under controlled environment. However, miRNA-mediated regulation of disease development throughout life cycle of plants under the influence of abiotic stresses, such as drought has not been studied so far. Since, stem rot becomes severe at maturity under hot and humid weather, despite the colonization of roots by M. phaseolina at early growth stages of jute, under high soil temperature and low soil moisture (drought) conditions, the jute–M. phaseolina interaction system could provide a good model to study whether physiological alterations induced by pathogen colonization or drought stress facilitate the development of disease. Sequencing of miRNAs during this could elucidate molecular interactions between plant biotic and abiotic stress. http://dx.doi.org/10.1016/j.nbt.2014.05.1743 www.elsevier.com/locate/nbt S57 SYMPOSIUM 15: RECOMBINANT PROTEIN PRODUCTION Symposium 15: Recombinant protein production O15-1 Getting a grip on complexes: tools and technologies for multiprotein complex research Imre Berger EMBL, Grenoble Most eukaryotic proteins exist as large multicomponent assemblies with many subunits, which act in concert to catalyze specific cellular activities. Many of these are only present in low amounts in their native hosts, impeding purification from source material. Unraveling their mechanisms of action will therefore often depend on heterologous overproduction. Complex proteins, involved in disease causing processes, are also entering center-stage as key drug targets of the future. In addition to their essential role as tools for drug discovery, such complex biologics themselves are increasingly being employed as medicines, for example in the form of multicomponent vaccines. These are predicted to dominate the next generation of drugs. My laboratory develops advanced methods for producing multicomponent protein biologics for studying their structure and function at molecular resolution. We have created MultiBac, a baculovirus/insect cell system designed for high-quality multiprotein complex production, and have installed MultiBac as a platform technology at the Eukaryotic Expression Facility (EEF) in our laboratory. More recently, in the context of the EC FP7 project ComplexINC, we have extended our technology concept to multiprotein production in other host systems including mammalian cells. Our technologies and successful applications in academic and industrial R&D will be presented [1–3]. New Biotechnology · Volume 31S · July 2014 interest in the field and as MPs are extremely important drug targets they attract the attention of the pharmaceutical industry. For characterization of MPs and acquisition of information for the rational design of drugs large amounts of protein are required. As their natural abundance is usually very low, MPs are produced by overexpression in heterologous hosts. However, they are notoriously difficult to express as the yields per cell are typically low and MP production is highly toxic to cells. The aim of the present work is to engineer strains of Escherichia coli to overcome MPoverexpression toxicity. The ultimate goal is to use these strains for large-scale production of MPs. Also, our goal is to investigate the mechanism behind the observed cytotoxicity. For this purpose we have generated libraries of mutant bacteria carrying different types of genetic modifications and used appropriate genetic screens to isolate the desired clones. The human GPCR bradykinin receptor 2 that shows very high overexpression toxicity in E. coli was used as our model MP. We have identified a number of strains that overcome the toxicity upon BR2 overexpression while more overexpressed protein is produced. The strains have also been shown to have a more general application for several GPCRs in E. coli. The clones are being characterized using various methods. A potential physiological role of the identified mutations is proposed. http://dx.doi.org/10.1016/j.nbt.2014.05.1745 O15-3 Population heterogeneity in Pseudomonas putida analyzed on the single cell level using proteomics and digital PCR Michael Jahn 1,∗ , Carsten Vorpahl 1 , Dominique Türkowsky 1 , Martin Lindmeyer 2 , Bruno Bühler 2 , Hauke Harms 1 , Susann Müller 1 1 2 References [1].Bieniossek, et al. The architecture of human general transcription factor TFIID core complex. Nature 2013;493(January (7434)):699–702. [2].Barford D, Takagi Y, Schultz P, Berger I. Baculovirus expression: tackling the complexity challenge. Curr Opin Struct Biol 2013;23(3):357–64. [3].Bieniossek C, Imasaki T, Takagi Y, Berger I. MultiBac: expanding the research toolbox for multiprotein complexes. Trends Biochem Sci 2012;37(February (2)):49–57. http://dx.doi.org/10.1016/j.nbt.2014.05.1744 O15-2 Making life better for Escherichia coli cells that produce toxic membrane proteins Dimitra Gialama 1,∗ , Fragiskos Kolisis 2 , Georgios Skretas 1 1 2 National Hellenic Research Foundation, Greece National Technical University of Athens, Greece Membrane proteins (MPs) constitute an exciting world that includes protein families such as G protein-coupled receptors (GPCRs) and transporters. Currently, there is increasing scientific S58 www.elsevier.com/locate/nbt Helmholtz-Centre for Environmental Research - UFZ Leipzig, Germany TU Dortmund University, Germany Recombinant protein production using microbial cells is a key player for the chemical and pharmaceutical industry of the future. The efficiency of such biotechnological processes is usually benchmarked using bulk parameters. However, the catalytic unit driving a process is the single cell. Even clonal populations show high cellto-cell variability, e.g. caused by genetic mutations, cell cycling or regulatory decisions. As a model system for population heterogeneity, we use Pseudomonas putida with plasmid-based production of an EGFP fused recombinant protein (StyA). With EGFP as a marker, flow cytometry shows large proportions of cells (30–60%) not able to produce functional protein even under high induction regimes. We used cutting edge cell sorting of fluorescent (EGFP+) and non-fluorescent cells (EGFP−) to identify the cause for impaired protein production. Sorted sub-populations were analyzed by protein mass spectrometry to reconstruct pathways, and by digital PCR to determine the plasmid copy number (PCN) with extraordinary precision. In summary, only minor changes in the protein inventory were found, most of them related to stress as a result of protein aggregation. In contrast to that, we identified unequal distribution of plasmids as a major cause for heterogeneity: The non-fluorescent SYMPOSIUM 15: RECOMBINANT PROTEIN PRODUCTION New Biotechnology · Volume 31S · July 2014 sub-population was almost plasmid-free, although some cells refused gene expression despite presence of the plasmid. http://dx.doi.org/10.1016/j.nbt.2014.05.1746 O15-4 Synthesis of antibacterial bacteriophage proteins in microalgae Laura Stoffels 1,∗ , Bambos Charalambous 2 , Saul Purton 3 1 University College London, UK Research Department of Infection, University College London Medical School, UK 3 Institute of Structural and Molecular Biology, University College London, UK 2 Widespread antibiotic resistance among pathogenic bacteria and the low specificity of these drugs cause a pressing need for the development of novel antibacterial agents. Endolysins are antibacterial proteins that are produced by bacteriophages to digest the bacterial cell wall for phage progeny release at the end of the lytic cycle. These efficient enzymes are highly specific for the cell wall of the target bacteria without affecting other species. Development of resistance against endolysins is very rare, because they evolved to target molecules in the cell wall that are essential for bacterial viability. Taken together, this makes them promising novel antibacterial agents. The eukaryotic microalga Chlamydomonas reinhardtii offers already established techniques for the expression of foreign genes in the chloroplast and is an attractive expression platform for therapeutic proteins, due to the lack of endotoxins and potentially infectious agents. Furthermore it can be inexpensively cultivated in full containment and under sterile conditions in simple photobioreactors. Two bacteriophage endolysins targeting two major human pathogens were successfully expressed in the chloroplast of C. reinhardtii, purified and their specificity and efficiency in killing the target bacteria was assayed in vitro. Furthermore the cyanobacterium Synechocystis sp. PCC6803 was investigated as an alternative expression platform for endolysins. http://dx.doi.org/10.1016/j.nbt.2014.05.1747 O15-5 Integrative ‘-omic’ approach to explore molecular mechanism of miRNA engineered Chinese hamster ovary cell Vaibhav Jadhav 1,∗ , Matthias Hackl 1 , Deniz Baycin-Hizal 2 , Gerald Klanert 3 , Michael Betenbaugh 2 , Johannes Grillari 1 , Nicole Borth 3 1 University of Natural Resources and Life Sciences, Vienna, Austria Johns Hopkins University, Baltimore, USA 3 Austrian Center for Industrial Biotechnology (ACIB), Austria 2 MicroRNAs (miRNA) are noncoding regulators of translation, controlling a broad range of physiological functions. They have been shown to regulate large number of mRNA targets simultaneously, thus acting as global regulators to alter cellular functions. The application of miRNAs to engineer Chinese hamster ovary (CHO) cells is an emerging strategy to improve the bioindustrially relevant characteristics of CHO cells, which are one of the most important biopharmaceutical cell factories. In this regard, recently we have developed stable engineered miR-17 overexpressing CHO cells, which exhibit both enhanced growth performance and increase in specific productivity. This lead to overall 3-fold increase in recombinant protein (EpoFc) titers, this parallel enhancement of both cell-specific growth rate and productivity is very unique. The underlying molecular mechanism that controls such phenotype is generated by co-regulations various biological networks and understanding such molecular regulation is hindered by the lack of a miRNA-mRNA interaction database for CHO cells. For this reason, we applied transcriptomics (mRNA and miRNA) using microarray platform and quantitative proteomics using iTRAQ technology. These data sets are ultimate readout of miRNA activity and developed a bioinformatics pipeline to handle such datasets where the rules of interaction are not a priori known. This novel inter-omics approach is described as well as an interpretation of the effects caused by miR-17 overexpression that led to the interesting phenotype described above. http://dx.doi.org/10.1016/j.nbt.2014.05.1748 O15-6 Investigating the physiological effect of increased heterologous gene dosage in Pichia pastoris using transcriptomics Elena Camara 1,∗ , Landes Nils 2 , Lluís Revilla Sancho 3 , Joan Albiol 4 , Diethard Mattanovich 2 , Pau Ferrer 4 1 UAB, United States Department of Biotechnology, University of Natural Resources and Life Sciences Vienna/Austrian Centre of Industrial Biotechnology (ACIB GmbH), Austria 3 School of Bioengineering, University of Applied Sciences FH Campus Vienna, Austria 4 Department of Chemical Engineering, Autonomous University of Barcelona, Spain 2 The alcohol oxidase (AOX1) promoter of Pichia pastoris is one of the strongest promoters for heterologous gene expression in methylotrophic yeast, allowing the methanol-regulated expression of the gene of interest. Previous studies have shown that increased heterologous gene dosage often leads to the overload of the secretory pathway and metabolic burden. The expression of Rhizopus oryzae lipase (Rol) in P. pastoris has been previously shown to trigger the unfolded protein response (UPR) [1]. To assess that physiological response in P. pastoris is related to the increased ROL gene dosage, a subset of strains with 1, 2, 4, 8 and 15 copies was constructed and tested in carbon-limited chemostat cultures, using a mixed glycerol:methanol substrate feed. Once the system was at steady state a transcriptomic analysis was performed using DNA microarrays. The macroscopic physiological parameters revealed that growth yield and carbon uptake rate are gene dosage dependent, and the highest productivity was found at 2 copies of ROL. These results were supported by the transcriptomic data, showing a correlation between the regulation of central carbon metabolism and the gene copy number. The number of regulated genes increased with gene dosage. In addition to carbon metabolism, transcriptional changes www.elsevier.com/locate/nbt S59 SYMPOSIUM 15: RECOMBINANT PROTEIN PRODUCTION in other cellular processes such as peroxisome biogenesis and stress responses involving the UPR were observed. Reference [1].Resina D, Bollók M, Khatri NK, Valero F, Neubauer P, Ferrer P. Transcriptional response of Pichia pastoris in fed-batch cultivations to Rhizopus oryzae lipase production reveals UPR induction. Microb Cell Fact 2007;6:21. http://dx.doi.org/10.1016/j.nbt.2014.05.1749 O15-7 Enhanced membrane protein expression by engineering increased intracellular membrane production Katrien Claes 1,2,∗ , Mouna Guerfal 1,2 , Nico Callewaert 1,2 1 Unit for Medical Biotechnology, Inflammation Research Center (IRC), VIB, Belgium 2 Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, Belgium Membrane protein research is frequently hampered by low natural abundance of these proteins in cells and typically requires recombinant expression. Different expression systems have been used to date, but only little research is directed towards the specific customization of the host cell itself. We hypothesized that increasing the intracellular membrane content would increase the membrane area available to accom- S60 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 modate recombinant membrane proteins. Hereto, we inactivated the phosphatidic acid phosphatase gene, PAH1, which codes for a key enzymatic and regulatory factor in lipid biosynthesis. The result is a shift of lipid metabolism away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis. We chose Yarrowia lipolytica as a test organism, as this yeast preferentially grows on fatty acids and a compatible strong inducible protein expression system is available. Electron microscopic imaging of the knock-out revealed strong membrane proliferation upon growth on oleic acid, without any signs of ER-phagy. Guided by this, we analyzed membrane protein expression in the PAH1 knock out strain of Yarrowia lipolytica. Analysis of eight representatives of different integral membrane protein families showed enhanced protein accumulation levels for all of them and in some cases also reduced proteolysis. Complimentary to this improvement, UPR co-induction further enhances the quality of the membrane proteins in terms of proper folding and biological activity. These results allow to conclude that re-routing of lipid metabolism is a valid strategy to increase membrane protein production and quality. As this pathway is conserved in eukaryotes, similar strategies can be explored in other frequently used expression organisms. http://dx.doi.org/10.1016/j.nbt.2014.05.1750 SYMPOSIUM 16: BIOPROCESSING New Biotechnology · Volume 31S · July 2014 The lignin content of lignocellulose and lignin-containing wastes represents a possible resource for production of aromatic chemicals, if efficient biocatalytic routes for lignin degradation can be found. The enzymology of fungal lignin degradation is well studied, but the enzymology of bacterial lignin degradation is much less well known. In Rhodococcus jostii RHA1 we have identified a dyp-type peroxidase DypB as a lignin peroxidase that is activated by Mn2+ , and shows activity with a ␤-aryl ether lignin model compound, with Kraft lignin, and with lignocellulose. Using a colorimetric assay as a screen, we have also identified a number of novel lignin-degrading bacteria from soil samples, which show higher activity for lignin degradation, including three strains of Microbacterium and a thermotolerant strain of Sphingobacterium, in which strains we are currently investigating further enzymes for lignin degradation. Deletion of the gene encoding vanillin dehydrogenase in Rhodococcus jostii RHA gives a mutant strain which, when grown on minimal media containing wheat straw lignocellulose, accumulates up to 96 mg/L of vanillin, a high value chemical, highlighting the potential for pathway engineering to be used for conversion of lignin into renewable aromatic chemicals [1–4]. tion. It was shown that the extent of heterogeneity is strongly linked to frequency and amplitude of physicochemical changes in the extracellular environment. Hence not only the identification of individual physiological phenotypes, but also the ability to analyze single cells in a controlled and steady environment is a fundamental requirement for the accurate description of cellular features. We tackle the challenge of creating controlled physicochemical microenvironments for a precise analysis of individual cellular physiology with the Envirostat single cell analysis system [1,2]. In contrast to most other microfluidic systems for SCA, which mostly rely on the mechanical trapping of cells, the Envirostat uses contactless retention via negative dielectrophoresis, which excludes cell-surface interaction and resultant changes in the cellular phenotype. Furthermore, the immediate removal of potentially inhibiting metabolites and unlimited availability of nutrients and dissolved oxygen is guaranteed by a continuous medium flow. CFD simulations indicate that the microenvironment of the target cell has indeed a virtually static composition [1]. We experimentally validated the Envirostat principle with single cell growth studies [3]. We observed significant differences between specific growth rates of single cells and populations in bulk cultivations. Two yeast species and one bacterial strain consistently exhibited increased specific growth rates of up to 120% when cultivated with the Envirostat system [3]. Our results imply that the extracellular environment can dictate the specific growth rate of unicellular microbial eukaryotes and prokaryotes and a constant extracellular environment diminishes the influence of physiological cell-to-cell differences. Moreover, our experiments demonstrate the Envirostat as a platform for systems biology that may be used for disclosing the impact of controlled perturbations on cellular physiology unbiased by population activity. References References Symposium 16: Bioprocessing O16-1 Bacterial enzymes for lignin degradation: production of aromatic chemicals from lignocellulose Timothy D.H. Bugg University of Warwick, Department of Chemistry, Coventry CV4 7AL, UK [1].Ahmad M, Taylor CR, Pink D, Burton K, Eastwood D, Bending GD, et al. Mol Biosyst 2010;6:815–21. [2].Ahmad M, Roberts JN, Hardiman EM, Singh R, Eltis LD, Bugg TDH. Biochemistry 2011;50:5096–107. [3].Taylor CR, Hardiman EM, Ahmad M, Sainsbury P, Norris PR, Bugg TDH. J Appl Microbiol 2012;113:521–30. [4].Sainsbury PD, Hardiman EM, Ahmad M, Otani H, Seghezzi N, Eltis LD, et al. ACS Chem Biol 2013;8:2151–6. [1].Kortmann H, et al. Lab Chip 2009;9:576. [2].Fritzsch F, et al. Lab Chip 2013;13:397. [3].Dusny C, et al. Appl Environ Microbiol 2012;78:7132. http://dx.doi.org/10.1016/j.nbt.2014.05.1752 O16-3 http://dx.doi.org/10.1016/j.nbt.2014.05.1751 O16-2 Quantitative single cell analysis of isolated microbes in controlled microenvironments Christian Dusny ∗ , Frederik Fritzsch, Katrin Rosenthal, Andreas Schmid ∗ TU Dortmund University, Laboratory of Chemical Biotechnology, 44227 Dortmund, Germany Single cell analysis (SCA) has been recognized as the key technology for an unbiased disclosure of cellular functionality. In contrast to conventional bulk analysis strategies, SCA grants access to mechanistic data of individual cells–the minimal functional unit of cell based bioprocesses. These data are usually masked behind an average value of a clonal, but heterogeneous popula- Continuous precipitation of recombinant antibodies from CHO cell culture supernatant by calciumphosphate flocculation and cold ethanol precipitation Nikolaus Hammerschmidt 1,∗ , Anne Tscheließnig 2 , Bernhard Helk 3 , Alois Jungbauer 2 1 Centre of Industrial Biotechnology (ACIB), Austria University of Natural Resources and Life Sciences Vienna, Austria 3 Novartis Pharma, Austria 2 We have developed two precipitation steps that could replace the costly protein A affinity chromatography as capture step in the purification of recombinant antibodies, relying solely on cheap mineral salts (CaCl2 ) and organic solvents (ethanol). Both steps can be performed in continuous mode using tubular reactors without changes in performance compared to batch mode. The startup time until steady state conditions were reached was very short and both reactors were operated for several hours at steady state withwww.elsevier.com/locate/nbt S61 SYMPOSIUM 16: BIOPROCESSING out manual intervention, delivering antibody at constant yield and purity. An overall yield of >90%, a host cell protein reduction to 9000 ppm and a DNA reduction to 7 ppm (for a titer of 7.7 g/L) could be achieved for the antibody investigated. The cold ethanol precipitation step can be used for concentrating the antibody to >45 g/L, thereby making a subsequent concentration step unnecessary. Furthermore, the antibody can be readily dissolved in a variety of buffers of high and low ionic strength optimized for the next purification step. Cell culture supernatants with high antibody titer can be processed with constant tubular reactor size and without changing any parameters or increasing precipitant consumption. Continuous precipitation allows the use of disposable reactors, allowing flexible operation. http://dx.doi.org/10.1016/j.nbt.2014.05.1753 O16-4 Modelling of mixing and microbial growth in bubble column bioreactors using computational fluid dynamics Dale McClure ∗ , John Kavanagh, David Fletcher, Geoffrey Barton The University of Sydney, Australia Bubble columns are widely used in the bio-processing industry for large-scale aerobic fermentations. In order to maximise the yield of many bioprocesses, it is desirable to achieve a homogeneous distribution of substrate; a task of some complexity at the industrial scale. Computational Fluid Dynamics (CFD) offers a promising approach as a tool to model such processes, as it enables flow patterns inside the reactor to be readily visualised, something which is very difficult to achieve experimentally. A further advantage of a CFD model is that it allows different designs and operating conditions to be evaluated in silico, minimising the need for costly and time-consuming experimentation. By including growth kinetics into the CFD model, it becomes possible to quantify the effect of poor mixing on the overall yield of the process. As part of a longterm industrial collaboration we have developed and validated a CFD model of the complex and time-varying hydrodynamics (including mixing) inside bubble columns operating at industrially relevant superficial velocities. Here, we extend this model by including the growth kinetics for two processes of industrial interest; namely the production of baker’s yeast and the production of a recombinant protein using Escherichia coli. The impact of substrate gradients on the yield of each process is examined, as well as the potential for employing CFD as a tool to model industrial bio-processes. http://dx.doi.org/10.1016/j.nbt.2014.05.1754 S62 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 O16-5 Bioprocess strategies for production of xylanase on agroresidual products with Aureobasidium pullulans Sirma Yegin 1,∗ , Sayit Sargin 2 , Yekta Goksungur 1 1 2 Ege University, Food Engineering Department, Turkey Ege University, Bioengineering Department, Turkey Xylanases are mainly used in pulp and paper industry and recently found widespread applications in food and feed industries. They are also used in waste clarification processes and bioethanol production. The aim of this study was to enhance production of xylanase with A. pullulans by evaluating the effects of different fermentation parameters. Among the A. pullulans strains tested, NRRL Y2311-1 provided the highest activity on both xylose and xylan grown cultures. Maximum activity was observed after 96 h of cultivation at 28 ◦ C. The optimized initial medium pH and shaking speed for cultures grown on xylan were 3.0 and 200 rpm, respectively. After elucidating the effects of fermentation parameters on xylanase production in synthetic medium, the potential of several agro-residual products were examined for the production of xylanase. The highest activity was obtained when wheat bran was utilized. Further optimization studies were performed by response surface methodology for cultures grown on wheat bran. By fitting the experimental data to second-order polynomial equation, the optimum levels of initial medium pH (4.2), temperature (30.3) and incubation time (126.7 hours) were determined. The maximum xylanase activity at optimum process conditions reached to 851.95 ± 28.70 U/ml. The model obtained satisfactorily explained the effect of process variables on enzyme production. Acknowledgements: This study was supported by TUBITAKTOVAG 112O521 and COST TD1203. http://dx.doi.org/10.1016/j.nbt.2014.05.1755 O16-6 Bioconversion of lignocellulosic hydrolysates: strategies to overcome the inhibitory effects at high gravity processes Charilaos Xiros ∗ , Lisbeth Olsson Chalmers University of Technology, Sweeden High-gravity (HG) technology aims at generating final ethanol concentrations above 50 kg m−3 in order to reduce the cost of the distillation step. The generation of higher amounts of inhibitors during the pretreatment step is one of the challenges that accompany the increase in initial dry matter. Detoxification of spruce hydrolysate, adaptation of the cells before fermentation, supplementation with nutrients, and washing of solids were the strategies compared in this study. They represent different approaches to cope with the inhibitory effects, and we compared their efficiencies using a thermotolerant strain of Saccharomyces cerevisiae at temperatures from 30 ◦ C up to 40 ◦ C. New Biotechnology · Volume 31S · July 2014 The dilute acid-pretreated spruce used as substrate in this study was not fermentable under HG conditions (200 g kg−1 water-insoluble solids) when no improvement method was applied. In HG simultaneous saccharification and fermentation at 30 ◦ C combined with a 24 h pre-hydrolysis step, the detoxification of pretreated spruce with reducing agent (Na2 S2 O4 ) gave the best result with an ethanol yield of 57% (on total sugars) of the maximum theoretical and a volumetric productivity of 1.58 g dm−3 h−1 . In HG separate hydrolysis and fermentation, SYMPOSIUM 16: BIOPROCESSING nutrients supplementation gave better final ethanol yields than detoxification of the material, reachingan ethanol yield of about 60% of the theoretical (on total sugars). The results obtained, showed an increase in severity of inhibitory effects with temperature increase. Improved cell viability was observed when detoxified material was used and also when yeast extract addition was coupled with adaptation of the cells to the hydrolysate. http://dx.doi.org/10.1016/j.nbt.2014.05.1756 www.elsevier.com/locate/nbt S63 SYMPOSIUM 21: BIODEGRADATION AND BIOMEDIATION Symposium 21: Biodegradation and bioremediation O21-1 Bioremediation for resource recovery Louise Horsfall University of Edinburgh, United Kingdom In order to move towards a sustainable and circular economy we need to start viewing waste differently. Through biotechnology research we have the potential to use waste as a feedstock rather than it being an end product. A limiting factor in using waste in this way is the presence of minor amounts of metal contaminants, which inhibit bioprocesses and kill bioremediating microorganisms. There are, however, bacteria that are tolerant to high concentrations of metal ions due to a resistance mechanism that involves metal reduction and nanoparticle formation. We are investigating this pathway to enable the bioremediation of waste, water and land; employing the techniques, tools and principles provided by synthetic biology to increase the value of the metal recovered. To achieve this we are taking a modular approach to identify and optimise genetic elements that have transferable metal ion use, with an aim to control production, size, shape and homogeneity, tailoring the nanoparticles to their ascribed use. Our particular case studies under investigation are platinum group metals, nickel, copper and arsenic. However, since we are attempting to develop a flexible process, there is huge potential to transfer the knowledge gained in our biomineralisation studies to other metal waste streams. http://dx.doi.org/10.1016/j.nbt.2014.05.1757 O21-2 Reduction of hexavalent chromium using combination of nanoscale zero-valent iron and biological treatment in situ Tomáš Cajthaml 1,∗ , Jan Němeček 2 , Petr Pokorný 2 , Lenka Lacinová 3 , Miroslav Černík 3 , Zuzana Masopustová 3 , Ondřej Lhotský 4 1 Institute of Microbiology v.v.i., ASCR, Czech Republic ENACON s.r.o, Czech Republic 3 Technical University of Liberec, Czech Republic 4 DEKONTA a.s., Czech Republic 2 Hexavalent chromium is considered as a priority pollutant due to its high toxicity and mobility. The aim of this study was to set up a pilot-scale in situ remediation experiment in the saturated zone of a historically Cr(VI)-contaminated site. Two geofixation methods were used – chemical reduction of Cr(VI) with commercially available nanoscale zero-valent iron (nZVI) and consequent biological reduction of Cr(VI). Combination of the methods resulted in a rapid decrease of Cr(VI) concentrations in the groundwater. The process was monitored using standard chemical analyses, measurement of redox potential, standard ecotoxicity tests and phospholipid fatty acid analysis (PLFA) supported with 454 pyrose- S64 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 quencing. The result of PLFA revealed that the application of nZVI stimulated significantly growth of autochthonous microorganisms indicating general reduction of toxicity of the site in the first phase. The consequent injection of an organic substrate enabled further transformation of Cr(VI) below the respective quantification limit even after 9 months of the single organic substrate injection. The results of the pilot test indicated a synergic effect of both of the abiotic and biotic phases. nZVI that had been oxidized during the abiotic phase was afterwards partially recovered during the biotic phase due to the substantial decrease of the redox potential. The immobilized Fe (III) was probably microbially reduced to Fe(II) that acted further as the reducing agent for Cr(VI) even when microbial density was already low due to depletion of the organic substrate. http://dx.doi.org/10.1016/j.nbt.2014.05.1758 O21-3 Isolation of PAH dwelling Penicillium for application in bioremediation processes Elisabet Aranda 1,∗ , Patricia Godoy 1 , Rocio Reina 1 , Marina BadiaFabregat 2 , Mónica Rosell 3 , Regina Michela Wittich 1 , Ernest MarcoUrrea 2 , Inmaculada García-Romera 1 1 Spanish National Research Council (CSIC), Spain Universitat Autònoma de Barcelona, Spain 3 Universitat de Barcelona, Spain 2 Fungi represent the living dominant biomass in soils and are abundant in aqueous systems. In addition, they possess a high potential for degrading environmental organic chemicals. The aim of this study is to find polycyclic aromatic hydrocarbon (PAH) degrading fungi, which are adapted to polluted environments, using culturing-based techniques. In this study, a total of 12 fungal cultivable species have been isolated from a PAH contaminated pond. The isolated fungi were genetically identified by amplifying, cloning and further sequencing fragments corresponding to the ITS1-5.8S-ITS2 (internal transcribed spacer ITS) region of each cultivable fungal strain. We tested their ability to convert anthracene, in time courses of 42 days. Among the 12 screened fungal species, Penicillium oxalicum showed remarkable conversion ability, degrading 100 ␮M in 5 days in a rich carbon source medium. The use of a defined mineral medium with 13C-labelled anthracene showed that P. oxalicum can cometabolize anthracene, leading to the formation of anthraquinone, anthrone and hydroxyderivatives, as revealed by nuclear magnetic resonance (NMR) analysis. The last metabolites could indicate the further ring cleavage of anthracene that could be mediated by hydrolase or dioxygenase enzymes. The conversion level of anthracene was reduced to 50% in presence of an inhibitor of cytochrome P450 monooxigenase, suggesting its participation in the first oxidation step. Our results show the high effectiveness in PAHs conversion by the isolated fungi P. oxalicum by cometabolic strategies, indicating its potential application in biotechnological pollutant removal processes. http://dx.doi.org/10.1016/j.nbt.2014.05.1759 SYMPOSIUM 21: BIODEGRADATION AND BIOMEDIATION New Biotechnology · Volume 31S · July 2014 O21-4 O21-5 Potential of ectomycorrhizal fungus Pisolithus tinctorius to tolerate and to degrade trifluoroacetate into fluoroform Evaluation of the biological sulfamethoxazole degradation mechanism for biotechnological applications Paula Castro los Afonso 3 1,∗ 2 2 3 , Albina Franco , Miguel Ramos , Sara Cravo , Car- 1 CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa/Porto, Portugal 2 CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Portugal 3 2CEQUIMED-UP, Laboratório de Química Orgânica e Farmacêutica, Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), Departamento Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Portugal Trifluoroacetate (TFA) is a persistent fluorinated organic compound originated from the degradation of fluorinated compounds, such as HCFC and isoflurane, or as a side product from the thermolysis of fluoropolymers, like Teflon. TFA can reach soil through precipitation, where it persists in water and soil, and may contribute to forest decline. In this study, we assessed the capacity of P. tinctorius, an ectomycorrhizal fungus (ECMF), to tolerate and/or degrade TFA. In vitro studies in glucose-supplemented solid medium showed that the fungus tolerated up to 8.77 mM TFA. P. tinctorius also degraded 88.3%, 89.9%, and 42.1% of 0.88, 2.39, and 4.39 mM TFA, respectively, in liquid cultures. No TFA accumulation was detected on the fungus mycelium, suggesting that TFA depletion was due to fungal degradation. Defluorination was not detected. A volatile compound with a structure and behavior compatible with fluoroform (CHF3 ), a potent greenhouse gas, was detected using GC-MS/MS, only in the gas phase of sealed P. tinctorius cultures supplied with TFA. Further confirmation of this compound is needed. Nevertheless, the study shows that P. tinctorius was capable to degrade TFA possibly through a similar pathway to that found on marine sediments. The results evidence the role of ECMF may play in the degradation of fluorinated organic compounds, enhancing their potential contribution on establishing tree growth in soils exposed to organic contamination. Acknowledgements: A. Franco thanks FCT the grant SFRH/BD/47722/2008. This work was supported by FCT Project -PTDC-AGR-CFL-111583-2009 and PEst-OE/EQB/LA0016/2011. http://dx.doi.org/10.1016/j.nbt.2014.05.1760 Benjamin Ricken 1,∗ , Markus Lenz 1 , Danuta Cichocka 1 , Hans-Peter Kohler 2 , Boris Kolvenbach 1 , Philippe Francois-Xavier Corvini 1 1 Institute for Ecopreneurship/University of Applied Sciences and Art Northwestern Switzerland, Switzerland 2 Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Microbiology, Switzerland Sulfonamide antibiotics are of rising concern as their release into the environment has been suspected to enhance the formation of resistant pathogenic bacterial strains [1]. Among sulfonamides, antibiotics such as sulfamethoxazole (SMX) are arousing public interest since they are photo- and thermostable as well as recalcitrant to traditional, biological waste water treatment processes [2]. Our studies focused on SMX as it is the most used sulfonamide antibiotic in human medicine. Even though extensive research has been done on SMX degradation by bacterial consortia or isolates, the complete pathway is not yet understood, let alone the proteins involved. We were able to identify the first crucial degradation step of several sulfonamide antibiotics [3] by means of isolating Microbacterium sp. strain BR1, proven to partially mineralize SMX for the first time [4]. Concerning the pathway, molecular oxygen is incorporated into the sulfonamide antibiotic at its aniline moiety through ipso-substitution, which is an uncommon process for biodegradation. This leads to the release of the verified metabolites: 3-amino-5-methylisoxazole, p-aminophenol and sulfite. Further investigations are currently under way in order to identify the involved protein and its cofactor dependencies. To conclude, it is postulated that there is one common sulfonamide degradation mechanism among various bacteria [3]. Its elucidation and the determination of involved proteins are essential for upcoming biological sulfonamide removal approaches. References [1].Hruska, Franek. Vet Med 2012;2012:1–35. [2].Gros, et al. Environ Int 2010;36:15–26. [3].Ricken, et al. Appl Environ Microbiol 2013;79:5550–8. [4].Bouju, et al. Appl Environ Microbiol 2012;78:277–9. http://dx.doi.org/10.1016/j.nbt.2014.05.1761 www.elsevier.com/locate/nbt S65 WEDNESDAY 16 JULY SYMPOSIUM 17: DEVELOPMENT OF NEW VACCINES AND ANTIMICROBIALS New Biotechnology · Volume 31S · July 2014 Wednesday 16 July O17-2 Symposium 17: Development of new vaccines and antimicrobials Engineering of factor H binding protein, a key vaccine antigen for the prevention of meningococcal disease O17-1 Hayley Lavender ∗ , Susan Lea, Christoph Tang University of Oxford, United Kingdom Insect cell technology as a vaccine producing platform Paula Marques Alves IBET/ITQB-UNL, Apartado 12, Oeiras, Portugal The insect cell-baculovirus system is today a well-accepted universal manufacturing platform as demonstrated by the number of approved veterinary and human vaccines. The key advantage of this platform is that a universal “plug and play” process may be used for producing a broad range of biologicals while offering the potential for low manufacturing costs. Its major downside resides on the quality and quantity of the generated product, two critical variables that seldom pose problems to the translation of the production process from lab to industrial scale. Aiming at closing this gap, our group has been focusing on (1) the application of systems biology tools and (2) the development of stable insect cell lines to better understand and optimize this producing platform. Taking advantage of recent advances in systems biology tools for insect cells we have conducted a metabolomics study with the two most used cell lines, Sf9 and Hi5 cells, with the objective of fine-tuning the insect cell-baculovirus system for production of enveloped virus-like particles (VLPs) and retrovirus like particles (unpublished data). Results demonstrate that although Sf9 cells have improved growth and metabolic efficiency, Hi5 cells were better recombinant protein producers for all tested targets with productivities 3- to 4-fold higher. In addition, data points out towards the potential of media manipulation strategies as a way to reinforce specific cellular pathways associated with higher productivity and thus optimize the production of complex biologicals. In parallel, we have been developing stable insect cell lines using targeted approaches based on the flp recombinase-mediated cassette exchange (RMCE) system. Sf9 and Hi5 cells populations after flp-mediated cassette exchange were compared for complex proteins expression, including influenza VLPs. Specific protein productivities in Hi5 cells were markedly higher than in Sf9 cells for all proteins tested. Noteworthy, the metabolic efficiency of Sf9 cells allowed to extend the production phase and to deliver similar protein titers at the end of the process. Overall, a comparison of Sf9 and Hi5 cells expression capabilities with our flexible plug-and-play Flp-RMCE platform will be discussed. http://dx.doi.org/10.1016/j.nbt.2014.05.1762 Neisseria meningitidis is a leading cause of bacterial sepsis and meningitis in children. There are effective vaccines available to prevent meningococcal disease caused by strains expressing certain polysaccharide capsules. However, capsule-based approaches cannot be used against serogroup B N. meningitidis because its capsule is identical to a molecule found in the developing human brain. Therefore there have been intense efforts to identify protein antigens that provide protective immunity against this important human pathogen. Meningococcal factor H binding protein (fHbp) is a surface lipoprotein that elicits serum bactericidal responses in mice and human volunteers. This antigen is a key component of Bexsero, a licensed vaccine for the prevention of meningococcal disease. There is considerable sequence variation of fHbp among strains of N. meningitidis, affecting predicted coverage of fHbp-based vaccines. Also fHbp binds the human complement regulator factor H (fH); there is evidence that fH binding impairs immunogenicity of fHbp. Finally, certain fHbps display inherent instability, making them unsuitable as vaccine antigens. Based on the structure of the fHbp:fH complex and mutagenesis studies, we have identified non-functional fHbps for all three variant groups, and generated stable versions of the proteins. Additionally, we found that a homologue of fHbp in Neisseria gonorrhoeae fails to bind fH. Given the specificity of fHbp for human fH, the vaccine candidacy of these antigens has been evaluated in a transgenic mouse model. In conclusion, we have employed structure-based design to modify and characterise novel fHbps as antigens in next generation vaccines against meningococcal disease. http://dx.doi.org/10.1016/j.nbt.2014.05.1763 O17-3 Antimicrobial properties of sophorolipids produced by Candida Bombicola ATCC 22214 against gram positive and Gram-negative bacteria Mayri Alejandra Diaz De Rienzo 1,∗ , Ben Dolman 1 , Fernando Guzman 1 , Candice Kaisermann 1 , James Winterburn 1 , Ibrahim M. Banat 2 , Peter Martin 1 1 2 University of Manchester, United Kingdom University of Ulster, United Kingdom Biosurfactants are amphipathic, surface-active molecules of microbial origin which accumulate at interfaces, decreasing surface and interfacial tensions and forming aggregated micellular structures in solution. Some are reported to have antimicrobial properties and anti-adhesive/disruption abilities on biofilm formation. Sophorose lipids are glycolipids Biosurfactant con- S66 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 WEDNESDAY 16 JULY SYMPOSIUM 17: DEVELOPMENT OF NEW VACCINES AND ANTIMICROBIALS sisting of a dimer sugar, sophorose and a long-chain fatty acid that are produced by yeasts belonging to the Candida genus. Antimicrobial properties and the ability to disrupt biofilms using sophorolipids produced by Candida bombicola ATCC 22214 during a fed-batch fermentation, using glucose 100 g/L as carbon source and glucose/rapeseed oil as feed at 47 and 120 h and sodium dodecyl sulfate (SDS) at different concentrations were investigated. Growth of Ralstonia eutropha ATCC 17699 was inhibited by sophorolipids and SDS at concentrations > 1% (v/v) and the growth of B. subtilis BBK006 was inhibited by sophorolipids and SDS at concentrations > 0.5%, v/v. Sophorolipids were able to disrupt biofilm formation (at concentrations higher than 1%, v/v) and the effect was evidenced through fluorescence microscopy. It is concluded that sophorolipids are promising compounds for the inhibition/disruption of biofilms formation by Gram-positive and Gram-negative microorganisms which could be enhanced by the presence of a combination of biosurfactants. http://dx.doi.org/10.1016/j.nbt.2014.05.1764 O17-4 Tailoring Streptomyces: producing novel minor groove binder antibiotics Emilio Cortes Sanchez 1,∗ , Sara De Ornellas 2 , John May 2 , Glenn Burley 2 , Paul Hoskisson 3 1 University of Strathclyde, United Kingdom University of Strathclyde, Chemistry, United Kingdom 3 University of Strathclyde, SIPBS, United Kingdom 2 The rise of antibiotic resistant strains and the decline of antibiotic discovery have resulted in an urgent need to develop and discover novel antimicrobials. Minor groove binders (MGBs) are compounds that shown antibiotic, anticancer and antiviral activity by binding to the minor groove of the DNA helix. MGBs are naturally produced by Streptomyces, and recently some of the clusters encoding for those antibiotics have been characterised. In order to produce a new drug, we are using synthetic biology and combinatorial biosynthesis to modify a pathway of a Streptomyces strain, forcing the assembly of a new antibiotic. For this we transformed several Streptomyces strains with an MGB producing biosynthetic cluster containing four independent non-ribosomal peptide synthetases. These strains were fermented and sampled at different time points and the organic extractions were analysed looking for the production of novel compounds. The HPLC, LCMS and HRMS data suggest the assembly of a novel compound, which exhibits antibiotic activity according to our bioassays. Further characterisation of the molecule will be done by NMR .We have also cloned one of the non-ribosomal peptide synthetases in order to engineer this enzyme to broaden its substrate specificity. http://dx.doi.org/10.1016/j.nbt.2014.05.1765 O17-5 Progesterone biosynthesis by combined action of adrenal steroidogenic and mycobacterial enzymes in fast growing mycobacteria Nicolai Strizhov 1,∗ , Victoria Fokina 1 , Galina Sukhodolskaya 1 , Dmitry Dovbnya 1 , Mikhail Karpov 1 , Andrey Shutov 1 , Ludmila Novikova 2 , Marina Donova 1 1 Institute of Biochemistry & Physiology of Microorganisms, Russian Academy of Sciences, Russia 2 Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Russia We have for the first time demonstrated the use of mycobacteria as a harbor for synthesis of C21-steroids by heterologous expression of mammalian steroidogenesis key proteins. Progesterone biosynthesis was successfully reconstructed in recombinant mycobacterial cells. The first stage of mammalian steroidogenesis, cholesterol conversion to pregnenolone is carried out by heterologously expressed cytochrome P450scc cleaving the substrate side chain. cDNA copies of the CYP11A, Adx and AdR genes encoding mature forms of cytochrome P450 cholesterol hydroxylase/20, 22-lyase (P450scc), adrenodoxin (Adx) and adrenodoxin reductase (AdR) from bovine adrenal cortex were transferred with pNS11 construct to Mycobacterium smegmatis mc2 155. Acetamide chemoinduction resulted in high expression level of all three proteins. Pregnenolone oxidation to progesterone in the subsequent step is mediated by innate mycobacterial 3␤-hydroxysteroid dehydrogenase (3␤-HSD). However, no progesterone synthesis was detected in mycobacteria with pNS10 construct expressing P450scc alone without Adx and AdR redox partners. We concluded that bacterial ferredoxins and ferredoxin reductases ubiquitously present in mycobacteria fail to deliver electrons to P450scc. Progesterone was also produced by recombinant pNS11 mycobacterial cells with sitosterol as a substrate for bioconversion though with lower yields. The highest progesterone production level achieved is 25 mg/l at 7.5 mM cholesterol load. This yield is 60 fold higher than the maximum pregnenolone concentration we obtained earlier with recombinant Escherichia coli (pBar Triple). The level difference between two hosts clearly reveals prospects of the mycobacterial progesterone biosynthesis for biotechnological purposes. Our findings pave the way for future exploration of these lipophilic bacteria for production of valuable active pharmaceutical ingredients. http://dx.doi.org/10.1016/j.nbt.2014.05.1766 O17-6 DNA vaccine expressing ubiquitin-conjugated multifragments antigens protects BALB/c mice against Toxoplasma gondii infection Hua Cong ∗ , Quan Yuan Shandong University, China Toxoplasma gondii, an intracellular parasite with a complex life cycle, is highly prevalent in humans and animals and causes www.elsevier.com/locate/nbt S67 WEDNESDAY 16 JULY SYMPOSIUM 17: DEVELOPMENT OF NEW VACCINES AND ANTIMICROBIALS zoonotic toxoplasmosis. In order to prevent from I infection, an ideal vaccine that can elicit protective cell-mediated immune responses is greatly needed. In our study, we selected seven fragments derived from seven T. gondii surface antigens (SAG3, ROP18, MIC6, GRA7, MAG1, BAG1, SPA), which was linked to ubiquitin protein. DNA vaccines encoding 7 fragments and ubiquitin protein were constructed. BALB/c mice were immunized with p-Ub p-Tgag, pVAXI, or PBS and challenged with highly virulent T. gondii RH strain or the cyst of T. gondii genotype II strain of PRU. Vaccination with p-Ub and p-Tgag both showed high ability to generate a strong Th1 cell response with significant production of IFN-␥, IL-2 and low levels of IL-10, and protected mice against high parasite burden when challenged with T. gondii compared with PBS and pVAX1. Furthermore, the result of vaccination with p-Ub demonstrated that it was more effective than p-Tgag in protection of T. gondii infection. We conclude that the DNA vaccine we constructed in this study is efficacious and in particular, ubiquitin-conjugated vaccines showed a stronger Th1-type immunity and significant resistance to parasite challenge. http://dx.doi.org/10.1016/j.nbt.2014.05.1767 O17-7 Proteome-wide analysis of the functional roles of bacilysin biosynthesis in Bacillus subtilis Gulay Ozcengiz 1,∗ , Asli Aras Taskin 2 , Mustafa Demir 1 , Ayten Yazgan Karatas 3 1 Department of Biol. Sci., METU, Turkey University of Freiburg, Germany 3 Mol. Biol. Gen. Department, Istanbul Technical University, Turkey 2 Bacilysin, being produced and excreted by certain strains of Bacillus subtilis, is a dipeptide antibiotic composed of L-alanine and L-anticapsin. We previously showed that the biosynthesis of bacilysin is positively regulated by quorum sensing regulatory cir- S68 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 cuit and sporulation regulator Spo0A while negatively regulated by transition regulators CodY, ScoC and AbrB. In this study, a comparative proteomic analysis of the bacilysin producer B. subtilis PY79 and its bacilysin non-producer derivative OGU1 (bacA::lacZ::erm) was performed in order to gain a deeper insight into the functional roles of bacilysin biosynthesis in its producer. 2-DE gel electrophoresis coupled to MALDI-TOF/MS within different pH ranges separated more than 1900 protein spots. Of these, 159 protein spots were identified which corresponded to 123 distinct proteins. 60 out of 123 distinct proteins were down-regulated in OGU1 strain as compared to the wild-type. Thirty-five of 123 protein spots were expressed more abundantly while 19 protein spots were found to be absent and one protein spot was newly induced in the mutant strain. To avoid any experimental limitations, we also performed 1-DE gel electrophoresis coupled to LC–MS/MS analysis which led to the identification of 1282 proteins from the total soluble proteome of PY79 and OGU1. 76 of those proteins were found to be differentially expressed as deduced from their relative abundance. The analysis of the results obtained from 2-DE gel electrophoresis coupled to MALDI-TOF/MS and GeLC–MS/MS revealed the impact of the absence of bacilysin biosynthesis on the expression of sporulation, two component-regulatory system, peptide transport, stress and global repressor CodY-regulated proteins. http://dx.doi.org/10.1016/j.nbt.2014.05.1768 New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 18: EXPLOITATION OF METAGENOMICS FOR ENVIRONMENTAL AND BIOCATALYTIC APPLICATIONS Symposium 18: Exploitation of metagenomics for environmental and biocatalytic applications O18-1 Metagenomics: mining for novel catalysts Elizaveta Bonch-Osmolovskaya Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia About three decades back the analysis of environmental 16S rRNA clone libraries revealed that roughly 95% of microorganisms in natural microbial communities had never been obtained in laboratory cultures, and so their catalytic capacities were not known. The metagenomic approach, actively developing over the last 10 years, provides access to genes of microorganisms that might never be isolated. This became possible as a result fast progress of the sequencing technologies, leading to their significant cost reduction. The contemporary state of technology makes feasible efficient sequencing of environmental DNA with multiple coverage, allowing consequent alignment and assemblage of large genome fragments. Metagenomic libraries became an object for gene mining, both for predicting novel metabolic pathways, not known in cultured prokaryotes, and for searching new catalysts for biotechnology. For the latter purpose, microbial communities of extreme environments have been investigated, including hot springs, acidic mines, soda lakes, and polar soils; and new enzymes – highly stable hydrolases, esterases, DNA polymerases with new capacities have been cloned and expressed. One example of such work is a current FP7 project “HotZyme – Systematic screening of novel hydrolases from hot environments”, in which an international team covers ground from environmental samples to purified and crystallized new proteins with target activities. http://dx.doi.org/10.1016/j.nbt.2014.05.1769 O18-2 Metagenomics unveils bacterial and fungal communities response to mycoremediation of polychlorinated biphenyl-contaminated soil Tatiana Stella 1,∗ , Stefano Covino 1 , Monika Čvančarová 1 , Maurizio Petruccioli 2 , Alessandro D’Annibale 2 , Tomas Cajthaml 1 1 2 Institute of Microbiology AS CR, v.v.i., Czech Republic Tuscia University, Italy The remediation of sites contaminated by polychlorinated biphenyls (PCBs) is a major priority due to the teratogenic, carcinogenic and endocrine-disrupting features of these xenobiotics. Within this frame, the present work was aimed at assessing the technical feasibility of both bioaugmentation (white-rot fungi Irpex lacteus and Pleurotus ostreatus) and biostimulation treatments of three different samples of a historically PCB-contaminated soil (bulk soil, topsoil and rhizosphere soil). The highest PCB depletion yields (41 and 51%) were observed in P. ostreatus-augmented micro- cosms with topsoil and rhizosphere soil, respectively. Several PCB degradation intermediates were detected (i.e. chlorobenzoates, hydroxylated and methoxylated PCBs) and the initial acute toxicity was reduced mainly in the rhizosphere soil. Furthermore, to gain new insights into the biota composition throughout the remediation processes, the diversity and dynamics of both bacterial and fungal communities were estimated via high-throughput 454-pyrosequencing method. Metagenomic analysis showed that Firmicutes relative abundance increased when the bulk and rhizosphere soils were treated with P. ostreatus. Conversely, in all soil samples augmented with I. lacteus an initial rise in the relative abundance of the Proteobacteria phylum was observed, whereas Bacteroidetes predominate in the topsoil and rhizosphere soil at the end of incubation. P. ostreatus was able to compete with the autochthonous fungi, its relative abundance being higher than 90% along the whole incubation period. By contrast, the abundance of I. lacteus sequences tended to decline during the last 6 weeks of incubation. In biostimulated microcosms, the large majority of detected sequences belonged to the phyla Ascomycota and Zygomycota. http://dx.doi.org/10.1016/j.nbt.2014.05.1770 O18-3 Mining alginate lyases in sediment metagenomes from four geographically distant cold coastal environments Hebe Dionisi 1,∗ , Marina Matos 1 , Luciano Anselmino 1 , Mariana Lozada 1 , Walter Mac Cormack 2 , Jolynn Carroll 3 , Leif Lundgren 4 , Sara Sjöling 5 , Krystle Chavarría 6 , Bernard Henrissat 7 , Janet Jansson 6 1 Patagonian National Research Center (CENPAT-CONICET), Argentina Argentinean Antarctic Institute and National University of Buenos Aires, Argentina 3 University of Tromsø, Norway and Akvaplan-niva AS, FRAM – High North Research Centre for Climate and the Environment, Tromsø, Norway 4 Stockholm University, Sweden 5 Södertörn University, Sweden 6 Lawrence Berkeley National Laboratories, USA 7 Centre National de la Recherche Scientifique, Marseille, France 2 Brown macroalgae are considered an attractive option as sustainable feedstock for the production of biofuels and commodity chemicals due to their high carbohydrate content. Microbial communities from cold coastal environments represent promising sources of novel enzymes depolymerizing brown algal polysaccharides such as alginates, as these organisms constitute a large primary biomass in these environments. We used a nested sampling strategy to obtain sediment samples from four high-latitude coastal environments (Svalbard Archipelago, Norway; Baltic Sea, Sweden; Ushuaia Bay, Argentina and Potter Cove, Antarctica). Twenty-three samples were sequenced using Illumina HiSeqTM 1500, assembled and annotated using the IMG/M pipeline. The complete assembled metagenome dataset contains 5.6 Gb and 1.4 × 107 protein coding genes. With the goal of identifying alginate lyase homologs in the metagenomes, we mined this dataset using both blastp searches and product names assigned in the functional annotation. We retrieved 2,705 sequences between www.elsevier.com/locate/nbt S69 SYMPOSIUM 18: EXPLOITATION OF METAGENOMICS FOR ENVIRONMENTAL AND BIOCATALYTIC APPLICATIONS 100 and 1,166 amino acids in length, mostly belonging to the CAZy polysaccharide lyase families PL17 (30.4%), PL7 (28.2%) or PL6 (22.1%). When normalized with the protein coding gene numbers of the assembled metagenomes, Antarctic samples contained the highest abundance of identified sequences, and Baltic Sea samples contained a larger proportion of novel sequences (Kruskal–Wallis test, p < 0.05). Different levels of gene order conservation were found among scaffolds containing these genes, and with genomes of isolated alginate-degrading bacteria. This study revealed a large diversity of alginate lyase homologs from yet-to-be cultured marine microorganisms, which could aid in the engineering of microbial platforms for biorefineries. http://dx.doi.org/10.1016/j.nbt.2014.05.1771 O18-4 Designed sensor cells for direct detection of microbial colonies with target enzyme activities on solid plates Haseong Kim ∗ , Eugene Rha, Bong-Hyun Sung, Seung-Goo Lee KRIBB, Republic of Korea Screening of new enzymes from vast genetic resources is indispensable for environment-friendly, cost-effective, and sustainable chemical industry. Here is a new method to explore the variety S70 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 of microbial colonies on solid plates for the existence of target enzyme activity using designed sensor cells. When an effector, such asp-nitrophenol, is released from a microbial colony, it will diffuse out to the vicinity surrounding the colony and the sensor cells in the vicinity turn on the expression of a fluorescence and an auxotrophic genes. The expression of these reporters enables the sensor cells to grow rapidly and to display strong fluorescence signals near the original colonies. So, by simply co-culturing these sensor cells with any microbes from nature, the sensor cells will directly mark the locations of microbial colonies with target enzyme activity, which is responsible for the buildup of pnitrophenol. We tested this proof-of-concept using Escherichia coli sensor cells with a phenol-sensitive promoter, dmpR, to detect Citrobacter freundii cells expressing tyrosine phenol-lyase. Also, the sensor cell was applied to examine diverse microbial cells from soils, to find a cellulase activity using p-nitrophenyl-cellobioside as a substrate. We isolated seven cellulase producing bacteria and identified one of them identified as a novel species of Pseudomonas, based on 16s rRNA/NGS analysis along with morphological, biochemical, and physiological properties. Lastly, the sensor cell was used to isolate new cellulases from metagenome libraries, which were confirmed to have the hydrolyzing activity and specificity on cellulose by conventional methods. http://dx.doi.org/10.1016/j.nbt.2014.05.1772 New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 19: EVOLUTIONARY STRATEGIES FOR CELL FACTORY DEVELOPMENT Symposium 19: Evolutionary strategies for cell factory development O19-1 Evolutionary and reverse metabolic engineering of Saccharomyces cerevisiae Jack T. Pronk Department of Biotechnology, Delft University of Technology, Delft, The Netherlands Laboratory evolution is a powerful, versatile approach for improving and expanding the capabilities of industrial microorganisms which, in contrast to targeted genetic modification, does not require a detailed a priori understanding of the molecular basis for the trait of interest. Cultivation in bioreactors offers many options to design and implement culture conditions that maximize the selective advantage of spontaneous mutations that confer a specific, industrially relevant trait. I will briefly discuss applications of this ‘evolutionary engineering’ approach to the yeast Saccharomyces cerevisiae for optimizing its sugar fermentation kinetics and for increasing its robustness to industrially relevant stresses. Until recently, expression profiling with DNA micro-arrays was the only cost-affordable genome-wide analytical technique for analysing the molecular basis for improved performance of yeast strains derived from evolutionary engineering experiments. However, such transcriptome analyses tended to generate large numbers of targets, often without a clear lead to the responsible mutation(s). Based on recent studies from the Delft yeast group, I will illustrate how the advent of whole-genome sequencing has transformed the molecular analysis of evolved genotypes. Analysis of multiple, independent evolution experiments and integration of classical genetics approaches were shown to greatly amplify the power of whole-genome resequencing of evolved strains. http://dx.doi.org/10.1016/j.nbt.2014.05.1773 O19-2 Adaptive evolution of saccharomyces cerevisiae to early stage of an alcoholic fermentation Ana Mangado ∗ , Pilar Morales, Jordi Tronchoni, Ramon Gonzalez ICVV/CSIC, Spain Experimental evolution was used to identify genes involved in the adaptation to the early stages of wine fermentation. Evolution experiments were performed in continuous culture for 150-250 generations, in conditions emulating the initial stages of alcoholic fermentation. We performed three independent experimental evolution experiments of a haploid laboratory strain BY4741 and one evolution with a haploid strain (not adapted to winemaking growth conditions) obtained by meiotic segregation of the wine yeast EC1118. By the end of the experiments, colonies were phenotypically characterized, and strains that showed improved initial growth rates were selected. We used next generation sequenc- ing techniques in order to find the genetic changes. Four strains were selected from the evolution of BY4741. The mutations found, pointed to the Rsp5p-Bul1/2p ubiquitin ligase complex as the preferred evolutionary target under these experimental conditions. Rsp5p is a multifunctional enzyme able to ubiquitinate target proteins participating in different cellular processes, while Bul1p is an Rsp5p substrate adaptor involved in the ubiquitin-dependent internalization of Gap1p and other plasma membrane permeases. Our results might be related to increased halftime of plasma membrane amino acid permeases. Apparently the genetic background of the laboratory strain is conditioning the result. However, we have shown the strength of this approach. The evolution of the haploid segregant of EC1118 was run for 250 generations, three adapted strains were selected. Preliminary bioinformatic analysis highlighted changes in chromosome numbers in mutant strains. We are currently testing these results by karyotyping, qPCR, flow cytometry and other techniques. http://dx.doi.org/10.1016/j.nbt.2014.05.1774 O19-3 Genome dynamics of the human embryonic kidney 293 (HEK293) lineage in response to cell biology manipulations Morgane Boone 1,2,∗ , Yao-Cheng Lin 3,4 , Leander Meuris 1,2 , Irma Lemmens 5,6 , Nadine Van Roy 7 , Arne Soete 8 , Joke Reumers 9 , Matthieu Moisse 9,10 , Stephane Plaisance 11 , Radoje Drmanac 12 , Jason Chen 12 , Frank Speleman 7 , Diether Lambrechts 9,10 , Yves Van de Peer 3,4,13 , Jan Tavernier 5,6 , Nico Callewaert 1,2 1 Unit for Medical Biotechnology, Inflammation Research Center (IRC), VIB, Belgium 2 Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, Belgium 3 Department of Plant Systems Biology, VIB, Belgium 4 Department of Plant Biotechnology and Bioinformatics, Ghent University, Belgium 5 Department of Medical Protein Research, VIB, Belgium 6 Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Belgium 7 Center for Medical Genetics, Ghent University Hospital (MRB), Belgium 8 Bioinformatics Core Facility, Inflammation Research Center (IRC), VIB, Belgium 9 Laboratory for Translational Genetics, Department of Oncology, KULeuven, Belgium 10 Vesalius Research Center, VIB, Belgium 11 VIB Bioinformatics Training and Services (BITS), VIB, Belgium 12 Complete Genomics, USA 13 Genomics Research Institute, University of Pretoria, South Africa The HEK293 human cell lineage is widely used in cell biology and biotechnology. We used whole genome resequencing methods in six 293 cell lines to study the dynamics of this aneuploid genome in response to the cell biology manipulations that were used to generate common derivatives of 293 cells, such as transformation and stable clone generation (293T); suspension growth adaptation (293S) and cytotoxic lectin selection to isolate a glycosylation-homogenous clone (293SG). While the chromosomal structure of single 293 cells within a culture appears to be extremely diverse, our analysis suggests that standard cell culture www.elsevier.com/locate/nbt S71 SYMPOSIUM 19: EVOLUTIONARY STRATEGIES FOR CELL FACTORY DEVELOPMENT procedures (passaging and cell banking) do not affect the ‘average’ genome structure and sequence to a great extent. The extraordinary chromosomal plasticity of this genome, however, seems to be the driving adaptive force when cells are put through a bottleneck. This feature underlies a novel application for which we provide proof of concept here: selection of 293 clones surviving stringent selective conditions (eg ricin toxin), followed by whole-genome analysis of copy number alterations, can effectively pinpoint the genomic region(s) that contain the gene(s) required for adaptation to those selective conditions. Furthermore, up to the level of sensitivity afforded here (single copy plasmid insertions were easily detected), these cell lines have no inadvertent virus insertions. In terms of tools, we optimized a workflow to detect human/vector genome breakpoints, and enabled visualization of the 293 genome data both through a user-friendly visualization web page, as well as through the Integrative Genome Browser (IGV) for rich data mining. http://dx.doi.org/10.1016/j.nbt.2014.05.1775 O19-4 Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16 Steffen Gruber , Jeremias Hagen, Helmut Schwab, Petra Koefinger ∗ Graz University of Technology, Austria The gram-negative ␤-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. Up to now some basic systems for targeted genetic manipulation of this bacterium were already established. However, the majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmids loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16 a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96 hours. The utility of the new vector family is demonstrated by providing expression data with different model proteins. http://dx.doi.org/10.1016/j.nbt.2014.05.1776 S72 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 O19-5 Novel human kidney epithelial cell line in pharmaceutical biotechnology Lukas Fliedl 1,∗ , Matthias Wieser 1 , Gabriele Manhart 1 , Matthias P. Gerstl 1 , Florian Kast 1 , Abdulhameed Khan 2 , Renate Kunert 2 , Johannes Grillari 2 , Regina Grillari-Voglauer 2 1 ACIB, Austria Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Austria 2 Mammalian cells are used as model systems, products themselves and as producers of recombinant proteins and vaccines. In these different applications a variety of different cell lines are used and although all have proven valuable for their specific application, there is still room for improvement in terms of posttranslational modifications. Especially novel human cell lines are of ever increasing importance since they ideally represent the in vivo situation and might produce high quality biopharmaceuticals as similar to endogenous proteins as possible. Therefore we established a novel human continuously growing renal proximal tubular epithelial cell line (RPTEC) that has maintained many differentiated characteristics of the normal nontransduced counterpart and tested its performance in the different fields of pharmaceutical biotechnology. A complex model protein, erythropoietin, was stably produced in this cell line and the quality of the recombinant protein was compared to CHO derived product by analysis of isoforms as well as specific non-human glycopatterns. Additionally, we used our cell line to produce influenza virus and proved high capabilities of our cell line in this application. Finally, we used our cell line to get insights into nephrotoxicity induced by cisplatin, which has important implications as a chemotherapeutic drug and hypothesize that especially epithelial barrier formation and polarity of RPTECs need to be considered in toxicity models to validly predict the in vivo situation. Therefore, the here established kidney epithelial cells combine applicability in various fields of pharmaceutical biotechnology, as they are capable of production as well as of pre-clinical testing of biopharmaceuticals. http://dx.doi.org/10.1016/j.nbt.2014.05.1777 O19-6 De novo production of geranic acid with Pseudomonas putida Jens Schrader ∗ , Jia Mi, Daniela Becher, Patrice Lubuta, Markus Buchhaupt, Dirk Holtmann DECHEMA Research Institute, Germany Production of plant terpenes by engineered microbes has become a prime example of applied synthetic biology with tremendous progress being made during the last decade. Whereas sesquiterpene titers reported have already reached g/L values in the bioreactor, efficient monoterpene production seems to be more New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 19: EVOLUTIONARY STRATEGIES FOR CELL FACTORY DEVELOPMENT difficult with conventional host strains due to product toxicity. Hence, we set out to investigate to potential of solvent tolerant Pseudomonas putida for monoterpene production. P. putida DSM 12264 shows a pronounced robustness in the presence of monoterpenes. Recent results revealed that the wildtype strain efficiently converts geraniol to geranic acid. The monoterpenoic acid shows interesting properties as a fungicidal agrochemical and may be used as a natural preservative for foods and cosmetics. Comparing the growth pattern with E. coli and S. cerevisiae, P. putida revealed a several times higher tolerance towards geranic acid. Following the cell factory idea, we functionally expressed plant geraniol synthase in P. putida, which led to the production of small amounts of the desired geranic acid from glycerol as the C-source indicating exploitation of terpene precursors derived from the endogenous DXP pathway. The cellular precursor supply was improved by expression of the mevalonate (MVA) pathway from Myxococcus xanthus. With this P. putida strain, a product concentration of ca. 230 mg/L was obtained in a fed-batch bioreactor. This is the first example of de novo monoterpenoic acid production with an engineered microbe. We intend to further improve the product titers by pathway engineering to eventually take advantage of the host’s monoterpene tolerance. http://dx.doi.org/10.1016/j.nbt.2014.05.1778 tions despite an indisputable potential. While peptide engineering protocols allow for the generation of thousands of novel, putative active antimicrobial peptides at ease, the assessment of their activity is a rather laborious procedure limiting the assay throughput to 102 variants per day. We will present a platform based on nL-sized reaction vessels (nL-reactors) that are used for peptide production and activityscreening in a single step and at rates of 105 variants per day. During screening, library cells are grown to microcolonies within nL-reactors along with a sensor strain serving as a model for a pathogen. Library cells secreting an active antimicrobial peptide, will deactivate the sensor cells within the encircling nL-reactor. Clearance of an nL-reactor from the sensor thus indicates the presence of a strain secreting a highly active peptide. We use large particle flow cytometry and fluorescently labeled cells in order to isolate promising candidates. The power of the technology will be demonstrated by screening of libraries containing ∼105 peptide variants, generated by sitesaturation mutagenesis or synthetic biology approaches based on the blueprint of natural antimicrobial peptides. We show that the assay has not only a high throughput but also a high accuracy, making this technique a valuable contribution for future approaches targeting the design of highly active antimicrobial compounds. http://dx.doi.org/10.1016/j.nbt.2014.05.1779 O19-7 High-throughput nL-reactor screening for antimicrobial peptides Steven Schmitt 1,∗ , Manuel Montalban Lopez 2 , Oscar Kuipers 2 , Sven Panke 1 , Martin Held 1 1 2 ETH Zürich, Department of Biosystems Science and Engineering, Switzerland University of Groningen, Molecular Genetics Group, Netherlands The number of multi drug resistant pathogens is constantly growing and novel antibiotic substances are desperately needed in order to at least maintain the status quo. Ribosomally synthesized antimicrobial peptides are not yet exploited for human applica- www.elsevier.com/locate/nbt S73 SYMPOSIUM 20: SYSTEMS BIOCATALYSIS Symposium 20: Systems biocatalysis O20-1 Combination of the two ‘worlds’ chemo- and biocatalysis towards multi-step one-pot processes Harald Gröger Faculty of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany Multi-step one-pot processes represent an attractive synthetic concept for the improvement of overall process efficiency by decreasing the required number of work up and purification steps. By avoiding such time-, capacity- and solvent-intensive process steps, multi-step one-pot syntheses contribute to a significantly improved process economy as well as to more sustainable synthetic routes. A key criterion for multi-step one-pot processes is the compatibility of the individual reaction steps with each other. Accordingly, most of today’ known multi-step one-pot processes are based on either chemocatalytic multi-step reactions or ‘pure’ biotechnological processes such as, for example, fermentation. In contrast, successful combinations of chemo- and biocatalytic reactions, in particular in aqueous reaction media, are much less widely known. In this contribution strategies for the combination of chemoand biocatalysts towards the development of multi-step onepot processes in aqueous reaction media are presented. Since palladium-catalyzed cross-coupling reactions are of particular importance in the field of metal catalysis, as enzymatic reductions are in the field of biocatalysis, we were interested in the investigation of the compatibility of these types of reactions with each other in water. As an example for such a one-pot process the synthesis of chiral biaryl-containing alcohols via Suzukicross-coupling reaction and subsequent asymmetric enzymatic reduction is shown [1]. A further example for the combination of palladium catalysis and a biotransformation is a one-pot process comprising a Wacker oxidation and subsequent enzymatic reduction [2]. Very recently we could also demonstrate the compatibility of a metal-catalyzed cross-metathesis reaction with a biotransformation [3]. A further research focus is on the combination of enzyme-compatible organocatalytic reactions with biotransformations towards multi-step one-pot syntheses. It turned out that a reaction mixture resulting from an asymmetric organocatalytic aldol reaction is compatible with a direct subsequent enzymatic reduction without the need for a work-up step of the aldol reaction [4,5]. In addition, an organocatalytic nitroalkene synthesis has been successfully combined with its subsequent ene reductasecatalyzed asymmetric reduction, leading to the corresponding nitroalkane with high enantioselectivity [6]. References [1].Burda E, Hummel W, Gröger H. Angew Chem 2008;120:9693–6; Angew Chem Int Ed 2008;47:9551–4. [2].Schnapperelle I, Hummel W, Gröger H. Chem Eur J 2012;18:1073–6. [3].Tenbrink K, Seßler M, Schatz J, Gröger H. Adv Synth Catal 2011;353:2363–7. S74 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 [4].Baer K, Kraußer M, Burda E, Hummel W, Berkessel A, Gröger H. Angew Chem 2009;121:9519–22; Angew Chem Int Ed 2009;48:9355–8. [5].Rulli G, Duangdee N, Baer K, Hummel W, Berkessel A, Gröger H. Angew Chem 2011;123:8092–5; Angew Chem Int Ed 2011;50:7944–7. [6].Burda E, Ress T, Winkler T, Giese C, Kostrov X, Huber T, et al. Angew Chem 2013;125:9493–6; Angew Chem Int Ed 2013;52:9323–6. http://dx.doi.org/10.1016/j.nbt.2014.05.1780 O20-2 Engineering artificial metabolisms in vitro Wolf-Dieter Fessner Technische Universität Darmstadt, Institut für Organische Chemie und Biochemie, Alarich-Weiss-Str. 4, 64287 Darmstadt, Germany Systems Biocatalysis is a new concept [1] of organizing best enzymes in vitro to construct novel artificial metabolisms for an efficient, sustainable synthesis of valuable chemical products [2]. The strategy merges the synthetic focus of chemistry with the modular design of biological systems, which is similar to Synthetic Biology but can be realized at a far lower level of complexity from a true reductionist approach. A particular advantage of the concept arises from the inherent potential to construct novel biocatalytic reaction systems for the efficient synthesis of non-natural products by using enzymes engineered for non-natural substrate promiscuity. Such operations are free from material erosion by competing metabolic pathways and from kinetic restrictions by regulating circuits, which are notorious problems in cellular production systems (cell factories). Examples illustrating the technology will be discussed. References [1].http://www.cost.eu/domains actions/cmst/actions/CM1303. [2].Fessner W-D, Walter C. “Artificial metabolisms” for the asymmetric one-pot synthesis of branched-chain saccharides. Angew Chem Int Ed Engl 1992;31:614–6. http://dx.doi.org/10.1016/j.nbt.2014.05.1781 O20-3 Expanding the diversity of diketopiperazines biosynthesized by cyclodipeptide synthases Isabelle Jacques ∗ , Jérôme Seguin, Mireille Moutiez, Emmanuel Favry, Muriel Gondry, Pascal Belin CEA, iBiTec-S, Service d’Ingénierie Moléculaire des Protéines (SIMORPO), France Cyclodipeptides and more complex diketopiperazines (DKPs) are secondary metabolites mostly synthesized by microorganisms. They are well known for their wide range of noteworthy biological activities including antibacterial, antifungal, antiviral and anticancer effects [1]. In the last decade, an important effort has been made to elucidate the biosynthesis pathways of these compounds. Our group and others characterized five novel pathways [2–4] that SYMPOSIUM 20: SYSTEMS BIOCATALYSIS New Biotechnology · Volume 31S · July 2014 are dependent on cyclodipeptide synthases (CDPSs) [5,6], enzymes that catalyze the formation of the DKP scaffold by condensing two aminoacyl-tRNAs. Herein, I will describe our medium-high throughput method to characterize 46 putative CDPSs identified by bioinformatics. Our work confirms thereby 40 new CDPSs and reveals a large variety of produced cyclodipeptides. This study opens the ways to a future characterization of the CDPS-dependent pathways, enabling combinatorial biosynthesis to produce unnatural natural DKPs. References [1].Borthwick AD. Chem Rev 2012;112:3641–716. [2].Belin P, et al. Nat Prod Rep 2012;29:961–79. [3].Giessen TW, et al. Chem Biol 2013;20:828–38. [4].Giessen TW, et al. Biochemistry 2013;52:4274–83. [5].Gondry M, et al. Nat Chem Biol 2009;5:414–20. [6].Seguin J, et al. Chem Biol 2011;18:1362–8. http://dx.doi.org/10.1016/j.nbt.2014.05.1782 O20-4 Improving the performance of coupled racemase/acylase systems: new structural insights and novel tools for high-throughput screening Guiomar Sanchez Carron 1,∗ , Dominic Campopiano 1 , Toni Fleming 2 1 2 University of Edinburgh, United Kingdom Dr. Reddys Laboratories, India A coupled enzymatic dynamic kinetic resolution (DKR) system to allow the preparation of synthetically useful amino acids has been developed and improved. We combined a previously engineered N-acetyl amino acid racemase (NAAAR G291D/F323Y) with an L- or D-specific acylase and used them in a preparative scale DKR to generate enantiomerically pure amino acids. In this work we describe the latest efforts to expand the synthetic utility of this process. We present two novel spectrophotometric assays to measure NAAAR activity towards different substrates, replacing the existing HPLC and In vivo selection assays. One of them links the NAAAR/acylase couple to an L-amino acid oxidase and peroxidase. The other uses a D-amino acid dehydrogenase with broad substrate specificity. These assays allow us to identify novel NAAAR substrates and facilitate the high-throughput screening of NAAAR saturation mutagenesis libraries. X-ray structural analysis of NAAAR mutants in complex with N-acetyl-D-naphthylalanine reveals active site residues involved in the accommodation of bulkier substrates. These assays, combined with structural insights, guide the engineering of new NAAARs with catalytic potential across a larger range of amino acids. Acknowledgements: This work has been funded by the BBSRC and Dr Reddys Laboratories. O20-5 Artificial enzyme cascade to the polymer building block ␻-amino caproic acid Wolfgang Kroutil 1,∗ , Johann Resch 1 , Joerg Schrittwieser 1 1 2 Sattler 2 , Michael Fuchs 1 , Verena University of Graz, Austria ACIB GmbH, Austria The monomer units for polyamides may be ␻-amino-carboxylic acids, lactams or diamines and dicarboxylic acids. Caprolactam, for example, is produced chemically at 4.2 million ton/year mainly from cyclohexanone via its oxime and Beckmann rearrangement. Biocatalytic cascades involving redox steps [1] allow to perform oxidation and reduction reaction in the linear sequence simultaneously. [2,3] Oxidation and reduction reactions might be coupled in that way that the electrons gained in the oxidation step are consumed in the reduction step turning the overall process redox neutral and cost efficient. Here we report a biocatalytic redox cascade starting from cyclohexanol to yield ␻-amino caproic acid, thus the hydrolysed equivalent to caprolactam. The designed cascade involves four redox steps and additional hydrolytic steps, whereby the cascade was designed in that way, that no external redox reagents were required, thus the cascade was redox neutral or redox selfsufficient. Since an intermediate in the reaction sequence caused inhibition for a later enzyme in the cascade an in situ functional group protection strategy was established to avoid the formation of the inhibiting compound. References [1].Schrittwieser H, Sattler J, Resch V, Mutti FG, Kroutil W. Curr Opin Chem Biol 2011;15:249–56. [2].Staudt S, Burda E, Giese C, Müller CA, Marienhagen J, Schwaneberg U, et al. Angew Chem Int Ed 2013;52:2359–63. [3].Sattler JH, Fuchs M, Tauber K, Mutti FG, Faber K, Pfeffer J, et al. Angew Chem Int Ed 2012;51:9156–9. http://dx.doi.org/10.1016/j.nbt.2014.05.1784 O20-6 Uncovering the broader roles of redox partner proteins for cytochrome P450 enzymes Shengying Li 1,∗ , Wei Zhang 1 , Yi Liu 1 , Yojiro Anzai 2 , Fumio Kato 2 , Larissa Podust 3 , David Sherman 4 1 Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, China 2 Toho University, Japan 3 University of California, San Francisco, USA 4 University of Michigan, Ann Arbor, USA http://dx.doi.org/10.1016/j.nbt.2014.05.1783 The superfamily of cytochrome P450 enzymes is one of the most versatile biocatalytic systems in nature. P450 enzymes are capable of catalysing many distinct types of industrially important reactions such as regio- and stereoselective oxidation of unactivated C−H bonds, dealkylation, decarboxylation, and aromatic coupling. For most P450 enzymes, redox partner proteins www.elsevier.com/locate/nbt S75 SYMPOSIUM 20: SYSTEMS BIOCATALYSIS are required for the electron transfer during catalysis. Conventionally, it has been thought that these auxiliary proteins only influence catalytic efficiency and/or product distribution, but not the type and selectivity of reactions catalysed by P450s. However, our recent study (J. Am. Chem. Soc. 2014, 136, 3640) on MycG, which is the multifunctional P450 monooxygenase involved in the biosynthetic pathway of 16-membered ring macrolide antibiotics mycinamicins in the rare actinomycete Micromonospora griseorubida, has provided solid evidence to challenge this generally accepted “postulate”. Moreover, we have found that some redox S76 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 partner proteins are able to support the activity of a biofuel related P450 peroxygenase from Jeotgalicoccus sp. ATCC 8456 that normally uses hydrogen peroxide as cofactor (Biotechnol. Biofuels 2014, 7, 28). These results highlight broader roles of redox partner proteins for modulating the catalytic activity of P450 enzymes via alternative protein-protein interactions that have not been well understood so far. http://dx.doi.org/10.1016/j.nbt.2014.05.1785 New Biotechnology · Volume 31S · July 2014 BIO-BASED PRODUCTION OF CHEMICALS, FUELS AND MATERIALS BY METABOLICALLY ENGINEERED MICROORGANISMS Bio-based production of chemicals, fuels and materials by metabolically engineered microorganisms PL3-1 Bio-based production of chemicals, fuels and materials by metabolically engineered microorganisms Sang Yup Lee Department of Chemical and Biomolecular Engineering (BK21+ Program), BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, Republic of Korea Many chemicals, fuels and materials we use every day are derived from fossil resources. Relying on the current production system is not sustainable and has been raising concerns due to the climate change and other environmental problems. To address this issue, there has recently been much interest in developing bio-based processes for the production of chemicals, fuels and materials from renewable non-food biomass. In order to maximize the efficiencies of bioconversion process, metabolic engineering has become an essential practice. Metabolic engineering has recently become more powerful through the integration with systems biology, synthetic biology and evolutionary engineering, which led to the birth of systems metabolic engineering. In this lecture, systems metabolic engineering approaches taken to develop strains capable of efficiently producing various chemicals, fuels and materials will be described together with several example cases. Through systems metabolic engineering, it is possible to achieve cost-effective production of desired bioproducts, which are either natural or nonnatural products. Acknowledgements: This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries of Ministry of Science, ICT & Future Planning. http://dx.doi.org/10.1016/j.nbt.2014.05.1786 www.elsevier.com/locate/nbt S77 BIOCATALYSIS New Biotechnology · Volume 31S · July 2014 Posters Biocatalysis PA-02 PA-01 Simultaneous improvement of specific catalytic activity and thermal stability of MBP fusion heaprinase II by modifying amino acid residues in 758 site Enhancing of enzymatic palmitoylation of racemic 9(2,3-dihydroxypropyl)adenine in co-solvent mixture as the reaction media Nan Su ∗ , Jingjun Wu, Chong Zhang, Xin-Hui Xing Krecmerova 1 , Marie Key Laboratory for Industrial Biocatalysis, Ministry of Education of China, Department of Chemical Engineering, Tsinghua University, Beijing, China Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague 2 Departamento de Biocatálisis. Instituto de Catálisis (CSIC).Campus UAM Cantoblanco, Madrid, Spain Heparinase II (HepB) is an important polysaceharide lyase, which plays an vital role in the development of anticancer drugs, production of low molecular weight heparin (LMWH) and quality control of heparins. At present, the main technical bottleneck which has restrained its industrial application is the high production cost, the poor level of heterologous recombinant expression and lack of the thermal stability. In a systematic design for construction of fusion expression system with the MBP tag in E.coli, we found that heparinase II could be expressed efficiently with high activity in the soluble form, and firstly observed that the amino acid residues of the 758 site in the HepB sequence greatly affected the catalytic activity and thermal stability of this enzyme, which were closely related with the protein dimmer formation of HepB. Furthermore, we systematically studied the fusion protein design by changing the linker, indicating that linker property could significantly affect both the fusion enzyme activity during the expression and the thermo stability. As a result, the total MBPHepB activity reached 3674.82IU/L, and half-life of the enzyme in 30 ◦ C reached more than 250 h which increased 25 times compared with the wild HepB. Jana Brabcova 1,∗ , Jiri Blazek 1 , Marcela Zarevucka 1 , Jose M. Palomo 2 1 A comparative study of racemic 9-(2,3-dihydroxypropyl) adenine (DHPA) palmitoylation, a potential prodrug, catalysed by several lipases in DMF/co-solvent mixtures and in pure organic solvent was performed. The optimal conditions were investigated as follows: optimal co-solvent mixture, initial aw (water activity), vinyl palmitate/DHPA molar ratio, temperature, enzyme dosage, and chemical modification of biocatalysts. It was shown that an enhancement in the substrate conversion could be achieved with DMF/hexane (4:1) co-solvents as the reaction medium instead of pure organic solvent with immobilized Candida antarctica B (CALB) lipase, although in very low yield. The chemical modification of the lipase had strong positive effect on its activity. The reaction yield was successfully increased (50% after 24 hours) when CALB glycosylated with dextran polymer was used under optimal conditions. The results described further highlight the versatility of lipases and the potential of rational substrate, solvent and enzyme engineering for modulating enzymatic reactions. Acknowledgments: The authors are grateful for the financial support of the Academy of Sciences of the Czech Republic (project No. M200551203). http://dx.doi.org/10.1016/j.nbt.2014.05.1788 http://dx.doi.org/10.1016/j.nbt.2014.05.1787 S78 www.elsevier.com/locate/nbt 1871-6784/$ — see front matter BIOCATALYSIS New Biotechnology · Volume 31S · July 2014 PA-03 Effective encapsulation of biocatalysts in sol–gel silica nanosheet prepared by peptide catalysts Katsuya Kato ∗ , Hitomi Nakamura, Fukue Nagata National Institute of Advanced Industrial Science and Technology (AIST), 226698 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560, Japan Encapsulation restricts the mobility of the biomolecules contained within a confined space and can prevent denaturation even in harsh environmental conditions related to temperature, pH, and solvents. For this reason, many biomolecules have been trapped within sol–gel silica, and this research has led to some of the most interesting and important applications of these materials, paving the way for their use in the design and fabrication of biocatalysis and biosensing devices [1]. Very recently, we reported [2] the mesoporous silica (MPS) sheet (thickness < 100 nm) was synthesized using a novel approach for the immobilization of enzyme. Enzyme immobilized on the MPS sheet exhibited a remarkably higher catalytic oxidation activity than native enzyme. From these results, the sheet morphology significantly influences the activity of immobilised enzyme. In this report, we present the details of our investigation on the immobilization of the enzyme, glucose oxidase (GOX), in a sol–gel silica matrix using poly-L-lysine assisted direct condensation reactions of silicon oxide, trimethoxysilane. The resulting GOX-silica composites with hexagonal sheet morphologies were thoroughly characterized in terms of morphology, size and surface structure. The activity and stability of the immobilized GOX were also investigated in details. References [1].Kato K, et al. J Asian Ceram 10.1016/j.jascer.2013.12.004. [2].Nakanishi K, et al. RSC Adv 2014;4:4732. Soc 2014. DOI: http://dx.doi.org/10.1016/j.nbt.2014.05.1789 PA-04 Novel mesoporous silica sheet as a protein carrier for enhancement of catalytic activity Kazuma Nakanishi 1,2,∗ , Masahiro Tomita 1 , Katsuya Kato 2 1 Department of Chemistry for Materials, Mie University, 1577 Kurimamachiyacho Tsu city, Mie 514-8507, Japan 2 National Institute of Advanced Industrial Science and Technology (AIST), 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560, Japan Porous materials have much attention because they can interact with atoms, ions, molecules, and nanoparticles, at their surfaces as well as throughout the bulk of the material. Mesoporous silica (MPS) (pore size 2–50 nm) with great mechanical stability has been applied in many fields including enzyme immobilization, catalysis and chromatography. Recently, we reported the enhanced activity and stability of enzymes and proteins on MPS as an effective carrier. [1] The presence of pore in nanostructured materials for biological applications must be promoted. Then we focused on the morphologies of materials in order to increase the mass trans- fer of substrate, which have important role in immobilized enzyme activity. Only few MPS sheet have been reported because they are quite difficult to prepare. On these bases, we introduce a new synthetic approach to MPS sheets (thickness under 100 nm) by using the carboxylate surfactant (N-palmitoyl-L-alanine) and Pluronic P123 as dual-templating agents. Cytochrom c (cyt c, molecular size of 2.5 × 2.5 × 3.7 nm) immobilised on the MPS sheet exhibited a remarkably higher catalytic oxidation activity than native cyt c. Reference [1].Nakanishi K, et al. Mater Chem B 2013;1:6321. http://dx.doi.org/10.1016/j.nbt.2014.05.1790 PA-05 Physiological responses of Pinus sylvestris var. mongolica seedlings to the interaction between Suillus luteus and Trichoderma virens Ruiqing Song 1,∗ , Dachuan Yin 1 , Xun Deng 2 1 2 Northeast Forestry University Forestry Protection Institute, Heilongjiang Academy of Forestry The effects of the interaction between Suillus luteus (L.) Roussel and Trichoderma virens (J.H. Mill., Giddens & A.A. Foster) Arx on Pinus sylvestris var. mongolica Litv. were studied using plant physiology, mycorrhizal science, forest pathology and biochemistry. Seedling growth and physiological parameters were determined, including the colonization rate of mycorrhizal fungi, biomass, root activity, photosynthetic pigment content, soluble protein content, antioxidant enzyme activities, rhizosphere soil enzyme activities and protective enzyme activities. In addition, an optimal resistance system involving T. virens, mycorrhizal fungus (Suillus luteus) and P. sylvestris var. mongolica seedlings was constructed. Synergies between S. luteus and T. virens were observed and most of the parameters of Pinus sylvestris var. mongolica seedlings inoculated with S. luteus 30 days + T. virens were higher than other treatments. After three months, when compared the control, the S. luteus 30 days + T. virens treatment gave increases in: height (42.3%); collar diameter (66.7%); fresh weight (54%); dry weight (50%); soluble protein content (69.86%); root activity (150%); chlorophyll a (77.6%); chlorophyll b (70.5%); carotenoids (144%); CAT activity (876.9%); POD activity (268.3%); SOD activity (66.18%); ␤-1,3-glucanase activity (125.8%); chitinase activity (40%); rhizosphere soil catalase activity (97.8%); and phosphatase activity (266.7%). These results indicate that there may be a stimulating factor between S. luteus and T. virens when they are inoculated together (S. luteus 30 days + T. virens) Keywords: Suillus luteus; Trichoderma virens; interaction; synergies; Pinus sylvestris var. mongolica http://dx.doi.org/10.1016/j.nbt.2014.05.1791 www.elsevier.com/locate/nbt S79 BIOCATALYSIS PA-06 Purification and characterisation of a ␤-galactosidase from the thermoacidophilic bacterium Alicyclobacillus vulcanalis DSM 16176 Gary Walsh ∗ , Jayne Murphy University of Limerick Thermoacidophiles are microorganisms capable of optimum growth under a combination of high temperature and low pH. These microorganisms are a rich source of thermo- and acidactive/stable glycosyl hydrolases. Such enzymes could find use as novel biocatalysts in some industrial processes, as operation at elevated temperature can increase substrate solubility, decrease viscosity and reduce the risk of microbial contamination [1,2]. We report the purification and characterisation of an intracellular ␤-galactosidase from the thermoacidophile Alicyclobacillus vulcanalis DSM 16176. The enzyme was purified 110-fold, with a 5% yield. Denatured (84 kDa) and native (179 kDa) molecular masses were determined by SDS-PAGE and gel filtration, respectively and suggest the enzyme functions as a homodimer. Highest activity was measured at 70 ◦ C and pH 6.0. The Km on the substrates ONPG and lactose were, respectively, 3.8 and 425.3 mM. This enzyme is thermostable, retaining 76, 50 and 42% relative activity after 30, 60 and 120 min, respectively, at 70 ◦ C. This property could lend its use to high-temperature industrial processes requiring a thermo-active ␤-galactosidase, including the production of lactulose and in the manufacture of lactose-free milk and dairy products. To date, there have been no reports in the literature on the characterisation of a glycosyl hydrolase from A. vulcanalis DSM 16176. Funded by IRCSET EMBARK Initiative. Key-words., ␤-galactosidase, thermoacidophile, protein purification. References [1].Niehaus F, Bertaldo C, Kahler M, Antranikian G. Applied Microbiology and Biotechnology 1999;51(6):711–29. [2].Turner P, Mamo G, Karlsson EN. Microbial Cell Factories 2007;6:9. http://dx.doi.org/10.1016/j.nbt.2014.05.1792 PA-07 Regioselective hydroxylation of aromatic carboxylic acids by cytochrome P450 CYP199A2 and its mutants Toshiki Furuya ∗ , Kuniki Kino Waseda University Hydroxy-aromatic carboxylic acids are practically or potentially important industrial chemicals since the functional groups play key roles in biological activities and physical properties and also can be utilized for further modification of the chemicals. Cytochrome P450 monooxygenases are promising catalysts for use in the hydroxylation of chemicals. We found that CYP199A2 from Rhodopseudomonas palustris has catalytic activity for the hydroxylation of 2-naphthoic acid to 7- and 8-hydroxy-2S80 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 naphthoic acids. [1]. Furthermore, CYP199A2 was able to catalyze the regioselective hydroxylation of hydroxy-2-naphthoic acids, indolecarboxylic acids, and a quinolinecarboxylic acid [2,3]. These results indicate that CYP199A2 should be an efficient biocatalyst for the hydroxylation of various aromatic carboxylic acids [4]. The crystal structure of CYP199A2 determined by Bell et al. (J. Mol. Biol., 383, 561 (2008)) shows that Phe at the 185 position is situated directly above the heme iron. When this Phe185 residue was replaced with hydrophobic or hydroxylated amino acids, several mutants predominantly produced 5-hydroxy-2-naphthoic acid from 2-naphthoic acid. Interestingly, these Phe185 mutants also exhibited novel substrate specificities. In particular, the Phe185Leu mutant exhibited high hydroxylation activity for cinnamic and p-coumaric acids [5]. The Phe185Leu whole-cell catalyst achieved gram-per-liter-scale production of caffeic acid from pcoumaric acid. References [1].Furuya T, Kino K. ChemSusChem 2009;2:645–9. [2].Furuya T, Kino K. Biosci Biotechnol Biochem 2009;73:2796–9. [3].Furuya T, Kino K. Appl Microbiol Biotechnol 2010;85:1861–8. [4].Furuya T, Kino K. Appl Microbiol Biotechnol 2010;86:991–1002. Review. [5].Furuya T, et al. Appl Environ Microbiol 2012;78:6087–94. http://dx.doi.org/10.1016/j.nbt.2014.05.1793 PA-08 New biotransformation process for production of ␥lactones from hydroxy fatty acids by permeabilized Waltomyces lipofer cells induced with oleic acid Jung ung An ∗ , Deok kun Oh Konkuk university The production of the flavor lactone was developed by using permeabilized Waltomyces lipofer, which was selected as an efficient ␥-lactones producing yeast among 10 oleaginous yeast strains. The cells were permeabilized using 50% ethanol and 0.5% Triton X-100, sequentially. Among several fatty acids tested, oleic acid was selected as the most efficient inducer for the production of ␥-lactones. The cells were induced by incubation for 12 h in a medium containing 10 g l−1 yeast extract, 10 g l−1 peptone, 5 g l−1 oleic acid, 1 g l−1 glucose, and 0.05% (w/v) Tween 80. The optimal reaction conditions for ␥-lactones production by whole Waltomyces lipofer were pH 6.5, 35 ◦ C, 200 rpm, 0.71 M Tris, 60 g l−1 hydroxy fatty acid, and 20 g l−1 cells. Under these conditions, non-treated cells produced 30 g l−1 ␥-dodecalactone from 60 g l−1 10-hydroxystearic acid after 30 h, with a conversion yield of 63% (w/w) and a productivity of 1.3 g l−1 h−1 , whereas treated cells produced 51 g l−1 ␥-dodecalactone from 60 g l−1 10-hydroxystearic acid after 30 h, with a conversion yield of 85% (w/w) and a productivity of 1.7 g l−1 h−1 . The conversion yield and productivity of treated cells were 22% and 1.3-fold higher, respectively, than those of non-treated cells. The treated cells also produced 28 g l−1 ␥-decalactone and 12 g l−1 ␥-butyrolactone. These are the highsest BIOCATALYSIS New Biotechnology · Volume 31S · July 2014 reported concentration, conversion yield, and productivity for the production of bioflavor lactone. http://dx.doi.org/10.1016/j.nbt.2014.05.1794 plemented with 4.5 U ml−1 SA-bglu. Under optimum conditions, major protopanaxadiol ginsenosides in ginseng root extract were completely converted to compound K after 20 h with the respective productivities of 144 mg l−1 h−1 . http://dx.doi.org/10.1016/j.nbt.2014.05.1796 PA-09 Bioconversion of linoleic acid to 5,8-dihydroxy9,12(Z,Z)-octadecadienoic acid by 5,8-diol synthase from Aspergillus nidulans Seo Min-Ju ∗ , Shin Kyung-Chul, Oh Deok-Kun Konkuk university PA-11 Structural conformation of enzyme in ionic liquids: molecular dynamic simulation study Yoon-Mo Koo Inha University The fungus diol synthase from Aspergillus nidulans was cloned and expressed in Escherichia coli. Recombinant E. coli cells converted linoleic acid to 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid, which was identified by LC-MS/MS. The recombinant cells and the purified enzyme showed activity for linoleic acid. The reaction using purified diol synthase stoppedafter 15 min, whereas the reaction using recombinant cells expressing diol synthase from A. nidulans continued over 60 min, indicating that recombinant cells expressing diol synthase were more stable than the purified enzyme. The optimal reaction conditions for the production of linoleic acid to 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid using whole recombinant E. coli cells were pH 7.5, 35 ◦ C, 250 rpm, 23 g l−1 cells, 5 g l−1 linoleic acid, and 20% (v/v) dimethyl sulfoxide in a 250 ml-baffled flask. Under these optimized conditions, whole recombinant cells produced 4.98 g l−1 5,8-dihydroxy-9,12(Z,Z)octadecadienoic acid for 150 min, with a conversion yield of 99% (w/w) and a productivity of 2.5 g l−1 h−1 . This is the biotechnological production of dihydroxy fatty acid using whole recombinant cells expressing diol synthase. Enzymatic reactions in ionic liquids have been gaining increasing interests during the last decade due to their unique properties. It is well documented that, in some cases, enzymes showed enhanced activity, selectivity and stability in ionic liquids. However, there were very few studies investigating the structure of enzymes in ionic liquids. In this study, the conformational structure changes of Candida antarctica lipase B (CALB) in different imidazolium-based ionic liquids observed by molecular dynamic simulation are discussed. The results showed that two isoleucines, ILE-189 and ILE-285, in CALB played critical role in the openclosed conformations of the catalytic cavity. The ILE-285 situated on ␣-10 helix region (residues 268-287) where its conformation can be significantly changed in different solvents. For example, in 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) conformation of ␣-10 helix changed to a turn as a result of direct interactions with chlorine anions gave rise to a closed conformation of the catalytic cavity, corresponding to lower enzyme activity. http://dx.doi.org/10.1016/j.nbt.2014.05.1797 http://dx.doi.org/10.1016/j.nbt.2014.05.1795 PA-12 PA-10 Production of compond K from major protopanaxadiol ginsenosides by combined enzymes, including ␣-Larabinofuranosidase and ␤–galactosidase from Caldicellulosiruptor saccharolyticus and ␤–glucosidase from Sulfolobus acidocaldarius Kyung-Chul Shin ∗ , Hye-Jin Oh, Baek-Joong Kim, Deok-Kun Oh Konkuk university The ginsenoside compound K has diverse pharmaceutical activities such as anti-tumor, anti-inflammatory, anti-allergic, and hepatoprotective effects. To increase the production of compound K from major protopanaxadiol ginsenosides in ginseng root extract, ␣-l-arabinofuranosidase (CS-abf) and ␤–galactosidase (CSbgal) from Caldicellulosiruptor saccharolyticus were mixed with ␤–glucosidase (SA-bglu) from Sulfolobus acidocaldarius. The optimum conditions for the production of ginsenoside compound K from major protopanaxadiol ginsenosides in ginseng root extract were determined to be pH 6.0 and 75 ◦ C with 10% (w/v) ginseng root extract and 10.5 U ml−1 CS-abf and 10.5 U ml−1 CS-bgal sup- Conjugation of chitooligosaccharides and glucosamine to bovine trypsin. Effects on stability and functionality Hörður Filippusson ∗ , Jóhann G.K. Gizurarson University of Iceland The improvement of protein stability is important in relation to the use of enzymes as practical biocatalysts and in the use of proteins as pharmaceuticals. All proteins are unstable, especially when in solution. Among the processes that can affect protein stability are proteolytic degradation in vitro or in vivo, thermal denaturation, and antigenicity after injection. Attempts to improve protein stability include protein engineering, immobilization, chemical modification and the use of cosolutes. In this study bovine trypsin was modified by coupling the enzyme to either D-glucosamine or a partially acetylated chitoologosaccharide via binary carbodiimide/succinimide ester conjugation. D-glucosamine was found to conjugate, on average, to 12 residues on trypsin. The oligosaccharide coupling produced cross-linked polydisperse complexes with hydrodynamical radii www.elsevier.com/locate/nbt S81 BIOCATALYSIS from, on average, from 218 to 330 nm for 1:5 and 1:10 cross-linked trypsin, repectively. The physical properties of the enzyme species were studied by electrophoresis, gel filtration, circular dichroism, nanoprticle tracking analysis and MALDI-TOF mass spectrometry. The stability of these enzyme species was studied by activity assays, urea denaturation, diffferential scanning calorimetry and autolysis. The modified trypsin showed increased resistance against thermal inactivation and autolysis, better storage stability but stability against urea inactivation was unchanged. The proteolytic activity against azocasein improved for the cross-linked trypsins but was slightly reduced for D-glucosamine conjugated trypsin. The species were found to become basophilic upon conjugation and cross-linking. D-glucosamine conjugated trypsin was found to be slightly structurally altered which and as consequence displayed 1.2 times higher catalytic efficiency (kcat /Km ) than native trypsin against the substrate L-BAPNA. http://dx.doi.org/10.1016/j.nbt.2014.05.1798 PA-13 1,8-cineole-hydroxylating cytochrome P450s from Sphingobium yanoikuyae Birgit Unterweger 1,∗ , David J. Midgley 2 , Paul Greenfield 3 , Dieter M. Bulach 4 , Dena Lyras 5 , Priscilla Johanesen 5 , Geoffrey J. Dumsday 6 1 Department of Microbiology, Monash University, Clayton, VIC 3800, Australia and CSIRO Future Manufacturing Flagship, Clayton, VIC 3168, Australia 2 CSIRO Animal, Food and Health Sciences, North Ryde, NSW 1670, Australia 3 CSIRO Computational Informatics, North Ryde, NSW 1670, Australia 4 Victorian Bioinformatics Consortium, Monash University, Clayton, VIC 3800, Australia 5 Department of Microbiology, Monash University, Clayton, VIC 3800, Australia 6 CSIRO Materials Science and Engineering, Clayton, VIC 3168, Australia In Australia, extensive Eucalyptus plantations have been established to prevent dry land salinity and novel applications for this renewable resource are being sought. One approach is to add value to the leaf oil from these plantations via the oxyfunctionalisation of its chemically unreactive component 1,8-cineole. Hydroxyl-groups create starting points for further modification and/or incorporation into more complex molecules such as polymers. Biooxidation of 1,8-cineole promises a more sustainable route to hydroxylated intermediates with greater stereospecificity compared to conventional chemistry. The availability of biocatalysts known to catalyse the oxidation of 1,8-cineole, however, is limited. To expand the range of biocatalysts, several microbes capable of hydroxylating 1,8-cineole were isolated including a Sphingobium yanoikuyae strain. Genome sequencing revealed the presence of multiple genes encoding P450s and potential electron transport partners. To confirm the identity of genes encoding 1,8-cineole-hydroxylating P450s, proteins exhibiting the characteristic spectroscopic shift upon addition of 1,8-cineole were purified from S. yanoikuyae cultivated in the presence of 1,8cineole. In addition to this the genes were cloned and the gene products heterologously expressed in Escherichia coli and purified from clarified cell lysates. Spectroscopic characterisation and whole-cell biotransformation have demonstrated that the proteins S82 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 can hydroxylate 1,8-cineole. Compared to the well characterised P450cin from Citrobacter braakii, the S. yanoikuyae enzymes have limited amino acid sequence identity, appear to have differing stereo- and regioselectivity and based on testing completed so far the preferred substrate is 1,8-cineole. Current investigations involve mining the S. yanoikuyae genome for the natural electron transport partners to optimise the hydroxylation process. http://dx.doi.org/10.1016/j.nbt.2014.05.1799 PA-14 Human flavin monooxygenase 2: Heterologous expression in E. coli and API modification Margit Winkler 1,∗,1 , Thorsten Bachler 2,2 , Martina Geier 2,2 , Steven P. Hanlon 3,3 , Matthias Kittelmann 4 , Anton Glieder 2,2 , Stephan Lütz 4 , Beat Wirz 3,3 1 ACIB GmbH c/o Institute of Molecular Biotechnology, Graz University of Technology 2 ACIB GmbH 3 F. Hoffmann-La Roche Ltd 4 Novartis Pharma AG Abstract The flavin monooxygenase (FMO) isoenzyme 2 is known as the “pulmonary” FMO because it is expressed predominantly in the human lung. FMO2*1 is the active FMO2 allele found in Africans and Hispanics [1]. The enzyme is membrane associated and oxidizes xenobiotics with soft nucleophiles such as sulfur and nitrogen at the expense of NADPH and oxygen. The aim of the current study was to generate simple and efficient whole cell biocatalysts for the preparation of FMO2 drug metabolites on the multi-milligram scale. Recently, we demonstrated the possibility to express human FMOs in E. coli, however, the expression level of the FMO2*1 isoform was very low in comparison to FMO3 and FMO5 [2]. This prompted us to investigate truncated variants of FMO2*1 and to compare their performance in whole cell biotransformations of different active pharmaceutical ingredients such as e.g. ethionamide–an antitubercular drug [3] Key-words: Biocatalysis, flavin monooxygenase, drug metabolites, oxidation, membrane protein. References [1].(a) Dolphin C, et al. J Biol Chem 1998;273(46):30599–607; (b) Krüger SK, et al. Drug Metabol Disposit 2004;32(12):1337–40. [2].Hanlon SP, et al. Chem Commun 2012;48(48):6001. [3].Francois AA, et al. Drug Metabol Disposit 2009;37(1):178–86. http://dx.doi.org/10.1016/j.nbt.2014.05.1800 1 2 3 ACIB GmbH, Petersgasse 14/III, 8010 Graz, Austria F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland Novartis Pharma AG, 4070 Basel, Switzerland BIOCATALYSIS New Biotechnology · Volume 31S · July 2014 PA-15 Co-factor regeneration at the cell surface–upgrading the biotechnological potential of whole cell biocatalysts Jan Schüürmann ∗ , Joachim Jose Westfälische Wilhelms Universität Münster Whole cell biocatalysts offer significant advantages compared to purified enzymes, specifically cheap and efficient production combined with obsolete purification. However, there are limitations to the applicability of whole cells. Cross reactions with endogenous enzymes and membrane impermeability of substrates and products might complicate their use. Autodisplay presents enzymes on the surface of E. coli cells eliminating mass transfer problems and possible side reactions. The technique replaces the passenger domain of a native autotransporter protein by a peptide or protein of choice. However, a major challenge for some enzymes to be used in biotechnological applications is the regeneration of co-factors, which are too expensive to be added stoichiometrically. Here, we report the development of a cell surface based NADPH regeneration system. To circumvent laborious protein purification and keep the advantages of surface displayed catalysts we used the Autodisplay technology to present various dehydrogenases on the cell surface of E. coli. Surface display was confirmed by protease accessibility tests and FACS analysis. NADPH production was measured photometrically at 340 nm. We first concentrated our efforts on an engineered formate dehydrogenase. The enzyme showed similar specific activity compared to the soluble enzyme, but the efficiency of the system was limited by the number of enzymes on the bacterial surface and high amounts of whole-cell catalysts were needed for effective production of NADPH. Thus, we are currently exploring alternative enzymes with higher specific activities for surface display and cell associated co-factor regeneration. http://dx.doi.org/10.1016/j.nbt.2014.05.1801 PA-16 Surface display of enzymes on Zymomonas mobilis and Zymobacter palmae using the autotransporter secretion pathway Iasson E.P. Tozakidis 1,∗ , Annika Meyers 1 , Tatjana Brossette 2 , Joachim Jose 1 1 Institute of Pharmaceutical and Medicinal Chemistry, Westfälische WilhelmsUniversität Münster 2 Autodisplay Biotech GmbH from a reaction mixture is often difficult. The gram-negative bacteria Zymomonas mobilis and Zymobacter palmae are promising host organisms for industrial applications due to their high ethanol productivity and resistance towards rough reaction conditions. Until now, no functioning surface display system for these organisms has been reported. For this reason, we tested if the autotransporter secretion pathway can be utilized in Z. mobilis and Z. palmae. Here we present the surface display of two industrially relevant enzymes, namely Burkholderia gladioli esterase EstA and Bacillus subtilis endoglucanase Cel5A, and demonstrate that the enzymes retain their activity on the cell surface of both organisms. This work represents the first step towards using Z. mobilis and Z. palmae as expression platforms for surface display applications. http://dx.doi.org/10.1016/j.nbt.2014.05.1802 PA-17 Combined Surface Display of Cytochrome P450 1A2 and its Reductase on Escherichia coli Paul Quehl ∗ , Joachim Jose University of Münster Human cytochrome P450 monooxygenases (CYPs) play a prominent role in drug metabolism as these enzymes are involved in the breakdown of literally every drug. They catalyze a variety of oxidations of a broad range of substrates and have a major impact on bioavailability and drug-drug interactions. CYPs obtain the electrons from the NADPH-dependent cytochrome P450 reductase (CPR). However, recombinant expression of both enzymes in bacteria is often not feasible and they exhibit poor stability after purification. Moreover, the membrane associated proteins CPR and CYPs require membrane surroundings for activity. CYPs alone can be displayed functionally active on the surface of Escherichia coli with externally added CPR. Here, we tested the co-expression of CYP1A2 and its NADPH dependent Cytochrome P450 reductase (CPR) on the E.coli outer membrane. Surface display is facilitated by usage of the autotransporter secretion pathway for which the enzyme of interest is combined with an N-terminal signal peptide, a C-terminal linker and a beta-barrel domain. Surface presentation was confirmed by FACS analysis and protease accessibility tests. Both CYP1A2 and CPR bind their cofactors and the surface displayed NADPH-dependent cytochrome P450 reductase is able to react with Cytochrome C. The functional interaction of CYP1A2 with the CPR is currently under investigation by testing the enzymatic activity towards the substrates 7-ethoxyresorufin and phenacetin. http://dx.doi.org/10.1016/j.nbt.2014.05.1803 Displaying enzymes on a microbial cell surface has the potential to significantly reduce the costs of large scale biocatalytic conversion processes. In contrast to intracellularly expressed enzymes, surface displayed enzymes do not have to be purified prior to their use. Instead, the host cells can directly be employed in a reaction, and neither substrates nor products need to cross a membrane barrier. Additionally, whole cells can easily be harvested and used in multiple process cycles, whereas the recovery of free enzymes www.elsevier.com/locate/nbt S83 BIOCATALYSIS New Biotechnology · Volume 31S · July 2014 PA-18 PA-19 Biochemical and Structural Characterization of a Thermophilc L-Arabinose Isomerase from Geobacillus kaustophilus Functional Characterization of Putative UDP-Glucose 4-Epimerase (TM0509) from the Hyperthermophilic Eubacterium Thermotoga maritima Dong-Woo Lee 1,∗ , Yong-Jik Lee 1 , Jin Myung Choi 2 , Sun-Mi Shin 1 , Sang-Jae Lee 3 , Han-Seung Lee 3 , Sang Jun Lee 4 , Sung Haeng Lee 2 Sun-Mi Shin 1,∗ , Jin Myung Choi 2 , Yong-Jik Lee 1 , Sang-Jae Lee 3 , Sang Jun Lee 4 , Sung Haeng Lee 2 , Dong-Woo Lee 5 1 1 2 2 Kyungpook National University Chosun University 3 Silla University 4 KRIBB Thermophilic L-Arabinose isomerase (AI), that catalyzes the interconversion of L-arabinose to L-ribulose, can also isomerize D-galactose to D-tagatose as a natural sugar substitute, which is of commercial interest in the food and healthcare industries.Recently, biochemical and mutational studies revealed that unlike mesophilic AIs, thermophilic AIs showed the distinct metal dependence for their catalytic activity and thermostability at elevated temperatures. However, it still remains unclear how mesophilic and thermophilic AIs showed different substrate preferences and metal requirements at molecular levels. Herein we characterized a thermophilic AI from Geobacillus kaustophilus (GKAI) and presented the first crystal structures of the apo and holo forms of GKAI by X-ray crystallography to 2.40 and 2.30 Å, respectively. We also determined the crystal structure of holo enzyme bound to L-arabitol as a substrate analog at 2.25 Å resolution.In combination with biochemical and site-directed mutagenesis studies, the structures identified the structural elements of metal binding and substrate recognition.In comparison with the crystal structures of Escherichia coli AI (ECAI) as a mesophilic counterpart, the GKAI structures revealed quite conserved structural features for substrate and metal binding, except forsubtle interactions of a few polar residues with water molecules near the substrate binding region.Our comparative analysis proposes a metal-mediated substrate binding model for the isomerization reaction at elevated temperatures, providing a versatile strategy to engineer the promiscuity of substrate specificity for sugar isomerases as well as thermostability for mechanistic studies and industrial applications. Kyungpook National University Chosun University 3 Silla University 4 Korea Research Institute of Bioscience and Biotechnology 5 Kyungpook National Univ UDP-glucose 4-epimerase (GalE; EC 5.1.3.2) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal), which is a pivotal step in the Leloir pathway for galactose metabolism. Although GalEs are widely distributed in Bacteria and Eukaryotes, there is little information on hyperthermophilic GalE. Herein we cloned and overexpressed the TM0509 gene encoding a putative GalE from Thermotoga maritima (TMGalE) as a fusion protein containing an N-terminal hexa-histidine sequence in Escherichia coli. This gene encodes a 309-amino acid protein with a calculated molecular weight of 34899 and a theoretical pI of 5.72.The recombinant protein was purified to homogeneity by heat precipitation, Ni2+ affinity chromatography followed by size-exclusion chromatography. The native enzyme was estimated to be a homodimer with a molecular mass of 70 kDa. The recombinant TMGalE could reversibly catalyze the epimerization of UDP-Gal and UDP-Glc in the presence of NAD+ at elevated temperatures. The apparent optimal temperature and pH for epimerization activity were 85 ◦ C and pH 7.0, respectively. In order to further characterize TMGalE at molecular levels, we determined not only the crystal structure of TMGalE at 1.9 Å resolution, but also the co-crystal structure of TMGalE bound to UDP-glucose at 2.0 Å resolution. These biochemical and structural data showed that TM0509 is an UDP-galactose 4-epimerase involved in galactose metabolism, which is the first detailed characterization of a thermostable GalE from hyperthermophilic bacterium. http://dx.doi.org/10.1016/j.nbt.2014.05.1805 http://dx.doi.org/10.1016/j.nbt.2014.05.1804 PA-20 Microscale tools for evaluating the biological and process options of alkane biooxidations Johannes Kolmar 1,∗ , Frank Baganz 1 , Philip Engel 2 1 2 University College London Evonik Industries AG The direct ␻-oxyfunctionalisation of aliphatic alkanes in a regio- and chemoselective manner remains difficult to perform by industrial organic chemistry. Monooxygenases such as the AlkB enzyme complex from Pseudomonas putida efficiently catalyse these readily available substrates to primary fatty alcohols and acids under mild conditions. These are of considerable interest as potential intermediates in the chemical, pharmaceutical and cosmetics industry. S84 www.elsevier.com/locate/nbt BIOCATALYSIS New Biotechnology · Volume 31S · July 2014 The ability to rapidly screen biotransformation reactions for characterisation and optimisation is of major importance in process development and biocatalyst selection. Studies at lab scale are time consuming and labour intensive with low experimental throughput. The feasibility of a parallel microwell platform for two-liquid phase whole-cell bioconversions has previously been shown for longer chain alkane substrates [1]. However, excessive evaporation has limited the use of the microscale approach for volatile substrates. This study developed a microwell system for use with highly volatile n-alkane substrates. Particular attention was paid to material compatibility, evaporation and oxygen transfer. It was shown that the use of 24 deep square microwell plates machined from a fluoropolymer alleviates problems of organic phase permeation and absorption. In combination with a new sealing approach to avoid substrate evaporation, the reproducibility of results was increased. However, a comparison with unsealed plates revealed that, despite similar cell growth the bioconversion productivity was lower in sealed plates. The effect of sealing the plates on oxygen transfer will be discussed. Reference [1].Grant C, Pinto A, Lui H, Woodley J, Baganz F. Biotechnol Bioeng 2012;109(9):2179–89. http://dx.doi.org/10.1016/j.nbt.2014.05.1806 PA-21 Chemical modification of lipase B from Candida antarctica for improving biochemical properties of activity, stability and selectivity Rodrigo Torres Sáez 1,∗ , Claudia Ortiz López 2 , Oveimar Barbosa 3 , Roberto Fernández-Lafuente 4 1 School of Chemistry. Universidad Industrial de Santander School of Bacteriology and Clinical Laboratory. Universidad Industrial de Santander, Colombia 3 School of Chemistry. Universidad Industrial de Santander, Colombia 4 Department of Biocatalysis. ICP-CSIC, Spain 2 Chemical modification of enzymes can be used to modulate enzyme properties by means of modification of protein surface or key residues from the enzyme structure [1,2]. In this work, it was carried out chemical modifications of Candida antarctica lipase B (CALB) preparations immobilized on octyl-agarose, BrCN-agarose and Eupergit-C supports using different chemical compounds, e.g. ethylenediamine (EDA), succinic anhydride (SA) and 2,4,6trinitrobenzensulfonic acid (TNBS). These modifications of the enzyme surface caused changes in physical properties such as charge (isoelectric point) or hydrophobicity (solubility), and proved to be practical methods to enhance the biocatalyst performance (stability, activity and enantio-selectivity) when the enzyme preparations were submitted to different ranges of pH (4-9) and temperature (25-70 ◦ C) and organic co-solvents such as acetonitrile or tetrahydrofuran at 50% (v/v). These immobilized and chemically modified CALB preparations displayed high enantioselectivity during the kinetic resolution of (R/S)-methyl mandelate in aqueous solution and esterification of beta-blocker drugs such as atenolol and propranolol. These alterations in enzyme properties by chemical modification should be due to changes in the structure of the active form of CALB. Therefore, solid phase chemical modification of immobilized lipases may become a powerful tool in the design of lipase libraries with very different properties. References [1].Rodrigues RC, Ortiz C, Berenguer-Murcia A, Torres R, FernándezLafuente R. Modifying enzyme activity and selectivity by immobilization. RSC 2013;42:6290–307. [2].Davis BG. Chemical modification of biocatalysts. Curr Opin Biotechnol 2003;14:379–86. http://dx.doi.org/10.1016/j.nbt.2014.05.1807 PA-22 Newly engineered diol dehydrogenase from Clostridium butyricum as promising agent in cell-free biosystems for biomanufacturing Marta Jankowska ∗ , Włodzimierz Schmidt, Marcin Schmidt Grajek, Agnieszka Olejnik- Poznań Uniwersity of Life Sciences Economical and environmentally friendly production of biochemicals and biotechnology-based polymers is a critical goal of modern biotechnology. The use of enzymes as catalysts for chemical transformations has emerged as more ecological synthesis. Generally, naturally occurring enzymes do not have appropriate features necessary for their use in chemical industry. The development in protein engineering allows optimization of particular enzyme traits which make them more suitable to chemical process. 1,3-Propanediol is a key chemical bulk for the synthesis of polytrimethylene terephthalate with desired properties for large volume markets. 1,3-Propanediol dehydrogenase (PDOR, EC 1.1.1.202) encoded in Clostridium butyricum by dhaT gene is involved in the conversion of glycerol into 1,3-propanediol. The study aimed to improve enzyme activity by directed evolution and rational design methods to generate a more efficient 1,3-propanediol production. Error-prone PCR, employed to re-engineer PDOR resulted in enzyme variants with higher enzymatic activity. Variants with highest activity showed twice higher oxidative activity and twelve times higher reductive activity, respectively. Subsequently threedimensional structure of PDOR was predicted by homology modeling with Lactaldehyde reductase from Escherichia coli as template. Location of the mutations, important for activity and their potential impact on the structure and function of the enzyme were specified. Results of study indicate mutations that might influence the effectiveness of cofactor binding to protein surface as well as on the enzymatic activity of PDOR. This work was part of POIG 01.01.02-00-074/09 project cofunded by The European Union through The European Regional Development Fund within the framework of the Innovative Economy Operational Programme 2007-2013. http://dx.doi.org/10.1016/j.nbt.2014.05.1808 www.elsevier.com/locate/nbt S85 BIOCATALYSIS PA-23 Sequence and function relationship of Escherichia coli flavin mononucleotide binding fluorescent protein Byung-Kwan Cho ∗ , Hyeonseok Shin Korea Advanced Institute of Science and Technology Flavin mononucleotide (FMN)-binding fluorescent proteins can provide in vivo reporter system, without oxygen. Here, we present the functional landscape of individual amino acid sequence of Escherichia coli FMN-binding fluorescent protein (EcFbFP). We used random mutagenesis to generate the mutant libraries, which were screened by function loss or retained. The function and sequence relationship of the mutants were analyzed in single high-throughput sequencing, resulting in 329 tolerant mutations and 259 sensitive mutations that show retained fluorescence or loss of fluorescence respectively. In addition, the enrichment of tolerant or sensitive mutations in each amino acid residues were weighed to find functionally important residues n EcFbFP. The mutation enrichment analysis show that the positions critical to the function of EcFbFP lies among the FMN binding pocket, turns and loops of the protein where dynamic conformational changes occur and the Glu56-Lys97 salt bridge which is critical to structural stability of EcFbFP. Collectively, the mutational scanning results provide a functional landscape of each amino acid of EcFbFP. This work was supported by Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031957, 2011-0031962). http://dx.doi.org/10.1016/j.nbt.2014.05.1809 PA-24 Analysis and Optimisation of the Physiology of Engineered Biofilms for Biotransformations James Thomas Leech 1,∗ , Isaac Vizcaino-Caston 1 , Tania Barberi 2 , Rebecca Goss 2 , Mark Simmons 1 , Tim Overton 1 1 2 University of Birmingham University of St Andrews Engineered biofilms formed from reaction-competent recombinant Escherichia coli provide a useful platform for biotransformation reactions [1,2]. Biofilms form in response to chemical and physical stresses such as organic solvents and shear, and thus are able to resist conditions encountered in flow reactor biotransformation reactions far better than planktonic cultures [3]. Improving attachment and maturation of engineered biofilms is paramount to the optimisation of their use in biotransformations. Through the use of fluorescent reporter genes, flow cytometry and confocal laser scanning microscopy, the physiology and composition of the biofilm has been studied, in addition to the spatial and temporal expression of biofilm constituents such as curli, poly-N-acetlyglucosamine (PGA/PNAG) and colanic acid. Molecular biology techniques have been used to bypass normal biofilm signals, creating increased biofilm formation. Furthermore, the physiological effects of biotransformation reactions on the biofilm S86 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 are being studied with an aim to optimise biotransformation processes, and to observe the effect of increased biofilm production on biotransformations. References [1].Tsoligkas AN, Winn M, Bowen J, Overton TW, Simmons MJH, Goss RJM. Engineering Biofilms for Biocatalysis. ChemBioChem 2011;12:1391–5. [2].Tsoligkas AN, Bowen J, Winn M, Goss RJM, Overton TW, Simmons MJH. Characterisation of spin coated engineered Escherichia coli biofilms using atomic force microscopy. Colloids and Surfaces B: Biointerfaces 2012;89:152–60. [3].Perni S, Hackett L, Goss R, Simmons M, Overton T. Optimisation of engineered Escherichia coli biofilms for enzymatic biosynthesis of l-halotryptophans. AMB Express 2013;3:66. http://dx.doi.org/10.1016/j.nbt.2014.05.1810 PA-25 Library sequencing strategies for comparative analysis of stress resistance mechanisms in Escherichia coli strains Rebecca Lennen ∗ , Ida Bonde, Anna Koza, Markus Herrgård Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark Transposon insertion sequencing (Tn-Seq) has recently emerged as a powerful next-generation sequencing method that enables querying the contributions of all genes in a bacterial genome toward the fitness of a growing organism. In this method, transposon insertion mutant libraries are constructed and subjected to growth selections. Following selection, the locations of all insertions in the population are counted and can be compared between a control and a target condition, enabling the identification of genes that are both conditionally essential and conditionally detrimental. We have exploited Tn-Seq to probe the basis for the large variations in osmotic and acetate stress tolerance of different laboratory strains of Escherichia coli (K-12 MG1655, BL21(DE3), W, and Crooks). Little is currently known to explain the source of this variation and to enable rational engineering to impart stress tolerance. Tn-Seq revealed many differences and similarities in resistance mechanisms at the genetic level across strains, allowing correlations to be made with growth phenotypes. Cross-strain comparisons of conditionally essential genes and their relative essentiality also suggest a large degree of variation in metabolic flux distributions and regulation of gene expression between strains. A number of direct targets for metabolic engineering of stress resistance via loss-of-function mutations were also discovered, and we show that deletion of a selection of these genes results in improved growth under the original selection condition. http://dx.doi.org/10.1016/j.nbt.2014.05.1811 BIOCATALYSIS New Biotechnology · Volume 31S · July 2014 PA-26 Microorganisms respond in different ways to oscillations in large-scale bioreactors: Conclusions from scale-down approaches Peter Neubauer 1,∗ , Anja Lemoine 2 , Sergej Trippel 2 , Eva Brand 2 , Robert Spann 2 , Dennis Runge 2 , Ping Lu 2 , Basant El Kady 2 , Christian Reitz 2 , Stefan Junne 2 1 2 TU Berlin, Chair of Bioprocess Engineering TU Berlin Gradients of various growth related parameters are observed in large scale bioreactors by the limited mass transfer and are enhanced by the application of the substrate limited fed-batch technology. Consequently, cells are exposed to oscillations, which cause a specific physiological adaptation. Our studies with different scale-down approaches indicate that microorganisms adopt with different strategies to oscillations, which is closely connected to their response to oxygen limitation. While Escherichia coli, similar to Saccharomyces cerevisiae reacts with increased glycolytic fluxes, Bacillus subtilisdecreases the maximum glucose uptake capacity. In contrast, Corynebacterium glutamicum shows a high robustness to oscillations. In all cases, the oscillations influenced the pools of various amino acids, which are closely connected to the central carbon metabolism. When we connected the two-compartment scaledown bioreactor with the rapid sampling unit Bioscope to use 13 C-labelling for a study of the metabolic fluxes in E. coli, we observed that the preferred route towards the synthesis of branchchain amino acids after a pulse depends on the history of growth conditions. The presented methodology, which also includes other novel analytical instruments, provides a bridge between systems biology for the investigation of the cellular regulatory networks under industrially relevant conditions. http://dx.doi.org/10.1016/j.nbt.2014.05.1812 PA-27 Exploring the new threonine aldolases with broad donor specificity Kateryna Lypetska (Fesko) ∗ , Gernot Strohmeier, Rolf Breinbauer Graz University of Technology Threonine aldolases have great biotechnological potential, as they catalyze the formation of unnatural amino acids with high enantioselectivity. The enzymes have been efficiently applied for the aldol condensation of an aldehyde and glycine to produce Land D-␤-hydroxy-␣-amino acids.[1] Aldolases are tolerant towards acceptor aldehyde, but they are quite rigid for the amino acid donor. In our previous work we have isolated two natural threonine aldolases, which were able to accept alanine and serine as donor.[2] Here we present the identification and characterization of a range of L- and D-threonine aldolases with broad donor specificity. The kinetic properties and the substrate specificity of new enzymes were investigated and the biocatalytic method for the stereoselective synthesis of a-quaternary a-amino acids was developed. Acknowledgements: The research leading to these results has received funding from the Innovative Medicines Initiative Joint Undertaking under grant agreement n◦ 115360, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution. References [1].(a) Steinreiber J, Fesko K, Reisinger C, Schurmann M, van Assema F, Wolberg M, Mink D, Griengl H. Tetrahedron 2007;63:918–26; (b) Steinreiber J, Fesko K, Mayer C, Reisinger C, Schürmann M, Griengl H. Tetrahedron 2007;63:8088–93. [2].Fesko K, Uhl M, Steinreiber J, Gruber K, Griengl H. Angew Chem 2010;122:125–8. Angew. Chem. Int. Ed. 2010, 49, 121–124. http://dx.doi.org/10.1016/j.nbt.2014.05.1813 PA-28 The effect of salt-preconditioning Torulaspora delbrueckii cells on fermentation performance Stilianos Logothetis 1,∗ , Fotini Drosou 1 , Arhodoula Hatzilazarou 1 , Panagiotis Tataridis 1 , Anastasios Kannelis 2 , Elias Nerantzis 1 , Graeme Walker 2 1 2 TEI of Athens Department of Enology and Spirit Technology Abertay University of Dundee Abstract This paper concerns research into the influence of salt on physiology of the yeast, Torulaspora delbrouekii. Specifically, the work focused on how NaCl affected the growth, viability and fermentation performance of this yeast in laboratory-scale experiments. One of the main findings of the research presented involved the influence of salt “preconditioning” of yeasts which represents a method of pre-culturing cells in the presence of salt in an attempt to improve subsequent fermentation performance. Such an approach resulted in preconditioned T. delbruekii yeasts having an improved capability to ferment high-sugar containing media (up to 30% w/v of glucose) with increased cell viability and with elevated levels of produced ethanol. Salt-preconditioning most likely influenced the stress-tolerance of yeasts by inducing the synthesis of key metabolites such as trehalose and glycerol which act to improve cells’ ability to withstand osmostress and ethanol toxicity. Overall, this research has demonstrated that a relatively simple method designed to physiologically adapt yeast cells–by salt-preconditioning–can have distinct advantages for alcohol fermentation processes. http://dx.doi.org/10.1016/j.nbt.2014.05.1814 www.elsevier.com/locate/nbt S87 BIOCATALYSIS PA-29 Protein engineering of arylmalonate decarboxylase variants with promiscuous racemising activity Robert Kourist ∗ , Sarah Gaßmeyer, Nadine Hülsemann, Robin Dorau Ruhr-Universität Bochum Enzymatic racemization allows the smooth interconversion of stereocenters under very mild reaction conditions. Racemases find frequent applications in deracemization and dynamic kinetic resolutions [1]. Arylmalonate decarboxylase (AMDase) from Alcaligenes bronchosepticus [2] has high structural similarity to cofactor-free amino acid racemases. The racemase-like catalytic machinery of mutant G74 C conveys it a unique activity in the racemisation of pharmacologically relevant derivates of 2-phenylpropionic acid (profenes), which makes AMDase G74 C an interesting object for the mechanistic investigation of cofactor-independent racemases. While the racemase has high activity towards small arylaliphatic acids, larger substrates such as ibuprofen are converted much slower. Objective of this study is the extension of the substrate range by rational design, either by creating space in the binding pocket or by stabilizing the substrate by the introduction of hydrophobic interactions. References [1].Felfer U, Goriup M, Koegl ME, et al. Adv Synth Catal 2005:951–61. [2].Kourist R, Miyauchi Y, Uemura D, Miyamoto K. Chem Eur J 2011:557–63. http://dx.doi.org/10.1016/j.nbt.2014.05.1815 PA-30 Enzymatic Hydrolysis of PET: Structural Diversity and Kinetic Properties of Cutinases from Thermobifida Altijana Hromic 1,∗ , Doris Ribitsch 2 , Andrzej Lyskowski 1 , Georg Steinkellner 1 , Helmut Schwab 3 , Georg Gübitz 2 , Karl Gruber 1 1 ACIB GmbH c.o. IMB Graz ACIB GmbH 3 ACIB GmbH c.o. Institute of Molecular Biotechnology, Graz University of Technology 2 Poly(ethyleneterephthalate) (PET) is one of the most widely used polymers worldwide. Its use ranges from different medical and thermoforming applications to fibers for textiles. It is highly hydrophobic which makes this polymer difficult to be functionalized. Therefore, industrial applications often involve surface activation prior to final treatment. Plasma or aggressive chemistry methods are energy consuming or environmentally harmful and in some cases they lead to a decrease in polymer weight or strength. The exchange of unpleasant chemical by enzymatic processes would avoid these problems. Cutinases from Thermobifida cellulosilytica DSM44535 (Thc Cut1 and Thc Cut2) hydrolyze PET. Their ability to hydrolyze the polymer was compared with other enzymes hydrolyzing natural polyesters including the PHA depolymerase from Pseudomonas fluorescens and two other cutinases from Thermobifida fusca KW3. S88 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 The two isolated Thermobifida cutinases are very similar but had different kinetic parameters on soluble substrates. We determined the structure of both enzymes. Structural analysis revealed surface regions of Thc Cut1 and Thc Cut2, which differ in electrostatic and in hydrophobic properties. Modeling studies suggest that these regions interact with PET during turnover which may explain the differences in hydrolysis efficiencies which were observed for the two enzymes. http://dx.doi.org/10.1016/j.nbt.2014.05.1816 PA-31 Characterisation of a recombinant patchoulol synthase for the biocatalytic production of high valuable sesquiterpenes Thore Frister 1,∗ , Steffen Hartwig 2 , Katharina Schnatz 2 , Thomas Scheper 2 , Sascha Beutel 2 1 2 Leibniz University Hannover, Institute of Technical Chemistry Leibniz University Hannover Sesquiterpenes are a structurally diverse class of secondary metabolites, which are mainly produced by various plants. Due to their special odor sesquiterpenes are widely used as fragrance compounds in exclusive perfumes, scented household goods and as naturally flavor additives in food. These days the majority of sesquiterpene production is still based on isolation techniques such as solvent extraction and steam distillation. Recent biotechnological approaches for the production of sesquiterpenes require time and cost-extensive pathway engineering. We hereby present a biocatalytic method for the production of high valuable sesquiterpenes such as patchoulol and germacrene A by converting synthetic farnesyl diphosphate (FPP) with a recombinant patchoulol synthase (PTS). [1] For the production of the PTS we have established a bioprocess combined with a reliable purification strategy. The product spectrum, substrate scope and important parameters concerning the kinetic properties and enzyme stability have been studied. FPP is produced in a short and fast synthesis starting from chemically derived, cheap farnesol using an optimized method according to Keller et al. [2] The bioconversion of FPP was performed in different multi-phase model reactor in order to increase sesquiterpene yield and maintain an easy product recovery. References [1].Deguerry F, Pastore L, Wu S, Clark A, Chappell J, Schalk M. Archives of Biochemistry and Biophysics 2006;454:123–36. [2].Keller R, Thompson R. Journal of Chromatography A 1993;645:161–7. http://dx.doi.org/10.1016/j.nbt.2014.05.1817 BIOCATALYSIS New Biotechnology · Volume 31S · July 2014 PA-32 Nuclear Magnetic Resonance Spectroscopy: An Alternative Fast Tool for Quantitative Analysis of the Solvent-free Ethanolysis of Coconut Oil Using Fungal Resting Cells Ramon Canela Garayoa 1,∗ , Edinson Yara-Varón 2 , Mercè Balcells 2 , Mercè Torres 2 , Jordi Eras 2 1 2 The University of Lleida-DBA Center The University of Lleida Ethyl fatty esters (EFE) from coconut oil have broad applications in the flavouring and fragrance industries. A solvent-free synthesis of EFE using fungal resting cells was conducted. A reliable and fast analytical method was needed to optimize the biocatalytic process. The method had to allow the quantification of the starting material (TAG), the various intermediates (monoacylglycerols–MAG- and diacylglycerols–DAG-) and the EFE. The analysis of acylglycerols and EFE has been carried out by employing analytical techniques such as high performance liquid chromatography (HPLC), and gas–liquid chromatography (GLC). However, these methods are time-consuming. Recently, nuclear magnetic resonance spectroscopy (NMR) has been proposed in the preparation of biodiesel and DAG as an alternative analytical tool [1,2]. The usefulness of NMR has been increasingly recognized for its non-invasiveness and rapidity to detect a wide range of compounds that can be detected in a single measurement (spectrum), whereas little or no need for sample pre-treatment is required. In the present study, a single NMR experiment was used as a fast and effective method for studying the progress of the biocatalytic synthesis of EFE. A new algorithm was developed to determine the contents of TAG, DAG, MAG, EFE and free fatty acids in the crude samples resulting from various experimental conditions. Results were in accordance with those resulting from GLC. References [1].Hatzakis E, Agiomyrgianaki A, Kostidis S, Dais P. J Am Oil Chem Soc 2011;88:1695–708. [2].Prakash P, Aulakh SS. J Basic Microbiol 2011;51:607–13. http://dx.doi.org/10.1016/j.nbt.2014.05.1818 PA-33 Enhancing hydrocortisone transformation to 16a-hydroxy hydrocortisone by Streptomyces roseochromogenes Paola Diana ∗ , Odile Francesca Restaino, Mariacarmela Marseglia, Maria Giovanna Borzacchiello, Chiara Schiraldi activity. Streptomyces roseochromogenes could be used as microbial whole cell catalyser for the biotechnological transformation of hydrocortisone to 16a-hydroxy hydrocortisone by using fermentation technologies. Previous studies demonstrated the possibility to obtain 0.508 ± 0.01 g·L−1 of 16␣-OH-HC in 2-L pulsed batch fermentations at 30 ◦ C and pH 7, by using a malt extract- and yeast extract-based medium. In this study the influence on the conversion ratio and the by-product formation of growing conditions, like pH and temperature, was investigated in both shake flask experiments and 2-L batch fermentations. Once determined the optimal conditions for both bacterial growth and steroid conversion,different feeding strategies and substrate addition profiles were exploredin pulsed batch fermentations, and a 16␣-OH-HC maximum production of 0.630 ± 0.02 g·L−1 was reached. Fedbatch experiments, carried out scaling-up the process in a 15-L fermentor, allowed to reach a maximum of 0.804 ± 0.05 g·L−1 of 16␣-OH-HC, while further investigations will be performed to drive the process on industrial scales. http://dx.doi.org/10.1016/j.nbt.2014.05.1819 PA-34 Immobilization of cells and enzymes to PVA gel Martin Rebros 1,∗ , Michal Rosenberg 1 , Radek Stloukal 2 1 2 Slovak University of Technology LentiKats Compared to conventional free cell and enzyme processes immobilized one offers several important advantages such as: the possibility of repetitive use of immobilized cells and enzymes, the improvement of enzyme stability, the reduction of non-productive cell growth phases, faster reaction rates at increased cell density and higher yields. A veryeffective and useful matrix for entrapment immobilization is polyvinyl alcohol (PVA). The gelation of PVA hydrogel is based on partial drying at room temperature and therefore is very gentle to both types of biocatalysts. Due to cell growth within the immobilizates the volumetric productivities may gradually increase and reached higher values compared to free cell processes. By immobilization enzymes improved their stability. These phenomena were successfully applied to various whole-cell and enzyme biocatalysts. Acknowledgement: This work was done during implementation of the project Development of Competence center for research and development in molecular medicine, ITMS code 26240220071, supported by the Research and Development Operational Program funded by ERDF. http://dx.doi.org/10.1016/j.nbt.2014.05.1820 Second University of Naples In the last years the biotechnological production of steroids by microbial transformations has became a common practice and a reliable method to produce structural tailored-cut molecules also in large scales. Hydrocortisone is a pharmaceutical active molecule generally used as drug; its hydrolylated forms, like 16a-hydroxy hydrocortisone, have higher and more efficient anti-inflammatory www.elsevier.com/locate/nbt S89 BIOCATALYSIS New Biotechnology · Volume 31S · July 2014 PA-35 Screening of Lipase Microorganisms Production Among Different ¸ ın 2 , Nihat Alpagu Sayar 1 Duygu Elif Yılmaz 1,∗ , H. Tansel Yalc 1 2 Marmara University Ege University The design and manifecture of single enantiomers of chiral intermediates have been attracting attention in the pharmaceutical industry during the last decades. Single enantiomers that are potential biocatalysts due to their higher degree of enantioselectivity and regioselectivity are usually prepared by chemical catalysis. L-amino acid esters are promising intermediates for various active drug components and their catalytic biosythesis through transesterification reactions could be a rational alternative way. Besides, they can be effectively producted by using lipase from various microorganisms as a biocatalyst. The selection of the most appropriate organism among different strains can be a valuable starting point for this study. Various isolates from various potantial microorganisms which differ significantly from each other, may exhibit different catalytic effectiveness. The microbial isolates for this study are maintained by monthly transfers on MGYP agar slants and stored at + 5◦ C. For the production stage, microorgansims are transferred to MGYP preculture medium. After overnight incubation, this culture is inoculated to production medium including different types of oil. The culture is incubated in appropriate conditions. Samples are taken at different time intervals to determine the growth and lipase activity. The enzyme activity is shown by p-nitrophenyl palmitate (pNPP) as substrate. P-nitrophenol is obtained from pNPP and absorbance is measured spectrophotometrically against an enzyme free sample. One unit of lipase activity (U) is defined as the amount of enzyme which liberates 1 ␮mol p-nitrophenol/min under assay conditions. Protein is measured by the Coomassie Blue G-250 binding method using bovine serum albümin as standard. http://dx.doi.org/10.1016/j.nbt.2014.05.1821 PA-36 A cutinase from Fusarium oxysporum with potential for PET surface modification Evangelos Topakas ∗ , Efstratios Nikolaivits, Maria Kanelli, Paul Christakopoulos National Technical University of Athens Cutinases are small extracellular serine hydrolases whose natural function is the hydrolysis of the polyester cutin. Their ability to hydrolyze a wide range of substrates (from low molecular weight esters to high molecular weight polymers) makes cutinases very useful biocatalysts for various applications. In the present study, a cutinase (FoCut5a) from the ascomycete fungus Fusarium oxysporum was functionally overexpressed in Escherichia coli BL21, harboring pET22b(+)-cut16606. In order to improve enzyme sta- S90 www.elsevier.com/locate/nbt bility, the folding of the expressed protein took place also in the periplasm in addition to cytosolic protein production by cloning cut16606 gene downstream of the pelB signal peptide. The heterologous expression was induced with IPTG in 16о C for 20 h of incubation. The recombinant cutinase was purified from the cytosol or culture supernatant using immobilized-metal affinity chromatography (IMAC), resulting in a monomeric protein of ca. 23 kDa that is optimally active at 40 ◦ C. FoCut5a cutinase was tested for its potential use in surface modification of PET fabrics, with the intention to increase their hydrophilicity and to improve their properties. Towards this direction, the enzymatic hydrolysis of two model substrates bis(benzoyloxyethyl)terephthalate (1) and commercial bis(2-hydroxyethyl)terephthalate (2) was attempted, in order to evaluate the capability of the recombinant cutinase in PET modification. FoCut5a succeeded in hydrolyzing both models, releasing bis(2-hydroxyethyl)terephthalate and benzoic acid from substrate 1 and a bis(2-hydroxyethyl)terephthalate derivative in case of substrate 2. Experiments are in progress for the surface modification and functionalization of PET fibers. http://dx.doi.org/10.1016/j.nbt.2014.05.1822 PA-37 Synthesis of biological active compounds using carbohydrate esterases as biocatalysts Paul Christakopoulos 1,∗ , Io Antonopoulou 1 , Evangelos Topakas 2 1 2 Luleå University of Technology National Technical University of Athens Various fungal and bacterial carbohydrate esterases represent appealing biocatalysts that have the ability not only to deconstruct plant biomass but also to modify compounds with a potential use in food, cosmetic and pharmaceutical industries. Feruloyl esterases (FAEs, E.C. 3.1.1.73) have been proved promising candidates for the enzymatic synthesis of antioxidants allowing more flexible process configurations. Among the advantages they provide are use of lower temperatures (50-60 ◦ C) comparing to the counterpart chemical process (150о C), one step production of one product instead of mixtures and no need of by-product and catalyst residues removal in order to produce clean and high quality substances. Glucuronoyl esterase (GE) synthetic ability needs to be explored towards the production of alkyl branched glucuronic acid derivatives which are non-ionic surfactants and have good surface properties, including biodegradability. In addition, due to their tastelessness, non skin-irritation and non toxicity, these bioactive compounds find diverse uses in the cosmetic and pharmaceutical industries. Aim of this work is the development of competitive and eco-friendly bioconversions based on transesterification reactions catalyzed by FAEs and GEs, for the production of molecules with antioxidant activity, such as phenolic fatty and sugar esters. The synthesis of four biological active compounds (prenyl ferulate, prenyl caffeate, 5-O-(trans-feruloyl)-arabinofuranose, and glyceryl ferulate) was evaluated using recombinant FAEs from Myceliopthora thermophila and Fusarium oxysporum, while the BIOCATALYSIS New Biotechnology · Volume 31S · July 2014 synthesis of benzyl D-glucuronate and prenyl-D-glucuronate was evaluated using recombinant GEs from M. thermophila. All reactions were carried out in ternary systems of nhexane/alcohol/water forming surfactantless microemulsions. http://dx.doi.org/10.1016/j.nbt.2014.05.1823 PA-38 Biodegradation of esfenvalerate by bacteria from Brazilian biome mangrove André Luiz Meleiro Porto 1,∗ , Willian G. Birolli 2 , Eloá B. Meira 2 , Nitschke Marcia 2 , Luciene P.C. Romão 3 1 Institute of Chemistry of São Carlos - University of São Paulo Instituto de Química de São Carlos, Universidade de São Paulo 3 Departamento de Química, Universidade Federal de Sergipe 2 Esfenvalerate is a widely used pyrethroid insecticide. This work aimed the biodegradation of the commercial formulation of esfenvalerate (EsfCom) by environmental bacteria isolate from Brazilian biome mangrove, especially from turfa soil. The turfa soil is a material of plant origin, partially decomposed, found in layers, usually in swampy areas and mountains. The four bacterial strains (P5CBNB, P5MNB, P8CNB, R5RaNB) assessed in this work were able to grow in a solid and liquid culture medium (nutrient agar) in presence of esfenvalerate (100 mg.L−1 ). The insecticide and its main metabolites [3-phenoxybenzoic acid (PBAc), 3-phenoxybenzaldehyde (PBAld), and 2-(4-chlorophenyl)3-methylbutyric acid (ClAc)] were quantified. It was observed that all the evaluated strains promoted the esfenvalerate biodegradation after 14 days.The residual esfenvalerate ranged between 103.0 mg.L−1 (similar to method recovery) and 41.6 mg.L−1 , while the PBAc formation was 8.1-1.2 mg.L−1 , ClAc was 10.6-0.0 mg.L−1 . PBAld was not detected in any of the biodegradation experiments. The EsfCom biodegradation promoted the accumulation of PBAc and ClAc, which are considered toxic compounds. The formed PBAld was completely transformated by strains and it was the main metabolite in the abiotic degradation. It was observed that some strains were efficient in esfenvalerate biodegradation and might be used in bioremediation. A biodegradation pathway for esfenvalerate by bacteria isolated from turfa soil was proposed. Acknowledgements: FAPESP, CNPq, CAPES, USP-SGA http://dx.doi.org/10.1016/j.nbt.2014.05.1824 PA-39 Lipase Catalyzed Esterification Reactions–A Kinetic Model Ceyda Kula ∗ , Nihat Alpagu Sayar Marmara University Bioengineering Department Biocatalysis has attracted important interest in chemistry and engineering fields. Substrate specificity, enantioselectivity, regiospecificity, chemoselectivity and soft reaction conditions are important advantages for enzyme-based synthesis in an indus- trial process. Compared to chemical catalysts, the limited rate and low throughputs are amongst the challenges which need to be addressed for broader application of biocatalysis. And also biocatalyst and process development require expensive and time consuming experimentation. Mathematical modeling and simulation tools will be used as solution for process development duration problems. The use of model based techniques will facilitate the reduction of unnecessary experimentation accelerate optimisation and automation of processes, resulting in a reduction in cost and time. Enzyme-catalyzed esterification has gained increasing attention in many applications, due to the significance of the derived products. More specifically, the lipase-catalyzed esterification reactions have attracted research interest during recent years, due to an increased use of organic esters in biotechnology and the chemical industry. Selective substrate and desired product have also significant value for scientific field. The final products may be new molecules with different characteristics and properties. We aim to focus on kinetic and mathematical modeling of a lipase catalyzed esterification reaction as a model system. Esterification will be carried out with various substrates such as different amino acids and carbohydrates. The kinetic model will be developed using Matlab programming environment with extra possible use of Mathematica software. Depending on the type of process designed for the esterification reaction a process model will be developed using the kinetic model as a basis. http://dx.doi.org/10.1016/j.nbt.2014.05.1825 PA-40 Gluconobacter oxydans used to production of natural aroma - 2-phenylacetic acid in immobilized system (LentiKats form) Monika Vidová 1,∗ , Ivana Slezáková 2 , Martin Krištofíková 2 , Michal Rosenberg 2 Rebroš 2 , L’udmila 1 Institute of Biotechnology and Food Science, Slovak University of Technology Institute of Biotechnology and Food Science, Faculty of Chemical and Food Technology, Slovak University of Technology 2 System of generation of natural aromas using G. oxydans in interesting in many cases. Acetic acid bacteria G. oxydans specifically oxidates in unresting condition 2-phenylethanol PEA into phenylacetic acid PA on membrane surface, so product is easily remove to reaction mixture which significantly facilitates the separation. Both PEA and PA have aromatic properties and in biotransformation step with whole cells biocatalysts change kind of flavor (from rose to honey like) and physical properties also. For highlighting is the fact that biomass in fermentation step is obtaied by using renewalbe source, cheap waste product - glycerol. Immobilization of G. oxydans into biocompatible polyvinyl alcohol gel in form of LentiKats may increase the benefits of preparing natural flavorings repeated using of biocatalysts cells. The work presents comparison of repeated bioconversion with free and immobilized cells, benefits of immobilized system and measured www.elsevier.com/locate/nbt S91 BIOCATALYSIS technologically important factors - the effect of pH, oxygen, different concentrations of the substrate and also scale-up experiments in controlled fermentation unit. Keywords: G. oxydans, 2-phenylethanol, 2-phenylacetic acid, immobilization, LentiKats, glycerol Acknowledgment: This study was supported by the grant of Agency for Research and Development of the Slovak Republic ITMS APVV-0302-10 and by the grant of VEGA - Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic VEGA 1/0229/12. http://dx.doi.org/10.1016/j.nbt.2014.05.1826 PA-41 Enzyme mixture production from Pycnoporus sanguineus DMSZ 3024 using a lignocellulosic waste, Hazelnut husk: A case study for laccase and cellulase ¸ ak Koc ¸ han 2 , Nihat Alpagu Sayar 2 , Kübra KaraosOrkun Pinar 1,∗ , Bas manoğlu 2 , Dilek Kazan 2 1 2 Marmara University, Bioengineering Department Marmara University Introduction: Hazelnut husk is a promising important lignocellulosic material for fermentable sugar production. According to Fiskobirlik, 774,000 to 1,032,000 tons of hazelnut husk are left in fields as waste or incinerated. In different methods, cellulose and hemicellulose are converted into fermentable sugars by the S92 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 pretreatment of lignocellulosic waste and hydrolysis. In this context, hydrolysis must be successful, inexpensive, and have low energy requirements with lower fermentation time and less byproducts. In hydrolysis, it is very difficult to obtain higher yield of fermentable sugars due to lignin. Sugars obtained with enzymatic hydrolysis can be fermented easily because of the relative absence of by-products and avoidance of undesirable operating conditions. Pycnoporus sanguineus is a white rot fungus with high lignocellulolytic potential. Aim: Production of enzyme mixture (laccase and cellulase) from P. sanguineus DMSZ 3024 by the hydrolysis of hazelnut husk is aimed in this study. Methods: Both laccases that catalyze the oxidation of lignin related compounds and cellulases that hydrolyse cellulose to obtain smaller ␤-glucose oligomers and ␤-D-glucose were produced from P. sanguineus by the hydrolysis of hazelnut husk. Enzyme mixture and hazelnut husk concentration for hydrolysis conditions were optimized at different ratios and reaction times. All enzyme activities were measured. Sugar and total sugar content were determined by the phenol-sulfuric acid and DNS method. Results and Conclusion: An appropriate method for hydrolysis of hazelnut husk with enzyme mixture using, P. sanguineus was obtained to overcome hydrolysis problems. Acknowledgement: This work was supported by the Turkish Scientific and Technical Research Council (113M053). http://dx.doi.org/10.1016/j.nbt.2014.05.1827 BIOFUELS, BIOCHEMICALS AND BIOENERGY New Biotechnology · Volume 31S · July 2014 Biofuels, biochemicals and bioenergy PB-01 Minimization of Bacterial Contamination with High Solid Loading during Ethanol Production from Lignocellulosic Materials Mofoluwake Taherzadeh 2 1 2 Ishola 1,∗ , Tomas Brandberg 2 , Mohammad Swedish Centre for Resource Recovery, University of Borås University of Borås Abstract Ethanol is the most important renewable fuel in the transportation sector. Its production from lignocellulosic materials, commonly referred to as second generation ethanol, is considered more attractive than production from starch and sugar crops. Bacterial contamination by lactic acid-producing bacteria is still a major problem during ethanol production processes. Bacteria compete with the yeast by consuming the sugars and the nutrients required by the yeast for efficient ethanol production. This often causes substantial economic losses at industrial fermentations. In this study, without any sterilization of the substrate, simultaneous saccharification and fermentation (SSF) was performed using cellulase Cellic® Ctec2 enzyme for hydrolysis and Baker’s yeast, Saccharomyces cerevisiae, was used as the fermenting organism with different loads of suspended solids - 8%, 10% and 12%. With 8% and 10% SS, there was a significant contamination, which caused consumption of both hexoses pentose sugars in the fermentation medium, this resulted in lactic acid concentrations of 43 g/L and 36 g/L from 10% SS and 8% SS respectively. In contrast, only 2.9 g/L lactic acid was observed with 12% SS. An ethanol concentration of 47 g/L was produced from high solid loading of 12% SS while just 26 g/L and 23 g/L were produced from 10% and 8% SS respectively. Our results show that SSF with 12% SS has an increased concentration of inhibitors, particularly acetic acid which selectively inhibited the bacterial growth without affecting the metabolic activities of the yeast during the fermentation Keywords Bacterial contamination; Lignocellulosic ethanol; Saccharomyces cerevisiae, lactic acid. http://dx.doi.org/10.1016/j.nbt.2014.05.1828 PB-02 Breeding low temperature resistant Camelina sativa for biofuel production Florentina Jurcoane 2 Matei 1,∗ , Florentina Sauca 2 , Paul Dobre 3 , Stefana 1 University of Agronomical Sciences and Veterinary Medicine Bucharest, Faculty of Biotechnologies 2 Centre of Microbial Biotechnology Biotehgen, Bucharest, Romania 3 University of Agronomical Sciences and Veterinary Medicine Bucharest, Faculty of Agriculture, Bucharest, Romania In the European effort to produce sustainable aviation fuel our team is involved in a FP7 project (ITAKA) in breeding activities of Camelina sativa L. as main feedstock. One of our purposes it is to obtain a variety with high productivity and resistant to low temperatures specific to continental winters as can occur in Romania. This variety may be seeded as autumn culture under minimal tilling conditions. As starting material have been used a local camelina cultivar resistant to low temperatures and an international variety GP202 with high productivity and oil content. It has been taken into account classical approach, the use of immature embryo rescue technique. The first hybrid generation was obtained by castration and pollination; the immature embryos were cultivated in MS medium w/o hormones; new plantlets were cultivated till full maturity under greenhouses conditions. The obtained seeds have been used in open field for other randomized hybridization. The new hybrid registered the following characteristics: the plant lengths and the branching level are non-significantly different compared to the parental lines (ANOVA tests); in terms of productivity, GP202 variety kept its top position, while the new hybrid has proven top position for oil content (2.13% higher in total fat). Regarding the winter resistance, the hybrid has registered 10% more survived plants in open land. As a remark, during the experiments GP202 showed the highest sensibility to the mildew attack. The further aim of the work is to fix the new characters in order to homologate the new variety. http://dx.doi.org/10.1016/j.nbt.2014.05.1829 PB-03 Advanced Biomass Value: Microalgae biomass as a new source of sustainable aviation biofuels and lubricant production Felix Bracharz ∗ , Jan Lorenzen, Farah Qoura TUM, Industrial Biocatalysis The aviation industry grows 5% p.a. and by 2020 has to comply with strict governmental emission standards that mandate a 20% CO2 reduction compared to the emission levels measured at 2005. Together with the eminent end of fossil resources these drivers force the development of sustainable aviation fuel alternatives that are carbon neutral and in compliance fuel standard regulations such as Jet A. In this study a new, mass-and energy efficient algae biorefinery concept for the integrated production of aviation fuels, industrial lubricants and CO2 adsorbing building materials has been developed. The process chain is based on production of fast growing microalgae biomass containing up to 20%w/w lipids. Subsequently, algae lipids are separated from the biomass fraction and converted to high performance, high value lubricants by targeted functionalisation using a cascade of optimized biocatalytic processes. The biomass residue is enzymatically hydrolysed and used as a fermentation substrate for oleaginous yeast strains. Since these fast growing, oleaginous yeasts can accumulate up to 80% w/w lipids, they are the ideal biomass base for the production of drop-in aviation fuels. Conversion of wet yeast biomass is accomplished using a streamlined thermochemical process that features optimized heterogenous catalysts. In this process carbon rich coke is a residue of the thermochemical biomass processing. This residue stream is used as a settling modifier in the production www.elsevier.com/locate/nbt S93 BIOFUELS, BIOCHEMICALS AND BIOENERGY of CO2 adsorbing building materials. The integrated biorefinery process does not produce any waste streams and adds value to every process intermediate. http://dx.doi.org/10.1016/j.nbt.2014.05.1830 New Biotechnology · Volume 31S · July 2014 PB-05 The development of biodiesel production using lipase from mutant and selected yeast Aree Rittiboon ∗ , Wannisa Pansuk, Marisa Jatupornpiput King Mongkot’ s Institute of Technology, Ladkrabang PB-04 Enzymatic production of biodiesel that avoids glycerol as byproduct, by using immobilized Rhizopus Oryzae lipase Carlos Luna 1,∗ , Cristobal Verdugo 2 , Enrique D. Sancho 3 , Diego Luna 4 , Juan Calero 4 , Alejandro Posadillo 5 , Felipa M. Bautista 4 , Antonio A. Romero 4 1 University of Cordoba Crystallographic Studies Laboratory, Andalusian Institute of Earth Sciences, CSIC 3 Department of Microbiology, University of Cordoba 4 Department of Organic Chemistry, University of Cordoba 5 Seneca Green Catalyst S.L 2 Immobilized Rhizopus oryzae lipase (ROL) was used as biocatalyst on different supports in the selective transesterification reaction of sunflower oil with ethanol to generate a new second generation biodiesel. As ROL it was applied a low cost powdered enzyme preparation from Biocon®-Spain (BIOLIPASE-R), a multipurpose additive used in food industry. In this respect, it was carried out a study to optimize the support used and the immobilization process, as well as the best pH conditions in each case. It has been also evaluated the physical adsorption of this enzyme on a demineralised sepiolite as well as the covalent immobilization of this lipase on amorphous AlPO4 support by using two different linkers (p-hydroxybenzaldehyde and benzylamine-terephthalic aldehyde, respectively).On the other hand, the resulting new biofuel, already patented as ECODIESEL® is composed by a mixture of fatty acid ethyl esters and monoacylglycerols (FAEE/MG) blended in a 2/1 molar relation. This novel biofuel, which present the advantage of integrating glycerol as monoacylglycerols (MG) into biofuels composition, exhibits similar physicochemical properties than the conventional biodiesel and it is produced through a process which minimizes waste generation and maximizes efficiency. Finally, one of these immobilization processes of lipases not only was effective with vegetal oils, it has been even successfully carried out the transesterification reaction with animal fat from butchery wastes, using this biocatalyst covalently immobilized with p-hydroxybenzaldehyde, achieving a viable and functional biofuel from low quality raw materials as animal waste. http://dx.doi.org/10.1016/j.nbt.2014.05.1831 S94 www.elsevier.com/locate/nbt The aim of our research was to select and characterise yeast isolates from soil and other materials produced in the oil palm industry. Of the strains isolated from palm kernels after compression, strain SLP27 produced the highest lipase activity (0.28 unit/ml). Strain SLP27 was mutated sequentially with ultraviolet light for 38 seconds. The resulting strain, UV79, was mutated with ethyl methane sulfonate for 58 minutes to produce strain EM107, which was then mutated with gamma rays at 2 kGy to yield strain GAM47. The maximum lipase activities of these strains were 0.30, 0.36 and 0.70 unit/ml, respectively. Lipase from GAM47 could be used as a catalyst in the transesterification reaction for biodiesel production. This selected strain was identified to species level by analysis of the D1/D2 domain of 26S ribosomal RNA sequence as Candida orthopsilosis. http://dx.doi.org/10.1016/j.nbt.2014.05.1832 PB-06 Production of 3-hydroxybutyrate from waste biomass by metabolically engineered Escherichia coli Johan Jarmander 1,∗ , Mónica Guevara 1 , Mariel Perez Zabaleta 1 , Gustav Sjöberg 1 , Jaroslav Belotserkovsky 1 , Jorge Quillaguamán 2 , Gen Larsson 1 1 Industrial biotechnology, School of biotechnology, KTH Center of biotechnology, Faculty of science and technology, San Simón University 2 There is a vast interest in establishing biorefineries that rely on microbial conversion of biomass for production of fuels and value-added compounds. Especially waste biomasses, such a lignocellulose waste from e.g. agriculture, and municipal food waste are, due to their low cost, attractive substrates for production of biochemicals. Escherichia coli is one of the most commonly used organisms for production of recombinant products since it is well documented, easily manipulated and fast growing in simple media. In this work we aim to use E. coli for production of the chiral compound 3-hydroxybutyrate from waste biomass. This was done by introducing part of the pathway to poly-hydroxybutyrate from the halophilic bacterium Halomonas boliviensis into E. coli. 3-hydroxybutyrate is an industrially important compound as it can be utilized as a drug, as well as in synthesis of various co-polymers. By employing flux modelling and metabolic engineering, the objective is to identify how the internal fluxes and concentrations of metabolites and co-factors can be shifted towards an increased productivity of 3-hydroxybutyrate. This knowledge is also applied in the process design, where limiting one or several elements in the cultivation medium BIOFUELS, BIOCHEMICALS AND BIOENERGY New Biotechnology · Volume 31S · July 2014 can have the desired effect of increased carbon flux towards 3-hydroxybutyrate. http://dx.doi.org/10.1016/j.nbt.2014.05.1833 PB-08 The usage of carrot pomace as a feedstock for bioethanol production Ekin Demiray ∗ , Sevgi Ertugrul Karatay, Gönül Dönmez, Sedat Dönmez PB-07 Achievement of a biofuel-like biodiesel by regioselective transesterification of sunflower oil with mucor miehei lipase Juan Calero 1,∗ , Juan Calero 2 , Diego Luna 2 , Enrique D. Sancho 3 , Carlos Luna 2 , Cristóbal Verdugo 4 , Alejandro Posadillo 5 , Felipa M. Bautista 2 , Antonio A. Romero 2 1 University of Cordoba Department of Organic Chemistry, University of Córdoba 3 Department of Microbiology, University of Córdoba 4 Crystallographic Studies Laboratory, Andalusian Institute of Earth Sciences, CSIC 5 Seneca Green Catalyst S.L 2 In previous researches, we have developed a biofuel that avoid the production of glycerol as by-product. This biofuel is similar to the conventional biodiesel, being in the same way applicable to diesel engines. Thus, glycerol is kept as monoglyceride (MG), together to two fatty acid ethyl esters (FAEE) molecules. In this respect, this biofuel is obtained by a partial ethanolysis of sunflower oil with Mucor Miehei lipase as biocatalyst. Results obtained by using M Miehei lipase have shown that this lipase is an efficient biocatalyst in the 1,3-selective enzymatic ethanolysis reaction of triglycerides. Thus, reactions were performed to determine the optimal conditions, such as amount of lipase, volume of NaOH 10 N aqueous solution, temperature and oil/ethanol molar ratio. It was always used 12 mL of sunflower oil, in a 25 mL round bottom flask, with a conventional magnetic stirrer at 300 rpm, during 2 h. Finally, a study of reuses is also carried out. The optimized conditions obtained were 3.5 mL of absolute ethanol (oil/ethanol molar ratio 1/6), 37.5 ␮l of NaOH 10 N solution, temperature of 30 ◦ C and 15 mg of M Miehei lipase. Operating under these experimental conditions, eighteen successive reactions were efficiently carried out after recovering the lipases by centrifugation. Acknowledgements Grants from the Spanish Ministry of Economy and Competitiveness (Project ENE 2011-27017), Spanish Ministry of Education and Science (Projects CTQ2010-18126 and CTQ201128954-C02-02), FEDER funds and Junta de Andalucía FQM 0191, PO8-RMN-03515 and P11-TEP-7723. http://dx.doi.org/10.1016/j.nbt.2014.05.1834 Ankara University Keywords: Bioethanol; Saccharomyces cerevisiae In today’s world there is urgent need for alternative energy sources due to rapid depletion of the fossil fuels. Renewable sources are good candidates instead of fossil fuels because of their environmentally friendly and less toxic properties. It has been estimated that bioethanol will be the most widely used renewable source in the near future. The usage of agricultural wastes for bioethanol production has some advantages such as lower production costs. Therefore in this study we investigated the potential of carrot pomaces as a feedstock for bioethanol production by using Saccharomyces cerevisiae. For obtaining fermentable sugars carrot pomaces were hydrolysed in 1.5% H2 SO4 (v/v). The yeast growth, initial and consumed sugar concentrations were monitored periodically. The bioethanol concentration was determinated with gas chromotography. The microbial growth media containing carrot pomace as a carbon source and different nitrogen sources were prepared to increase the bioethanol production. It has been observed that yeast cells used 35.4 g/L sugar in the medium containing 0.5 g/L KH2 PO4 and1 g/L (NH4 )2 SO4 . As a result the media that were prepared with carrot pomace sugars supported the growth of Saccharomyces cerevisiae. These results show that carrot pomaces are suitable feedstocks for bioethanol production. http://dx.doi.org/10.1016/j.nbt.2014.05.1835 PB-09 Lactic acid production from lignocellulosic hydrolysates under non-sterilized conditions using Bacillus coagulans IPE22 Yinhua Wan ∗ , Yuming Zhang, Xiangrong Chen, Benkun Qi, Yi Su Institute of Process Engineering, Chinese Academy of Sciences A thermophilic lactic acid (LA) producer was isolated and identified as Bacillus coagulans strain IPE22. The strain showed remarkable capability to ferment pentose, hexose and cellobiose, and was also resistant to inhibitors from lignocellulosic hydrolysates. Based on the strain’s promising features, it was used to produce lactic acid (LA) from mixed sugar and wheat straw hydrolysates under non-sterilized conditions. In order to eliminate the sequential utilization of mixed sugar and feedback inhibition during batch fermentation, membrane integrated repeated batch fermentation (MIRB) was used to improve LA productivity. With MIRB, a high cell density was obtained and the simultaneous fermentation of glucose, xylose and arabinose was successfully realized. The separation of LA from broth by membrane in batch fermentation also decreased feedback inhibition. MIRB was carried www.elsevier.com/locate/nbt S95 BIOFUELS, BIOCHEMICALS AND BIOENERGY out for 5 cycles repeated culture using wheat straw hydrolysates (29.72 g/L glucose, 24.69 g/L xylose and 5.14 g/L arabinose) as carbon source, a 2.33-folds increase of LA productivity (2.35 g/L/h) was obtained as compared with conventional batch fermentation. http://dx.doi.org/10.1016/j.nbt.2014.05.1836 PB-10 Solid state fungal fermentation as a biological pretreatment strategy to convert lignocellulosic raw material into fermentable sugars Chenyu Du ∗ , Nattha Pensupa, Siobhan Knight, Jwan Abdullah The University of Nottingham Pre-treatment is one of the key challenges in the conversion of lignocellulosic raw materials into bioethanol. Various pre-treatment strategies have been developed, such as dilute acid pre-treatment, alkali pre-treatment, steam explosion and ammonia explosion pre-treatment. These methods are energy intensive as these are normally operated at high temperature, high pressure or high chemical loading rate. Inhibitor formation during the pre-treatment is another concern in these methods, which significantly impacts the efficiency of the following fermentation processes. In the University of Nottingham, we developed a solid-state fungal fermentation-based pre-treatment strategy to convert lignocellulosic raw materials into a fermentable hydrolysate. Aspergillus niger was firstly cultured on biomass for production of cellulolytic enzymes and then the biomass was hydrolyzed by the enzyme solution into a fermentable hydrolysate. Various lignocellulosic raw materials have been tried, including wheat straw, willow, miscanthus, palm oil tree branches, napier, sago extract and municipal solid waste. In solid-state fermentations of most of these raw materials, around 4-10 U/g cellulase activity could be obtained after 5 days’ culture. The acid or alkali modification of biomass at mild condition improved the cellulase production to over 10 U/g. The addition of yeast extract (0.5% w/v) and minerals significantly improved the cellulase production, for examples, to 24 U/g in wheat straw, 22 U/g in napier. The fungal culture filtrate from the solid-stage fermentation showed higher cellulolytic hydrolysis ability then the commercial cellulase Ctec2 at the same enzyme loading rate. Moreover, no inhibitor was detected in the hydrolysate examined. http://dx.doi.org/10.1016/j.nbt.2014.05.1837 PB-11 Metabolic Engineering of E. coli for the production of alkanes Yong Jun Choi ∗ , Sang Yup Lee Korea Advanced Institute of Science and Technology Our increasing concerns on limited fossil fuels and global environmental problems are urging us to develop sustainable biofuels S96 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 from renewable resources. Although microbial production of diesel has been reported, production of another much demanded transport fuel, gasoline, has not yet been demonstrated. Here we report the development of platform Escherichia coli strains that are capable of producing short chain alkanes (gasoline). The b-oxidation pathway was blocked by deleting the fadE gene to prevent the degradation of fatty acyl-CoAs generated in vivo and the activity of 3-oxoacyl-ACP synthase (FabH) was enhanced to promote the initiation of fatty acid biosynthesis by deleting the fadR gene. A modified thioesterase was employed to convert short chain fatty acyl-ACPs to the corresponding FFAs, which were consequently converted to short chain alkanes by the sequential reactions of fatty acyl-CoA synthetase, fatty acyl-CoA reductase and fatty aldehyde decarbonylase. The final engineered strain produced up to 580.8 mg l−1 of SCAs consisting of nonane (327.8 mg l−1 ), dodecane (136.5 mg l−1 ), tridecane (64.8 mg l−1 ), 2-methyl-dodecane (42.8 mg l−1 ) and tetradecane (8.9 mg l−1 ) together with small amounts of other hydrocarbons. [This work was supported by the Advanced Biomass Research and Development Center of Korea (ABC-2010-0029799) through the Global Frontier Research Program of the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF). Systems metabolic engineering work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012-C1AAA001-2012M1A2A2026556) by MSIP through NRF]. http://dx.doi.org/10.1016/j.nbt.2014.05.1838 PB-12 Statistical optimization of critical parameters for alkaline treatments of canola agricultural residue by advanced regression model Seung Wook Kim 1,∗ , Hah Young Yoo 1 , Da Un Jung 1 , Sung Bong Kim 1 , Ja Hyun Lee 1 , Chulhwan Park 2 1 2 Korea University Kwangwoon University In this study, canola agricultural residue from Honam area of Korea was pretreated by alkaline reagents (ammonium and sodium hydroxide) in order to enhance the enzyme accessibility, and the treatment conditions were optimized by statistical method. Generally, most researches only focused on the enhancement of enzymatic digestibility in the process but if not mentioned about solid recovery then the overall sugar yield can be reduced. Therefore, a novel experimental response (biomass to glucose conversion; BtG) which modified from solid recovery and enzymatic digestibility is applied toregression analysis, in current study. As a result, the optimal conditions were carried out by BtG model and the sugar yield was improved compared with the previous work. In optimal conditions, the predicted solid recovery, enzymatic digestibility and BtG were 75.8%, 78.3% and 25.7% at ammonium hydroxide treatment and 49.2%, 86.4% and 24.3% at sodium hydroxide treatment, respectively. The experimental results show BIOFUELS, BIOCHEMICALS AND BIOENERGY New Biotechnology · Volume 31S · July 2014 numerically over 90% in general. Finally, under the overall mass balance, glucose yield was about 3 fold increased by the treatments. Biogas production from 3 strains of Napier grass of the red fluorescence appeared to change following the affinities between the LZs, as observed by fluorescence imaging and flow cytometry. Currently, we are applying the proposed method to construct an in vivo matrix to entrap different enzymes in E. coli cytosole while maintaining their catalytic activities. In addition, easy detection of localization to IBs provides a unique platform for the engineering and analyses of protein-protein interactions in E. coli. Pramote Sirirote 1,2,∗ , Farida Promma 2 , Dusanee Thanaboripat 2 http://dx.doi.org/10.1016/j.nbt.2014.05.1841 1 King Monkut’Institute of Technology Ladkrabang, Biogas production from 3 strains of Napier grass, (Pennisetum purpureum) 2 Department of Biology, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand PB-15 http://dx.doi.org/10.1016/j.nbt.2014.05.1839 PB-13 Keywords: Biogas; Napier grass; Batch anaerobic digestion Direct biocatalytic conversion of methane-to-methanol using methane and ammonia-oxidizing bacteria Eun Yeol Lee ∗ , In Yeub Hwang Abstract The aim of this research was to study the potentials of biogas production from 3 strains of Napier grass (Pennisetum purpureum); i.e, King grass, Napier Pakchong1 and Alafal. Batch anaerobic digestion was performed on five ratios of different grass and inoculum volume at 1:1, 1:2, 1:3, 2:1 and 3:1, and performed on working volume of 5 L digestors for 45 days at room temperature (29-34 ◦ C). The results showed that the highest cumulative biogas production was 22.45, 26.25 and 24.29 L at the ratios of 1:3, 1:2 and 1:2 for King grass, Napier Pakchong1 and Alafal, respectively. Initial pH was 6 - 6.5 and biogas yield were 0.37, 0.53 and 0.47 L biogas/g VS. The efficiencies for the COD removal were 82.8, 76.9 and 85.0% at the best grass inoculum ratios, respectively.Therefore, Napier Pakchong1 and Alafalat grass to inoculum ratio of 1:2 were selected for further experimentation.The digestion was performed on working volume of1 L and adjusted initial pH to 7.1 for 36 day. The resulst showed that the cumulative biogas production were 2.46 and 6.97 L for Napier Pakchong1 and Alafal, respectively. Therefore, the results indicated that Alafal has higher potential for biogas production than King grass and Napier Pakchong1. http://dx.doi.org/10.1016/j.nbt.2014.05.1840 PB-14 Controlled localization of functionally active enzymes to inclusion bodies using leucine zippers Seung-Goo Lee ∗ , Haseong Kim, Bong-Hyun Sung Korea Research Institute of Bioscience and Biotechnology Inclusion bodies (IBs) are typically non-functional particles of aggregated proteins. However, some proteins in fusion with amyloid-like peptides, viral coat proteins, and cellulose binding domains (CBDs) generate IB particles retaining the original functions in cells. Here, we attempted to generate CBD IBs displaying functional leucine zipper proteins (LZs) as bait for localizing cytosolic proteins in E. coli. When a red fluorescent protein was tested as a target protein, microscopic observations showed that the IBs red-fluoresced strongly. When different LZ pairs with KD s of 20–1,000 mM were tested as the bait and prey, the localization Kyung Hee University Production of methanol from methane is the first step for achieving methane-based production of a wide range of chemicals. Methanol is a precursor to various chemicals and a liquid fuel that can be blended with gasoline. In this study, we employed methane and ammonia-oxidizing bacteria to partially oxidize methane to methanol. To prevent methanol catabolism, thus allowing methanol accumulation in the medium, various inhibitors for methanol dehydrogenase were used. Further, sodium formate was supplied as reducing power regeneration because methane moonoxygenase-catalyzed oxidation of methane to methanol was limited by reducing equivalents supply. Acknowledgment This work was supported by the New & Renewable Energy of the Korea Insitute of Energy Technology Evaluation and Planning (KETEP) grant funded by the Korea government Ministry of Knowledge Economy (No. G031595311). http://dx.doi.org/10.1016/j.nbt.2014.05.1842 PB-16 Encapsulation of Candida rugosa lipase in chitosan beads as biocatalyst for biodiesel production via nonalcohol route Heri Hermansyah ∗ , Merisa Bestari Faiz, Intan Sipangkar, Rita Arbianti Department of Chemical Engineering, Universitas Indonesia, Depok 16424, Indonesia Lipase-catalyzed biodiesel production offers many advantages such as high level of selectivity and it has good ability to catalyze organic reactions in aqueous or non-aqueous media. Unfortunately, lipase is expensive and it cannot be reused because it is dissolved in reaction media. To overcome the problem, in this research, we immobilized Candida rugosa lipase (CRL) in chitosan beads through encapsulation method. The optimum condition of www.elsevier.com/locate/nbt S97 BIOFUELS, BIOCHEMICALS AND BIOENERGY enzyme to support mass ratio, immobilization time and crosslinking agent concentration were investigated in the range of 1:4 to 1:8, 50 to 150 minutes and 0.6% to 3%w/v, respectively. Enzyme concentration was measured through spectrophotometry method. The highest enzyme loading was 97.24% when the operating conditions were 1:6 mass ratio of lipase to chitosan, 120 minutes immobilization time and 0.6%w/v of cross-linking agent concentration. The immobilized enzyme then was used as biocatalyst in biodiesel production. Non-alcohol route was used as a route for biodiesel production because it can prevent enzyme deactivation which is commonly found in conventional route. The operating condition for biodiesel production process was 37◦ C, 4%w/w biocatalyst and 1:12 mole ratio of used cooking oil (as triglyceride source) to methyl acetate (as acyl acceptor). The biodiesel, fatty acid methyl ester (FAME), concentration was determined using High Liquid Performance Chromatography (HPLC). Through 50 hours batch production process, 94.06% biodiesel yield was achieved. Whereas through continuous process in a packed bed reactor sized 11 mm ID and 150 mm length with 1ml/hour flow rate, 93.67% biodiesel yield was achieved. Keywords: Biodiesel, immobilization, biocatalyst http://dx.doi.org/10.1016/j.nbt.2014.05.1843 PB-17 Enhancing energy yield through saccharification of sorghum bagasse and second generation bio-ethanol production Teodor Vintila ∗ , Adrian Trulea, Daniela Vintilă, Georgeta Pop, Iosif Gergen, Kornel Kovacs University of Agricultural Science Timisoara Sweet sorghum is an important source of sugar that can be utilized for ethanol production. Sorghum bagasse resulted after sweet juice extraction can be further processed to obtain more energy as lignocellulosic ethanol. In our study, we used six commercial products consisting of biomass degrading enzymes to hydrolyze cellulose from pretreated sorghum bagasse and obtain fermentable sugars. Tests containing combinations of steamalkaline, mechanical pretreated bagasse and different enzymes cocktails were conducted. The results indicated the combination of steam-alkaline pretreatment and NS22086 cellulase complex (Novozymes) as the most efficient. Next, these conditions were applied to hydrolyze three types of Sorghum bicolor bagasse (Sugar Graze, Jumbo and Fundulea FT132). The hydrolysis rates obtained in the three sorghum types are between 32% and 40%. Concentration of total sugars and glucose released in hydrolysis buffer was monitored. The conversion process continued with fermentation of hydrolyzates by Saccharomyces cerevisiae in 500 ml fermenters equipped with NIR sensors (BlueSens) to evaluate in real time the ethanol and CO2 concentration. Ethanol concentrations between 1.65 g·ml−1 and 1.96 g·ml−1 were obtained in fermentation media of the hydrolyzed bagasse from three sorghum varieties, representing ethanol yields between 330 g·g−1 and 392 g·g−1 reported at DM bagasse. Applying biotechnology developed in this study in combination with current sugar extraction method applied in sorghum, S98 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 the overall ethanol production efficiency and energy yield would increase. http://dx.doi.org/10.1016/j.nbt.2014.05.1844 PB-18 Effect of hydraulic retention time on the performance of a novel tubular MFC fed with petroleum hydrocarbons Oluwaseun Adelaja ∗ , Godfrey Kyazze, Taj Keshavarz University of Westminster Pollution of groundwater by petroleum hydrocarbons is a serious threat to human health as the hydrocarbons are toxic, mutagenic and carcinogenic. Microbial fuel cells (MFCs) could be employed in the treatment of these recalcitrant pollutants with concomitant bioelectricity generation. For practical application the MFCs would have to be effective, efficient and robust. This study investigated the performance of a novel tubular MFC, operated in a continuous mode at different hydraulic retention times, HRT, in the range 2.5 to 10 days at room temperature. Bromate was employed as the catholyte and the inoculum was an adapted anaerobic microbial consortium. A mixture of benzene and phenanthrene was used as the substrate. Total chemical oxygen demand removal efficiencies and peak power densities decreased from 74 to 57% and 3.4 to 1.1mW/m2 respectively when HRT was decreased from 10 to 2.5 days.The removal efficiencies were higher than 90% for both phenanthrene and benzene.Bromate removal efficiencies increased from 52.5-78.6% as HRT was raised from 2.5-10d. The outcome of this study suggests the application of MFCs in the simultaneous removal of petroleum hydrocarbons and bromates (with concomitant power production) in anoxic environments, especially deep groundwater reservoir. MFC technology could possibly be a substitute for the more expensive conventional technologies such as permeable reactive barrier (PRB) and electroremediation which are currently employed in remediation of hydrocarbon pollutants in subsurface environments. http://dx.doi.org/10.1016/j.nbt.2014.05.1845 PB-19 An integrated lignocellulose-based bioprocessing for the production of a generic microbial feedstock Chen-Wei Chang ∗ , Colin Webb University of Manchester Conversion of ligno-cellulosic materials to sugars through a purely biological treatment is a key to sustainable chemicals production. Sequential solid-state fermentation of sugarcane bagasse with soybean hull and enzyme hydrolysis using Trichoderma longibrachiatum was performed. Microorganism incubation through solid-state fermentation resulted in process, not only producing an abundant enzyme complex, but also degrading the recalcitrant structure of the ligno-cellulosic materials. BIOFUELS, BIOCHEMICALS AND BIOENERGY New Biotechnology · Volume 31S · July 2014 The process includes three stages: (1) Simultaneous deconstruction of recalcitrant lignocellulosic materials and cellulolytic enzyme production through filamentous fugal bioconversion usingTrichoderma longibrachiatum; (2) generic fermentation feedstock production through enzymatic hydrolysis of fermented solid associated with fungi autolysis; (3) subsequent fermentation by native Saccharomyces cerevisiaefor ethanol production. This process provides cost-competitive and environmental friendly alternative to produce chemical and biofuel from lignocellulosic wastes. http://dx.doi.org/10.1016/j.nbt.2014.05.1846 PB-20 A synthetic biology approach towards improved cellulolytic activity of Clostridium acetobutylicum ATCC 824 Katalin Kovacs ∗ , Benjamin Willson, Nigel Minton University of Nottingham Several Clostridia species employ large, self-assembled multienzyme complexes, or cellulosomes, for efficient decomposition of the plant cell wall cellulosic polysaccharides cellulose and hemicellulose. Clostridium acetobutylicum, the best studied of the butanol producing Clostridia, produces small amounts of a non-functional cellulosome and is therefore unable to grow on cellulose. In our studies, we employed synthetic biology approaches to create stable C. acetobutylicum strains with the potential to utilise various cellulosic substrates by genome integration of synthetic cellulosomal subunits derived from various cellulosic degrading bacterial species (Clostridia and other species) as well as integrating noncellulosomal cellulolytic enzymes (bacterial and other sources). We use standardised synthetic parts (optimized DNA sequences) in BioBrick2 format to assemble a range of synthetic genes encoding cellulosomal scaffoldin proteins, glycoside hydrolases (GHs) and synthetic cellulosomal operons. All synthetic genes and operons are integrated into the C. acetobutylicum genome using the recently developed allele-coupled exchange (ACE) technology. Heterologous protein expression levels of the synthetic genes and the self-assembly of the mini-cellulosomes are assayed by Western blot, native PAGE and enzyme activity. We have demonstrated the successful expression, secretion, self-assembly and activity of the mini-cellulosomes produced by recombinant C. acetobutylicum strains, providing a platform for the construction of novel strains with finely tuned cellulolytic properties http://dx.doi.org/10.1016/j.nbt.2014.05.1847 PB-21 Studies on the role of different culture conditions on the growth and biochemical composition of marine microalgae Nannochloropsis sp Maria Savvidou ∗ , Fragiskos Kolisis National Technical University of Athens The climate change, the rising fossil-fuel prices and overall the societal concerns have raised attention to the sustainable production of advanced biofuels, as well as high added value bioproducts such as fatty acids (␥-linolenic, eicosapentaenoic, docosahexaenoic acids, etc.) pigments (carotenoids, ficobilins), vitamins and metabolites. The rapid growth rates, the accumulation of large quantity of lipid and other compounds of interest and the ability to be cultivated in seawater or brackish water on non-arable land make algae an exciting addition to the sustainable fuel portfolio. Microalgae are sunlight-driven cell factories that use photosynthesis to efficiently and quickly convert CO2 and other greenhouse gasses into potential biofuels, foods, animal feeds and bioactive compounds, the cultivation conditions and the role of nutrient media characteristicsare still under investigation. Amongst various microorganisms, Nannochloropsis sp., an oleaginous eukaryotic marine alga, is well appreciated in aquaculture due to its nutritional value and the ability to produce valuable chemical compounds, such as pigments, proteins and polyunsaturated fatty acids. The aim of the present study is to quantify some biochemical compounds (lipids, chlorophyll a, carotenoids, proteins, total cell biomass) in cultures of Nannochloropsis sp. grown in different culture conditions. The microorganism is cultivated in photobioreactors containing sterilized seawater enriched with f/2 medium nutrients under standard illumination conditions. The effect of parameters such light intensity, pH, sodium bicarbonate supplementation and sodium nitrate concentration on growth and biochemical composition of Nannochloropsis sp. are extensively studied. http://dx.doi.org/10.1016/j.nbt.2014.05.1848 PB-22 Effect of different components of media prepared with sugar beet hydrolysate on cell growth and ethanol production Meltem Yesilcimen Akbas ∗ , Sar Taner Gebze Institute of Technology Ethanol is an alcohol made from the fermentation of the carbohydrate or sugar fraction in biomass materials. Sugar beet composition makes it a potential and attractive raw material for the production of the second generation bioethanol. The aim of this research was to assess the usefulness of dilute enzymatic hydrolysis of sugar beet molasses and the different media components for enhancement of cell growth and ethanol production of www.elsevier.com/locate/nbt S99 BIOFUELS, BIOCHEMICALS AND BIOENERGY ethanologic E. coli strain (FBR5). Sugar beet molasses was hydrolysed with a dilute sulfuric acid at room temperature overnight to convert sucrose to glucose and fructose, followed by autoclaving. The enhancement of growth and ethanol production of strain FBR5 were investigated in a variety of growth media components including nutrients (peptone and yeast extract) or thiamin or/and trace elements. The growth properties of the strain were also evaluated before and after overliming with Ca(OH)2 treatment. Overliming treatment did not affect the growth and the ethanol production. The hydrolysate (diluted to 20%; v/v) medium including nutrients, thiamin and trace elements enhanced the cell biomass and ethanol yield. http://dx.doi.org/10.1016/j.nbt.2014.05.1849 PB-23 Potential use of different hydrolyzing methods of potato and corn processing industry wastes for ethanol production New Biotechnology · Volume 31S · July 2014 Such characteristics have increased the interest in these molecules however, their large scale production is currently more expensive comparatively to synthetics. The use of cellulosic material can improve economics of biosurfactant production and also contribute to reducing greenhouse gas emissions. This work investigates the production of surfactin by Bacillus subtilis using the liquor from sisal (Agave sisalana) pulp hydrolysis as substrate. The liquor deriving from acid and enzymatic hydrolysis of sisal cellulose was utilized as carbon source in culture media. The partially-purified product obtained using acid hydrolysate showed a surface tension (ST) of 29.8 mN/m, interfacial tension (IT) against hexadecane of 5.7 mN/m and a critical micelle concentration (CMC) of 1394.0 mg/L whereas when enzymatic hydrolysate was utilized the product showed a ST of 28.7 mN/m, IT of 3.8 mN/m and a CMC of 64.0 mg/L. The enzymatic derived liquor demonstrates to be more suitable generating the BS with the best surface active properties. In conclusion, the liquor from sisal cellulose hydrolysis can be explored as an alternative sustainable substrate for biosurfactant production. http://dx.doi.org/10.1016/j.nbt.2014.05.1851 Meltem Yesilcimen Akbas ∗ , Fatma Sumer Gebze Institute of Technology Bioethanol produced from renewable sources, such as starch or lignocellulosic materials are promising sources of alternative energy resources. Potato and corn processing industry waste (PCW) is a zero value waste rich in starch and lignocellulosic material.The purpose of present research was to investigate the potential use of different hydrolyzing methods of PCW. The cell growth and ethanol production of ethanologic E. coli strain (FBR5) were compared. PCW was hydrolyzed with different methods by using different concentrations of dilute acid and/or enzymes such as xylanase, cellulase or both enzymes. The sugar contents (glucose, xylose and arabinose) of different hydrolysates were investigated by Thin Layer Chromatography. The dilute acid treatment methodology yielded the highest levels of glucose, xylose and arabinose; 7.6%, 2.7% and 1.8%, respectively. The growth properties and ethanol production of the strain FBR5 were evaluated by using hydrolysate medium including nutrients, thiamin and trace elements. The experiments resulted in higher cell numbers and ethanol yield. http://dx.doi.org/10.1016/j.nbt.2014.05.1850 PB-24 Potential application of liquor from sisal pulp hydrolysis as alternative substrate for biosurfactant production Marcia Nitschke ∗ , Claudia Marin Abadia, Joice Kaschuk, Elisabete Frollini University of São Paulo Microbial-derived surfactants are more environmentallyfriendly, biocompatible and biodegradable in comparison with conventionally produced detergents from petroleum sources moreover, their production supports the concept of biorefinery. S100 www.elsevier.com/locate/nbt PB-25 Use of hardwood sulphite spent liquor for acclimating a polyhydroxyalkanoate storage capacity of a mixed microbial culture Diogo Queirós 1 , Luísa Seuanes Serafim 1,∗ , Simona Rossetti 2 1 2 CICECO, Chemistry Department, University of Aveiro Water Research Institute, CNR Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible biopolymers that emerge as a possible solution as substitutes of petroleum-based plastics. PHAs can be produced within the Biorefinery concept, in which wastes and by-products of numerous industries are used as carbon source. The effectiveness of PHAs production process includes a first stage of selection of a MMC with a stable PHAs-producing capacity, which determines the success of subsequent PHA accumulation step. This could be done resorting to hardwood spent sulphite liquor (HSSL) which is a complex feedstock originated from the pulp industry and meets the concept of a lignocellulosic-based biorefinery, due to its abundance and affordability, wide variety and good marketing of the bio-based products. To complement the selection of the PHAstoring populations, the evolution of microbial community must be evaluated in order to identify the best producers and determine the individual relative abundance, allowing for the design of operating conditions favoring the most important PHA-accumulating microorganisms. In this project, a MMC collected in a wastewater treatment plant was submitted to Aerobic Dynamic Feeding (ADF) in a Sequencing Batch Reactor (SBR) in order to select PHAaccumulating organisms using HSSL. The reactor has been operating for near 500 days in a steady state condition, producing a copolymer, poly(hydroxybutyrate-co-hydroxyvalerate). The medium, rich in xylose, acetic acid and lignosulphonates, led to a selection of a co-dominant culture between Alphaproteobac- BIOFUELS, BIOCHEMICALS AND BIOENERGY New Biotechnology · Volume 31S · July 2014 teria and Betaproteobacteria, determined by fluorescent in situ hybridization. A clone library was constructed and genera like Agrobacterium, Rhizobium, Brachimonas, Acidovarax, Flavobacterium, Leadbetterella and Dyadobacter were identified. http://dx.doi.org/10.1016/j.nbt.2014.05.1852 PB-26 Bioethanol Production: Adaptation of Scheffersomyces stipitis to Hardwood Spent Sulfite Liquor Ana M R B Xavier 1,∗ , Cláudio J R Frazão 1 , Susana R Pereira 2 , Violeta Sanches i Nogué 3 , Luísa S Serafim 2 , Marie F Gorwa-Grauslund 3 1 CICECO, Chemical Department, University of Aveiro, Portugal Chemical Department, University of Aveiro, Portugal 3 Division of Applied Microbiology, Department of Chemistry, Lund University 2 Biofuels, which consist of fuels produced from biomass, are suitable renewable alternatives to conventional motor fuels (e.g. gasoline, diesel). Bioethanol and biodiesel are the most promising biofuels. Biofuels can be generated from various raw materials, like lignocellulosic biomass and its derivatives. Hardwood Spent Sulfite Liquor, HSSL, is the side-product of the acidic sulfite pulping process, and it is rich in sugars (40–45 g.L−1 ), mainly the pentose sugar xylose that can be converted to ethanol by yeast Scheffersomyces stipitis. However, HSSL contains toxic compounds (e.g. acetic acid and phenolics) that inhibit yeast metabolism. The aim of this study was the use of evolutionary engineering to generate a S. stipitis strain with increased tolerance to HSSL inhibitors and maintained xylose conversion rate. A continuous reactor with increasing HSSL concentrations (2060% (V/V)) was operated. The final population, POP, obtained after 382 generations in HSSL, was characterized and compared to the parental strain, PAR. By using this approach, improved fermentation performance was obtained. POP showed a higher xylose consumption rate (0.33 g.L−1 .h−1 ) and maximum ethanol concentration (6.93 g.L−1 ) than PAR (0.10 g.L−1 .h−1 and 1.76 g.L−1 , respectively). http://dx.doi.org/10.1016/j.nbt.2014.05.1853 PB-27 Microbial Consortium with High Cellulolytic Activity (MCHCA) for enhanced biogas production Lukasz Drewniak 1,∗ , Krzysztof Poszytek 2 , Martyna Ciezkowska 2 1 Faculty of Biology, University of Warsaw Laboratory of Environmental Pollution Analysis, Faculty of Biology, University of Warsaw 2 hydrolyzer of agricultural biogas plant, cattle slurry and manure, and represents the following genera: Bacillus, Providencia, Ochrobactrum. The main aim of this study was to verify whether the constructed consortium can degrade lignocellulosic material and enhance the production of biogas from maize silage and cattle manure. The enhancement of the methane production by supplementation with the MCHCA consortium was monitored in batch bioreactors with the use of maize silage as substrate and cattle manure as methanogens inoculum. In the first step of the process, maize silage (1% d.m.) was pretreated by 5% (v/v) or 10% (v/v) MCHCA consortium (with a density ∼108 cells/ml) for 72 h at 300 C, and then pretreated substrate was subjected to anaerobic digestion with cattle manure (20% v/v) for 21 days at 370 C. During the experiment the following parameters were monitored: cellulolytic activity, pH, level of volatile fatty acids and stability of the consortium structure by DGGE (denaturing gradient gel electrophoresis) analysis. Methane concentration and volume of gas in the culture was measured after 7, 14 and 21 days. The experimental results showed that constructed MCHCA consortium can increase the efficiency of biogas production up to 20-30%, when used 5% addition of the consortium. http://dx.doi.org/10.1016/j.nbt.2014.05.1854 PB-28 Production of 1,3-propanediol from glycerol by C. butyricum: Optimization of medium composition and kinetic studies Wojciech Białas ∗ , Marta Pikuła, Katarzyna Mroczyk, Włodzimierz Grajek Poznan University of Life Sciences A two-step approach was employed to optimize culture medium composition for enhanced production of 1,3-propanediol (1,3PD) by Clostridium butyricum. In the first step, a simple two-level screening experimental design was used to evaluate the influence of individual components of the medium on 1,3-PD production. In the second step, a central composite experimental design and response surface methodology were employed to derive a statistical model describing the impact of yeast extract, potassium phosphate dibasic and iron (II) sulfate on the 1,3-PD production. The final titer of 1,3-PD produced by C. butyricum cultured in the optimized medium, with the starting glycerol concentration of 80 g/L, was of 38.8 ± 1.23 g/L, resulting in the process yield of 0.49. The results obtained under the optimal fermentation conditions were used as a starting point for development of a kinetic model describing the glycerol utilization, 1,3-PD and biomass production rates. http://dx.doi.org/10.1016/j.nbt.2014.05.1855 In our previous work we have constructed Microbial Consortium with High Cellulolytic Activity (MCHCA). This consortium involves twenty two bacteria strains, which were isolated on minimal salt medium containing carboxymethylcellulose as the sole carbon source. Isolates were derived from: sewage sludge, www.elsevier.com/locate/nbt S101 BIOFUELS, BIOCHEMICALS AND BIOENERGY PB-29 Lignin utilization by Bacillus sp. associated with the growth enhancement and the molecular weight distribution change of lignin Gyeongtaek Gong 1,∗ , Han Min Woo 1 , Youngsoon Um 1 , Tai Hyun Park 2 1 2 Korea Institute of Science and Technology School of Chemical and Biological Engineering Lignin is one of the major components in lignocellulosic biomass and is the most abundant natural aromatic polymer. To utilize lignocellulosic biomass efficiently, lignin-degradation is an important issue; however, lignin is known to be recalcitrant for biodegradation. Previously, we isolated Bacillus sp. GWD275 from mud flat thorough the culture in kraft lignin agar plate and the Azure B decolorization screening method for lignin degradation. In this study, lignin utilization by Bacillus sp. GWD275 was investigated by observing growth enhancement in the presence of lignin. Also, the molecular weight distribution of lignin in culture media was analyzed by Gel Permeation Chromatography (GPC) during the growth of Bacillus sp. GWD275 in the presence of lignin. The growth of GWD275 was compared with Luria-Bertani (LB) and LB supplemented with lignin (1 g/L). The growth in LB supplemented with lignin was increased by 1.8-fold compared with LB medium only, implying lignin utilization by Bacillus sp. GWD275. Furthermore, when the defined medium with glucose or xylose without complex nitrogen source was used, the growth of GWD275 was also enhanced in the presence of lignin (0.5 g/L), possibly by using lignin as a co-substrate for growth. Interestingly, the molecular weight distribution of lignin in the cultures with the defined medium was shifted to a lower molecular weight portion, while it was shifted to a higher molecular weight portion in cultures with LB medium. The results presenting here would provide an insight for efficient use of limited lignocellulosic bio-resources by lignin utilization using Bacillus sp. GWD275. http://dx.doi.org/10.1016/j.nbt.2014.05.1856 PB-30 Biosynthesis of nylon precursor dodecanedioic acid from fatty acid Liang-Jung Chien ∗ , Szu-Min Yu Ming Chi University of Technology Aliphatic a,w-dicarboxylic acids (DCA) of the type addressed by this program are used in a wide variety of plastics and other chemical applications. The DCA12 produced in the largest quantity (>40 MM lb/yr) as a pure chemical intermediate is dodecanedioic acid (C12); it is used in polyamides such as nylon 6,12, which is noted for high moisture resistance. The dodecanedioic process is based on non-renewable petrochemical feedstocks. The multistep conversion process produces unwanted byproducts such as cyclooctadiene and vinyl cyclohexene, which result in yield losses. The nitric acid oxidation step yields NOx, which is either released S102 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 to the atmosphere or must be destroyed in a reduction furnace. Biotechnology offers an innovative way to overcome the limitations and disadvantages of the chemical processes to make diacids. Yarrowia biocatalyst are able to convert long-chain fatty acids directly to long-chain diacids through overexpressing ␻-oxidation pathway and blocking the ␤-oxidation pathway. This study was developed and demonstrated key biocatalyst to produce costcompetitive long-chain dodecanedioic acid, DCA12. The focus of this study was to increase the rate of conversion of glucose into the corresponding fatty acid feedstocks through overexpress the gene of fatty acid synthesis, Acetyl-CoA carboxylase (AccD) and Fatty acid synthase (FA-1, FA-2, FB-1), in the yeast biocatalyst (Yarrowia lipolytica). In order to enhance DCA12 content, the Yarrowiacodon acyl-carrier protein thioesterase gene, BTE from Umbellularia califoenica, FatB3 from Cocos nucifera were also expressed. Finally, the RNA inference technology was also used to enhance DCA12 production in this research. http://dx.doi.org/10.1016/j.nbt.2014.05.1857 PB-31 Butanol production by metabolically engineered Clostridium acetobutylicum with in-situ butanol removal Sang-Hyun Lee 1,∗ , Min-A Kwon 1 , Yong-An Shin 1 , Kyoung Heon Kim 2 1 2 GS Caltex Corporation Korea University Clostridium acetobutylicum is industrially important strain for butanol production. However, economically feasible production of butanol by wild type Clostridium acetobutylicum is still limited by butanol toxicity and by-product formation, resulting in low butanol yield and productivity. To overcome the cellular toxicity of butanol, we developed fermentation strategy with in-situ butanol recovery (ISBR) by addition of butanol selective synthetic resin to the bioreactor. Also, we constructed the recombinant strain, which are deletion mutant of acid formation pathways to reduce by-products, acetic acid and butyric acid. Fed batch ISBR fermentation profiles showed the increse in yield, butanol selectivity and productivity of butanol production. In continuous fermentation with ISBR for 140 h, the recombinant strain showed that the overall yield, butanol selectivity and volumetric producivity were 35%, 78%, and 2.6 g/L/h, respectively, when feeding 200 g/L glucose solution containing 3% corn steep liquor as a nutrient Acknowledgement: This work was supported by the Center for Organic Wastes to Energy Business under the Environmental Technology Development Program funded by the Ministry of Environment, Korea. http://dx.doi.org/10.1016/j.nbt.2014.05.1858 BIOFUELS, BIOCHEMICALS AND BIOENERGY New Biotechnology · Volume 31S · July 2014 PB-32 PB-33 Production of gaseous biofuels and fine chemicals from food industrial wastes The role of adhe in thermophile ethanol production 1,∗ 2 2 3 , Balazs Balint , Rita Beres , Agnes Kis , Kornel Gabor Rakhely Kovacs 2 , Krisztian Laczi 2 , Andrea Nyilasi 4 , Andras Fulop 2 , Zoltan Bagi 2 , Etelka Kovacs 2 , Gergely Maroti 5 , Katalin Perei 6 1 Institute of Environmental Sciences and Department of Biotechnology, University of Szeged 2 Department of Biotechnology, University of Szeged 3 Institute of Biophysics, Biological Research Center, Insitute of Environmental Sciences, University of Szeged 4 Institute of Biophysics, Biological Research Center 5 Institute of Biochemistry, Biological Research Center 6 Department of Biotechnology, Institute of Environmental Sciences, University of Szeged Food industrial activity is accompanied by the emission of various kinds of organic wastes including protein, fatty and sugarbased materials. These substrates have distinct values, some of them could be utilized as animal nutrients but this is often limited by public sanitation. Protein containing wastes could be used for both biohydrogen and biogas production. Keratin was a substrate of a two-stage procedure for biohydrogen production. Alternatively, various protein substrates could be converted into biogas via a microbial adaptation processes which were monitored by metagenomic approach. Sugar-based biowastes could also be used for either biohydrogen or biogas production and the yield could be stimulated by addition of either nutrients or properly chosen microbes. Both processes were monitored by new generation sequencing based approaches. Fatty acids could be degraded by several microbes. Unctuous wastes were effectively utilized by Rhodococcal strains and the process was monitored by whole cell transcriptome analysis. During fermentation, surfactant molecules were produced which might be utilized in e.g. cosmetics. Food industry and dark fermentation processes produced numerous organic acids which can be utilized either for biohydrogen production or for synthesizing bioplastics. The bioplastic metabolism - also related to CO2 capture - was shown to be in connection with hydrogen metabolism in purple phototrophic cells. Thus, the overall process has benefits in recovery of food industrial wastes in valuable products and also in CO2 capture. Tianyong Zheng ∗ , Daniel Olson Dartmouth College Clostridium thermocellum is a thermophilic, gram-positive obligate anaerobe that is a candidate organism for converting cellulosic biomass into ethanol through consolidated bioprocessing. C. thermocellum has one of the highest rates of cellulose utilization known, but ethanol production in wild type C. thermocellum was reported to be < 30 g/L. This is relatively low comparing to an engineered strain of Thermoanaerobacterium saccharolyticum, which has acheived theoretical yields of ethanol. However, because T. saccharolyticum cannot use cellulose as an energy source, we seek to reconstruct the T. saccharolyticum ethanol production pathway in C. thermocellum. The bi-functional alcohol/acetaldehyde dehydrogenase AdhE was shown to be a key enzyme in ethanol production in many organisms, but this enzyme has never been individually studied in C. thermocellum or T. saccharolyticum. Therefore, this study characterized and compared the activity of AdhEs in different strains of C. thermocellum or T. saccharolyticum, through expression in E. coli and purification. The Km values for substrates and product inhibition were measured, the enzyme specificity for co-factors (NADH/NADPH) were also determined. The end goal of this study is to find a better performing AdhE that can be expressed in C. thermocellum to generate higher yields of ethanol. http://dx.doi.org/10.1016/j.nbt.2014.05.1860 Acknowledgments “The Project is supported by the European Union and co-financed by the European Social Fund (grant agreements no. TÁMOP-4.1.1.C-12/1/KONV-2012-0012, TÁMOP-4.1.1.C12/1/KONV-2012-0014).” http://dx.doi.org/10.1016/j.nbt.2014.05.1859 www.elsevier.com/locate/nbt S103 STEM CELLS, GENE THERAPY, BIOMARKERS AND DIAGNOSTIC TOOLS Stem cells, gene therapy, biomarkers and diagnostic tools PC-01 Lysophosphatidic acid enhanced angiogenic capability of chondrocytes by regulating Gi/NF-kB-dependent angiogenic factor expression Hung-Chih Hsu 1,∗ , Pey-Jium Chang 2 , Chang-Zern Hong 3 1 Chang Gung Memorial Hospital Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University 3 Department of Physical Therapy, Hung Kuang University, Taichung, Taiwan 2 Lysophosphatidic acid (LPA) regulates myeloid differentiation, osteogenesis, cell proliferation and migration, and inhibits apoptosis in chondrocytes. We investigated the effect on the angiogenic capability of human chondrocytes and the underlying mechanism using human chondrocyte cell line, CHON-001, and human vascular endothelial cells (HUVECs). Angiogenic capability was determined by capillary tube formation, monolayer permeability, cell migration and cell proliferation. Angiogenesis protein array kit used to evaluate the angiogenic factors secretion. Angiogenin, insulin-like growth factor-binding protein 1 (IGFBP-1), interleukin (IL)-8, chonocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGF) mRNA and protein expression were evaluated by EIA and Q-RT-PCR, respectively. LPA receptor (LPAR) expression was determined by RT-PCR. Signalling pathways were clarified using inhibitors, Western blot analysis and reporter assays. LPA promotes the angiogenic capability of CHON-001 cells, resulting in enhanced HUVEC capillary tube formation, monolayer permeability, migration and cell growth. Angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9 and VEGF mRNA and protein expression were significantly enhanced in LPA-treated chondrocytes. In addition, LPA2, 3, 4 and 6 were expressed in CHON-001 cells. Pre-treatment with the Gi/o type G protein inhibitor, pertussis toxin (PTX) and the NF-kB inhibitor, PDTC, significantly inhibited LPA-induced angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9 and VEGF expression. PTX pre-treatment also inhibited LPA-mediated NFkB activation, suggesting the presence of active Gi/NF-kB signaling in CHON-001 cells. The effect of LPA on inducing chondrocyte angiogenesis may be due to increased angiogenesis factor expression via the Gi/NF-kB signaling pathway. http://dx.doi.org/10.1016/j.nbt.2014.05.1861 New Biotechnology · Volume 31S · July 2014 PC-02 Genotyping of Campylobacter jejuni using a novel Polymerase-Chain-Reaction-Microsphere approach Ross Barnard 1,∗ , Fang Liang 2 , Lawrence Wong 2 , Anna Weis 2 , Jillian Templeton 3 , Patrick Blackall 4 1 The University of Queensland School of Chemistry and Molecular Biosciences, The University of Queensland 3 Department of Agriculture Fisheries and Forestry 4 Queensland Alliance for Agriculture and Food Innovation, The University of Queensland 2 Campylobacter is a major cause of foodborne disease, with Campylobacter jejuni contributing more than 90% of reported cases. For diagnosis and monitoring of transmission, several genotyping methods have been developed, including multi-locus sequence typing (MLST) [1]. However, there is still a need for fast, cost effective methods for routine analysis. We developed a technique combining allele-specific PCR with a microsphere-flow cytometer system [2]. Seven loci with single-nucleotide polymorphisms (SNPs) with the highest Simpson’s index of diversity (D) were selected from an MLST database. With these loci as a target, multiplex allele-specific PCR was conducted on microspheres in a single reaction and fluorescence signal was detected in a flow cytometer. The signal on the microspheres indicated which allele specific primer was consumed in the PCR. By this means all seven loci could be determined. This approach has a turnaround time of 4 h. Results To date the method has been tested with two strains of Campylobacter jejuni possessing known SNP patterns and six of seven loci have been correctly determined for these strains in a single PCR reaction. Conclusion A new allele specific PCR-microsphere-based method for genotyping Campylobacter jejuni is under development. This approach will be useful in laboratories utilizing PCR and flow cytometers. References [1].Cornelius AJ, et al. Applied and environmental microbiology 2010;76:1533–44. [2].Liang F, et al. Anal Biochem 2013;432:23–30. http://dx.doi.org/10.1016/j.nbt.2014.05.1862 PC-03 Immunocapture and labeling of cd133- and cd54-positive cells by using magnetic microspheres coupled with antibody via oriented immobilization Wen-Chien Lee 1,∗ , Wei-Chih Kuan 1 , Daniel Horák 2 1 National Chung Cheng University Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic 2 Magnetic microspheres coupled with cancer biomarkers can be very useful for clinical cancer diagnosis. Cancer stem cells (CSCs) have been identified as a subpopulation of cells within tumor which are responsible for tumor growth and metastasis. CD133 is considered a marker of CSCs in many malignant S104 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 tumors. Furthermore, to detect the presence of circulating tumor cells (CTSs) in cancer patients, ICAM-1 (intercellular adhesion molecule-1) also known as CD54 is a potential target since CD54 is expressed on endothelial cells in attracting CTCs in blood. CD54 was also found to be highly expressed on cell surface in hepatocellular carcinoma. In the present work, anti-CD133 and anti-CD54 antibodies were separately immobilized on the aminocontaining magnetic poly(glycidyl methacrylate) (NH2 -PGMA) microspheres. Antibody molecules were oxidized on their carbohydrate moieties and bound to the magnetic microspheres via a site-directed (oriented) procedure. Experimental results suggest that the orientation immobilization could preserve the antigen recognizable site of antibody for binding with its antigen. Also, results indicate that antibody molecules were evenly fixed on the surface of magnetic microspheres. A bound antibody density up to 104.2 mg/g-magnetic microspheres was achieved. The anti-CD133 antibody-immobilized microspheres were found effectively for capturing CD133 positive cells from human cancer cell lines. Flow-cytometric analysis confirmed the immunolabeling of CD54-positive cell and the enrichment of CD133-expressing cells by using these antibody-bound magnetic microspheres. STEM CELLS, GENE THERAPY, BIOMARKERS AND DIAGNOSTIC TOOLS improvements over standard PCR in terms of minority profile signal. Results and discussion Our preliminary data show that COLD PCR principle is not fully transferable from SNP (single nucleotide polymorphism) typing to STR typing. Acknowledgements This project was partially supported by grants CZ.1.07/2.3.00/ 30.0004, CZ.1.07/2.3.00/30.0041,and CZ.1.05/2.1.00/01.0030. http://dx.doi.org/10.1016/j.nbt.2014.05.1864 PC-05 The detection of TNT by the periplasmic ribose binding protein RbsB of Escherichia coli is not yet solved Artur Reimer ∗ , Shantanu Roy, Manupriyam Sentchilo, Jan Roelof van der Meer Dubey, Vladimir UNIL http://dx.doi.org/10.1016/j.nbt.2014.05.1863 There exists a wide range of naturally occurring biological sensing systems, which can be combined in biosensors with versatile application possibilities. In addition, it has been proposed that PC-04 the natural range of biological sensing proteins can be expanded through predictions from computational simulation algorithms. Application of CO-amplification at Lower Denaturation A landmark paper in 2003 described the computational simutemperature (COLD) PCR principle to DNA profiling by lation and subsequent construction of mutant periplasmic ribose STR genotyping binding protein from Escherichia coli to create a biosensor capable Tereza Tichá 1,∗ , Jiří Drábek 2 , Filip Kokáš 1 , Karolína Burdová 1 , Jana of detecting TNT (trinitrotoluene). The results from this work sugStránská 1 , Veronika Holinková 2 , Miroslava Rabčanová 2 gested that periplasmic binding proteins can be used as universal 1 scaffolds for new target specificities, which could then be plugged Palacký University, Olomouc 2 Institute of Molecular and Translational Medicine into a unique single E. coli host cell that carries a hybrid chemotaxis/osmoregulation cascade by which de novo gene expression Introduction and aims The sensitivity of DNA profiling can be induced. methods based on microsatellite genotyping process has increased Here we describe an independent reconstruction and examisince the introduction of the first commercial fluorescent mulnation of the scaffold idea and of the quality of the produced tiplex kit through improvements in PCR (polymerase chain TNT biosensor. The reconstructed E. coli hybrid scaffold is an excelreaction) or primer chemistry and low-template modifications lent biosensor for ribose in the nM-range, using wild-type RbsB. In of protocol (i.e. increase of cycle number, post-PCR cleaning of contrast, both in vitro and in vivo results suggest that the mutant amplicons, and/or increase of injection time). These changes with RbsB protein is not able to bind TNT, nor able to induce reporter various successes tried to faithfully generate electrophoreogram gene expression in presence of TNT. In order to better understand representing signals from minute amounts of single or mixed biothe importance of the various amino acid residues in protein foldlogical sample. However, it is sometimes desirable to increase the ing, stability and receptor binding, we substituted each residue of minor profile in the mixture (i.e. to boost a signal of felon over sigRbsB by alanine and measured gene expression through the hybrid nal of victim). In this project, we tested whether CO-amplification signaling pathway. This information will be implemented in the at Lower Denaturation temperature (COLD) PCR that is used in used simulation algorithm to increase the predictive power of the clinical genetics settings able to improve the detection of single calculations. nucleotide mutant tumour DNA in a surplus of wildtype DNA, http://dx.doi.org/10.1016/j.nbt.2014.05.1865 and if so,whether it is amenable to STR (short tandem repeats) typing. Materials and methods DNA was extracted from cell lines and/or EDTA (ethylenediaminetetraacetic acid) treated blood using QiaGen kits, amplified in mixture or unblended either by homemade fluorescent singleplex (locus DXS101) or by commercial multiplex GenomeLab Human STR Primer Set (Beckman Coulter, USA) to see if COLD PCR protocol bring any www.elsevier.com/locate/nbt S105 STEM CELLS, GENE THERAPY, BIOMARKERS AND DIAGNOSTIC TOOLS PC-06 High Performance Computing Based Smart Scan for the Identification of Species Based Unique DNA Sequences Kivanc Bilecen 1,∗ , Behnam Rahnama 2 , Ender Altiok 2 1 2 (1) Okan University, (2) Duzen Laboratories Group Okan University Nucleic acid based tools and techniques such as PCR, RTPCR, DNA microarrays and lately DNA-hybridization-on-a-chip devices provide reliable detection and identification of microorganisms. These platforms rely on the presence of target specific DNA sequences to be known in advance. These sequences, however, can be hard to identify in close species or on the strain level. In this work we have developed a parallel algorithm to accurately specify and classify species and strain specific DNA sequences. The parallel implementation of the intelligent search algorithm runs on the parallel GPGPU cores on an HPC server. Each Kepler K20X computational card provides 2688 fine cores accessing to 6 GB of DDR5 shared memory. Such massive parallelization allows us to compare variable window size of base pairs in an unmatched performance in comparison with conventional methods. The intelligent scan results bidirectional and circular stream search as well as comparing not only the same instance but also similarity scan up to a defined threshold. Well-established CUBLAS library allows comparison of the determinant of sample matrices in portion of microseconds rather than sequential scanning of each base pare. Our algorithm successfully identified unique markers (70 to 120 bp) to differentiate Bacillus cereus and B. subtilis. Locations of these markers on the chromosome have also been taken into account. Our second set will include Salmonella group as this group is highly important for the food industry. Our results will compare marker selection and validation studies, and also their identification power in PCR and RT-PCR. http://dx.doi.org/10.1016/j.nbt.2014.05.1866 PC-07 Influence of analgesia on circulating tumor cells in patients with colorectal carcinoma Hanuš Slavík 1,∗ , Emil Berta 2 , Josef Srovnal 2 , Andrea Prokopova 2 , Lenka Radova 2 , David Vrana 3 , Marian Hajduch 2 1 Palacký University Olomouc Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Czech Republic 3 Department of Oncology, Faculty of Medicine and Dentistry, Palacky University, Czech Republic 2 Introduction: Circulating tumor cells (CTC) can create metastases, which are responsible for 90% causes of deaths at patients with a cancer. CTC are located in a blood stream or bone marrow and they are most common cause of cancer recurrence after surgical resection of the primary tumor. Presence of CTC has become an important prognostic and predictive factor, which is possible to study by molecular methods such as polymerase chain reaction S106 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 (PCR). A correlation between recurrence of disease after surgery and analgesics or anesthetics used during or after surgery was also found. Analgesia and anesthesia can affect amount of CTC because of their immunomodulatory effects. Methods and patients: Influence of morphine and piritramide was studied in 121 patients with colorectal carcinoma in our research. Detection of CTC was based on real-time PCR in samples from peripheral blood and bone marrow with using epithelial genes as markers (carcinoembryonic antigen - CEA and cytokeratin 20 - CK20). Afterward, the presence of CTC was evaluated depending on the type of analgesia and disease-free and overall survival of patients. The main objective was to optimize analgesic techniques after surgery to decrease risk of cancer recurrence. Results: One month after surgery, morphine-based analgesia usually induced higher level of CTC. Disease-free survival of patients was also shorter in case of morphine. Conclusion: Piritramide seems to be better analgesic technique than morphine, which negatively influences prognosis of patients with colorectal carcinoma after surgery. Acknowledgement: This study was supported by grants IGA UP LF 2014 019, CZ.1.05/2.1.00/01.0030 and CZ.1.07/2.3.00/30.0004. http://dx.doi.org/10.1016/j.nbt.2014.05.1867 PC-08 Differential Expression of Circulating miRNAs Following Resistance Exercise and Carbohydrate/Protein Supplementation Foued S. Espindola 1,∗ , Olga Bocanegra 2 , Renata Teixeira 2 , Maria Siqueira 2 , Matheus Gomes 2 , Miguel Diaz 2 1 2 Universidade Federal de Uberlândia Institute of Genetics and Biochemistry - Universidade Federal de Uberlândia We investigated the levels of expression of 12 circulating miRNAs (c-miRNAs) involved in cell proliferation, differentiation, angiogenesis, inflammation and glycemic control in response to resistance exercise (RE) and dietary supplementation. Twelve subjects performed 10 sets of 10 repetitions with 80% of their respective 1RM followed by either carbohydrate or carbohydrate/protein supplementation in a randomized single-blind counter-balanced design. Samples of blood were collected before RE, 03 and 24 hours afterwards. The relative expression data of all of the genes were analyzed using a two-way analysis of variance with repeated measures. The molecular response in the group that supplemented with protein was more pronounced for c-miRNAs involved in the regulation of myogenesis, particularly hsa-miR133a and 503. Both treatments revealed a differential expression of the c-miRNAs hsa-miR-126 and 16, which are associated with angiogenesis. We argue that hsa-miR-133a might be associated with satellite cell proliferation, and possibly partially responsible for muscle hypertrophy following RE. Further, both the up- and down-regulation of hsa-miR-126 and 16, respectively, are likely to reflect neovascularization. These findings support the hypothesis that circulating miRNAs bear paracrine-like functions and thus, are involved in cell-to-cell crosstalk. STEM CELLS, GENE THERAPY, BIOMARKERS AND DIAGNOSTIC TOOLS New Biotechnology · Volume 31S · July 2014 Support: FAPEMIG, CNPq and CAPES http://dx.doi.org/10.1016/j.nbt.2014.05.1868 PC-09 The relationship between fragile sites gene of FHIT in type II pneumocytes and idiopathic pulmonary fibrosis Fei Duan ∗ , Duan Fei, Gu Yu Hebei University Objective: The relationship between fragile sites gene of FHIT in type II pneumocytes and idiopathic pulmonary fibrosis(IPF). Methods: With the techniques of cellular biology, molecular biology and immunocytochemisty (ICC), changes in expression of fragile sites genes, FHIT and WWOX, were investigated to provide a foundation for gene therapy against IPF. Results: 1.WWOX and FHIT were found to be expressed highly in type II pneumocytes of rat in sham-operated group. Compared with sham-operated group, no significant difference in the protein expression of WWOX and FHIT were found in model group for 7 days (P>0.05). After 14 and 28 days, expression of WWOX and FHIT at the protein level was reduced greatly in model group (P<0.05). Nevertheless, tetramethylpyrazine significantly enhanced FHIT and WWOX expression, compared to model group (P<0.05). 2. After 7 days, no significant difference in the mRNA expression of WWOX and FHIT were found between all groups. After 14 days, the expression of WWOX and FHIT at the mRNA level was reduced greatly in model group when compared with shamoperated group. Nevertheless, tetramethylpyrazine significantly enhanced mRNA expression of FHIT and WWOX, compared to model group. Results obtained after 28 days of administration were similar to observation after 14 days. Conclusion: We demonstrate that the expression of fragile sites gene of FHIT and WWOX is altered during IPF. Tetramethylpyrazine suppresses the alteration in FHIT and WWOX, suggesting the medication could play a protective role for IPF. http://dx.doi.org/10.1016/j.nbt.2014.05.1869 PC-10 Phage display as a tool for rapid in vitro cell characterization by fluorescence imaging and Raman spectroscopy Laura De Plano 1,∗ , Federica Calabrese 1 , Germana Lentini 1 , Marco Nicolò 1 , Domenico Franco 1 , Enza Fazio 2 , Sebastiano Trusso 3 , Alessandro Allegra 4 , Fortunato Neri 2 , Salvatore Guglielmino 1 1 Department of Biological and Environmental Sciences, University of Messina Department of Physics and of Earth Sciences, University of Messina 3 C.N.R. Chemical-Physical Processes Institute (Messina) 4 Department of General Surgery, Oncology and Pathological Anatomy, University of Messina 2 The discovery of new markers for the identification and discrimination of cell types such as neoplastic cells is one of the principal objectives in diagnostics. Using random M13 phage display libraries on the whole U937 cells, we selected a clone, named EIII6, displaying a peptide RKIVHAQTP that preferentially recognizes these cells. The human promonocytic cell line U937 is a model for leukemia, cancer therapeutics and in vitro hematopoietic cell differentiation. Therefore, we directly labeled the phage clone EIII6 with fluorescein isothiocyanate (FITC) for microscopy imaging application. This method allowed to identify fixed U937 cells, while preserving binding affinity of labeled phage clones. Microscopic observations showed that U937 cells were fluorescently labeled. In order to further investigate the interaction between U937 cells and EIII6, we utilized Raman Spectroscopy. Spectral peaks observed at about 980, 1008, 1185, 1215, 1320,1340,1455, 1589 and 1660 cm−1 , are commonly assignable to proteins, saccharides and lipids components of U937. These Raman peaks in U937-EIII6 complex are shifted in position or reduced in intensity when compared to U937 alone, suggesting that phage-probe were selectively localized at cell membrane. Thereafter, we realised networks consisting of EIII6 phage clone and Ag-Nanoparticles (AgNPs) as signal reporters in Surface Enhanced Raman Spectroscopy (SERS) to better discriminate U937 cells. With this approach, U937-EIII6 Raman spectral features become sharper and more intense, confirming the selective phage-cell interaction. Both methodological approaches, proposed in this work, allows to quickly and selectively identify U937 and could be extended to the identification of other types of neoplastic cells. http://dx.doi.org/10.1016/j.nbt.2014.05.1870 PC-11 Glycosphingolipids as specific markers during the differentiation of mini-pig bone marrow mesenchymal stem cells toward neuronal cells Young-Kug Choo 1,∗ , Malg-Um Lim 1 , Dong Hoon Kwak 1 , Ju-Hyoung Lee 1 , Sung-Youn Heo 1 , Ji-Su Kim 2 , Sun-Uk Kim 2 , Kyu-Tae Chang 2 1 2 Wonkwang University Korea Research Institute of Bioscience and Biotechnology(KRIBB) For development of specific markers during the differentiation of mini-pig bone marrow mesenchymal stem cells (mpBMSCs) toward neuronal cells, we studied their glycosphingolipid pattern, with particular attention to gangliosides. mpBMSCs contained GD3 etc as major gangliosides. In order study, their distribution in mpBMSCs and its possible change during the differentiation of neuronal cells. When mpBMSCs were cultured under neural differentiation media contained BME/DMSO/BHA, most of mpBMSCs acquired the distinctive morphological features like neural cells. In differentiated cells, expression of neural markers such as neural precursor marker (nestin), neuronal markers (␤-tubulin, neurofilament-M) and astrocyte marker (GFAP) were further demonstrated by revered transcription-PCR, western blotting and immunofluorescence. Specifically, we find that a significant increase in GM1 and GD3 expression was observed during neural differentiation of mpBMSCs. These results suggest that GM1 www.elsevier.com/locate/nbt S107 STEM CELLS, GENE THERAPY, BIOMARKERS AND DIAGNOSTIC TOOLS and GD3 may play a role in the neural differentiation process of mpBMSCs. http://dx.doi.org/10.1016/j.nbt.2014.05.1871 PC-12 AKT and ERK dependent differentiated Mesenchymal Stem Cells survival promoted by Pulse Electromagnetic Field Jung-Keug Park , Enerelt Urnukhsaikhan, Hyunjin Cho Dongguk University Researchers have reported that BM-MSC have ability to differentiate into neuron cells in induction media. Also some studies reported that magnetic field has a role in promoting BM-MSCs differentiation into neural cells. However from in vitro and in vivo studies it is not clear how the methods used induce BMMSCs to differentiate into neural cells. Successfully differentiated the BM-MSCs would be useful for developing clinical stem cell transplantation strategies against central nervous system diseases. After differentiation, cells are no longer live, especially after chemical differentiation. We focus on maintaining cellular survival through the AKT, ERK pathway. In this work, we show the effect of Pulsed Electromagnetic Field (PEMF) on cell survival and differentiation potential of hBM-MSC in vitro. hBM-MSC were cultured with induced medium, and they displayed neuron-like morphology. The cells viability was increased after exposure to 10 mT PEMF for 24 h. Differentiation medium treatment combined with PEMF exposition reduced the death cells rate. NeuroD1, NF-L proteins are expressed those early stage markers of neuronal differentiation and p-ERK, p-AKT after induced by PEMF. Our result is shown that PEMF could play a role in regulating ERK and AKT pathway. Also phosphorylation of AKT and ERK promote a survival effect by inhibiting or regulating the pro-apoptotic, anti-apoptotic proteins such as BAX, BAD and Bcl-xL. Furthermore PEMF exposure could be inducing differentiation of hBM-MSCs. http://dx.doi.org/10.1016/j.nbt.2014.05.1872 PC-13 The dynamics of biofilm formation in clinical strains of Staphylococcus epidermidis and Staphylococcus haemolyticus associated with neonatal infections Monika Grzebyk ∗ , Anna Piotrowska, Monika Brzychczy-Włoch, Piotr Heczko Jagiellonian University Medical College, Chair of Microbiology Introduction: Staphylococcus epidermidis and Staphylococcus haemolyticus are major causes of device-related infections in Neonatal Intensive Care Units. The main virulence factor in S. epidermidis is biofilm formation and S. haemolyticus also form biofilm, but this process haven’t been well decribed jet. Biofilm formation is usually tested in 24- or 48-hour cultures, but the dynamics and the early stages of biofilm formation are still unlcear. S108 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 Aim: To test and analyse dynamics of biofilm formation in clinical strains of S. epidermidis and S. haemolyticus Matherials and Methods: The study covered 10 strains of each S. epidermidis and S. haemolyticus. Overnight cultures in Tryptic Soy Broth (TSB) were diluted 1:100 in fresh TSB and placed in sterile 24-well plates and incubated in 37 ◦ C. After 2 h, 6 h, 12 h, 24 h or 48 h of incubation, the contents of each well were aspirated and the remain biofilm was washed three times with 200 ␮l of phosphate-buffered saline. Biofilms were fixed, dried and then stained with crystal violet. At the end, 96% ethanol was added to each weel and the optical density (OD) of the stained biofilms was measured at wavelength of 600 nm. The measurement was repeated two times and averaged. Fresh uninoculated TSB treated with the same procedure as test samples was used as negative control. Strong biofilm producing Staphylococcus epidermidis strain RP62A was used as positive control. Study was supported by K/DSC/001393 grant. Results and conclusions: Tested strains differed in dynamics and strength of biofilm formation. S. epidermidis strains were stronger biofilm-producers than S. haemolyticus. http://dx.doi.org/10.1016/j.nbt.2014.05.1873 PC-14 A simple pre-analytical tool to enrich bacterial DNA: implications for blood sepsis diagnosis application Ngo Tat Trung , Le Huu Song Tran Hung Dao University Hospital Despite of improved modern medicine, blood sepsis is still remaining as a high risk of mortality. One barrier to therapeutic advancement of the disease is the challenges to accurately identify causative pathogens in a limited time frame. In this content, blood culture is still widely accepted as routine detection approach. However, this conventional method embeds various drawbacks. Nevertheless, the multiplex PCR based detection art, although is considered to be a complimentary approach, it is still confronted by many challenges; of that, the most important factor is the inhibitory effect of human DNA: Imagine that an optimized PCR reaction using DNA extracted by common DNA extraction kits can only sense pathogen’s ribosomal 16S pieces if the bacterial load exceeds roughly 500 CFU/ml, whereas typical blood sepsis patients would have bacterial density of 100–10000 CFU/ml blood, therefore in many case, the PCR is unable to detect pathogen’s genetic materials. To circumvent this problem, various technical prompts have been resorted to deplete human DNA prior to the PCR reaction. A common DNAse-based bacterial DNA isolation Kit (MolYsis) can acquire an enrichment ratio of 40.000 hence allowing to detect 50 S. aureus CFU/ml blood, however, its price is hardly to be accepted in lower income community (about £10/extraction). In the recent study, we introduce a simple pre-analytical protocol that combines the use of polar detergent solvent and basic pH to remove 99% human DNA from patients’ specimens hence making our downstream PCR assays’ technical sensitivity of 10 to100 CFU bacteria/ml blood. http://dx.doi.org/10.1016/j.nbt.2014.05.1874 BIOMEDICAL MATERIALS New Biotechnology · Volume 31S · July 2014 Biomedical materials PD-02 PD-01 Putative motility-related genes in Gluconacetobacter xylinus. Initial verification of their influence on BioNanoCellulose biosynthesis Role of clpP and tpi genes in bionanocellulose biosyntehesis by Gluconacetobacter xylinus Marzena Jedrzejczak-Krzepkowska , Malgorzata Parniewska, Klaudia Jadczak, Michal Rozanski, Katarzyna Kubiak, Przemyslaw Rytczak, Karolina Ludwicka, Marek Kolodziejczyk, Stanislaw Bielecki ∗ Institute of Technical Biochemistry, Lodz University of Technology Among the microorganisms that produce bionanocellulose, acetic bacteria of the species Gluconacetobacter xylinus are the most efficient producers. Bacterial cellulose produced by these organisms have unique properties such as high hydrophilicity, the lack of cytotoxicity, excellent biocompatibility, is non-allergic, not mutagenic nor teratogenic. Due to these properties and possibility of modification the bionanocellulose has found wide application in the food, paper, textile and chemical industry as well as in medicine, e.g. production of wound dressing. This biopolymer has been tested for usefulness as implants of the ears, cartilage implants, a mesh for hernia operation, as well as a material for tracheal reconstruction [1]. Industrial-scale production of bionanocellulose is still difficult and not sufficiently cost effective. These bacteria are susceptible to phenotypic variation, which very often decreases the ability for the biosynthesis of cellulose. Unfortunately, current knowledge about the molecular mechanisms of cellulose biosynthesis and its regulation is insufficient to effectively affect the intensification of biosynthesis (increase efficiency or accelerate the process of production) and change the properties and structure of the obtained cellulose membranes. The aim of current research is to verify the effect of genes such as clpP (encoding ATP-dependent Clp protease proteolytic subunit) and tpi (encoding triosephosphate isomerase) selected in previous studies of transcripts differentiating the cells of G. xylinus synthesizing cellulose (Cel + ) from these non producing cellulose (Cel-) on cellulose biosynthesis. Reference [1].Kowalska-Ludwicka K, Cala J, Grobelski B, Sygut D, JesionekKupnicka D, Kolodziejczyk M, Bielecki S, Pasieka Z. Arch Med Sci 2013;9(3):527–34. Marzena JedrzejczakKatarzyna Kubiak ∗ , Bartłomiej Porebski, ˛ ˛ Krzepkowska, Przemysław Rytczak, Karolina Ludwicka, Marek Kołodziejczyk, Stanisław Bielecki Institute of Technical Biochemistry, Technical University of Lodz BioNanoCellulose (BNC) synthesized by Gluconacetobacter xylinus is intensively investigated as biomaterial for medical applications (best known as wound healing and bone regeneration material) and as a drug delivery system [1,2] but also has potential in various materials preservation (such as skin, paper and textile) and in electronics. The best known commercial usage of BNC is Nata-de Coco dietary dessert popular in Asia. Scientifically BNC gained an attention of tissue engineers, since its features make it a suitable material for scaffolds production [1,3]. However, in some applications porosity of BNC is not accurate for mammalian cells ingrowth (pores diameter < 20 ␮m). One approach to address this issue is genetic modification of G. xylinus in order to obtain strain capable to synthesize BNC with desired features. The aim of this project is to influence the motility of the cells secreting cellulosic fibers. After sequencing the G. xylinus genome [4] we have searched for genes involved in auxiliary motility mechanism and identified two encoding proteins potentially related to it. Subsequently, adequate disruption mutants of G. xylinus ATCC53582 strain were obtained. Motility test, conducted on semi-solid agar plates, showed less intense colony spreading. The initial analysis of mutants’ phenotype revealed inhibition and limited BNC production. Furthermore structural changes in BNC membranes were investigated. References [1].Arch Med Sci 2013;9:527. [2].J Pharm Sci 2012;102:579. [3].Tissue Eng Part A 2010;15:1091. [4].J Biotechnol 2014;176:18. http://dx.doi.org/10.1016/j.nbt.2014.05.1876 http://dx.doi.org/10.1016/j.nbt.2014.05.1875 www.elsevier.com/locate/nbt S109 BIOPHARMACEUTICALS Biopharmaceuticals PE-01 Functions of natural pigments on gastric ulcer and cancer Hyo-Ihl Chang 1,∗ , Sun-Joong Kim 2 , Jee Min Kim 1 1 2 Korea University UC burkeley The Natural pigments have many applications in inflammatory, and oxidative related damage as well as in cancer chemotherapy. Recently, precise cellular roles of natural pigments, such as modulator of key cellular signaling pathway on variety diseases, are elucidated. On based on antioxidant, anthocyanins reduced naproxen-induced gastric ulcer. Anthocyanins reduced the level of lipid peroxidation and increased the level of the antioxidant enzymes. Anthocyanins increased the expression of Nuclear factor E2-related factor 2 (Nrf2) which is transactivator for cellular defense genes. Interestingly, anthocyanins induced gastrointestinal-glutathione peroxidase expression via Nrf2 that bind to regions of antioxidant response element (ARE) in GI-GPX promoter. Otherwise, Shikonin, and genipin stimulates production reactive oxygen species (ROS) in gastric cancer cells. They induced apoptotic cell death in gastric cancer cells in a caspase dependent manner. They also induced cell cycle arrest at G2/M phase via regulation of p21 by early growth response1 (Egr1). The p21 contains promoter region of Egr1 binding motif. Transient expression of Egr1 in AGS cells enhanced shikonin and genipin-induced p21 promoter activity, whereas suppression of Egr1 expression by small interfering RNA attenuated the ability of shikonin and genipin induced p21 promoter activity. Anthocyanins improve gastric ulceration through Nrf2 associated with antioxidant enzymes, such as GI-Gpx pathways. And, shikonin and genipin induced cell damage in AGS cells through the Egr1/p21 pathways. http://dx.doi.org/10.1016/j.nbt.2014.05.1877 PE-02 Effect of P-Protein Inhibition by si-RNA on Melanogenesis in Melan-A Cells Eunki Kim ∗ , E. Noh Inha University Background; Hyperpigmentation diseases, especially melasma and lentigines, are major psychological diseases in most of the societies. The obsession for the clean, fair and healthy skin is one of the basic instincts of every individual. For such diseases, a number of melanogenesis inhibitors have been screened. They were effective but their side effects cause many complications. Pink eye dilution protein (P-protein) is a structural protein in the melanosome that plays a critical role in cellular melanogenesis. S110 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 Objective: The aim of the present study was to investigate the effect of P-Protein inhibition, by using P-protein small interfering RNA (siRNA), on melanogenesis in Melan-a melanocyte. Methods: si-RNA for p Protein was introduced into Melan-A cells. Melanin content, cell viability, PCR and western blot for tyrosinase were performed. Results and Conclusions; It has been observed the both Pprotien and mRNA level were significantly lowered by the siRNA treatment. siRNA of P-Protein also suppressed melanin synthesis without any cytotoxicity in the melan-a melanocyte cells. These results suggest that molecular approaches using siRNA targeting P-protein may provide a novel approach for the control of the melanogenesis http://dx.doi.org/10.1016/j.nbt.2014.05.1878 PE-03 Sex hormones regulate gender-dimorphic hepatic fetuin expression in rats Jong Won Yun ∗ , Sang Woo Kim, Jae Heon Choi, Jung Won Choi Daegu University To date, there are limited studies on the sex-specific relationship between fetuins (Ft-A and Ft-B) and metabolic diseases. Our recent proteomic study has shown that fetuins may play sexdependent roles in obesity and diabetes. In the present study, we investigated the expression of hepatic fetuins with respect to the effects of sex hormones both in vivo and in vitro. A sex hormonetreated rat model was established in order to study the effects of sex hormones on hepatic fetuin expression. Animal experiments revealed that 17␤-estradiol (E2)- and dihydrotestosterone (DHT)treated rats showed opposite effects in terms of body weight gain in both genders. Interestingly, Ft-A and Ft-B were sex-dependently expressed in the livers of rats, responding to different regulatory modes of sex hormone receptors (ER␣, ER␤, and AR). To validate in vivo data, rat normal liver cells were treated with E2 or DHT at different concentrations, and similar expression patterns as those in the animal-based experiments were confirmed. We found that these changes were mediated via sex hormone receptors using antagonist experiments. The results of the present study indicate that sex hormones induce gender-dimorphic expression of hepatic fetuins directly via sex hormone receptors. To the best of our knowledge, this is the first approach to address the effects of sex hormones on hepatic fetuin expression as well as possible roles of fetuins in gender-dimorphic obesity development. http://dx.doi.org/10.1016/j.nbt.2014.05.1879 BIOPHARMACEUTICALS New Biotechnology · Volume 31S · July 2014 PE-04 Immunomodulatory activity of natural polyamines in murine macrophages Sun Chul Kang ∗ , Anil Kumar Chauhan, Souren Paul, Rekha Jakhar Daegu University Macrophages are the key players of innate immunity, defending the body from foreign invaders through phagocytosis. In the present study we investigated the potent role of natural polyamines in modulation of macrophage activity by analysing all the sequential steps involved during phagocytosis. Initially, we treated splenocytes with three natural polyamines (PUT, SPD and SPM) to test their ability in proliferation of cells and involvement in mitosis. Thereafter, we detected their role in promoting membrane fluidity on RAW 264.7 cells, which is essential during uptake of particles. Phagolysosome fusion activity of macrophages in the presence of polyamines was then evaluated by measuring acid phosphatase through p-nitro phenyl phosphate. Further, we determined the capacity of polyamines in generation of superoxide anion to create respiratory burst inside the macrophages. We find decrease in the proliferation of splenocytes in the presence of polyamines which proved their potent role as strong immunomodulator. Uptake capacity of macrophages was observed to be enhanced after treatment with polyamines and also increased lysosomal activity of macrophages was detected at a concentration dependent manner. Percentage of NBT reduction calculated for superoxide anion generation revealed that polyamines potentiated this property of macrophages too. We also determined the anti-complementory activity of polyamines by detecting classical pathway of complement which showed to be effective, suggesting its command over unwanted activation of complement. This study presents the potential role of polyamines as an immunostimulatory drug, which could be effective to treat various immunological disorders. http://dx.doi.org/10.1016/j.nbt.2014.05.1880 PE-05 Thymol protects gastric ulcer induced by alcohol through regulation of matrix metalloprotein 9 activity Anil Kumar Chauhan ∗ , Sun Chul Kang Daegu University In gastrointestinal disorders, ulcer is a common disease with multiple etiologies and most of them are observed due to high consumption of alcohol. Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes capable of degradation of extracellular matrix and are key players in various inflammatory diseases and among them MMP-9 is found to play major role during gastric inflammation. In this study, we aimed to determine the roles of thymol in expression of MMP-9 during ethanol induced gastric ulcer model in vivo. Sprague-Dawley rats, pretreated with thymol (10 mg/kg) or normal saline as control were subjected to intragastric administration of 95% ethanol (5 ml/kg). Morphological examination includes ulcer index for hemorrhage and hematoxylin and eosin staining was performed to analyze the severity of ulceration. Gelatin zymography was done to determine the expression of MMP-9 and a number of biological and immunological tests were carried out to determine the antioxidant and cytokine levels. Rats challenged with alcohol, developed severe injury in gastric mucosa with increased level of pro-inflammatory mediators like TNF-␣, Prostaglandin E2 and nitric oxide. Expression of MMP-9 was up-regulated and less production of antioxidant enzymes (SOD and GSH) was documented after treatment with alcohol. In thymol pretreated rats, a significant decrease in ulcer index, level of pro-inflammatory cytokines and nitric oxide was observed. Expression of MMP-9 was downregulated and antioxidant enzymes production was increased in thymol pretreated group. In conclusion, study suggests that thymol protects gastric mucosa injury from ethanol consumption by down-regulation of MMP-9 and inflammatory cytokines. http://dx.doi.org/10.1016/j.nbt.2014.05.1881 PE-06 7-Hydroxydehydronuciferine induces human melanoma A375.S2 autophagy and apoptosis and inhibits metastasis in vitro and in vivo Hui Min Wang Kaohsiung Medical University Melanoma is the deadliest cancer. We identified 7hydroxydehydronuciferine (7-HDNF) isolated from the leaves of Nelumbo nucifera Gaertn cv. Rosa-plena to be a bio-active agent against human melanoma A375.S2 cells. 7hydroxydehydronuciferine (7-HDNF) was known to induce autophagy and apoptosis response mechanisms, and antimigratory activity of melanoma in vitro and in vivo. Cell proliferation assay was used to test cell viability. Acridine orange (AO) staining and flow analysiswere applied to observe cell morphology. The apoptotic cell death ratio was measured via two-dimensional flow cytometry by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double stained. Western blot was applied to examine protein expressions whereas wound healing assay was to examine cell activity. Strong anticancer effects of 7-HDNF were exhibited in a dose-dependent mannerand displayed minor cytotoxicities on normal human skin cells.7-HDNF induced the formation of intracellular vacuoles and the augmentation of acidic vesicular organelles (AVO). 7-HDNF increased the cellular arrest in cell cycle at G2/M phase. Cellular membrane asymmetry loss was confirmed. Protein expressions were discovered to verify autophagy and apoptosis response mechanisms sharing the associated pathways. 7-HDNF presented the high-quality anti-migratory activity. 7-HDNF inhibited melanoma tumor growth in mice xenograft model, accompanied with a decrease of phosphorylation of AKT. We demonstrated the mechanism of this compound starting with the formation and accumulation of AVO leading to autophagy. 7-HDNF caused the cellular membrane asymmetry loss, triggering the G2/M cell cycle arrest in caspase-dependent apoptosis. 7-HDNF presented highwww.elsevier.com/locate/nbt S111 BIOPHARMACEUTICALS New Biotechnology · Volume 31S · July 2014 quality anti-migratory bio-functions and inhibited melanoma tumor growth in mice xenograft model. http://dx.doi.org/10.1016/j.nbt.2014.05.1882 PE-07 Oleuropein attenuates visceral adiposity in high fat dietinduced obese mice through the modulation of WNT10band galanin-mediated signalings Taesun Park ∗ , Narae Kuem, Sujin Song Yonsei University The aim of the present study was to investigate the antiobesity effect of oleuropein on high-fat diet (HFD)-induced body weight gain and visceral adiposity in mice, and to explore the underlying mechanisms involved. C57BL/6 N mice were fed with the normal diet (ND), high-fat diet (HFD; 40% fat of total energy) and HFD-supplemented with 0.03% oleuropein (OD) for 10 weeks. OD-fed mice significantly reduced HFD-induced body weight gain and visceral adiposity. Oleuropein also significantly reversed the HFD-inducedelevations of adipogenic-related gene expression involved in WNT10b- and galanin-mediated signalingsin adipose tissue of mice. Consistent with in vivo findings, oleuropein dose-dependently suppressed lipid accumulation in 3T3-L1 cells during preadipocyte diffentiation. Additionally, exposure of the 3T3-L1 preadipocytes to oleuropein resulted in a marked attenuation of the galnon (galanin receptor agonist) or SFRP2 (WNT inhibitor)-induced cellular lipid accumulation. This study demonstrated that oleuropein reduced body weight gain and visceral adiposity in HFD-fed mice. The protective effect of oleuropein against HFD-induced adiposity in mice appeared to be mediated through the upregulation of genes involved in WNT10bmediated signaling and downregulation of genes involved in galanin-mediated signaling cascades. http://dx.doi.org/10.1016/j.nbt.2014.05.1883 PE-08 Biologically active sycamore bark triterpenoids from birch and Lucie Borkova 1,∗ , Milan Urban 2 , Marian Hajduch 3 , Jan Sarek 1 1 Department of Organic Chemistry, Faculty of Science, Palacky University in Olomouc, Czech Republic 2 Department of Organic Chemistry, Faculty of Science and Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University in Olomouc, Czech Republic 3 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University in Olomouc, Czech Republic Triterpenes are natural compounds usually occurring in plants or marine animals and are often used in natural medicine in Asia. They have various biological activities (e.g. anti-HIV).[1] A large number of triterpenes are cytotoxic against wide range of tumor cell lines, their anticancer activity was often observed in preclinical S112 www.elsevier.com/locate/nbt animal models.[2,3] Therefore, they become promising candidates for new drug development. The main interest of our group is to modify lupane and oleanane derivatives in order to improve their pharmacological properties and to increase their therapeutic index. Whereas des-E ␤-ketoacid (des-E lupane derivative) is our most active semisynthetic triterpenoid (cytotoxic on a set of 30 cell lines including MDR phenotypes with IC50 0.32 ␮mol/L on K562) causing fast and selective apoptosis, the A-ring modified betulin and betulinic acid derivatives that we prepared later have a completely different mechanism of action. In order to obtain another hit compounds, we synthesized a set of derivatives with various substituents in the position 2 of the skeleton and found them to be highly cytotoxic on T-lymphoblastic leukemia CCRF-CEM cell line. The mechanizm of action is still being investigated; however, we are able to give some basic assumption about the structure–activity relationships. References [1].Dzubak P, Hajduch M, Vydra D, Hustova A, Kvasnica M, Biedermann D, Markova L, Urban M. J Sarek Nat Prod Rep 2006;23:394–411. [2].Salvador J-A-R. Pentacyclic Triterpenes as Promising Agents in Cancer. Hauppauge NY: Nova Science Publishers, Inc; 2010. [3].Murph M. Research on Melanoma. InTech; 2011. http://dx.doi.org/10.1016/j.nbt.2014.05.1884 PE-09 Intranasal Delivery of Nanoparticles Containing nButylidenephthalide to Treat Malignant Brain Tumor Tzyy-Wen Chiou 1,∗ , Yu-Shuan Chen 1 , Yu-han Chiu 1 , Yuan-Sheng Li 1 , Dean-Kuo Hsieh 2 , Horng-Jyh Harn 3 1 Graduate Institute of Biotechnology,National Dong Hwa University, Hualien, Taiwan 2 Department of Applied Chemistry, Chaoyang University of Technology, Taichung, Taiwan 3 Department of Medicine, China Medical University, Taichung, Taiwan Malignant brain tumor is a highly invasive disease with a very high death rate. The effective treatment method for this disease is still an unmet medical need. Intranasal delivery method is a noninvasive administration route, may bypass the blood brain barrier and reduce the required drug dosage. n-Butylidenephthalide (BP) has previously been shown as a drug candidate for treating glioblastoma multiforme (GBM) and temozolomide-resistant GBM. However, its hydrophobic property may limit some of the applications. Therefore, in this study, emulsification method was used to prepare BP-containing nanoparticles in order to provide desirable features. The obtained nanoparticles showed high permeation ability as tested by artificial cellulose membrane or nasal septum squamous cells. The BP-containing nanoparticles also exhibited cytotoxic effect in human brain glioblastoma multiforme (GBM 8401) with IC50 vaule of 85 ␮g/ml. Moreover, the nanoparticles delivered by aerosol could reduce the tumor size in transgenic GBM mice model after the treatment for 30 days. Based on these results, it is suggested that the preparation of BP-containing nanoparticles and its application in nasal BIOPHARMACEUTICALS New Biotechnology · Volume 31S · July 2014 adminstration to treat GBM may have the potential in further development for clinical use. http://dx.doi.org/10.1016/j.nbt.2014.05.1885 PE-11 Investigation on preventive measures of virus diseases Zinaida Klestova 1 , Marina Marchenko 2 , Viktor Tashuta 2,∗ , Alla Voronina 3 PE-10 The chemical synthesis of sulfur analogues of lysophospholipids and cyclophospholipids Przemyslaw Rytczak ∗ , Maria Koziołkiewicz, Andrzej Okruszek Lodz University of Technology, Institute of Technical Biochemistry Development of new synthetic methods for the preparation of biologically active phospholipid derivatives is a challenging problem of membrane-chemistry and biochemistry today. In fact structural and dynamic studies of biomembranes for the establishment of structure-activity relationships, phospholipids-proteins interactions and mechanisms of action of phospholipids metabolizing enzymes require the preparation of a great number of phospholipids derivatives as the key step in advancing membrane biochemistry. Lysophospholipids have recently become the focus of special attention since it was discovered that in addition to their role in phospholipid metabolism they function as second messengers, exhibiting a broad range of biological activities in their own right. The chemical synthesis of new sulfur analogues of lysophospholipids has been described, including phosphorothioate/phosphorodithioate derivatives of lysophosphatidic acids (LPA), phosphorothioate/phosphorodithioate derivatives of cyclic phosphatidic acids (cPA) and phosphorothioate/phosphorodithioate derivatives of lysophosphatidylcholine (LPC). For the preparation of LPA, cPA and LPC derivatives both oxathiaphospholane and dithiaphospholane approaches have been employed. Each lysophospholipid analogue has been synthesized as a series of five compounds, bearing five different fatty acid residues, both saturated (12:0, 14:0, 16:0, 18:0) and unsaturated (18:1). Acknowledgements: This work was supported by a grant (PBZ-MNiSW-07/I/2007) from the Polish Ministry of Science and Higher Education and by a grant (011/01/B/ST5/06383) from the National Science Center (NCN-Poland). http://dx.doi.org/10.1016/j.nbt.2014.05.1886 1 State scientific-control institute of biotechnology and strains of microorganisms 2 the Institute of veterinary medicine 3 The Institute of pharmacology and toxicology Viruses that caused diseases worldwide spread and have possibility for mutations. More dangerous viruses attack animal and human organisms, causing economic losses. Many countries are creating biosafety systems for preventing viral infections, for example, by developing new prophylaxis and antiviral methods. We investigated the possibility of using some plants and dosage forms in anti-viral therapy in sensitive model systems. In the investigation we used: continuous cell culture of versenised swine embryonic kidney and BHK-21; test-model virus-member of Coronaviridae family; propylenglycol extracts of plants- Aloe vera, Camellia sinensis var. ɑssamica, Echinacea purpurea, Húmulus lúpulus. Results of experimental research of cytotoxic and antiviral action of preparations of a different origin are presented. MTD is10−5 mkl/ml. Index of CC50 -10−4 mkl/ml. In experiments on cell cultures we obtained the data that showed antiviral activity to 1,8 ± 0,04lg TCID50 /ml. http://dx.doi.org/10.1016/j.nbt.2014.05.1887 PE-12 In vitro assay by bioengineering of new antiviral drugs Viktor Tashuta 1,∗ , Zinaida Dgebuadze 4 Klestova 2 , Alla Voronina 3 , Shota 1 Institute of Veterinary Institute The State scientific control institute of microorganisms strains 3 The Institute of pharmacology and toxicology 4 Georgian Technical University 2 The viral diseases are still increasing. There is a wide spectrum of tested antiviral compounds, application of which inhibits or stops viral activity, but universal antiviral means are absent. The investigation was aimed to finding the new approaches of the infection eradication. Methods: test-model viruses–members of Herpesviridae and Coronaviridae family; continuous animal cell cultures:–cell culture of versenised swine embryonic kidney (CCVSEK), BHK-21, Vero, SK-6. For investigation of anti-virus properties we have applied a complex of standard methods. Results of experimental research of cytotoxic and antiviral actions of 10 new substances from indol derivates are presented. We show the data about all examined means that significantly reduced the infection activity of tested virus strains in vitro systems in different ways. We tested different schemes of antivirus actions of tested substances in creation of treatment by viral infection process, as preventive as treatment. But the highest antiviral activity www.elsevier.com/locate/nbt S113 BIOPHARMACEUTICALS among tested substances was shown by decreased viral infection activity to 7,04 ± 0,04 lg TCID50 /ml. Conclusion: All tested means are suitable in using for clinical research for testing in eradication virus infections. The results confirm the perspective of tested means for production of new antiviral drugs. S114 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 The results were obtained through STCU grant # p450 by financial support of KCP. http://dx.doi.org/10.1016/j.nbt.2014.05.1888 BIOPROCESSING AND ENGINEERING New Biotechnology · Volume 31S · July 2014 Bioprocessing and engineering PF-01 RAMOS: New applications of an established online technology for shaken vessels Tibor Anderlei 1,∗ , Tim Bürgin 2 , Andreas Richter 2 , Cedric Bürki 3 , Wolf Klöckner 4 , Kristina Meier 4 1 Forum Shaking Technology Kühner AG 3 ExcellGene SA 4 AVT Aachen 2 RAMOS determines the oxygen transfer rate (OTR), the carbon dioxide transfer rate (CTR) and the respiratory quotient (RQ) of microbial, plantal and cell cultures online. The respiration rates (OTR, CTR) are the most suitable measurable variables to quantify the physiological state of fermented cultures. RAMOS is the right tool to meet the PAT initiative of the FDA regarding shaken bioreactors. On the one hand RAMOS was made to measure in 250 mL glass shake flasks. Therefore the application field was limited. To overcome this bottle neck Kühner developed three new add-ons: - MicrOTR: With this tool the user is able to measure OTR and CTR in microtiter plates. The main application of this tool is to find the right fermentations conditions for microtiter plates. - Adapter for disposable flasks: With this new development disposable flasks can be directly attached to the RAMOS-System. Therefore the RAMOS system can now easily be used for the application field cell cultivation. On the other hand the application field was enlarged by applying the RAMOS technology for testing of plastic components to determine their impact on the cultivation. All the new features and their advantages will be presented and results from microbial, plant and mammalian cell cultures will be shown. http://dx.doi.org/10.1016/j.nbt.2014.05.1889 PF-02 Online Monitoring of pH, Oxygen and Backscattered Light during Heterologous Protein Production in Disposable Shake Flasks Gernot Thomas John 1,∗ , Christian Ude 2 , Thomas Scheper 2 , Sascha Beutel 2 , Michael Findeis 1 , Damian Andrzejewski 1 1 2 PreSens Precision Sensing GmbH Institut für Technische Chemie, Leibnitz Universität Hannover Keywords Single-Use shake flask, online backscattered light, shakes flask reader, protein production, inclusion bodies Shake flask cultivation is one of the classical methods to perform proliferation of cells with low effort and cost. It provides precultures for the scale up and is suitable for standard screening routines like the evaluation of new media or cultivation conditions. Estimation of the optimal culture conditions needs a continuous analysis concerning biomass data, growth rate, oxygen concentration and pH. In the context of this work a multisensory platform (shake flask reader) was evaluated by monitoring of three basic cultivation parameters (pH, pO2 , and biomass) [1]. The biomass sensor is detecting the cell growth by backscattered light through the flask wall at a wavelength of 625 nm. The measurement is fully non-invasive and the measuring interval can be reduced to a minimum of 7 s. The sensor signal is calibrated against CDW or OD600 by application of logarithmic functions. With the given calibration it is used e. g. for screening procedures in order to determine the adequate moment of induction. Backscattered light is also sensitive to changes in particle size and opacity. Thus there is evidence that inclusion body formation within the cells can be monitored online. [1] Ude C., Beutel S., Findeis M., Andrzejewski D., John G. T., Scheper T. (2013): Online biomass monitoring of shake flask cultures via an optical multisensory platform, Dechema Frühjahrstagung 2013, Frankfurt am Main, Germany 2013 http://dx.doi.org/10.1016/j.nbt.2014.05.1890 PF-03 Developing strategies to improve the recovery of a periplasmicaly expressed recombinant protein by manipulation of fermentation conditions Ioannis Voulgaris 1,∗ , Alex Chatel 1 , Gary Finka 2 , Mark Uden 2 , Mike Hoare 1 1 2 University College London GlaxoSmithKline A significant problem for the clarification of E.coli broths is the amount of cytoplasmic DNA which may have been released into the extracellular space by cell lysis. This leads to large increases in viscosity which for example can make removal of cell solids difficult. E.coli is not a natural protein secretor; hence, a degree of cell lysis is required for the product to be released from the periplasm to the extracellular space. Changes in the relationship between protein and DNA release favouring protein release should favour the performance of the subsequent separation stages. Therefore, the balance between product and DNA release must be carefully monitored and if possible controlled during the fermentation. We present strategies to facilitate a better recovery of an antibody fragment from recombinant E.coli whilst diminishing the extent of the effect of nucleic acid release. These include by: (i) the manipulation of the cell growth rate in order to increase the outer cell membrane permeability (ii) the incorporation of reagents within the broth designed to increase the cell membrane permeability, (iii) the incorporation of reagents within the broth designed to remove selectively nucleic acids released during cell lysis. Using an ultra-scale-down approach we have determined the maximum level of DNA release into the culture while still allowing satisfactory removal of cell solids by continuous flow pilot scale centrifugation. In this way a strategy may be developed integrating www.elsevier.com/locate/nbt S115 BIOPROCESSING AND ENGINEERING fermentation operation and cell recovery to optimise the recovery of protein product in a well-clarified broth suitable for subsequent processing. http://dx.doi.org/10.1016/j.nbt.2014.05.1891 PF-04 Aqueous Phase Partitioning–from Analytics to Process Jonathan Huddleston ∗ , Rana Hameed, Derek Fisher, Svetlana Ignatova Brunel Institute for Bioengineering The presentation will focus on aspects of our work on the application of Aqueous Phase Partitioning both to the development of liquid-liquid extraction based approaches to the recovery of biopharmaceuticals and to the development of a simple bioprocess analytical technology. We will consider the recovery of monoclonal antibodies using Counter Current Chromatography and highlight important differences in the dynamic flow regimes of different configurations of equipment and their impact on the structural integrity of biomolecules. In addition, consideration will be given to some of the different operational regimes available in this type of equipment and how these can impact upon overall process efficiency. In contrast we will also present some aspects of our work to understand the molecular basis of phase partitioning in the development of partitioning based assays having a strong structurally related component. We will show from a theoretical and practical perspective how the technique of Analytical Phase Partitioning may be applied to the quantitative determination of process derived changes and post-translational modifications of biopharmaceutical products. http://dx.doi.org/10.1016/j.nbt.2014.05.1892 PF-05 Simulation of butanol production by an integrated fermentation process using supercritical fluid extraction with carbon dioxide Bernadete Delgado 1,∗ , Fernando Luiz Pellegrini Pessoa 2 1 2 Federal University of Rio de Janeiro UFRJ Petroleum is becoming a scarce resource and alternative biofuels has been studied as biobutanol. It can be blended directly with standard oil-based fuels and has the advantage of been produced from glycerol. Nowadays glycerol is an excellent source of raw material derived from biodiesel production. Clostridium pasteurianum uses glycerol as the main source of carbon but is productivity and butanol concentration is low. The main problem related to fermentation for butanol production is its toxicity to the microorganism Clostridium. Downstream process for recovering solvents in the diluted fermentation broth may increase butanol productivity by reducing the toxicity of solvents to the S116 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 cells and minimize the energy consumption in the process. In this work a conceptual design of downstream process using supercritical fluid extraction with carbon dioxide and solvent extraction was compared with traditional distillation process for butanol recovery in the broth by simulation. The raw material used in fermentation was glycerol and the main products of the metabolism of C. pasteurianum were butanol and 1,3 propanediol. Supercritical extraction with carbon dioxide was applied to remove 1,3 propanediol and glycerol then solvent extraction using n-butyl-butyrate was applied for butanol recovery. An integrated process using single supercritical extraction was also developed. Simulations were performed using SuperPro Design®. An economic evaluation was carried out to compare these systems. The integrated process using supercritical fluid extraction with carbon dioxide was an economically attractive scenario, although, butanol has a lower purity than this process followed by solvent extraction with butyl butyrate. http://dx.doi.org/10.1016/j.nbt.2014.05.1893 PF-06 Assessing the volumetric productivity of an ultrafiltration membrane bioreactor during the synthesis of galacto-oligosaccharides Andres Córdova ∗ , Carolina Astudillo, Andres Illanes, Cecilia Guerrero Pontificia Universidad Católica de Valparaíso Galacto-oligosaccharides (GOS) are a potent prebiotic which allows the upgrade of underutilized lactose of the dairy industry. However, this industry is somehow reluctant to use enzymes, due to their high-cost owing to the high-volumes of lactose processing that entails. Ultrafiltration membrane bioreactor (UF-MBR) allows the bioconversion of lactose into GOS, removing the reaction products while retaining the enzyme for re-use in a single step. The objective of this research was to evaluate the effect of operational variables on volumetric productivity (␲) of GOS synthesis when using an UF-MBR. A tubular ceramic-membrane (50 kDa) and retentate recirculation mode was used. The reacting mixture was a lactose solution (40%w/w, pH 4.5) containing Aspergillus oryzae ␤-galactosidase dosed at 50IU/glactose . The temperature (40 ◦ C to 60 ◦ C), the transmembrane pressure (PT) (2.5 to 4 bar) and cross-flow velocity (CFV) (3.5 to 7 m/s) were varied according to a 2k factorial design. All reactions were conducted for 240 min. Lower conversions were obtained at the higher levels of CFV and PT, and at 40 ◦ C.This can be attributed to a greater shear-force on the enzyme, and more compaction of the solute on the membrane which may adsorb the biocatalyst, decreasing its activity. At 40 ◦ C partial precipitation of lactose and a higher permeate viscosity may explain this situation. An optimized ␲ value of 30.53 (g GOS/L x h) was achieved at 60 ◦ C, 3.5 m/s and 2.5 bar, increasing ␲ by 318% (compared to a conventional batch-synthesis) by using enzyme in two reaction-cycles. BIOPROCESSING AND ENGINEERING New Biotechnology · Volume 31S · July 2014 Work funded by Grants 1130059 and 11110402 FONDECYT, and CONICYT-PhD-Scholarship, Chile http://dx.doi.org/10.1016/j.nbt.2014.05.1894 PF-07 Continuous photofermentative production of hydrogen with Rhodobacter sphaeroides DSM 158 bio- Karsten Helbig ∗ , Rico Hellwig, Felix Krujatz, Thomas Bley, Jost Weber TU Dresden Because of the coming lack of fossil energy sources and to mitigate the global climate change there is a need for an environmentally friendly fuel. Hydrogen could be produced from nearly omnipresent water and its utilization does not cause the emission of environmentally harmful pollutants (like CO2 ). Light is a highly available source of energy. Thus, hydrogen production with phototrophic microorganisms has a potential to make a significant contribution to use this energy and produce a renewable fuel. For industrial application there is a need to establish a continuous process. The aim of this study was to examine conditions for a continuous hydrogen production with purple-non-sulfur bacteria. Rhodobacter sphaeroides is able to produce molecular hydrogen with the enzyme nitrogenase. For the study of production conditions a 1-L stirred glas bioreactor was operated as a chemostat and equipped with 12 radial installed 50-w-tungsten lamps for light supply. The dilution rate has been varied to determine its influence onto the hydrogen production rate. Throughout the experiments the influence of different dilution rates onto hydrogen production and the biomass concentration has been determined. Dilution rates have been regulated from 0.024 to 0.216 h−1 . At a dilution rate of 0.12 h−1 the highest production rate (152 mL h−1 L−1 ) and a biomass concentration of 2.15 g L−1 were observed. Neither higher nor lower dilution rates improved productivity. The higher the dilution rate the smaller was the concentration of biomass. The shown data show the potential of Rhodobacter sphaeroides for a continuous photo-biological hydrogen production. http://dx.doi.org/10.1016/j.nbt.2014.05.1895 PF-08 Optimization of medium composition for the novel pectin lyase producer Rhizomucor pusillus DSM 1331 through response surface methodology Amira Rizk 1,∗ , Sonja Diercks-Horn 2 , Mahmoud Yousef 2 , Marcelo Fernández-Lahore 2 1 Jacobs university Bremen Downstream Bioprocessing Laboratory, School of Engineering and Science, Jacobs University, Campus Ring 1, D-28759 Bremen, Germany 2 Pectin lyase is a member of pectinases that plays an important role in food processing industries (e.g. juice clarification). In this study, the influence of solid state fermentation medium compositions containing wheat bran, sugar cane bagasse and lemon peel powder as agricultural by-products for pectin lyase production using Rhizomucor pusillus DSM 1331 were accessed. Response surface methodology (RSM) was used for medium optimization, fermentation conditions, and the interactions among all the experimental parameters at flask level. Several physico-chemical parameters: pH, temperature, moisture content and fermentation time of the production media were optimizedby using the Doptimal Design. The results show that pectin lyase production was significantly affected by fermentation time and moisture content which stimulates themaximal enzyme production to 100 U/mL and specific activity of 45.24 U/mg at 30 ◦ C temperature; 6 days of fermentation and moisture content of 120%. Under these optimized conditions, the predicted maximal activity was 107 U/mL. The obtained activity was two times higher than some of the common pectin lyase producers. Additionally, the fermentation process was scaled up from 10 g to 1 kg using a rotating drum type solid-state bioreactor in order to evaluate the difference between pectin lyase production in flask and at bioreactor level. In conclusion, the application of response surface methodology had a noteworthy enhancement in pectin lyase production indicating this method was a promising approach for enzyme production and cost reduction http://dx.doi.org/10.1016/j.nbt.2014.05.1896 PF-09 Advanced clarification of cell culture supernatant by 3MTM EmphazeTM AEX Hybrid Purifier for fast and economic bioprocessing of recombinant proteins Michael Maurer 1,∗ , Frederik Hoppe 2 , Harald Schillinger 3 , Melanie Hutter 2 , Renate Kunert 4 , Kurt Eyer 3 1 FH Campus Wien/School of Bioengineering FH Campus Wien/School of Bioengineering/ACIB 3 3 M Alpine/3 M Purification 4 University of Applied Life Sciences an Natural Resources Vienna 2 Mammalian cells are the most important expression platform for the production of biopharmaceutical proteins. More than 50% of all registered products are produced in those expression systems according to Ferrer et al. During this work, we used a CHO cell line secreting the fusion protein EPO-Fc for the simulation of a biopharmaceutical manufacturing. For pharmaceutical products it is of particular interest to reduce and eliminate biological contaminates, such as genomic DNA, host cell protein, endotoxins and viruses by efficient DSP operations. On the other hand, biopharmaceutical companies are in a harsh economic competition, what generates a strong need for shorter, save and economical processes (reduction of unit operations and the rise of single use systems). In this study we evaluated the new 3MTM EmphazeTM AEX Hybrid Purifier, which combines depth filtration with an allsynthetic construction, substantial chromatographic capability, and a defined 0.2 ␮m pore size in one single-use cartridge, for clarification and purification very early in the manufacturing process. www.elsevier.com/locate/nbt S117 BIOPROCESSING AND ENGINEERING Further we compare the performance to a state of the art depth filter, 3MTM ZetaPlusTM EXT, and show the scalability from lab scale (25 cm2 ) to pilot scale (340cm2 ). Data on a dramatic reduction of gDNA and a significant reduction of HCP by the new 3MTM EmphazeTM AEX Hybrid Purifier will be presented. The advantage of early removal of biological contaminates and the protection of chromatographic material will be discussed. Ferrer-Miralles N., Domingo-Espín J., Corchero J., Vázquez E., Villaverde A. Microbial factories for recombinant pharmaceuticals. Microbial Cell Factories 2009, 8:17 http://dx.doi.org/10.1016/j.nbt.2014.05.1897 PF-10 Adhesion of anaerobic microbial beer spoilers to stainless steel Gita Prochazkova ∗ , Milan Bittner, Martina Brozova, Tomas Branyik Institute of Chemical Technology Prague To reduce the public health threat posed by food/beverage pathogens or spoilers many sources of contamination have to be considered. Increased focus is necessary in the case of anaerobic microorganisms. In the case of breweries, the highest risks are associated with the genus Pectinatus, but also the genus Megasphaera must not be neglected. Due to their ability to form or to be a part of microbial biofilms that are present on the surfaces of pipelines, floors, machinery etc. these microorganisms pose a continuous threat to beer contamination. The focus of the presented work is help localize conditions/materials prone to colonization by anaerobic bacteria and thus to prevent undesirable biofilm formation in breweries. The work can be divided into three parts: (i) characterization of the physicochemical properties (contact angles and zeta potentials) of two anaerobic bacteria (Pectinatus frisingensis and Megasphaera cerevisiae) and of typical construction material’s surfaces (stainless steel), (ii) prediction of the cell adhesion to stainless steel according to thermodynamic, classical and extended colloidal models, and (iii) comparison of model predictions with experimental data from real adhesion tests carried out in test tubes with model surfaces (stainless steel microparticles) under model conditions. The (in)consistency between model predictions and adhesion experiments can help identify the crucial interactions for microbial adhesion. http://dx.doi.org/10.1016/j.nbt.2014.05.1898 S118 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 PF-11 Whisky by-products: a valuable source of protein and potential applications in aquaculture Jane White ∗ , Julio Traub, Dawn Maskell, Paul Hughes, Alan Harper, Nik Willoughby Heriot-Watt University Scotland is famous for its whisky, a tradition stretching back over 500 years. There are over 100 malt distilleries in Scotland with the capacity to produce 280 million litres of pure alcohol annually. This results in the generation of by-products, one of which is pot ale, the liquid residue remaining after the first distillation stage. At least 8 litres of pot ale is produced with every litre of alcohol. It contains over 30% protein on a dry matter basis, originating from yeast and barley residues and its main value is as pot ale syrup, an animal feed produced by evaporation. Evaporation is energy intensive, the high temperatures have a deleterious effect on protein quality, all components in addition to the protein are concentrated and it is only an option for larger distilleries. New markets for pot ale could be realised if cost-effective methods to separate the protein components were available. These proteins are a good match with the protein requirements of salmon feed and may offer a local, sustainable and secure supply of protein for Scottish salmon. The work presented here demonstrates the development of novel processes for the energy-efficient extraction and recovery of valuable proteins. The composition of pot ale, methods for producing novel by-products and potential applications in aquaculture are discussed. http://dx.doi.org/10.1016/j.nbt.2014.05.1899 PF-12 Pilot-scale biotrickling filter for hydrogen sulfide removal from biogas under continuous discharge flow Martín Ramírez ∗ , Fernando Domingo Cantero Almenglo, José Manuel Gómez, University of Cadiz Biogas is a valuable renewable energy source. However, the average H2 S concentration (0.1-2%) into the biogas stream limits many applications. Biotrickling filters (BTFs) have been studied under aerobic and anoxic (nitrate like electron acceptor) conditions. In the conventional mode operation, the liquid phase is recirculated from bottom to the top of the column and a portion volume or small split of the flow is discharge to help to remove the oxidation products (sulfate and elemental sulfur). In this study, a pilot-scale BTF (diameter column 0.5 m, bed height 0.85 m, packing material: open-pore polyurethane foam) was feed with real biogas and treated water (TW) from a wastewater treatment plant (WWTP). The aim of this work was study the performance under discharge flow equal to the recirculation flow. Moreover, the mass transfer coefficient was determined. Nitrate concentrate solution was mixed with TW stream at the top of the BTF. The trickling flow rate was set to 1.7 m3 h−1 , biogas BIOPROCESSING AND ENGINEERING New Biotechnology · Volume 31S · July 2014 flow rate were of 1, 2 and 3 Nm3 h−1 and nitrate mass flow rate were of 20 and 70 g N-NO3 2– h−1 . The elimination capacities (EC) were equal under both nitrate mass flow rate. The critical EC (H2 S removal efficiency (RE) of 99%) was 37.9 gS m−3 h−1 and the maximum EC was 88.4 gS m−3 h−1 . However, the nitrate consumption was superior at the highest nitrate mass flow rate, probably due to the higher gradient concentration and therefore the higher mass transfer to the biofilm. http://dx.doi.org/10.1016/j.nbt.2014.05.1900 PF-13 Chromatographic Separation of Mono-PEGylated Teriparatide Isomers Wen-Yih Chen 1,∗ , Ching-Wei Chang 2 , Ruoh-Chyu Ruaan 1 1 2 Tasi 1 , Wei-Hung Kao 1 , Li-Chiao National Central University Scinopharm Inc., Taiwan Peptide drugs covalently conjugated with polyethylene glycol (PEG) polymers can effectively prevent protease digestion and prolong the circulation half-life. Chromatographic purification of PEGylated peptide drugs is essentially critical in pharmaceutical industry, especially for the positional PEGylated peptide isomers. In this study, we aimed to interpret the separation mechanisms of mono-PEGylated Teriparatide isomers by reversed-phase chromatography (RPC) and further to provide guidance for a better chromatographic separation of PEGylated isomers. Two monoPEGylated Teriparatide isomers, N-terminal and Lys13 PEGylated Teriparatide, were synthesized through the succinimidyl ester functionalized methoxy PEG (5 KDa) by controlling the pH of PEGylated reaction buffer. Both two PEGylated Teriparatide isomers exhibit high protease stability against trypsin, but their structural helicity decrease dramatically. However, two PEGylated Teriparatide isomers are co-eluted by acetonitrile/H2 O containing of 0.1% (v/v) trifluoroacetic acid. To separate these isomers, two approaches were carried out. One is to tune the pH value of mobile phase. The results showed that two positional isomers are gradually separated as the pH value increased from 2.0 to 9.0. The other is to alter the eluent composition. Based on the solubility parameters theory (thermodynamics theory), we changed the eluent composition from acetonitrile/H2 O to tetrahydrofuran/H2 O, resulting in baseline separation of PEGylated peptide isomers. Both approaches revealed that the difference of polarity term (Ddp ) between peptide isomers plays an important role for baseline separation. Consequently, we suggested that the tuning of mobile phase composition based on solubility parameters theory makes it possible to achieve the separation of PEGylated isomers in chromatographic operation. http://dx.doi.org/10.1016/j.nbt.2014.05.1901 PF-14 A comparative study of chromatographic matrices for affinity chromatography of the diphtheria toxin variant Cross-Reacting Material 197 Alessandra Stefan ∗ , Mattia Hochkoeppler Boiani, Luca Longanesi, Alejandro University of Bologna Cross-Reacting Material 197 (CRM197) is a variant of the diphtheria toxin characterised by a single mutation, a glycine-glutamic acid substitution at position 52 [1]. This mutation reduces its toxicity but maintains the same inflammatory and immunostimulant properties. The conventional industrial-scale production of CRM197 is performed using cultures of Corynebacterium diphtheriae, but recently we have proposed an alternative process for its over-expression in Escherichia coli [2]. Accordingly, the recombinant protein, bearing a short artificial histidine tag, was purified by a metal chelating affinity chromatography (IMAC), performed either under denaturing and native conditions. In order to investigate possible non-specific interactions between CRM197 and the matrix employed for the IMAC, we compared three types of polymers: a matrix consisting of agarose beads (GE Healthcare Life Sciences), a macroporous silica matrix (Machery-Nagel) and the Profinity IMAC resin, based on UNOsphere beads (Bio-Rad). The agarose-based matrix showed the higher non-specific sorption of the CRM197 protein, leading to a low final recovery. On the contrary, the Profinity IMAC resin proved the maximum yield, likely due to a low retention of the protein in the column via non-specific interactions with the matrix. Moreover, the Profinity resin was found to be suitable for both the purification and the refolding of CRM197 and, accordingly, the recovered protein featured enzymatic activity. References [1].Uchida T, Pappenheimer Jr AM, Greany R. J Biol Chem 1973;248:3838–44. [2].Stefan A, Conti M, Rubboli D, Ravagli L, Presta E, Hochkoeppler A. J Biotechnol 2011;156:245–52. http://dx.doi.org/10.1016/j.nbt.2014.05.1902 PF-15 A two-step one-pot bioprocess for production of 11␣hydroxyandrost-4-ene-3,17-dione from phytosterol Dmitry Dovbnya 1 , Vyacheslav Kollerov 2 , Sergey Danila Malov 1 , Marina Donova 2,∗ Khomutov 1 , 1 G.K. Skryabin’s Institute of Biochemistry and Physiology of Microorganisms RAS 2 Pharmins Limited 11␣-Hydroxyandrost-4-ene-3,17-dione (11␣-HAD) is a primary adrenal steroid in mammalians and the key precursor in the syntheses of halogenated corticoids (pharmaceutically valuable analogs of natural corticosteroids). Conventional routes for its obtaining are based on chemical synthesis, or microbial hydroxwww.elsevier.com/locate/nbt S119 BIOPROCESSING AND ENGINEERING New Biotechnology · Volume 31S · July 2014 ylation of androst-4-ene-3,17-dione (AD). AD in turn is produced primarily with microbial biotransformation of natural sterols by some actinobacteria. The aim of this work was to develop a bioprocess for obtaining of 11␣-hydroxyandrost-4-ene-3,17-dione from phytosterol. Specific biochemical activities of two microbial strains were used as a basis for the two-stage bioprocess. On the first stage phytosterol was converted to AD by Mycobacterium sp. NRRL 3805B. The conditions of biotransformation were optimized to get approx. 70% molar yield of AD from 12 g/l of the substrate. On the second stage AD accumulated in the biotransformation broth was regioand stereo-specifically hydroxylated into C-11-alpha position by the mycelial fungus Aspergillus ochraceus VKM F-830Y. Cultivation conditions for preparation of the fungal biocatalyst with higher specific activity and mode of the biocatalyst application were optimized. Both biotransformations were carried out in a single laboratoryscale bioreactor thus allowing exclude AD isolation and purification procedures. The two-stage bioprocess provided 6568% molar yield of 11␣HAD from phytosterol for 65-72 h. The product was separated and purified to 95% by step-wise crystallization and re-crystallizations from a system of polar organic solvents. For our knowledge, microbial production of 11␣-HAD from phytosterol was not so far reported. http://dx.doi.org/10.1016/j.nbt.2014.05.1903 PF-16 Plasmid DNA purification by integrating membrane technology with arginine affinity chromatography João Queiroz ∗ , Catherine António Morão, Fani Sousa Nunes, Ângela Sousa, José Nunes, University of Beira Interior The implementation of clarification and purification processes to isolate the supercoiled (sc) plasmid isoform at industrial scale becomes crucial. In the present study, membrane filtration technology was performed to isolate and clarify the sc plasmid DNA (pDNA) from lysates. Microfiltration process was implemented to eliminate the suspended solids and to perform a diafiltration of the solution, followed by an ultrafiltration technique to concentrate the plasmid and to remove the different types of RNA [1]. Finally, a suitable chromatographic strategy is essential to remove residual impurities and to obtain the sc pDNA as a highly pure product. Affinity chromatography with amino acids as ligands, such as arginine, has been employed for this objective due to its high selectivity for the sc isoform and also because of the mild elution conditions required to achieve its purification [2]. Thereby, the sample resultant from the ultrafiltration process was applied in the arginine chromatographic matrix, to attain an adequate strategy for sc pDNA purification. The nature of the arginine support and the use of moderate salt concentrations render this operation more economically sustainable and viable to be used in large scale systems. The separation of sc isoform was proved by electrophoretic and HPLC analysis. Overall, the integration of membrane technol- S120 www.elsevier.com/locate/nbt ogy with affinity chromatography to efficiently purify the pDNA results in a powerful tool for industrial manufacturing. References [1].Nunes JC, et al. Journal of Membrane Science 2012;415-416:24–35. [2].Sousa A, et al. Journal of Separation Science 2009;32:1665–72. http://dx.doi.org/10.1016/j.nbt.2014.05.1904 PF-17 Establishment of Efficient Microalgal Harvesting Technique by the Concomitant Application of Red or Blue Light Wavelength with Chitosan Dae Geun Kim 1,∗ , Yoon-E. Choi 2 1 Department of Bioprocess Engineering, Chonbuk National University LED Agri-bio Fusion Technology Research Center, Chonbuk National University 2 Microalgae are considered to be one of the most promising feedstocks for biodiesel, due to their rapid growth and high lipid content. However, microalgal harvesting is one of indispensable step claiming almost 20-30% of total biomass production cost. Chitosan is a natural, non-toxic, polycationic polymer with multiple applications in pharmaceuticals, food, agricultural, and chemical industries. There are multiple lines of reports that chitosan can also be a promising alternative flocculants for microalgal harvesting. In our previous study, light-emitting diodes(LEDs) especially red and blue color were demonstrated to govern the specific microalgal cell biology. Blue light illumination led to significantly increased cell size, whereas red light resulted in small-sized cell with active divisions. Based on that, in this study, we attempted to establish a novel harvesting strategy of microalgal biomass by the concomitant applications of both blue light illumination and chitosan. We successfully proved that microalgal cells cultivated under blue light settled more rapidly than those of biomass cultivated under red light illumination. Next, the combinationary effects of different light wavelengths and chitosan concentration were thoroughly tested. The data suggested that the optimal harvest condition under the blue light with chitosan were significantly deviated from those under the red light with chitosan. Our strategy, based on the illumination of blue light in conjunction with chitosan, will contribute to setting up the future biomass harvest process using microalgae. http://dx.doi.org/10.1016/j.nbt.2014.05.1905 PF-18 Optimization of an industrial primary protein recovery process by Design of Experiment Tanja Buch ∗ , Ian Marison Dublin City University The primary recovery of proteins at the end of a fermentation process can be considered as a keystone for the overall protein loss during purification. Recombinant proteins are widely produced in BIOPROCESSING AND ENGINEERING New Biotechnology · Volume 31S · July 2014 batch and fed-batch fermentations at industrial scale since the early 1980’s using Escherichia coli or other microbial cells. The main challenge remains in the isolation and recovery of the protein from the fermentation broth at the end of the fermentation, which leads to increase loss in the protein of interest, especially at manufacturing scale. The primary protein recovery process of an industrial E.coli fermentation at large scale was characterised based on the protein concentration and protein mass balances were set up to identify critical process parameters. A loss of more than 80% of the recombinant protein was observed occurring at two main critical process steps (CPS). A major problem at the critical process steps was identified to be the solubility levels of the recombinant protein. The protein solubility at the CPS was optimized using Design of Experiment (DoE) with a central composite face-centred design. Statistical analysis was applied to determine significant factor interactions and to identify optimal process conditions. The optimization of the solubility of the recombinant protein led to a four-fold improvement in the recombinant protein recovery and an enhancement of the overall process yield. http://dx.doi.org/10.1016/j.nbt.2014.05.1906 PF-19 Production of ethanol and biomass from thin stillage using edible Neurospora intermedia Jorge Ferreira ∗ , Patrik R. Lennartsson, Mohammad J. Taherzadeh University of Borås Thin stillage is a prime candidate for improvement of the industrial ethanol process. Production of biogas, cell oil, eicosapentaenoic acid and biomass for animal feed has been investigated using thin stillage. In this work, the edible ascomycete fungus Neurospora intermedia was investigated for production of ethanol and biomass from mostly wheat-derived thin stillage. Aeration rate influenced the production of ethanol and biomass during cultivation in a 26 L capacity airlift reactor; the highest amount of ethanol (3.2 g/L) was obtained at lower aeration rate of 0.5 vvm, while the highest amount of biomass (9.2 g/L) was obtained at 2 vvm. The reactor was also used as a bubble column. Similar amounts of ethanol (3.5 g/L) and biomass (5.0 g/L) were obtained. N. intermedia was also investigated in continuous mode in the bubble column; dilution rates up to 0.2 h−1 could be used without cell wash-out. At dilution rate of 0.1 h−1 , 5 g/L of ethanol and 4 g/L of biomass containing 50% protein were obtained. The solid content in the thin stillage was reduced by 18%. The inclusion of this process using N. intermedia can lead to a 5.5% improvement on ethanol production considering a facility producing 200,000 m3 ethanol/year. The produced ethanol can be sent to the beginning of the process and follow the main stream towards the distillation column. The high value biomass can be used for animal feed e.g. fish feed, while the reduction of solids can have positive effects on energy savings and water recirculation. http://dx.doi.org/10.1016/j.nbt.2014.05.1907 PF-20 Sustainable Manufacture of Industrially Relevant Platform Chemicals Using Microbial Bioprocesses Laura Jeffrey 1,∗ , Alison Arnold 2 , Brian McNeil 1 , Linda Harvey 1 1 2 University of Strathclyde Ingenza Ltd As petroleum stocks decrease on a global scale, it is essential that industry decreases its dependency on oil and petroleum based materials in favour of more sustainable resources. Biologically produced chemicals could provide a sustainable route for the manufacture of high value monomers. Such biological processes should have high productivities, use simple media, be operable at large scale, produce a final process fluid with a high concentration of suitable bio-product which can easily be recovered and easily integrated into further chemical conversions. At present, no such system is in place within the industrial biotechnology sector. This presents the unique opportunity to develop a novel process wherein amino acids can be converted into high value chemicals. Corynebacterium glutamicum, a Gram positive, non-sporulating bacterium was chosen as a model organism. Since its discovery it has become an industrial workhorse in the production of amino acids, especially L-glutamate. For this process to be industrially competitive, efficiency is paramount. Therefore, production of L-glutamate was examined in batch processes using standard Lglutamate induction conditions including; biotin limitation, heat induction and ethambutol addition. As biotin limitation exhibited a 10 fold yield increase during batch conditions compared to other chosen methods a fed-batch process was developed where balancing carbon and nitrogen was crucial. Strain screening under optimal production conditions was employed to increase productivity further. Once a sufficient titre was achieved, downstream processing on the culture broth could be examined, converting this relatively low value amino acid into a high value, desirable platform chemical for industrial purposes. http://dx.doi.org/10.1016/j.nbt.2014.05.1908 PF-21 Overproduction of glutathione by recombinant Escherichia coli expressing bifunctional glutathione synthetase Zhimin Li East China University of Science and Technology Glutathione is an important bioactive substance being applied in pharmaceutical and food industries widely. Traditionally, the production of glutathione was conducted in yeast system. In the present study, a recombinant Escherichia coli strain expressing the gene gshF coding for bifunctional glutathione synthetase was used as a producer and the fed-batch cultivation was investigated. During this process, glucose was the sole carbon and energy source. Without extra addition of three amino acids, 0.79 g/l of glutathione was obtained and the productivity was 56 mg/l/h. With www.elsevier.com/locate/nbt S121 BIOPROCESSING AND ENGINEERING the addition of 75 mM glutamic acid, cysteine and glycine, 11.3 g/l glutathione was formed with a productivity of 628 mg/l/h, which were 13 and 10 times respectively higher than those in the absence of amino acids addition. http://dx.doi.org/10.1016/j.nbt.2014.05.1909 New Biotechnology · Volume 31S · July 2014 PF-23 The use of enzymes in the coating industry– environmentally friendly strategies for curing and hydrolysing coatings Katrin Greimel 1,∗ , Veronika Perz 1 , Karolina Haernvall 1 , Enrique Herrero Acero 1 , Georg Guebitz 2 PF-22 1 acib Gmbh University of Natural Resources and Life Sciences, Institute of Environmental Biotechnology 2 Isolation of microorganisms for biosurfactant production Franco Liporace 1 , Carla Quevedo 1,∗ , Ana María Giulietti 2 , Juan Olivera 1 1 2 Facultad Regional Delta-UTN-Argentina Facultad de Farmacia y Bioquímica-UBA-Argentina Microorganisms with surfactant producing ability were isolated from hydrocarbon-contaminated soil and water in order to carry out processes for biosurfactant production.The samples were obtained from a petroleum distillery (RHASA) located at Campana, (Argentina). Isolation was performed by enrichment cultures in a mineral salt medium (MSM) containing 4,5% of a mixture of three different hydrocarbons (HC) or polluted lagoon water (AgLag) as both carbon and energy source. The strains isolated were cultured in 250 ml erlenmeyer flasks containing 25 ml of MSM supplemented with HC (pH 7.00) and incubated at 25 ◦ C ± 2 ◦ Cin orbital shaker at 150 rpm for 7 days. The biosurfactant producing ability of isolated microorganisms was estimated by emulsification capacity, surface tension measurement using Du Nouy ring tensiometer, oil spreading techniques, drop collapse method and haemolytic activity. Only six of the isolated strains were able to reduce the growth media surface tension in more than 40%. One of them, Ag.A.1HC strain, was cultured in a stirred tank bioreactor (NewBrunswick BioFlo115) containing 2500 ml MSM medium with a mixture of HC, operating at 200 rpm and 25 ◦ C ± 2 ◦ C. Biomass concentration and surface tension were evaluated during the process. A decrease in the surface tension value of the cellfree supernatant was observed. The initial and final values were 46,85mN/m and 34,90mN/m, respectively. The highest cell concentration was found between day 5 and 7 of culture. According to these results the Ag.A.1 HC strain, isolated from hydrocarboncontaminated water would have a potential use in both production of biosurfactant and bioremediation processes of environments contaminated with hydrocarbons. http://dx.doi.org/10.1016/j.nbt.2014.05.1910 The use of enzymes in industrial processes has become more and more common. For the coating industry, it could be envisaged that enzymes will replace siccatives, paint removers or acticides. We investigated the potential use of laccases as siccatives for coatings containing alkyd resins as well as the potential use of hydrolases as paint removers. Alkyd resins are binders for coating formulations containing unsaturated fatty acids. During the hardening process the fatty acids are cross-linked. Cobalt complexes are the most widely used siccative systems, but because they are suspected to be carcinogenic, they need to be replaced. The potential of a laccase from Trametes hirsuta in combination with two mediators was evaluated regarding its capability to crosslink the unsaturated fatty acids [1]. The drying reaction was evaluated using different methods, like FTIR spectroscopy, oxygen measurements, GC chromatography and drying time recorder measurements. The removal of coatings is usually performed using harsh chemicals or mechanical strength. Also in this case the development of environmentally friendlier methods will sooner or later be of great importance. We investigated the possibility of using enzymes for the hydrolysis of polyester based coatings [2]. Two enzymes - a lipase from Thermomyces lanuginosus and a cutinase from Humicola insolens–showed different activities in hydrolysing two different model substrates for polyester based coatings. References [1].Greimel, et al. Green Chemistry 2013. [2].Greimel, et al. Reactive and Functional Polymers 2013. http://dx.doi.org/10.1016/j.nbt.2014.05.1911 PF-24 Hollow fibre membranes as a high mass transfer gas diffusing system for microbial CO fermentation Muhammad Yasin ∗ , Shinyoung Park, Yeseul Jeong, In Seop Chang Gwangju Institute of Science and Technology (GIST) Recent research on the biological conversion of carbon monoxide (CO) and hydrogen (H2 ) into multi-carbon compounds have revealed that syngas fermentation is one of the potential alternatives for the production of more sustainable fuels and chemicals. However, poor mass transfer of the sparingly soluble gaseous substrates (CO and H2 ) and low cell density in the fermentation media are the big hurdles in the commercialization of the technology. These issues can be resolved by using the membrane based S122 www.elsevier.com/locate/nbt BIOPROCESSING AND ENGINEERING New Biotechnology · Volume 31S · July 2014 reactors (MBR) instead of most widely employed stirred tank reactors (STR) and less common bubble column reactors (BCR). The purpose of this study was to develop a simple lab scale hollow fiber membrane bioreactor (HFMBR) for addressing the issues of mass transfer and kinetic limitations in syngas fermentation. The first phase of the research has been completed and a hydrophobic PVDF (Polyvinylidene fluoride) membrane has been successfully utilized to achieve high mass transfer. The performance of the system has been examined by measuring the gas-liquid volumetric mass transfer coefficient (kL a). Pressure and membrane surface area have been used as two controllable factors to achieve high mass transfer. We have found a kL a of 135.72 h−1 under 13.6psi transmembrane pressure at AS /VL (membrane surface area/working volume of the liquid) = 0.27 cm−1 . High kL a of 155.16 h−1 was achieved by increasing AS /VL to 0.62 cm−1 under lower transmembrane pressure of 5.4psi. that can be obtained in characterizing microbial populations using multiple molecular methods. http://dx.doi.org/10.1016/j.nbt.2014.05.1912 Overflow metabolism is a significant problem in many industrial bioprocesses, which can lead to decreased productivity as well as total process failure. Avoiding excessive overflow metabolism while maintaining a high feed rate and hence productivity is therefore highly desirable. This is complicated by difficulties in modelling and online sensing in industrial processes using complex media. The current study employs a method for online estimation of the metabolic state in relation to overflow metabolism, based on the response to sinusoidal perturbations in the feed rate. This method requires only measurement of dissolved oxygen, for which robust and precise measurement devices are universally available in industrial bioprocesses. The method is based on directly studying the effects of the saturation in oxidative metabolism causing overflow, meaning that it allows estimation of metabolic state regardless of which substrates are used. The study includes a number of experiments in industrial production-scale fermentations (>100 m3), using an industrial Bacillus licheniformis strain and a complex starting medium. Firstly, these have shown that for feed rate perturbations in a certain frequency interval fermentor mixing dynamics can be approximated by a simple model and that the perturbations do not have a negative impact on growth and productivity. Secondly, they have shown that the method’s estimation of the current metabolic state is consistent with offline measurements of main substrate and byproduct. The method therefore provides a useful measure of the metabolic state, which can be used for both online diagnosis and closed-loop control in industrial fermentations. PF-25 Evaluation of dynamic microbial communities in a styrene-degrading biotrickling filter using 16S rDNA tag pyrosequencing and denaturing gradient gel electrophoresis Kevin Portune ∗ , María Carmen Pérez, Francisco Javier ÁlvarezHornos, Carmen Gabaldón University of Valencia Accurately characterizing microbial communities within bioreactors undergoing dynamic operating conditions is an essential first step towards understanding the relationship between microbial community structure and bioreactor performance. A detailed assessment of the changes in microbial populations within a styrene-degrading biotrickling filter was carried out using samples collected at multiple time points ranging from 21 to 155 days of biotrickling filter operation. Examination of microbial populations was conducted by 16S rDNA tag pyrosequencing and denaturing gradient gel electrophoresis (DGGE). Validation of pyrosequencing results was performed by quantitative polymerase chain reaction (qPCR) in order to examine the relative changes in percentages of selected taxonomic groups. Pyrosequencing results revealed a predominance of bacteria assigned to the phylum Proteobacteria for all sampling time points in the bioreactor. Relative fluctuations in percentages of total bacterial sequences assigned to selected taxonomic groups detected by pyrosequencing during biotrickling filter operation were confirmed by qPCR. Pyrosequencing revealed substantial changes in the community structure between sampling time points, with observed differences in microbial diversity indices and operational taxonomic units (OTUs) among certain samples. DGGE further revealed shifts in the dominant microbial species during changes in biotrickling filter operational parameters. The application of several different molecular tools to examine changes within microbial populations from bioreactors allows a more detailed view of the community structure as compared to using only one molecular method. This study highlights both the complementary as well as contrasting information http://dx.doi.org/10.1016/j.nbt.2014.05.1913 PF-26 Online estimation of metabolic state in industrial-scale bioreactors through the dissolved oxygen response to feed rate perturbations Ola Johnsson 1,∗ , Jonas Hägglund 1 Andersson 2 , Gunnar Lidén 3 , Tore 1 Lund University, Department of Automatic Control Novozymes A/S 3 Lund University, Department of Chemical Engineering 2 http://dx.doi.org/10.1016/j.nbt.2014.05.1914 www.elsevier.com/locate/nbt S123 BIOPROCESSING AND ENGINEERING PF-27 Improved scale down of industrial fermentations by application of oxygen enrichment on lab scale Rogier Meulenberg ∗ , Rogier Meulenberg, Jeroen Van Santen, Erik Van der Lucht, Wouter Van Winden DSM Biotechnology Center Lab scale improvement programs of industrial fermentations require a good scale down of the process. Only then it is possible to test process adaptations under representative conditions and, moreover, scale up of the improved process will be faster and more successful. In the absence of headspace overpressure (in case of glass fermenters) and negligible hydrostatic pressure, oxygen transfer capacity in lab scale fermenters is often lower than on industrial scale. Therefore, process intensity needs to be decreased for proper scale down. For such a scale down approach it is crucial that the limiting factor on industrial scale is the same as on lab scale. If not, production strain physiology may be different and representativeness of the lab scale process is lost. However, in some cases it turned out to be difficult to confirm that the same limiting factor was maintained after process intensity scaling. In contrast to this, lab scale oxygen transfer capacity can also be increased to industrial levels by application of oxygen enrichment of the sparged air. With this approach, the saturating oxygen solubility on lab scale can be increased to similar levels as on industrial scale at overpressure. In some cases this prevents the need for decreasing process intensity for a proper downscale. Examples of different scale down approaches will be discussed. http://dx.doi.org/10.1016/j.nbt.2014.05.1915 PF-28 Rapid prototyping meets bioreactor–a novel small-scale organic light emitting diode based photobioreactor with integrated sensor systems Felix Krujatz 1,∗ , Karsten Fehse 2 , Matthias Jahnel 2 , Thomas Bley 1 , Jost Weber 1 1 2 TU Dresden Fraunhofer COMEDD Microalgae represent a promising raw material for various industries due to their wide range of valuable ingredients. By photosynthetic processes carbon dioxid is fixed under the influence of light and converted into products like proteins, fats, oils or pigments. Despite the great potential of algae research only 150 of the estimated 400,000 algae strains are used for industrial applications. State of the art of microalgal cultivation is the illumination with inorganic semiconductor elements known as light emitting diodes (LEDs). However, this technology has disadvantages like an inhomogeneous ligth distribution (point light source), a strong self-heating and thus the need to integrate cooling elements. Because of these limitations no phototrophic screening system S124 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 for the small-scale cultivation under defined conditions could be developed so far. Rapid prototyping allows the design and manufacturing of finestructured reactor components. Here, we present the first rapid manufactured cultivation system for phototrophic microorganisms with a working volume of 12 milliliters equipped with organic light emitting diodes (OLED) as lighting source and an optical process monitoring sensor system. OLED light technology is characterized by a very homogenous light distribution (area light source) and low self-heating. Important process parameters of microalgal cultivation like pH, pO2 or pCO2 can be monitored online by integrated optical sensor spots. Moreover, cell-specific parameters likeoptical density and fluorescence signals (e.g. marker proteins or chlorophyll fluorescence) are detected online by optical readout approaches (LED-excitation and spectral sensitive photodiodes). Thus, this reactor-setup represents an ideal screening and process optimization tool for photo-biotechnological processes. http://dx.doi.org/10.1016/j.nbt.2014.05.1916 PF-29 Bacterial growth modeling for rapid fermentation process development using a parallel mini-bioreactor system Aarron Erbas 2,∗ , Tony Allman 1 , Frank Baganz 2 1 2 Infors AG UCL In order to establish a generic framework for the rapid development and optimisation of scalable fermentation processes a novel methodology will be explored which integrates small scale fermentations with model-based experimental design and predictive control strategies. In the first instance mathematical models will be developed to predict microbial growth kinetics. Most commonly the first order kinetics logistic and Gompertz models are comparatively used to assess the model fit to empirical data. Optical density measurements may provide a quick off-line analysis of the growth curve of microbial populations, as compared to cell plate counts or dry weights that require more time. Here we propose a modified four parameter logistical model for batch growth of E. coli w3110 in a minimal medium. The mean square error (MSE) was used to measure the fit of the empirical model to the experimental data that were obtained using a quadruple parallel mini-bioreactor system (Multifors). The average optical density after 23 hours was 4.46 + /- 0.09 (n = 4) demonstrating excellent reproducibility. The best MSE for the model was MSE = 0.034 showing specifically a good fit to the log and stationary phases of bacterial growth but also enabling fitting to the lag phase with a best MSE = 0.0028. Such models have the potential to predict typical microbial cell growth and thereby aiding the development of advanced fermentation control strategies. http://dx.doi.org/10.1016/j.nbt.2014.05.1917 BIOPROCESSING AND ENGINEERING New Biotechnology · Volume 31S · July 2014 PF-30 A novel lab-scale two-stage reactor for biogas production through the use of efficient and stable microbial consortia Martyna Ciezkowska 1,∗ , Krzysztof Poszytek 1 , Otton Roubinek 2 , Jacek Palige 2 , Aleksandra Sklodowska 1 , Lukasz Drewniak 1 1 Laboratory of Environmental Pollution Analysis, Faculty of Biology, University of Warsaw 2 Institute of Nuclear Chemistry and Technology The anaerobic digestion is very fragile and sensitive process, that requires to maintain the balance among different microbial populations. To achieve stable biogas production different source of microorganism were tested and a variety and numerous technical solutions were developed. The main aim of this work was verification and optimization of a novel anaerobic digesters for laboratory studies of two-phase biogas production with the use of efficient and stable microbial consortia. The developed biogas production system consists of two reactors, one dedicated to hydrolysis process (2L tank operated in 30 ◦ C) and second designed to fermentation (25L tank operated in 37 ◦ C). Both reactors use hydraulic agitation and operate in a quasi-continuous mode. The optimization of biogas production with the constructed reactors was carried out with the use of stable microbial consortia isolated from fermentation tank of biogas plant and was conducted in four phase until the stabilization of methane production. For each phase the bioreactors were supplied with maize silage from 1 to 5% DM increasing during process. During methane fermentation the following parameters were analyzed: CH4 content; biogas yield, concentration of VFAs and COD, and microbial community structure and activity changes. The conducted analyses showed that the microbial consortia have effective and stable production of biogas after 20 days of processes and constructed reactors are perfect for the analyses of each step (hydrolysis/acetogenesis and methanogenesis) of two phase biogas production process. http://dx.doi.org/10.1016/j.nbt.2014.05.1918 PF-31 Methods for removing endotoxin contaminants from biotechnological chondroitin Mariacarmela Marseglia ∗ , Paola Diana, Alberto Alfano, Katia Della Corte, Rosaria Di Nuzzo, Chiara Schiraldi seconda università degli studi di Napoli Endotoxins released from the cellular membranes of gram negative bacteria during their growth constitute dangerous pyrogens to be removed in case of biotechnological production of pharmaceuticals, expecially in case of parenteral drugs. Polysaccharide-based pharmas have wide biomedical applications but also these products have to be endotoxin free to become suitable for specific uses. Purification of mannans, for example, was performed with size separation techniques (gel filtration or ultrafiltration). Bearing in mind the importance of biotechnological production of glycosaminoglycans the aim of this research was to develop new methods for purifying the capsular polysaccharide of Escherichia coli K4 (chondroitin-like) in order to obtain a pyrogenfree product with high recovery yields and purity grade. This product may than be suitable for preclinical in vitro evaluation of potential biological activity. During fermentation these bacteria release into the culture media, together with the K4 capsular polysaccharide, the lipopolysaccharide molecules that result the main contaminant during the downstream process. The removal of the lipopolysaccharides is difficult because of their amphiphilic nature and because of structural similarities between its oligosaccharidic portion and the capsular polysaccharide chain. Strategies based on activated charcoal either in powder form and/or packed disks where compared to newly developed solvent phase separation processes. In both case pyrogen free chondroitin of 30 KDa, with a purity >98% was recovered. http://dx.doi.org/10.1016/j.nbt.2014.05.1919 PF-32 Single-use bioreactors for microbial application Nico Oosterhuis 1,∗ , Stefan Junne 2 , Peter Neubauer 2 1 CELLution Biotech Chair of Bioprocess Engineering, Institute of Biotechnology, Technische Universität Berlin, Germany 2 Nowadays single-use bioreactors are fully accepted in the biopharmaceutical industry. Reactors up to 2000 L working volume are commonly used. However, these bioreactors are limited in terms of mass-transfer and mixing capabilities and therefore only suited for application in mammalian cell culture. Single-use processing offers same benefits for microbial processes as for mammalian processes. As microbial expression systems are widely use in the biopharma industry, there is a strong need for single-use bioreactors applicable for microbial processes as well. The CELL-tainer® technology, based on a 2-dimensional rocking motion is available now with a working volume from 0,15–25 L in one and the same bag as well as from 10 – 150 L in one bag size. kL a values of 300 hr−1 and above have been reported for both sizes of reactors. Recently achieved culture data of E.coli and of a Rhodutorula yeast show that the CELL-tainer® single-use bioreactor is comparable to stirred fermenters and thus suitable for microbial cultivations. Application in both the seed train as well as cultivation system for small batches in GMP-production is possible now. http://dx.doi.org/10.1016/j.nbt.2014.05.1920 www.elsevier.com/locate/nbt S125 BIOPROCESSING AND ENGINEERING PF-33 Butanol production by fermentation of Clostridium acetobutylicum: Solventogenic kinetics Alessandra Procentese 1 , Francesca Raganati 1,∗ , Giuseppe Olivieri 1 , Maria Elena Russo 2 , Piero Salatino 1 , Antonio Marzocchella 1 1 Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriali - Università degli Studi di Napoli Federico II 2 Istituto di Ricerche sulla Combustione, Consiglio Nazionale delle Ricerche The awareness of fossil supply depletion and the impact of petroleum fuel emissions have increased the research for alternative fuel sources. Particular relevant are the fuels produced from renewable resources such as rapid growth biomasses. Among biofuels, butanol is particularly valuable because it possesses many favourable physical properties [1]. Acetone–butanol–ethanol (ABE) fermentation from renewable resources is one of the biotechnological routes to butanol production. The potential of cheese-whey as feedstock for butanol production has been pointed out by several authors (e.g. [2]). The traditional batch fermentation process for butanol production suffers from two major issues: i) low butanol specific productivity, which means large fermentors and long fermentation periods; ii) severe product inhibition, which limits the butanol concentration and increases the industrial cost for solvent recovery. Continuous operation mode provides several advantages over batch processes. Increased productivity was also achieved by increasing cell concentration by cell recycling or immobilization [2]. The objective of this study was to characterize a continuous butanol production system by means of Clostridium acetobutylicum adopting lactose as the sole carbon source. A cell recycling system (CSTR equipped with a microfiltration unit) has been adopted. Continuous cultures were carried out under a wide interval of operating conditions (dilution rate and recycle flux) in order to characterize the fermentation process under solventogenesis phases. References [1].Cascone. Chem Eng Prog 2008 Aug:S4–9. [2].Raganati, et al. Bioresour Technol 2013;138:259–65. http://dx.doi.org/10.1016/j.nbt.2014.05.1921 PF-34 Evaluation & optimization of yield, productivity, and sustainability of the EBA process; A surface energetics approach Prasad Babu Kakarla ∗ , Roy D’Souza, Marcelo Fernández Lahore Jacobs University Bremen Expanded bed adsorption (EBA) chromatography is an advanced integrated unit operation for the downstream processing of biomolecules. Biomass-adsorbent interactions not only lead to unstable hydrodynamics in these systems, but also to the elutriation of adsorbent beads, and the deposition of intact cell particles, S126 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 cell debris, and suspended materials on the stationary phase. We have employed extended Derjaguin– Landau–Verwey–Overbeek (xDLVO) theory, based on colloid theory principles, as an effective tool in predicting these unfavourable interactions. We have adapted capillary rise method to characterize the surface thermodynamics of EBA adsorbents, and have used streaming potential measurements to describe surface electrostatics. Both these methods now avoid mechano-chemical alterations in surface properties associated with the bead disruption, which was required for earlier characterization methods. The corresponding xDLVO interactions energies calculated were used to generate a minimum adsorbentbiomass interaction energy surface as a function of pH and ionic strength, which led to the predictive optimization of EBA process conditions in terms of solution chemistry. Consequently, product recovery was shown to increase without compromising on purity (99%). http://dx.doi.org/10.1016/j.nbt.2014.05.1922 PF-35 High throughput systems and mathematical models for prediction of protein renaturation processes Bernhard Mißbichler 1,∗ , Cornelia Walther 2 , Sabrina Dorota Antos 4 , Alois Jungbauer 3 , Astrid Dürauer 3 Mayer 3 , 1 ACIB GmbH University of Natural Resources and Life Sciences Vienna, Department of Biotechnology, Muthgasse 18, 1190 Vienna, Austria 3 1ACIB Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria 4 Rzeszow University of Technology, Chemical and Process Engineering Department, a. Powstancow warszawy 6, 35-959 Rzeszow, Poland 2 Heterologous recombinant expression of biopharmaceutical products in E. coli often leads to high density protein aggregates in inclusion bodies. In contrast to empirical strategies for process development we developed high throughput screening methods for inclusion body solubilization and subsequent refolding on ␮scale. Thereof parameters were extracted to predict this crucial process steps in a laboratory scale stirred tank reactor controlled by inline monitoring. Mathematical modelling using solubility curves and dissolutions kinetics was carried out. The solubilization process could be described by a homogeneous layer model with a high order of reaction which enables evaluation of the process on a numerical level. Hence, solubilization conditions and process parameters can be related to the rate and yield of the solubilization. Approximation of the resistances potentially affecting the IB solubilization showed that the process is controlled predominantly by pore diffusion. Maintaining the homogeneity of the IB suspension is sufficient for efficient solubilization, and further power input will not improve the process. The HTS on ␮-scale can predict solubilization in STR over a range of 500 and can be used to determine optimal solubilization conditions for laboratory and industrial scale. Furthermore, kinetics of the refolding process determined in different scales – from static vial over beaker stirred by magnetic stirrer to laboratory scale STR were equivalent keeping different scale-up criteria such as phase number and Reynolds number BIOPROCESSING AND ENGINEERING New Biotechnology · Volume 31S · July 2014 within a certain range. The established models and prediction enable the engineering based and thus material and cost efficient process development of IB solubilization and refolding steps. http://dx.doi.org/10.1016/j.nbt.2014.05.1923 PF-36 Gamma ray-mediated functionalization of monolithic cryogels for macro-biomolecule purification Naveenkumar Singh 1,∗ , Roy Marcelo Fernández-Lahore 1 1 2 N. Dsouza 1 , Mariano Grasselli 2 , Jacobs University Bremen gGmbH Universidad Nacional de Quilmes, Bernal, Argentina Recently introduced megaporous cryogels are relatively new and have not been fully exploited for the downstream processing of macro-biomolecules. Weak anion-exchange functionality was incorporated by gamma irradiation-induced grafting. The total ionic capacity obtained for DEAE-functionalized cryogel was 0.59 meq/g. The resulting weak anion-exchange cryogels showed a pore size distribution of up to 100 ␮m with dynamic binding capacities of ca. 27 ± 3 mg/mL. The adsorbents presented in here showed better column efficiency when compared to packed-bed adsorbents. The dynamic binding capacities of monolithic cryogels were found to be independent of the applied flow rate. Scanning electron microscopy confirmed that the porous nature of the cryogels remained unaffected by the irradiation-grafting procedure. Due to the large pores, high permeability, disposability, and cost-efficient nature, megaporous cryogels are an attractive alternative for the purification of macro-biomolecules like mAbs, mRNA, pDNA and viruses. http://dx.doi.org/10.1016/j.nbt.2014.05.1924 PF-37 High throughput systems and mathematical models for prediction of protein renaturation processes Bernhard Mißbichler 1,∗ , Cornelia Walther 2 , Sabrina Dorota Antos 3 , Alois Jungbauer 1 , Astrid Dürauer 1 Mayer 1 , 1 ACIB GmbH BOKU 3 Rzeszow University of Technology 2 Heterologous recombinant expression of biopharmaceutical products in E. coli often leads to high density protein aggregates in inclusion bodies. In contrast to empirical strategies for process development we developed high throughput screening methods for inclusion body solubilization and subsequent refolding on ␮scale. Thereof parameters were extracted to predict this crucial process steps in a laboratory scale stirred tank reactor controlled by inline monitoring. Mathematical modelling using solubility curves and dissolutions kinetics was carried out.The solubilization process could be described by a homogeneous layer model with a high order of reac- tion which enables evaluation of the process on a numerical level. Hence, solubilization conditions and process parameters can be related to the rate and yield of the solubilization. Approximation of the resistances potentially affecting the IB solubilization showed that the process is controlled predominantly by pore diffusion. Maintaining the homogeneity of the IB suspension is sufficient for efficient solubilization, and further power input will not improve the process. The HTS on ␮-scale can predict solubilization in STR over a range of 500 and can be used to determine optimal solubilization conditions for laboratory and industrial scale. Furthermore, kinetics of the refolding process determined in different scales – from static vial over beaker stirred by magnetic stirrer to laboratory scale STR were equivalent keeping different scale-up criteria such as phase number and Reynolds number within a certain range. The established models and prediction enable the engineering based and thus material and cost efficient process development of IB solubilization and refolding steps. http://dx.doi.org/10.1016/j.nbt.2014.05.1925 PF-38 Precipitation: A powerful tool for continuous purification of monoclonal antibodies Ralf Sommer 1,∗ , Anne Tscheliessnig 1 , Henk Schulz 2 , Bernhard Helk 2 , Alois Jungbauer 3 1 University of Natural Resources and Life Sciences Vienna/Department of Biotechnology 2 Novartis Pharma AG 3 University of Natural Resources and Life Sciences Vienna Currently, recombinant biopharmaceutical protein production operates discontinuous, in batch processes. Till 2012, the number of approved mAbs increased to 30. These trends require a more economical mAb production processes which only can be achieved with a change from batch to continuous production. Most promising way for improvement of mAb production is the implementation of novel continuous downstream processes. For mAb purification a combination of caprylic acid (CA) and polyethylene glycol (PEG) precipitation was developed. Target of novel continuous precipitation purification was to reach the highest possible purity values. Caprylic acid precipitation reduced both HMWI and HCP contents. For IgG capturing a PEG precipitation was performed, which further reduces HCP content. Adequate yield and purity of the final product showed that CA/PEG precipitation was competitive with affinity chromatography. Furthermore, additional combination of precipitation methods such as of CA, PEG, CaCl2 , and cold ethanol precipitation were tested to determine whether biopharmaceutical purity could be achieved with a continuous performable process. The most promising combination provided a low HCP content (300 ppm), without aggregates, and a yield of approximately 70%. That purity is nearly suitable for a biopharmaceutical agent and the solitary use of precipitation methods lead to a continuous performable process. http://dx.doi.org/10.1016/j.nbt.2014.05.1926 www.elsevier.com/locate/nbt S127 BIOPROCESSING AND ENGINEERING New Biotechnology · Volume 31S · July 2014 PF-39 Development of a rapid and low-cost tool for biopharmaceutical formulation screening Amanda Quigley 1,∗ , Stuart Hassard 1 , Dan Bracewell 2 1 2 deltaDOT Ltd University College London One of the most significant challenges in protein formulation is the tendency of proteins to aggregate. Aggregation can have severe implications for the safety and effectiveness of biopharmaceuticals therefore methods to predict solution conditions that limit aggregation would be of great industrial value. However, very little progress has been made in this area in recent years and current formulation screening methodologies are lengthy, outdated and empirical and often incompatible with high protein concentrations. This has generated great interest in the measurement of the weak protein-protein interactions (quantified by the osmotic second virial coefficient, B22 ) involved in protein aggregation as a method for rapidly predicting protein stability. Measurement of protein-protein interactions also has a potential method for predicting other factors of importance to formulation such as solubility and viscosity. This experimental study reports on the development of a novel screening platform to measure B22 . The advantages of this methodology over other techniques include significantly reduced experimental time and consumption of materials including valuable protein. In addition to increased resolution of peak shape due to deltaDOT’s multipixel detection and algorithms, providing important extra information on protein stability. B22 data is presented on a formulation screen of varying buffer type, pH and NaCl concentration for lysozyme as a model protein, as well as 2 industrial monoclonal antibodies. This data is compared to that obtained via benchmark techniques for stability screening; size exclusion chromatography (SEC) and dynamic light scattering (DLS). This work establishes the decisive importance of B22 as a predictor of protein aggregation. http://dx.doi.org/10.1016/j.nbt.2014.05.1927 PF-40 Extraction of compounds from the cultivation of Arthrospira (Spirulina) platensis using ion exchange Fernando Lisboa 1,∗ , Luana Agassi 2 , Evelin Barbosa 2 , Mônica Okura 2 , Lúcia Pelizer 2 Brandão 2 , Marlei 1 Instituto Federal de Educação, Ciência e Tecnologia do Triângulo Mineiro Campus Uberlândia - Brazil/Universidade Federal do Triângulo Mineiro - Brazil 2 Universidade Federal do Triângulo Mineiro - Brazil Arthrospira (Spirulina) platensis is known because of its high concentration of compounds, so separating them is necessary to promote their use, increasing the viability of the process. In this context, the aim of this study was to analyze the extraction of phycocyanin, chlorophyll, amino acids and beta carotene in the liquid phase of this cyanobacterium’s cultivation using ion exchange. S128 www.elsevier.com/locate/nbt For this, inoculants were prepared in Erlenmeyer flasks, grown in ‘shaker’ for 7 days, with temperatures of 30 ◦ C, 6 Klux illuminance and agitation of 150 rpm, with an initial concentration of 50 mg L−1 in standard mineral medium. After that, the microorganism was separated by filtration. The permeate was adsorbed on an ion exchange column using the resin IRA 402 (Rohm and Haas), washed with distilled water and eluted with a NaCl gradient of 0.5 to 5.0% (w/v). The content of phycocyanin, chlorophyll, beta-carotene and amino acid present in the solution were evaluated by spectrophotometry UV. As a result, it was observed that the greatest concentration values were obtained for elution with 5% NaCl. It was possible to obtain high concentrations of amino acids 13 times, chlorophyll 16 times, phycocyanin 5 times and betacarotene in 4 times at the exit of the column. Therefore, according to the experimental conditions under which this work was performed, the ion exchange showed promise in the extraction of the compounds analyzed in Arthrospira (Spirulina) platensis cultivation. http://dx.doi.org/10.1016/j.nbt.2014.05.1928 PF-41 Development of microfluidic system for screening and reaction optimization for lipase Hsiang-Yu Wang ∗ , Chong-Yi Ho National Cheng Kung University This study presents microfluidic platforms for the rapid screening of lipase and the corresponding optimization of lipid hydrolysis reaction. The conventional screening methods of lipase involve time-consuming processes and high-end instrument, making the analysis not accessible. The proposed microfluidic systems utilize microdroplet (either dynamic or stationary) to enable rapid analysis of the hydrolysis reaction and use instrument which is readily accessible in common laboratories. Due to the merit of high surface area per volume (SAV) ratio of microdroplet, which can be as high as 2.5 × 106 m−1 in this study, the lipid hydrolysis reaction occurring on the interface of droplet is extremely fast. The analysis for hydrolysis of soybean oil by the Burkhoderia sp. lipase can be accomplished within 5 min. Reaction temperature and pH can be easily adjusted via hydrodynamic control for reaction optimization. In the dynamic microdroplet system, droplets containing lipase and fluorogenic reagents that react with glycerol are generated at T-junction and flow through a serpentine microchannel to allow lipase to react with the continuous phase (soybean oil). The retention time of the droplet inside the device can be adjusted from less than 1 sec to 10 min; therefore, this system is suitable for analyzing the initial rate to obtain the maximum reaction rate and Michaelis constant. The stationary droplet system traps the droplet in an indentation for observation as long as 30 days; therefore, it is applied on long-term examinations such as screening of microorganisms producing lipases. http://dx.doi.org/10.1016/j.nbt.2014.05.1929 BIOPROCESSING AND ENGINEERING New Biotechnology · Volume 31S · July 2014 PF-42 From small synthetic ligands to small protein scaffolds: Affinity reagents for biologics purification Ana Roque 1,∗ , Ana Pina 1 , Cláudia Fernandes 1 , Ricardo Branco 1 , Ana Dias 1 , Iris Batalha 1 , Olga Iranzo 2 , Christopher Lowe 3 1 FCT-UNL University of Marseille 3 University of Cambridge 2 Over the past 40 years monoclonal antibodies and derived structures became the standard binding proteins representing powerful tools in biotechnology and biomedicine, namely on protein purification, biocatalysis, diagnostic imaging and targeted therapy. Other protein binding scaffolds, with the robustness and versatility required, are recently being explored. We employed biological and chemical combinatorial libraries supported by computational design tools to develop robust peptidomimetics based on different scaffold molecules. The scaffold molecules ranged from small synthetic ligands based on the triazine and Ugi reactions, to peptide-based ␤-hairpin engineered scaffolds and larger natural scaffolds. We studied the potential of these scaffold affinity reagents to find binding partners against recombinant proteins (de novo designed fusion proteins), phosphorylated peptides and viral particles. These affinity reagents were employed as ligands for the purification of the target biomolecules using standard affinity chromatography and magnetic fishing methodologies. The performance of the new affinity adsorbents was benchmarked with currently available purification and enrichment methodologies, showing high-performance and re-usability potential at low costs. http://dx.doi.org/10.1016/j.nbt.2014.05.1930 PF-43 Optimization of lipase extraction/recovery produced by the psychrotrophic yeast Leucosporidium scottii L117 using aqueous two-phase micellar systems Alysson Duarte 1,∗ , André Pessoa 2 , Lara Sette 3 Lopes 2 , João Molino 2 , Adalberto 1 UNICAMP USP 3 UNESP 2 This study aims to evaluate the lipase extraction by micellar system using nonionic detergent Triton X-114 (TX-114)/McIlvaine buffer systems. Additionally, the parameters affecting the extraction/recovery of lipase by ATPMS were optimized by Central Composite Design (CCD23 ). The micellar system extraction was prepared with TX-114 at 25 ◦ C and 28 ◦ C, both with 2.0% TX114/McIlvaine buffer systems (pH 7.0). The variables optimized were pH (3.5–7.5), temperature (19–31 ◦ C) and TX-114 (2–14% (%w/w). The volume of the top and bottom phases were recovered (with sterile disposable syringes) and evaluated. The lipase activity was measured using the synthetic substrate p-nitrophenyl palmitate (p-NPP). The three variables were statistically significant (p < 0.05) when the response variable was of lipase recovery (%RECbot ) in bottom phase (r2 = 0.93). Similar result was obtained when the variable was partition coefficient (K). For the partition coefficient of lipase and recovery lipase (%RECbot ) in bottom phase, the influence of pH and temperature was negative and the influence of concentration of TX-114 was positive. The majority of lipase partition was in the micellar-rich phase (bottom phase) (K = 7.7 and 4.7; 93.8 and 73.5% recovery in bottom phase and purification factor PF = 1.2 and 1.97, respectively). The greater extraction of lipase was in the bottom phase, this result was probably influenced by the hydrophobic residues present on lipases. The micellar-rich phase showed to be very clear and transparent. Thus, this micellar ATPMS presents an alternative to the conventional purification/extraction of lipase from L. scottii L117. http://dx.doi.org/10.1016/j.nbt.2014.05.1931 PF-44 Risk assessment of feed additives of microbial origin in the European Union Jaime Aguilera ∗ , Montserrat Anguita, Rosella Brozzi, Jaume Galobart, Paola Manini, Jordi Tarrés-Call, Claudia Roncancio Peña European Food Safety Authority, Italy Microorganisms can be used in animal nutrition as probiotics or as silage agents, and are the source of other feed additives like enzymes, amino acids and vitamins. In the European Union, all feed additives need, by law, to undergo a risk assessment, which is conducted by the European Food Safety Authority. A proper characterisation of the microorganism is fundamental for its safety assessment. This includes an unequivocal identification of the species, consideration of its pathogenic or toxigenic potential and the presence of resistance to antimicrobials, the genetic basis of which may need to be established. Genetically modified strains require particular attention, including a full molecular characterisation of the modification. If the microorganism is the source of an additive which is purified after fermentation, it is necessary to test whether the final product is free from the production organism and from its DNA if it encodes antimicrobial resistances. The characterisation of the microorganism determines the nature and extent of further tests to be done to establish the safety of the product. EFSA grants a qualified presumption of safety (QPS) status to certain species having well documented safety. Strains fulfilling the requirements for QPS are considered safe for target species, consumers and the environment without the need of further testing. EFSA has published a complete set of guidance stating the data needed for the safety evaluation. The conclusions of the assessments are published in form of scientific opinions, and allow the European regulatory authorities to make scientifically sound decisions regarding market authorisations. http://dx.doi.org/10.1016/j.nbt.2014.05.1932 www.elsevier.com/locate/nbt S129 CELL FACTORIES Cell factories PG-01 Fine-tuning of defined media for chemical production by lactic acid bacteria Kadri Aller ∗ , Kaarel Adamberg, Veronica Timarova, Andrus Seiman, Raivo Vilu The Competence Center of Food and Fermentation Technologies The lactic acid bacterium Lactococcus lactis has emerged as an effective cell factory for recombinant protein production and secretion [1] and for the synthesis of various biochemicals [2]. In order to produce chemicals of interest, the growth physiology of the expression host has to be well characterized [3]. Chemically defined media (CDM) are required for studying the metabolism of cells as well as producing recombinant proteins [3]. However, if the medium contains an excess of nutrients, analytical measurements are rendered imprecise and the interpretation of metabolic data is aggravated. We have previously elucidated the growth requirements of L. lactis IL1403 and developed several CDMs where the utilization of each amino acid is larger than the measurement error (5%). Now we compare the growth of IL1403 on 3 of those media in continuous cultivation experiments and show the differences in metabolic behavior. Naturally, decrease in the concentrations of components leads to lower maximum specific growth rate values. Nevertheless, biomass yield and productivity were higher and energy spilling was lower in media with reduced concentrations of nutrients, e.g. the metabolism of IL1403 is more efficient in minimalized media. Thus, the importance of fine-tuning a growth medium for a specific strain and process cannot be underestimated. References [1].Morello, et al. J Mol Microbiol Biotechnol 2008;14(1–3):48–58. [2].Gaspar, et al. Biotechnol Adv 2013;31(6):764–88. [3].Marreddy, et al. PLoS One 2010;5(4):e10317. http://dx.doi.org/10.1016/j.nbt.2014.05.1933 PG-02 Development of a yeast cell factory for production of aromatic products Angelica Rodriguez Prado 1,∗ , Kanchana Kildegaard 1 , Mingji Li 1 , Irina Borodina 1 , Jens Nielsen 2 1 Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark 2 Department of Chemical and Biological Engineering, Chalmers University of Technology There is much interest in aromatic chemicals in the chemical industry as these can be used for production of dyes, anti-oxidants, nutraceuticals and food ingredients. Yeast is a widely used cell factory and it is particularly well suited for production of aromatic chemicals via complex biosynthetic routes involving P450 enzymes. In Saccharomyces cerevisiae the fluxes towards aromatic S130 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 acids (L-tryptophan, L-tyrosine and L-phenylalanine) are strictly controlled on transcriptional and kinetic levels and therefore are difficult to manipulate. We engineered S. cerevisiae for increased production of aromatic compounds by eliminating degradation, up-regulating the key enzyme encoding genes, and removing feed-back inhibition in the pathway. In order to test the strain performance we overexpressed heterologous pathway for coumaric acid production. We obtained 4-fold higher concentrations of coumaric acid in the engineered strain compared with the reference strain. Overexpression of the enzymes prephenate dehydrogenase and transketolase had a negative effect on production of coumaric acid. In summary, we developed a S. cerevisiae platform strain suitable for production of aromatic amino acids-derived compounds. http://dx.doi.org/10.1016/j.nbt.2014.05.1934 PG-03 Homologous and heterologous expression of dehydrogenases and oxidoreductases of Ralstonia eutropha H16 Steffen Gruber ∗ , Petra Koefinger, Helmut Schwab Graz University of Technology Ralstonia eutropha is a Gram-negative, strictly respiratory facultative chemolithoautotrophic bacterium which can use H2 and CO2 as sole sources of energy and carbon in the absence of organic substrates. It has attracted great interest for its ability to degrade a large list of chloroaromatic compounds and chemically related pollutants. Furthermore it was already applied for the production of biodegradable polymer polyhydroxyalkanoates on an industrial scale. R. eutropha serves as a model organism for the mechanisms involved in the control of autotrophic carbon dioxide fixation, hydrogen oxidation and denitrification. In our project we are interested in establishing specialized R. eutropha based cell factories by genetic engineering. The particular interest is constructing cells efficiently performing oxidoreductase reactions by overexpression of homologous and/or heterologous enzymes. One of the main types of oxidoreductase reactions is performed by dehydrogenases, particularly alcohol dehydrogenases, which have a wide range of possible biotechnological applications. Biotransformations involving the interconversion of alcohols, aldehydes and ketones have great potential for the commercial production of pure optically active compounds and also for other processes such as the treatment of industrial effluents. The genome of R. eutropha H16 contains a remarkable diversity of oxidoreducteases. A selection of alcohol dehydrogenases as well as short chain dehydrogenases of R. eutropha H16 was cloned and expressed in native versions in Escherichia coli. Their activity was analyzed by NAD/NADH dependent enzyme activity assays with different substrates. Currently we are working on the homologous expression of these enzymes in R. eutropha H16 and their functional analysis. http://dx.doi.org/10.1016/j.nbt.2014.05.1935 CELL FACTORIES New Biotechnology · Volume 31S · July 2014 PG-04 Applicability of a mechanosensitive channel Corynebacterium glutamicum as a versatile exporter in Ken-ichi Hashimoto ∗ , Tomoyuki Konichi, Isamu Yabe, Tsuyoshi Nakamatsu, Hisashi Kawasaki Tokyo Denki University Corynebacterium glutamicum is used worldwide in the industrial fermentative production of glutamic acid. In 2007, the involvement of the NCgl1221 gene, which encodes a homolog of the mechanosensitive channel of small conductance protein, in glutamic acid overproduction was reported [1]. We used electrophysiological methods to obtain direct evidence of the excretion of glutamic acid through the NCgl1221 protein channel. We showed that the NCgl1221 protein functions as a mechanosensitive channel [2], and we found direct evidence of glutamic acid excretion through this channel by passive diffusion [3]. Furthermore we found that aspartic acid, phenyl propionic acid and lysine were also transported across the cytoplasmic membrane via NCgl1221 by passive diffusion. We further evaluated the possibility of the effective application of this mechanosensitive channel as a versatile exporter, for the creation of microbial cell factories for the production of valuable chemicals other than glutamate. If successful, this would be a substantial development in exporter engineering (ExE). A gain-of-function mutation in NCgl1221 was expressed in a phenylalanine producer (Escherichia coli AJ12741) and an inosine producer (Escherichia coli FADR add edd AJ13473). These strains displayed remarkably high productivity compared with a control strain. These data suggest that utilizing the mechanosensitive channel as a versatile exporter could be an effective method for improving fermentation yield. References [1].Nakamura J, et al. Appl Environ Microbiol 2007;73:4491–8. [2].Hashimoto K, et al. Biosci Biotechnol Biochem 2010;74:2546–9. [3].Hashimoto K, et al. Biosci Biotechnol Biochem 2012;76:1422–4. http://dx.doi.org/10.1016/j.nbt.2014.05.1936 PG-05 Research on the Relationship between Pyruvate Kinase and the Biosynthesis of Epothilones in Sorangium cellulosum 2161 Xinli Liu ∗ , Lin Zhao, Xiaona Wang Qilu University of Technology Pyruvate kinase (PK) catalyzes the formation of pyruvate and ATP from phosphoenolpyruvate and ADP, and it is a key enzyme of glycolysis. Pyruvate has been one of most important substrates for the biosynthesis of epothilones. There are two coding genes of PK in Sorangium cellulosum 2161: pyk sce5197 and pyk sce4540. In this paper, we analyzed the relationship between PK activity, epothilone production, and the gene expression quantity of pyk sce5197 and pyk sce4540 in S. cellulosum 2161 incubated with vitamin K3 , which is a specific inhibitor of PK. Results showed that PK activity significantly influenced epothilone production. In addition, the expression of pyk sce4540 and putative oxidase gene (OX) in S. cellulosum 2161 treated with vitamin K3 was remarkably inhibited, while the response of pyk sce5197 was not sensitive. These results indicate that PK is one of the key enzymes in epothilone biosynthesis across the metabolic network in S. cellulosum 2161. It might also play an important role in the biosynthesis of epothilones. Keywords: Epothilones, pyruvate kinase, inhibitor, Sorangium cellulosum 2161, qRT-PCR http://dx.doi.org/10.1016/j.nbt.2014.05.1937 PG-06 Engineering of the UDP-precursor biosynthesis pathway to enhance the production of capsular polysaccharide in E. coli K4 Elisabetta Carlino ∗ , Chiara Schiraldi, Ottavia Argenzio, Ileana Dello Iacono, Donatella Cimini Seconda Università degli Studi di Napoli Chondroitin sulphate is a linear polysaccharide chain made of alternating units of glucuronic acid and N-acetyl-galactosamine, modified by sulfation in various positions depending on both the animal source and tissue of origin [1]. Besides its established use in the treatment of osteoarthritis, potential applications as an antiinflammatory drug and surprising antiviral properties have been suggested. E. coli K4 produces a capsule that belongs to the class of GAG-like polymers; in fact the molecule can be described as a chondroitin backbone decorated with fructose side branches on the GlcA residues. The synthesis of the necessary sugar precursors for chain elongation is performed by the cells through two different metabolic pathways. The first starts from glucose 6-phosphate and ends with the production of UDP-GlcA. The second one starts from fructose 6-phosphate and ends with the synthesis of UDP-GalNAc. In the present work two E. coli K4 recombinant strains were engineered to improve the productive yields of K4 capsular polysaccharide (CPS) by overexpressing genes involved in the biosynthesis of one of the UDP-sugar precursor. The E. coli wild type strain 05:K4:H4 and EcK4r3 strain (previously obtained in our laboratories) were both transformed with the construct. In order to evaluate the production of K4 CPS by each strain, shakeflask and microbioreactor experiments were performed. Pathway modifications were also analysed to correlate the productive yields of K4 CPS to gene overexpressions. Reference [1].Cimini D, et al. Homologous overexpression of RfaH in E. coli K4 improves the production of chondroitin-like capsular polysaccharide. Microb Cell Fact 2013;12:46, 2013 May 9. http://dx.doi.org/10.1016/j.nbt.2014.05.1938 www.elsevier.com/locate/nbt S131 CELL FACTORIES PG-07 The isolation of novel marine yeasts; a new procedure Abdelrahman Saleh Zaky ∗ , Chenyu Du University of Nottingham The recent research on marine yeasts highlighted that they are able to produce many bioactive substances including enzymes, amino acids, killer toxins, and vitamin C with many potential applications in food, pharmaceutical, fuel, cosmetics and chemical industries [1]. However, the employment of marine yeast in research and industry is limited due to the lack of suitable isolation methods. Current methods suffer from fungus interference and/or low number of yeast isolates. In this report, a novel 3-step isolation method has been developed. Samples were taken from seawater, sea sand or seaweed which could potentially contain S132 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 marine yeasts. These samples were enriched using a nutrient rich media with antibiotics for three cycles as the first step. In the second step, single colonies with different morphologies were isolated on plates of agar medium lacking antibiotic after 24–48 h of incubation at 30 ◦ C. These isolates were further confirmed to be pure cultures of yeast by streaking them on agar plates and checking their purity and morphology under a microscope. In this study, 14 samples from different marine habitats in Egypt, UK and USA were used for the isolation. 122 isolates were obtained and confirmed to be pure yeast cultures. Our 3-step isolation method for marine yeast has demonstrated high level of efficiency comparing with two reference methods. Reference [1].Zaky, et al. Marine Yeast Isolation and Industrial Application FEMS Yeast Research 2014 (accepted). http://dx.doi.org/10.1016/j.nbt.2014.05.1939 ENVIRONMENTAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 Environmental biotechnology PH-01 Formate oxidation-driven calcium carbonate precipitation by Methylocystis parvus OBBP for a concomitant building material surface protection and atmospheric methane removal Giovanni Ganendra 1,∗ , Giovanni Ganendra 2 , Adrian Ho 3 , Nico Boon 2 1 Ugent Ghent university 3 Netherlands Institute of Ecology 2 Microbially Induced Carbonate Precipitation (MICP) is the basis for several biotechnological applications in the construction sector e.g., concrete surface protection. The typically used urea based MICP poses several disadvantages, such as ammonia release to the air. Therefore, an alternative MICP for the application on building materials should be investigated. In this study, MICP was driven by formate oxidation by Methylocystis parvus OBBP, a methanotrophic bacteria. Up to 91.4 ± 1.6% of the initial calcium was precipitated in the methane amended cultures, but only a maximum of 35.1 ± 11.9% when methane was not added. Because the bacteria could only utilize methane but not formate for growth, a higher culture density and subsequently a higher calcium removal was exhibited by the bacteria when methane was added. The methane oxidation rate (MOR) of the bacteria decreased from 3.15 ± 0.32 ␮g CH4 (ml h)−1 when formate was not added to 0.52 ± 0.01 ␮g CH4 (ml h)−1 when 2.88 g L−1 of formate was added (i.e., the maximum formate addition). An optimum 0.67 ± 0.03 g CaCO3 g Ca(CHOOH)2 −1 calcium carbonate precipitate yield was obtained when 109 cells ml−1 and 2.5 g L−1 of calcium formate was used. Compared to the currently used biogenic urea degradation as the basis for MICP, several advantages are presented here: the prevention of ammonia release to the air and nitric acid production and the atmospheric methane removal. http://dx.doi.org/10.1016/j.nbt.2014.05.1940 PH-02 Activity of Bdellovibrio on Sludge bacteria and its potential use for cleaning of Membrane Bioreactors Melek Özkan ∗ , Merve Akay ¸Celik, Pınar Karagöz, Hilal Yılmaz, ¸Cisel ¸Sengezer Gebze Institute of Technology, Environmental Engineering Department, 41400 Gebze Kocaeli, Turkey Bdlelovibrio bacteriovirus is a gram negative predator bacterium feeding on other gram negative bacteria. Its activity on biofilms formed by different bacterial species especially pathogenic ones has been investigated in recent years. One of the recent technologies for wastewater treatment is Membrane bioreactors(MBRs) which have high effluent capacity and good disinfection capa- bility. One important problem of MBR technology is membrane fouling. In this study, Bdellovibrio activity was analysed for reducing membrane fouling caused by sludge bacteria, and potential of B. bacteriovirus as a biological cleaning method for MBR systems was investigated. For this aim, nineteen bacteria were isolated from wastewater sludge and characterized by 16SrRNA analysis. Bacteria were mostly found to be a member of Gammaproteobacteria, Betaproteobacteria and Actinobacteria families. B. bacteriovirus showed high lysis activity on Aeromonas, Proteus, Alcaligenes and Bordatella species. B. bacteriovirus activity was also tested on membranes plugged after filtration of wastewater. Pore size of the membranes was found to be important for B. bacteriovirus activity. Flux through Poly(ether)sulphone (PES) membrane with a pore size of 0.05 micrometer was improved after cleaning by predator bacteria. B. bacteriovirus successfully degraded the biofilm formed by sludge bacteria on the membrane surface. Acknowledgement We thank Scientific and Technological Research Council of Turkey for supporting this study (Project no: 112Y156). http://dx.doi.org/10.1016/j.nbt.2014.05.1941 PH-03 Comparison of inoculums in the removal of 2butoxyethanol from air emissions by biotrickling filter: Performance and microbial monitoring Maria del Carmen Perez 1,∗ , Francisco Javier Alvarez-Hornos 1 , Daniel Dobslaw 2 , Karl-H. Engesser 2 , Carmen Gabaldon 1 1 2 University of Valencia University of Stuttgart 2-butoxyethanol is one of the most used glycol ether in industrial activities and the treatment of air 2-butoxyethanol-emissions become necessary. Biotechnologies are potential treatment technologies due to their low operational costs. The use of two inoculums in the treatment of 2-butoxyethanol by biotrickling filters (BTFs) packed with polyurethane-foam was studied. A pure culture of Pseudomonas putida, previously adapted to 2butoxyethanol, was used as inocula in a BTF operated in the University of Stuttgart. Fresh activated sludge from a municipal waste water treatment plant was used as inocula in a BTF operated in the University of Valencia. An empty bed residence time of 12.5 s and inlet concentrations of 400 and 800 mg/Nm3 were applied. After 40 days of operation at 400 mg/Nm3 , the BTF inoculated with Pseudomonas putida reached removal efficiencies (REs) ∼ 80%, whereas the BTF inoculated with activated sludge presented REs ∼ 60%. At 800 mg/Nm3 , the BTF inoculated with Pseudomonas putida reached REs ∼ 60%. Microbial community was monitored in both BTFs by using denaturing gradient gel electrophoresis analysis (DGGE) with subsequent 16S sequencing and plating methods using 2-butoxyethanol as sole carbon source. Acknowledgements The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European www.elsevier.com/locate/nbt S133 ENVIRONMENTAL BIOTECHNOLOGY Union’s Seventh Framework Programme FP7/2007-2013/under REA grant agreement n◦ 284949. Financial support from Ministerio de Ciencia e Innovación (CTM2010-15031/TECNO) and Generalitat Valenciana (PROMETEO/2013/053), is also acknowledged. Ma Carmen Pérez acknowledges her FPU contract http://dx.doi.org/10.1016/j.nbt.2014.05.1942 New Biotechnology · Volume 31S · July 2014 theticum, Brevibacillus laterosporus and Pantoea ananatis) were isolated from an agricultural soil located on Chihuahua state, Mexico. The most intense effects were observed on C. indotheticum and on B. laterosporus, e.g., on C. indotheticum superficial damages (cavities and pits) were observed on its membrane, and on B. laterosporus severe oxidative stress and the presence of Cu in the outer cell membrane were detected. References PH-04 Diversity of fungi present in the Sossego copper mine in Pará State, Brazil Luciana Jandelli Gimenes ∗ , Bruno Karolski, Claudio Nascimento, Elen Perpetuo, Ingrid Avanzi, Louise Gracioso, Marcela Baltazar, Marcela Veiga, Tatiana Reis, Benedito Correa Universidade de São Paulo Industrial growth, which results from technological development and activities considered essential to human life, has caused serious environmental problems. For some time, fungi have been used in organic waste bioremediation. Recently, a good alternative in the bioremediation of heavy metals has been discovered. This work aims to isolate, identify and characterize fungi and evaluates mechanisms and strategies for remediating areas contaminated by copper. Soil and water samples have been collected from Sossego Mine (Pará State, Brazil) and have been grown on PDA culture medium, incubated at 28 ◦ C for 5 days. After this period, the isolates were identified by morphologic and molecular methods. Since the project started, 49 fungi were isolated, and various species among 12 different genders were identified: Aspergillus, Aureobasidium, Bionectria, Eupenicillium, Eurotium, Fusarium, Lentinus, Microspheropsis, Nigrospora, Penicillium, Purpureocillium and Rhizopus. This work has great importance due to the low cost of repair systems compared to conventional ones. It also allows a better use of copper wastes and, consequently, a better mining economic return. Moreover, the main advantage of this technology is further reduction of environmental impact by the mining activity. http://dx.doi.org/10.1016/j.nbt.2014.05.1943 PH-05 CuO nanoparticles toxicity against bacteria strains isolated from agricultural soil Sandra I. Concha-Guerrero a , Elcia M.S. Brito b , Hilda A. PiñónCastillo a , M. Antonia Luna-Velasco a , Erasmo Orrantia-Borunda a,∗ a b Centro de Investigación en Materiales Avanzados, SC, Chihuahua, México Universidad de Guanajuato, Guanajuato, México The intensified use of nanoparticles by human society brings out the risk of exposure to these particles. In fact, some kinds of nanoparticles were found to effects on the organisms or on their cells [1–3]. In the present work, we studied the interaction and effects produced by oxide copper nanoparticles (CuONP) to native bacterial strains. Selected strains (Chryseobacterium indoS134 www.elsevier.com/locate/nbt [1].Gajjar P, Pettee B, Britt DW, Huang W, Johnson WP, Anderson AJ. Antimicrobial activities of commercial nanoparticles against an environmental soil microbe, Pseudomonas putida KT2440. Journal of Biological Engineering 2009;3(9). [2].Fent K. Ecotoxicology of engineered nanoparticles. F.F.H. Berlin: SpringerVerlag; 2010. [3].Das M, S.K.H., An SSA, Yi DK. Review on gold nanoparticles and their applications. Toxicol Environ HealthSci 2011;3(4):193–205. http://dx.doi.org/10.1016/j.nbt.2014.05.1944 PH-06 The use of dairy processing waste as a media for growth of recombinant microorganisms Michael Ryan ∗ , Gary Walsh University of Limerick A laboratory-based study was undertaken to assess the potential of whey waste as a media for the growth of recombinant Escherichia coli, which remain a preferred choice for process-scale manufacture of many recombinant proteins. Growth characteristics of a recombinant strain of Escherichia coli (MC1061) was assessed on 2 whey-based media, and compared to growth on standard LB (Luria broth) media, known to achieve high cell densities. Whey-based media were: whey only (WM), and whey mixed with LB media (9:1 ratio; W:LB). Media pH was adjusted to 7 prior to autoclaving. All experiments entailed media inoculation (100 ml) with 1.0 ml of the recombinant strain (grown in Luria-Bertani broth [LB] to OD600 of 1.5) at 37 ◦ C in a shaking incubator (250 rpm). Growth kinetics and maximum biomass yield was followed by absorbance at 600 nm and dry cell weight determination, respectively. 6h cultures, n = 3) E. coli growth rates (A600 , were: WM (0.257 ± 0.0448) < W/LB (0.348 ± 0.0246) + LB (1.178 ± 0.0872). Dry cell weights attained after 24 hrs were: WM (4.73 mg ± 0.001387) < W/LB (19.8 mg ± 0.00593) + LB (31.9 mg ± 0.00699). Un-supplemented whey waste is a poor media for E. coli growth. However, optimally nutrient supplemented whey may yet prove a viable and inexpensive media for E. coli fermentation to high cell densities, converting a potentially waste product into a valuable commodity. This work is funded by the Irish EPA under the Science, Technology, Research & Innovation for the Environment (STRIVE) Programme 2007–2013. 2012-WRM-MS-9 http://dx.doi.org/10.1016/j.nbt.2014.05.1945 ENVIRONMENTAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 PH-07 Effects on Tomato Growth and Soil Bacterial Community by Application of Arthrobacter woluwensis ED Immobilized in Alginate Beads Hong-Gyu Song ∗ , Seung-Tak Kwon Kangwon National University For the promotion of plant growth and increase of persistence of plant growth promoting rhizobacteria (PGPR) in rhizpsphere, tomato growth was examined after application of PGPR Arthrobacter woluwensis ED immobilized in alginate bead. When tomato seedlings were treated with A. woluwensis ED of 1 × 106 cells g soil−1 and incubated for 30 days in a plant growth chamber, shoot and root length, fresh weight and dry weight of the grown tomato plants treated with the suspended inoculants significantly increased by 36.2, 59, 51.1 and 37.5%, respectively compared to the uninoculated control. The treatment of the immobilized bacteria increased those by 42, 67.4, 62.5 and 60.4%, respectively compared to the uninoculated control. Therefore, the enhancement of tomato growth by the treatment of the immobilized bacteria was higher than those by the suspended inoculants. The effects of the inoculation on soil bacterial community and the fate of the inoculated bacteria were monitored by DGGE analysis. The DNA band intensity of A. woluwensis ED in the tomato rhizosphere treated with the suspended inoculants continuously decreased after inoculation, but the band intensity in the tomato rhizosphere soils treated with the immobilized inoculants showed the maximum at 1 week after inoculation and the decreasing rate was less than that of the suspended inoculants, which indicated the longer maintenance of the immobilized bacteria at rhizosphere. Therefore, encapsulation of PGPR in alginate beads may be more effective than liquid inoculant for the plant growth promotion and survival of PGPR at plant rhizosphere. http://dx.doi.org/10.1016/j.nbt.2014.05.1946 PH-08 Bioremediation of heavy metal contaminated soil using plant extract as biomaterial In-Hyun Nam ∗ , Chul-Min Chon, Jae-Gon Kim Korea Institute of Geoscience and Mineral Resources (KIGAM) An indigenous plant extract was used to produce calcite from Canavalia ensiformis as effective biomaterial, and its ability to form under stable conditions was compared to that of purified urease. X-ray diffraction and scanning electron microscopy were employed to elucidate the mechanism of calcite formation from the crude plant extracts. The results revealed that urease in the plant extracts catalyzed the hydrolysis of urea in liquid state cultures and decreased heavy metal amounts in the contaminated soil. The heavy metal amounts were decreased in the leachate from the treated mine soil; 55.4% of Pb, 35.6% of Cu, 33.6% of Mn, 32.0% of As, and 25.6% of Fe, respectively. The procedure described herein is a simple and beneficial method of calcite biomineraliza- tion without cultivation of microorganisms or further purification of crude extracts. This study suggests that crude plant extracts of Canavalia ensiformis have the potential to be used in place of purified forms of the enzyme during remediation of heavy metal contaminated soil. Thus, we report the molecular characterization of the microbial diversity and composition change of the mine impacted soil or waste ore taken from the site during bioremediation processes are presented. To evaluate the stability of DGGE patterns in remediated mine soils in comparison with before or after plant extract treatment, a number of soil samples with time interval in the mine impacted soil were compared using PCRDGGE of 16S rDNA. http://dx.doi.org/10.1016/j.nbt.2014.05.1947 PH-09 Investigation of organic wastes from Mediterranean plants to produce biogas by anaerobic digestion Camille Menard ∗ , Anais Fantoni, Pascale Bradesi, Eric Leoni, Dominique Cancellieri Universite de Corse The University of Corsica is contributing to the research of both efficiency and integration of renewable energy in the main electrical grid. Since 2013, a scientific program concerning the valorization of biomass energy has been developed to investigate the methane potential through the anaerobic digestion process of lignocellulosic resources. In Corsica, due to the clearing brush policy to prevent forest fires, cellulosic wastes are generated. Besides, Corsica is an important producer of essential oil from Mediterranean species thanks to a process generating an important amount of dried vegetation as waste every year. The aim of this preliminary study on biomass as renewable energy was to characterize and to select the most appropriate substrates for the anaerobic digestion process. Fiber contents and Biochemical Methane Potential (BMP) were performed on five substrates. Heather, rockrose and strawberry tree were chosen as representative of forest fuels. Water distillation residues of laurel and immortelle were considered as natural resource wastes. On this work, we focus on the three species which had the best BMP. Rockrose, dry residue of immortelle and strawberry tree produced 139, 124 and 87 Nm3 CH4 per grams of volatile solids, respectively. The ratio holocellulose: lignin, thanks to the Van Soest method of fiber determination, was determined for those species: 3.8, 3.4 and 1.7. This parameter is a key factor to take into account for the correlation of the BMP with the compositional characteristics. Further work will be performed on these chosen substrates to optimize the anaerobic digestion process in 15 L-reactors. http://dx.doi.org/10.1016/j.nbt.2014.05.1948 www.elsevier.com/locate/nbt S135 ENVIRONMENTAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 Ian Sofian Yunus ∗ , Shen-Long Tsai can decolorize RBBR more efficiently in continous mode than in batch mode. Acknowledgements: The research wassupported by the following projects: OPVK CZ 1.07/2.3.00/30.0019, FP7-KBBE-2012-6-singlestage no. 312100 BIOCLEAN, CZ.1.05/2.100/03.0100 (IET) and CR National Feasibility Programme I no.LO1208. National Taiwan University of Science and Technology http://dx.doi.org/10.1016/j.nbt.2014.05.1950 PH-10 Biomolecules for Environmental Application: Direct Use of Biorecovered Precious Metal Catalyst from Artificial Wastewater for Chlorinated Environmental Pollutant Degradation In the last decades, precious metals have been extensively studied for their potential use as catalysts for chlorinated pollutant degradations. These pollutants are resulted from various industrial processes, groundwater, pharmaceuticals wastewater treatment plant (WWTP) effluents, and many other sources. In this study, a new recovery method of precious metal catalyst for dechlorination of environmental pollutant was examined. The precious metal catalyst was produced by reduction of its ionic state, which was recovered from artificial wastewater, using biomolecules based adsorbent. This study demonstrates that the newly discovered biorecovery concept of precious metal catalyst from artifical wastewater using biomolecules based adsorbent and its direct use as catalyst for chorinated pollutant degradation are both feasible and applicable. http://dx.doi.org/10.1016/j.nbt.2014.05.1949 PH-11 Degradation of recalcitrant polymers and synthetic dyes by microrganisms isolated from polluted soils Ashutosh Kumar Verma 1,∗ , Dornakova Veronika 2 , Hana Kotulova 2 , Katerina Malachova 2 , Cenek Novotny 3 1 University of Ostrava Ostrava University 3 Academy of Sciences of the Czech Republic 2 An attempt was made to isolate microorganisms which may have potential to degrade polymer plastics and recalcitrant organopollutants. The prescreening was carried out on agar plates containing 100 ppm of a model dye, Azure B, whose decolorization was considered to be a tool indicating an enhanced degradation capacity towards recalcitrant polymers and xenobiotics. The compost, sludge and polymer-polluted soils had been used for isolation. More than 50 monocultures including bacteria, yeast and fungi belonging to various genera showed efficient decolorization of Azure B. Identification of the isolates was carried out using ribosomal molecular markers. A few isolates had been used for the degradation of virgin and pre-treated plastic polymers with limited success. Culture-independent approach was applied to assess the diversity of microbial communities in polymer-polluted soil(s). A fungal isolate affiliated with Trametes sp. had been further used for the decolorization of an anthraquinone dye, Remazol Brilliant Blue R (RBBR) at a concentration of 50 ppm. Our aim was to evaluate the efficiency and behaviour of the fungus under the conditions of rotating biological contactor reactor in both batch and continuous mode. The results indicated that the selected fungal species S136 www.elsevier.com/locate/nbt PH-12 Composting of creosote-impregnated wood via composting with green wastes: Ecotoxicity and microbial community dynamics during polycyclic aromatic hydrocarbons degradation process Stefano Covino ∗ , Zdena Filipová, Tomaš Cajthaml Křesinová, Monika Čvančarová, Alena Institute of Microbiology AS CR, v.v.i Composting has been shown to be a suitable bioremediation method for the clean-up of polluted matrices. In our pilot-scale test, creosote-impregnated wood with an overall polycyclic aromatic hydrocarbons (PAH) contamination of 26498 mg kg-1 was the target material and two different substrates were used as bulking agents, namely grass cuttings and pre-treated broiler litter. Incubation took place in 400 l static composters over a period of 240 days (40 days active composting followed by 200 days maturation). The effectiveness of the two composting processes was comparatively evaluated throughout the whole incubation period by means of contaminant degradation analyses and toxicological testing whereas shifts in microbial community structure were assessed via phosholipid fatty acid analysis (PLFA) and 454pyrosequencing. The grass substrate promoted an almost complete removal (i.e. 97%) of the total PAH content in creosote wood, while overall PAH depletion using broiler litter as bulking agent was 81% of the original concentration. The acute toxicity test towards the luminescent bacterium Vibrio fischeri and the phytotoxicity test based on germinability of barley seeds (Hordeum vulgare L.) showed that the PAH degradation process was accompanied by a significant drop in toxicity. Regardless of the treatment typology, PLFA profiling highlighted an increased incidence of Gram- bacteria and fungi in the crucial phases of PAH dissipation. Morevoer, fungi appeared to be dominant also in the maturation phase, especially when broiler litter was the substrate. Pyrosequencing analyses of 16S rRNA and ITS gene sequences for bacteria and fungi respectively, are in progress. http://dx.doi.org/10.1016/j.nbt.2014.05.1951 ENVIRONMENTAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 PH-13 PH-14 Selection and characterization of indigenous hydrocarbon-degrading bacteria from tourist ports in the Mediterranean Sea Basin Characterization of sulphate reducing bacteria communities in sediments from tourist ports in the Mediterranean Sea Basin Enrica Bullita 1 , Claudio Ruggeri 1 , Simona Sergi 1 , Laura Serreli 1 , Giovannimatteo Erby 2 , Alessio Nieddu 2 , Alessandra Carucci 2 , Elena Tamburini 1,∗ Claudio Ruggeri 1,∗ , Paolo La Colla 2 , Enrica Bullita 2 , Simona Sergi 2 , Francesco Vitali 3 , Giorgio Mastromei 3 , Elena Tamburini 2 1 2 University of Cagliari - Department of Biomedical Sciences 2 University of Cagliari - Department of Civil-Environmental Engineering and Architecture Pollution by petroleum hydrocarbons is one of the major environmental problems in ports and it is mainly associated with ship/boat traffic and related facilities. Ports are not closed systems and their pollution may impact adjacent coastal areas. Hydrocarbon degraders and particularly the obligate hydrocarbonoclastic bacteria carry out a fundamental and global activity in biological removal of hydrocarbons in marine habitats. This study was carried out within MAPMED, a multidisciplinary project aimed to improve the environmental sustainability of tourist ports in the Mediterranean Sea with regard to monitoring and reduction of hydrocarbon pollution. Three tourist ports were selected as case study sites: Cagliari (Italy), El Kantaoui (Tunisia), and Heraklion (Greece). The degradation potential of the autochthonous bacterial communities was evaluated enumerating heterotrophs and hydrocarbon degraders by MPN in the surface seawater. Heterotrophs were significantly more abundant in seawater from Cagliari port as compared to El Kantaoui and Heraklion ports. On the contrary, higher viable titles of diesel- and phenanthrene-degraders were found in seawater from El Kantaoui port compared to the other two areas. Hydrocarbon-degrading bacteria were isolated and characterized regarding their phylogenetic position and catabolic abilities. The hydrocarbon degradation activities were evaluated by GC-MS in aerobic batch reactors on diesel as carbon source. The majority of degraders from Cagliari were assigned to Pseudomonas whereas strains from El Kantaoui and Heraklion were assigned to Alcanivorax and Marinobacter. The selection of the most appropriate methodologies for the eco-efficient remediation of petroleum-hydrocarbon contamination of selected sites is currently in progress. 1 University of Cagliari University of Cagliari - Department of Biomedical Sciences 3 University of Florence - Department of Biology Ports receive pollution from land, ships and port facilities. Furthermore, tourist ports are subject to seasonal anthropogenic impacts. Hydrocarbon contamination associated with port activities poses major concerns for human health and coastal ecosystems. This study was carried out within MAPMED, a multidisciplinary project aimed to improve the environmental sustainability of tourist ports in the Mediterranean Sea with regard to monitoring and reduction of hydrocarbon pollution. Three tourist ports were selected as case study sites: Cagliari (Italy), El Kantaoui (Tunisia), and Heraklion (Greece). In each port, sampling stations were located in different sectors according to their different uses. Three sampling campaigns were carried out in winter, spring at the beginning of tourist season and late summer at the end of tourist season. Samples of surface and anoxic sediments were collected at different stations. The dsrAB gene was chosen as genetic marker to specifically characterise sulphate reducing bacteria (SRB). It codes for the dissimilatory sulphite reductase catalysing the last step in the sulphate reduction pathway. Hydrocarbon degradation under sulphate-reducing conditions is an important process in marine sediments in anoxic environments. T-RFLP analysis of dsrAB gene was employed to elucidate the community structure of SRB. This work provides a spatial comparison of SRB at two scales, different port sectors within each port area and different port areas across the Mediterranean Sea, as well as a temporal comparison among different seasons. The selection of the most appropriate methodologies for the eco-efficient remediation of petroleum-hydrocarbon contamination of selected sites is currently in progress. http://dx.doi.org/10.1016/j.nbt.2014.05.1953 http://dx.doi.org/10.1016/j.nbt.2014.05.1952 www.elsevier.com/locate/nbt S137 ENVIRONMENTAL BIOTECHNOLOGY PH-15 Experimental design in the degradation of pyrene by marine-derived fungi Chaunopycnis alba CBMAI 1346 and Xylaria sp. CBMAI 1464 Maria Vasconcelos 1,∗ , Rafaella Bonugli-Santos 1 , Marili Rodrigues 2 , Sinésio Boaventura 2 , Lara Sette 3 1 Center of Chemical, Biology and Agricultural Research Organic Chemical and Pharmaceutical Division 3 São Paulo State University 2 Marine-derived filamentous fungi are considered strategic for biotechnological applications, including bioremediation of environmental pollutants under saline conditions. Chaunopycnis alba CBMAI 1346 and Xylaria sp. CBMAI 1464 isolated from marine sponges were subjected to experimental design in order to optimize their ability to degrade pyrene. At first a Plackett-Burman (PB) model was used, with nine variables, providing 20 assays in total. The matrix was analyzed statistically using STATISTICA 7.0. After 7 days C. alba CBMAI 1346 and Xylaria sp. CBMAI 1464 reached 79.39% and 46.00% of pyrene degradation, respectively. For the fungus C. alba CBMAI 1346, salinity, concentration of malt extract, peptone and yeast extract and the presence of MnSO4 presented a positive effect on the pyrene degradation, being significant at 90% (p < 0.1). For Xylaria sp. CBMAI 1464, pH, salinity, concentration of malt extract, peptone and yeast extract and inoculum presented a positive effect on the pyrene degradation, however, none of them was significant at 90% (p < 0.1). In the second PB composed by 5 variables (total of 16 assays) the fungus C. alba CBMAI 1346 reached 58.28% of pyrene degradation. The variables riboflavin and bibasic potassium phosphate (KH2 PO4 ) presented a negative effect on the process, suggesting that the inducers might work as inhibitors. C. alba CBMAI 1346 was subjected to fractional factorial (FF) using four variables, reaching 94.13% of pyrene degradation. Based on these results a central composite rotational design (CCRD) was performed. However, a higher percentage of degradation was not obtained and the result from the FF was validated. http://dx.doi.org/10.1016/j.nbt.2014.05.1954 PH-16 Heterologous expression of three laccases from different origin in Saccharomyces cerevisiae and their use for environmental applications Klara Richterova 1,∗ , Zuzana Antosova 1 , Jiri Dostal 2 , Iva Pichova 2 , Hana Sychrova 1 1 Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i Department of Biochemistry, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic v.v.i 2 Laccases are “eco-friendly” oxidoreductases with a wide range of biotechnological applications [1]. Three laccase genes were cloned into the S. cerevisiae expression vectors under various constitutive promoters. The genes originated from Myceliophthora thermophila [2], Trametes versicolor [3], and Trametes trogii [4]. FuncS138 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 tional expression of laccases in yeast was detected as an ability to convert ABTS (2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) to a green product. The level of expression and the composition of the expression media were optimized. All recombinant laccases were produced as secreted proteins due to their native Nterminal signal sequences, and thus they were easily isolated from the medium by ion-exchange and gel chromatography. The highest specific activity was found for the laccase from Trametes trogii, which, moreover, was the only laccase showing the ability of dye decolorization. This work was supported by TA CR grant TA0101 1461. References [1].Riva S. Trends in Biotechnology 2006;24(5):219–26. [2].Bulter T, et al. Applied and Environmental Microbiology 2003;69(2):987–95. [3].Cassland P, Jonsson LJ. Applied Microbiology and Biotechnology 1999;52(3):393–400. [4].Colao MC, et al. Microbial Cell Factories 2006;5(31). http://dx.doi.org/10.1016/j.nbt.2014.05.1955 PH-17 Expression analysis of APX and CAT genes in eggplants subjected to Cu + 2 and Zn + 2 heavy metals İlker Büyük , Semra Soydam Aydın, Demet Cansaran Duman, Sumer Aras ∗ Ankara University Stress could be called as any change in unfavorable growing conditions that disrupts homeostasis in plants and can lead to lower yields and possible crop failure. Biotic and abiotic stress factors are the sources of environmental stress in plants and heavy metal toxicity is one of the global environmental problems. To overcome negative effects of heavy metal contaminations on plant health, evaluation of the genes responsible for stress tolerance is the first stage of producing stress-tolerant plants. For this purpose, Solanum melongena L. plants were exposed various concentrations of Cu+2 and Zn+2 heavy metals (blank, 80 ␮M, 160 ␮M, 320 ␮M, 640 ␮M, 1280 ␮M) for 24 h. Gene expression analysis of catalase (CAT) and ascorbate peroxidase (APX) genes were performed using Light Cycler Nano Real-Time PCR instrument with cDNAs which were synthesized from mRNAs isolated from the leaf tissues of eggplant samples. Results indicated that all concentrations of Cu+2 and Zn+2 triggered the expression changes of the CAT and APX genes. Almost all concentrations of both metal contaminations led to increases in mRNA levels of CAT and APX genes and this increment pointed to the importance of these genes for stress defence in eggplant. The overexpression and silencing of the APX and CAT genes in eggplants under Cu+2 and Zn+2 stresses will be identified in further studies. http://dx.doi.org/10.1016/j.nbt.2014.05.1956 ENVIRONMENTAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 PH-18 Oxidation of low density polyethylene by a laccasemediator system Guillermo Huerta ∗ , Marcela Ayala Instituto de Biotecnología, Universidad Nacional Autónoma de México Synthetic polymers such as low density polyethylene (LDPE) represent a big ecological problem in today′ s environment. Scientists have explored and proposed a variety of possible solutions to alleviate this problem [1]. Biological degradation is an eco-friendly process in plastic waste management, first, the polymer must suffer physical and chemical changes to facilitate the mineralization or degradation process by microorganism [2]. Such changes become possible when the plastics are oxidized by abiotic factors or by catalysts. Laccases are oxidoreductases catalyzing the oxidation of organic substrates. Laccases have been described as efficient catalysts for many applications, from paper bleaching, decolourization of textile effluents, biosensors and bioremediation [3]. In this work we investigated the use of a laccase and mediators, to modify LDPE films. We observed changes in chemical composition of enzymetreated LDPE films, mostly the presence of carbonyl groups in the infrared region of 1720 cm−1 ; along with these oxidation signals, there was a loss of surface hydrophobicity (measured by contact angle) and changes in mechanical properties, such as relative elongation. The enzyme-treated LDPE films were incubated in the presence of selected microorganisms in order to evaluate their potential biodegradability. Acknowledgments. Authors acknowledge the financial support of Conacyt 179241. References [1].Panda AK, Singh RK, Mishra DK. Renewable and Sustainable Energy Reviews 2010;14:233–48. [2].Bonhomme S, Cuer A, Delort AM, Lemaire J, Sancelme M, Scott G. Polymer Degradation and Stability 2003;81:441–52. [3].Rodriguez-Cuoto S, Toca-Herrera JL. Biotechnology Advances 2006;24:500–13. http://dx.doi.org/10.1016/j.nbt.2014.05.1957 PH-19 Probing the formation of secondary minerals in the bioleaching reactions of chalcopyrite by Raman and FTIR microspectroscopy a diffusion barrier to fluxes of reactants and products. We will present Raman and FTIR microspectroscopic evidence toward the identification of secondary minerals formed during chalcopyrite passivation in the presence of iron and sulphur-oxidizing bacteria isolated from the mines of HCM. Potassium jarosite is the initial product followed by the formation of ammonia-jarosite. Covellite and elemental sulphur are detected in the passivation layer. The mechanism of passivation and testing strategies to minimize it will be presented. http://dx.doi.org/10.1016/j.nbt.2014.05.1958 PH-20 Characterization of a bacterial collection isolated from a extreme environment with potentiality of use in biotechnology industry Jose Manuel Gomez 1,∗ , Arelys Diaz 2 , Jeannette Marrero 2 , Gema Cabrera 1 , Orquidea Coto 2 1 2 University of Cadiz University of Havana Heavy metal pollution is still a worldwide problem. The metallic elements can not be degraded but only could be transformed from an oxidation state or organic compound to another. Therefore, bioremediation is a attractive option to be applied for cleaning up the environment and recovery of soils. Microbiote isolated from extreme environmemts is considered as a source of enzymes and active metabolites which could be used in the biotechnology industry. In this work bioactive compounds and remotion capacity were evaluated in seven strains ofSerratia marcencensisolated from nickel lateritic ores located in Moa, Cuba. 100% of the strains produced extracellular enzymes including DNases, proteases, lipases, lecitinases and caseine hydrolases as well as were able to solubilize inorganic phosphorus. The strains were classified as highly resistant strains based on the minimal inhibitory concentration (MIC) of Ni(II) (>25 mM) and Co(II) (>12 mM). Biomasses of the strainsS. marcescensC-1, 16 and 19 showed removal capacity Ni(II) and Co(II) from solution until values higher than 100 ppm in two hours of biomass - metal contact. Removal capacity of strain 16 in bioreactor reached values of 36% of Co(II) and higher than 50% of Ni(II), Cu(II) and Zn(II) from multimetallic solutions. The results presented here suggest the potentiality of the use of theses strains in the medical and biotechnology industry. http://dx.doi.org/10.1016/j.nbt.2014.05.1959 Constantinos Varotsis 1,∗ , Chrystaleni Giangou 1 , Sotiris 1 1 1 Papadatos , Elena Xenofontos , Ioannis Vyrides , Giorgos Manos 2 , Nicolas Messios 2 , Constantinos Xydas 2 1 2 Cyprus University of Technology Hellenic Copper Mines Chalcopyrite passivation reduces the yields from leaching and bioleaching. Despite the great efforts the problem has not been successfully resolved. Passivation involves the formation of a layer of secondary minerals on chalcopyrite surface which becomes www.elsevier.com/locate/nbt S139 ENVIRONMENTAL BIOTECHNOLOGY PH-21 Harnessing microbial communities in tropical peatlands as resources for novel biocatalysts for plant biomass deconstruction Nicole Chua 1,∗ , Pui Yi Yung 1 , Shivshankar Umashankar 1 , I. Made Sudiana 2 , Sanjay Swarup 1 1 2 National University of Singapore LIPI Large amounts of plant biomass are generated annually, usually from factory and municipal wastes, forest- and agriculturalresidues. Plant-derived lignocellulosics represents a major source of renewable organic matter as raw material for fuel, macromolecules and aromatics. However, lack of efficient technologies to convert these “waste” into useful product resulted a great deal of them being burnt for heat generation, or merely to reduce the volume of biomass. Common rate limiting steps during biological degradation of biomass include accumulation of microbial growth inhibitors during treatment process, and the lack of diverse enzymes available for degradation of the heterogenic lignin- and lignin-bounded chains. Microbial enzymes are well known for their vast functional diversity, which have increasing recognitions from industries. We investigated the functional capabilities of the microbial communities in tropical peatlands for their ability to degrade lignocellulosic compounds. Using both culturing and culture-independent techniques, enzymes capable of lignocellolytic activities were screened. To date, we have found microbial isolates capable of hydrolyzing hemicelluloses such as xylan and arabinoxylan. The same isolates are also capable of growth in high concentration of growth inhibitors such as syringaldehyde, hydroquinone and furfural. Culture-independent, sequence-guided metagenomic approaches have revealed the microbial communities in tropical peatlands contain several classes of multi-copper oxidases (e.g. laccases), dyedecolorizing peroxidases, and feruloyl esterases. Sequence analysis revealed these enzymes are distinct from existing enzymes in the database. http://dx.doi.org/10.1016/j.nbt.2014.05.1960 PH-22 Functional and genomic analysis of the plant growth promoting bacterium I Pui Yi Maria Yung ∗ , Wei Ling Ng, Yong Jian Lee, Shao Bing Johanan Aow, Boon Kiat Lennon Lim, Nicole Chua, Shivshankar Umashankar, Sanjay Swarup National University of Singapore Plant growth promoting Pseudomonas species are of great agricultural interest, especially in food crops. Using biological agent to enhance plant growth is both environmentally friendly (reduce use of chemicals and fertilizers) and economical in a long term. We studied both the physiological and genomic properties of our model plant growth promoting bacterium Pseudomonas putida S140 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 PNL-MK25. The genome of P. putida PNL-MK25 strain has been sequenced and annotated, coupling with biological assays relating to plant growth promoting properties. Results indicated that the estimated genome size of the bacterium is around 5.8Mb, encoding 5313 putative open reading frames. Whole genome sequence comparison between strain MK25 to other sequenced genomes placed it closest to Pseudomonas fluorescens Pf01, sharing ∼82% of ORFs. Substrate utilization revealed that the strain is capable of utilizing a range of carboxylic acids, esters and fatty acids. It is also resistant to several classes of known antibiotics. Plant growth promoting assays have revealed that strain MK25 promotes lateral root formation in model plant Arabidopsis. Genes potentially responsible for plant growth promotion, for example the production of antimicrobial compounds and auxin, has been found and/or functionally confirmed. http://dx.doi.org/10.1016/j.nbt.2014.05.1961 PH-23 ’Biodesalination’: a synthetic biology approach for the use of photosynthetic bacteria in water treatment Annegret Honsbein 1,∗ , Mary Ann Madsen 2 , Jaime M. Amezaga 3 , Catherine A. Biggs 4 , Tom Bond 5 , Catherine J. Gandy 3 , Esther Karunakaran 4 , Linda Lawton 6 , Konstantinos Minas 6 , Michael R. Templeton 5 , Anna Amtmann 2 1 University of Glasgow Institute of Molecular, Cell and Systems Biology, University of Glasgow 3 School of Civil Engineering and Geosciences, Newcastle University 4 Department of Chemical and Biological Engineering, University of Sheffield 5 Department of Civil and Environmental Engineering, Imperial College London 6 Institute for Innovation, Design and Sustainability, Robert Gordon University, Aberdeen 2 Shortage of freshwater is a serious global problem, and expected to become even more urgent over the next decades. Many of the driest regions worldwide are close to the sea, but irrigation of fields with seawater–even if diluted–leads to the build-up of salt levels in the soil that are toxic to all common food crops (http://www.unwater.org). Current desalination technologies such as membrane-based reverse osmosis, are successfully used in large-scale desalination plants, however, they are expensive and energy inefficient [1]. Our multi-disciplinary team of biologists and engineers from 5 UK universities is working on an innovative desalination technology based on biological processes [2]. The “Biodesalination” strategy envisions the use of photosynthetic cyanobacteria modified with light-driven ion transport proteins to function as ion exchangers that selectively remove sodium chloride from seawater. This process would harness solar energy to provide a more cost effective and energetically sustainable desalination process. References [1].Plappally AK, Lienhard JH. Costs for water supply, treatment, enduse and reclamation. Desalination and Water Treatment 2013;51: 200–32. [2].Amezaga JM, Amtmann A, Biggs CA, Bond T, Gandy CJ, Honsbein A, Karunakaran E, Lawton L, Madsen MA, Minas K, Templeton ENVIRONMENTAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 MR. ’Biodesalination’: a case study for applications of photosynthetic bacteria in water treatment. Plant Physiology 2014;164: 1661–76. http://dx.doi.org/10.1016/j.nbt.2014.05.1962 PH-24 Characterisation of Metal Transport Proteins for providing metal stress tolerance in green microalgae Aniefon Ibuot ∗ , Andrew Dean, Jon Pittman University of Manchester Characterisation of Metal Transport Proteins for providing metal stress tolerance in green microalgae The Cation Diffusion Facilitator (CDF) family is known for providing significant tolerance in metals such as Mn2+ and Zn2+ . These proteins are well characterised in organisms such as human, yeast and plants but not in unicellular green algae. There are five CDF genes in Chlamydomonas reinhardtii. MTP1 to MTP4 were cloned and functionally characterised by yeast heterologous expression using a Zn2+ and Co2+ sensitive mutant yeast strain zrc1cot1 and a Mn2+ sensitive yeast strain pmr1. The MTP-expressing yeast strains were screened for suppression of metal sensitivity to ascertain the transport function each MTP protein. MTP1 was able to strongly rescue the Zn2+ and Co2+ sensitivity of the zrc1cot1 strain, while MTP3 could weakly mediate Zn2+ and Co2+ growth. MTP2 and MTP4 appeared to have no Zn2+ or Co2+ transport activity. MTP2 to MTP4 but not MTP1 could strongly rescue the Mn2+ sensitivity of the pmr1 strain. This clearly confirms the metal transport functions of the MTP family in Chlamydomonas. Further studies are over-expressing these MTP proteins in Chlamydomonas to determine whether enhanced metal uptake and tolerance of specific metals can be achieved. This research could increase our understanding of how yeast and algae could be utilized as a potential environmentally sustainable tool for the remediation and decontamination of metal-polluted wastewater. Keywords: Metal transport proteins, metals, yeast, Chlamydomonas reinhardtii http://dx.doi.org/10.1016/j.nbt.2014.05.1963 PH-25 Engineering high-redox potential laccases in the lab to aid biomass conversion into chemicals, materials and biofuels Susana Camarero 1,∗ , Ana I. Vicente 1 , Miguel Alcalde 2 , Isabel Pardo 1 1 2 Centro de Investigaciones Biológicas, CSIC Instituto de Catálisis y Petroleoquímica, CSIC Laccases catalyze the oxidation of a variety of aromatic compounds without any other requirement than oxygen from air. In previous studies we have highlighted the biotechnological potential of fungal laccases to improve the utilization of plant biomass in the modern biorefineries [1]. Lignin derived compounds released during lignocellulose processing can be used as redox mediators of laccases or as bioactive precursors for the enzymatic synthesis of natural products. However, the industrial implementation of fungal laccases is hampered by the lack of powerful expression systems and the need for active and stable enzymes under the harsh operational conditions. Recently we developed fungal laccases of high-redox potential functionally expressed in S. cerevisiae by directed evolution [2,3]. Using these platforms as the starting points and ad-hoc high-throughput screening methods [4,5] we developed a more robust laccase with improved catalytic activity towards phenolic compounds under preferred pH conditions, which represents a step forward to aid the conversion of lignocellulosic biomass into chemicals, materials and biofuels. This work has been funded by the Spanish Project BIO201019697. References [1].Cañas AI, Camarero S. Biotechnol Adv 2010;28:694–705. [2].Maté D, García-Burgos C, García-Ruiz E, Ballesteros A, Camarero S, Alcalde M. Chem Biol 2010;17:1030–41. [3].Camarero S, Pardo I, Cañas AI, Molina P, Record E, Martínez AT, Martínez MJ, Alcalde M. Appl Environ Microbiol 2012;78:1370–84. [4].Pardo I, Chanaga X, Vicente AI, Alcalde M, Camarero S. BMC Biotechnol 2013;13:90. [5].Pardo I, Vicente AI, Alcalde M, Camarero S. Biotechnol Bioeng 2012;109:2978–86. http://dx.doi.org/10.1016/j.nbt.2014.05.1964 PH-26 Investigation of polyvinylchloride biodegradation by microbial consortia enriched from digested sludges Fabio Fava ∗ , Noura Raddadi, Lucia Giacomucci, Nadia Lotti University of Bologna During last decades, production of synthetic plastics has increased dramatically to reach approximately 280 million tonnes in 2011. The accumulation of plastic waste in the environment is raising concerns about its effects both on human and the environment. In EU, about 40% of plastic waste is currently disposed of in landfills, where partially undergoes photodegradation, producing microplastics which can absorb toxins and toxic chemicals and together with plasticizers enter the marine environment and thus the food chain, where they exert toxic effects. Furthermore, colonization of plastics by sessile organisms may permit transport of alien species in the ocean environment and may threaten marine biodiversity. Therefore it is necessary to find new eco-friendly techniques for safe handling and degradation of plastic wastes. In this work, ten microbial communities enriched from waste plastics from digested sludges were screened for their capability of degrading non-pretreated films of polyvynil chloride (PVC) and polypropylene (PP). After six months of anaerobic incubation in the presence of the plastic films as main carbon source, growth of microbial community was recorded in all enriched consortia. Thermogravimetric analysis (TGA) performed on PP and PVC films www.elsevier.com/locate/nbt S141 ENVIRONMENTAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 showed biodegradation of only PVC plastic film by 5 communities. Further analyses, including ATR-FTIR and SEM analyses to analyze surface film modifications and GPC for the evaluation of the reduction of the polymer molecular weight on biodegraded PVC films are ongoing. Acknowledgments. The support of the FP7 EU project BIOCLEAN (GA-312100) is acknowledged. PH-28 http://dx.doi.org/10.1016/j.nbt.2014.05.1965 1 Analysis of the effectiveness of a cereal milling by product monocomponent medium for the low cost production of Bacillus thuringiensis Gihane Rahbany 1,∗ , Dominique Roger Lteif 1 2 PH-27 Electrical conductivity in granular biomass. Standardization and evaluation Diego Andres Suarez Zuluaga ∗ , Sebastian Canizales, Annemiek ter Heijne, Jan Weijma, Cees J.N. Buisman Wageningen University Biologically produced oxidation-reduction reactions in anaerobic granules require electron transfer between bacteria and archaea cells. Anaerobic types of biomass perform this electron transfer by mean of different mechanisms. Understanding how this process occurs would help in the optimization and development of processes for anaerobic wastewater treatment. So far, electron transfer via outer-surface c-type cytochromes, long-range electron transfer via nanowires, electron flow through a conductive biofilm matrix containing cytochromes and soluble electron shuttles are the mechanisms proposed for explaining such process. A simple method, based on the one developed by Morita et al. (2011) [1], for the measurement and analysis of electrical conductive of biomass granules is assessed in this study. By means of electrochemical interface equipment and a probe consistent of two electrodes separated by a non-conductive gap, voltages were applied to granular biomass and current was measured. Different physical and microbiological factors that could affect the measurement were studied and their influence determined. A methodology was standardized and voltage vs. current graphs were generated. From them, conductance (and conductivity) was calculated. Biomass granules originated on Emmtec (sulphate reducing sludge) and Eerbeek (methanogenic sludge) were used for the standardization of the parameters of the tests and presenting conductance up to 625.86 and 46.94 nS respectively. By relating the measured conductance with the origin, composition and specific activity of each granule a further understanding of the mechanisms used to transfer electrons was obtained. Reference [1].Morita M, et al. mBio 2011;2(4):1–6. http://dx.doi.org/10.1016/j.nbt.2014.05.1966 S142 www.elsevier.com/locate/nbt Salameh 1 , Cedric Brandam 2 , Université Saint-Joseph Liban Université de Toulouse; INPT, UPS; Laboratoire de Génie Chimique France Bacillus thuringiensis is a facultative anaerobe, gram positive, spore forming bacterium. The biotechnological importance of this bacterium resides in its ability to produce, during sporulation, crystal proteins known as ␦-endotoxins which express specific insecticidal activity. At industrial scale, the culture media represents an important part of B. thuringiensis based biopesticides production cost. According to the literature, different agro-industrial residues and byproducts were used as sources of proteins in order to reduce the cost of B. thuringiensis culture medium, but carbohydrates (glucose, starch or molasses) and/or mineral sources were added. In this work, a cereal milling by-product (CMB) as a monocomponent medium was investigated and compared to synthetic mediums in terms of ␦-endotoxin yield and productivity in submerged fermentation of different strains of B. thuringiensis. The CMB was shown efficient to be used as a complete substrate (source of proteins, carbohydrates and minerals) for B. thuringiensis production. The optimal CMB ratio in the culture medium was found to be 6% in shake flasks experiments. The consumption of the CMB sugars by the bacteria was analyzed. Production of the bio-insecticide in lab-bioreactor in controlled conditions was equally performed to give basic elements for extrapolation in industrial conditions. http://dx.doi.org/10.1016/j.nbt.2014.05.1967 PH-29 NaCl addition for increased polyhydroxyalkanoate production by Cupriavidus necator Pearl Passanha ∗ , Gopal Kedia, Richard Dinsdale, Alan Guwy, Sandra Esteves University of South Wales The effect of five different NaCl concentrations, namely 3.5, 6.5, 9, 12 and 15 g/l NaCl on PHA productivity using Cupriavidus necator has been investigated alongside a control (no added NaCl) when acetic acid was used as the sole carbon source. A dielectric spectroscopy probe was used to measure real-time PHA accumulation online in conjunction with the chemical offline analysis of PHA. The highest PHA production was obtained with the addition of 9 g/l NaCl, which yielded 30% higher PHA than the control. Increasing the addition of NaCl to 15 g/l was found however to inhibit the production of PHA. NaCl addition can be used as a simple, low cost, sustainable, non toxic and non reactive external stress strategy for increasing PHA productivity when compared New Biotechnology · Volume 31S · July 2014 to previous increased temperature and other types of chemical stress such as ethanol, hydrogen peroxide or metal stress. The order of PHA accumulation for the above salt concentrations were 9 g/l > 6.5 g/l > 3.5 g/l > control > 12 g/l > 15 g/l NaCl, which clearly indicates that addition of NaCl, which up to 9 g/l enhanced PHA production. http://dx.doi.org/10.1016/j.nbt.2014.05.1968 PH-30 Enhancement of treatment of poly aromatic hydrocarbon contaminated water using new biosurfactant producing marine bacterium Tarek Taha 1,∗ , ELsayed Hafez 1 , Hesham Mahdy 2 , Saad Alamri 3 1 City for Scientific Research and Technology Applications Alazhar University 3 King Khalid University ENVIRONMENTAL BIOTECHNOLOGY phenanthrene as sole carbon and energy source. The bacterial isolates were grouped and classified using ISSR-PCR technique through four different primers. The best phenanthrene degrader was chosen and identified using 16S rRNA gene sequencing as Lysinibacillus fusiformis EHTH1 and was able to degrade up to 150 mg/l phenanthrene. The bacterial isolate was able to degrade another single, double and triple ring containing hydrocarbons as sole carbon and energy source. The strain was able to produce rhamnolipid biosurfactant which hasn’t been detected for this genus before. The 800 bp amplified fragment of RhlA gene was successfully cloned into TOPO vector with subsequent transformation into E. coli (Top10 strain). The results showed successful cloning and transformation and showed the ability of the cloned strain to degrade phenanthrene at higher concentrations as sole carbon and energy source. http://dx.doi.org/10.1016/j.nbt.2014.05.1969 2 Poly Aromatic Hydrocarbons (PAH) are well known for their slight solubility in water which considered one of the most obstacles that face the bacterial isolates to degrade hydrocarbons. Our study succeed to isolate nine bacterial isolates able to consume www.elsevier.com/locate/nbt S143 GENERAL BIOTECHNOLOGY General biotechnology PI-01 G-protein alpha subunit gene, CGA1, is involved in the resistance against heat and osmotic stress in Chlamydomonas reinhardtii Changsu Lee 1,∗ , Yoon-E Choi 2 1 Department of Bioprocess Engineering, Chonbuk National University LED Agri-bio Fusion Technology Research Center, Chonbuk National University, 79 Gobong-ro, Iksan-si, Jeollabuk-do 2 The function of heterotrimeric GTPase (G-protein) has been characterized in eukaryotic cells. G-proteins are composed of three subunits, the alpha (␣), beta (␤), gamma (␥) subunits. The general GTPase activity of G␣ induces the hydrolysis of the bound GTP, thereby playing pivotal roles in relaying the signals. However, microalgal G-proteins including G-protein alpha subunit have remained largely unknown. In this study, we characterized a gene, CGA1, encoding G-protein alpha subunit in Chlamydomonas reinhardtii. Independent knock-down mutants of CGA1 were generated via the RNA interference (RNAi) approach. CGA1 expressions consistently reduced significantly in all of the CGA1 mutants (cga1). Interestingly, all of the cga1 displayed higher survival rate at 35 ◦ C, compared to wild type. In addition, all of the cga1 has an enhanced survival rate at high osmotic stress, compared to that of the wild type. To further understand mechanisms of CGA1-mediated resistance, the extent of reactive oxygen species (ROS), the expressions of several putative downstream genes including heat shock proteins(HSPs) and MAP kinases were thoroughly analyzed. Our data indicated that CGA1 is associated with the regulation of resistance against heat or osmotic stress in C. reinhardtii by affecting multiple downstream genes. Since a G-protein alpha subunit is highly conserved across microalgal species, our results will shed light on future biotechnological applications of microalgae under the extreme environmental conditions. http://dx.doi.org/10.1016/j.nbt.2014.05.1970 PI-02 Adaptive laboratory evolution study of Corynebacterium glutamicum resistant to oxidative stress and their application to develop an artificial oxidative stress-resistant strain Joo-Young Lee 1,∗ , Jiyoon Seo 1 , Eung-Soo Kim 2 , Heung-Shick Lee 3 , Pil Kim 1 1 The Catholic University of Korea Inha University 3 Korea University 2 In this study, we performed adaptive laboratory evolution (ALE) study of Corynebacterium gluiamicum in chemostat for 1,900 hours with gradual increase of oxidative stress-intensity to understand the oxidative stress response and develop C. glutamicum as a platform microbe. Through ALE experiment, C. S144 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 glutamicum acquired an ability to grow under stress of 10 mM H2 O2 . In the transcriptome results of the adapted strain, a total of 1,180 genes (38.6% of the genome) were up-regulated more than 2-fold, and 126 genes (4.1% of the genome) were down-regulated less than 2-fold under oxidative stress condition compared with those of wild-type (WT) strain under non-stress condition. Especially the genes encoding enzymes in the ␤ketoadipate pathway (pca genes) were up-regulated more than 3-fold. To develop an artificial oxidative stress-resistant strain, the pca gene clusters in the ␤-ketoadipate pathway were expressed in the WT strain. WT strain was unable to grow under 2 mM H2 O2 , while the strains expressing pca gene clusters restored growth. The expressions of pca gene clusters also enabled the WT strain to increase its resistance against various oxidative stressors including H2 O2 . The oxidative stress resistance of those strain was correlated to the reactive oxygen species (ROS)scavenging activity of the cytosol. These findings would be useful to develop an oxidative stress-resistant synthetic strain for industrial applications. [This study was financially supported by the Korean Ministry of Science, ICT & Future Planning (Intelligent Synthetic Biology Center of Global Frontier Project 2012M3A6A8054887)] http://dx.doi.org/10.1016/j.nbt.2014.05.1971 PI-03 Symbiosis Mechanisms of Lactic Acid Bacteria and Yeasts in Inner Mongolian Traditional Fermentative Milk Products Yinfeng He ∗ , Jianjun Tian, Minmin Liu, Bin Yan Inner Mongolia University of Agriculture The majority of the traditional dairy products in Inner Mongolia, China, are co-fermented with lactic acid bacteria and yeasts. So far, forty-seven strains of lactic acid bacteria and nineteen strains of yeast have been isolated and identified from koumisses, ripened creams, and yogurts. A systematic study was performed to understand the co-culture properties and cofermentation characteristics between them. The results show: (1) The lactic acid bacteria and the yeasts found in the traditional dairy products in Inner Mongolia have symbiotic effects that can increase active strain numbers and enhance acid productivity; (2) The metabolites of the yeasts can act as pH buffer in the broth of the lactic acid bacteria, reduce acid inhibition, enhance the growth of the strains, and increase titration acidity. In the meanwhile, the metabolites of the lactic acid bacteria can also promote the growth of the yeasts, increase titration acidity, and decrease the pH value to an optimum growth range; (3) When the lactic acid bacteria and the yeasts are co-cultured in skimmed milks, both small molecular acidic substances, such as the lactic acid and propionic acid, and flavor components that can form aromatic substances such as ethyl acetate can be produced easily; and (4) The metabolites produced from the yeasts after a culture time of 60 hours are the best for the lactic acid bacteria, and the optimal amount of the metabolites in the culture GENERAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 medium is 40 wt%. Details of this study will be discussed in the presentation. http://dx.doi.org/10.1016/j.nbt.2014.05.1972 PI-04 Construction, expression and renaturation UBI::human insulin analog single chains of Diana Mikiewicz ∗ , Anna Wojtowicz-Krawiec, Natalia Lukasiewicz, Iwona Sokolowska, Agata Jagiello, Piotr Borowicz, Grazyna Plucienniczak, Andrzej Plucienniczak Institute Biotechnology and Antibiotics Insulin is a hormone secreted by the pancreas in response to high blood sugar levels that induces hypoglycemia. Insulin regulates the body’s use of glucose and the levels of glucose in the blood. Insulin and its various derivatives are used in large amounts in treatments of diabetes. The aim of this work was using a deubiquitinating protease analogue - UBP1 to obtain purified single chain A and B of human insulin analog in E. coli. We constructed fusion genes - Ub::ins and protease analogue - UBP1 in one vector together. The region that codes for the recombinant fusion gene is under control deoP1P2 promoter. The protease analogue - UBP1 gene which is under the control of pms promoter(WO05066344 A2) was discovered and described in our Institute. The recombinant human insulin chain genes present downstream of ubiquitin gene was used as a signal protein. Ubiquitin is composed of 76 amino-acid residues with a total molecular mass of 8.6 kDa. This protein is an element of the universal protein modification in eukaryotes called ubiquitination, a phenomenon which does not occur in bacteria. In spite of that, it has been shown that proteins fused to ubiquitin undergo greater expression in E coli and are easier to purify and renaturate than nonhybrid foreign proteins. We obtained a high level of expression of fusion proteins Ub::ins analog A chain and Ub::ins analog B chain in E. coli. After cleavage of the ubiquitin by UBP1 analog, good yields of thepurified insulin chains were produced. http://dx.doi.org/10.1016/j.nbt.2014.05.1973 PI-05 The crude lecithin gum as a substrate for lipase production by Pseudomonas sp Maria Antonia Celligoi 1,∗ , Cristiani Baldo 1 , Marcos Oliveira 1 , Lilian Baggio 1 , Fabiana Gasparin 1 , Marcelo Melo 1 , Dionisio Borsato 2 1 2 of this study was to optimize the production of lipase by a strain isolated from a slaughterhouse effluent, identified as Pseudomonas sp, using the CLG as a carbon source. The lipase activity at 233,44 U/mL was obtained by growing the microorganism on minimum medium containing 1% (w/v) of CLG, after 24 hours. The highest enzymatic activity (400,22 U/mL) was observed when 2% (w/v) of CLG was used. The optimization of the temperature levels, pH and agitation rate, by Box-Benhken design, resulted in the maximum enzyme activity of 517,00 U/mL, obtained at 32 ◦ C, pH 9.0 and 200 rpm. These data showed the possibility of using the CLG as substrate for lipase production by Pseudomonas sp and can contribute in the reduction of production cost of lipases in industrial scale, and increase the market value of soybean oil byproducts. Financial support: CAPES/PNPD http://dx.doi.org/10.1016/j.nbt.2014.05.1974 PI-06 Production of anti-cancer compound by microbial process Changhyun Roh KAERI In this study, we elucidated that the production of anti-cancer agent from microbial process for regio-specific hydroxylation of resveratrol. Among the strains examined, a strain showed high regio-specific hydroxylation activity to produce Piceatannol. In a 5 L (w.v. 3 L) jar fermentation, the wild type Streptomyces sp. in batch system produced 205 mg of Piceatannol (i.e., 60% yields) from 342 mg of resveratrol in 20 h. This biotransformation result demonstrates that regio-specific hydroxylated resveratrol exhibiting more potent anti-cancer activity could be produced on a large scale using microbial biotransformation. Using biotransformed piceatannol, the in vitro anti-cancer study was performed against human cancer cell line (HeLa) and MTT assay was used to analyze the cell growth inhibition. The results showed that the biotransformed piceatannol possessed a significant anticancer activity. This is first report elucidating production of anti-cancer compound using microbial process for regio-specific hydroxylation of resveratrol. This study has significant scope that a biotransformation result provides one initiative example such that regio-specific hydroxylated compound from resveratrol showing more potent biological activity could be produced in large scale using microbial biotransformation. Thus, microbial processes become a good model system to develop an industrial biotransformation system to produce drugs for treasure islands. http://dx.doi.org/10.1016/j.nbt.2014.05.1975 State University of Londrina - Department of Biochemistry and Biotechnology State University of Londrina - Department of Chemistry The refining of soybean oil involves the degumming process which results in byproducts with variety of quality grades. The crude lecithin gum (CLG) contains a mixture of phospholipids and oil, and may be submitted to a series of solvent extraction and precipitation processes to produce the commercial lecithin. The aim www.elsevier.com/locate/nbt S145 GENERAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 PI-07 Induced differential metaproteomics: identification of cellulases in a methanogenic microbial community at thermophilic conditions Jutta Speda 1,∗ , Bengt-Harald Jonsson 1 , Martin Karlsson 2 1 2 Linköping University Rational Enzyme Mining AB The identification of novel enzymes for use in industrial biotechnology is an important goal in enzyme discovery. Most industrially relevant enzymes to date have been isolated from pure cultured microorganisms. For future discovery of novel enzymes this is however a major bottleneck since it is well established that only a small fraction of all microorganisms can be obtained in pure cultures. The possibility to identify enzymes directly from complete microbial communities would therefore give access to a huge number of novel enzyme candidates. Metaproteomics has hitherto mainly been used to understand ecosystem functions. We have instead used the dynamics of proteomics to develop a method based on “induced differential metaproteomics”, by which a desired enzyme activity is induced in a full microbial population and compared to a non-induced reference of the very same population. In a first example the goal was to induce, select and identify cellulases from a thermophilic methanogenic community. Out of several hundred detectable proteins in a 2D-DIGE experiment, 24 proteins could be identified as at least two-fold up-regulated upon induction. For some proteins spots, the cellulolytic activity was further validated by activity staining using 2D-zymography. Mass spectrometry analysis revealed that 21 out of the 24 up-regulated proteins are cellulases or associated to cellulolytic activity giving a remarkable hit-rate of 88%. This demonstrates the high efficiency and precision of the method, by which a much wider span of the microbial world can be scanned for novel and targeted enzymes. http://dx.doi.org/10.1016/j.nbt.2014.05.1976 PI-08 Effect of lemongrass oil and powder on growth and aflatoxin production by Aspergillus flavus IMI242648 in maize Dusanee Thanaboripat 1,∗ , Yaowapa Kurdhom 1 , Suchada Rakphung 1 1 2 Suvathi 2 , Manatsanun King Mongkut’s Institute of Technology Ladkrabang Government Pharmaceutical Organization Aflatoxin is one of the most important mycotoxins producing in nature and poses health hazard to human and animals. Control of aflatoxin producing fungi and aflatoxin production in various commodities by various means have been investigated. Natural plant extracts may provide an alternative way to prevent food or feed from fungal contamination. The objective of this study is to compare the effect of lemongrass (Cymbopogon citratus (DC.) S146 www.elsevier.com/locate/nbt Staph), an edible herb, in the forms of oil and powder on the growth and aflatoxin production of Aspergillus flavus IMI 242684 in maize. Lemongrass oil at concentrations of 1, 2, 3, 4 and 5% and lemongrass powder at 1, 2, 3, 4, 5, 10, 20, 30, 40 and 50% were applied in maize with aflatoxin producing fungus for 7 days at ambient temperature. The results show that lemongrass oil at all concentrations inhibited fungal growth whereas lemongrass powder at 40 and 50% could inhibit fungal growth. For the ability to inhibit aflatoxin production, it was found that lemongrass powder at 10-30% and at 40-50% could inhibit aflatoxin production for 3 and 7 days, respectively. http://dx.doi.org/10.1016/j.nbt.2014.05.1977 PI-09 Bioconversion of Ginsenosides from Red Ginseng Extract Using Candida allociferrii JNO301 Isolated from Meju Sulhee Lee 1,∗ , Yong-Hun Lee 1 , Jung-Min Park 2 , Dong-Hoon Bai 3 , Young-Seo Park 1 1 Gachon University Korean Culture Center of Microorganisms 3 Dankook University 2 Red ginseng (Panax ginseng), a Korean traditional medicinal plant, contains a variety of ginsenosides as major functional components. It is necessary to remove sugar moiety from the major ginsenosides to make them aglycone form due to their low absorption rate into the intestine. To screen the microorganisms which show the bioconversion activity on the ginsenosides from red ginseng, total 50 yeast strains were isolated from Korean traditional meju (a starter made with soybean and wheat flour for the fermentation of soybean paste). Twenty strains which form the black zone around the colony on the esculin-YM agar plate were screened first, and among them 5 strains which have high β-glucosidase activity on p-nitrophenyl-β-D-glucopyranoside as a substrate were then selected. Strain JNO301 was finally chosen as a bioconversion strain in this study on the basis of high bioconversion activity against red ginseng extract by TLC analysis. The selected bioconversion strain was identified as Candida allociferrii JNO301 by the nucleotide sequence analysis of 18S rRNA gene. The optimum temperature and pH for the cell growth were 20-30 and pH 58, respectively. By the analysis of TLC, it was confirmed that C. allociferrii JNO301 converted ginsenoside Rb1 into Rd and then into F2, Rb2 into compound O, Rc into compound Mc1, Rf into Rh1. Quantitative analysis using HPLC showed that bioconversion of red ginseng extract resulted in the increase in the concentration of Rd, F2, compound O, compound Mc1, and Rh1 with 2.73-, 3.32-, 33.87-, 16-, 5.48-fold, respectively. http://dx.doi.org/10.1016/j.nbt.2014.05.1978 GENERAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 PI-10 Construction of a genetically encoded binary counting module in Escherichia coli Jia Zhao 1,∗ , Sean Colloms 2 , Susan Rosser 3 1 Institute of Molecular, Cell and Systems Biology, University of Glasgow University of Glasgow 3 University of Edinburgh was noticed between the extraction with isopropanol and acetone. Samples extracted with ethanol had higher antioxidant activity than the others and exhibited the rice fragrance. Keywords: Gamma-oryzanol, Antioxidant activity; Rice bran oil; conventional solvent extraction http://dx.doi.org/10.1016/j.nbt.2014.05.1980 2 PI-12 Binary systems have been applied extensively in computer science because they allow large numbers to be encoded. Introducing a similar counting system into living cells will be a fundamental step towards building cellular computers. One way to do this is to encode information in the DNA sequence using site-specific recombination to invert the orientation of a DNA segment, making the information heritable and easily detectable. Serine phage integrases catalyse recombination between specific DNA sites, attP and attB, producing sites attL and attR which are no longer substrates for integrases alone. However, in the presence of a recombination directionality factor (RDF) this directionality is reversed so that attL and attR recombine to recreate attP and attB. If two att sites are placed in inverted repeat, recombination flips the orientation of the intervening sequence between two possible states, which can represent a single binary digit (0 or 1) heritably stored in the DNA. Based on this principle, a toggle switch has been constructed in E.coli. By first expressing the integrase, and then the integrase and the RDF together, its state can be changed between 0 and 1 under the control of different signals. This switch will be developed to a binary counting module by placing the RDF under the control of the sequence state and using a single chemical signal to control the expression of the integrase. By linking N units together, each using a different phage integrase, a binary counter can be made to help cells count to 2N -1. Isolation and characterization of antibiotic producing Micromonospora from Thai Jasmine rice Chitti Thawai King Mongkut’s Institute of Technology Ladkrabang Thirty eight actimonycete strains were isolated from tissue of root and stem of Thai jasmine rice (Oryza sativa). These strains were grouped using phenotypic, chemotypic and genotypic characteristics into 5 groups. Phylogenetic position, chemotaxonomic analyses including some phenotypic characterisation revealed that the representative strains in each group belonged to the members of the genus Micromonospora. The fermentation broths of these isolates were extracted with ethyl acetate and were tested for antimicrobial activity. The results showed that more than 60% of Micromonospora strains inhibited the growth of pathogenic agents of leaf blight disease (Xanthomonas oryzae) and blast disease (Pyricularia grisea). Based on these results, we conclude that endophytic Micromonospora strains are great and should represent an excellent source for discovery of the bioactive compounds. Keywords: Actinomycetes, Micromonospora, Antibiotic, Blight and Blast disease. http://dx.doi.org/10.1016/j.nbt.2014.05.1981 http://dx.doi.org/10.1016/j.nbt.2014.05.1979 PI-13 PI-11 Evaluation of gamma-oryzanol in Thai rice bran oil extracted by conventional solvent extraction method and its antioxidant activities Androgen’s effect on MCP-1 and monocyte attraction to prostate cancer cells Eun-Kyung Kim ∗ , Eun-Ju Choi Konkuk University Sujitra Sukonthamut ∗ , Chitti Thawai, Duangkamol Ruen-ngam King Mongkut’s Institute of Technology Ladkrabang Rice bran oil was extracted by solvent-assisted extraction with hexane, ethylacetate, acetone, isopropanol and ethanol using a solvent-to-rice bran ratio of 4:1 (w/w). The experiments were done in triplicate at room temperature (30 ◦ C) with a total extraction time of 2 hours/sample. The oil components were separated by reverse-phase HPLC and quantified with a fluorescence detector. The radical scavenging capability of the oil was tested with DPPH and was expressed as ␮mol Trolox Equivalent Antioxidant Activity. Among these solvents, ethanol was the best solvent for the extraction of gamma-oryzanol as compared with the others solvents for conventional solvent extraction while isopropanol was better for oil yield extraction at room temperatures. No difference in oil yield Trigger of inflammatory monocytes to the tumor site is mediated by monocyte chemoattractant protein-1 (MCP-1) through binding to its receptor. We assumed that androgen could modulate MCP-1 expression in hormone-responsive prostate cancer cells and then promote recruitment of monocytes. Dihydrotestosterone (DHT) stimulated a time-dependent (0–72 hours) and concentration-dependent (0–1 nmol/L) increase in MCP1 mRNA levels in androgen-responsive human prostate cancer cells (LNCaP). This increase in MCP-1 mRNA corresponded with increased secretion of MCP-1 protein. The effect of DHT was mediated through an androgen receptor (AR)-dependent pathway as small inhibitor RNA (siRNA) against AR negated the induction of MCP-1. Although DHT also induced TWIST1 mRNA, an epithelial–mesenchymal transition (EMT)–related factor, and purported inducer of MCP-1, blocking its expression with siRNA www.elsevier.com/locate/nbt S147 GENERAL BIOTECHNOLOGY did not inhibit DHT induction of MCP-1 mRNA. Moreover, conditioned media from androgen-treated cells promoted human monocyte THP-1 cell migration and this effect was inhibited by antibody against MCP-1. These results indicate that androgen may regulate MCP-1 and promote inflammatory microenvironment in prostate cancer. http://dx.doi.org/10.1016/j.nbt.2014.05.1982 New Biotechnology · Volume 31S · July 2014 used CotE, CotG, CotY spore coat protein as anchoring motives for the successful display of beta-galactosidase, spreptavidin, and other interesting target protein with biotechnological application such as laccase for the environmental usages. Surface localization using flow cytometry and enzymatic activity on the Bacillus subtilis spore was examined for the successful confirmation of surface localization of target proteins. Further usage and other application will be discussed. http://dx.doi.org/10.1016/j.nbt.2014.05.1984 PI-14 Suppression of dust mite extract and 2,4dinitrochlorobenzene-induced atopic dermatitis by the water extract of DA-9601 Choi Eun-Ju ∗ , Kim Eun-Kyung Konkuk University DA-9601 is a novel anti-peptic formulation prepared from the ethanol extracts of Artemisia asiatica possessing anti-oxidative, anti-allergic and anti-inflammatory activities. However, their effect on atopic dermatitis (AD) has not been studied yet. In this study, we report that topical application of DA-9601 suppressed house dust mite extract (Dermatophagoides farinae extract, DFE) and 2, 4-dinitrochlorobenzene (CDNB)-induced AD-like skin lesions in BALB/c mice model. We established atopic dermatitis model in BALB/c mice by repeated local exposure of DFE/CDNB to the ears. Repeated alternative treatment of DFE/CDNB caused ADlike lesions. DA-9601 reduced AD-like skin lesions based on ear thickness and histopathological analysis, and serum IgE levels. DA9601 inhibited mast cell infiltration into the ear and elevation of serum histamine in AD model. In addition, DA-9601 suppressed DFE/CDNB-induced expression of IL-4, IL-13, IL-31, and TNF-␣ in the ears. Taken together, our results showed that topical application of DA-9601 exerts beneficial effects in animal model of AD, suggesting that DA-9601 might be a candidate for the treatment of AD. http://dx.doi.org/10.1016/j.nbt.2014.05.1983 PI-15 Biotechnological application of Bacillus subtilis spore display system June Hyung Kim Dong-A University Bacterial surface display finds its important biotechnological application in the fields of screening tools of evolved enzyme, bioremediation, whole cell bioconversion and tool for live vaccine production. For the functional bacterial surface display of active enzyme of multimeric form, which is generally impossible due to molecular assembly of the monomer subunit subsequent to the secretion of displayed target protein outside the cell, a new surface display system based on Bacillus subtilis spore is developed. Here, we tried to develop a new bacterial surface display format for the efficient expression of multi-subunit enzyme. We S148 www.elsevier.com/locate/nbt PI-16 Production of 3-hydroxybutyrate by E. coli: Application of Nitrogen and Phosphorous limitation to steer fluxes to product formation Monica Guevara 1,∗ , Johan Jarmander 1 , Mariel Perez-Zabaleta 1 , Jorge Quillaguamán 2 , Gen Larsson 1 1 Industrial biotechnology, School of biotechnology, KTH, Stockholm Center of Biotechnology, Faculty of Science and Technology, San Simón University, Cochabamba Bolivia 2 Polyhydroxyalcanoates are polyesters produced in large amounts by different microorganisms, but not by E.coli. Here we expressed the bacterial polyhydroxybutarate (PHB) pathway of Halomonas boliviensis in E. coli using phbA and phbB genes to produce the monomer 3-HB. Acetyl coenzyme A (AcCoA) is the intermediate of the carbon metabolism and precursor of the PHB pathway. The objective is to design a process to produce 3-HB in E.coli with high productivity which is a first step to further produce different quality polyesters. Production of the wild type microorganisms takes place under nutrient deficient conditions and excess of a carbon source. The hypothesis is that directing the flux to the desired product (3-HB) by cultivations with controlled feed of nitrogen or phosphorous will increase the flux to the precursor (AcCoA) and minimize the formation of byproducts This will be done by: Fed-Batch cultivations with an excess of glucose and nitrogen limitation. Excess glucose will give a surplus of NADH that will inhibit citrate synthase and the AcCoA produced will instead be used for 3-HB production Fed-Batch cultivations with an excess of glucose and phosphorous limitation. Excess glucose will give a surplus of NADH that will inhibit citrate synthase and the AcCoA produced will instead be used for 3-HB production. Also the limitation of phosphorous activates the methylglyoxal pathway also leading to the more energy favorable production of AcCoA than central glycolisis. http://dx.doi.org/10.1016/j.nbt.2014.05.1985 GENERAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 PI-18 Development of a flow cytometer-based in vitro compartmentalization screening platform for directed protein evolution Georgette Wirtz ∗ , Christian Pitzler, Ljubica Vojcic, Ronny Martinez, Ulrich Schwaneberg Institute of Biotechnology, RWTH Aachen We describe the development of an ultra high throughput screening (uHTS) flow cytometer based in vitro compartmentalization (IVC) screening platformfor directed enzyme evolution. The system combines IVC (water-oil-water emulsion) and the detection of enzyme activity by fluorescence-activated cell sorting (FACS),offering an advanced uHTS screening platform (∼108 events/day) for the discovery and reengineering of industrially attractive enzymes [1].uHTS offers a qualitative differentiation between positive or negative enzymatic activity, enabling the enrichment of active variants by screening thousands of events per second. In IVC, single cellulase gene variants are transcribed and translated within a double emulsion compartment and upon translation, a fluorogenic substrate is converted by active variants and sorted using FACS [2,3]. IVC systems enable linking phenotype and genotype, which is a key requirement in directed evolution experiments. Additionally, IVC allows the expression of larger libraries (> 1010 ) by overcoming challenges such as loss of gene diversity due to low cloning and transformation efficiency and protein production avoiding technical limitations (e.g. inclusion bodies, toxicity). IVC also dramatically reduces the time required for screening enzyme libraries, allowing a greater number of directed evolution rounds per campaign, increasing the chance of generating and identifying improved enzyme variants. References [1].Ruff AJ, Dennig A, Wirtz G, Blanusa M, Schwaneberg U. ACS Catalysis 2012;2:2724–8. [2].Mastrobattista E, Taly V, Chanudet E, Treacy P, Kelly BT, Griffiths AD. Chem Biol 2005;12:1291–300. [3].Tu R, Martinez R, Prodanovic R, Klein M, Schwaneberg U. J Biomol Screen 2011;16:285–94. http://dx.doi.org/10.1016/j.nbt.2014.05.1986 PI-18 Development of a flow cytometer-based in vitro compartmentalization screening platform for directed protein evolution Georgette Wirtz ∗ , Christian Pitzler, Ljubica Vojcic, Ronny Martinez, Ulrich Schwaneberg Institute of Biotechnology, RWTH Aachen We describe the development of an ultra high throughput screening (uHTS) flow cytometer based in vitro compartmentalization (IVC) screening platformfor directed enzyme evolution. The system combines IVC (water-oil-water emulsion) and the detection of enzyme activity by fluorescence-activated cell sor- ting (FACS),offering an advanced uHTS screening platform (∼108 events/day) for the discovery and reengineering of industrially attractive enzymes [1].uHTS offers a qualitative differentiation between positive or negative enzymatic activity, enabling the enrichment of active variants by screening thousands of events per second. In IVC, single cellulase gene variants are transcribed and translated within a double emulsion compartment and upon translation, a fluorogenic substrate is converted by active variants and sorted using FACS [2,3]. IVC systems enable linking phenotype and genotype, which is a key requirement in directed evolution experiments. Additionally, IVC allows the expression of larger libraries (> 1010 ) by overcoming challenges such as loss of gene diversity due to low cloning and transformation efficiency and protein production avoiding technical limitations (e.g. inclusion bodies, toxicity). IVC also dramatically reduces the time required for screening enzyme libraries, allowing a greater number of directed evolution rounds per campaign, increasing the chance of generating and identifying improved enzyme variants. References [1].Ruff AJ, Dennig A, Wirtz G, Blanusa M, Schwaneberg U. ACS Catalysis 2012;2:2724–8. [2].Mastrobattista E, Taly V, Chanudet E, Treacy P, Kelly BT, Griffiths AD. Chem Biol 2005;12:1291–300. [3].Tu R, Martinez R, Prodanovic R, Klein M, Schwaneberg U. J Biomol Screen 2011;16:285–94. http://dx.doi.org/10.1016/j.nbt.2014.05.1987 PI-19 A fluorescent polymer shell-based enzyme screening platform for hydrolases using flow cytometry Christian Pitzler 1,∗ , Georgette Wirtz 1 , Ljubica Vojcic 1 , Stephanie Hiltl 2 , Alexander Böker 2 , Ronny Martinez 1 , Ulrich Schwaneberg 1 1 2 RWTH Aachen University, department of biotechnology DWI-Leibniz Institut für Interaktive Materialien A novel whole-cell enzyme screening platform based on a coupled reaction of glucose-oxidase and a hydrolase (Yersinia mollaretii phytase, YmPh) was developed and validated in a directed phytase evolution experiment. The coupled reaction produces hydroxyl radicals through Fenton’s reaction (hydrogen peroxide and Fe2+ ) which initiate poly(ethyleneglycol) diacrylate-based hydrogel polymerization, including the fluorescent Polyfluor-570acrylate. As consequence, fluorescent hydrogel is formed around E. coli cells that express active YmPh variants. Formation of the fluorescent hydrogel was confirmed by confocal microscopy. In addition, scanning force microscopy was used to visualize the hydrogel on the cell surface, revealing a distinct structure enclosing the cells. Labeled cells were analyzed and sorted by flow cytometry with a throughput of 1.8 × 107 events/h. Further sorting of mixed populations with a defined content of active/inactive cells yielded an enrichment factor of up to 5. Finally, the fluorescent polymer shell technology was validated by analyzing an error-prone PCR mutant library (4.8 mutations/gene). Screening of 107 events by flow cytometry yielded variant M1 with 97 U/mg increased specific activity compared to YmPh wild type www.elsevier.com/locate/nbt S149 GENERAL BIOTECHNOLOGY (315 U/mg). This screening platform is a promising approach for screening mutant and metagenome libraries for novel or improved hydrolases, by using a variety of glucose-derived substrates. http://dx.doi.org/10.1016/j.nbt.2014.05.1988 PI-20 Rye as substrate for L-lactic acid production Rygala, Katarzyna Dybka, Patrycja Piotr Walczak ∗ , Anna Pietraszek, Agata Czyzowska, Anna Otlewska, Elzbieta OltuszakWalczak Faculty of Biotechnology and Food Sciences The aim of work was L-lactic acid (LA) fermentation process using enzymatic rye hydrolyzates as an inexpensive carbon source and nutrients supporting growth of lactic acid bacteria. Rye grits or rye flour was enzymatically hydrolyzed in the low temperature process (90 - 50 ◦ C) with thermostable ␣–amylase Aquazym ATL, glucoamylase Spritase GA 14400L and malt prepared from naked oat Avena nuda as a source of pullulanase. Obtained hydrolyzates were diluted to the glucose concentration of about 120 g/l, supplemented with mineral salts (K+ , Na+ , Mg2+ , Mn2+ , PO4 3− ) and yeast extract (3 g/l) and used as the fermentation medium. L-lactic acid fermentation processes were carried out in BioFlo 415 bioreactor (5 l working volume, 42 ◦ C, 44 h) with Lactobacillus rhamnosus as producing organism. pH was controlled at 5,5 by automatic addition of 12.5% ammonia water. At the end of fermentation (44 h), 109.2 g × l−1 of glucose was utilized and 88.5 g × l−1 of lactic acid containing 97.5% of L-isomer was produced. Dry mass of bacterial cells reached value of 2.06 g × l−1 and average productivity was 2 g × l−1 × h−1 . It was proved that rye may replace corn and be competitive substrate for microbial production of L-lactic acid in rye belt countries with cold climate. The work was supported by the POIG 01.01.02-10-123/09 Project partially financed by the European Union within the European Regional Development Fund, Grants for Innovation. http://dx.doi.org/10.1016/j.nbt.2014.05.1989 New Biotechnology · Volume 31S · July 2014 medium containing 93.8 g × l−1 of reducing sugars, or glucose medium containing 120 g × l−1 of this saccharide. Biomass, reducing sugars and lactic acid concentrations were estimated during fermentation process. In the first 24 h of fermentation, specific production rate of lactic acid was 5.18 g × l−1 × h−1 for juice based medium and 4.77 g × l−1 × h−1 for glucose medium. Biomass concentration at the stationary phase reached value of 3.9 g × l−1 irrespectively of used medium. Specific substrate uptake rate was 3.98 g × l−1 × h−1 for juice and 2.80 g × l−1 × h−1 for glucose medium. Reducing sugars were completely utilized after 31 h of process in juice medium. In glucose based medium after 72 h of fermentation 29.5 g × l−1 of sugar was left unused. Final concentration of L-lactic acid after 31 h of fermentation reached value of 92 g × l−1 for juice and 62 g × l−1 for the glucose medium respectively. The optical purity of produced L-lactate was 99.13%. The work was supported by the POIG 01.01.02-10-123/09 Project partially financed by the European Union within the European Regional Development Fund, Grants for Innovation. http://dx.doi.org/10.1016/j.nbt.2014.05.1990 PI-22 A synthetic biology approach to characterise cyanobacterial promoters Mary Ann Madsen ∗ , Anna Amtmann University of Glasgow Cyanobacteria are a phylum of bacteria with the ability to perform oxygenic photosynthesis using minimal nutrient requirements: mainly sunlight, water and CO2 . Cyanobacteria thus provide a cost-effective and energetically sustainable chassis for biotechnological applications. Cyanobacteria are relatively new to the field of biotechnology, however, and the ‘tools’ available to genetically modify them are currently very limited. We therefore describe an approach to identifying promoters activated in high density cultures and characterization using a synthetic biology approach. http://dx.doi.org/10.1016/j.nbt.2014.05.1991 PI-21 Dynamics of calcium L-lactate fermentation by Lactobacillus rhamnosus in sugar beet thick juice and glucose based media Elzbieta Oltuszak-Walczak ∗ , Piotr Walczak, Agata Czyzowska, Anna Rygala, Patrycja Pietraszek, Katarzyna Dybka, Anna Otlewska Faculty of Biotechnology and Food Sciences PI-23 Differential Adaptive Fermentation of E. coli K-12 and B Strains in the Absence of adhE Gene Under Anaerobic Condition S Lee 1,∗ , Hyun Ju Kim 1 , Haeyoung Jeong 1 , Dong-Woo Lee 2 1 2 Production of pure L-lactate stereoisomer from renewable carbon sources is required for the manufacturing of green biodegradable tactic polymer poly-L-lactide (PLLA).The aim of work was evaluation of process dynamics of calcium L-lactate fermentation using sugar beet thick juice or glucose as substrates. Fermentation processes with industrial strain of Lactobacillus rhamnosus were carried out at 42 ◦ C for 48-72 h in the juice based S150 www.elsevier.com/locate/nbt Korea Research Institute of Bioscience and Biotechnology Kyungpook National University Alcohol dehydrogenase (AdhE) plays a key role in the maintenance of redox balance in E. coli under anaerobic condition. However, in the absence of AdhE, oxidized redox cofactors such as NAD+ could not be efficiently recycled in cells. Therefore, highly-reduced status could be alternatively oxidized by lactate dehydrogenase in E. coli adhE mutant cells. Indeed, under anoxic GENERAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 condition E. coli K-12 adhE mutant cells rapidly adapted to the stressed condition and grew with lactate fermentation, whereas E. coli BL21(DE3) adhE mutant cells begun very slowly adapting to anaerobic growth. It was observed that the recycling of NAD+ was very poor in E. coli BL21(DE3) adhE mutant cells during anaerobic culture, which caused severe redox stress and in turn decreased cellular viability. In contrast, K-12 adhE mutant cells maintained cellular redox balance (NAD+ /NADH ratio) and viability under anoxic condition. This difference allows K-12 and B cells to undergo differential adaptations to anaerobic stress. http://dx.doi.org/10.1016/j.nbt.2014.05.1992 accompanied by aberrant chromosomal alignment. Remarkably, the mitotic defects caused by ADD1 depletion were rescued by re-expression of ADD1 but not of an ADD1 mutant defective in Myo10 binding. Together, our findings unveil a novel function for ADD1 in mitotic spindle assembly through its interaction with Myo10. http://dx.doi.org/10.1016/j.nbt.2014.05.1994 PI-26 Degredable polymers of D-lactate from renewables Ozlem Osmanagaoglu 1,∗ , Haluk Hamamcı 2 , Onlu Harun 3 PI-24 Dihydroxyacetone-catalyzed phosphorylation Roland Wohlgemuth 1,∗ , Dominik Gauss 1 , Israel Sanchez-Moreno 2 , Eduardo García-Junceda 2 1 2 Sigma-Aldrich CSIC Biocatalytic phosphorylations have been shown to provide a number of advantages over chemical phosphorylations like increased selectivity and high step economy [1,2]. The use of recombinant dihydroxyacetone kinase from Citrobacter freundii in biocatalytic asymmetric phosphorylation has been analyzed by direct quantitative 31P-NMR analysis of the kinetics. The phosphoenolpyruvate/pyruvatekinase-system was thereby selected for the regeneration of the cofactor ATP. This system looks promising for further exploration. References [1].D. Gauss, B. Schönenberger, R. Wohlgemuth, Carbohydrate Research, in press (2014). [2].Matsumi R, Hellriegel C, Schönenberger B, Milesi T, van der Oost J, Wohlgemuth R. RSC Advances 2014;4(25):12989–94. http://dx.doi.org/10.1016/j.nbt.2014.05.1993 PI-25 Adducin-1 is essential for mitotic spindle assembly through its interaction with myosin-X Hong-Chen Chen ∗ , Po-Chao Chan National Chung Hsing University 1 Ankara University Middle East Technical University Department of Food Engineering 3 Ankara University Faculty of Sciences Department of Biology 2 The production of biodegradable plastics is of increasing interest because of environmental concerns. For the same reasons the utilization of renewable materials for the production of such materials is also being supported. Lactic acid polymers are among the biodegradable materials that are being produced and whose production capacity is increasing. Lactic acid has two chiral forms, D (-) and L (+); both of which can be produced by fermentation. These chiral forms, when polymerized may have distinct properties; so both are important from the materials science point of view. In this work we attempted to produce D (-) lactic acid from renewable materials like sunflower seed hulls or wood shavings by using Lactobacillus delbrueckii subsp. bulgaricus strains isolated from different home-made yogurts. The strains were tested for their chiral lactate production and the better producers were chosen and subjected to fermentation tests. By several methods of neutralization, N-source supplementation and Na-Acetate addition so far we could reach a level of 80 g/L D (-) lactate production. http://dx.doi.org/10.1016/j.nbt.2014.05.1995 PI-27 The Effect of Oral Administration of Pediococcus pentosaceus OZF Harun Onlu 1,∗ , Zhannatgul Sakyp 2 , Fadime Kıran 2 , Harun Onlu 2 , İlker Büyük 2 , Sümer Aras 2 , Özlem Osmanağaoğlu 2 1 2 Mitotic spindles are microtubule-based structures, but increasing evidence indicates that F-actin and F-actin-based motors are components of these structures. Adducin-1 (ADD1) is an actinbinding protein that has been shown to play important roles in the stabilization of the membrane cortical cytoskeleton and cellcell adhesions. In this study, we show that ADD1 associates with mitotic spindles and is crucial for proper spindle assembly and mitotic progression. Phosphorylation of ADD1 at Ser12 and Ser355 by cyclin-dependent kinase 1 enables ADD1 to bind to myosin-X (Myo10) and therefore to associate with mitotic spindles. ADD1 depletion resulted in distorted, elongated, and multipolar spindles, Ankara University Faculty of Science Department of Biology Ankara University The intestinal effects of Pediococcus pentosaceus OZF, a promising probiotic strain and its encapsulated form was evaluated in TNBS model of rat colitis. To increase the viability of OZF strain under gastrointestinal conditions, encapsulation was carried out by whey proteins and calcium alginate. Female Wistar rats (n = 6) were treated daily with oral administration of OZF strain (7 log cfu/ml) and its encapsulated form for 14 days, starting one week before the treatment of TNBS. The body weight, water and food intake and the appearance of feces were recorded www.elsevier.com/locate/nbt S151 GENERAL BIOTECHNOLOGY daily throughout the study. One week after induction of colitis, all animals was killed and colonic damage was evaluated both histologically and biochemically including the determination of glutathione, superoxide-dismutase, malondialdehyde (MDA) and catalase contents. MDA level, as a marker of oxidative stress was significantly increased in the colitis group. However, OZF administration markedly decreased the MDA contents in colonic tissue. In addition, pro-inflammatory cytokines (IL-1beta and IL-6) in serum and colonic tissue were assessed by Real-Time PCR and a significant reduction was detected when compared to TNBS control animals. In conclusion, P. pentosaceus OZF could be a beneficial adjuvant/agent in the treatment of inflammatory bowel diseases due to its intestinal anti-inflammatory activity. http://dx.doi.org/10.1016/j.nbt.2014.05.1996 PI-28 Application of confocal microscopy methods for estimation of lipid and proteins dynamics in thylakoid membranes Marketa Husakova 1,∗ , Karolina Ditrychova 2 1 2 University of Palackiana in Olomouc, Faculty of Science Charles University in Prague The mobility of photosynthetic proteins is an important factor for light-energy conversion in photosynthesis. It has an important role in regulation of photosynthesis and in protein transport after its synthesis or repair. Mobility of photosynthetic proteins outside the thylakoid membrane is less understood. Cyanobacterial phycobilisomes attached to the thylakoid membrane can move relatively fast. We can measure specific feature of photosynthetic proteins mobility in vivo using microscopic methods such as FRAP (Fluorescence Recovery After Photobleaching) which allows to observe autofluorescence. In our study we used procaryotic photosynthetic cyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803 as model organisms. To measure mobility on membranes we used confocal microscopy and FRAP method as mentioned. FRAP method is based on laser technology, thus we used stronger laser to bleach spots on the membrane and observed recovery of fluorescence caused by mobility of proteins in membrane. We used three different sizes of bleaching spots to compare rate of proteins mobility. We also studied mobility of lipids, therefore we used fluorescent dye BODYPI because lipids are not autofluorescent whereas phycobilisomes are. Main question of this study was, whether is FRAP limited by diffusion or phycobilisome binding to the photosystem. http://dx.doi.org/10.1016/j.nbt.2014.05.1997 New Biotechnology · Volume 31S · July 2014 PI-29 Characterization of Pichia pastoris Golgi and plasma membrane Andreas Grutsch 1,∗ , Karlheinz Grillitsch 1 , Pablo Tarazona 2 , Erich Leitner 3 , Ivo Feussner 2 , Günther Daum 4 1 Austrian Centre of Industrial Biotechnology (ACIB) Department for Plant Biochemistry, University of Göttingen 3 Institute of Analytical Chemistry and Food Chemistry, TU Graz 4 Institute of Biochemistry,TU Graz/Austrian Centre of Industrial Biotechnology (ACIB) 2 Pichia pastoris has a prominent status regarding expression of recombinant proteins and is worldwide used as a highly efficient cell system for the production of heterologous proteins. Despite the importance of Pichia pastoris in biotechnology, little information is available about the cell biology of this microorganism. This tempted us to intensify our studies on Pichia pastoris organelles with special emphasis on subcellular fractions involved in the classical secretory pathway. The Golgi harbors many processes of post-translational protein modifications and is a major branching point within the secretory pathway. The plasma membrane is important for protein secretion as it is the last barrier on the way of polypeptides to be externalized. We have established protocols to isolate Pichia pastoris Golgi and plasma membrane at sufficient yield and high purity. Lipid profiling of the isolated membranes highlighted characteristics of the specific organelles. Whereas the phospholipid patterns were organelle specific, the fatty acid composition was similar in all membranes. By contrast to evidence from Saccharomyces cerevisiae, a sterol gradient along the secretory route was not found in Pichia pastoris. Sphingolipids of Pichia pastoris showed a clear organelle dependent distribution pattern. Ceramides and hexosyl-ceramides were predominantly found in Golgi fractions, whereas inositol containing phosphorylceramides were almost exclusively enriched in plasma membrane fractions. Similarities of Pichia pastoris and Saccharomyces cerevisiae became evident, although certain differences have to be kept in mind. Comparison of the two yeasts is important for understanding different properties and designing new strategies to improve Pichia pastoris strains for industrial applications. http://dx.doi.org/10.1016/j.nbt.2014.05.1998 PI-30 Somatic embryogenesis from cultured zygotic explants of some olive cultivars (Olea europaea L.) Sara Oulbi 1,∗ , Ibrahim Toufik 2 , Ilham Belkoura 3 1 National School of Agriculture, Meknes, Morocco Department of Biology, Faculty of Sciences, University Moulay Ismail, Meknès, Morocco 3 Department of basic sciences, National school of Agriculture, Meknes, Morocco 2 Mature zygotic embryos taken from three olive cultivars (Dahbia cv, Moroccan Picholine (PM) cv and Arbequine cv) were used to test the embryogenic capacity of their juvenile S152 www.elsevier.com/locate/nbt GENERAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 explants (cotyledons and radicles). These explants were cultured in Olive Medium (OM) (Rugini, 1984) supplemented with following combinations of auxin and cytokinin: AIB and 2iP (6-dimethylallylaminopurine), AIB (Indole-3-butyric acid) + TDZ (Thidiazuron) and AIB + Zeatin for 3 weeks; they were then transferred on OM free hormone. Calluses formation began during the first subculture in the three media; in term of the 3 weeks of culture in the first medium, the highest rates of callus were obtained when medium supplemented with 2iP for Arbequine cotyledons (100%) and Moroccan Picholine radicles (81.5%) and TDZ for radicles of Dahbia (92%). However, somatic embryogenesis was observed on Dahbia and Arbequine cotyledons and radicles cultivars during the second week of culture on OM free hormone when calluses were formed on AIB + 2iP, whereas Picholine explants induced nodular calluses without somatic expression. Maturation of somatic embryos was observed when OM medium was supplemented with 1 g of activated charcoal; indeed, globular somatic embryos were transformed to cotyledonary embryos (16.7% and 7.8% for Dahbia and Arbequine respectively). On attempt to germination, somatic embryos were cultured on various media in a 16/8 photoperiod. Neoformation of buds was observed on somatic cotyledons (37.5%) whereas rooting of somatic embryos was more difficult to obtain. http://dx.doi.org/10.1016/j.nbt.2014.05.1999 PI-31 Effects of 4 essential oils on growth of afaltoxin producing fungi Sittichai Chareonsettasilp 1,∗ , Dusanee Thanaboripat 1 , Chanita Sarutipaisan 1 , Chutima Puangtong 1 , Phurin Chatpongsatorn 1 , Yaowapa Suvati 2 1 2 King Mongkut’s Institute of Technology Ladkrabang Government Pharmaceutical Organization There have been records that many essential oils shown antimicrobial properties. Some essentials oils can inhibit the growth of aflatoxin producing fungi and aflatoxin production. In this study we compare the ability of 4 plant essential oils. i.e. ginger oil, anise star oil, cajuput oil and cinnamon oil for controlling aflatoxin producing fungi. The oils at concentrations of 0, 0.5, 1, 2, 3, 4 and 5% were tested against Aspergillus flavus IMI 242684 and A. parasiticus IMI 102566 on Potato Dextrose Agar (PDA). The fungi were cultured and incubated at 30 C for 7 days. The results show that anise star oil at all concentrations had the most inhibitory effect on both Aspergillus flavus IMI 242684 and A. parasiticus IMI 102566 with significant difference followed by cinnamon oil and cajuput oils. Ginger oil had the least inhibition effect. http://dx.doi.org/10.1016/j.nbt.2014.05.2000 PI-32 RNA-Seq analysis of the degradation of haloacetate by Burkholderia caribensis MBA4 Jimmy Tsang ∗ , Yanling Pan, Nan Zheng School of Biological Sciences, The University of Hong Kong Burkholderia caribensis strain MBA4 was isolated for its ability to utilize monobromoacetate as carbon and energy source. This bacterium produced an inducible haloacid dehalogenase that transforms monohaloacetate to glycolate and to glyoxylate by glycolate oxidase. Genomic analysis of the bacterium showed that it contains three glycolate oxidases: ETY79679-81, ETY80271-3 and ETY84258-60. Transcriptomic analysis showed that ETY79679-81 was expressed constitutively to a reads per kilobase transcript per million reads (RPKM) value of around 100 no matter the substrate was pyruvate, glycolate or chloroacetate. ETY80271-3 gave values of 7 in pyruvate-, 867 in chloroacetate- and 1260 in glycolate-grown cells. ETY84258-60 gave values of 20 in pyruvate, 1880 in chloroacetate- and 178 in glycolate-grown cells. A malate synthase G gene, ETY84261, was found downstream of ETY84258-60. Apparently, ETY80271-3 converted glycolate to glyoxylate and through the glycerate pathway to pyruvate. When MBA4 was grown on chloroacetate, gene products of ETY8425861 were mainly used and malate was generated. Putative regulator GlcC genes, ETY80275 and ETY84257, can be found upstream of ETY80271-3 and ETY84258-60, respectively. The expression profiles of these glycolate oxidases suggested that both GlcC were activated by glycolate and chloroacetate with ETY80275 more responsive to glycolate and ETY84257 more reactive towards chloroacetate. While the relative transcript levels of ETY80275 were rather stable, expression of ETY84257 was enhanced in glycolate- and even more in chloroacetate-grown cells. The characterization of the degradation of haloacetate by B. caribensis MBA4 is made possible with the use of RNA-seq analysis. http://dx.doi.org/10.1016/j.nbt.2014.05.2001 PI-33 Analysis of the physiology of an Aspergillus nidulans mutant lacking the aoxA (cyanide-resistant alternative oxidase encoding) gene Erzsébet Fekete ∗ , Ákos P. Molnár, Mojtaba Asadollahi, Szabina Balázsi, Erzsébet Sándor, Levente Karaffa University of Debrecen One of the most characteristic features of fungal mitochondria is the presence of an additional terminal oxidase, the alternative oxidase, the activity of which is insensitive to inhibitors of cytochrome c oxidase and the bc1 complex. Responsible for this activity in the model fungus Aspergillus nidulans is the cyanideresistant enzyme alternative oxidase, a quinol oxidase localized in the inner mitochondrial membrane encoded by the nuclear gene aoxA. www.elsevier.com/locate/nbt S153 GENERAL BIOTECHNOLOGY Fungal alternative oxidase was reported to be involved in a variety of stress-related cellular responses. Typically, specific activity of the alternative oxidase as well as the level of transcription of the encoding gene increased upon stress events. In this study we aimed for a quantitative characterization of the physiological consequences of the deletion of the aoxA gene in A. nidulans in comparison with the wild-type reference strain. Growth properties on various carbon sources at different concentrations were compared. Furthermore, osmotic, heat and stress tolerance of the two strains were analysed. Acknowledgement The research was supported by the EU and co-financed by the European Social Fund under the project ENVIKUT (TÁMOP-4.2.2.A-11/1/KONV-2012-0043), and also by the Hungarian Scientific Research Fund (OTKA Grant K1006600 to Dr. Erzsébet Fekete). http://dx.doi.org/10.1016/j.nbt.2014.05.2002 PI-34 Insights into diversity and specificity of heavy metal resistance and efflux systems in Bacillus oceanisediminis 2691 Hyun Ju Kim 1,∗ , Yong-Jik Lee 2 , Haeyoung Jeong 3 , Dong-Woo Lee 2 , Sang Jun Lee 4 1 Biosystems & Bioengineering Program, University of Science and Technology (UST) 2 School of Applied Biosciences, Kyungpook National University, Daegu, Republic of Korea 3 Korean Bioinformation Center, Korea Research Institute of Bioscience and Technology (KRIBB), Daejeon, Republic of Korea 4 Korea Research Institute Bioscience & Biotechnology We previously determined the genome sequence of aerobic, endospore-forming, Gram-positive Bacillus oceanisediminis 2691 that was isolated from marine sediment of the South Korean coast (Lee YJ et al., 2012 J Bacteriol.). Many genes encoding heavy metal resistance and efflux systems were found in the genome. Genes encoding putative cadmium efflux pumps, arsenic efflux pumps, a chromate transporter, and lead-, cadmium-, zinc-, and mercury-transporting ATPases were found. Putative resistance proteins of copper, cobalt-zinc cadmium, and tellurium were also identified. Apparently, transcriptions of those genes are controlled by CadC homologous metal responsive repressors. The transcription profiles of CadC-controlled genes were monitored in the presence of various heavy metals in B. oceanisediminis 2691. Furthermore, six cadC promoter-operator-structural genes were transcriptionally fused with egfp gene in E. coli. A variety of heavy metals were treated and specific fluorescence intensities were measured. Taken together, the results showed that CadC proteins specifically respond to heavy metals and may play separate roles in heavy metal resistances, which have been evolved in the heavy metal abundant marine sediment milieu. In addition, CadC-controlled transcriptional modules S154 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 could be used in the development of harmful heavy metal biosensor. http://dx.doi.org/10.1016/j.nbt.2014.05.2003 PI-35 High cell density cultivation of the chemolithoautotrophic bacterium Nitrosomonas europaea in a dialysis membrane bioreactor Levente Karaffa 1,∗ , Tibor Török 2 , Antal Kökényesi 1 , István Kolláth 1 , Erzsébet Sándor 1 , Zoltán Németh 1 , Noémi Lipták 1 , Erzsébet Fekete 1 1 University of Debrecen TEVA Pharmaceutical Ltd., Safety and Environmental Department, Debrecen, Hungary 2 Nitrosomonas europaea is a chemolithoautotrophic nitrifier Gram-negative bacterium that gains all of its energy for growth from the oxidation of ammonia into nitrite ions. A major prerequisite to produce a high cell density N. europaea culture is to ensure that inhibitory metabolic by-products remain at minimal concentrations. The principal inhibitory metabolite being continuously formed during N. europaea fermentation is nitrite. As a consequence, N. europaea cannot grow into high cell density under conventional batch conditions. A suitable method presented in this study to overcome this problem is the application of a single-vessel dialysis membrane bioreactor system. Fermentations were performed in a reactor with 2 L total/1.5 L useful and 6 L total/5.5 L useful volumes of medium in the inner and outer chambers, respectively. Growth of N. europaea was monitored via cell density determinations and by the measurement of nitrite formation. Metabolic activity was also visualized by Acridine Orange staining. Maximal cell density and maximal calculated specific growth rate were over five times of the value achieved under conventional batch conditions. We concluded that dialysis fermentors are suitable tools to overcome growth limitations observed in standard batch cultures of N. europaea. Acknowledgement The research was supported by the EU and co-financed by the European Social Fund under the project ENVIKUT (TÁMOP-4.2.2.A-11/1/KONV-2012-0043), and also by the Hungarian Scientific Research Fund (OTKA Grant K1006600 to Dr. Erzsébet Fekete). http://dx.doi.org/10.1016/j.nbt.2014.05.2004 GENERAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 PI-36 Offer, demand, and needs in training and education: a study focusing on microbial culture collections within the MIRRI Consortium André Antunes 1 , Veerle Piessens 2 , Nelson Lima 1 , The MIRRI Consortium 1 Micoteca da Universidade do Minho, Centre of Biological Engineering, University of Minho, Braga, Portugal 2 BCCM/LMG Bacteria Collection, Faculty of Sciences, Ghent University, B-9000 Ghent, Belgium The bioeconomy is fueled by Biological Resource Centers, which play a vital role in harnessing and preserving the world’s biodiversity [1]. MIRRI (the Microbial Resource Research Infrastructure: www.mirri.org) is an EU-project involving a total of 33 partners, aiming to provide facilitated access to microbial resources, associated data and expertise, and promote knowledge transfer and foster innovation. One crucial step is to properly define our stakeholder community and identify their current and future needs, and match them to our offer. In order to achieve these goals, MIRRI surveyed both its partners, and current and potential users of microbial resources and services. Here we present and analyze some of the results of this survey, focusing on training and education. Despite current trends and benefits in increased use and production of contents in new formats (e.g. video, interactive), and use of new tools and technologies (e.g. e-learning, b-learning), training within MIRRI still has an overwhelming dominance of classic content types and delivery methodologies. Also, we identified a much wider untapped market for education and training within our customer base, and we estimate a spike in demand of training from culture collections, particularly from the profit sector. Additional efforts are clearly necessary in adjusting our offer, adapting contents and content delivery and focusing on costefficiency and proper advertising to increase visibility, and better serve the needs of our customers. Reference [1].OECD. Underpinning the future of life sciences and biotechnology. OECD, Biological Resource Centres; 2001. p. 1–66. http://dx.doi.org/10.1016/j.nbt.2014.05.2005 www.elsevier.com/locate/nbt S155 GLYCOBIOLOGY Glycobiology PJ-01 Cloning and characterization of novel β-glucosidases from Aspergillus and their functional expression in methylotrophic yeast Pichia pastoris Richard Auta ∗ , Paul Hooley, Iza Radecka University of Wolverhampton The production of fermentable sugars from lignocellulosic material has attracted interest in the optimization of conditions related to conversion of cellulose to glucose for biofuel production. Aspergillus strains are known as efficient producers of β-glucosidase which is a rate limiting factor during enzymatic hydrolysis of cellulose.A bioinformatics based approach to characterize β-glucosidase encoding enzymes in the genus Aspergillus is described and the application of bioinformatics in the selection and expression of target genes is explained. Five Pichia clones (carrying A. nidulans AN2227.2, AN2612.2, AN0712.2, AN1551.2 and AN1804.2 in pPICZ vectors) that exhibit satisfactory levels of expression of recombinant β-glucosidase were obtained from the Fungal Genetics Stock Centre (FGSC). A study to compare their hydrolytic activities and relate these to their growth profiles using different media was carried out. The characteristics of these enzymes and the use of P pastoris in the expression of these proteins are discussed Keywords ␤-glucosidase; Aspergillus; Bioinformatics; Hydrolysis http://dx.doi.org/10.1016/j.nbt.2014.05.2006 PJ-02 Kinetic evaluation of carbohydrate-protein interaction using SPR Jeong Hyun Seo 1,∗ , Chang Sup Kim 2 , Hyung Joon Cha 2 1 2 Yeungnam university POSTECH, Korea Surface plasmon resonance (SPR) can provide kinetic information about an interaction, and it can also be used to rapidly monitor dynamic processes, such as adsorption and degrada- S156 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 tion, without the need for sample labeling. Here, we employed SPR to analyze carbohydrate-protein interactions, particularly GM1-related carbohydrate-Vibrio cholera toxin interactions. The interaction between cholera toxin subunits A (ctxA) and B (ctxB) was similar to general ligand-receptor interactions. After the direct immobilization of thiol-containing GM1 pentasaccharide on a gold surface, the GM1-ctxB interaction kinetics were evaluated, and they showed a similar degree of kinetics as reported in previous reports. We found that ctxA had a high affinity for the GM1ctxAB complex, although its equilibrium dissociation constant was 10-times lower than that of GM1-ctxB binding. Comparative analyses for GM1-related carbohydrates-ctxAB interactions were also conducted to determine the kinetic values of several GM1 analogues with different structures, although their kinetic values were one-order of magnitude lower than those of the GM1-ctxAB interaction. The kinetic analysis results for the interactions of GM1 analogues and ctxAB indicated that the sialic acid thumb is important for recognition, and the terminal galactose and Nacetylgalactosamine finger are required to stabilize the GM1-ctxAB interaction. Taken together, our results indicate that the direct immobilization of carbohydrate in an SPR-based analytical system can be used to evaluate the structural contribution of carbohydrate moieties in carbohydrate-protein interactions, as well as provide valuable information that can be used to understand the interactions. http://dx.doi.org/10.1016/j.nbt.2014.05.2007 GREENHOUSE GASES AND GLOBAL WARMING New Biotechnology · Volume 31S · July 2014 Greenhouse gases and global warming PK-02 PK-01 Emerging Biotechnologies For Landfill Greenhouse Gases’ Efficiency And Development Of Useful Web Drawing Utilities For Public Health Protection CO2 gas conversion to oxaloacetate by Escherichia coli harboring codon-optimized carbonic anhydrase and phosphoenolpyruvate carboxylase derived from marine bacteria Soohyun Park 1,∗ , Soohye Hong 1 , Sangwoo Kim 1 , Seung Pil Pack 2 , Jinwon Lee 1 1 2 Sogang university Korea University Increased emission of CO2 is a major problem which causes global warming throughout the world. Interest in bio-refinery process has increased remarkably because microbial conversion of CO2 to chemicals has large number of industrial applications, and also it can alleviate the increasing residual CO2 . Carbonic anhydrase (CA) is an enzyme, which plays a role in carbon sequestration process. And, phosphoenolpyruvate carboxylase (PEPC) is a biocatalyst which converts phosphoenolpyruvate to oxaloacetate. Oxaloacetate can be converted in to various valuable C4 chemicals (succinate, malate etc.) in TCA cycle. First, novel CA and PEPC genes were screened and found in marine bacteria by basic local alignment using the Escherichia coli PEPC amino acid (AA) sequence as a query. The CA gene from Hahella chejuensis and PEPC gene from Photobacterium profundum SS9 were selected. The codons of the heterologous CA and PEPC genes were synthesized using codon-optimization technique. Codon-optimized genes were cloned into pETDuet1 and transformed in E. coli BL21(DE3). Due to the SDS-PAGE results, it was confirmed that the codon-optimized genes were expressed as soluble protein forms in E. coli. The specific activity value of the codon-optimized enzymes were relatively high compared to the previously known CA and PEPC activities (codon-optimized CA (HC-aCA) = 478 ± 63 WAU/mg protein in the presence of Zn2+ , codon-optimized PEPC (OPPP) = 80.3 U/mg protein). In this study, a CO2 gas converting platform technology based on the co-expression system of CA and PEPC is developed and the potential of CO2 gas conversion platform technology is shown. Tilemachos Koliopoulos ∗ Director Telegeco - Scient. Colaborator Technological Educational Institute of Athens - This paper analyses the effects of different waste management landfill biotechnology techniques influencing on produced greenhouse emissions, leachate emissions, acids and landfill mass biodegradation stages. The biodegradation of Mid Auchencarroch experimental landfill project is studied in four different cells. The variations of the examining emissions are analysed in order to develop an efficient project management for the right operational measures for food safety in areas adjacent to landfill boundaries and public health protection. An analysis is made for emerging landfill biotechnology designs and modern spatial monitoring systems are developed utilizing properly web drawing utilities and associated information technologies (IT’s). Useful emerging landfill biotechnologies and simulation models are presented for produced landfill greenhouse gases making useful conclusions. Keywords landfill biotechnology; greenhouse gases; landfill emissions; experimental landfill design; public health protection; water resources; global warming; climate change; food security; emerging technologies. http://dx.doi.org/10.1016/j.nbt.2014.05.2009 http://dx.doi.org/10.1016/j.nbt.2014.05.2008 www.elsevier.com/locate/nbt S157 HIGH VALUE PLANT PRODUCTS High value plant products PL-01 Effect of pH on chromium and nickel biosorption by Litchi chinensis seeds in single and binary metal systems Liliana Morales Barrera ∗ , Liliana Morales-Barrera, Imelda GuerreroCoronilla, Víctor Zambrano-Pérez, Jessica Reyes-Ledezma, Griselda Chávez-Camarillo, Eliseo Cristiani-Urbina National Polytechnic Institute The extensive industrial use of hexavalent chromium [Cr(VI)] and divalent nickel [Ni(II)], as well as their improper disposal have led to heavy metal contamination of water [1]. Biosorption is an efficient and low-cost alternative for removing toxic metals from aqueous solutions. The purpose of this work was to evaluate the effect of pH on chromium and Ni(II) removal by Litchi chinensis seeds (LCS) in single and binary metal systems. LCS was able to remove chromium and Ni(II) from monometal solutions, and its removal capacity depended on the pH. LCS exhibited the highest removal capacity of Cr(VI) (86.79 mg/g), total chromium (64.74 mg/g) and Ni(II) (20.0 mg/g) at pH values of 1.0, 2.0 and 7.5, respectively. At a pH value of 1, a difference between Cr(VI) (86.79 mg/g) and total chromium removal capacity (34.4 mg/g) was observed and this was probably due to the fact that part of the Cr(VI) initially present in the solution was reduced to trivalent chromium [Cr(III)] by LCS. In the bimetal systems, Ni(II) did not have any effect on chromium removal at any pH value. Ni(II) biosorption capacity was not affected by the presence of chromium in acidic solutions; in contrast, at neutral pH values the capacity improved significantly. These results suggest that chromium and Ni(II) do not compete between them for the same LCS binding sites. Reference reached 75% and the clearance rate was above 60%. The solubility of the hydrolysate was as high as 93%. Emulsifying property and the emulsifying stability decreased in the pH range from 3.0 to 5.0, increasing with increased pH. Compared with the measured globulin, the foamability of hydrolysate was significantly increased, lowest at pH 5.0. The viscosity of hydrolysate was reduced during the whole process. http://dx.doi.org/10.1016/j.nbt.2014.05.2011 PL-03 Combinatorial optimization of synthetic operons for the microbial production of monolignols in Escherichia coli Philana van Summeren-Wesenhagen ∗ , Raphael Voges, Stephan Noack, Michael Bott, Jan Marienhagen Forschungszentrum Jülich The monolignol p-coumaryl alcohol is an important precursor of lignans and key building block of the plant polymer lignin, which is widely recognized as cheap source of aromatic compounds. However, due to its complex and irregular structure the uitilization of lignin is technically challenging. In contrast, microbial production of p-coumaryl alcohol and other monolignols represents a promising alternative. Recently, the first synthetic pathway for the production of p-coumaryl alcohol from L-tyrosine in E. coli was published [1]. Here we introduce a fast and robust method to optimize the product titers of this four gene pathway, based on the Phosphorothioate based Ligase-Independent Gene Cloning (PLICing) method [2]. An operon library was generated in which the translation efficiency of every gene was systematically varied by different spacings between the ribosomal binding site and the START-codon. Screening of this library yielded mutants producing up to 55 mg/L p-coumaryl alcohol, 7 times more compared to the starting strain under similar cultivation conditions. References [1].Park D, Yun YS, Yim KH, Park JM. Effect of Ni(II) on the reduction of Cr(VI) by Ecklonia biomass. Bioresource Technol 2006;97:1592–8. http://dx.doi.org/10.1016/j.nbt.2014.05.2010 PL-02 Preparation and Functional Characteristics of Peptide from Naked Oat Globulin by Protease Hydrolysis Meili Zhang , Rui Lin ∗ Inner Mongolia Agricultural University Globulin of naked oat was prepared by the Osborne methods and hydrolyzed by alkaline protease. The degree of protein hydrolysis and clearance rate of hydroxyl free radicals were used to identify the best enzyme hydrolysis process. Solubility, emulsifying property, viscosity and foamability of hydrolysate were tested. The result showed that the optimum conditions were as follows: dose of enzyme, 10000U/g; concentration of substrate, 5%; the temperature, 60◦ C; and pH 9.0. After 3 h, the degree of hydrolysis S158 New Biotechnology · Volume 31S · July 2014 www.elsevier.com/locate/nbt [1].Jansen F, Gillessen B, Mueller F, Commandeur U, Fischer R, Kreuzaler F. Metabolic engineering for p-coumaryl alcohol production in Escherichia coli by introducing an artificial phenylpropanoid pathway. Biotechnology and Applied Biochemistry 2014, doi: 10.1002/bab.1222. (published ahead of print). [2].Blanusa M, Schenk A, Sadeghi H, Marienhagen J, Schwaneberg U. Phosphorothioate based Ligase-Independent Gene Cloning - A method for cloning of mutant libraries in directed evolution experiments. Anal Biochem 2010;406:141–6. http://dx.doi.org/10.1016/j.nbt.2014.05.2012 HIGH VALUE PLANT PRODUCTS New Biotechnology · Volume 31S · July 2014 PL-04 In vivo and in vitro cultures of Lavandula angustifolia Mill. for essential oils production Pana Lohasupthawee ∗ , Prangmas Srisurat King Mongkut’s Institute of Technology Ladkrabang The aim of this research was to extract the essential oils of lavender (Lavandula angustifolia Mill.) from callus culture, multiple shoots culture and hydroponic culture. The chemical composition of each extract was analyzed by gas chromatography and mass spectrometry (GC-MS). In vitro culture of lavenders, multiple shoots were obtained in culture medium fortified with Murashige and Skoog (MS) nutrients and 1.5 mg/l benzyladenine which produced 12 shoots per explant. Callus was achieved by using leaf explants and showed best growth in MS medium supplemented with 0.5 mg/l 2,4-dicholorophenoxyacetic acid. In vivo culture, hydroponic lavenders were achieved by using deep flow technique system. The leaves and roots of hydroponic lavenders were extracted for essential oils separately. The GC-MS results showed that the leaves of hydroponic lavenders and in vitro shoots demonstrated the components of lavender essential oils whereas callus and roots of hydroponic lavenders showed no essential oils production. http://dx.doi.org/10.1016/j.nbt.2014.05.2013 PL-05 Fermentative Nisin Production in Tofu for its Preservation Nicole Illas ∗ , Raphael Cziskus, Caterina Hünniger, Myriam Bello, Xuan Zhu, Markus Fischer, Bernward Bisping University of Hamburg Tofu has a high nutritional content and serves as stable food in eastern Asia. In developing countries pasteurization is not affordable because tofu production takes place mostly in small factories. A cost-effective and natural preservation of tofu may be thenisin production in tofu cubes (2*2 cm) submerged in water by fermentation with Lactococcus lactis ssp. lactis DSM 20729. Optimal fermentation was conducted by adding 3.9% soy peptone. Furthermore it could be shown that there is a non-uniform distribution of nisin in tofu cubes. The nisin concentration decreases from the surface to the interior of tofu but it was sufficient to extend the shelf-life of tofu. Two methods for the detection of nisin in fermented tofu were tested. The comparison of an inhibition test (modified [1]), and LC-ESI-MS/MS method (based on ISO/TS 27106:2009 [2]) illustrates that the detection limit of the inhibition test was significantly lower. Concentrations of 0.19 mg/kg nisin in tofu and 0.06 mg/L in the supernatant could be determined. The detection limit for nisin using LC-ESI-MS/MS was 0.34 mg/kg in tofu. Matrix calibration of the liquid substrate could not be carried out by LC-ESI-MS/MS, because the background noise of the matrix was too high. It could be shown that the fermentation of tofu with L. lactis is an efficient and cost-effective alternative. References [1].Pongtharangkul T, Demirci A. Appl Microbiol Biotechnol: 2004;65:268–72. [2].International Organization for Standardization ISO/TS 27106 Geneva Switzerland 2009. http://dx.doi.org/10.1016/j.nbt.2014.05.2014 PL-06 Fermentation-like incubations of Theobroma cacao L. Good quality in shorter time Claudia Bahmann ∗ , Tumforde Thomas, Lieberei Reinhard University of Hamburg In the course of the post-harvest treatment of cocoa seeds (fermentation) the pulp is microbially degraded. During fermentation first ethanol, then acetic acid is produced in combination with a temperature increase. The conditions in the fermentation mass entail the acidification of the cotyledon tissue and an extensive proteolysis of storage proteins. The latter is carried out by endogenous proteases that are activated by acidic conditions. The cleavage activity of the proteases as well as the conservative amino acid sequences of storage proteins provide a defined pattern of flavor precursors of cocoa. In the study at hand seeds have been incubated under conditions analog to those of fermentation, because of the ability to control different parameters separately. Media with different pH-values, all in the acid range, and different organic acids have been used. The basic effects of these factors on the seed with regard to the proteolysis are examined. In order to imitate the temperature conditions of fermentation, incubations are carried out under different temperature sequences. In the course of the incubation process comparatively higher amounts of the amino acids and phenolic compounds examined are achieved already after three days. Comparable values in the fermentation procedures are achieved not until after six days. This illustrates the efficiency of the incubation procedure. In this study, an endogenic bioconversion has been proven to take place in cocoa seeds in the course of incubations. Furthermore, the striven accumulation of characteristic chocolate aroma precursors can be influenced by processing seeds under defined external conditions. http://dx.doi.org/10.1016/j.nbt.2014.05.2015 PL-07 Glucanocellulosic biomass: learning from marine biomass to optimize terrestrial biomass conversion Christian Voigt ∗ , Claudia Zwikowics University of Hamburg The recalcitrance of the plant cell wall is one of the obstacles in improving biomass conversion. We followed a strategy to enrich biomass with a polymer that is easily degradable and would www.elsevier.com/locate/nbt S159 HIGH VALUE PLANT PRODUCTS not impair the physiology of the plant. In marine biomass from the brown algae Fucus vesiculosus, the polymer (1,3)-β-glucan is a major biomass component showing these characteristics and is also present in terrestrial plants. We used this glucan-enriched, marine biomass to optimize hexose release and bioethanol production. The addition of a bacterial (1,3)-β-glucanase to a commercial enzyme cocktail as well as the usage of an optimized Saccharomyces cerevisiae strain that we engineered for fermenting glucan-enriched biomass resulted in a 50% increase in bioethanol production. To test the optimized processing of glucan-enriched biomass on terrestrial biomass, we screened for plants with a high (1,3)-β-glucan content and identified leaves from the energy crop Miscanthus x giganteus with an exceptionally high (1,3)-β-glucan content of 5%. S160 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 Applying the optimized processing for glucan-enriched biomass on M. giganteus leaf biomass, we increased bioethanol production by 13% compared to non-adapted conversion and fermentation strategies. To further improve bioethanol production with this adapted processing, we overexpressed a (1,3)-β-glucan synthase from Arabidopsis thaliana in M. giganteus, which further increased (1,3)-β-glucan content in leaf biomass to 8.5% and improved bioethanol production by 20%. Our results suggest that generation of glucan-enriched biomass via synthetic biology approaches combined with optimized processing for glucanocellulosic bioethanol production is a promising alternative in increasing efficiency of biomass conversion. http://dx.doi.org/10.1016/j.nbt.2014.05.2016 METABOLIC ENGINEERING New Biotechnology · Volume 31S · July 2014 Metabolic engineering PM-01 Sex-Dimorphic Expression Of Caveolin 1 Is Linked To Obesity Development In Rats Jae Heon Choi ∗ , Rajib Mukherjee, Sang Woo Kim, Jong Won Yun Daegu University Recently, it has been reported that CAV1 is an important target protein in sex hormone-dependent regulation of various metabolic pathways, particularly in cancer and diabetes. To clarify distinct roles of CAV1 in sex-dependent obesity development, we investigated the effects of high fat diet and sex steroid hormones on CAV1 expression in adipose tissues of male and female rats. Results of animal experiments revealed that estrogen (17-β-estradiol, E2) and androgen (dihydrotestosterone, DHT) had opposite effects on body weight gain as well as on the regulation of CAV1, hormone sensitive lipase (HSL) and uncoupling protein 1 (UCP1) in adipose tissues. Furthermore, sex hormone receptors and aromatase were differentially expressed in a sex-dependent manner in response to E2 and DHT treatments. In vivo data were confirmed using 3T3-L1 and HIB1B cell lines, where Cav1 knock down stimulated lipogenesis but suppressed sex hormone receptor signaling proteins. Most importantly, co-immunoprecipitation enabled the identification of previously unrecognized CAV1-interacting mitochondrial or lipid oxidative pathway proteins in adipose tissues. Taken together, current data showed that CAV1 may play important preventive role in the development of obesity, with more prominent effects in females, and proved to be an important target protein for the hormonal regulation of adipose tissue metabolism by manipulating sex hormone receptors and mitochondrial oxidative pathways. Therefore, we can report, for the first time, the molecular mechanism underlying the effects of sex steroid hormones in the sex-dimorphic regulation of CAV1. http://dx.doi.org/10.1016/j.nbt.2014.05.2017 PM-02 Metabolic engineering of Clostridium acetobutylicum for highly selective butyric acid production Yu-Sin Jang ∗ , Sang Yup Lee Korean Advanced Institute of Science and Technology (KAIST) Butyric acid, a saturated four-carbon carboxylic acid, has been widely used in chemical, food, pharmaceutical, and animal feed industries. The well-known clostridial native butyric acid producers include C. butyricum, C. thermobutyricum, C. tyrobutyricum, C. acetobutylicum and C. pasteurianum. A typical characteristic of such butyric acid-producing Clostridium is coproduction of both butyric and acetic acids. Increasing the butyric acid selectivity important for economical butyric acid production has been rather difficult in clostridia due to their complex metabolic pathways. In this work, C. acetobutylicum was metabolically engineered for highly selective butyric acid production. For this purpose, the second butyrate kinase of C. acetobutylicum encoded by the bukII gene instead of butyrate kinase I encoded by the buk gene was employed. Furthermore, metabolic pathways were engineered to further enhance the NADH-driving force. Batch fermentation of the metabolically engineered C. acetobutylicum strain at pH 6.0 resulted in the production of 32.5 g/L of butyric acid with a butyric-to-acetic acid ratio (BA/AA ratio) of 31.3 g/g from 83.3 g/L of glucose. These results suggested that the buk gene knockout was essential to get a high butyric acid selectivity to acetic acid in C. acetobutylicum. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012-C1AAA001-2012M1A2A2026556).] http://dx.doi.org/10.1016/j.nbt.2014.05.2018 PM-03 The parologues pyruvate kinases in Streptomyces coelicolor have distinct roles in growth and antibiotic production Jana Hiltner 1,∗ , Pablo Cruz-Morales 2 , Lorena Fernandez-Martinez 3 , Hrovje Petkovic 4 , Iain S. Hunter 1 , Francisco Barona-Gomez 2 , Paul A. Hoskisson 1 1 University of Strathclyde Langebio Cinvestav 3 John Innes Centre 4 Acies Bio Ltd 2 Streptomyces species are prolific producers of antibiotics, nevertheless analysis of complete genomes still shows that there are many biosynthetic clusters present that are silent under laboratory cultivation conditions. The current increase in clinical antibiotic resistance requires the discovery of new antibiotics, but also a greater understanding of antibiotic production for industrial exploitation. Our interest is in studying the transition of primary metabolites into secondary metabolism. We focus on the Phosphoenolpyruvate-Pyruvate-Oxaloacetate (PEP-PYR-OAA) node of central carbon metabolism using Streptomyces coelicolor as a model and have identified pyruvate kinase influences antibiotic production. The genome encodes two parologue pyruvate kinase genes - SCO2014 (pyk1) and SCO5423 (pyk2). Phenotypic analysis of the two mutants revealed differences in their physiological role, pyk2 exhibits altered growth on glucose, whereas pyk1 shows a difference in antibiotic production. We used crossspecies complementation experiments with E.coli pykF, pykA and pykApykF and complemented these with pyk1 and pyk2 from S. coelicolor on different media to clarify their physiological roles. Furthermore Pyk1 and Pyk2 were overexpressed in E. coli for a detailed characterisation of their biochemical properties. Our data show that gene parologues in primary metabolism have distinct physiological roles in Streptomyces that impact significantly on growth and the production of antibiotics, which could be used for industrial strain improvement in the biotechnology industry. http://dx.doi.org/10.1016/j.nbt.2014.05.2019 www.elsevier.com/locate/nbt S161 METABOLIC ENGINEERING PM-04 Re-distribution of carbon flux toward 2,3-butanediol production in Klebsiella by metabolic engineering Borim Kim ∗ , Soojin Lee, Daun Jeong, Jeongmo Yang, Jinwon Lee Sogang university As shown in previous studies, Klebsiella pneumoniae showed high potential as a 2,3-butanediol (2,3-BDO) producer, and exhibited great productivity. However, the accumulation of substrates other than 2,3-BDO such as; lactate, ethanol, and acetate, in the log-phase of cell growth remained as an obstacle for efficient large scale 2,3-BDO production. Hereby, in order to re-distribute the substrate-directed carbon flux to 2,3-BDO production metabolic engineering was done. Incorporation of the gene deletion method (deleting competitive NADH consuming pathway by ldhA gene deletion) and gene over-expression method (re-directing carbonflux toward 2,3-BDO biosynthesis by budA gene over-expression) were conducted for efficient utilization of glucose conversion to 2,3-BDO under slightly acidic condition(pH 5.5). The engineered strain SGSB105 showed a 40% increased 2,3-BDO production from glucose, compared to the wild-type strain SGSB100. Also the closely related genes in the 2,3-BDO biosynthesis pathway were observed at the gene transcription level by cultivating mutant strains under unify culture conditions. As a result, the gene expression levels of the budB, budA, and budC genes showed a 10% increased transcription level at the log-phase of the cell growth, compared to the SGSB100. Also the 2,3-BDO gene transcription levels of SGSB105 was maintained at a high level during the logand stationary-cell growth phase. By incorporating the gene deletion and over-expression method, the carbon flux was re-directed to a valuable biochemical producing process, and also by combining the batch culture data with the gene transcription data it shows an insight for improving the 2,3-BDO biosynthesis metabolic network for industrial application. http://dx.doi.org/10.1016/j.nbt.2014.05.2020 PM-05 Metabolic engineering of Corynebacteruim glutamicum for production of 5-aminovaleric acid and glutaric acid as C5 platform chemicals Jae Ho Shin 1,∗ , Si Jae Park 2 , Sang Yup Lee 1 1 2 Korea advanced institute of science and technology Myongji University The amino acid, L-lysine can be naturally degraded via multiple conduits including the cadaverine and 5-aminovaleric acid pathways. The degradative intermediates, cadaverine, 5-aminovaleric acid and glutaric acid are C5 platform chemicals that can be used for bio-polyamide production. Here we report the development of Corynebacterium glutamicum strains overproducing 5-aminovaleric acid and glutaric acid by metabolic engineering of a classical L-lysine producer and introducing novel synthetic pathways. The first synthetic novel pathway consists of the heterologously S162 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 introduced davB and davA genes from Pseudomonas putida ATCC 12633 together with the endogenous gabT and gabD genes in C. glutamicum. DavBA function as metabolic “gap” filling for glutaric acid production in engineered C. glutamicum since the non-engineered strain does not degrade L-lysine. This engineered strain cultured in a laboratory-scale bioreactor produced 24.9 g/L of 5-aminovaleric acid and 11.9 g/L of glutaric acid in 144.5 h using glucose as a sole carbon source. The second synthetic pathway comprises the codon-optimized davB and davA genes from P. putida ATCC 12633 and the codon-optimized davT and davD genes from P. putida KT2440. The engineered C. glutamicum harboring the second synthetic pathway produced 23.7 g/L 5-aminovaleric acid and 10.6 g/L glutaric acid in 150.5 h. These results suggest that C5 platform chemicals can be efficiently produced by metabolically engineered C. glutamicum. This study also presents a strategy for assembling and establishing synthetic pathways in C. glutamicum for the production of chemicals, which will be useful for designing other strains for the bio-based production of chemicals. http://dx.doi.org/10.1016/j.nbt.2014.05.2021 PM-06 Functional Expression and Characterization of Codon Optimized Proteorhodopsin in Escherichia coli Yong-Jik Lee 1,∗ , Sang Jun Lee 2 , Dong-Woo Lee 1 1 2 Kyungpook National Univ Korea Research Institute of Bioscience and Biotechnology Proteorhodopsin (pR) as an integral membrane light-harvesting proton pump generates proton motive force across the cellular membrane. A wide range of pRs have been studied on the ecological distribution and function, but the expression level of pR and its physiological role in nonphotosynthetic host strains still remain unclear. Here, we chemically synthesized the SAR 86 gene with codon optimization (co SAR86) and expressed the pR gene in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. The recombinant enzyme was purified to homogeneity by solubilization of E. coli membrane, Ni2+ affinity chromatography followed by gel filtration chromatography. Comparison of thephysicochemical properties of both membraneembedded and purified co SAR86 in the presence and absence of all-trans retinals established that these enzymes expressed in E. coli were integrated properly. To investigate whether the expressed co SAR86 can enhance the cellular energy production of host cells, we compared the growth phenotypes of co SAR86 expressing E. coli strain and the wild-type strain under various conditions. Here, we report successful production and initial characterization of a functional co SAR86 that supports extra energy production for the growth of E. coli cells under certain growth conditions, which may facilitate the exploitation of pR for commercial biotechnological applications. http://dx.doi.org/10.1016/j.nbt.2014.05.2022 METABOLIC ENGINEERING New Biotechnology · Volume 31S · July 2014 PM-07 Generation of Oxalic Acid Hyperproducers by Overexpressing the Oxaloacetate Hydrolase Gene in Aspergillus niger available for this purpose, and use several examples to demonstrate the application of protein engineering for metabolic engineering in amino acid overproduction. http://dx.doi.org/10.1016/j.nbt.2014.05.2024 Keiichi Kobayashi ∗ , Shotaro Watanabe, Kohtaro Kirimura Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University PM-09 The filamentous fungus Aspergillus niger is worldwide used in the industrial production of citric acid. On the other hand, under specific cultivation conditions, A. niger accumulates oxalic acid. Oxalic acid is one of the valuable chemicals used as a chelator, detergent, or tanning agent, and industrially produced by the chemical method, but not microbial one. In this study, to generate oxalic acid hyperproducers by metabolic engineering, we constructed transformants overexpressing the gene oahA encoding oxaloacetate hydrolase (OAH; EC 3.7.1.1) in citric acidproducing A. niger WU-2223L [1] as a host. The mRNA level of oahA and specific activity of OAH in strain EOAH-1, a representative oahA-overexpressing transformant, were higher than those in WU-2223L. [2] To examine the potentiality of oxalic acid production, EOAH-1 was cultivated in OAP30 medium containing 30 g/L glucose as a carbon source, (NH4 )2 SO4 as a nitrogen source, and 2-[N-morpholino] ethanesulfonic acid (MES) as a buffering substance, and produced 28.9 g/L oxalic acid during 12 days of cultivation. Moreover, by the use of NaNO3 and K2 HPO4 -KH2 PO4 buffer instead of (NH4 )2 SO4 and MES in OAP30 medium, EOAH1 produced 35.8 g/L oxalic acid during 9 days of cultivation. The yield of oxalic acid for EOAH-1 reached 79.6% of the maximum theoretical yield. Therefore, we succeeded in generating oxalic acid hyperproducers by overexpressing a single gene, i.e., oahA in citric acid-producing A. niger. Svyatoslav Kondrat ∗ , Olav Zimmermann, Eric von Lieres References [1].Kobayashi K, et al. Biosci Biotechnol Biochem 2013;77:1492–8. [2].K. Kobayashi, et al., J. Ind. Microbiol. Biotechnol., in press (2014). http://dx.doi.org/10.1016/j.nbt.2014.05.2023 PM-08 Protein engineering for strain engineering Ping Zheng ∗ , Jibin Sun Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences Engineering microbial strains to overproduce chemicals represents a promising topic in the field of industrial biotechnology. The production performance and even the production portfolio can be well reconfigured by the advanced metabolic engineering technology. Successful examples can be found in the area of antibiotics, amino acids, organic acid, biofuels, biopolymers, and recombinant plant-originated drugs. The gene expression by overexpression or disruption is often used tools. Further fine-tuning the activity of the enzymes in particular by structure-based approach is very powerful for global optimization of the metabolic pathway but not well-recognized in the literature. Here we discuss the toolboxes Diffusion in crowded cytoplasm-like environment Forschungszentrum Jülich We perform Brownian dynamics simulations to study shortand long-time diffusion of macromolecules in crowded environment of biological cells. We confirm that the diffusion slows down with increasing volume fraction, and focus on its dependence on macromolecular composition of cell’s cytoplasm. The effect of composition on different types of diffusion, and its importance for modelling metabolic pathways will be discussed. http://dx.doi.org/10.1016/j.nbt.2014.05.2025 PM-10 Application of a controllable degron strategy for metabolic engineering Christoph Knuf ∗ , Jérôme Maury, Simo Jacobsen, Jochen Forster Novo Nordisk Foundation Center for Biosustainability, DTU In numerous cases of metabolic engineering, metabolite pools have to be increased in order to obtain flux into heterologous pathways. A simple tool for this would be the deletion of genes that would practically lead to a block of the natural pathway, so that the carbon can flow into the heterologous pathway. Unfortunately some deletions are lethal, as end products of pathways are needed for cellular growth. One example of such a pathway is the mevalonate pathway in S. cerevisiae with ergosterol as one of the most important end products. A great number of bioactive compounds, like various terpenoids, can be produced from intermediates of this pathway. Different strategies have been applied in order to down-regulate the expression of enzymes involved in the mevalonate pathway. All these strategies work on the transcriptional level. This leads to a delay of the actual regulation, as the existing enzyme will still be active. We present a strategy for down-regulation that acts on the protein level and which can therefore be controlled in a more precise manner than the hitherto reported strategies. As a case study we show the action of the degron strategy for controlling the pools of intermediates of the mevalonate pathway around 2,3-oxidosqualene, which is the precursor for triterpenoids. Many triterpenoids are pharmaceutically relevant compounds which nowadays need to be extracted from plant material through an intricate and resource consuming process. http://dx.doi.org/10.1016/j.nbt.2014.05.2026 www.elsevier.com/locate/nbt S163 METABOLIC ENGINEERING New Biotechnology · Volume 31S · July 2014 Efficient GABA production system development via introduction of synthetic protein scaffold for the rational design of bFMP. This strategy can be applied to various enzymes for the identification of the potential target sites for the rational design, independent on the three-dimensional structure of target protein. Soonho Hong ∗ http://dx.doi.org/10.1016/j.nbt.2014.05.2028 PM-11 University of Ulsan Gamma-aminobutyric acid (GABA) is a precursor of one of the most promising heat-resistant biopolymers, Nylon-4, and can be produced by the decarboxylation of monosodium glutamate (MSG). In this study, a synthetic protein scaffold was applied to improve the GABA conversion in engineered Escherichia coli. Scaffolds were constructed by assembling a single protein–protein interaction domain SH3 to the glutamate decarboxylase (GadA and GadB) and attaching a cognate peptide ligand to the glutamate/GABA antiporter (GadC) at the N-terminus, C-terminus, and the 233rd amino acid residue. When GadA and GadC were cooverexpressed via the C-terminus scaffold, a GABA concentration of 5.65 g/L was obtained from 10 g/L MSG, which corresponds to a GABA yield of 93%. A significant increase of the GABA productivity was also observed where the GABA productivity increased 2.5 fold in the early culture period due to the introduction of the synthetic protein scaffold. The GABA pathway efficiency and GABA productivity were enhanced by the introduction of the scaffold between glutamate decarboxylase and glutamate/GABA antiporter. This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant number: PJ00954904) by RDA, and Basic Science Research Program by the MEST (2011-0022392). http://dx.doi.org/10.1016/j.nbt.2014.05.2027 PM-12 Relationship between amino acid properties and catalytic function in bacterial flavin-containing monooxygenase using deep mutational scanning approach Namil Lee ∗ , Jongoh Shin, Byung-Kwan Cho KAIST Flavin-containing monooxygenases (FMOs) are the promising enzymes that catalyze oxidation reactions of a wide array of substrates, including indole compounds. Thus, the engineered FMOs with broad substrate specificity, enhanced catalytic efficiency (kcat/Km), and increased thermal stability have great potential to produce value-added bio-chemicals. However, their rational design for obtaining desired changes in the functional parameters is hampered by the lack of information about functions of amino acid residues of the enzymes. In order to understand a relationship between the amino acid residues and function of the bacterial flavin-containing monooxygenase (bFMO), we performed a saturated mutagenesis that all amino acid residues of the enzyme were replaced by other amino acids, followed by deep sequencing. We classified amino acid residues of the enzyme into function-retained and function-loss subgroups, which are subsequently interpreted based on the three-dimensional structure. The functional relationship between amino acid residues and catalytic function was used S164 www.elsevier.com/locate/nbt PM-13 Statistical Optimization for Simultaneous Production of PLA Degrading and Raw Starch Degrading Enzymes by Thermophilic Filamentous Bacterium, Laceyella sacchari LP175 Using Agricultural Crops as Substrates Vichien Kitpreechavanich ∗ , Thanasak Lomthong, Srisuda Hanphakphoom Kasetsart University Optimization of medium using low cost agricultural products by statistical mixture design for simultaneous production of PLA degrading and raw starch degrading enzymes by thermophilic filamentous bacterium Laceyella sacchari LP175 was investigated to increase biodegradation of polylactide/starch blend bioplastics. Total 5 g of different amounts of between cassava chip, soy bean meal and corncob were used as substrates in 0.035% PLA basal medium with 7 experiment runs with triplicate in shaking flask at 50 ◦ C. The highest enzymes productions were obtained from the mixed substrates of cassava chip and soy bean meal in ratio 1:1 at 24 h cultivation. Cassava starch as the additional carbon source was found to be the best for both enzymes productions. The response surface methodology with central composite design was used for enhanced both enzymes production consisted of PLA powder and cassava starch in the mixture of cassava chip and soy bean meal as the basal medium. The maximum predicted activity of PLA degrading enzyme was 69.7 U/mL with 92.1 U/mL RSDE (raw starch digesting enzyme) activity from the basal medium consisted of 0.52 g/L PLA powder and 3.34 g/L cassava starch, while the maximum predicted activity of RSDE was 92.9 U/mL with 69.2 U/mL PLA degrading activity was obtained from the basal medium consisted of 0.52 g/L PLA powder and 3.04 g/L cassava starch. The validation results of both enzymes were 68.8 and 86.1U/mL PLA degrading and RSDE activities, respectively. http://dx.doi.org/10.1016/j.nbt.2014.05.2029 METABOLIC ENGINEERING New Biotechnology · Volume 31S · July 2014 PM-14 Study of the role of Escherichia coli central metabolism pathways related genes in the synthesis of hydrogen and ethanol by using glycerol as carbon source Jorge Bolivar 1,∗ , Antonio Cantero 3 Valle 2 , Gema Cabrera 3 , Domingo 1 University of Cadiz University of Cadiz-Department of Biomedicine, Biotechnology and Public Health-Biochemistry and Molecular Biology; Chemical Engineering and Food Technology 3 University of Cadiz-Department of Chemical Engineering and Food Technology 2 Earth’s climate warming as a result of anthropogenic emissions of greenhouse gases, particularly carbon dioxide (CO2 ) from fossil fuel combustion, has provoked an urgent need to develop clean and renewable energy sources. Bioenergy has emerged as an alternative source of fuel, which includes biodiesel, hydrogen and bioethanol. However, the biodiesel industry currently generates a huge amount of glycerol as a by-product in such a magnitude that it has become an environmental problem. An achievable solution to this problem is the use of waste glycerol as the main carbon source for microbial transformation to hydrogen and ethanol. This is an environmentally safe process that may lead to the production of renewable energy resources, which could contribute to the reduction of the CO2 emissions. Escherichia coli is a very promising alternative for glycerol utilization and it has the advantage that is commonly used for metabolic engineering in many biotechnological applications. In this work we study how the blockage of some enzymes by using E. coli single knock out strains affect to the H2 and ethanol productions as well as glycerol consumption when the cells grew in a glycerol-based medium. Due to the role that the central carbon metabolism plays in the hydrogen and ethanol synthesis, several mutants of these important pathways have been analysed. We describe here several novel mutant backgrounds that could be useful in order to enhance the ethanol and H2 productions in E. coli. http://dx.doi.org/10.1016/j.nbt.2014.05.2030 PM-15 Metabolic Engineering an ATP-neutral EMP pathway in C. glutamicum: adaptive point mutation in NADH dehydrogenase restores growth Gajendar Komati Reddy 1,∗ , Steffen N Lindner 2 , Volker F Wendisch 1 1 Chair of Genetics of Prokaryotes, Faculty of Biology and CeBiTec, University of Bielefeld 2 Chair of Genetics of Prokaryotes, Faculty of Biology University of Bielefeld Microorganisms produce ATP by substrate level phosphorylation in Embden–Meyerhof–Parnas pathway and/or by electron transport phosphorylation. By bypassing substratelevel phosphorylation via phosphorylating NAD+ -dependent glyceraldehyde-3-phosphate dehydrogenase(s) and phosphoglyceratekinase, we engineered a strain with ATP-neutral gly- colysis pathway in the industrially important and natural glutamate producer Corynebacterium glutamicum. To this end, a double deletion mutant devoid of the genes for phosphorylating NAD+ and NADP+ -dependent glyceraldehyde-3phosphate dehydrogenase gapA and gapB was constructed. Nonphosphorylating NADP+ -dependent glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum (encoded by gapNCac ) irreversibly oxidizes glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) in an NADP+ -dependent manner without addition of Pi , thus, bypassing ATP generation via phosphoglyceratekinase. The resulting recombinant strain C. glutamicum∆gapA∆gapB (pEKEx3-gapNCac ) was expected to oxidize glucose to pyruvate without net ATP yield whereas 2 mol of NADPH are formed. However, this strain did not grow in glucose minimal medium. Upon prolonged incubation suppressor mutants could be isolated. Further analysis of the suppressor mutants by genome sequencing analysis revealed a SNP shared by the four suppressor mutants. The SNP in the ndh gene (cg1656) encoding the non-proton pumping NADH dehydrogenase caused an amino acid exchange. This mutation increased NADPH oxidation by NDH and, thus, provides a unique solution to re-oxidize NADPH generated in the engineered ATP-neutral glycolytic pathway in C. glutamicum. http://dx.doi.org/10.1016/j.nbt.2014.05.2031 PM-16 Metabolic Engineering of Saccharomyces cerevisiae for Isoprenoid Production Stefan Tippmann ∗ , Sakda Nielsen Khoomrung, Verena Siewers, Jens Chalmers University of Technology This project attempts to establish a yeast cell factory for the production of isoprenoids, which were attributed a key function in the search for alternative transportation fuels. For this purpose, Saccharomyces cerevisiae was chosen as a host organism, whereas the main focus is set on sesquiterpenes such as farnesene, which can be used as diesel alternative in its hydrogenated form farnesane. In order to enable for efficient production of farnesene, two central aspects are being addressed, i.e. metabolic engineering for enhanced synthesis and analytical method development for accurate quantification of intra- and extracellular metabolites from two-liquid phase fermentations. In the first part, an existing platform optimized for sesquiterpene production was recently used for the integration of farnesene synthase genes from different plant sources to enable the one-step conversion from farnesyl pyrophosphate to farnesene. As a result, maximal titers of ∼1 g/L were attained in a comparative evaluation in fed-batch cultivations with exponential feeding. Enhanced synthesis, however, will not only involve heterologous expression of these enzymes, but it will also include further engineering of the endogenous mevalonate pathway as well as the integration of different ‘omics’ analysis to support the cycle of metabolic engineering. http://dx.doi.org/10.1016/j.nbt.2014.05.2032 www.elsevier.com/locate/nbt S165 METABOLIC ENGINEERING New Biotechnology · Volume 31S · July 2014 PM-17 PM-18 Identification of a new putative regulatory protein involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea by omics approaches Identification of engineering targets for improving putrescine production by Corynebacterium glutamicum Hrvoje Petkovic 1,∗ , Vasilka Magdevska 2 , Benjamin Kirm 2 , Miha Tome 2 , Marinka Horvat 2 , Katarina Karničar 2 , Robert Vidmar 3 , Špela Baebler 4 , Polona Jamnik 5 , Štefan Fujs 2 , Jaka Horvat 2 , Marko Fonovič 3 , Boris Turk 3 , Kristina Gruden 4 , Gregor Kosec 2 2 1 University of Ljubljana, Biotechnical Faculty Acies Bio d.o.o 3 Institut Jožef Štefan 4 Nacionalni Institut za Biologijo 5 Biotechnical Faculty, University of Ljubljana 2 Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. A significant increase in erythromycin yields has been achieved, compared to the wild type strain over decades of intensive strain improvement. Considering the annual world production and commercial importance of erythromycin and its semi-synthetic derivatives, current yields remain relatively low. Therefore, there is a clear commercial incentive to further improve erythromycin production technology. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. However, improving the understanding of regulatory elements involved in erythromycin biosynthesis in S. erythraea remains a challenging task because no regulatory genes are present inside the erythromycin gene cluster. The difficulty of identifying key regulatory genes, crucial for improvement of erythromycin yield, is reflected by the fact that among about 7000 predicted ORFs in S. erythraea genome 15.5% are putative regulatory genes. To address this issue, we have carried out a comprehensive comparative omics approach, comparing genome, transcriprome and proteome of erythromycin high-producing ABE1441 and WT S. erythraea strains during the bioprocess closely resembling industrial large-scale production process. Among others, we have identified a new putative regulatory gene, profoundly overexpressed in the industrial strain, which simultaneously influences sporulation during the life cycle of this actinomycete and importantly, significantly affects erythromycin yield of the WT and high-producing strains. Importantly, we have shown that “omics” approaches are valuable tools for identification of industrially relevant genes/pathways. http://dx.doi.org/10.1016/j.nbt.2014.05.2033 S166 www.elsevier.com/locate/nbt Anh Do Quynh Nguyen 1,∗ , Jens Schneider 2 , Volker Wendisch 3 1 Cebitec & Genetics of Prokaryotes, University of Bielefeld Evonic Industry 3 Chair of Genetics of Prokaryotes, University of Bielefeld Corynebacterium glutamicum shows great potential for the production of the polyamide monomer putrescine. Previously, we have constructed the putrescine producing strain PUT21 by deletion of argF, the gene for ornithine transcarbamoylase (OTC), and argR, encoding the L-arginine repressor combined with heterologous expression of the Escherichia coli gene for the L-ornithine decarboxylase SpeC and low-level argF expression from a plasmid addiction plasmid system. Acetylputrescine was detected as by-product in fermentations with PUT21 (39% of putrescine formed). Mutation analysis of 18 (putative) acetyltransferase genes revealed cg1722 to be responsible for putrescine acetylation. Subsequently, PUT21cg1722 was shown not only to produce acteylputrescine, but also 54% more putrescine than PUT21. To improve provision of L-glutamate as precursor, the activity of 2-oxoglutarate dehydrogenase complex (ODHC) was reduced by promoter exchange, which resulted in 27% higher putrescine production than PUT21. Similarly, replacing the translational start codon of proB encoding the first enzyme of L-proline biosynthesis from ATG to TTG resulted in 58% higher putrescine production than the parental strain. A transcriptome analysis revealed increased expression of the genes cgmR and cgmA during putrescine production. Overproduction of the exporter CgmA in PUT21 improved putrescine yield by 48% and productivity by 64%. Transcirional fusions and microarray analysis confirmed increased cgmA expression in the absence of the transcriptional repressor CgmR. Binding of CgmR to cgmO DNA was confirmed and shown to be counteracted by the putative inducers putrescine and cadaverine. The current work focusses on combining all identified targets to improve putrescine production in one strain. http://dx.doi.org/10.1016/j.nbt.2014.05.2034 METABOLIC MODELING New Biotechnology · Volume 31S · July 2014 Metabolic modeling PN-01 Effective Fast Filtration Method for Metabolomics Studies of Mammalian Cells Vinoth Shanmukam 1,∗ , Juan-Antonio Hernandez-Bort 2 , 2 3 Michael Hanscho , Christian Leitner , Stephan Hann 3 , Gunda Köllensperger 3 , Denise Sonntag 4 , Christine Heel 5 , Nicole Borth 3 1 Austrian Centre of Industrial Biotechnology Austrian Centre of Industrial Biotechnology, Graz, Austria 3 University of Natural Resources and Life Sciences, Vienna, Austria 4 Biocrates Life Sciences AG, Innsbruck, Tirol, Austria 5 Sandoz GmbH, Schaftenau, Austria 2 During the last two decades, the advent of metabolomics significantly increased the investigation of cellular processes. Detection, quantification and determination of intracellular metabolites highly rely on rapid inhibition of metabolic activity. Thus a quick and effective quenching process is essential to corroborate in vivo conditions. To address this challenge the conventional centrifugation method is replaced by a modified fast filtration protocol which uses commercially available components. This optimized protocol fulfills the requirement of quenching by avoiding cell leakage and contamination with extracellular metabolites, by preserving cell membrane integrity and by rapid inhibition of enzymatic activity without metabolite degradation. An added advantage of this method is that the whole quenching process can be performed in less than 15 seconds. To understand growth behavior, viability and metabolic activity, two batch fermentations were run with protein-free suspension cultures of CHO-K1 cells grown in medium containing 8 mM glutamine, and the same cell line adapted to growth in glutamine free medium. Samples were quenched with 13 C labelled internal standards and stored at -80 ◦ C. Metabolite extractions were performed with 80% cold methanol and samples analyzed by a targeted approach using LC/MS (HILIC and Atlantis) for the identification of metabolites. A comparison of a wide range of metabolites of the two fermentation setups will be presented. http://dx.doi.org/10.1016/j.nbt.2014.05.2035 PN-02 Identification of metabolic characteristics of liver cancer stem cells by integrative systems analysis Jae Yong Ryu ∗ , Hyun Uk Kim, Sang Yup Lee Korea Advanced Institute of Science and Technology Liver cancer stem cells are known to be responsible for cancer recurrence, metastasis, and various types of resistances. Especially, understanding mechanisms of their resistance to several cancer treatments are critical in combating cancers. To this end, integrative systems analysis involving constraint-based modeling and simulation was conducted to better understand metabolic characteristics of liver cancer stem cells and potential cues for their anticancer treatment resistances. Transcriptomic profiles of CD133-expressing liver cancer cells and exo-metabolic profiles of 60 cancer cell lines were integrated with a human generic metabolic model, Recon 2, to generate CD133+/- -specificliver cancer metabolic models; these two models were employed to simulate their metabolic states. In particular, we paid attention to: 1) metabolites differently consumed or secreted by each cell, 2) metabolic pathways whose overall fluxes appear to be different by more than 1.5-fold in the two cancer cells, and 3) potential roles of microRNA on the metabolic states of liver cancer stem cells. Experimental validation of the prediction results from the integrative systems metabolic analysis conducted herein will further contribute to elucidating metabolic aspects of cancer cell resistances. http://dx.doi.org/10.1016/j.nbt.2014.05.2036 PN-03 Design and flux modelling for recombinant production of 3-Hydroxybutyrate in Escherichia coli Mariel Perez-Zabaleta 1,∗ , Johan Jarmander 1 , Mónica Guevara 1 , Jorge Quillaguamán 2 , Gen Larsson 1 1 2 KTH UMSS Poly (3-hydroxybutyrate) (PHB) is accumulated intracellularly by microorganisms, usually under nutrient deficient conditions and excess of carbon source, is known to possess plastic properties, biodegradable and biocompatible. 3-hydroxybutyrate (3HB) is the monomer of PHB. It is believe that increasing the production of 3HB consequently can achieve high concentrations of PHB, because this monomer can be polymerized outside the cell by different methods. H. boliviensis, a native strain of Laguna Colorada, Bolivia, is known for produce large amounts of PHB. The PHB is synthesized by the successive action of b-ketoacyl-CoA thiolase (phbA), acetoacetyl-CoA reductase (phbB) and PHB polymerase (phbC). The genes phbA and phbB of the phbCAB operon from H. boliviensis were introduced into E. coli and the production of 3HB was achieved. The phbC gene was not cloned in E. coli because it has unspecified thioesterases that can remove the CoA part of 3HBCoA and excrete 3HB to the medium. Previous studies found that H. boliviensis posses 7 different genes involved in the formation acetoacetyl-CoA (phbA) and 3 genes able to produce 3HB-CoA (phbB). We want to determine the effect of the different combinations of these genes on the yield of 3HB attained by the recombinant E. coli. Wild type E. coli does not have the capacity to synthesize 3HBCoA but grows fast, at a higher temperature than H. boliviensis and it is easy to clone because is one best understood microorganisms. Recombinant E. coli metabolism will be modeled in order to obtain a high productivity of 3HB and co-polymers. http://dx.doi.org/10.1016/j.nbt.2014.05.2037 www.elsevier.com/locate/nbt S167 METABOLIC MODELING PN-04 Cotton Breeding Research Progress in China Wuwei Ye ∗ Institute of cotton research, CAAS, China Cotton production plays a significant role to the economy of china, because china is one of the largest cotton producers worldwide. In addition to these, china is also the largest consumer country of cotton. Therefore, a healthy and stable development trend of cotton production is important to promote the efficiency of china’s agriculture, the incomes of Chinese farmers, as well as S168 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 the stability of china’s rural economy. Due to the reduction of cultivated area, damages by pests and diseases, food safety issues, the ratio of grain to cotton prices, and other factors, the planting area of cotton in china cannot be increased substantially. How to coordinately develop the national cotton production to achieve high cotton quality, and production efficiency is the major and urgent issue for the healthy development of china’s cotton industry. In response to this issue, the overall situation of China’s cotton production, the cotton molecular assisted breeding progress in China, and the development directions of China’s cotton industry will be introduced. http://dx.doi.org/10.1016/j.nbt.2014.05.2038 METAGENOMIC APPLICATIONS New Biotechnology · Volume 31S · July 2014 Metagenomic applications PP-01 The impact of the compostable packaging material poly(lactic) acid on fungal communities in compost Mehlika Karamanlioglu ∗ , Geoff Robson The University of Manchester The compostable biopolymer, polylactic acid (PLA), is mostly derived from a renewable source, starch, and increasingly being used as an alternative to conventional plastics for short shelflife products, disposable bags and packaging materials as it decomposes at elevated temperatures during composting. Abiotic hydrolysis in the presence of water leads to a progressive decrease in the polymer molecular weight and ultimately the release of lactic acid. Despite the increase in the amount of PLA entering composting systems, few studies have examined the potential impact of PLA degradation on the compost microbial community. Thermophilic fungi play an import role in the composting process and in this study, the impact of PLA hydrolysis on the compost fungal community was examined by incubating PLA films and PLA granules in compost at different concentrations, 0-50% (w/w), by terminal restriction fragment length polymorphism (TRFLP) and 454-pyrosequencing. When PLA was incubated at 50 ◦ C, a discernible PLA disintegration occurred and TRFLP revealed that the fungal community profile initially changed but shifted back toward the initial compost community profile over time when PLA concentration was less than 50% (w/w) in compost. However, when PLA was at a concentration of 50% (w/w), fungal community profile did not shift back toward to the initial profile and the fungal diversity decreased due to a marked acidification of the compost. 454-pyrosequencing revealed that the presence of PLA enriched the thermophilic fungus, Thermomyces sp. in the compost population over time. http://dx.doi.org/10.1016/j.nbt.2014.05.2039 PP-02 Exploring biomass degrading communities for lignocellulolytic activities Senta Blanquet 1 , Agnès Hébert 1 , Franc ¸ oise Fayolle-Guichard 1 , 2 2 Pedro Coutinho , Bernard Henrissat 1 2 IFP Energies nouvelles AFMB CNRS/Université Aix-Marseille UMR6098 Present schemes for bioethanol production from lignocellulosic biomass, and especially the enzymatic hydrolysis step, are currently still too costly. An interesting alternative is the “consolidated bioprocessing” production scheme (CBP), which uses a single organism to catalyse both biomass hydrolysis and fermentation of the liberated sugars, and which therefore represents an important potential for cost reduction. The aim of the present project was therefore to identify new microorganisms and enzymes employable in such a process. Compost samples were collected and enriched for several months on lignocellulosic materials, such as wheat straw, poplar and Miscanthus. The most active cultures, as evidenced by CO2 production, were selected for further analysis. 16S sequence data showed that significant shifts in the microbial community composition occurred with time. Unexpectedly, important differences were noticed in submerged cultures compared to solid medium setups. Bacteriodetes and Actinobacteria prevailed in the solid medium, whereas Firmicutes and Proteobacteria were the dominating phyla in the submerged culture. In parallel, metagenomic DNA was extracted from two of the enrichments and sequenced. Analysis of Carbohydrate active enzymes (CAZymes) revealed that members of glycoside hydrolase (GH) families GH6, GH9 and GH48 (cellulases) as well as families GH27, GH29 and GH36 (xyloglucan side chain digestion) had accumulated after enrichment. Families of CAZymes for hemicelluloses and pectin breakdown, especially GH28, GH30, GH53, GH106 and polysaccharide lyase (PL) families PL1, PL4 and PL22 were also enriched. Thus, the difference in taxonomical composition between submerged and solid medium was also mirrored in the CAZyme family composition. http://dx.doi.org/10.1016/j.nbt.2014.05.2040 PP-03 Identification and functional characterization of Est16, an esterase isolated from a metagenomic library of microbe consortium specialized in diesel oil degradation Mariana Rangel Pereira 1,∗ , Gustavo Fernando Mercaldi 1 , Thaís Carvalho Maester 2 , Andrea Balan Fernandes 3 , Eliana G. de Macedo Lemos 2 1 Laboratório Nacional de Biociências UNESP 3 USP 2 Lipolytic enzymes have been attracting global market attention because they show enormous biotechnological potential such as production of detergents, processing of leather, production of cosmetics, perfumes and biodiesel. To search for novel lipolytic enzymes, a DNA metagenomic library was constructed from a microbe consortium isolated from oil contaminated soil at Ribeirão Preto, Brazil. After functional screenings using trybutirin, one clone referred to as Est16, was used for further analysis. The sequence analysis revealed that Est16 shares 87% with lipases/esterases (ADM63076.1) from uncultured bacterium in the database. After an amino acid sequence alignment of the Est16 with 34 sequences of members of the eight lipolytic families demonstrated that Est16 is a new member of family V. The catalytic triad (Ser, Asp and His), is highly conserved and the serine is located in the conserved motif GXSMGG. The est16 gene was cloned into the pET28a vector and expressed as a N-terminal fused His6tag protein in Escherichia coli BL21(DE3) cells. The recombinant protein was purified as active soluble form and used for activity assays. Est16 showed wide range of substrates and highest catalytic efficiency against p-nitrophenyl valerate (C5 ), optimum activities at mesophilic temperature ranges and the optimum pH of 9.0. In particular, Est16 showed an increase in reaction rate using up to 5% www.elsevier.com/locate/nbt S169 METAGENOMIC APPLICATIONS New Biotechnology · Volume 31S · July 2014 of DMSO, suggesting tolerance to the presence of organic solvents. Here, we demonstrated that metagenomic approach can be used as a DNA source to expand the lipolytic enzymes diversity and our results indicate that Est16 has potential for use in industrial process. PP-05 http://dx.doi.org/10.1016/j.nbt.2014.05.2041 1 Discovery of thermostable hydrolytic enzymes of industrial interest by metagenomic screening Dimitra Zarafeta 1,∗ , Georgios Skretas 2 , Fragiskos N Kolisis 3 National Technical University of Athens Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation, Athens, Greece 3 Laboratory of Biotechnology, School of Chemical Engineering, National Technical University of Athens, Athens, Greece 2 PP-04 ANASTASIA a versatile web platform for metagenomic analysis Efthymios Ladoukakis 1 , Eleftherios Chatziioannou 2 , Fragiskos Kolisis 1 1 2 Pilalis 2 , Aristotelis National Technical University of Athens National Hellenic Research Foundation In this work, ANASTASIA (Automated Nucleotide Aminoacid Sequences Translational plAtform for Systemic Interpretation and Analysis) web repository is presented. ANASTASIA enables automated massive metagenomic sequence assembly, analysis and interpretation tasks under the same workflow. It does so by providing a rich suite of computational tools integrated within numerous algorithmic pipelines implementing and integrating versatile data processing tasks for (meta)genomic sequencing, assembly and protein sequence datasets. The modules of these pipelines incorporate established bioinformatic algorithms like HMMER and BLAST as well as Python and Perl scripts, which perform annotation and classification tasks in synergy with stand-alone programs, while being integrated in an all-in-one inclusive solution exploiting the Galaxy workflow engine. ANASTASIA supports the labor-free denovo creation and operation of workflows able to handle the analytical challenge of large datasets (e.g. from metagenomic experiments) and store the generated annotation results automatically into a MySQL database running in the background. In addition it promotes workflows and large datasets sharing through its transparent shell which a user-friendly graphical interface. The current configuration of the ANASTASIA installation comprises a cluster of 36 CPU cores, 256 GB RAM and a fast storage machine with 32 TB capacity provided by the University of Copenhagen. ANASTASIA represents the bioinformatics core of the FP7 project HotZyme which targets to the exhaustive analysis of metagenomes in thermal springs, with the scope of tracing proteins with interesting enzymatic properties which will be indispensable in a wide range of biotechnological applications; from paper pulp bleaching to production of biofuel from biomass. http://dx.doi.org/10.1016/j.nbt.2014.05.2042 Enzymes are biocatalysts used in a wide range of industrial applications and provide a “green” alternative to chemical conversions. Hydrolases are a class of enzymes that exhibit high selectivity and potential synthetic ability when used in nonconventional media. These characteristics render this subcategory of enzymes very appealing to the industry, especially for the production of fine chemicals and pharmaceuticals. Despite their advantages, a very limited amount of hydrolases is currently being used in biotechnological applications, as many industrially relevant processes require high temperatures where conventional biocatalysts perform poorly. For such processes, thermoor hyperthermostable enzymes are required. Thermophilic organisms remain until today a largely unexplored source of such enzymes since the vast majority of those organisms (>99%) cannot be cultured using standard laboratory techniques. To address some of these issues, the international consortium HotZyme was formed with the aim of applying systematic metagenomic screening approaches in order to identify novel hydrolases from hot environments. Within the framework of this project, metagenomic libraries were constructed from samples collected from diverse high-temperature ecosystems around the world (Russia, Iceland, Italy, China, New Zealand, Japan and U.S.A). These libraries were then analyzed using bioinformatic tools and highthroughput functional screens in Escherichia coli cells to identify open reading frames with desired hydrolytic activities and stability at elevated temperatures. These approaches have led to the discovery of novel hydrolases of biotechnological interest. The characterization of these novel enzymes will be presented and their potential use for the synthesis of fine chemicals will be discussed. http://dx.doi.org/10.1016/j.nbt.2014.05.2043 PP-06 Functional metagenomic and proteomic characterization of soil microbial community associated with decomposing reeds Gaetano Perrotta ∗ , Linda Bianco, Fabrizio Daddiego, Paolo Facella, Loredana Lopez Carbone, Loretta ENEA Recent demands for the production of biofuels from lignocellulose biomass led to an increased interest in soil microbial communities. Ligninolytic microbes have developed a unique strategy to handle lignin degradation based on S170 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 a pletora of synergistically acting enzymes. Besides, specific biomasses can be characteristically affected by these networks of collaborative enzymes, deriving from typical soil microbial community. In order to unravel the microbial environment of plant litter, we collected soil samples from sites characterized by the presence of different decomposing plants, Arundo donax and Phragmites australis, DNA extracted from soil was used for meta-genomic analyses using Roche 454 platform. A total of 617,604 high quality reads have been collected, corresponding to more than 2000 microorganisms. Among them, a bacterial consortium potentially involved into biomass deconstruction was identified. A large number of bacteria belonging to this consortium are enriched in genes coding for ligno-cellulolitic enzymes. METAGENOMIC APPLICATIONS In parallel, decomposing leaves and stems of Arundo donax and Phragmites australis were collected and used for the isolation of cultivatable fungi. Molecular characterization were performed on the isolated fungi, leading to the identification of 7 fungal species. Among them, we isolated a white-rot fungus, belonging to the Polyporales order, showing outstanding metabolic activities in ligno-cellulose degradation. A proteomic characterization of this fungal secretome is currently under investigation. The integration of the data coming from these analyses is expected to point out a number of microorganisms, genes and proteins likely involved into lignocellulose degradation pathways that could be used to improve biomass deconstruction in industrial applications. http://dx.doi.org/10.1016/j.nbt.2014.05.2044 www.elsevier.com/locate/nbt S171 NANOBIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 Nanobiotechnology PQ-02 PQ-01 A pH-responsive high density lipoprotein-like nanoparticle of epothilone B The use of chimeric virus-like particles with inserted target peptides for tailored antibody production Aurelija Zvirbliene ∗ , Indre Kucinskaite-Kodze, Alma Gedvilaite Vilnius University, Lithuania Protein engineering provides an opportunity to generate new immunogens with desired features. Viral structural proteins with their intrinsic capacity to self-assemble to highly-organized virus-like particles (VLPs) have been shown to possess high immunogenicity and were exploited as potential vaccines. We have demonstrated that major capsid protein VP1 derivatives of hamster polyomavirus (HaPyV) harboring foreign sequences at certain surface-exposed regions allowed the formation of chimeric VLPs. The chimeric VLPs meet the requirements for a strong immunogen being able to activate both B cells recognizing the surface-located epitopes and T-helper cells providing the necessary signals for Ig class switching and affinity maturation. Moreover, the immunogenicity of inserted peptides is enhanced due to the repetitive multimeric structure of chimeric VLPs. We have exploited the VLP approach for generation of monoclonal antibodies against short non-immunogenic peptides of human proteins as well as difficult-to-express antigens such as viral glycoproteins. The chimeric HaPyV-VP1 VLPs have been shown to induce in mice strong insert-specific B- and T-cell responses. The generated antibodies were reactive with native full-length target proteins thus demonstrating the surface localization and proper folding of the inserted sequences. http://dx.doi.org/10.1016/j.nbt.2014.05.886 Woo-Jong Lee 1,∗ , Ji-Chun Lee 2 , Byoung-Jae Kong 2 , Jonghyeok Shin 2 , Sung-Gun Kim 3 , Heekyung An 1 , Chi-Heung Cho 1 , DaeHyuk Kweon 2 1 Korea Institute of Industrial Technology, South Korea Sungkyunkwan University, South Korea 3 Youngdong University, South Korea 2 Epothilone B (EpoB) is a paclitaxel (PTX)-like microtubulestabilizing agent; it induces mitotic arrest and apoptosis in cells. EpoB is a promising anti-tumor agent, and is thought to have the potential to overcome well-known PTX resistance. However, EpoB is barely soluble in water, and is fatal to normal cells due to extremely potent cytotoxicity. To reduce the unwanted cytotoxicity of EpoB to normal cells, a reconstituted high-density lipoprotein (rHDL) of EpoB (EpoB-rHDL) was generated using apolipoprotein A-I (apoA-I). The EpoB-rHDL, as well as PTX-rHDL (a HDL-like nanoparticle of paclitaxel), was indeed mild (nontoxic) to certain cell lines such as MCF7, MDA-MB-231, and SK-OV-3, while free EpoB and PTX were very toxic to these same cells. In contrast, the EpoB-rHDL and PTX-rHDL were very effective in killing the Caco-2 and ZR-75-1 while free drugs were less toxic to these cells. The susceptibility of cell lines to rHDLs was dependent on the expression of scavenger receptor class B type I (SR-BI), indicating that EpoB-rHDL selectively and efficiently kills only SR-BI-overexpressing cells. Furthermore, the EpoB-rHDL released EpoB only at acidic pH, which may facilitate the escape of drugs from acidic endosome. Thus, EpoB-rHDL shown in this study enables safe and targeted delivery of the potent EpoB to cancer cells in SR-BI-dependent manner. http://dx.doi.org/10.1016/j.nbt.2014.05.887 S172 www.elsevier.com/locate/nbt 1871-6784/$ — see front matter NANOBIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 PQ-03 PQ-05 A nanovesicle-based olfactory biosensor and its application to disease diagnosis and grain quality assessment Biosynthesis of single nanoparticles using various metal binding proteins Tai Hyun Park ∗ , Jong Hyun Lim, Jung Ho Ahn Yoojin Choi ∗ , Sang Yup Lee, Doh Chang Lee Seoul National University, South Korea Korea Advanced Institute of Science and Technology, South Korea We integrated the olfactory system to carbon nanotube platforms for biosensing applications. Human olfactory receptor (OR)-containing nanovesicles were produced from human embryonic kidney (HEK)-293 cells. The nanovesicles, which generate olfactory signals through a cAMP pathway, were integrated into single-walled carbon nanotubes field-effect transistors (SWNTFETs). The nanovesicles and SWNT-FETs play roles in perceiving specific odorants, and in amplifying cellular signals, respectively. In particular, this system can be used for the diagnosis of disease such as lung cancer and also for the real-time monitoring of fungal contamination in grain. Specific olfactory receptors recognizing the chemical biomarkers were first selected through screening a library of human olfactory receptors. The nanovesicle-integrated device was able to detect a lung cancer biomarker (heptanal) and a specific compound generated from contaminated grain (1-octen-3ol) with excellent sensitivity and selectivity, similar to the original olfactory system. Recently nanotechnology has attracted attention worldwide because of the interesting physicochemical properties of these particles. However, most nanoparticles are chemically synthesized and involve the use of expensive catalysts for reactions at high temperature and pressure. The environmental issues related to the synthesis of nanoparticles have motivated research toward greener methods that utilize microorganisms such as bacteria, yeast, and fungi for their ability to reduce metal ions. We synthesized various single nanoparticles using metal binding proteins on recombinant Escherichia coli (E. coli). The morphology and size of the synthesized nanoparticles was observed by low to high resolution transmission electron microscopy (TEM) at 200 kV and energy-dispersive X-ray (EDX) spectra. Finally, we suggest a possible mechanism of the biosynthesis process that might provide a guide for conditions required for the synthesis of various nanoparticles. http://dx.doi.org/10.1016/j.nbt.2014.05.890 http://dx.doi.org/10.1016/j.nbt.2014.05.888 PQ-06 PQ-04 Biological synthesis of silver nanoparticles using plant leaf extracts and their specific antimicrobial activity Beom Soo Kim ∗ , Bipinchandra Salunke, Shailesh Sawant, Bassam Alkotaini Chungbuk National University, South Korea Several plant leaf extracts (Kalopanax, Magnolia, Persimmon, Pine, Ginkgo, Platanus, etc.) were used for extracellular synthesis of silver nanoparticles. Stable silver nanoparticles were formed by treating aqueous solution of AgNO3 with the plant leaf extracts as reducing agent. The synthesized silver nanoparticles were characterized by UV-vis spectroscopy, FT-IR, inductively coupled plasma spectrometry, energy dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, high-resolution transmission electron microscopy, etc. Antimicrobial susceptibility tests of silver nanoparticle treatments revealed variability in sensitivity of Bacillus cereus and Saccharophagus degradans. Minimum inhibitory concentration (MIC) values of the silver nanoparticles for B. cereus and S. degradans were found to be 30 ␮g/mL and 10 ␮g/mL, respectively. The mixed culture of B. cereus and S. degradans treated with silver nanoparticles at 10 ␮g/mL after 24 h showed presence of only B. cereus colonies. This study suggests that plant leaf extract synthesized silver nanoparticles can selectively inhibit growth of the Gram negative S. degradans and retain the Gram positive B. cereus at MIC values of S. degradans. http://dx.doi.org/10.1016/j.nbt.2014.05.889 Electro-triggered, spatioselective, quantitative gene delivery into a single cell nucleus by Au nanowire nanoinjector Seung Min Yoo ∗ , Sang Yup Lee KAIST, South Korea Delivery of bioactive materials into a cell is highly important in the study of cell biology and medical treatments. Ideal nanoinjectors should be able to deliver biomaterials with high spatial resolution while causing minimum cell damage. We developed a Au nanowire (NW) nanoinjector that has the thinnest diameter among the DNA delivering devices as well as optimum mechanical properties, minimizing cell damage. Well-defined single-crystalline Au surface and high electric conductivity of a Au NW nanoinjector allow precisely timed and efficient electrochemical release of DNA molecules attached on a Au NW surface. Both linear DNA and plasmid DNA were delivered separately, and showed successful expression. The Au NW nanoinjector would find important biomedical applications in the fields such as gene therapy, DNA vaccination, targeted drug delivery, and probe/control of cell signaling events [1]. Acknowledgements: This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012C1AAA001-2012M1A2A2026556) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea. www.elsevier.com/locate/nbt S173 NANOBIOTECHNOLOGY Reference [1].Yoo SM, Kang M, Kang T, Kim DM, Lee SY, Kim B. Nano Lett 2013;13(6):2431–5. New Biotechnology · Volume 31S · July 2014 proteoliposome was increased to withstand up to ∼9 atm and water purification was demonstrated using our device. http://dx.doi.org/10.1016/j.nbt.2014.05.893 http://dx.doi.org/10.1016/j.nbt.2014.05.891 PQ-09 PQ-07 Nano-pattern integrated biomimetic system for wound healing assay A comparative study of the effectiveness of ␤-glucosidase immobilized on CNT-nanoparticles and Ca-alginate beads Sun Min Kim ∗ , Insu Lee, Galahm Park, Tae-Joon Jeon Ahmad Jameel ∗ , Labiba Mahmud, Faridah Yusof Inha University, South Korea International Islamic University Malaysia, Malaysia Wound healing process of damaged skin involves various steps of cellular behavior and complex combinations of signaling pathway. In this research, we simply fabricated nano-patterned surfaces with biocompatible PDMS (Polydimethylsiloxane) polymer and integrated a patterned surface with a microfluidic system which can mimic wound healing rad processes. To form wound damage to 3T3 fibroblast cell layer cultured on the surface, we generated layered flows of cell culture media and trypsin/EDTA in a microchannel. We monitored cell migration on the pattern during wound healing process and found that the patterned surface guided the migration of cells as well as the intercellular cytoskeleton structure. The results demonstrate that cellular behavior can be controlled for wound healing by mechanical stimuli. We expect that the developed 2D skin model can be integrated with various types of surface and used as a standard assay platform for wound healing research. Enzymes are extensively used in various industrial, biomedical and biopharmaceutical applications. However, enzymes in their free form are unstable and expensive besides being characteristically susceptible to inhibition by high product concentrations and are highly sensitive to pH and temperature changes. Immobilization technology offers solutions to these challenges besides enhancing operational stability, longevity and ease of separation. ␤-Glucosidase has been widely employed as model enzyme for enzymatic studies. Ca-alginate beads provide a gentle environment for immobilization, but have certain limitations such as low stability, high porosity and limitations in biocompatibility. Carbon nanotubes (CNTs) on the other hand have excellent mechanical, thermal and electrical properties, as well as dimensional and chemical compatibility with biomolecules like DNA and enzymes, suitable for biosensor design. Here, ␤-glucosidase was immobilized in Ca-alginate gel and multiwalled carbon nanotubes (MWCNT) using standard techniques and their activity was compared with that of free enzyme. The activity was found highest (12.53 U/mL) for the free enzyme and lowest (9.768 U/mL) for the immobilized Ca-alginate. The activity of immobilized MWCNT (12.20 U/mL) was close to the free enzyme activity. The enzyme reaction was found to follow Michaelis–Menten kinetics. The Michaelis constants, Km and Vmax , determined using Langmuir linearized plots are, respectively, 0.09048 ␮mol/mL and 0.00989 ␮mol/mL min for immobilized Ca-alginate; and 0.0985 ␮mol/mL and 0.01237 ␮mol/mL min for immobilized MWCNT. The corresponding values for the free enzyme are 0.0854 ␮mol/mL and 0.01263 ␮mol/mL min. Thus, the MWCNT appears to be a promising support material for enzyme immobilization. http://dx.doi.org/10.1016/j.nbt.2014.05.892 PQ-08 Water purification through cross-linked proteoliposomes using a preconcentrator Tae-Joon Jeon 1,∗ , Hyunil Ryu 2 , Huisoo Jang 2 , Insu Lee 2 , Ga Lahm Park 2 , Sun Min Kim 2 1 2 Inha University/Biological Engineering, South Korea Inha University, South Korea Aquaporin is the most efficient filter in nature due to its high water selectivity and permeability. However, the manufactural difficulties of efficient and large scaled aquaporin embedded membrane preclude its industrial applications. It is mainly attributed to the fragility of a lipid bilayer or other biomimetic membrane. We have created robust membranes by cross-linking liposomes with linkers. In addition, we concentrated liposomes effectively using a microfluidic preconcentrator which has nanochannels formed by the electrical breakdown of a polydimethylsiloxane (PDMS) membrane at a high electrical bias with no nano-lithography. Proteoliposomes were continuously concentrated at the target position by applying an electric field through the junction of micro- and nanochannels. Amine-terminated proteoliposomes embedded with aquaporin were conjugated to the surface of the PDMS device via EDC/NHS reaction. As a result, the durability of S174 www.elsevier.com/locate/nbt http://dx.doi.org/10.1016/j.nbt.2014.05.894 NANOBIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 PQ-10 PQ-11 Impacts of carbon nanotubes on biochemical reactions: insight into interaction between carbon nanotubes and DNA polymerase enzyme Iron-based nano-particles for Lipolase immobilization and stabilization Ebru Uysal 1,∗ 2 , Meral Yuce , Hasan Kurt 1 1 Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey 2 Nanotechnology Research and Application Center, Sabanci University, Istanbul, Turkey Recently, the Polymerase Chain Reaction technique has begun to benefit from nanotechnology. In this paper, effects of carbon nanotubes in the Polymerase Chain Reaction were investigated by Electrophoresis, Circular Dichroism Spectrometry and Dynamic Light Scattering Techniques. The unique ability to amplify low copy number DNA within minutes has made in vitro Polymerase Chain Reaction (PCR) one of the most essential techniques in modern biology. In order to harness this technique to its full potential, certain obstacles, such as nonspecific by-products, low yield, and complexity of GC rich and long genomic DNA amplification need to be surmounted. Nanomaterial-assisted PCR, so-called nanoPCR, is a new area in biotechnology that introduces nanostructured materials into PCR reaction to obtain improved results. Nanomaterials have unique physical and chemical properties, such as high thermal conductivity, stability and high surface to volume ratios. The effects of nanomaterials in PCR depend on their size, shape, concentration, heat conductivity, electron transfer properties and surface modifications. Carbon nanotubes are predicted to bind major PCR components, such as primers, template and polymerase enzyme, via specific or non-specific interactions. In this paper, we demonstrate the interaction of carbon nanotubes with wild type DNA polymerase enzyme, and the effect of this interaction in PCR for the first time. According to the results, chiral properties of the wild type DNA polymerase enzyme has changed after incubation with amine functionalized multiwall carbon nanotubes, which confirms direct interaction between the enzyme and tubes. Furthermore, this interaction has been found to be temperature dependent via dynamic light scattering spectroscopy. Surabhi Mehra ∗ , Shamsher S. Kanwar Himachal Pradesh University, Shimla, India In our study, a commercial Lipolase 100L (Novozymes, Bangalore, India) was covalently immobilized on silane coated modified magnetic nano-particles. These magnetic (Fe3 O4 or ␥-Fe2 O3 ) NPs were in the size range of 25–30 nm and their surface modification was carried out by coating with Tetra Ethoxy Silane (TEOS) by sol–gel reaction and these silane-coated magnetic NPs were then used for immobilization. Thereafter, Lipolase immobilized nano-particles [30 mg] were separated by magnetic decantation and suspended in 6 ml of phosphate buffered saline (pH 7.4). These Lipolase-bound NPs were kept in a homogeneous form [20 ␮l] and were assessed [∼2 mg = 118.29 U of enzyme in ml] for enzymatic activity, stability and reusability using 3 ml reaction system containing p-nitrophenyl palmitate as a substrate in 0.05 M Tris buffer pH 8.2. NPs-immobilized Lipolase showed enhanced activity (59.2 U/mg NPs) and stability. Effect of Cchain length/substrate specificity of NPs-bound Lipolase was also assessed and found maximum towards p-nitrophenyl palmitate [139.1 U/ml] followed by p-nitrophenyl myristate [87.8 U/ml], p-nitrophenyl caprylate [77.7 U/ml] and p-nitrophenyl laureate [75.3 U/ml]. The temperature, pH and reusability measurements showed that Lipolase immobilized on NPs were capable of working at broader pH and higher temperature ranges and was reusable up to several cycles. http://dx.doi.org/10.1016/j.nbt.2014.05.896 http://dx.doi.org/10.1016/j.nbt.2014.05.895 www.elsevier.com/locate/nbt S175 NATURAL AND SYNTHETIC POLYMERS Natural and synthetic polymers PR-01 A mathematical model for polyhydroxybutyrate production by a wild type Bacillus megaterium using raw glycerol from biodiesel industry as sole carbon source Paalo Andrea Moreno Yañez 1,∗ , Débora Jung Luvizetto Faccin 2 , Nilo Sérgio Medeiros Cardozo 2 , Humberto Escalante 1 , Marianny Y. Combariza 1 , Carolina Guzmán 1 1 Universidad Industrial de Santander, Colombia 2 Federal University of Rio Grande do Sul, Brazil Polyhydroxybutyrate (PHB) is a biodegradable, biocompatible and thermoplastic biopolymer, synthesized naturally as cytoplasmic inclusions by various genera of Gram-positive and Gram-negative bacteria. PHB shares similar properties with polypropylene, and could potentially replace it, but its industrial production is limited by its high cost and low productivity. We have previously reported [1] optimized growth conditions for a wild type Bacillus megaterium with the ability to produce PHB from raw glycerol byproduct of a Colombian biodiesel industry. Aiming for a better understanding of the PHB biosynthetic process with the referred carbon source and wild type Bacillus megaterium, in the present work we present a mathematical model that allows us to describe the kinetics of microbial growth, substrate consumption, product formation and also to simulate different cultivation strategies. In this model, microbial growth and product formation were described by the Monod and the Luedeking–Piret equations, respectively. Model implementation and parameter estimation were carried out in the EMSO process simulator. Key model parameters were estimated from experimental data obtained in batch cultivation using a submerged bioreactor. Experimentally, under optimized bioreactor conditions, a maximum PHB concentration of 0.96 g/L was reached at 12 h. The model provided excellent fitting of the experimental data previously obtained, providing correlation coefficients around 0.9. Reference [1].Moreno P, Yañez C, Tarazona N, Cardozo NSM, Escalante H, Combariza MY, Guzmán C. Statistical optimization of PHB production by a wild type Bacillus megaterium using raw glycerol as sole carbon source. Int J Biol Macromol 2014 (submitted for publication). http://dx.doi.org/10.1016/j.nbt.2014.05.897 PR-02 New Biotechnology · Volume 31S · July 2014 0.75 ng/mL of analogues were detected. The product extracted and isolated in its cyanide form had the similar UV spectrum as standard cyanocobalamin and as cobalamin produced by Lactobacillus reuteri DSM 20016. No cobalamin was detected in the fermentation broth containing 1% acetate, and less cobalamin was obtained when acetate started to be consumed. http://dx.doi.org/10.1016/j.nbt.2014.05.898 PR-03 Ultrasonic-assisted production of active polysaccharides from Crassostrea hongkongensis Bingna Cai 1,∗ , Jianyu Pan 2 , Huili Sun 3 1 No. 164, Xingangxi Rd, Haizhu District, P.O. Box 510301, China South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China 3 Chinese Academy of Sciences, Guangzhou, China 2 The beneficial effects of oyster extract against various disorders and diseases induced by oxidative stress have aroused great concern. Oyster polysaccharides, as immune nutrients, were conducted to provide nutrients for cell metabolism and prevent the side effects such as immunotoxicity and gastrointestinal toxicity caused by chemoradiotherapy. Ultrasonic-assisted enzymolysis was studied to increase the active polysaccharide yield and purity from Crassostrea hongkongensis, showing that it is more efficient than ultrasonic extraction or enzymatic hydrolysis alone. On the basis of Box-Behnken design and ridge analysis, the optimum conditions were obtained as ultrasonic treatment time of 24 min, power of 876 W, temperature of 49 ◦ C and material-solvent ratio of 1:6 (w/v). Furthermore, polysaccharide fraction (CHP), which was obtained by ultrasonic pretreatment and then alcalase hydrolysis at the conditions: 3000 U/g, 55 ◦ C, pH 8.0 for 4 h, exhibited obvious scavenging effect on DPPH and hydroxyl radical (98.48 ± 0.55% and 99.20 ± 0.12%, respectively) and linoleic acid peroxidation inhibition effect (85.48 ± 0.65%) at concentration of 5.0 mg/mL. Then the CHP was separated into three fractions by graded ethanol precipitation. The 30–60% ethanol precipitation fraction (C30–60% ) from CHP showed the highest activities, including promoting RAW 264.7 murine macrophage, tlymphocyte and IEC-6 cells proliferation (the highest cell proliferation rate was 137.10% at 0.0391 mg/mL, 160.48% at 0.0781 mg/mL and 153.70% at 0.0195 mg/mL, respectively). The activities of CHP might attribute to its uronic acid, sulfate composition and molecular weight. These results reveal the potential application of CHP in nutraceutical for cancer patients. Acetobacter pasteurianus DSM 3509 produces cobalamin Clemens Bernhardt ∗ , Xuan Zhu, Bernward Bisping Uni Hamburg, Germany A strain of acetic acid bacteria used in food applications was found to have the ability to synthesize cobalamin. A preliminary genetic study of the gene of uroporphyrinogen-III synthase and a survival test indicated the ability to synthesize cobalamin. By a modified microbiological assay based on Lactobacillus delbrueckii spp. lactis DSM 20355, 4.57 ng/mL of real cobalamin and S176 www.elsevier.com/locate/nbt http://dx.doi.org/10.1016/j.nbt.2014.05.899 NATURAL AND SYNTHETIC POLYMERS New Biotechnology · Volume 31S · July 2014 PR-04 New clues to design cell factories for tailor-made biopolymer production: Bacillus cereus as a source of polyhydroxyalkanoates biosynthetic proteins Marco Vastano ∗ , Angela Casillo, Maria Michela Cosaro, Giovanni Sannia, Cinzia Pezzella University of Naples Federico II, Italy Polyhydroxyalkanoates (PHAs) are biopolymers, accumulated as intracellular storage-granules by a variety of bacteria. The material properties and the potential biotechnological use of PHAs are strictly dependent on their monomeric composition. In PHAs producing Bacilli, the presence of a peculiar biosynthetic protein cluster, encoded by the operon phaRBC has been revealed [1]. In this work a functional study of the phaRBC operon from B. cereus has been carried out through its heterologous expression in Escherichia coli. Deletions mutants in phaR, phaB and phaC have been assayed for PHA accumulation in culture media boosted with a related (fatty acids) carbon source. PhaR function has been evaluated in its two potential active forms (LPhaR/sPhaR), which differ for the presence of an extra 16 aminoacids long N-terminal extension. This N-terminal tail seems to play a crucial role in determining PhaR function, promoting PhaR activity as transcriptional regulator in its longer form (LPhaR), and switching its role to PHA synthase (PhaC) activator/stabilizer in its shorter form (sPhaR). PhaB role was found to be crucial in determining biopolymer composition, influencing monomer chain length depending on its substrate specificity. Functional information obtained from this study represents the groundwork to design new cell factories for low-cost production of tailor-made PHAs. Reference [1].Rehm B. Polyester synthases: natural catalysts for plastics. Biochem J 2003;376:15–33. http://dx.doi.org/10.1016/j.nbt.2014.05.900 www.elsevier.com/locate/nbt S177 ANTIMICROBIALS, PATHOGENS AND DISEASE Antimicrobials, pathogens and disease PS-01 Genomics-based discovery of macrolide glycosyltransferase from Bacillus sp. Won-Gon Kim ∗ , Ji Yeong Park, Hyun Ju Kim Korea Research Institute of Bioscience and Biotechnology, South Korea Glycosylation of pharmacologically active secondary metabolites is an attractive method to improve their biological activity as well as pharmacokinetics. The availability of suitable glycosyltransferases for naturally unglycosylated compounds, however, is limited because of substrate specificity. Recently, Bacilli have been used as a source for the isolation of GT enzymes involved in the modification of aromatic or bulky substrates. In an effort to discover enzymes for the glycosylation of a macrolide compound, we identified putative glycosyltransferases by the genomic analysis of Bacillus sp. that produces various glycosylated macrolides. The selected glycosyltransferases were screened, leading to the identification of a glycosyltransferase that could catalyze the transfer of a glucose residue from UDP-␣-D-glucose to the macrolide compound. Using NMR and MS analysis, we determined the chemical structures of the new products to be a new glucosylated macrolide which exhibited improved water solubility. These data showed that genomics-based discovery of glycosyltransferase from Bacillus sp. was an effective strategy for the isolation of a suitable glycosyltransferase for naturally unglycosylated compounds. http://dx.doi.org/10.1016/j.nbt.2014.05.901 PS-02 Antifungal protein of seed coat extracts of Theobroma cacao L. during fermentation Fahrurrozi Fahrurrozi 1,∗ , Claudia Bahmann 1 , Nicolas Niemenak 2 , Reinhard Lieberei 1 , Bernward Bisping 1 1 2 University of Hamburg, Germany University of Yaounde I, Cameroon Seed coat is an important tissue for the regulation of imbibition and maintenance of the integrity of seed, and it is also the first seed barrier encountered by pests and pathogens. Seed cotyledons contain an array of proteins that may be involved in the protection of quiescent seeds against fungi. In a previous study (Fahrurrozi et al., 2013) we found that the seed coat from Theobroma cacao L. seeds contains an antifungal activity. Seed coat extract can inhibit growth of fungi (e.g. Aspergillus niger, Penicillium citrinum, Penicillium purpurogenum, Penicillium roquefortii) and yeasts (e.g. Candida lipolytica, Candida krusei, Rhodotorula rubra, Rhodotorula mucilaginosa, Saccharomyces cerevisiae). 25 and 10 mg/mL of seed coat extract can inhibit growth of fungi and yeasts respectively. By separation of seed coat proteins using SDS-PAGE 17 proteins bands were found. Analysis of the bands using mass spectrometry showed that 3 proteins have antifungal effect namely: glucanase, chitinase and osmotin (Bahmann, 2014). Further Niemenak (per- S178 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 sonal communication) found 4 proteins that have antifungal effect namely: glucan endo-1,3-beta-glucosidase, chitinase, 2S albumin storage protein, nonspecific lipid-transfer protein. During the fermentation process the seed coat proteins seem to be degraded, probably caused by an increase in temperature and by activity of proteases produced by microbes during the fermentation. To find out more details about the degradation of proteins during the fermentation, we are currently analyzing 28 protein bands, which we extracted during the fermentation. Temperature characterization showed that the seed coat protein is not stable at 40 and 50 ◦ C for 24 h. http://dx.doi.org/10.1016/j.nbt.2014.05.902 PS-03 Inactivation of microbial biofilms by visible light with a porphyrinic photosensitizer Sandra Beirão, Sara Fernandes, Joel Coelho, Adelaide Almeida, Maria da Graça Neves, Maria do Amparo Faustino, João Tomé, Angela Cunha ∗ University of Aveiro, Portugal Biofilms are aggregates of microbial cells imbedded in a matrix composed essentially by water and extracellular polymeric substances (EPS). The matrix provides a first line of defense against biological attack, environmental stress, and biocide diffusion, making biofilms a challenge to conventional antimicrobial approaches. The photodynamic inactivation (PDI) of microorganisms relies on the interaction of a non-toxic photosensitizer, molecular oxygen and light. This study aimed the assessment of the photodynamic effect on the matrix of model Pseudomonas aeruginosa biofilms, using the tetra-cationic porphyrin derivative tetra-iodide 5,10,15,20-tetrakis(1-methylpyridinium-4yl)porphyrin (Tetra-Py+ -Me) as photosensitizer (PS) and white light (380–700 nm) at an irradiance of 40 W m−2 . The photodynamic inactivation of imbedded cells, in single-species or mixed biofilms of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans was also determined. A reduction of 81% in the polysaccharides content of matrix of P. aeruginosa biofilms was observed after treatment with a light dose of 64.8 J cm−2 and 20 ␮M of Tetra-Py+ -Me. The photosensitization with Tetra-Py+ -Me also caused inactivation of cells in all tested biofilms. The maximum reduction factors were 3, 7 and 6 logs, for biofilms of P. aeruginosa, S. aureus and C. albicans, respectively. In mixed biofilms, the inactivation of S. aureus was as efficient as in single-strain biofilms (7 log reduction in colony counts) but was less efficient (5 log) for the yeast. Photosensitization with Tetra-Py+ -Me caused EPS destruction and a significant inactivation of cells. PDI may, therefore, be regarded as a promising approach for biofilm control, even in cases of bacteria-yeast mixed assemblages. http://dx.doi.org/10.1016/j.nbt.2014.05.903 ANTIMICROBIALS, PATHOGENS AND DISEASE New Biotechnology · Volume 31S · July 2014 PS-04 Antibacterial activity of marine Bacillus spp. isolated from mangroves of Saudi Arabian Eastern Province Abdurahman Hirad 1,∗ , Ali Bahkali 2 , Chandra Santhappa 2 1 King Saud University, Saudi Arabia College of Botany and Microbiology, King Saud University, Saudi Arabia 2 Bacteria that synthesize bioactive substances against known human pathogens were screened from water, sediment, and decomposing leaf litter samples collected from Tarut Island of east Coast of Saudi Arabia. Bacillus aquimaris KSAWD2 was selected as a potential strain that showed considerable bioactivity from amongst 320 isolates. This isolate was identified based on morphological, biochemical and gene sequence of 16sRNA. Culture filtrate of this bacterium showed satisfactory inhibition activity against the ATCC bacterial reference strains and clinical pathogens obtained from Military Hospital laboratory in Riyadh. The targeted pathogens included Salmonella enterica subsp. enterica serovar Typhimurium (ATCC 13311), Shigella sonnei (ATCC 11060), E. coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 15442), Staphylococcus aureus subsp. aureus (ATCC 6538P, 25923, and 33591), Streptococcus pneumoniae, Haemophilus influenzae, Campylobacter jejuni and Streptococcus pyogens. Inhibitory activity was tested employing cross streaking, agar well diffusion method, agar overlay method and assay of growth inhibition in liquid medium. This isolate showed a broad spectrum of bioactivity by recording inhibition of growth against more than one pathogen. It was observed that the culture filtrate of Bacillus aquimaris KSAWD2 showed considerable inhibition against the tested pathogens better than commercial antibiotic discs including chloramphenicol, gentamicin, tetracycline, penicillin, neomycin and ampicillin. Results indicate a scope for deriving potential bioactive compounds from this marine Bacillus aquimaris KSAWD2 against well-known human pathogens. http://dx.doi.org/10.1016/j.nbt.2014.05.904 PS-05 Development of recombinant baculovirus for high yield production of enterovirus 71 virus-like particle vaccine Yu-Chen Hu ∗ , Shi-Yeh Lin National Tsing Hua University, Taiwan Enterovirus 71 (EV71) is the major pathogen responsible for hand, foot and mouth disease prevalent in East Asia and can cause serious neurological complications and even death. Thus EV71 has posed a tremendous threat to children health. To develop the vaccine, we previously constructed recombinant baculovirus BacP1-3CD for the production of EV71 virus-like particle (VLP) that consists of EV71 capsid proteins. The VLP elicited potent humoral and cellular immune responses in mouse and monkey models. However, the previous generations of baculoviruses resulted in relatively low VLP yield and excessive degradation products upon production. In this study we constructed a new generation of baculovirus using the flashBACÔ system to improve the quantity and quality of EV71 VLPs. The VLP produced by this baculovirus reached a yield of about 200 mg/L (10-fold improvement) with lower amounts of degradation products, putatively thanks to the delayed cell death and reduced cellular protease activity. The purified VLP, which mainly consisted of VP0, VP1 and VP3 proteins, resembled the intact EV71 capsid in shape, size and composition. After injection into Balb/c mice, the VLP elicited promising titers of binding antibody (≫212 ) and neutralization antibody (≫27 ) against EV71 in immune serum with broad spectrum to crossneutralize the infection of different EV71 subtypes (C2, C4 and B5 subtypes). In conclusion, the improvement of EV71 VLP production using the 3rd generation recombinant baculovirus moves the EV71 vaccine development one step further towards commercialization and clinical applications. http://dx.doi.org/10.1016/j.nbt.2014.05.905 PS-06 Quantitative analysis of protein orientation in membrane environments by kinase activity Chunshan Quan ∗ , Wen Shengdi Fan Xiong, Wenzhong Hu, Aili Jiang, Dalian Nationalities University, China AgrC is a membrane-embedded histidine kinase in Staphylococcus aureus that is thought to act as a sensor for the recognition of environmental signals and the transduction of signals into the cytoplasm so as to regulate and control a series of related pathogenic gene expressions. However, due to the complexity of the cell membrane, it turns to be difficult to study AgrC on bacterial cell membrane directly. Many researches try to take advantage of proteoliposome which could provide an approximate natural membrane environment and keep the protein activity to study the structure and function of membrane proteins. Given that most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. Many studies have been carried out to determine membrane protein orientation, however, most of the methods are complicated and time consuming. In this study, AgrC orientation in liposomes was determined based on thiolreactive reagent labeling and kinase activity which showed that the method based on kinase activity of AgrC could get an accurate percentage of protein orientation and only costs nearly one-sixth of the time compared with the method based on thiol-reactive reagent labeling. The results showed that an efficient and rapid method was established to determine the orientation of membrane protein kinase, like AgrC. http://dx.doi.org/10.1016/j.nbt.2014.05.906 www.elsevier.com/locate/nbt S179 ANTIMICROBIALS, PATHOGENS AND DISEASE PS-07 The impact of MDCK cell source on the production of influenza and canine adenovirus Paulo Fernandes 1 , Rute Castro 2 , Tanja Laske 3 , Yvonne Genzel 3 , Eric Kremer 4 , Paula Alves 2,∗ , Ana Coroadinha 2 1 iBET, Portugal iBET/ITQB-UNL, Portugal 3 Max Planck Institute for Dynamics of Complex Technical Systems, Germany 4 Institut de Genetique Moleculaire de Montpellier, France 2 MDCK cells are widely used for the production of viral-based vaccines, namely influenza virus. We have shown that MDCK are also suitable for canine adenovirus type 2 vectors (CAV-2) manufacturing. Apart of their suitability for virus production, there are several sources and subclones from the original cell line obtained by Madin and Darby. Furthermore, given the well-documented heterogeneity of MDCK cell-line population, understanding the differences between MDCK cells currently available would help to better select producer cells and develop more reproducible bioprocesses. In this work, MDCK cell-lines from ECACC and ATCC suppliers were adapted to suspension and compared for the production of CAV-2 and Influenza. Despite similar cell growth, ATCC cells were more easily adapted to suspension. However, ECACC cells showed to be better CAV-2 producers, attaining productivities 2-9 fold higher than ATCC cells. ECACC cells also showed to be more infected, indicating that cells hold different susceptibilities to infection. Furthermore, the evaluation of infection progression and cell volume increase due to virus production indicates that virus replication kinetics and cell response to infection was compromised in ATCC cells. A more effective antiviral response negatively impacting CAV-2 propagation in ATCC cells is currently under evaluation. Similarly to CAV-2, Influenza productivities were 2-3 fold higher with ECACC cells in adherent cultures. To our knowledge, this is the first attempt to characterize MDCK cell-lines from different suppliers with respect to their performance. Our findings show that the cell source/supplier represents a key issue to be considered when implementing a robust bioprocess. http://dx.doi.org/10.1016/j.nbt.2014.05.907 PS-08 Resensitization of antibiotic-resistant bacteria using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)—–Cas9 system Da-Hyeong Cho ∗ , Jonghyeok Shin, Myungseo Park, Younghun Jung, Byoung-jae Kong, Junghoon In, Dae-Hyuk Kweon Sungkyunkwan University, South Korea Antimicrobial drug development is increasingly lagging behind the evolution of antibiotic resistance. As a result, there is pressing need for new antibacterial therapies that can be readily designed and implemented. In this study, a new antibacterial therapy S180 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 using CRISPR-Cas9 system was developed. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated (Cas) system are adaptive immune system that silences invading nucleic acid by using RNA-guide endonuclease activity in bacteria and archaea. Targeting of single guide RNA (sgRNA) with Cas9 directs sequence-specific double strands DNA cleavage. Using Larabinose induction of Cas9 and a constitutive sgRNAbla cassette, it is shown that cleavage of targeted double-strands DNA resulted in degradation of plasmids containing bla gene. Ampicillin-resistant cells could be re-sensitized by expressing Cas9/sgRNAbla , thereby reducing the number of resistant cells. The strategy demonstrated in this study enables the continuous use of old antibiotics, which are already developed and the safety issue was cleared, by reverting the resistant cells to sensitive cells. http://dx.doi.org/10.1016/j.nbt.2014.05.908 PS-09 Biosurfactant produced by marine bacteria interacts with diffusible pyoverdine produced by Pseudomonas aeruginosa M. Alejandro Dinamarca 1,∗ , Natalia Romo 2 , Claudia IbacacheQuiroga 3 1 University of Valparaíso, United States Universidad de Valparaíso, Chile 3 Centro Nacional de Biotecnología CNB-CSIC/Micromarine Biotech, Spain 2 Biosurfactants are surface-active molecules with a wide diversity of biological functions. Recently, we reported that specific surfaceactive compounds produced by marine bacteria are able to capture diffusible chemical signals from bacteria, affecting the cell-to-cell communication system based on quorum sensing. In this context, the goal of the present work was to study the interaction between marine surface-active molecules and pyoverdine, a diffusible siderophore produced by Pseudomonas aeruginosa for the acquisition of iron from the environment. In human hosts, pyoverdine production by this opportunistic pathogen promotes its virulence and the cooperation with other microorganisms, affecting health recovery in patients. In order to determine the interaction between the biosurfactant and pyoverdine, P. aeruginosa was exposed to different concentrations of the selected marine biosurfactant. At these conditions, pyoverdine presence was measured by fluorescence spectrophotometry (460 nm). Also, the transcriptional levels of PvdS (transcriptional regulator of the pyoverdine synthesis), and PvdQ (periplasmic acylase) were measured by qRTPCR. Results show that when P. aeruginosa was exposed to the marine surface-active compound at the critical micellar concentration, the fluorescence generated by pyoverdine was reduced in 81,97%, respect to the condition without biosurfactant. On the other hand, gene expression of pvdS and pvdQ were increased 1.85fold and 1.0-fold, respectively. Since the assays were performed in non-limiting iron conditions, we propose that the marine surfaceactive compound captures the extracellular pyoverdine, inducing or simulating an iron limiting condition in P. aeruginosa. http://dx.doi.org/10.1016/j.nbt.2014.05.909 New Biotechnology · Volume 31S · July 2014 PS-10 Analysis of 16S rDNA sequences of endophytic bacteria associated with Huanglong disease of citrus plants in Guangning County, China Chongbi Li ∗ , Feiyun Gao, Min Zhang, Yugan Hao Center of Biopharmaceutical Engineering in Zhaoqing University Citrus yellow shoot disease is an incurable disease of citrus plants that has long been recognised. It is a serious threat to the development of the local citrus industry. The reports a study of the Chinese citrus industry in Guangning County of Zhaoqing. Endophytes were isolated from the roots and stems. Endogenous DNA was extracted and bacterial 16S rDNA was amplified by PCR. Sequences were analysed and compared with those isolated from bacteria on both healthy and diseased plants. The results revealed differences between the endogenous dominant bacteria in phloem tissue of healthy plants and plants with Huanglongbing disease. Sphingobacterium multivorum, Acinetobacter junii, and Pseudomonas species similar to those that infect the fish, perch, were more abundant in healthy plants. In contrast, Exiguobacterium acetylicum, the plant pathogen Pantoea agglomerans and Acinetobacter baumannii were more abundant in infected plants. The discovery of bacteria associated with found only in citrus plants with Huanglongbing disease of citrus plants should provide a theoretical and experimental basis for the prevention and control of citrus yellow shoot disease. ANTIMICROBIALS, PATHOGENS AND DISEASE The dynamic study of TEM-1 ␤-lactamase by our group has shown that binding of the ␤-lactamase inhibitory protein causes changes in the flexibility of regions away from the ␤-lactam binding site. In addition to the previously identified H10 helix, which forms a lid over an allosteric inhibitor binding site, sequence conservation analysis has shown that Trp229 residue of H10 is highly conserved and it has a stacking interaction with Pro226 and Pro251 residues. In order to investigate the importance of Trp229, this residue has been mutated to alanine. To this end, R-TEM-1 ␤-lactamase gene of pUC18 was cloned into pET28a(+) with an C-terminal 6xHis tag. The mutation was performed using QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, USA). Enzyme expression was achieved in Escherichia coli BL21 (DE3) cells at 30 ◦ C with 0.1 mM IPTG. Using nickel affinity column chromatography ␤-lactamase was purified and its activity was measured with CENTA as the substrate. These measurements have shown that W229A mutation causes significant loss of activity, hence it could be an important residue that enables communication with the active site. Acknowledgements: This work was supported by TUBITAK project 113M533. http://dx.doi.org/10.1016/j.nbt.2014.05.911 http://dx.doi.org/10.1016/j.nbt.2014.05.910 PS-11 The importance of a conserved residue of the H10 helix in ␤-lactamase Fatma Gizem Avcı 1,∗ , Elif Özkırımlı Ölmez 2 , Berna Sarıyar Akbulut 3 1 Marmara University, Bioengineering Department, Turkey Bogazici University, Turkey 3 Marmara University, Turkey 2 The most common ␤-lactam resistance mechanism utilized by bacteria is the production of ␤-lactamase enzymes that cleave the amide bond in the ␤-lactam ring rendering the antibiotic inactive. ␤-Lactam antibiotics in combination with ␤-lactamase inhibitors are used to overcome bacterial resistance. Thus design of new inhibitors is a promising area. www.elsevier.com/locate/nbt S181 PLANT GENETIC ENGINEERING Plant genetic engineering PT-01 Identification and characterisation of key genes involved in fruit ripening of the Chilean strawberry Michael Handford ∗ , Analia Espinoza, Milagros Bracamonte, Aliosha Figueroa, Sara Zapata, Uri Aceituno, Lorena Norambuena Universidad de Chile, Chile The Chilean strawberry (Fragaria chiloensis L. (Duch.)) is a promising fruit product for Chile. The appeal of these whitishpink fruits is associated with their higher sweetness and stronger aroma compared to the red fruits of the commercial strawberry (F. x ananassa). Strawberries are non-climacteric fruits and little is known about the factors governing their ripening. Nevertheless, the ripening of these fruits is coupled with multiple changes in texture, aroma, sweetness and colour, which are affected by hormonal and environmental cues. Therefore, this work aims to generate a greater knowledge of these processes in the Chilean strawberry, for increasing our understanding of fruit ripening, for use in breeding programs and for maximising the commercial potential of this product. By contrasting four different developmental and ripening stages of the Chilean strawberry, including small green, large green, large white and ripe whitish-pink fruit (soft), six suppression subtractive hybridization (SSH) libraries were generated. Of the 1809 differentially expressed cDNAs (ESTs), we identified potential events which could be key in fruit development and ripening, including genes which code for proteins putatively involved in the resistance to oxidative and biotic stress, the perception and signalling of auxins and abscisic acid, vasculature development and in anthocyanin synthesis. The expression patterns obtained by quantitative real time PCR generally confirm the data obtained from the SSH libraries and phenotypic observations; for example transcripts of a potential anthocyanidin synthase gene peaked in whitish-pink fruits, consistent with pigment accumulation. Further findings will be discussed. Financial support: Anillo ACT-1110. http://dx.doi.org/10.1016/j.nbt.2014.05.912 PT-02 Improvement of Agrobacterium-mediated transformation in Hi-II maize (Zea mays L.) by heat-shock treatment with immature embryos Ya Liu ∗ , Jiuran Zhao Beijing Academy of Agricultural and Forestry Science, China Improving Agrobacterium-mediated transformation of Hi-II maize (Zea mays L.) would be beneficial for transgenic breeding. Although Hi-II maize is used in various maize transformation research efforts, improvements in the transformation frequency of this genotype are still needed. In the present study, the immature maize embryos were given heat shock treatment prior to Agrobacterium tumefaciens mediated transformation. The S182 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 immature embryos of maize were treated at different temperatures for different time durations. Our results showed that the peak GUS expression occurred when the embryos were subjected to a temperature of 42 ◦ C for 3 min or at 38 ◦ C for 9 min. A 9 percent (%) in the transformation frequency was achieved in a number of experiments. Heat shock pretreatment at 38 ◦ C for 9 min also resulted in a 7% increase in the “embryo positive callus”. Besides, it was found that 100 mg/L Casein Hydrolysate (CH) could accelerate the redifferentiation of somatic embryos; and 2 mg/L Multi-effect triazole (MET) promoted root elongation, lateral root formation and robust seedlings to improve the survival rate of the plantlets. Therefore, our protocol can help advance the overall breeding of transgenic maize. http://dx.doi.org/10.1016/j.nbt.2014.05.913 PT-03 A simple, novel and high efficiency transformation method to introduce foreign DNA into corn plants mediated by cell-penetrating peptides Zhongyi Wu ∗ , Jianhua Wei Beijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, China Genetic engineering breeding and identification of novel genes in maize are very dependent on high efficiency of corn transformation. The major deficiencies in current plant transformation systems include but are not limited to the production efficiency of the system, transformation variability due to genotype or species diversity and explant limitations and a long labor-intensive process requiring much skill. In particular, there is a continuing need in the field of plant biotechnology to provide more efficient, simple and low cost transformation methods suitable for high capacity production of economically important plants, particularly elite cultivars. Cell-penetrating peptides (CPPs) were discovered to protect DNA from degradation and deliver a variety of molecules including DNA into cell. Here, we studied the function of CPPs (Tat2) in improving maize transgenic efficiency through the pollen tube method. The transformation experiments in Jing 24 and 178 corn inbred lines were conducted under the different conditions of linear DNA concentration, ratio of Tat2, calcium, sucrose, ATP, and GTP concentration. Screened with herbicide for T0 seedlings, the optimal transformation system was obtained by the mediation of Tat2 through the pollen tube transformation method, its transgenic efficiency was up to 1–1.5%. Screened with herbicide for T1 seedlings, genetic inheritance and segregation of T1 progeny was revealed under this genetic manipulation, supporting a genetic ratio 3:1 of T1 progeny was about 16–18% in all transgenic lines. This is a novel transgenic method for corn with high efficiency, safe, low cost and no limitation of corn genotype. http://dx.doi.org/10.1016/j.nbt.2014.05.914 PLANT GENETIC ENGINEERING New Biotechnology · Volume 31S · July 2014 PT-04 Impact of non-edible transgenic plant on soil microbial communities Kijong Lee ∗ , Sung-Dug Oh, Tae-Hun Ryu, Soo-In Sohn, Jong-Bum Kim NAAS, South Korea Studies on the adverse effects of non-edible transgenic plants on soil microbial colonies are scarce. Here, we evaluated the effect of virus-resistant trigonal cactus on nearby soil microbial communities. We collected soil samples at the site of genetically modified (GM) and non-GM trigonal cactus cultivation. We collected soil samples during the vegetative growth period and the post-harvest period to provide the data for comparative evaluation. Ecoplate was used to evaluate the functional diversity of soil microbial communities. We found no significant difference between the GM and non-GM soil samples collected during the vegetative growth period. However, the post-harvest soil sample exhibited a marked difference in carbon substrate utilization between the study group and the control group, but the observed change was not permanent. Principal component analysis showed that similar soil sample groups were not formed based on GM or non-GM trigonal cactus cultivation, but based on the cultivation period. Denaturing gradient gel electrophoresis fingerprinting revealed that virus-resistant trigonal cactus cultivation had negligible effect on soil microbial communities including dominant rhizosphere bacteria, actinomycetes, and fungi. We found no clear evidence of GM trigonal cactus cultivation affecting the functional diversity of soil microbial communities. http://dx.doi.org/10.1016/j.nbt.2014.05.915 PT-05 Development of genetic resistance to plum pox virus in Prunus Aiming Wang Agriculture and Agri-Food Canada, Canada Plum pox virus (PPV) is the causal agent of the devastating viral disease known as Sharka in Europe, on many stone fruit spp. To engineer genetic resistance against PPV through the hairpin-mediated RNA silencing (RNAi) approach, previously two plant transformation vectors, pAWp1 and pAWcp were constructed by cloning two highly conserved regions of the PPV genome corresponding to portions of viral RNA coding for P1 and CP, respectively, into a Ti binary vector under the control of the double Cauliflower mosaic virus 35S promoter as inverted repeats spanned by an intron from the peach endo-polygalacturonase (endo-PG) genomic DNA. The resulting transgenic plants expressing hairpin RNAs either targeting the viral P1 or CP sequences showed resistance to PPV. Here we report construction of a new construct pAWp1-cp by cloning the P1 and CP hairpin sequences together into the Ti vector and the inserted DNA contains a triple-intron double-hairpin sequence simultaneously targeting the P1 and CP sequence of PPV. This vector was transformed into Nicotiana benthamiana and plum (Prunus domestica L.). Resistance assays showed the transgenic plants were efficiently resistant to PPV. http://dx.doi.org/10.1016/j.nbt.2014.05.916 PT-06 Inducible expression of Agrobacterium virulence gene VirE2 for stringent regulation of T-DNA transfer in biosafe plant transient expression systems Erna Denkovskiene 1,∗ , Sarunas Paskevicius 2 , Stefan Werner 3 , Anatoli Giritch 3 , Ausra Razanskiene 2 1 Vilnius University Institute of Biotechnology, UAB Nomads, Lithuania 2 UAB Nomads, Lithuania 3 Nomad Bioscience GmbH, Germany Agrobacterial delivery of viral expression vectors is a comfortable solution for the transient production of biomolecules in plants. Valuable proteins are successfully produced in plants in contained facilities, avoiding release of transfecting agrobacteria to the environment. However, field applications would be needed to gain the full economical advantage in the production of lower value and high-volume products, or transient molecular reprogramming of plants to resist stresses or modulate time to flower. The release of GM agrobacteria into open field environment requires appropriate addressing of biosafety issues. The safety of GM Agrobacterium could be increased by controlling its T-DNA transfer. We constructed chemically regulated T-DNA transfer based on inducible expression of the essential Agrobacterium virulence gene VirE2. Looking for the strongest and stringently regulated promoters in Agrobacterium, we evaluated IPTG inducible lac, tac, T7/lac, T5/lac promoters in ␤-galactosidase assays and also evaluated hybrid cumic acid inducible promoters lacUV5/CuO, tac/CuO, T5/CuO and VirE/CuO. For inducible T-DNA transfer tests, Nicotiana benthamiana plants were transfected with a VirE2deficient A. tumefaciens strain containing transient expression vectors harboring inducible VirE2 expression cassettes in vector backbone and a marker GFP gene in their T-DNA region. The efficiency of T-DNA transfer was evaluated by counting GFP expression foci on plant leaves. VirE2 expression regulated by tac/CuO, T5/CuO and VirE/CuO promoters resulted in 50-73% of T-DNA transfer efficiency in comparison with the wild type Agrobacterium. We present efficient and tightly regulated promoters for gene expression in A. tumefaciens and a very new approach to address plant transfection biosafety concerns in agrobiotechnology. http://dx.doi.org/10.1016/j.nbt.2014.05.917 www.elsevier.com/locate/nbt S183 PLANT GENETIC ENGINEERING New Biotechnology · Volume 31S · July 2014 PT-07 Quantitative determination of phytase activity and inorganic phosphorus of transgenic barley and dihaploid lines Tomas Vlcko 1,∗ , Marie Ohnoutkova 3 Hanakova 2 , Jana Vaskova 3 , Ludmila 1 Centre of the Region Hana for Biotechnological and Agricultural Research, Czech Republic 2 Department of Plant Biology, Faculty of Agronomy, Mendel University in Brno, Czech Republic 3 Institute of Experimental Botany AS CR, v. v. i. and Centre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacky University Olomouc, Czech Republic Barley is one of the most prominent crops in the world with thousands of square kilometers of sown fields. Harvested barley grains are commonly utilized as livestock feeding and in the brewing industry. Phytic acid (about 70%) is the main storage form of phosphorus in plant seeds. Owing to the acidic nature of the phytic acid molecule it binds with cations forming an insoluble complex. This is considered the main antinutritional factor for the availability of minerals (calcium, zinc and iron principally), because the salts of phytic acid are unfortunately indigestible for monogastric species such as swine, fish and fowl. Phosphorus in feces ultimately leads to environmental pollution. Phytase is an enzyme which catalyzes the degradation of phytate complexes thus providing necessary phosphate and cations during germination. This enzyme converts the indigestible form of phosphorus into a fully exploitable form. Transgenic barley line SCLW-GP-PHYA developed using biolistic transformation of immature embryos exhibits stable expression of the microbial [Aspergillus niger] phytase enzyme. This line was used for a hydrid programme, crossing with Czech barley cultivar Azit. The activity of phytase and content of digestible phosphorus in obtained lines were analyzed. The original transgenic line was also assessed in brewing trials. Acknowledgement: This work was supported by project OP VaVPI CZ.1.05/3.1.00/14.0327 – New biotechnological products of IEB ASCR. http://dx.doi.org/10.1016/j.nbt.2014.05.918 PT-08 Genetic modification to enhance breeding for improved postharvest quality of ornamental plants – barriers for commercial application Margrethe Serek Leibniz University Hannover, Institute for Horticulture Production Systems, Germany Development of novel varieties with improved postharvest characteristics is an important goal for the floriculture industry. Improved varieties can secure higher productivity for growers, increased marketing opportunities, reduced postharvest losses during storage and distribution and finally provide better products S184 www.elsevier.com/locate/nbt for consumers. While conventional breeding programs have been successful in achieving such objectives, directed genetic modification can provide additional ways to create new, improved ornamental products. In the last two decades the postharvest performance of a range of varieties has been successfully improved by genetic modification. In our laboratory we used the etr1-1 mutant gene for improvement of display life of several ornamentals, such as kalanchoe, campanula and orchid. Although significant progress has been made in developing improved new varieties using tools of plant biotechnology, most of such improved crops never reach consumers. While scientists are developing the products, they are not being released to the market because of legal obstacles, including lack of interest or financial incentive of patent owners in negotiating a license. The result is that such improved products never reach the market. Possibilities for ensuring that remarkable varieties do not become neglected and forgotten somewhere in an experimental greenhouse or ultralow freezer must be discussed. http://dx.doi.org/10.1016/j.nbt.2014.05.919 PT-09 Subcellular localization of maize cytokinin dehydrogenases using heterologous expression in Arabidopsis thaliana Ler cell suspension cultures Patricie Johnová 1,∗ , David Šamajová 2 , Petr Galuszka 1 Zalabák 1 , Ondřej Plíhal 1 , Olga 1 Department of Molecular Biology, Palacký University in Olomouc, CR Haná, Czech Republic 2 Department of Cell Biology, Palacký University in Olomouc, CR Haná, Czech Republic Cytokinins are plant hormones playing a key role during many plant developmental processes, such as shoot initiation, bud formation, apical dominance, delay of leaf senescence, etc. As the cytokinins act at the nanomolar concentrations, its homeostasis must be precisely controlled on the tissue as well as cellular and subcellular levels. One of the ways to control the cytokinin level is its irreversible degradation. This process is mediated via a class of flavoproteins, cytokinin dehydrogenases (CKX; EC 1.5.99.12). Plant genomes usually encode for small CKX gene families. So far, best characterized is the AtCKX gene family from Arabidopsis thaliana, comprising seven members. Individual CKX isoforms exhibit various biochemical features e.g. substrate specificity, distinct spatial and temporal distribution. Interestingly, respective AtCKX’s differ in their subcellular localization too. While four of AtCKX proteins are secreted to the apoplast, two are vacuolar and the last one is cytosolic (Werner et al., 2003; Kowalska et al., 2010). In contrast to Arabidopsis as the representative dicot, the information on the CKX subcellular localization in monocots is still not clear. So far, only two out of 13 maize (Zea mays) ZmCKX isoforms were studied in detail. It was shown that ZmCKX1 is apoplastic, while ZmCKX10 is cytosolic (Šmehilová et al., 2009). New Biotechnology · Volume 31S · July 2014 The goal of the presented research is to find out the localization of remainder ZmCKX isoforms, to complete the information on cytokinin catabolism in maize model. For this purpose, the ZmCKX-GFP fusions were prepared, heterologously expressed in Arabidopsis thaliana Ler cell suspension cultures and studied using live cell confocal microscopy. http://dx.doi.org/10.1016/j.nbt.2014.05.920 PT-10 Regulation of trichome density in Artemisia annua Xiaofen Sun ∗ , Fangyuan Zhang Shanghai Jiao Tong University, China Artemisia annua is the source of the most potent antimalarial drug, artemisinin, which is synthesized in multicellular glandular PLANT GENETIC ENGINEERING secretion trichomes of Artemisia leaf surface. To increase the trichome number is one of the efficient ways to increase artemisinin yield. To investigate the development of multicellular glandular trichomes in A. annua, AaMYB1, a R3 MYB family gene was cloned and analyzed. Overexpression of AaMYB1 in Arabidopsis led to the disappearance of trichome and seed mucilage, enhanced root hair number and reduced anthocyanin contents, showing that AaMYB1 had a similar function with AtCPC. Overexpression of AaMYB1 in A. annua reduced the artemisinin content as well as the trichome number. On the contrary, the suppression of AaMYB1 in A. annua increased the trichome number as well as the artemisinin content. This study indicates that AaMYB1 is a negative regulator of multicellular glandular trichome development in A. annua and could be useful in engineering of A. annua for increasing artemisinin content. http://dx.doi.org/10.1016/j.nbt.2014.05.921 www.elsevier.com/locate/nbt S185 RECOMBINANT PROTEIN PRODUCTION Recombinant protein production PU-01 Protein extraction by means of electroporation and bacterial viability of E. coli Saša Haberl Meglič ∗ , Tilen Marolt, Damijan Miklavčič University of Ljubljana, Faculty of Electrical Engineering, Slovenia Proteins extracted from recombinant bacteria have proved of great value in industry and medicine. Established processes to extract proteins from bacteria often include use of mechanical forces or chemicals, which cause complete disruption of the cell and release of membrane contaminants (endotoxins). Thus, additional purification steps are needed, which at large scales represents up to 80% of the production costs [1]. Extraction by means of electroporation is a quick, chemical free and cost efficient release of intracellular components from E. coli [2]. In order to avoid cell disruption and by that the release of membrane contaminants, and to obtain adequate quantity of proteins, electroporation pulse parameters need to be adjusted. Thus, we studied the influence of pulse strength, duration, number and repetition frequency on extracted proteins from E. coli and on bacterial viability. Our results show that by increasing electric field strength, pulse repetition frequency and/or pulse duration, also concentration of extracted proteins increases (maximum 15.29 ␮g/ml), while E. coli viability decreases (minimum 2.2 – log reduction). The correlation was expected, since more bacteria were destroyed and more proteins were released into surrounding media. The best choice under experimental conditions used and for our strain was a train of eight pulses with 1 ms duration, 1 Hz pulse repetition frequency and electric field strength of 5 kV/cm. Under these conditions the best ratio between extracted proteins and bacterial viability was observed. References [1].Assenberg R, et al. Curr Opin Struct Biol 2013;23:393. [2].Haberl S, et al. J Membr Biol 2013;246:861. http://dx.doi.org/10.1016/j.nbt.2014.05.922 PU-02 Production of an antibody fragment by transient gene expression in insect cells Hideki Yamaji ∗ , Tomohisa Katsuda, Hirotsugu Hamada, Keita Mori Department of Chemical Science and Engineering, Kobe University, Japan Monoclonal antibodies and their fragments have been used in a variety of diagnostic and therapeutic applications. Novel antibody-based biologics are currently selected from a large pool of lead candidates. High-throughput production systems for rapidly providing a large number of recombinant antibody molecules of high quality and in sufficient quantity are of major imporS186 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 tance. Insect cells have been shown to be an excellent platform for the production of functional recombinant antibodies [1]. In the present study, the production of an antibody Fab fragment by transient gene expression in insect cells was investigated. The cDNA fragments encoding the Hc (Fd fragment) and Lc genes of a mouse anti-bovine ribonuclease A Fab fragment with the Drosophila BiP signal sequence were cloned separately into the plasmid vector pIHAneo [2], which contained the Bombyx mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator, the BmNPV HR3 enhancer, and the B. mori actin promoter for high-level expression. After co-transfection with the resulting plasmids carrying the Hc and Lc genes, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were incubated with a serum-free medium in static or shake-flask cultures. Transfection conditions were optimized with flow cytometric analysis using the green fluorescence protein. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) of culture supernatants revealed that transfected cells secreted an Fab fragment with antigen-binding activity. Production of over 30 mg/L of Fab fragment was achieved in 6 days. References [1].Yamaji H, et al. Cell Eng 2011;7:53–76. [2].Yamaji H, et al. Biochem Eng J 2008;41:203. http://dx.doi.org/10.1016/j.nbt.2014.05.923 PU-03 RMCE reference sites – A valuable tool for comparing antibody expression capabilities in CHO cells Patrick Mayrhofer ∗ , Alexander Mader, Renate Kunert University of Natural Resources and Life Sciences, Austria Chinese hamster ovary (CHO) cells represent the major host for recombinant monoclonal antibody production for biotherapeutic applications. Traditional methods for generation of stable antibody producing cell clones are based on random integration of the gene of interest into unpredictable chromosomal loci. Therefore, much effort is undertaken for tedious and time consuming screening and selection procedures. Additionally, integration events into chromosomal loci with variable transcription rates and co-introduction of residual vector sequences triggers epigenetic mechanisms in various extent making prediction of expected expression levels and comparisons between cell lines a tedious task. The development of site-directed gene integration methods enables the integration of different transgenes into pre-determined chromosomal positions. Here we demonstrate that the application of recombinase mediated cassette exchange (RMCE) provides a valuable tool for introducing a re-targetable gene cassette into a stable and transcriptionally active chromosomal locus. A RMCE competent cassette was introduced into DUKX-B11 cells by two rounds of RMCE reactions leading to stable and homogenous gfp reporter gene expression. This RMCE cassette was replaced by two different anti-HIV antibody variants leading to cell RECOMBINANT PROTEIN PRODUCTION New Biotechnology · Volume 31S · July 2014 clones with reproducible expression behavior in T25 roux flasks and spinner vessels during batch cultivation. Intracellular product formation analyzed by flow cytometry and qPCR indicated a uniform antibody expression behavior of the established cell lines demonstrating the success of targeted integration by RMCE. The established RMCE host cell line enables the comparison of different cell clones and product specific expression patterns in future projects based on an identical chromosomal environment. http://dx.doi.org/10.1016/j.nbt.2014.05.924 PU-04 Development of a method to quantify genomic rearrangements in Chinese Hamster Ovary cells Inmaculada Hernandez Lopez 1,∗ , Vaibhav Jadhav 2 , Martina Baumann 1 , Norbert Auer 2 , Angelika Zotter 1 , Nicole Borth 3 1 Austrian Center of Industrial Biotechnology, Austria University of Natural Resources and Life Sciences, Austria 3 University of Natural Resources and Life Sciences/Austrian Center of Industrial Biotechnology, Austria 2 Chinese Hamster Ovary (CHO) cells are the preferred host cells for the production of therapeutic proteins and the most commonly used mammalian expression system due to properties such as an easy cultivation, fast growth, complex protein folding and humanlike post-translational modifications. Nevertheless, development of recombinant CHO cell lines is a slow and work intensive process that requires testing of thousands of clones as these tend to be heterogeneous in behaviour. While this is the source of easy adaption and the occurrence of extraordinarily high producers, it also means that frequently the most promising clones turn out to be unstable with respect to productivity and phenotype. Despite the high frequency of genomic rearrangements due to genomic instability, no methods are currently available to rapidly assess large scale chromosomal rearrangements, with the exception of FISH (fluorescence in situ hybridization) and karyotyping. The focus of the present work is to adapt a method for this purpose, Amplified Fragment Length Polymorphism (AFLP), which allows simple detection and quantification of genomic changes over time using standard laboratory equipment. The approach, which is already established in cancer research and plant breeding, was modified for use in CHO cells. It defines an initial pattern of electrophoretic bands that enables detection and quantification of changes over time and the determination of the degree of genomic differences between CHO strains and subclones and therefore can also be used for cell line identification and characterization. http://dx.doi.org/10.1016/j.nbt.2014.05.925 PU-05 Establishment of stable, high producing, recombinant CHO cell lines using Rosa26 Bacterial Artificial Chromosomes for transgene delivery Wolfgang Sommeregger 1,∗ , Andreas Gili 2 , Katalin Zboray 3 , Thoams Sterovsky 2 , Emilio Casanova 3 , Renate Kunert 1 1 VIBT/DBT/University of Natural Resources and Life Sciences, Vienna, Austria 2 Polymun Scientific Immunbiologische Forschung GmbH, Klosterneuburg, Austria 3 Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria Chinese Hamster Ovary (CHO) cells are the most frequently used mammalian host for the production of biopharmaceuticals. The achieved volumetric titers using CHO cell factories have increased significantly over the past two decades. However, the establishment of well-producing cell lines remains tedious. Many parameters like the host cell line, the genetic construct, the cell culture medium, the applied cultivation strategy and the product by itself are influencing volumetric titers. Bacterial Artificial Chromosomes (BACs) harbouring the Rosa26 locus show improvements concerning transcriptional efficiency when used as shuttle vectors for the delivery of transgenes. The high transcription rate of the Rosa26 BACs enables to use the expression system without gene amplification in auxotrophic and non-auxotrophic CHO hosts. In this work we combined the transcriptional efficiency of the Rosa26 BAC with CHO host cell lines growing to high cell densities in order to increase the volumetric product titers. The commonly used CHO hosts CHO-K1, -DUKX-B11, -DG44, and CHO-S were screened for their growth behaviour in batch experiments using optimized serum-free cultivation strategies. CHO-K1 and CHOS performed best and were used for the establishment of stable clones. The model proteins in this study were two anti–HIV-1 IgG-1 antibodies and the HIV-1 gp140 (CN54) envelope protein. For all products we could show that the combination of cells growing to high cell densities and the transcriptional efficiency of the Rosa26 BAC system leads to the accumulation of high product concentrations. Even for the highly glycosylated HIV-1 envelope protein we achieved titers above 1 g/L. http://dx.doi.org/10.1016/j.nbt.2014.05.926 PU-06 Modification of signal peptide for enhanced production of recombinant erythropoietin from animal cells Duk Jae Oh 1,∗ , Ji Hye Park 2 1 Sejong University, Department of Bioscience and Biotechnology, South Korea 2 Sejong University, South Korea Background and novelty: Various secretory proteins are synthesized as precursors with additional N-terminal signal peptide. The signal peptide is cleaved off by signal peptidase once it has www.elsevier.com/locate/nbt S187 RECOMBINANT PROTEIN PRODUCTION served its purpose of targeting the protein to, and importing it into, the ER. Recently, recombinant DNA research was used to study signal peptide and made it possible to show the efficient activity of a proposed signal peptide by fusing it to another protein. Experimental approach: In this study, signal peptide of human erythropoietin was replaced with the signal peptide of human IL-2, then the hydrophobic region of the signal peptide was modified and its effect on the production of the target protein (erythropoietin) was evaluated. The nucleotide sequence of modified signal peptide was transported directly into the upstream of the 5’end of the human erythropoietin gene by performing one round of amplification with pfu DNA polymerase. The gene of target protein with modified signal peptide was transiently expressed in HEK293/CHO cells and quantification of protein secretion was evaluated. Results and discussion: As a result, we could observe significantly increased protein secretion up to 4 fold higher by modifying the native signal peptide, in particular the hydrophobic region. This observation is believed to be applicable to improve the productivity of recombinant therapeutic proteins from animal cells. http://dx.doi.org/10.1016/j.nbt.2014.05.927 PU-07 Effect of gene copy number on production yield of recombinant ergopeptine hydrolase ErgA in Pichia pastoris Julia Panhölzl 1,∗ , Julia Lindenberger 1 , Patricia Menczik 1 , Markus Aleschko 1 , Irene Hahn 2 , Heidi Schwartz-Zimmermann 2 , Gerd Schatzmayr 1 , Michaela Thamhesl 1 , Wulf-Dieter Moll 1 1 BIOMIN Research Center, Austria Christian Doppler Laboratory for Mycotoxin Metabolism and Center for Analytical Chemistry, Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna, Austria 2 Our long term goal is to prevent ergot alkaloid poisoning in farm animals by providing enzymes for use as feed additives to degrade ergot alkaloids in the digestive tract. We isolated an ergot alkaloid degrading bacterial strain, Rhodococcus erythropolis MTHt3, from a soil sample, cloned the genes for ergot alkaloid degradation, and characterised the recombinant enzymes. In the present work, we attempted to optimise the production yield of the recombinant ergopeptine hydrolase ErgA in Pichia pastoris by increasing copy number of the heterologous gene. We cloned plasmids with one, two, and three cassettes of ergA fused to the secretion signal sequence of Saccharomyces cerevisiae alpha-mating factor under control of the GAP promoter. Plasmids were transformed into Pichia pastorisNRRLY-11430 (CBS 7435), recombinant Pichia strains were cultivated in YPD medium in Erlenmeyer shake flasks, and yield of secreted ErgA was measured by SDS-PAGE analysis and enzyme activity assay based on ergotamine hydrolysis and HPLC. We found that two copies of the ergopeptine hydrolase gave higher ErgA yield than one copy, but a third copy ofergA showed S188 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 little or no further increase. Further strain optimisation will be required to increase production yield of ergopeptine hydrolase ErgA to a level suitable for a technological enzyme production process. http://dx.doi.org/10.1016/j.nbt.2014.05.928 PU-08 Osmolality as a determining factor in screening for high performing clones of human hematopoietic expression systems designed for monoclonal antibody production in chemically defined media Galina Kaseko ∗ , Kim Lou, Derrick Theys, Tohsak Mahaworasilpa The Stephen Sanig Research Institute, Australia In cell line development for monoclonal antibody (mAb) production, the screening and selection of a highly productive and stable clone from transfectant population in a limited time frame remains a major challenge. The clone performance including product quality, productivity, cell growth and cell metabolic profile is often dependent on cell culture conditions. With industry being pushed towards chemically defined, non-animal components or low protein media, the selection of the best performing clone faces an additional challenge as not all antibody-producing cell lines can achieve high yields in such conditions. In attempt to evaluate a possibility of obtaining high performance clones of an in-house generated host cell line derived from human hematopoietic cells, the cells were cloned in four either chemically defined, low protein or animal component free media. We found that a key factor in establishing commercially viable clones was culture osmolality. Most cell culture media have an osmolality range 270–330 mOsm/kg. The impact of high osmolality seems to be cell line specific with reports of inhibition of cell growth and specific productivity and an increase in specific productivity balanced with reduction in cell growth to yield an increased volumetric productivity. In our experiments, osmolality above 300 mOsm/kg had deleterious effects on single cell cultures regardless of the types of culture media used. Reducing osmolality to 250–290 mOsm/kg lead to an improved clone performance. It remains to be determined whether these results are specific to our host cell line or have broader implications to other expression systems, particularly those based on human hematopoietic cells. http://dx.doi.org/10.1016/j.nbt.2014.05.929 RECOMBINANT PROTEIN PRODUCTION New Biotechnology · Volume 31S · July 2014 PU-09 Development of improved methanol dehydrogenases using directed evolution and biological methanol sensor system for the elimination of formaldehyde Bong Hyun Sung 1,∗ , Ji-Yeun Yi 1 , Seung-Goo Lee 1 , Sun Chang Kim 2 , Jung-Hoon Sohn 1 1 Korea Research Institute of Bioscience and Biotechnology, South Korea 2 KAIST, South Korea Formaldehyde is an important organic precursor to many materials and chemical compounds. Despite its widespread use, exposure to formaldehyde is a significant consideration for human health due to its toxicity and volatility. Methanol dehydrogenase (MDH) is an NAD+-dependent oxidoreductase that catalyzes formaldehyde to methanol reversibly. Reduction activity of MDH is noticeable in two aspects of the elimination of toxic formaldehyde and the production of methanol as an energy source. Herein, we screened improved mutants of Bacillus methanolicus MDH in Esehcichia coli that reduce formaldehyde to methanol effectively with directed evolution and biological methanol sensor system. To examine the resistance to formaldehyde, E. coli strains expressing each mutant were cultured in 3 mM formaldehyde, toxic concentration for E. coli. In the best mutant, three phenylalanine residues were substituted to leucine, valine and serine respectively and all the three substitutions were required to increase reduction activity for formaldehyde. Interestingly, the mutant which has F213V and F289L increased the reduction relative to the wild type MDH, on the other hand, the mutant that has F356S reduced the reduction. Based on these results, mutant MDH eliminates formaldehyde efficiently, therefore it could help solve environmental problem such as sick house syndrome. http://dx.doi.org/10.1016/j.nbt.2014.05.930 PU-10 Optimizing secretion efficiency of industrially relevant recombinant proteins in Pichia pastoris by means of yeast surface display and cytometric sorting Lars Toellner 1,∗ , Fabian Schneider 1 , Brigitte Gasser 2 , Diethard Mattanovich 2 1 2 ACIB GmbH, Austria BOKU Vienna/ACIB GmbH, Austria Pichia pastoris has become a competitive host system for the production of industrially relevant enzymes and biopharmaceutical proteins. Strong constitutive and inducible promoter systems and efficient export signals enable production of authentic soluble proteins in the range of grams per litre. Still, similar to other host systems, the expression and secretion of recombinant protein can be impaired drastically when the secretory capacity of the host is overloaded with the additional burden. Co-expression of proteins which support protein folding, ER transport or other relevant steps in expression and secretion, has been shown to potentially increase the yield of secreted recombinant protein. However, these factors have to be evaluated case-by-case for any new target protein and without any guarantee for improvement. Here, we describe the combination of a randomised library approach in conjunction with yeast surface display (YSD) and cytometric sorting with the aim of selecting producer clones with enhanced secretory capacities. In order to link the specific secretion rate to individual cells, the recombinant target protein is fused with a cell wall anchor sequence. Thus, the avidity of the recombinant protein on the cell surface reflects the secretory capacity of each cell clone. Using immunofluorescence staining, promising candidates can be sorted and enriched in consecutive rounds applying fluorescence-activated cell sorting (FACS). Finally, isolated clones are subjected to DNA sequence analysis and the identified genes are supertransformed in host cells secreting soluble target protein in order to achieve proof-of-principle. http://dx.doi.org/10.1016/j.nbt.2014.05.931 PU-11 Purification of tag-free recombinant fumonisin esterase FumD from Pichia pastoris culture supernatant for use as calibration standard for enzyme quantification Corinna Kern ∗ , Markus Aleschko, Wulf-Dieter Moll, Gerd Schatzmayr Biomin Holding GmbH, Austria Fumonisin esterase FumD is used as feed additive for gastrointestinal hydrolysis and detoxification of fumonisins, carcinogenic mycotoxins produced by Fusarium verticillioides, which are naturally contained in maize from warm growing regions, in the gastrointestinal tract of farm animals. The goal of our present work was to provide a preparation of pure, stable and active FumD for use as calibration standard for enzyme quantification and characterization. Instead of purifying FumD-6x His via affinity chromatography as before, we attempted purification of tag-free FumD from Pichia pastoris fermentation culture supernatant. We compared three different purification protocols: anion exchange (AIEX) versus cation exchange (CIEX) chromatography and hydrophobic interaction chromatography (HIC). Analysis was performed by SDS-PAGE, immunoblotting and/or gel filtration (GFC) on Superdex 200 resin. IEX led to higher purity of the FumD fraction, and HIC showed higher yield. We tested protein stability by storing a FumD preparation in different buffers at 4 ◦ C and -20 ◦ C (including freeze-thaw cycles) for defined time periods. After quantification via ELISA and activity analysis, Tris buffer pH 8 without any additives was selected for FumD storage. The final FumD preparation was generated by two-step purification via AIEX-GFC using Tris as final buffer, and offered the same specific enzymatic activity as the starting material, suggesting no loss during the purification process. After quantification via several spectrophotometrical methods (BCA-assay, Bradford, UV-absorption), ELISA and Amino Acid Analysis, the FumD calibration standard was available with well-defined concentration and specific activity. http://dx.doi.org/10.1016/j.nbt.2014.05.932 www.elsevier.com/locate/nbt S189 RECOMBINANT PROTEIN PRODUCTION PU-12 Developing a versatile tool set for heterologous gene expression in Pichia pastoris Helmut Schwab 1,∗ , Mudassar Ahmad 2 , Melanie Hirz 2 , Markus Kolmbauer 2 , Ingund Rosales Rodriguez 2 1 2 ACIB/Graz University of Technology, Austria Graz University of Technology, Austria Pichia pastoris has become an important microbial host for recombinant protein production. We have chosen as basis the strain P. pastoris CBS 7435 (also NRRL Y-11430), which was originally patented for single cell protein production via methanol utilization, and for which the patent protection has expired. A high quality genome sequence of this strain was recently determined [1]. On the one hand proper integration vectors based on different promoters and selection markers have been newly designed and constructed. For engineering P. pastoris strains, an efficient vector construct was developed for markerfree genome knock-out or integration of gene. Based thereon, a set of amino acid auxotrophic and protease deficient host strains was constructed. In addition, an AOX1 promoter based methanol inducible host system was developed allowing for efficient induction with low amounts of methanol. Moreover, a methanol-free alternative has been developed for induction of the AOX1 promoter. Reference [1].Küberl A, Schneider J, Thallinger GG, Anderl I, Wibberg D, Hajek T, Jaenicke S, Brinkrolf K, Goesmann A, Szczepanowski R, Pühler A, Schwab H, Glieder A, Pichler H. High-quality genome sequence of Pichia pastoris CBS7435. J Biotechnol 2011;154(4):312–20. http://dx.doi.org/10.1016/j.nbt.2014.05.933 PU-13 Temperature effect on recombinant protein production in continuous cultures of methylotrophic yeast Pichia pastoris: a comparative study of sorbitol and glycerol as a co-substrate Julio Berrios ∗ , Maria-Olga Flores, Alvaro Diaz-Barrera Pontificia Universidad Catolica de Valparaiso, Chile Pichia pastoris is a methylotrophic yeast that is widely used as an expression system of heterologous proteins. The use of cosubstrates (e.g. glycerol, sorbitol) together with methanol, and culture temperatures below 30 ◦ C are among the proposed strategies to improve recombinant protein productivity. Since these approaches normally affect the specific cell growth rate (µ), and it is well known that µ also affect the protein productivity, we have performed an experimental design based on continuous cultures operating at a steady-state, thus fixing µ by a constant dilution rate (D = µ = 0.05 h−1 ). Two sets of continuous cultures were run using either glycerol or sorbitol as co-substrates, exploring at 22 and 30 ◦ C, together with co-substrates and methanol mixtures with 30 and 60% of co-substrate in the mix. The recombinant protein Rhizopus oryzae lipase (ROL) was used as model. Our results indicate S190 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 that sorbitol as co-substrate leads to a higher specific productivity of ROL (qROL ) whereas glycerol allowed higher volumetric productivity of ROL (QROL ). Besides, there is a synergic effect of glycerol feeding and lowering the temperature on QROL . Controversially, we have found that decreasing the cultivation temperature from 30 to 22 ◦ C at constant µ did not result in a qROL increase, suggesting that the increase of specific productivity by decreasing the cultivation temperature previously reported elsewhere is actually a result of a decrease in µ rather than temperature itself. This may open a new approach to optimise production of heterologous proteins based on decreasing the cultivation temperature. http://dx.doi.org/10.1016/j.nbt.2014.05.934 PU-14 Hydrophobins class I versus class II: Trichoderma virens HFB9a and HFB9b (class I) are more effective as enhancing agents in enzymatic PET hydrolysis and surface modulators compared to HFB4 and HFB7 (class II) Agnieszka Przylucka 1,∗ , Doris Ribitsch 1 , Enrique Herrero-Acero 1 , Georg Gübitz 1 , Christian P. Kubicek 1 , Irina Druzhinina 2 1 2 ACIB GmbH, Austria Technical University of Vienna, Austria Poly(ethylene terephthalate) (PET) is a thermoplastic polyester with excellent tensile and impact strength, transparency, and appropriate thermal stability. Because of its high production (36 mio tons per year), PET constitutes a significant waste material, which – although not representing a direct hazard to the environment - is not readily decomposed in nature. Recently, microbial cutinases were shown to be capable of partial or complete hydrolysis of the synthetic polymer PET, therefore offering new possibilities in the processing and recycling of this highly inert material. We have demonstrated that the addition of class II hydrophobins of Trichoderma can enhance the rate of PET hydrolysis by cutinases. It is currently unknown whether this observed effect is a property of some specific hydrophobins, and whether the hydrophobins need to bind to the cutinases to enhance their action. In this work we therefore compared the class II hydrophobins (HFB4 and HFB7) to the Trichoderma-unique nonclass II hydrophobins (HFB9a and HFB9b) with respect to their activity as enhancers of PET hydrolysis by cutinase I (CutI) from Thermobifida cellulosilytica and surface modulators. Time scan analysis over 72 h revealed a hydrophobin-specific interaction with cutinases. Whereas the non-class II hydrophobins HFB9a and HFB9b where able to increase the rate of PET degradation by CutI, class II hydrophobins were less active: HFB4 showed an increased activity on the hydrolysis of PET at lower time points, an inhibition was observed when using HFB7. http://dx.doi.org/10.1016/j.nbt.2014.05.935 RECOMBINANT PROTEIN PRODUCTION New Biotechnology · Volume 31S · July 2014 PU-15 Strategy for on-column refolding of human recombinant His-tagged prethrombin-2 produced using the Escherichia coli-based expression system Michaela Osadska ∗ , Martin Šafranek, Hana Krahulec, Stanislav Stuchlík, Ján Turňa Boňková, Ján Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia One way to obtain an active human thrombin for pharmaceutical purposes is to prepare recombinant human thrombin with the use of an Escherichia coli expression system. Prethrombin-2, the smallest single chain thrombin precursor, modified with His-tag at N-termini forms inactive intracellular inclusion bodies during the expression. Therefore the refolding process to obtain an active conformation is necessary. Here we report two on-column refolding methods of His-tagged prethrombin-2. Immobilized metal ion affinity chromatography and hydroxyapatite chromatography for one-step protein purification and on-column refolding were used. Solubilization of aggregated structures was carried out with various buffers composition under the denaturing conditions, where higher yield of solubilized prehrombin-2 was obtained in the presence of 6.0 M guanidine hydrochloride compared to 8.0 M urea. After the solubilization step gradual refolding of the protein was performed on-column using a linear guanidine gradient from 6.0 M to 0.0 M in the presence of 50 mM Tris–HCl and 0.5 M NaCl in the case of IMAC and 1 mM KH2 PO4 if hydroxyapatite chromatography was used. There was one protein peak in the A280 profile during the refolding process, which means that protein was released during reduction of chaotropic reagent concentration. Final yield of refolded protein was therefore in minimal concentration and on-column refolding requires next optimization. Acknowledgements: This work is result of project implementation: “Production of biologically active agents based on recombinant proteins” (ITMS 26240220048) supported by the Research and Development Operational Program funded by the ERDF. http://dx.doi.org/10.1016/j.nbt.2014.05.936 PU-16 Preparation of human growth hormone in Pichia pastoris under a constitutive promoter Jirickova, Zdenko Diana Hopkova ∗ , Kristina Krahulec, Stanislav Stuchlik, Jan Turna Levarski, Jan Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Slovakia The human growth hormone (hGH) is the polypeptide used in modern medicine for a treatment of different diseases such as growth deficiency in children. Moreover, the marketed pharmaceuticals based on hGH got the approval for the treatment of Turner’s syndrome or cachexia associated with AIDS. Pichia pastoris was chosen as the host organism because of its ability to grow to high cell densities and its strong promoters capa- ble of the production of high yields of a recombinant protein. The system combines both advantages of prokaryotes – high expression, simple scale-up, cheap production media–, and advantages of eukaryotes – ability to perform various post-translation modifications. The hGH gene was integrated into genome of P. pastoris under the control of a constitutive promoter, in order to produce native hGH into the extracellular media. Then, the genome integration was validated through a PCR and expression in flasks. The SDSPAGE results were confronted with a Western blot analyses. hGH was then successfully produced in 1L fermenter and partially purified using anion-exchange chromatography. Acknowledgements: This publication is the result of the project implementation: “Production of biologically active agents based on recombinant proteins” (ITMS 26240220048) supported by the Research and Development Operational Program funded by the ERDF. http://dx.doi.org/10.1016/j.nbt.2014.05.937 PU-17 Isolation of novel lignin degrading enzymes and lignin degradation products from bacteria and fungi Fatai Bello ∗ , Takanori Furukawa, Louise Horsfall University of Edinburgh, United Kingdom Lignocellulosic biomass offers an alternative to non-sustainable fossil fuels for the production of useful chemical substances and is expected to become one of the key renewable energy resources in the near future. Lignin makes up as much as 30% of lignocellulosic biomass and is considered one of the most important limiting factors in the yield of bioethanol obtained from biomass fermentation. Currently, industries regard lignin as a nuisance in the utilization of cellulose and hemicellulose, which are the target starting materials for these industries. However, lignin is a potential source of a variety of aromatic chemicals, if a system for its controlled degradation can be developed. Several species of fungi and bacteria possess lignin-degrading ability through the production of different types of ligninolytic enzymes. This project aims to identify ligninolytic enzyme gene sequences from fungi and bacteria. The genes will then be cloned and expressed in bacterial and fungal hosts. The optimum expression conditions will be determined and the activity of the resulting enzymes on lignin and lignin model compounds will be determined. Structural analysis by X-ray crystallography will be performed on the best enzymes from the activity studies. The lignin degradation products will be analysed and methods for quantitative estimation are in development. The enzymes discovered from this study are intended to improve the current lignin enzyme treatments used in industry. http://dx.doi.org/10.1016/j.nbt.2014.05.938 www.elsevier.com/locate/nbt S191 RECOMBINANT PROTEIN PRODUCTION PU-18 Fusion protein and solubility enhancing strategies for heterologous expression of novel plant sesquiterpene synthases Steffen Hartwig ∗ , Thore Frister, Thomas Scheper, Sascha Beutel Leibniz University of Hannover, Germany Terpenes constitute the largest class of flavor and fragrance molecules in nature. Important representatives of this class are used widely in various foods, beverages, exclusive perfumes and personal care products. Biocatalytic production of terpenes is a promising method, but requires the availability of large amounts of soluble and active terpene synthases. Plant terpene synthases (TPS) exhibit large variations in amino acid sequence and protein fold. The soluble yield when expressed in E. coli varies substantially from enzyme to enzyme. In most cases, TPSs are considered hard to express in a heterologous host organism. We chose cDNAs of synthases, partially synthesized as well as extracted from native plants, which all produce chemically interesting and industrial relevant terpene products. We identified a cDNA variant of patchoulol synthase from Pogostemon cablin [1,2]. The active expression level of the enzyme variant was increased substantially by fusion to a thioredoxin moiety [3]. A previously not characterized zizaene synthase from Vetiveria zizanoides was assembled from artificial DNA strings. Because no soluble expression could be detected by conventional strategies, induction using a cold shock promoter as well as fusion to the highly soluble SUMO protein moiety were evaluated. The presented results enable other researchers in the field to decide what expression strategy might be most suitable for new TPSs, which have not been expressed in E. coli. References [1].Croteau, et al. Arch Biochem Biophys 1987;256:56–68. [2].Deguerry, et al. Arch Biochem Biophys 2006;454:123–36. [3].Hartwig, et al. Prot Expr Purif 2014;97:61–71. http://dx.doi.org/10.1016/j.nbt.2014.05.939 PU-19 Expression of soluble recombinant human growth hormone in Escherichia coli cultures using shake-flask, batch and fed-batch cultivation Jan Turna ∗ , Zdenko Levarski, Jan Krahulec, Kristina Jirickova, Diana Hopkova, Stanislav Stuchlik Comenius University in Bratislava, Department Mol. Biol., Slovakia Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. We have developed an efficient E. coli based expression system for the production of high levels of soluble hGH fused to S192 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 thioredoxin (Trx). In this work, we compare the expression level, portion of soluble product, efficiency and yield of Trx-hGH fusion protein using three cultivation methods – shake-flask, batch and fed-batch bioreactor runs. While more than 95% of the produced Trx-hGH was in soluble form when cultivated in shake-flask or bioreactor in batch mode, this number decreased to 47% when produced in fed-batch mode. We have been able to purify 1.045 g of soluble Trx-hGH from the liter of fed-batch culture and 0.8 g from the batch culture. These numbers represent a significant yield when compared to other published data and are of potential interest to the biotech industry. Acknowledgements: This work is the result of the project implementation: “Production of biologically active agents based on recombinant proteins” (ITMS 26240220048) supported by the Research and Development Operational Program funded by the ERDF. http://dx.doi.org/10.1016/j.nbt.2014.05.940 PU-20 Multiple chromosomal gene integration for production of pharmaceutical proteins in S. cerevisiae Malene Jensen 1,∗ , Uffe Mortensen 1 , Nina Gunnarsson 2 , Tomas Strucko 1 , Line Due Baron 2 1 2 Technical University of Denmark, Denmark Novo Nordisk, Denmark When studying protein folding and secretion the general conception is that all cells in a population express an equal amount of protein. Recent work has shown that expression levels vary greatly in cell populations which express proteins on plasmids. Hence a yeast expression platform has been developed at the Department of Systems Biology, DTU. The platform offers the opportunity to express genes on the chromosome in 1 to 10 copies. A comparison between the expression of CFP and RFP by the platform and by plasmids reveals the problems of plasmid expression. FACS analyses of two cell populations, expressing CFP and RFP on the separate plasmids or expressing CFP and RFP using the yeast expression platform shows expression varies greatly in a cell population based on plasmid expression compared to the yeast expression platform. When expressed on plasmids a few cells are high performers on both proteins but the largest fraction of cells is actually not expressing either of the proteins. The yeast expression platform is developed to facilitate stable expression of integrated genes. The integration sites are separated by essential genes which ensure that the integrated genes are not lost by recombination. An amplification method has been developed for the platform which enables fast integration of genes. Future perspectives involve exploring the capabilities of the platform for recombinant protein production including performance and stability studies. http://dx.doi.org/10.1016/j.nbt.2014.05.941 RECOMBINANT PROTEIN PRODUCTION New Biotechnology · Volume 31S · July 2014 PU-21 Biosynthesis, purification and biointeraction of human SCOMT with Parkinson’s disease inhibitors Luis Passarinha ∗ , Filipa Correia, Augusto Pedro, Diana Oliveira, João Queiroz CICS-UBI: Health Sciences Research Centre – Universidade da Beira Interior, Portugal Catechol-O-methyltransferase (COMT) is an important target in protein engineering due to its role in human neurological disorders such as Parkinson’s and Alzheimer’s disease [1]. Therefore, is relevant to study new COMT inhibitors since it will lead to an improvement in Parkinson’s therapy. So, it is necessary a biosynthesis process to produce and stabilize the human SCOMT and subsequently, a suitable purification strategy to obtain pure fractions for protein-inhibitors interaction studies. The biosynthesis process was performed based on pPICZ A and Pichia pastoris as a host cell where hSCOMT was synthesized with a hexahistidine tag. Moderate to high expression values were obtained for defined media at 24 h, 30 ◦ C and 250 rpm. Since COMT loses rapidly its activity during isolation and storage [2], stabilization assays were performed in order to improve the main isolation step by Immobilized-Metal Affinity Chromatography (IMAC) and further storage. Through a neuronal network we studied the protein stability in the presence of potential compounds, varying the time and temperature at which the protein is stored. The best input was selected and applied. Then, to obtain a purified fraction for inhibition trials, IMAC was applied and highly purified hSCOMT fractions were obtained. Finally, biophysical studies were performed by microcalorimetry in the presence of specific inhibitors in order to assess kinetic profiles for human SCOMT. Acknowledgements: A.Q. Pedro acknowledges a doctoral fellowship (SFRH/BD/81222/2011) from Fundação para a Ciência eTecnologia. This work was partially funded by FEDER funds through Programa Operacional Factores de Competitividade – COMPETE:FCOMP-01-0124-FEDER-027563 with the project EXPL/BBB478/BQB/0960/2012. http://dx.doi.org/10.1016/j.nbt.2014.05.942 PU-22 Directed evolution to remove expression and secretion bottlenecks of heterologous enterokinase in Escherichia coli Weiluo Lee ∗ , Paul Dalby University College London, Biochemical Engineering, United Kingdom Bovine enterokinase catalytic subunit (EKcat ) has significant industrial potential for selective modification of therapeutic proteins. The enzyme is typically expressed heterologously in eukaryotic cell strains such as Pichia pastoris to ensure proper glycosylation and folding into its soluble active form. EKcat production in E. coli offers several advantages (i.e. shorter fermentation times, undemanding growth conditions, and favourable pro- cessing economics) but is challenging due to the formation of inclusion bodies, leading to inactive enzymes. Engineering EKcat for periplasmic secretion generated a basal level of soluble and active enzyme. Directed evolution was then applied to screen for variants with improved total activities attained during expression. The generated library of variants contained mutations across the following gene sequences: EKcat , secretion signal peptide, promoter, lac operator, and ribosomal-binding site. The solubility, stability, and activity of the resultant EKcat variants were examined and hypotheses given based on sequence, structural, and biophysical findings. Furthermore, variants were also shown to adapt differently when the fermentation temperature was raised, providing another dimension for investigating the complex dynamics between expression and secretion. In addition to improving EKcat expression and activity in E. coli, the study presents insights into engineering proteins for high functional expression and periplasmic secretion. http://dx.doi.org/10.1016/j.nbt.2014.05.943 PU-23 siRNA-mediated engineering of DNA recombination for improved transgene integration and expression Kaja Kostyrko ∗ , Nicolas Mermod University of Lausanne, Switzerland Achieving high levels of stable heterologous gene expression in eukaryotic cells is limited in part by the efficiency of transgene integration into the genome. This likely relies on one of the cellular DNA repair pathways – non-homologous end-joining (NHEJ), homologous recombination (HR), or on less well characterized microhomology-mediated end-joining (MMEJ) pathways. However, the exact molecular basis of this process is still not fully understood. In the effort to identify the transgene genomic integration mechanism we silenced important DNA recombination repair components in Chinese hamster ovary (CHO) cells using small interfering RNA (siRNA), and analyzed the integration and expression of transgene-containing plasmid DNA in these cells. In addition, we used vectors bearing matrix attachment regions (MARs), genetic elements commonly used to increase and stabilize recombinant gene expression, which were recently proposed to also play a role in DNA recombination. We find that knock-down of the NHEJ pathway components does not affect transgene integration or expression, while silencing the HR pathway increases both. We also show that the use of a MAR element stimulates increased transgene integration and expression. However, in the absence of the HR pathway, MAR induces an even higher expression, without further increasing integration. Instead, we observe an increased expression per individual gene copy. Therefore, we propose that HR knock-down, as well as the MAR element, stimulate an alternative repair pathway, most likely MMEJ, which has a beneficial influence on transgene integration and resulting expression. This knowledge should help engineer cells for improved recombinant protein production. http://dx.doi.org/10.1016/j.nbt.2014.05.944 www.elsevier.com/locate/nbt S193 RECOMBINANT PROTEIN PRODUCTION PU-24 MAR elements and transposons for improved transgene integration and expression Déborah Ley 1,∗ , Valérie Le Fourn 2 , Pierre-Alain Girod 2 , Alexandre Regamey 2 , Nicolas Mermod 1 1 2 Institute of Biotechnology, University of Lausanne, Switzerland Selexis SA, Geneva, Switzerland A critical step for protein production in biotechnology or expression of a therapeutic gene in gene therapy is the ability to achieve consistent gene expression into the target cells. This implies transgene integration in a locus which allows its efficient and sustained expression. Among the wide variety of non-viral vectors used to deliver DNA, transposon systems have the advantage to provide increased frequencies of single-copy integration into the host genome. However, as with other methods of transgene delivery, the use of transposon-based systems are not immune to the risks of silencing and insertional mutagenesis. These unwanted events may be limited by the use of genetic elements, called matrix attachment region (MAR). Transgenes associated with MARs have been shown to have a higher and more stable expression and are less prone to silencing. Here, we assessed the effect a piggyBac (PB) transposon containing the MAR-1-68 in CHO cells. Using this combination, it was possible to obtain an enriched transgene-positive population harbouring few integrations sufficient for high transgene expression, without the need of selection pressure. As a proof of feasibility for biotechnology applications, we demonstrated the ability of the system to obtain significant amounts of therapeutics proteins from unselected cell populations 2–3 weeks after transfection. Since antibiotic-enforced selection protocols often result in a higher integrated copy number and mosaic expression patterns, this strategy could benefit many applications in which low integrated copy number and antibiotic-free conditions are desired. http://dx.doi.org/10.1016/j.nbt.2014.05.945 PU-25 Exploration and characterization of novel lignin degrading enzymes for a synthetic biology platform to aid enzymatic lignin disruption Takanori Furukawa 1,∗ , Aaron McKerracher 2 , Franck Escalettes 2 , Reuben Carr 2 , Loise Horsfall 1 1 2 The University of Edinburgh, United Kingdom Ingenza Ltd, United Kingdom Lignin is the most abundant aromatic polymer on earth derived from photosynthesis and considered to be potential feedstock for the production of renewable aromatic chemicals. However, due to its structural nature, it exhibits high resistance towards chemical and biological degradation, and this is a major obstacle for achieving efficient conversion of lignin into valueadded products. Many microorganisms including both fungi and bacteria have been reported to produce a wide variety of oxidative enzymes that are involved in the degradation of lignin. These include lignin peroxidases, manganese peroxidases, versatile S194 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 peroxidases and laccases, which act cooperatively to degrade lignin macromolecules. In this research project, we aim to develop a synthetic biological platform to optimize the degradation process and products of lignin disruption. To this end, several nucleotide sequences encoding a putative lignin-degrading enzyme were identified from GeneBank at the National Center for Biotechnology Information (NCBI) based on its putative domain structure and sequence similarity to reported lignolytic enzymes. Codon usage of the identified genes was optimized for Pichia pastoris and Saccharomyces cerevisiae to enable high-level protein expression, and heterologous production of the optimized genes was investigated in the two different yeast expression platforms. Furthermore, enzymatic properties of the expressed proteins were characterized in order to select lignolytic enzymes with the desired properties for the future development of synthetic microbes for controlled lignin degradation. http://dx.doi.org/10.1016/j.nbt.2014.05.946 PU-26 Metabolic model-based prediction of engineering targets for increased production of heterologous proteins Justyna Nocon 1,∗ , Matthias G. Steiger 2 , Martin Pfeffer 3 , Seung Bum Sohn 4 , Tae Yong Kim 4 , Hannes Rußmayer 5 , Stefan Pflügl 3 , Christina Haberhauer-Troyer 6 , Karin Ortmayr 6 , Gunda Köllensperger 6 , Brigitte Gasser 2 , Sang Yup Lee 7 , Diethard Mattanovich 5 1 University of Natural Resources and Life Sciences, Austria Austrian Centre of Industrial Biotechnology, Vienna, Austria 3 Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Austria 4 Bioinformatics Research Center, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, South Korea 5 Centre of Industrial Biotechnology, Vienna, Austria 6 Department of Chemistry, University of Natural Resources and Life Sciences Vienna, Austria 7 Korea Advanced Institute of Science and Technology (KAIST), Daejeon, South Korea 2 The production of recombinant proteins can be enhanced by influencing transcription, optimizing codon usage, increasing protein folding and secretion. Overproduction of heterologous proteins, however, also directly affects the primary metabolism of the host organism. The incorporation of recombinant protein production into the genome scale metabolic model of the yeast Pichia pastoris, allowed the simulation of the effects of overproduction. Gene targets for deletion or overexpression for enhanced productivity were predicted. Overexpression targets were localized in the pentose phosphate pathway and the TCA cycle, while knockout targets were found in several branch points of glycolysis. Five out of 9 tested targets led to an enhanced production of cytosolic human copper/zinc superoxide dismutase (hSOD). Beneficial mutations were mainly related to reduction of the NADP/H pool and the deletion of fermentative pathways. Overexpression of the hSOD gene itself had a strong impact on intracellular fluxes, RECOMBINANT PROTEIN PRODUCTION New Biotechnology · Volume 31S · July 2014 which were predicted with high accuracy by the model. Genome scale metabolic modeling is shown to predict overexpression and deletion mutants which enhance recombinant protein production with high accuracy. PU-28 http://dx.doi.org/10.1016/j.nbt.2014.05.947 Alexander Büttner 1,∗ , Simon Stammen 1 , Mark Dürkop 1 , Artur Schuller 1 , Lina Heistinger 2 PU-27 Generation and application of tools for increasing periplasmic soluble product yields of recombinant therapeutic proteins in Escherichia coli 1 2 The production of recombinant cationic a-helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum Cristina Ruiz Ramirez 1,∗ , Nuri Company 2 , Anna Nadal 2 , JoseLuis La Paz 3 , Sílvia Martinez 3 , Stefan Rasche 4 , Stefan Schillberg 4 , Maria Pla 2 1 Institute for Food and Agricultural Technology (INTEA), University of Girona, Spain 2 Institute for Food and Agricultural Technology (INTEA), Spain 3 Center for Research in Agricultural Genomics (CRAG), Spain 4 Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Aachen, Germany Synthetic linear antimicrobial peptides with cationic a-helical structures, such as BP100, are valuable as novel therapeutics and preservatives. However, they tend to be toxic when expressed at high levels as recombinant peptides in plants, and they can be difficult to detect and isolate from complex plant tissues because they are strongly cationic and display low extinction coefficient and extremely limited immunogenicity. We therefore expressed BP100 with a C-terminal tag which preserved its antimicrobial activity and demonstrated significant accumulation in plant cells. We used a fluorescent tag to trace BP100 following transiently expression in Nicotiana benthamiana leaves and showed that it accumulated in large vesicles derived from the endoplasmic reticulum (ER) along with typical ER luminal proteins. Interestingly, the formation of these vesicles was induced by BP100. Similar vesicles formed in stably transformed Arabidopsis thaliana seedlings, but the recombinant peptide was toxic to the host during latter developmental stages. This was avoided by selecting active BP100 derivatives based on their low haemolytic activity even though the selected peptides remained toxic to plant cells when applied exogenously at high doses. Using this strategy, we generated transgenic rice lines producing active BP100 derivatives with a yield of up to 0.5% total soluble protein. http://dx.doi.org/10.1016/j.nbt.2014.05.948 Boehringer Ingelheim RCV GmbH & Co KG, Austria University of Natural Resources and Life Sciences Vienna, Austria Soluble production of properly folded target proteins in Escherichia coli can avoid cumbersome protein refolding steps when compared to inclusion body (IB) production processes. Due to several advantages over the cytoplasm, the periplasmic space of E. coli is the compartment of choice for production of soluble and correctly folded disulfide-bond containing proteins. To address bottlenecks that can prevent efficient production, secretion and folding, a toolbox consisting of different host cell modifications, expression cassettes and process control strategies was created and tested for applicability. Cell engineering approaches comprised, e.g., different bacterial strains, genomic integration of the target gene expression cassette, its organization within the integration locus as well as the co-synthesis of helper factors, which improve secretion or folding of the target proteins. A range of inducible and constitutive promoter variants were tested concerning their applicability to control helper factor gene co-overexpression. Different fermentation processes with regard to temperature, feeding and induction mode and time were applied. For several therapeutically relevant proteins of interest, we showed that application of one or a combination of several of the developed methods increased the overall yield of the desired product. Additionally, the ratio of recombinant protein was shifted from insoluble (cytoplasmic and/or periplasmic aggregates) to soluble and correctly folded product in several cases. http://dx.doi.org/10.1016/j.nbt.2014.05.949 PU-29 Enhanced periplasmic production of therapeutic proteins in Escherichia coli Simon Stammen 1,∗ , Alexander Buettner 1 , Lina Heistinger 2 , Mark Duerkop 1 , Arthur Schuller 1 , Franz Kollmann 1 1 2 Boehringer Ingelheim RCV GmbH, Austria University of Natural Resources and Life Sciences, Austria For manufacture of therapeutic proteins that do not require translational modification (e.g. glycosylation) for their biological activity, the production in Escherichia coli is still the host system of choice. Its well-known biology, the ability to grow on inexpensive media, the availability of genetic tools for host cell modifications and last but not least its long history as pharmaceutical work host are only the most prominent benefits. However, pharmaceutical proteins became more and more complex and therefore the requirements on the expression systems grew. Many modern, biologically active proteins contain multiple disulphide bridges, www.elsevier.com/locate/nbt S195 RECOMBINANT PROTEIN PRODUCTION what makes classical production routes (e.g. production as inclusions bodies and subsequent in vitro refolding) more and more challenging. Even though recent advances in folding technology (e.g. high pressure refolding) enable refolding in commercially relevant yields for many target proteins, Boehringer Ingelheim (BI) also significantly increased its efforts in production of properly folded target proteins in the E. coli periplasm. We present several approaches to increase the amount of soluble therapeutic protein within the periplasmic space of the bacterial cell. Application of BI’s expression system, which makes use of a genomically integrated product gene significantly increased the titre of properly folded target protein when compared to one of the typically used plasmid-based expression systems. Furthermore, the secretion and refolding machinery was supported by co-overproduction of different helper proteins and chaperones to enhance cellular productivity. The fermentation process was optimized with regard to process variables to enhance the yield of properly folded target protein. http://dx.doi.org/10.1016/j.nbt.2014.05.950 PU-30 Identification of a novel master regulator of cellulase and hemicellulase production in Trichoderma reesei using genome-wide approach Mari Häkkinen, Mari Valkonen ∗ , Ann Westerholm-Parvinen, Nina Aro, Marika Vitikainen, Merja Penttilä, Markku Saloheimo, Tiina Pakula VTT Technical Research Centre of Finland, Finland Trichoderma reesei (anamorph Hypocrea jecorina) is an efficient producer of enzymes degrading lignocellulosic biomass. The cellulases and hemicellulases produced by the fungus are widely employed in industry, and this production system has a central role in biorefinery applications. Various environmental and metabolic factors together with the physiological state of the cell affect the enzyme production in T. reesei. In previous studies, both positively and negatively acting regulatory factors for cellulase and hemicellulase genes have been characterised in T. reesei. In this study, an expression microarray data on T. reesei cultivated in the presence of different carbon sources was analysed in order to identify additional regulatory genes for cellulase and hemicellulase production. In total, 28 putative regulatory factors were chosen to be over-expressed in T. reesei based on the fact that they were induced by lignocellulosic substrates. In the primary screening, over-expression of seven of these factors led to increased production of cellulases and/or xylanases. Among these factors is a novel master regulator of the cellulose and hemicellulose genes designated ace3. Its over-expression increased cellulase production 2 to 4-fold and also enhanced hemicellulase production. Its deletion abolished cellulase production totally, decreased hemicellulase production, and in the ace3 deletion strains the major cellulase gene transcripts were at extremely low levels. Interestingly, the modifications of ace3 also affected the mRNA levels of the previously identified hemicellulase and S196 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 cellulase master regulator xyr1, suggesting interplay between these factors. http://dx.doi.org/10.1016/j.nbt.2014.05.951 PU-31 Effects of K87 mutations in the alpha-crystallin domain of Tpv sHSP 14.3 on oligomerization and in vitro chaperone activity Semra Kocabiyik ∗ , Ilir Sharej Middle East Technical University, Turkey Small heat shock proteins (sHSPs) act as molecular chaperones by binding denaturing proteins and protecting them from aggregation before refolding. Their level is up-regulated in particular events, such as stress and pathological conditions. ACD is the signature of the sHSP monomer which is flanked by N-terminal arm of variable sequence and C-terminus with a conserved I/L-X-I/L motif. Seven or eight ␤-strands in ACD arranged into antiparallel sheets constituting ␤-sandwich which mediates dimerization. The sHSPs form reversible homo- or hetero-oligomers indicating the dynamic plasticity of the quartenary structure that modulate the recognition of substrate. Mutations in the ACD occur in positions that would have impact on this dynamic behavior. One of the well studied mutations is R120G in human CRYAB that causes congenital cataract and desmin related myopathy. At the dimer interface R120 makes two interface ion pairs with D109. The aim of this study is to determine if the topological equivalent of this residue has the same impact on oligomeric structure of non-metazoan sHSPs. We investigated the effect of substitutions at K87 position of Tpv HSP14.3 (an archaeal sHSP) on oligomerization and chaperone activity. The K87R and R87I mutations resulted in increased chaperone activity when citrate synthase was used as the client. Increased substrate binding capacity due to R87I mutation can be explained by increased hydrophobicity. The oligomere size of all mutant sHSPs increased at the temperature from 25 to 60 ◦ C. The notable increase was observed at 25 ◦ C for the K87I mutant particles. http://dx.doi.org/10.1016/j.nbt.2014.05.952 PU-32 Development of a “toolbox” approach for recombinant protein production Tania Selas Castiñeiras 1,∗ , Steven G. Williams 2 , Jeff A. Cole 1 , Tim W. Overton 1 , Anthony Hitchcock 2 1 2 University of Birmingham, United Kingdom Cobra Biologics Ltd, United Kingdom The high demand of recombinant proteins produced in microbial hosts, which accounts for more than 30% of the biological products in the market, have reinforced the necessity of the development of more efficient processes for the production of recombinant proteins. Higher production efficiencies and lower cost have become essential pre-requisites for a commercially viable RECOMBINANT PROTEIN PRODUCTION New Biotechnology · Volume 31S · July 2014 process. Optimisation of fermentation conditions to decrease cell stress has been shown to favour the accumulation of soluble and active recombinant proteins [1]. We have shown that shake-flask studies are an appropriate tool for the optimisation of fermentation conditions, reducing the time and cost involved in process development. This approach was used for the production of tumour necrosis factor ␣ (TNF␣) in E. coli using the arabinose-inducible T7 expression system. A volumetric yield of 3.82 g L−1 of TNF␣ was achieved by fermentation, 92% being soluble and active. The proven success of this approach can be now applied to a broader range of recombinant proteins. We will discuss the selection of adequate expression systems and approaches for the optimisation of cultivation conditions as key factors for the production of recombinant proteins. The main goal of this project is to test and integrate fermentation conditions allowing the design of platforms following a “toolbox approach” for protein production. Reference [1].Sevastsyanovich Y, Alfasi S, Overton T, et al. Exploitation of GFP fusion proteins and stress avoidance as a generic strategy for the production of high-quality recombinant proteins. FEMS Microbiol Lett 2009;299:86–94. http://dx.doi.org/10.1016/j.nbt.2014.05.953 PU-33 In vivo reconstitution of membrane protein by caveolin1 co-expression Jonghyeok Shin ∗ , Paul Heo, Joon-Bum Park, Myungseo Park, Younghun Jung, Da-Hyeong Cho, Byoung-jae Kong, Junghoon In, Jichun Lee, Dae-Hyuk Kweon Sungkyunkwan University/Bioengineering Department, South Korea Caveolae is a membrane-budding structure which exists in many animal vertebrate cells. One of the important functions of caveolae is to form membrane curvature and endocytic vesicle. Recently, It was shown that caveolae could be formed in Escherichia coli by expressing caveolin-1. The heterologous caveolae may host other membrane proteins overexpressed inside the cell. We utilized this system for construction of proteo-liposome in Escherichia coli. SNARE proteins (Syntaxin1a, SNAP25, VAMP2) were introduced to prove our in vivo reconstitution system. Here, we show that the purified heterologous caveolae indeed contain the co-expressed membrane protein and the membrane proteins were facing outward. The size of the purified caveolae with membrane protein reconstituted were measured by dynamic light scattering. The presence of VAMP2 & Syntaxin1a on this proteoendosome was confirmed by Western blot analysis. Furthermore, membrane proteins (VAMP2 & Syntaxin1) embedded in caveolae retained the ability to form SNARE complex. Our study proposes an in vivo membrane protein reconstitution system. http://dx.doi.org/10.1016/j.nbt.2014.05.954 PU-34 Genetic engineering of Saccharomyces cerevisiae for efficient IgG-assembly and secretion Essi Koskela ∗ , Alexander Frey Aalto University, Finland IgG-antibodies are complex molecules that require multiple elements to assemble efficiently, including folding factors and an oxidative environment. The growing demand for biosimilars and alternative treatments makes the production process of human antibodies an intriguing area of research. Although simple microbial expression platforms such as Saccharomyces cerevisiae are not intrinsically suited for IgG-production, the tools to genetically modify the yeast cells for this purpose are versatile. To improve the secretion of produced protein, we focus on modifying ER luminal environment and protein folding process. One important approach is increasing the size of ER, which can be achieved with a single gene knock-out [1]. The extra space is supplemented with additional folding factors important in IgG-tetramer assembly including molecular chaperones, PDI and PPIases. These factors are tested in different amounts and combinations and the strains producing the highest yields are identified with high-throughput screening. Genetic engineering requires a substantial amount of laborintensive cloning work. To optimize this aspect, we recently discovered a new approach to molecular cloning, which outcompetes many of the existing cloning methods in simplicity and affordability. This novel protocol is presented along with preliminary results of genetic modifications on IgG-secretion. With suitable high-throughput methods in hand, the microbial platform for IgG-production is optimized at the level of the whole system. Reference [1].Schuck S, a Prinz W, Thorn KS, Voss C, Walter P. Membrane expansion alleviates endoplasmic reticulum stress independently of the unfolded protein response. J Cell Biol 2009;187:525–36. http://dx.doi.org/10.1016/j.nbt.2014.05.955 PU-35 Study on the domain function of Listeria monocytogenes p60 protein Minliang Guo ∗ , Hao Gu, Qian Xu, Jinrong Zuo College of Bioscience and Biotechnology, Yangzhou University, China Listeria monocytogenes p60 protein is an autolysin that can hydrolyze the peptidoglycans of bacterial cell walls. L. monocytogenes p60 protein was required for L. monocytogenes virulence. Besides the importance of p60 protein in bacterial pathogenesis, p60 protein can also be developed as a new proteinaceous antimicrobial if its activity to hydrolyze the peptidoglycans can be greatly improved. It contains two independent structural domains, Nterminal LysM domain and C-terminal NlpC/P60 domain, which www.elsevier.com/locate/nbt S197 RECOMBINANT PROTEIN PRODUCTION can be separated at the amino acid residue 270. However, the active functions of these two domains in p60 protein and their influences on the substrate recognition and catalytic activity of p60 protein are unknown. Here we identified both of the functional hot spot and the mutational hot spot amino acid residues in these two structural domains by means of the amino acid sequence alignment of different p60 variants, including two p60 variants screened in our lab. The functional hot spot and the mutational hot spot amino acid residues were substituted to alanine (A) by using site-directed mutation to construct p60 variants. These p60 variants in combination with some truncated p60 proteins were used to unveil the molecular mechanism of substrate recognition and catalysis of p60 protein in the domain level. Results confirmed that the N-terminal LysM domain in p60 protein could bind to the bacterial cell wall tightly, whereas the C-terminal NlpC/P60 domain showed slight ability to hydrolyze the cell walls. These fundamental studies on p60 protein variants will provide strong support for engineering the p60 protein molecular. http://dx.doi.org/10.1016/j.nbt.2014.05.956 PU-36 GlycoDelete technology: shortcutting mammalian cell Nglycosylation Francis Santens ∗ , Leander Meuris, Morgane Boone, Nico Callewaert VIB Ghent University, Belgium Mammalian complex-type N-glycan synthesis is a multi-step process that results in heterogeneous glycosylation of proteins. Heterogeneity in therapeutic glycoproteins causes difficulties for protein purification and process reproducibility and can lead to variable therapeutic efficacy. Here we report engineered mammalian cell lines that have a shortened Golgi N-glycosylation pathway, which leads to the expression of proteins with small, sialylated trisaccharide N-glycans. This glycoengineering strategy, which we call GlycoDelete [1], results in proteins with substantially reduced glycan heterogeneity. To assess the potential of these GlycoDelete glycans and their influence on glycosylated pharmaceutical proteins, human GM-CSF and an anti-CD20 antibody were produced in 293s and 293sGlycoDelete cells. Both proteins were purified and thoroughly analysed. For hGM-CSF we did not see a significant influence of the GlycoDelete sugars on the activity of the protein. GlycoDelete anti-CD20 on the other hand has a significantly reduced Fc␥R affinity and an increased circulation times in mice compared to 293S produced anti-CD20. Reference [1].Meuris L, Santens F, Elson G, et al. GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins. Nat Biotechnol 2014, advance online publication. http://dx.doi.org/10.1016/j.nbt.2014.05.957 S198 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 PU-37 Extracellular transaminases for biocatalysis Katrin Weinhandl 1,∗ , Margit Winkler 1 , Anton Glieder 1 , Andrea Camattari 2 1 2 Austrian Center of Industrial Biotechnology (ACIB), Austria TU Graz, Austria Branched chain aminotransferase (BCAT, EC 2.6.1.42) of Escherichia coli is an intracellular protein and an interesting tool for the production of chiral amines or amino acids. Secretion of BCAT to the culture supernatant was the method of choice to facilitate industrial enzyme applications and downstream processing by whole cell applications while counteracting limited cell permeability for target substrates. Pichia pastoris was chosen as expression host because of its positive characteristics, such as the ability to reach high biomass levels as well as the lack of background proteins in the extracellular environment during expression. Although secretion of intracellular proteins was reported to be problematic in the past, we were able to secrete BCAT in Pichia pastoris and obtained a maximum activity level in the supernatant of 150 ␮mol/min/mg total protein (L-leucine conversion in a coupled enzymatic assay [1]). In order to improve the expression level, several approaches were investigated: on the one hand we examined different Pichia strains. On the other hand, alternative signal peptides and different promoters were evaluated for improved expression and secretion of BCAT. In our hands methanol-induced expression lead to a higher activity in the supernatant, compared to constitutive expression which still allowed satisfying BCAT secretion. Reference [1].Weinhandl, et al. Tetrahedron 2012;68(37):7586–90. http://dx.doi.org/10.1016/j.nbt.2014.05.958 PU-38 A method to stably integrate multiple genetic elements into Chinese hamster ovary (CHO) cells Sabine Vcelar 1,∗ , Martina Baumann 1 , Nicole Borth 2 1 ACIB - Austrian Centre of Industrial Biotechnology, Austria Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria 2 CHO cells are the preferred host cells for the production of therapeutic proteins and the most commonly used mammalian expression system. Advantages such as an easy cultivation, fast growth, complex protein folding and human-like posttranslational modifications are in part set of by slow cell line and process development. These constraints lead to an increased requirement for CHO cell line modification tools. The present work focuses on the integration of up to four genetic elements into CHO cells. Two different approaches were established. The principle of both systems is the Recombinasemediated cassette exchange (RMCE). Systems A is engineered on the basis of transfection vectors comprised of different resistance RECOMBINANT PROTEIN PRODUCTION New Biotechnology · Volume 31S · July 2014 markers and two cloning sites. The incorporation of the genetic elements of choice is mediated by RMCE at two specific genomic locations. The second system established combines the RMCE system with the R4 Integrase System to enable integration of up to four genes at a single specific genomic location. The stable integration of DNA elements of choice at specific genomic locations and the testing of candidate genes for phenotypic effects without disturbance by clone specific variation is getting feasible with these methods. http://dx.doi.org/10.1016/j.nbt.2014.05.959 PU-39 Construction of pH-sensitive Her2 binding antibody fragment by directed evolution using yeast display Elisabeth Lobner ∗ , Michael Traxlmayr, Florian Rüker, Christian Obinger University of Natural Resources and Life Sciences, Vienna, Austria The half-life of therapeutic antibodies, which are internalized together with their antigenic receptor, can be increased by decreasing their affinity at acidic endosomal conditions. Here, a directed evolution protocol was developed for construction of pHdependent binding sites by using yeast display. The C-terminal structural loops of an antigen binding crystallizable fragment of immunoglobulin G1 (Fcab) [1] have been engineered for reduced binding to the extracellular domain of human epidermal growth factor receptor 2 (Her2-ECD) at pH 6 compared to pH 7.4. A library based on a Her2-ECD binding lead Fcab was constructed by parsimonious mutagenesis and displayed on yeast. Alternating selections for binding at pH 7.4 and non-binding at pH 6.0 were performed by FACS probing the binding to the antigen as well as a structurally specific ligand. The three best performing variants (P1, P2, P3) were selected. Displayed on yeast they showed clear pH-dependent binding to soluble Her2-ECD (decrease in affinity at pH 6.0 compared to pH 7.4). Additionally, solubly expressed P1, P2 and P3 exhibited pH-dependent interactions with Her2positive cells whereas their conformational and thermal stability was pH-independent. The interaction of P1, P2 and P3 with the neonatal Fc receptor remained wild-type like showing the inverse pH-dependence compared to Her2-ECD binding. Interestingly, two of the three Fcabs did not contain a single histidine mutation but all of them contained variations next to histidines that already occurred in loops of the lead Fcab. Reference [1].Wozniak-Knopp, et al. Protein Eng Des Sel 2010;23:289–97. http://dx.doi.org/10.1016/j.nbt.2014.05.960 PU-40 The employment of a heterologous yeast expression system for production of VP1-derived virus-like particles originated from novel human polyomaviruses Alma Gedvilaite ∗ , Milda Norkiene, Rita Lasickieke Vilnius University Institute of Biotechnology, Lithuania Virus-like particles (VLPs) resemble their parent virion in structure, immunogenicity, tropism and transduction efficiency, but do not contain any viral genetic material. They can be used for diagnostic purposes, vaccination and gene therapy. Polyomaviridae is a growing family of naked, double-stranded DNA viruses that infect birds and mammals. The major capsid protein VP1 of all polyomaviruses (PyV) is sufficient for assembly of VLPs and represents the major immunogenic protein of PyV. In the last few years, the human polyomavirus (HPyV) family has expanded to 12 members. Serological studies are the primary tool to investigate the prevalence of various polyomaviruses in human populations. The recombinant VP1 VLPs are particularly valuable for the serological detection of these viruses as many PyV cannot be easily cultured. The earlier discovered PyV VP1-derived VLPs were successfully produced using different eukaryotic and prokaryotic expression systems including yeast. Here, we report that the galactoseinducible yeast S. cerevisiae expression system is efficient for high-level production and self assembly of VP1 derived from new HPyV: KIPyV, WUPyV, Merkel cell PyV, HPyV6 and HPyV7. The formation of empty VP1-derived VLPs was confirmed by cesium chloride ultracentrifugation, agarose gel electrophoresis and electron microscopy. Yeast-generated VP1 VLPs were free of toxins, host cell DNA and proteins. The purified VP1 VLPs originating from KIPyV, WUPyV, Merkel cell PyV, HPyV6 and HPyV7 were successfully used for generation of monoclonal antibodies and might be useful for the generation of new diagnostic tools, antiviral vaccines or gene delivery systems. http://dx.doi.org/10.1016/j.nbt.2014.05.961 PU-41 Purification by affinity chromatography of recombinant L-asparaginase I from Saccharomyces cerevisiae expressed in Escherichia coli Adalberto Pessoa-Jr 1,∗ , Gisele Monteiro 1 , Joao Santos 2 , Johanna Oses 1 , Albert Peixoto 3 , Juan Santos 1 , Joao Molino 1 , Laura Oliveira 4 , Joao Coutinho 2 , Sonia Ventura 2 , Andre Lopes 1 1 University of Sao Paulo, Brazil University of Aveiro, Brazil 3 Universidade Estadual do Sudoeste da Bahia, Brazil 4 University of Campinas, Brazil 2 L-Asparaginase is known by its capacity to catalyse the hydrolysis of L-asparagine into L-aspartic acid and ammonia. This enzyme has been clinically acceptable as an anti-tumour agent for treatment of acute lymphoblastic leukemia and lymphosarcoma. This biopharmaceutical is produced by fermentation processes and subsequently it needs to be correctly separated from the contaminants www.elsevier.com/locate/nbt S199 RECOMBINANT PROTEIN PRODUCTION from the fermentation broth. Here, Affinity Chromatography is applied as a novel approach to purify (His)6-tagged recombinant L-asparaginase I from Saccharomyces cerevisiae expressed in Escherichia coli. The Affinity Chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using a Ni2+ charged, 5 mL HiTrap IMAC FF. A linear gradient of 0 mM to 500 mM of imidazole at 5.0 mL min−1 was applied in order to identify the lowest imidazole concentration of the elution buffer that extracts the higher amount of L-asparaginase. This concentration range was further used in a step imidazole gradient. Two-step concentrations of the initial imidazole concentration (32.0 and 54.4%) were applied. Polyacrylamide gel electrophoresis combined with silver staining and Nessler activity assay were used to confirm the presence of the purified L-asparaginase (≈45 kDa). In the gradient step, the eluted recombinant L-asparaginase showed a high specific activity of 110.1 ± 0.3IU mg−1 . Furthermore, a recovery of 81.00 ± 0.01% was obtained resulting in a purification factor of 17. In this respect, the FPLC process has undoubtedly proved to be an efficient tool for the purification of this enzyme. Acknowledgements: This research was supported by CAPES, CNPq (301248/2010-9) and FAPESP (2013/08617-7), Brazil; FEDER funds through the program COMPETE/FCT (PestC/CTM/LA0011/2013); SFRH/BPD/79263/2011; and Santander. http://dx.doi.org/10.1016/j.nbt.2014.05.962 New Biotechnology · Volume 31S · July 2014 heterologous protein secretion, possibly by cluttering the ER with an overload of unfolded proteins. http://dx.doi.org/10.1016/j.nbt.2014.05.963 PU-43 Expression of C-terminal Fab fragment variants: the host determines the best variant Brigitte Gasser 1 , Rebecca Diethard Mattanovich 1 Goengrich 1,∗ , Christoph Kiziak 2 , 1 Department of Biotechnology, BOKU University of Natural Resources and Life Sciences, Vienna, Austria 2 Lonza AG, Visp, Switzerland Expression of antibody fragments is routinely done in microbial host cells such as the bacterium Escherichia coli or the yeast Pichia pastoris. In yeast, Fabs are usually secreted to the cell supernatant after passing the secretory pathway, while Fab expression in E. coli is usually done in the periplasm. Different Fab variants have been reported in literature, however, it has not yet been comprehensively assessed which work best in which host. Here we compare different Fab variants expressed in E. coli and P. pastoris, which resulted in a preference for different variants in each host. http://dx.doi.org/10.1016/j.nbt.2014.05.964 PU-42 Analysis of modifications in the ER protein quality control system on heterologous protein expression in Saccharomyces cerevisiae PU-44 Jorg de Ruijter ∗ , Essi Koskela, Alexander Frey Cell-free incorporation of unnatural amino acids for cloning-independent engineering of elastin-like polypeptides Aalto University, Finland Dong-Myung Kim 1,∗ , Su-Jin Oh 2 , Young-A Son 3 The yeast Saccharomyces cerevisiae is a widely used host in the production of therapeutic glycoproteins. However, the expression of high levels of heterologous proteins is known to interfere with various processes in the cells and can trigger stress responses. High volume production of heterologous glycoproteins is prone to overflow the ER with unfolded proteins. Glycoproteins that remain unfolded in the ER for too long are predisposed to be targeted for degradation by components of the ER associated degradation (ERAD) pathway. In this present study, the ERAD pathway was modified by deleting genes encoding various components, with and without a blocking of the unfolded protein response pathway (UPR). In addition, overexpression of the folding chaperones KAR2, PDI1, ERO1 and CPR5 was introduced. These proteins are expected to assist in the quick folding of proteins to decrease the blocking of the ER by unfolded proteins. The production levels of a human IgG1 molecule of these mutant strains were analyzed using ELISA. Since changing the ER quality control system might lead to a decreased quality of proteins, the quality of the produced IgG1 was analyzed by PAGE. The results at hand indicate that deletion of early ERAD factors is detrimental for protein production, as that removal of later stages leads to an increase. For the folding chaperones, control of expression levels is key, as high level expression tend to decrease 1 S200 www.elsevier.com/locate/nbt Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, South Korea 2 Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University of Korea, South Korea 3 Department of Advanced Organic Materials Engineering, Chungnam National University of Korea, South Korea The physicochemical nature of a protein is determined by the collective properties of consisting amino acids and their sequential order in the protein molecule. Although natural proteins exhibit a wide array of structure and biological functions with the concise set of 20 canonical amino acids, the ability to introduce unnatural amino acids into protein molecules will further expand the diversity, creating protein species that have never been explored. In this study, we demonstrate the use of a cell-free protein synthesis system as a versatile platform for synthesis of engineered bio-materials through the incorporation of unnatural amino acids into recombinant proteins. Elastin-like-polypeptides (ELPs) were chosen as a model protein to investigate the effects of unnatural amino acid incorporation. Herein, we discuss on the manipulation of the transition temperature of ELPs through cell-free incorporation of proline analogues. Through the replacement of proline RECOMBINANT PROTEIN PRODUCTION New Biotechnology · Volume 31S · July 2014 with a variety of its structural analogues, we were able to tune the transition temperature of ELPs. http://dx.doi.org/10.1016/j.nbt.2014.05.965 PU-45 Enhancing recombinant protein production with an Escherichia coli host strain lacking insertion sequences Sun-Chang Kim , Myung Keun Park, Jun Hyoung Lee, Kyung Seok Yang ∗ Korea Advanced Institute of Science and Technology, South Korea The genomic stability and integrity of host strains are critical for the production of recombinant proteins in biotechnology. Bacterial genomes contain numerous jumping genetic elements, the insertion sequences (ISs) that cause a variety of genetic rearrangements, resulting in adverse effects such as genome and recombinant plasmid instability. To minimize the harmful effects of ISs on the expression of recombinant proteins in Escherichia coli, we developed an IS-free, minimized E. coli strain (MS56) in which about 23% of the genome, including all ISs and many unnecessary genes, was removed. Here, we compared the expression profiles of recombinant proteins such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and bone morphogenetic protein-2 (BMP2) in MG1655 and MS56. Hopping of ISs (IS1, IS3, or IS5) into the TRAIL and BMP2 genes occurred at the rate of ∼10−8 /gene/h in MG1655 whereas such events were not observed in MS56. Even though IS hopping occurred very rarely (10−8 /gene/h), cells containing the IS-inserted TRAIL and BMP2 plasmids became dominant (∼52% of total population) 28 h after fermentation due to their growth advantage over cells containing intact plasmids, significantly reducing recombinant protein production in batch fermentation. Our findings clearly indicate that IS hopping is detrimental to the industrial production of recombinant proteins, emphasizing the importance of the development of IS-free host strains. http://dx.doi.org/10.1016/j.nbt.2014.05.966 PU-46 Analysis of amino acid residues in the N-terminal region of the beta-neurotoxin CssII and its specificity for voltage-gated Na+ channel subtypes Rodrigo Ibarra Vega 1,∗ , Juana María Jiménez Vargas 2 , Rita Restano Cassulini 2 , Georgina Estrada 3 , Lourival D. Possani 2 , Gerardo Corzo 2 1 Instituto de Biotecnología, Universidad Nacional Autónoma de México, UNAM, Mexico 2 Instituto de Biotecnología de la Universidad Nacional Autónoma de México, UNAM, Mexico 3 Centro de investigación Científica de Yucatán, AC. (CICY), Mérida, Yucatán, México, Mexico The scorpion toxin CssII from the scorpion Centruroides suffusus is a peptide that affects the voltage-gated sodium channel Nav 1.6. The CeII9 peptide toxin of Centruroides elegans is 86% identical to CssII but selectively affects Nav 1.4. The most significant variation between these two peptide toxins is observed at the N-terminus, specifically in residues 7 and 8. Therefore, these two residues in CssII were substituted for residues corresponding to the peptide CeII9 to evaluate if the CssII variants keep or shift their selectivity for the Nav1.6 or Nav1.4, respectively. Three CssII variants were generated by site-directed mutagenesis, named CssII K8H, CssII S7N and CssII S7N/K8H. Since the CssII native toxin has an amidation at the C-terminal and this amidation is important for binding the Navs, the aforementioned variants were constructed but including basic charges at the C-terminal (T64R/N66R). These substitutions improve the affinity of the CssII recombinant toxins for the Nav1.6. The synthetic genes were cloned into the pQE30 vector and expressed in E. coli BL21 strain. The recombinant variants had an N-terminal fusion peptide composed of a 6His-tag and a FXa proteolytic cleavage region. The mutants were purified, folded in vitro, digested with FXa and electrophysiological assayed on HEK293 cells stably expressing the Nav1.4 and Nav1.6 channels. http://dx.doi.org/10.1016/j.nbt.2014.05.967 PU-47 Expression of recombinant formate dehydrogenase in Escherichia coli and its immobilization onto magnetic nano-particles Stanislav Stuchlik 1,∗ , Lukas Hason 2 , Zdenko Levarski 2 , Lucia Bocanova 2 , Kristina Jirickova 2 , Diana Hopkova 2 , Pavol Kois 3 , Jan Turna 2 1 Comenius University in Bratislava, Department of Molecular Biology, Slovakia 2 Comenius University in Bratislava, Faculty of Natural Sciences, Department of Molecular Biology, Slovakia 3 Comenius University in Bratislava, Faculty of Natural Sciences, Department of Organic Chemistry, Slovakia The enzymatic bioconversion of aromatic compounds in food and cosmetic industry is often accompanied by the limitation in www.elsevier.com/locate/nbt S201 RECOMBINANT PROTEIN PRODUCTION terms of co-factor depletion or its insufficient regeneration. To address this issue, we have constructed an efficient E. coli based expression system capable of producing functional yeast formate dehydrogenase (FDH), an enzyme used for the regeneration of oxidized NAD+, during conversion of trans-2-hexenal to trans2-hexenol catalyzed by NAD-dependent alcohol dehydrogenase (ADH). To further improve this reaction, we have immobilized FDH onto magnetic nano-particles and tested the ability of produced enzyme to improve activity of ADH by NAD+/NADH conversion. The activity of FDH has been tested by applying various physical and chemical condition changes. The results show that although the activity of the immobilized FDH is decreased compared to the free enzyme, its stability is increased significantly allowing prolonged incubations or repeated use of a single batch. Acknowledgements: This work was supported by the Slovak Research and Development Agency grant APVV-0061-11 and is also result of the “Production of biologically active agents based on recombinant proteins” (ITMS 26240220048) project implementation supported by the Research and Development Operational Program funded by the ERDF. http://dx.doi.org/10.1016/j.nbt.2014.05.968 PU-48 Production of selenoproteins in yeast via genetically encoded incorporation of a photocaged selenocysteine Rasa Rakauskaite ∗ , Viktoras Masevicius, Giedre Urbanaviciute, Audrone Ruksenaite, Saulius Klimasauskas Vilnius University, Institute of Biotechnology, Lithuania Recent advancements in protein engineering materialized in a number of valuable products for pharmaceutical, industrial, and research applications. One promising way to achieve advanced engineered protein products is based on targeted incorporation of rare and unnatural amino acids. For instance, selenocysteine (Sec) provides a selenium atom with unique chemical characteristics (higher nucleophilicity, lower pKa, and lower redox potential) not attainable in common proteins. Although Sec is of significant technological importance as a component of both natural proteins and designed biocatalysts, the availability of such proteins is hampered by technical limitations. Here we report a universal method for production of recombinant selenoproteins in Saccharomyces cerevisiae cells via genetically encoded SECIS-independent incorporation of a photocaged unnatural amino acid, 4,5-dimethoxy-2-nitrobenzyl selenocysteine (DMNBSec). Photodeprotection of the incorporated residue in the target selenoprotein can be performed under mild conditions. The new approach was validated by inserting a Sec residue i) at a permissive position (Tyr39) of enhanced green fluorescent protein (EGFP) and ii) replacing an essential catalytic cysteine (Cys103) of a bacterial DNA cytosine-5 methytransferase, M.HpaII. Photodeprotection of the incorporated DMNB-Sec residue in the model EGFP protein demonstrated light-controlled protein dimerization and efficient Sec-specific covalent labeling in vitro. This method has a potential S202 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 to simplify the synthesis of selenoproteins and therefore expand their biotechnological and biopharmaceutical utilization. http://dx.doi.org/10.1016/j.nbt.2014.05.969 PU-49 Develop new tools for investigation of albumin transportation Dinh Khoi Nguyen Ly 1,∗ , Philip Poronnik 2 , David NikolicPatterson 3 1 HiRi RMIT Australia University of Sydney Australia 3 Monash Medical Center 2 Albumin is most abundant protein in blood and plays important homeostatic roles in the regulation of oncotic pressure and as a carrier of many small proteins, peptides and hormones. Albumin in the circulation can be covalently modified at surface lysine residues and thus the circulation contains a mixture of native albumin (i.e. not modified) and modified albumin. A significant amount of albumin is lost from the blood each day as a result of the filtration occurring in the capillaries of the renal glomeruli. This filtered albumin in the early filtrate is thought to be mostly taken up by and degraded in the early renal proximal tubular epithelial cells, while a small proportion is excreted in the final urine. This degradation of albumin by proximal tubules operates via the megalin scavenger receptor and an endocytic pathway. A major gap in our knowledge is whether the megalin-based albumin uptake is a non-selective process, or whether megalin has a significantly different affinity for modified versus native albumin. Using yeast expression system, we develop radioactive native recombinant albumin. The uptake experiment using 14 CrHA suggest the uptake pathway of native and modified albumin are different in opossum kidney cell line. The uptake of 14 C-rHA is not megalin dependent. http://dx.doi.org/10.1016/j.nbt.2014.05.970 STRESS RESPONSES New Biotechnology · Volume 31S · July 2014 Stress responses PV-02 PV-01 Overview of the biomedical research in the newly established Laboratory of Genome Integrity at the Palacky University, Olomouc Preliminary analysis of DNA isolated from flax grown in the radio-contaminated Chernobyl area for six generations suggests the stability of fatty acid desaturase genes Veronika Lancíková 1,∗ , Jana Žiarovská 2 , Maksym Danchenko 3 , Valentyna Berezhna 4 , Milan Bežo 2 , Katarína Ražná 2 , Namik Rashydov 4 , Martin Hajduch 5 1 Institute of Plant Genetics and Biotechnology of Slovak Academy of Sciences, Slovakia 2 Department of Genetics and Plant Breeding, Faculty of Agrobiology and Food Resources, Slovak University of Agriculture, Slovakia 3 Institute of Virology, Slovak Academy of Sciences, Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Slovakia 4 Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Slovakia 5 Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Institute of Virology, Slovak Academy of Sciences, Slovakia Plants have the ability to survive and reproduce under changed environmental conditions which were induced by biotic and abiotic stress. The area of the Chernobyl nuclear accident is a very interesting example of plant adaptation to abiotic stress in the form of increased radiation. Two experimental fields were established in the Chernobyl region – radio-contaminated and non-radioactive. Flax (Linum usitatissimum L., variety Kyivskyi) was cultivated in these experimental fields since 2007, and during the 2012 the sixth generation of seeds was collected. The aim of the presented study was to analyze fatty acid desaturase (FAD) genes in the sixth generation of flax, specifically FAD3A and FAD3B, using the restriction fragment length polymorphism technique. In a biosynthetic pathway of fatty acids, these genes perform an important function because they encode conversion of linoleic acid to ␣-linolenic acid. This study analyzed three parts of FAD3A gene with following length of the nucleotide sequences 1201, 1500, 1924 base pairs. Also, FAD3B gene was divided into three parts with length of the nucleotide sequences 1246, 1330, 1279 base pairs. For restriction analysis four different restriction enzymes were applied – MnlI for restriction digestion of the first part of FAD3A and FAD3B genes, NlaIII for the second and third part of FAD3A gene, AciI for the second and Hpy188I for the third part of FAD3B gene. Based on these preliminary data, no polymorphism of FAD genes in sixth flax generation was detected. However, the stability of FAD genes will be further verified by more detailed analyses. http://dx.doi.org/10.1016/j.nbt.2014.05.971 Zuzana Loubalová ∗ , Kamila Jahodíková, Martin Liptay Institute of Molecular and Translational Medicine Palacky University Olomouc, Czech Republic The Laboratory of Genome Integrity (LGI) is part of the recently opened Institute of Molecular and Translational Medicine belonging to the Palacky University in Olomouc, Czech Republic. The institute was built with the aim to conduct cutting edge research in the field of drug design and discovery. The collaborative network at the institute covers all crucial phases of the drug design process including search for novel drug targets. The research in the LGI is focused mainly on various mechanistic aspects of the DNA damage response and DNA repair pathways. Both pathways play a key role in the onset and progress of cancer and thus represent ideal targets for novel anti-cancer drugs. In addition, we have been trying to understand how elevated replication stress in rapidly growing pre-cancerous and cancerous cells affects the progression of the disease. We would also like to understand in detail how a cell copes with the replication stress at the molecular level. Our next long term goal is to set up specific DDR response based high throughput screens capable of assessing effect of different compounds and environment on DNA integrity in human cells. Apart from basic research, we have been also developing novel research and diagnostic tools, such as devices for more efficient chromosome spreading and non-laborious mammalian cell synchronization. http://dx.doi.org/10.1016/j.nbt.2014.05.972 PV-03 Acanthopanax sessiliflorus stem confers increased resistance to environmental stresses and lifespan extension in Caenorhabditis elegans Sang-Kyu Park, Jin-Kook Park ∗ , Chul-Kyu Kim, Sang-Ki Kong, AReum Yu, Mi-Young Lee Soonchunhyang University, South Korea Acanthopanax sessiliflorus is a native Korean plant and used as traditional medicine or an ingredient in many Korean foods. The free radical theory of aging suggests that cellular oxidative stress caused by free radicals is the main cause of aging. Free radicals can be removed by cellular anti-oxidants. Here, we examined the anti-oxidant activity of Acanthopanax sessiliflorus extract and its lifespan-extending effect in Caenorhabditis elegans. Oxidative DNA damage was reduced and survival under oxidative-stress conditions was significantly enhanced by Acanthopanax sessiliflorus stem extract. In addition, Acanthopanax sessiliflorus stem increased resistance to other environmental stresses, including heat shock and ultraviolet irradiation. Treatment with Acanthopanax sessiliflorus stem extract significantly extended both mean and maximum www.elsevier.com/locate/nbt S203 STRESS RESPONSES lifespan in C. elegans. However, fertility was not affected by Acanthopanax sessiliflorus stem. In conclusion, Acanthopanax sessiliflorus stem had strong anti-oxidant activity and conferred a longevity phenotype without reduced reproduction in C. elegans, which provides conclusive evidence to support the free radical theory of aging. http://dx.doi.org/10.1016/j.nbt.2014.05.973 New Biotechnology · Volume 31S · July 2014 PV-05 Determination of reliable house keeping gene(s) for qPCR in maize under different boron dosages Hasan Can 1,∗ , Mehmet Hamurcu 1 , tijen Demiral 2 , Anamika Pandey 1 , Mohd Kamran Khan 1 , Seyit Ali Kayıs 1 , Zuhal Zeynep Avsaroglu 1 , Nimet Can 2 , Sait Gezgin 1 , Erdogan Esref Hakki 1 1 2 PV-04 Transcriptome characteristic of echiuran worm Urechis unicinctus exposed to sulfide by digital gene expression analysis Zhifeng Zhang ∗ , Litao Zhang, Xiaolong Liu Ocean University of China, China Sulfide is a well-known toxicant. However, some organisms can tolerate and utilize sulfide. In this study, Urechis unicinctus, inhabiting coastal sediment and tolerating high concentration sulfide, was used to examine its transcriptional profile in response to 50 ␮M sulfide for 24 h by digital gene expression analysis. A total of approximately 16 million cDNA tags were sequenced and 3,909,160 and 4,057,279 clean tags were obtained in the control and 24 h libraries. Compared with the 0 h library, 1181 tag-mapped genes were detected as differentially expressed genes (DEGs) in the 24 h library. The DEGs were further subjected to GO and pathway analysis. More than 80% pathways were rarely reported to be related to sulfide stress before. Three key physiological actions induced by sulfide involving in energy metabolism, DNA damage response and inflammation were discussed in details. To our best knowledge, this study is the first transcriptome-wide effort to reveal the transcriptional response to sulfide stress by digital gene expression analysis and the identified DEGs can serve as a basis for further understanding of sulfide roles in organisms [1,2]. Acknowledgement: This work was supported by the Natural Science Foundation of China (31372506). Selcuk University, Turkey Harran University, Turkey Maize is one of the major cereal crops in the world. In Turkey a production of 5.9 million tons was recorded in the year 2013 and became third most important crop of the country. Production of maize has recently increased in Central and South Anatolian regions of Turkey. However, boron toxicity has emerged as a major crisis of the Central Anatolian Soil affecting the maize yield and product quality. For solving this important issue, different approaches, including boron-tolerant variety development and use, are implemented. Understanding the mechanism of boron tolerance in plants is an active research area. Different genes are known to be involved in this mechanism. Tracking properly the expression patherns of these genes requires normalization of the expressions using constantly expressed reference genes. The main aim of our research was to determine reliable house keeping genes for qPCR in maize under the effect of various boron concentrations. Initially, we focussed on the determination of boron tolerant commercial hybrid varieties. Further, experiments have been made for the determination of the gene/genes with constant expression level among the 6 candidates reference genes namely, S18, alpha-tubulin, globulin S, GAPDH, ubiquitin, beta2 tubulin. Results were computed using efficient software programs like geNorm and NormFinder. Two genes, S18 and GAPDH, were found to be the most consistently expressed genes under implemented boron stress conditions. Acknowledgement: This study was financially supported by The Selcuk University Scientific Research Projects Funding Unit (Project No: 13401089). References [1].Tamizhselvi R, et al. Preprotachykinin-A gene deletion regulates hydrogen sulfide-induced toll-like receptor 4 signaling pathway in cerulein-treated pancreatic acinar cells. Pancreas 2011;40:444–52. [2].Janssens S, Tinel A. The PIDDosome, DNA-damage-induced apoptosis and beyond. Cell Death Differentiation 2012;19:13–20. http://dx.doi.org/10.1016/j.nbt.2014.05.974 http://dx.doi.org/10.1016/j.nbt.2014.05.975 PV-06 Gene expression profiling of Thermoplasma volcanium under extreme stress conditions Sema Zabcı ∗ , Semra Kocabiyik Middle East Technical University, Turkey Identifying the stress response mechanisms in Archaea might have potential applications considering the industrial importance of their enzymes. In this study, as an extension of our research on anti-stress mechanisms, we have carried out genome wide transcriptome analysis of Thermoplasma volcanium (grows optimally at pH 2.7 and 60 ◦ C) under extreme stress (i.e., heat- shock at 68 ◦ C, hydrogen peroxide, 0.03 mM H2 O2 and pH stress at pH 5.0) using Agilent Custom Gene Expression Microarray. The data was analyzed by using GeneSpring Ver 12.6 Software. S204 www.elsevier.com/locate/nbt STRESS RESPONSES New Biotechnology · Volume 31S · July 2014 With a pH shift from 2.7 to 5.0 most of the gene expression was repressed: of 47 genes differentially expressed by ≥2-fold, 66% were up-regulated and 34% were down-regulated. Among the notably induced genes, some code for membrane proteins, sugar and amino acid transporters, cation transporter and antiporters which are crucial for maintaining cellular pH homeostasis. Oxidative stress mostly resulted in decreased gene expression; out of 452 genes expressions of which changed ≥2 fold, 75% were down regulated. Up regulation of ferrodoxins, oxidoreductases, thioredoxin, Fe-S oxidoreductases, aldo/keto reductases and dehydrogenases as a response to H2 O2 may be important for maintaining intracellular redox potentials and detoxifications. Temperature up-shift favored the expression of heat-shock proteins (including GroEL, sHSP, Dna K, DnaJ, GrpE) as well as genes related to carbohydrate metabolism (e.g., sugar permease). Acknowledgement: Supported by the Intelligent Synthetic Biology Global Frontier Program, and the Next-Generation BioGreen 21 Program. http://dx.doi.org/10.1016/j.nbt.2014.05.977 PV-08 The role of S-nitrosoglutathione reductase in defence response of tobacco plants and cells to elicitins Tereza Jendrišáková 1,∗ , Pavla Moricová 1 , Martina Železná 1 , Lenka Luhová 1 , Jan Lochman 2 , Tomáš Kašparovský 2 , Marek Petřivalský 1 1 2 http://dx.doi.org/10.1016/j.nbt.2014.05.976 PV-07 Stress responsive global regulatory mechanisms in the methylotrophic yeast Hansenula polymorpha Ohsuk Kwon 1,∗ , Eun Hye Kim 1 , Doo-Byoung Oh 1 , Hyun Ah Kang 2 1 Korea Research Institute of Bioscience and Biotechnology, South Korea 2 Chung-Ang University, South Korea The thermotolerant methylotrophic yeast Hansenula polymorpha has been regarded as an attractive model organism for fundamental studies of methanol metabolism, peroxisome biogenesis and function, nitrate assimilation, and protein glycosylation. H. polymorpha has become one of the promising hosts for the production of recombinant proteins on an industrial scale owing to the availabilities of strong inducible promoters and a multicopy integration system for target protein expression cassettes into the genome. In addition, recently H. polymorpha is gaining increasing interest for its several peculiar physiological characteristics, such as resistance to heavy metals and oxidative stress, and thermotolerance since these properties are advantageous for various biotechnological applications. However, only a limited amount of information is available for the global regulatory mechanisms for stress response in this organism. In the present study, we investigated the roles of representative signaling and regulatory proteins to understand the regulatory mechanisms governing the osmotic and oxidative stress responses of H. polymorpha. The hybrid histidine sensor kinases Sln1 and Nik1, histidine-containing phosphotransfer protein Ypd1, response regulator proteins Skn7 and Ssk1, high osmolarity glycerol pathway regulator Hog1, and oxidative stress response regulator Yap1 were functionally characterized by mutant construction, growth phenotype comparison, in vitro protein phosphorylation, and comparative transcriptome analysis. Obtained results indicate that the Skn7/NiK1-Ypd1-Skn7/Ssk2 two-component signal transduction pathway plays a critical role in oxidative, osmotic, and cell wall stress responses in H. polymorpha. Palacky University Olomouc, Czech Republic Masaryk University Brno, Czech Republic Nitric oxide (NO) is an important signalling molecule which participates in the plant immune responses. S-nitrosoglutathione reductase (GSNOR) is known as key enzyme in NO metabolism through cysteine S-nitrosylation, the post-translational modification mediated by reactive nitrogen species (RNS). GSNOR belongs to the family of alcohol dehydrogenases class III (ADH3; EC 1.1.1.1). GSNOR is highly specific for the S-nitrosoglutathione substrate and was demonstrated to indirectly control the total level of protein S-nitrosothiols. Elicitins, extracellular proteins secreted by oomycete pathogens of Pythium and Phytophthora spp., represent effective elicitors that trigger defence responses in plant-pathogen interactions. The aim of this work was to study a possible GSNOR role in defence response of tobacco plants and cell culture to selected elicitins. We tested cryptogein and its mutated forms V84F and L41F with different ability to trigger the formation of reactive oxygen species (ROS) and cell necrosis. Oligandrin, another elicitin from Pythium oligandrum, does not elicit hypersensitive reaction in plant tissue. The production of RNS and ROS was determined after elicitin application using specific fluorescent probes. Cryptogein and V84F mutant triggered significantly increased ROS level, which correlated with decreased cell viability. On the contrary, little effect of oligandrin and mutant L41F on growth was observed, whereas higher NO production was induced. Increased activity and expression of GSNOR were detected after application of cryptogein and V84F mutant. The results bring new insight into the involvement of ROS and RNS in plant response to elicitors and possible regulatory role of GSNOR in control of protein S-nitrosylation. http://dx.doi.org/10.1016/j.nbt.2014.05.978 www.elsevier.com/locate/nbt S205 STRESS RESPONSES PV-09 Sensitivity of selected root hair mutants of Arabidopsis thaliana to abiotic stress Lenka Vaškebová 1,∗ , Barbora Šatná 1 , Miroslav Ovečka 2 , Jozef Šamaj 2 1 2 Palacky University in Olomouc, Czech Republic Centre of the Region Haná, Olomouc, Czech Republic Plant growth requires the continuous uptake of solutes and minerals from the rhizosphere by the root as well as maintenance of ion homeostasis and avoidance of intracellular ion toxicity. Plants also possess effective molecular, physiological and developmental mechanisms to minimize harmful effects of external abiotic stress factors. Root hairs are tubular extensions of root epidermal cells, and they are formed by specialized tip growth. Since root hairs effectively extend surface area of the root, they could play significant role in plant sensitivity to abiotic stresses originating from the soil. Arabidopsis thaliana root hair mutants such as rhd2(root hair defective 2, lacking the activity of the NADPH oxidase AtRBOH C, Takeda et al., 2008) and tip1 (tip growth defective 1, inactive in S-acyl transferase, Hemsley et al., 2005) are defective in the tip growth. This suggests their possible involvement in root stress sensitivity. We studied effects of abiotic stresses on root growth and root hair development in certain stages of stress perception in these mutants. In general, root growth of rhd2-1 mutant was well comparable to Col-0 wild type, while root growth of tip1-1 mutant was weaker. Upon application of diverse abiotic stresses, rhd2-1 mutant showed similar sensitivity as Col-0 and all lines showed a comparable sensitivity to salt and oxidative stress. Stress responses were tested also in Arabidopsis plants stably overexpressing mitogenactivated protein kinase kinase SIMKK from Medicago sativa. These results contribute to better elucidation of root hairs as integral parts of the root developmental program in changing environment. http://dx.doi.org/10.1016/j.nbt.2014.05.979 PV-10 Sources and repair of nitrosative stress in Escherichia coli Basema Balasiny ∗ , Claire Vine, Jeff Cole University of Birmingham, United Kingdom Escherichia coli is a Gram negative, facultatively anaerobe. Inside the mammalian gut, E. coli utilises various alternative electron acceptors to respire efficiently when oxygen is unavailable. To survive, it must defend itself against reactive nitrogen species (RNS) generated as products of its own metabolism, or the innate immune response of the host. Under anaerobic conditions, nitrate reductase NarGHI generates nitric oxide (NO) as a side product during reduction of nitrate via nitrite to ammonia. However, some NO is formed from nitrite even by mutants that lack NarGHI. Nitric oxide is an extremely toxic gas that damages metal centres of proteins, especially iron-sulphur proteins such as aconitase and fumarase. Several studies have reported that the di-iron protein S206 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 YtfE has a crucial role in repairing iron-sulphur centres damaged by nitrosative stress conditions [1–3]. This poster will show that a major role of YtfE is to release nitric oxide that has become bound to proteins such as aconitase and fumarase, thereby restoring their activity under conditions of nitrosative stress. References [1].Justino MC, Almeida CC, Goncalves VL, Teixeira M, Saraiva LM. Escherichia coli YtfE is a di-iron protein with an important function in assembly of iron-sulphur clusters. FEMS Microbial Lett 2006;257:278–84. [2].Justino MC, Almeida CC, Teixeira M, Saraiva LM. Escherichia coli diIron YtfE protein is necessary for the repair of stress-damaged ironsulphur clusters. J Biol Chem 2007;282:10352–9. [3].Overton TW, Justino MC, Li Y, Baptista JM, Melo AMP, Cole JA, Saraiva LM. Widespread distribution in pathogenic bacteria of di-iron proteins that repair oxidative and nitrosative damage to ironsulphur centers. J Bacteriol 2008;190:2004–13. http://dx.doi.org/10.1016/j.nbt.2014.05.980 PV-11 Nitrosative stress response in Escherichia coli Jing Wang ∗ , Claire Vine, Jeff Cole University of Birmingham, United Kingdom Bacteria encounter various stress as the living environment changes. For instance, in anaerobic growth conditions when nitrite or nitrate is used as electron acceptor, nitric oxide (NO) is generated during the reduction process. Up to present, three NO reductases, Nrf, Nor and Hmp have been reported in the bacterium Escherichia coli. A recent microarray study revealed several genes that are highly up-regulated during nitrosative stress, one of which was hcp, a gene that encodes a hybrid cluster protein (HCP) with a unique structure. We tested two E. coli strains, one lacking all the above mentioned NO reductases and one with a further deletion of hcp. The hcp mutation decreased nitrosative stress tolerance of E. coli. A second gene, hcr, is co-transcribed with and located in the same operon as hcp. We detected a positive protein interaction between HCR and HCP in vivo using the bacterial two-hybrid system. This supports the hypothesis that HCP might function as another NO detoxifying enzyme, with HCR being a reductase of HCP. We subjected the two strains to NO stress and observed that in our control strain NO was reduced to N2 O, probably via a reaction catalysed by HCP. Our data demonstrated that HCP is essential for the reduction of low concentrations of NO to N2 O. http://dx.doi.org/10.1016/j.nbt.2014.05.981 STRESS RESPONSES New Biotechnology · Volume 31S · July 2014 PV-12 Molecular markers based approach for genetic improvement of tea (Camellia sinensis) Rahul Kumar 1,∗ , Himanshu Sharma 2 , Sanatsujat Singh 2 , Rakesh Kumar Sud 2 , Arvind Gulati 2 , Paramvir Singh Ahuja 2 , Ram Kumar Sharma 2 1 2 DAV University, India CSIR-IHBT, Palampur, HP, India Tea, due to multiple health benefits with various flavors and antioxidant properties is the most consumed beverage across the globe and becomes an important agro-based revenue source for many countries in the world including India, which ranks first in black tea production. Genetic improvement studies, however, have been limited in tea due to non-availability of sequence based co-dominant microsatellite markers. Novel microsatellite markers were identified through construction and sequencing of enriched genomic clones and mining of public expressed sequence data in tea. These markers along with other dominant DNA markers like AFLP and RAPD were utilized for genetic mapping using pseudo-test cross population comprising of 212 individuals derived through crossing between parental clones SA6 (resistant to blister blight disease) and Asha (susceptible). Of one thousand microsatellite markers identified, 576 recorded successful amplification in selected tea accessions including parental lines. Informative 80 SSR markers along with 102 AFLP and 160 RAPD were used for construction of linkage map in tea. Following a pseudo test cross approach, in total, 400 markers loci (292 AFLP, 81 RAPD, 27 SSR) were found to be segregating in a test cross ratio and 127 of these could be linked to 17 linkage groups covering a total length of 1659 cM. Novel SSRs enriched the limited repertoire of co-dominant markers in tea and will be further utilized in saturating the linkage map and subsequently in QTL analysis and MAS in tea. tolerance in monocot. Here, we report a new member of the chitinase family 19, designated as LcChi2, isolated from Chinese wildrye (Leymus Chinensis). Sequence analysis demonstrated that LcChi2 belonged to chitinase class II subgroup containing two conserved domains, i.e. Cys49 Gly71 and Val161 Met171 . In comparison with wild-type tobacco, the transgenic tobacco (Nicotiana tabacum) overexpressing LcChi2 showed increased tolerance to fungal and bacterial pathogens stresses. Semi-quantitative RT-PCR analysis indicated that the expression of LcChi2 in Chinese wildrye was up-regulated with the treatment of 400 mM NaCl, 100 mM Na2 CO3 and 20% PEG, so was the activity of LcChi2 product coincidently in similar treatments. Transgenic yeast (Saccharomyces cerevisiae) overexpressing LcChi2 exhibited an enhanced tolerance to 1.6 M NaCl, 10 mM Na2 CO3 , 1.5 M sorbitol, and 10 mM ZnSO4 stresses respectively. Interestingly, LcChi2-overexpressed transgenic tobacco presented increased tolerance to 200 mM NaCl, 3 mM Na2 CO3 and 500 mM sorbitol stresses during germination and to 600 mM NaCl stress during seedling development. This suggested that LcChi2 is likely an interesting candidate gene to be used in transgenic breeding to improve broad-spectrum tolerance in crop. Current results added information to plant chitinase family in abiotic stress tolerance over its existing functions in biotic stress tolerance. http://dx.doi.org/10.1016/j.nbt.2014.05.983 http://dx.doi.org/10.1016/j.nbt.2014.05.982 PV-13 A novel chitinase gene homologue from Chinese wildrye enhances tolerance to biotic and abiotic stress in transgenic tobacco Dongyun Hao 1 , Xiangguo Liu 2,∗ , Ying Yu 3 , Yang Liu 2 , Yinning Qu 3 , Shuo Yang 3 , Siping Han 2 , Shudan Feng 4 1 Institute of Agricultural Biotechnology, India Jilin Academy of Agricultural Sciences (JAAS), India 3 Jilin University, India 4 Harbin Normal University, India 2 Plant chitinase is a glycosyl hydrolase that is believed to be responsible exclusively for pathogens stress. However, there has been no report suggesting its function in relation to salinity stress www.elsevier.com/locate/nbt S207 SYNTHETIC BIOLOGY Synthetic Biology PW-01 Design of the novel catalytic units by key motif-directed domain recombination Yan Feng 1,∗ , Xiaoli Zhou 2 , Yuan Xie 3 , Guangyu Yang 3 1 State Key Lab of Microbial Metabolism, Shanghai Jiao Tong University, China 2 Jilin University, China 3 Shanghai Jiao Tong University, China Diverse functional domains in proteins provide a resource for designing novel biocatalysts. Recombination of more distant sequences in evolution offer greater opportunity for function leaps, but also introduces more disruptions in the chimeras. Here, we report two lipase-like chimeras engineered from a mesophilic lipase and a hyperthermophilic peptidase/esterase with only 14% or 21% amino acid identity. Recombination was at the conserved key motif regions which are part of the hard cores of the proteins instead of the flexible loops between protein functional domains. The resulting chimeric lipases retained the desirable preference for long-chain acyl ester substrates of their mesophilic parents, with pNP-C14 as the best substrate. Meanwhile, compared to the mesophilic parent, the chimeras exhibited more than 100fold increased thermostability at 50 ◦ C and the optimum catalytic temperature improved 40 ◦ C. These results suggest that the key motif-directed recombination (KMDR) approach promises for the efficient construction of robust hybrid enzymes. http://dx.doi.org/10.1016/j.nbt.2014.05.984 PW-02 Ether-lipid membrane engineering of Escherichia coli Antonella Caforio ∗ , Samta Jain, Arnold Driessen University of Groningen, The Netherlands The membrane lipid composition of archaea differs from bacteria and eukarya in having an ether-linked, isoprenoid hydrocarbon S208 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 chain with an enantiomer sn-glycerol-1-phosphate backbone. This unique structure is believed to be vital for the adaptation of these organisms to extreme conditions and the lipid divide is considered significant in a split of prokaryotes into archaea and bacteria. The aim of this project is to functionally introduce the archaeal ether lipid biosynthetic pathway into E. coli to examine the properties of mixed membrane lipids, to study the archaeal lipid biosynthetic pathway and to understand evolutionary aspects associated with the lipid divide. Using a synthetic biology approach, different modules of the pathway were designed for the synthesis of the isoprenoid chain and CDP archaeol, the precursor for polar head group attachment. The latter ether-lipid is produced by an uncharacterized enzyme, whose gene has not yet been identified in Archaea. Several in vivo and in vitro assays were performed to access the enzymatic activities and the hypothetical gene encoding for CDP-archaeol synthase was expressed in E. coli and validated. A membrane integrated precursor of the archaeal lipid biosynthetic pathway was produced in E. coli and formation of CDP archaeol can be reconstituted in vitro. http://dx.doi.org/10.1016/j.nbt.2014.05.985 USE OF ORGANIC WASTES New Biotechnology · Volume 31S · July 2014 Use of organic wastes PX-01 Integral exploitation of olive tree pruning in the paper industry Luis Jiménez 1,∗ , Alejandro Rodríguez 1 , Juan Domínguez 2 , Antonio Rosal 2 , Gustavo Cordero-Bueso 2 , Eva Valero 2 1 2 University of Córdoba, Spain University Pablo de Olavide, Spain This work seeks to obtain valuable products through an integral exploitation of lignocellulosic residues generated by agriculture, such as olive tree prunings. These sorts of residues are studied as alternative sources for lignocellulosic raw material in the paper industry and the black liquors generated could be used for several purposes, including the production of bioethanol. To reach these goals, a central composite factorial design was used to study the influence of operational variables [temperature (155, 170 and 185 ◦ C), cooking time (40, 65 and 90 min) and soda concentration (10, 14 and 18%)], on pulps and paper sheets properties obtained from olive tree prunings, likewise black liquors generated in these pulping processes were set up to enable both yeast strains (Saccharomyces cerevisiae CECT 1170 and Pichia stipitis CECT 1922) to be capable of doing an alcoholic fermentation at 30 and 26 ◦ C respectively, 5.5 pH, and under 150 rpm shaking. The results were similar to those obtained with other agricultural residues which are alternative sources for the paper industry. The best values of the physicochemical properties of cellulose pulps, such as Kappa number (31.98) or viscosity (716.11 mL/g) and the paper sheets, such as tensile index (608 m) or tear index (1.655 mNm2 /g), were reached operating to high values of soda concentration and temperature and slight cooking times. However, the black liquors of this study were not an optimal medium for the bioethanol production. It might be due to the important presence of inhibitors in the fermentation processes, such as 5hydroxymethyl-furfural. http://dx.doi.org/10.1016/j.nbt.2014.05.986 PX-02 Utilization of agroindustrial residues for hydrolytic enzymes production Ana Belen Diaz, Ana Blandino ∗ , Ignacio de Ory, Ricardo Martin, Maria Jose Munoz, Ildefonso Caro University of Cadiz, Spain Agricultural and agroindustrial residues are the most abundant resources on earth, which have significantly increased as a result of industrialization. In recent years there has been a growing trend towards efficient utilization and value-addition of these residues. Biotechnological processes, especially solid state fermentation (SSF), have contributed enormously to such reutilization as it can convert these agro-feedstocks into a wide variety of valuable chemical products. Agroindustrial residues are generally considered the best substrate for the solid state processes. Given their composition, rich in sugars such as cellulose, hemicellulose and pectin, they can be easily assimilated by microorganisms. Our research group has demonstrated that high levels of hydrolytic enzymes are produced by SSF of Aspergillus awamori on a mixture of grape pomace and orange peels. Furthermore, these enzymes could be safely added to food preparations for this fungus is considered a GRAS (Generally Recognized as Safe) microorganism. However, higher enzyme activities could be achieved by using the same methodology but growing different fungi. In fact, it would be interesting to be able to produce these enzymes from different agroindustrial residues but in the same conditions, as they could be processed in the same manufacturing facilities. Moreover, if possible and considering seasoning crops as raw material, it would be profitable for having the production plant operating for a longer time. Thus, the aim of this work is to evaluate the production of hydrolytic enzymes by SSF of Botryotinia fuckeliana grown in several agroindustrial residues with different composition in the same conditions of fermentation. http://dx.doi.org/10.1016/j.nbt.2014.05.987 PX-03 Liquid chromatography/mass spectrometry based identification, cloning and characterization of thermostable bacterial enzymes useful for the production of longchain oligosaccharides from agro-industrial wastes Nomeda Kuisiene ∗ , Raimonda Petkauskaite, Dangiras Lukosius, Andrius Jasilionis Vilnius University, Lithuania Oligosaccharides are important ingredients of the functional foods. The development of novel and highly functional oligosaccharides with physiological properties is now continuing. Over the past few years, long-chain oligosaccharides have evoked a great interest. Such oligosaccharides are absorbed to a much lower degree and persist longer in the colon than the shorter ones. Consequently, beneficial effect of the long-chain oligosaccharides is greater. Thermophilic microbial enzymes have potential to be used for the production of the long-chain oligosaccharides. We report identification of thermostable bacterial enzymes involved in the degradation of starch and pectin using liquid chromatography/mass spectrometry (LC/MS) based analysis. Zymographic analysis was combined with LC/MS. A range of polysaccharide degradation associated enzymes was identified using LC/MS of zymographic samples. The obtained peptides were used for the construction of primers in order to clone selected enzymes. Recombinant proteins were expressed, purified and characterized. The length of oligosaccharides produced by recombinant enzymes was determined. In order to determine conditions to monitor the chain length of obtainable oligosaccharides, we also tested activity of recombinant enzymes at suboptimal pH and temperature values. The potential of recombinant thermophilic bacterial enzymes in the degradation of agro-industrial wastes was also evaluated. It was shown, that long-chain oligosaccharides can be produced from starch and pectin (including that www.elsevier.com/locate/nbt S209 USE OF ORGANIC WASTES New Biotechnology · Volume 31S · July 2014 from apple pomace) using recombinant thermostable bacterial enzymes. Acknowledgement: The authors are thankful to the Lithuanian Science Council for the financial support (project No. SVE-08/2011). http://dx.doi.org/10.1016/j.nbt.2014.05.988 PX-04 Solubilization of animal bone char by Yarrowia lipolytica on medium containing glycerol Nikolay Vassilev 1,∗ , Bettina 1 Martos , Maria Vassileva 1 1 2 Eichler-Löbermann 2 , Vanessa University of Granada, Spain University of Rostock, Spain Phosphorus (P) is accepted as an essential element for all living organisms. P is mainly derived from mined rock phosphate, which is a non-renewable resource. It has been suggested that agricultural demand for P will outstrip mineable resources in 50–100 years and economically mineable resources will be depleted before the end of this century. Biotechnology offers a number of sustainable solutions that can mitigate these problems by using various waste materials as a source of P and, on the other hand, their solubilization by selected microorganisms. In this work we present results on solubilization of animal bone char (HABO) with a high content of phosphate by Yarrowia lipolytica. Free yeast cells were cultivated using different combinations of glycerol and HABO, the latter being simultaneously solubilized by the released citric acid. Biomass accumulation of 6.1 g/l, citric acid (8.7 g/l) and soluble phosphate (379 mg/l) concentrations were measured at 50 g/l glycerol and 6 g/l HABO after 96 h of shake-flask cultivation. However, the highest percentage of soluble phosphate of the total P of 44.8% was obtained at 30 g/l glycerol and 2 g/l animal bones char. Employing the latter combination, a separate experiment was carried out in fermenter where the solubilization efficiency was enhanced to 345 mg soluble phosphate/l (61.6% of the total phosphate) after 40 h of cultivation. The main advantage of HABO as an alternative P source is its abundance and low price. The EU produces about 3 million tons of meat and bone residues. http://dx.doi.org/10.1016/j.nbt.2014.05.989 PX-05 Plant growth enhancement by biotechnological tools Maria Vassileva 1,∗ , Massimiliano Fenice 2 , Antonia Galvez 1 , Nikolay Vassilev 1 1 2 University of Granada, Spain University of Viterbo, Spain An alternative, more environmentally friendly strategy for plant growth promotion was developed which include various types of plant-soil improvers and low-cost P-bearing material. Two types of microbial P-solubilizers were employed: a lactic S210 www.elsevier.com/locate/nbt acid producing bacterium and the filamentous fungus Aspergullus niger. The latter was introduced into a typical Mediterranean degraded soil as a biotechnological product containing partiallysolubilized phosphate (hydroxyapatite of animal bone origin, HABO), mineralized organic matter and mycelial mass. The lactic acid bacterium was entrapped in alginate beads and further inoculated in the soil, before transplanting plant seedlings (Lavender spica), to serve as a slow-release microbial formulation. Culture filtrate of Piriformospora indica was added to the above system in the beginning of the experiment and after two weeks. All treatments were supplemented with HABO. Results showed a significant plant growth promotion in treatments with immobilized bacterial cells (IC). Similar effect was observed when organic matter was used as an amendment. The highest plant growth was however registered in the treatment with both IC and microbiallytreated organic matter. This effect was more pronounced in the presence of filtrate of P. indica. Our results confirmed the beneficial effect of root-colonizing fungi and PGP microorganisms on plant growth. P. indica is a mycorrhiza-like root-colonizing fungus which can be cultivated in fermentation systems. In our study, the combination between P. indica filtrate, P-solubilizing IC, and, on the other hand, organic matter/soluble P product resulted in a highly effective promotion of plant growth in degraded soil as an alternative of chemical-fertilizer-based conventional scheme. http://dx.doi.org/10.1016/j.nbt.2014.05.990 PX-06 Production of carotenoids, ergosterol and other lipidic compounds by red yeasts cultivated on lignocellulose waste substrates Ivana Marova ∗ , Andrea Obruca, Iveta Kostovova Haronikova, Sinisa Petrik, Stanislav Brno University of Technology, Faculty of Chemistry, Czech Republic Carotenogenic yeasts are a diverse group of unrelated organisms (mostly Basidiomycota), that can be found in soil, fresh and marine water, on plants and also in foods. Due to its ubiquitous and world-wide occurrence, these yeasts have been able to assimilate various carbon sources, such as glucose, xylose, cellobiose, sucrose, glycerol, etc. Therefore, agro-industrial waste materials including lignocellulose materials can be used as cheap substrates. Presented work is focused on growth and production activity of red yeast strains Rhodotorula, Sporobolomyces and Cystofilobasidium cultivated on some lignocellulose wastes: pre-treated whey straw, pine hydrolyzates, SCG and rapeseed waste. The main aim of the current investigation was to assess the potentialities of red yeasts to transform these waste substrates to high-value products as carotenoids (about 1–3 mg/g CDW), ergosterol (2–4 mg/g CDW), coenzyme Q (0.5–1 mg/g CDW), lipids (11–21% of dry mass) and fatty acids as well as enriched red yeast biomass. Production properties of red yeasts were compared between tested strains and studied also during scale-up process in 5-L laboratory fermentor. The yields of about 30 g per liter of biomass enriched by 30–50 mg USE OF ORGANIC WASTES New Biotechnology · Volume 31S · July 2014 of total carotenoids and 60 mg of ergosterol were obtained by the most producing strains. Such biomass which is efficiently enriched for provitamins A, D and CoQ could serve as an additional natural source of significant nutrition factors in feed and food industry. Acknowledgement: This work was supported by project “Materials Research Centre at FCH BUT-Sustainability and Development, REG LO1211 NPS I MEYS CR”. PX-08 http://dx.doi.org/10.1016/j.nbt.2014.05.991 Food processing activities produce in Europe large amounts of by-products and waste. Such waste streams are only partially valorized at different value-added levels (spread on land, animal feed, composting), whereas the main volumes are managed as waste of environmental concern, with relevant negative effects on the overall sustainability of the food processing industry. The main focus of NOSHAN1 is to investigate the process and technologies needed to use food waste for feed production at low cost, low energy consumption and with maximal valorisation of starting wastes materials. Nutritional value and functionality as well as safety and quality issues are investigated and addressed as main leading factors for the feed production using food derived (fruit/plant and dairy). According to this not only wastes are characterized for their nutritional potential, but suitable technologies to stabilize them and convert them into suitable raw materials for bulk feed are researched. The topic of this communication is the characterisation, at molecular level, of the different waste streams selected. In particular a detailed composition of the nitrogen fraction has been performed. Determination of amino acids was performed by chromatography methods and racemisation was studied by GC–MS. Peptide and protein profiles were determined by LC/MS. Waste streams turned out to be quite promising to be transformed in feed or to be exploited as a source of feed additives, as far as their content in desirable compounds was concerned. PX-07 Fruit residues: low cost substrates for development of new food products Ninna Granucci ∗ , Silas G. Villas-Boas Centre for Microbial Innovation, School of Biological Sciences, The University of Auckland, New Zealand In recent years, there has been increasing interest in developing alternative uses for agricultural residues for production of higher added-value products. Bioconversion of these substrates using microbial fermentation could be an attractive option. Therefore, the main goal of my project is to optimise a fermentation process for bioconversion of fruit residues developed at laboratory scale using apple pomace as model. In our previous work, Candida utilis and Pleurotus ostreatus were employed for bioconversion of apple pomace, resulting in a nutritious enriched whitish substrate (flour-like) with attractive walnut/hazelnut aroma that contained high protein and low sugar. We have data demonstrating that the sequential fermentation of apple pomace with C. utilis and P. ostreatus resulted in 400% enrichment of protein content in the substrate in addition to increased availability of phosphorus, calcium and potassium as compared to nonfermented substrate. Moreover, the flavor and appearance of the processed substrate was very attractive, which made it ideal for a human consumption product. Thus, we envisage the following main steps for further research: 1 Assess other fruit residues to be bioconverted; 2 Scale up fermentation; and 3 Nutritional assessment of fermented product. This same strategy can be used for the bioconversion of different agriculture wastes, such as residues of other fruit processing and/or dairy industries. http://dx.doi.org/10.1016/j.nbt.2014.05.992 NOSHAN EU-project (Sustainable Production of Functional and Safe Feed from Food Waste): molecular characterization of selected food waste materials Tullia Tedeschi ∗ , Mariangela Bencivenni, Chiara Bottesini, Judith Muller-Maatsh, Federica Meli, Arnaldo Dossena, Stefano Sforza University of Parma, Italy http://dx.doi.org/10.1016/j.nbt.2014.05.993 PX-09 Production of polyhydroxyalkanoates from anaerobic digested grape pomace by employing a pure culture of Cupriavidus necator Gonzalo Martinez 1,∗ , Lorenzo Bertin 1 , Joana Domingos 1 , Gerhart Braunegg 2 , Fabio Fava 1 1 2 University of Bologna, Italy Technical University of Graz, Italy The present work was dedicated to verify the possibility of replacing the carbon source commonly employed for the biotechnological production of polyhydroxyalkanoates, i.e., glucose or fructose, with grape pomace (GP), which is a solid organic waste of the winery industry. To this aim, a grown culture was fed with the volatile fatty acids (VFAs)-rich effluent obtained by fermenting 1 Project full title: “Sustainable Production of Functional and Safe Feed from Food Waste” Grant agreement no.: 312140. www.elsevier.com/locate/nbt S211 USE OF ORGANIC WASTES GP under acidogenic conditions (GPAcid ). GPAcid mainly contained (g/L) acetic (14.69 ± 0.57), propionic (0.77 ± 0.04), iso-butyric (0.83 ± 0.03), butyric (4.67 ± 0.21) and caproic (0.55 ± 0.02) acids. Experiments were carried out in 500-mL Erlenmeyer flasks (working volume: 150-mL) at 30 ◦ C and 180 rpm. The whole polyhydroxyalkanoates production process was separated into two stages, namely: a balanced cell growth phase (by using DSMZ81 mineral medium amended with 5 g/L of fructose) and an accumulation phase, where harvested cells were suspended in a NH4 free-DSMZ-81 medium containing 20 or 40% of GPAcid (v/v). Higher VFAs concentrations were observed to inhibit polyhydroxyalkanoates accumulation. A control experiment aimed at indirectly evaluate water matrix inhibition was performed by substituting GPAcid with a VFAs water solution, wherein VFAs concentrations were the same of GPAcid . First results showed a higher polymer production when employing 40% of GPAcid , with a final polyhydroxybutyrate content of 60% (cell dry weight bases). Thermo-gravimetric analyses confirmed gas-chromatography ones and identical results were obtained in the control experiment. To our knowledge, this work represents the first attempt to produce polyhydroxyalkanoates with a pure culture of Cupriavidus necator and a fermented GP as alternative carbon source. http://dx.doi.org/10.1016/j.nbt.2014.05.994 PX-10 Composition of pectins from food waste to be used in bulk feed and as feed additives Stefania Baldassarre 1,∗ , Stefano Sforza 1 , Barbara Prandi 1 , Marcela Santarelli 1 , Neha Babbar 1 , Kathy Elst 2 , Monica Gatti 1 , Stefano Sforza 1 1 2 University of Parma, Italy University of Antwerp, Italy Pectins are heterogeneous carbohydrate polymers found in the cell wall of higher plants; they are formed by a backbone of (1->4)␣-D-galacturonic acid units interspersed with rhamnose residues linked to neutral sugar side chains. They contribute to primary wall functions such as cell strength, cell adhesion, stomatal function and defense response. Furthermore pectic oligosaccharides (POS) have been proposed as a new class of prebiotics able to exert several health-promoting effects. The main objective of this work is the characterization of pectins in vegetal food waste, in order to ensure their suitability as raw material for feed production. Two raw materials were considered: bulk feed prepared from vegetal food waste (rapeseed and malted barley mixed with dairy waste) and pectins extracted from vegetal waste to be used as feed additive .In the first case, pectic substances are precipitated with ethanol and the total alcohol insoluble residue is used for the determination of total pectins by quantitatively measuring the total uronic acid through a colorimetric assay after acid hydrolysis. In this way it is possible to assess the technological impact of the processing on the composition and the extractability of pectins. In the latter case, samples of pectic oligosaccharides (POS) extracted by enzymatical means, are analyzed in order to deterS212 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 mine the content of galacturonic acid, their composition in POS and their prebiotic activity, in order to define structure–function relationships. http://dx.doi.org/10.1016/j.nbt.2014.05.995 PX-11 Fungal bioconversions of various lignocellulosic byproducts to edible biomass Georgios Koutrotsios ∗ , Konstantinos Mountzouris, Georgios Zervakis Agricultural University of Athens, Greece The world-wide trend for a continuous increase in the amount of food produced leads to the accumulation of huge quantities of plant residues. Controlled solid state fermentation of various agroindustrial and forestry residues rich in lignocellulosics were treated by selected strains of Agrocybe cylindracea, Ganoderma lucidum, Hericium erinaceus and Pleurotus ostreatus (phylum Basidiomycota), the processes were monitored, and composition of end-products was comparatively evaluated. Olive mill waste (composted or not), olive prunings and grape marc were among the most promising substrate ingredients for mushroom production since their use contributed at obtaining significantly higher yields than the conventional cultivation media. In most cases, mushroom productivity correlated with substrates nitrogen, lignin, hemicelluloses, and residual mycelial content. Spent substrates exhibited high reductions in hemicelluloses and cellulose, and low in lignin; these values in conjunction with elevated protein content advocated their use in animal feeds. Mushroom proximate analysis showed correlations of protein and crude fat with substrates nitrogen and lipids, whereas total carbohydrates, crude fiber and ash content demonstrated relatively less variability among strains and cultivation media tested. Mushroom nutritional value could be suitably enhanced by appropriate selection/modification of the growth substrates. Exploitation (incl. detoxification) of waste streams could be combined with the generation of value-added products and provide a viable solution for the sustainable use of such materials. Acknowledgements: This research has been co-financed by European Union (ESF) and Greek national funds (NSRF) through the project titled “Metagenomics of ligninolytic microorganisms – Bioconversion of plant by-products into high-added value products” (THALIS–UOA–MIS377062). http://dx.doi.org/10.1016/j.nbt.2014.05.996 USE OF ORGANIC WASTES New Biotechnology · Volume 31S · July 2014 PX-12 Influence of cell size on performance of microbial fuel cells of single chamber using organic solid waste with Amazonian and High Andeans soils from Ecuador Washington Logroño, Celso Recalde, Magdy Echeverria Escuela Superior Politécnica de Chimborazo, Ecuador It was the first work done in Ecuador where compared three volumes to produce bioelectricity in Microbial Fuel Cells of Single Chamber, employing organic solid waste as substrate with 50:50 relationships (vegetables and fruits) to enrich electrogenic bacterias, without renovation of microbial fuel and over a testing time of 171 days. The study used soils from Kaiptach Achuar Community – AMAZON to 1000 m.a.s.l. and the same procedure with the Pichan Central Community-ANDES to 4000 m.a.s.l. The samples were taken among 20–40 cm depth of natural soils, each treatment have used polyethylene containers as bioreactors and carbon fiber was used as both electrodes with different surface areas. The initial physical chemical analysis indicated differences in parameters like: P, K, CaO, MgO, Rel.C/N, %H; while the initial heterotrophs counted in the Andean soil showed higher amount, but smaller microbiological diversity, in accordance to morphological description done under aerobic conditions. The means comparison test has indicated that there are significant differences among sizes both studies cases, at the ANDES the best treatment was the intermediate, with a mean generation of 317 mV, moreover at the AMAZON was the smallest size, with 270 mV. It was observed that the cell size is a parameter influencing at performance and stability of Microbial Fuel Cells in accordance to the ecosystem. http://dx.doi.org/10.1016/j.nbt.2014.05.997 MFCs provide a dual benefit: wastewater treatment and direct electricity generation. For industrial scale-up, the approach in this work is through plurality. Multiple units of relatively small scale can be connected electrically and hydraulically to achieve the necessary capacity [2]. Initially a single chamber open air cathode MFC of 170 ml volume treating spent wash subsequent to anaerobic digestion treatment of 2 g/l Chemical Oxygen Demand demonstrated an average voltage of 0.4 V in open circuit and a COD reduction of 70%. Scaling up, two 100 lt units connected in parallel and operating in directly diluted spent wash at 26.4 g COD/l.d demonstrated 84% COD removal, maximum voltage 0.9 V and current 160 mA. Ongoing experiments using this configuration and previously anaerobically treated spent wash demonstrate encouraging results, bringing practical industrial scale Microbial Fuel Cell treatment closer. References [1].Mohana S, Acharya K, Madamwar BD. Distillery spent wash: treatment technologies and potential applications. J Hazard Mater 2009;163:12–25. [2].Gálvez A, Greenman J, Ieropoulos I. Landfill leachate treatment with microbial fuel cells; scale-up through plurality. Bioresour Technol 2009;(100):5085-509. http://dx.doi.org/10.1016/j.nbt.2014.05.998 PX-14 New application of agro-industrial waste to prevent melanosis of Mediterranean pink shrimp Giorgio Rizza ∗ , Giuseppina Rosaria Antonella Alberio, Rosa Palmeri, Aldo Todaro, Giovanni Spagna University of Catania, Italy PX-13 Optimisation of scale-up of microbial fuel cell for sustainable wastewater treatment with positive net energy generation Ourania Dimou 1,∗ , John Andresen 2 , Veyacheslav Feodorovich 3 , Igor Goryanin 4 , Alan Harper 2 , David Simpson 5 1 Heriot-Watt University, UK Department of Chemical Engineering, Heriot-Watt University, Edinburgh Campus, EH14 4AS, UK 3 M Power World, Vavilova str. 5/3, Moscow, Russia 4 Informatics Life-Sciences Institute, Edinburgh University, Edinburgh EH8 9AB, UK 5 Biological Systems Unit, Okinawa Institute of Science and Technology, Okinawa 1919-1, Japan 2 From eight to fifteen litres of liquid by-products are generated for every litre of grain whisky produced. ‘Spent Wash’is the main liquid stream. If discharged untreated into the environment it might contribute to pollution such as eutrophication [1]. Microbial Fuel Cells (MFCs) are a natural bio-technology solution to the issue working either independently or in conjunction with established wastewater treatment technologies. Utilising metabolic reactions of electrochemically active microorganisms, Food byproducts represent an environmental and economic problem because peel, pulp, pulp wash, and yellow water are difficult to digest. Additionally, citrus byproducts are relatively resistant to microbial degradation (high COD and BOD5 indexes) due to their high content of bioactive compounds with antimicrobial activity, e.g. ascorbic acid, limonoids, and polyphenols. In addition, agro-industrial wastes are potential sources of biophenols that have proven antioxidant, anti-inflammatory and anticancer properties. Aims of this work is to test how natural extracts obtained from vegetable waste, in particular orange and lemon peel, may inhibit the process of melanosis in pink shrimp species (Parapeneus longirostris). The treatment with orange peel, extracted in hot water, significantly reduced (p < 0.05) the enzymatic activity of the PPO at the level of the cephalothorax of shrimp. Ethanolic extract of lemon peels was also effective for inhibition of melanosis. The results were related to the evaluation of the polyphenol quality index (QI) that confirmed the efficacy of treatment of the orange and lemon peel extracts. The addition of these natural extracts in pink shrimp samples can be considered a valid alternative to the use of agro-industrial waste. http://dx.doi.org/10.1016/j.nbt.2014.05.999 www.elsevier.com/locate/nbt S213 VETERINARY BIOTECHNOLOGY Veterinary biotechnology PY-01 Expression and bioactivity analysis of the expression of PEDV COE gene in Pichia pastoris Lumu Li ∗ , Fazhi Xu, Qingsong Hu, Xiaoling Ding, Xiongyuan Si Anhui Agricultural University, China The purpose of this study was to express the porcine epidemic diarrhoea virus (PEDV) COE protein in Pichia pastoris, and evaluate the neutralizing ability of mice serum after immunization. A pair of primers was designed based on the cloned PEDV S nucleotide sequences. Intestinal tissues were collected from piglet suffering from PEDV, and the mRNA of PEDV was extracted by the TRIzol reagent. The PEDV COE gene was amplified by RTPCR. Furthermore, the PEDV COE gene was inserted into pPIC9K, and the recombinant plasmid of pPIC9K-COE was transformed into P. pastoris GS115 by electroporation. High copy recombinant strains were screened and then expression was induced by addition of methanol. SDS-PAGE and Western blotting were used to analyse the immunogenicity of recombinant protein. The neutralizing ability of mice serum antibodies by PEDV COE recombinant was analyzed by virus neutralization test. PEDV COE gene was amplified by RT-PCR, and it was 530 bp in length .The PEDV COE protein was expressed in P. pastoris. The MW of the proteins was about 30 kDa as analyzed by SDS-PAGE, and the concentration was 30 mg/L. Western blotting showed that the protein had immunogenicity. The virus neutralization test result showed that the mice immunized with the recombinant PEDV COE protein produced specific serum antibodies with neutralizing activity, the neutralization titer reached 1:36. PEDV COE protein was correctly expressed in P. pastoris and the protein had a good biological activity. http://dx.doi.org/10.1016/j.nbt.2014.05.1000 PY-02 Protective effects of catalpol against hydrogen peroxide induced oxidative stress on preimplantation porcine embryos Deog-Bon Koo ∗ , Yong-Hee Lee, Sung-Hun Min, Jin-Woo Kim, JaeHyun Ahn, Geon-Yeop Do, Sung-Kyu Chae Daegu University, South Korea Catalpol, an iridoid glucoside, isolated from the root of Rehmannia glutinosa, possesses a broad range of biological and pharmacological activity including anti-tumor, anti-inflammation and anti-oxidant by acting as a free radical scavenger. Therefore, in the present study, the effects of catalpol on blastocyst development and expression levels of reactive oxygen species (ROS) were investigated in preimplantation porcine embryos. After in vitro maturation and fertilization, porcine embryos were cultured for 6 days in porcine zygote medium 3 (PZM-3) with catalpol (0, 100, 200 and 400 ␮M respectively). Blastocyst development was not significantly improved in the catalpol treated groups when compared the control group. However, subsequent evaluation of S214 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 the intracelluar levels of ROS and numbers of apoptotic nuclei in catalpol (100 ␮M) treated blastocysts revealed that ROS levels of catapol-treated porcine blastocyst were decreased (P < 0.05) and the numbers of apoptotic nuclei were reduced by catalpol treatment in porcine embryos. Moreover, the blastocyst development and total cell numbers of blastocysts were significantly increased in the catalpol treated group relative to the untreated catalpol group under H2 O2 (200 ␮M) induced oxidative stress (P < 0.05). Furthermore, the intracellular levels of ROS in catalpol-treated group were significantly decreased in the untreated catalpol group under H2 O2 induced oxidative stress (P < 0.05). In conclusion, our results suggest that catapol improves the developmental competence of porcine embryos via modulation of intracellular levels of ROS and the apoptotic index during the preimplantation stage. http://dx.doi.org/10.1016/j.nbt.2014.05.1001 PY-03 Study on the establishment of the animal model infected by influenza virus in Microtus brandti Defeng Wu ∗ Fujian Agriculture and Forestry University, China In this study, the brandti voles were infected by influenza virus A/WSN/33(H1N1) intranasally, in order to assess the susceptibility of influenza virus on brandti voles. The brandti voles showed high susceptiblity to infection. The infection susceptibilty was further evaluated to establish a possible susceptibility index for brandti voles model infected by influenza virus. By means of the method of infecting brandti voles, influenza virus was separated and identified successfully. The result showed that the virus was widely distributed in brandit vole tissues. which also indicated that the brandti vole are highly sensitive to influenza virus, and the major target organ was lung. The animal model of brandti voles infected by influenza virus has been established successfully by means of some model indexes, such as clinical symptoms, body weight, mortality, the index of the separation of influenza virus and so on. http://dx.doi.org/10.1016/j.nbt.2014.05.1002 PY-04 A Salmo salar diet based on a marine biosurfactant for the profilaxis and treatment of Pisciricketsia salmonis Claudia Ibacache-Quiroga 1,∗ , M. Alejandro Dinamarca 2 , Juan Ojeda 2 , José Miguel Troncoso 3 1 Centro Nacional Biotecnología/Micromarine Biotech, Spain Universidad de Valparaíso, Chile 3 Ewos, Chile 2 Aquaculture is a fast growing economic activity due to an increase of food demand worldwide, which main challenges are bacterial infections that causes significant biomass and economic losses. Although classic antibiotic therapy was effective against New Biotechnology · Volume 31S · July 2014 bacterial infections in aquaculture, nowadays these therapies are unsuccessful due to an increase of antibiotic resistance. We recently reported that a specific marine biosurfactant (BS) interferes with quorum sensing (QS) cell-to-cell communication system, reducing virulence of bacterial fish pathogens. BS do not act modifying bacterial cellular processes, therefore no resistance to BS is developed, making these molecules an alternative to the use of antibiotics in aquaculture. The aim of this study was to incorporate the biosurfactant into fish food and to evaluate the effect of the supplemented diet on fish survival to Pisciricketsia salmonis. BS was incorporated by emulsification into fish food. The effect of the supplemented diet was evaluated through a challenge assay, VETERINARY BIOTECHNOLOGY in which Salmo salar was directly exposed to the fish pathogen P. salmonis. Fish were fed with food supplemented with BS at a final concentration of 400 mg/kg of fish/10 days, during thirty days. Fish diet with BS showed a survival rate 15% higher than the control diet (without BS). The immune makers IL-1␤ and IL-8 evaluated by qRT-PCR showed an increase in gills and intestine respect to the control diet. The food containing the BS and serum samples form treated fish were active for the inhibition of QS and growth of P. salmonis, demonstrating the presence of the active principle. http://dx.doi.org/10.1016/j.nbt.2014.05.1003 www.elsevier.com/locate/nbt S215 YEAST AND FILAMENTOUS FUNGI Yeast and filamentous fungi PZ-01 New source of hyaluronidase – Fistulina hepatica Lenka Bobková 1,∗ , Dzianis Smirnou 1 , Martin Krčmář 1 , Veronika Moravcová 2 , Martina Hermannová 2 , Vladimír Velebný 3 1 Contipro Biotech s.r.o, Czech Republic Contipro Pharma s.r.o, Czech Republic 3 Contipro Group s.r.o, Czech Republic 2 Hyaluronidases are a group of enzymes capable of hyaluronic acid (HA) depolymerization. These enzymes can be divided into groups according to the mechanism of HA cleavage or source of origin. Bovine testicular hyaluronidase (hydrolase) and bacterial hyaluronidases (lyases) are the main ones used in cosmetic or medicinal applications nowadays. Fungi as a new source of hyaluronidases were not authentically described until recently [1]. This work was focused on novel hyaluronidase production by the Basidiomycete Fistulina hepatica. The enzyme activity was detected in culture medium. Characterization of the enzyme was performed after chromatographic purification. Products of HA cleavage were identified by HPLC and MS. The reaction proceeded in an eliminative manner and unsaturated hyaluronan oligosaccharides were formed. This type of mechanism was so far described only for hyaluronan-lyases produced by bacteria. Optimal conditions for HA–oligosaccharide production were pH 4 and 20 ◦ C. The enzyme was stable for 8 days under these conditions. Reference [1].Bakke M, Kamei J, Obata A. Identification, characterization, and molecular cloning of a novel hyaluronidase, a member of glycosyl hydrolase family 16, from Penicillium spp. FEBS Lett 2011;585:115–20. http://dx.doi.org/10.1016/j.nbt.2014.05.1004 PZ-02 Particles prevent pellets: microparticle enhanced cultivation MPEC increases 2-phenylethanol production in Aspergillus niger Maria M.W. Etschmann ∗ , Ina Huth, Jens Schrader, Dirk Holtmann DECHEMA Research Institute, Germany Adding microparticles to fungal shake flask cultivations prevents pellet formation and leads to finely dispersed and highly productive mycelia. This addresses a problem occurring in many industrial processes using filamentous fungi in submerged cultures: formation of pellets. Fungi tend to grow in agglomerates which can be as large as several centimeters in diameter. The center of the pellets is badly supplied with oxygen and nutrients and can consist of essentially dead biomass. As only the biomass in the outer shell is active, productivity of these processes can be low. Techniques to increase productivity are therefore in high demand. S216 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 The production of the rose-like aroma compound 2phenylethanol (2-PE) by Aspergillus niger spp. is used as an example to investigate the effect of microparticle enhanced cultivation MPEC on the production of fungal secondary metabolites. The production of the higher alcohol 2-phenylethanol from Lphenylalanine was up to now mainly investigated in yeasts. However, Aspergillus niger DSM 821 is also capable of synthesizing 2-PE. It yields product concentrations of up to 700 mg/L, which is twice as much as previously investigated strains. Screening of 16 types of particles of different chemical composition showed that the application of MPEC increased the final concentration by a further 30%. Experiments for downscaling the technique to 48 well microtiter plate scale is under way, results we be available by the time of the congress. http://dx.doi.org/10.1016/j.nbt.2014.05.1005 PZ-03 The cellulase induction system in Trichoderma reesei strains remains well conserved despite several generations of random mutagenesis and screening Dante Poggi-Parodi 1,∗ , Aurelie Pirayre 1 , Thomas Portnoy 1 , Hugues Mathis 1 , Thiziri Aouam 1 , Frédérique Bidard 1 , Stéphane Le Crom 2 , Antoine Margeot 1 1 IFP Energies Nouvelles, France Université Pierre et Marie Curie, Laboratory of Developmental Biology, CNRS UMR7622, Paris, France 2 Trichoderma reesei is the main industrial producer of enzymes degrading cellulosic and hemicellulosic biomass because of its high protein secretion capacity. This outstanding level of cellulases production is the product of several rounds of random mutagenesis and screening in the past. In this study, using a well-studied lineage of improved strains from T. reesei, NG14 and RUT C30, we investigated the links between the mutations generated during the mutagenesis screens, that allowed development of these strains, and the transcriptome differences in the first hours of the cellulases induction process. Despite a high number of reported mutations and very different productivities, the transcriptome from both strains were surprisingly similar, with few genes being over or under expressed in the higher producer strain. Comparison of transcriptomes of the mother strain QM6a and RUT C30 before induction of cellulase production revealed that most of observed changes between NG 14 and RUT C30 were probably due to basal changes to the cell physiology, and that the cellulase production system was essentially intact. Accordingly, very few mutated genes seem to belong to the cellulase induction system, suggesting that there is room for improvement regarding this system. This study shows that systems biology methods can gather information about cellular systems that cannot be obtained by traditional techniques, and that modern strain development programs should include both approaches to construct competitive strains. http://dx.doi.org/10.1016/j.nbt.2014.05.1006 YEAST AND FILAMENTOUS FUNGI New Biotechnology · Volume 31S · July 2014 PZ-04 Characterization of the relevant genes and development of salt-tolerant yeast strains by transposon mutagenesis Hyun-Soo Kim ∗ , Hye-Min Kang Department of Food Science and Industry, Jungwon University, South Korea Kimchi is a traditional, fermented Korean food prepared with different vegetables, spices, and ingredients and is an important dietary source of vitamins, minerals, and other nutrients. The waste brine from manufacturing process of kimchi is released and eventually results in serious environmental pollution. Saccharomyces cerevisiae strains tolerant to salt stress are important for the production of single cell protein by using kimchi waste brine and removal of potential pollutants from the waste water. In this study, approximately 3000 transformants of the mTn3mutagenized genome library were selected as leucine prototrophs and replica-plated to both YPD agar and YPD agar containing 10% NaCl. Three strains (Tn 1–3) tolerant to up to 10% NaCl were isolated by screening a transposon-mediated mutant library. Three transposon mutants showed higher ethanol production and grew faster than control strain when cultured in rich media containing 5%, 7.5%, and 10% NaCl respectively. The determination of transposon insertion sites and Northern blot analysis identified putative genes and revealed simultaneous down-regulations and disruptions of the genes indicating that salt tolerance can be conferred. The genes identified in this study may provide a basis for the application in developing industrial yeast strains. http://dx.doi.org/10.1016/j.nbt.2014.05.1007 PZ-05 Epicoccum purpurascens (Didymellaceae, Ascomycota) as a non-model source of novel bioactive compounds: biotechnological perspectives Mikhail Fokin 1,∗ , Bevan Weir 2 , Ting-Li Han 3 , Neethu Arun 3 , Lydia Yamaguchi 4 , Massuo Jorge Kato 4 , Silas Villas-Boas 3 1 University of Auckland, New Zealand Landcare Research, New Zealand 3 Centre for Microbial Innovation, School of Biological Sciences, The University of Auckland, New Zealand 4 Chemistry Institute, University of São Paulo, Brazil 2 Filamentous fungi have been widely used as a source of valuable bioactive compounds for medicine, agriculture and white biotechnology. Recently, genomic studies have significantly improved our knowledge of the functional background for known compounds and revealed a vast amount of novel secondary metabolites not commonly expressed in the laboratory. Among fungi these studies have been focused mostly on a few model species. Epicoccum purpurascens is a widespread member of Dothideomycetes class, which consists of over 20 thousands species. E. purpurascens has been isolated from different substrates such as plants, soil, and marine invertebrates. Several bioactive compounds were isolated from different E. purpurascens strains, including epicoccaene, a broad-range fungicide, discovered and patented by our group. It is remarkable that the biology of this fungal species has not been much studied, and we have very little knowledge on its genetic and phenotypic diversity. Only recently the genus Epicoccum was placed in the newly described family Didymellaceae, and great intraspecific diversity of E. purpurascens has been reported. We determined the genetic and metabolite profiles of different E. purpurascens strains isolated in New Zealand, and we have found that metabolite profile was closely associated with the ability of each strain to produce biologically active compounds, which did not match their similarities based on their genetic profile. We have now de novo sequenced and assembled the draft genome of our working E. purpurascens strain. Further annotation of the genome and specific mining for secondary metabolite gene clusters will strongly facilitate biotechnological use of E. purpurascens. http://dx.doi.org/10.1016/j.nbt.2014.05.1008 PZ-06 The effect of carbon sources on upstream regulatory regions controlling the expression of the Candida utilis maltase gene Ján Krahulec ∗ , Hanka Boňková, Veronika Osadská, Stanislav Stuchlík, Ján Turňa Lišková, Michaela Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia In the last few years it has been demonstrated that the industrially important yeast system Candida utilis represents a promising expression host, generating relatively high levels of recombinant proteins. Nevertheless, basic knowledge of its gene structure and regulation of gene expression, which is needed to allow more extensive use of this organism, is lacking. The current study presents preliminary characterization of the carbon sourcedependent expression of the C. utilis maltase gene in order to compare its regulation. Our results showed that C. utilis maltase is able to hydrolyze cellobiose and soluble starch, and its affinity for cellobiose is even three times higher than for maltose. To further investigate the function of maltase and the use of the maltase promoter for the heterologous protein expression, we successfully applied the Cre-loxP system to acquire a null mutant strain with disrupted maltase gene and promoter in the polyploid yeast C. utilis. The first step was to introduce a mutagenesis cassette harboring a marker gene between two loxP sites into the target cells. After identification of a partial mutant strain, the same mutagenesis cassette was repeatedly used and the null mutant strain was selected on higher antibiotic concentration. The last step was to introduce a Cre-expression plasmid and screen for marker rescue strains. Acknowledgements: This work is result of project implementation: “Production of biologically active agents based on recombinant proteins” (ITMS 26240220048) supported by the www.elsevier.com/locate/nbt S217 YEAST AND FILAMENTOUS FUNGI Research and Development Operational Program funded by the ERDF. http://dx.doi.org/10.1016/j.nbt.2014.05.1009 New Biotechnology · Volume 31S · July 2014 PZ-08 Selection of preservation conditions for Debaryomyces hansenii yeasts killer toxins Barbara Zarowska 1 , Lukasz Bobak 2 , Piotr Regiec 3 , Adam Figiel 4 , Monika Grzegorczyk 5 , Marek Szołtysik 2 , Maria Wojtatowicz 5 , Xymena Polomska 5,∗ PZ-07 Characterizing the sterol specificity of microbial and mammalian acyltransferases Holly Stolterfoht 1,∗ , Barbara Petschacher 1 , Katharina Littringer 1 , Martin Lehmann 2 , Hans-Peter Hohmann 2 , Harald Pichler 3 1 ACIB GmbH, Austria DSM Nutritional Products Ltd, Austria 3 Institute of Molecular Biotechnology, TU Graz, Austria 2 Sterols are essential lipid components of eukaryotic membranes influencing membrane fluidity, membrane permeability as well as the activity of membrane-bound proteins. In case of excessive sterol formation, sterols are stored as sterol esters in lipid particles as energy reservoir or stock of membrane building blocks. The free sterol 3-OH group is acylated by the integral membrane proteins acyl coenzyme A: sterol O-acyltransferases (ACATs). This storage mechanism is conserved from yeasts to plants and mammals, although the main membrane sterols differ between the kingdoms of life. In yeasts, ergosterol is formed while mammalian membranes contain mainly cholesterol. For the two S. cerevisiae acyltransferases, differences in sterol specificity have been shown for Are2p preferring ergosterol and Are1p esterifying also ergosterol precursors [1]. Here, we compare in a cross-species study the sterol specificities of ACATs. Therefore, we expressed endogenous acyltransferases Are1 and Are2, and several heterologous microbial and mammalian acyltransferases, in a S. cerevisiae are1are2 knockout strain. ACAT substrate specificities were determined in vitro by activity assays using microsomal preparations, radioactively labeled oleoyl-CoA and ergosterol- and cholesterol-like sterol substrates. Reference [1].Zweytick D, Leitner E, Kohlwein SD, Yu Chunjiang, Rothblatt J, Daum G. Contribution of Are1p and Are2p to steryl ester synthesis in the yeast Saccharomyces cerevisiae. Eur J Biochem 2000;267:1075–82. 1 Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland 2 The Department of Animal Technology and Quality Management, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland 3 The Department of Food Storage and Technology, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland 4 The Institute of Agricultural Engineering, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland 5 The Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland Killer toxins of Debaryomyces hansenii yeast species are proteins with an ecological regulation function: they are active against other yeast species and filamentous fungi. The study investigated the possibility of protein concentration by ultrafiltration or evaporation under vacuum, and then drying by means of spray drying and lyophilization. Ultrafiltration on a membrane with 18 kDa cut-off point has proved to be an effective method for killer protein concentration of the tested strains. The use of membranes with a higher cut-off resulted in the activity decrease of the killer protein in the obtained concentrates, and at the same time, relatively high activity in filtrates. Comparably good results, as in the case of the ultrafiltration with membrane of 18 kDa cut-off, were obtained using the vacuum evaporator. Spray drying resulted in the loss of activity of lethal preparations. The smallest losses of 15% were observed in preparations dried at 100 ◦ C of inlet air. The best method to stabilize the killer toxin was freeze-drying. Immediately after lyophilization and after three months of storage killer toxins exhibited activity similar to that before preservation. Acknowledgements: This work was supported by research grant POIG.01.03.01-02-080/12, co-financed by the European Union from the European Regional Development Found. http://dx.doi.org/10.1016/j.nbt.2014.05.1010 http://dx.doi.org/10.1016/j.nbt.2014.05.1011 PZ-09 Evaluation of Geotrichum candidum deacidification potential for yoghurt keeping quality Imran Muhammad, Saima Riaz, Nawaz Farah ∗ Department of Microbiology, Islamabad, Pakistan Quaid-i-Azam University, Lactic acid bacteria have received a lot of research attention due to their diverse technological and metabolic characteristics in fermented dairy products. The present study was designed to study the impact of microbial population on maintaining the quality of S218 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 fermented milk. Post fermentation acidification is a serious concern in dairy industry owing to its potential detrimental effects on yogurt safety and quality. In order to rectify post acidification problem in yogurt, the deacidifying ability of G. candidum was utilized. G. candidum and starter culture when used in combination were found to have positive influence on the stability and quality of the yogurt. Growth parameters such as pH, acidity, syneresis, total solid content, sensory evaluation and viscosity were analysed. Results have shown that G. candidum remarkably maintained pH to 3.75 from 48 until 196 h as compared to control yogurt. Similarly solid contents were higher and reduced syneresis was recorded for yogurt with G. candidum. In the light of above findings it could be inferred that this mix culture starter could be a better option for industry for controlling the post fermentation acidification problem in fermented dairy products thus contributing towards safety and stability of final product. http://dx.doi.org/10.1016/j.nbt.2014.05.1012 PZ-10 YEAST AND FILAMENTOUS FUNGI molecules of different size. Those plasmids replicate independently from cell nucleus due to the encoding of their own DNA polymerases together with the terminal protein being a very important element of replication initiating complex. In this work seven new D. hansenii plasmid systems (four double and three triple) were sequenced by NGS and the sequences of genes encoding DNA polymerases were compared to each other and to previously known plasmid sequences from D. hansenii and other yeast species. All genes of D. hansenii DNA polymerases shared very high homology with corresponding plasmids (the smallest or the largest plasmids from the systems) with identity on the level of 95–96%. Also high homology, about 72% of identity, was observed with genes of plasmids from other yeast species like Kluyveromyces lactis, Babjeviella inositovora, MIllerozyma acaciae, Schwanniomyces etchellsii. Within one plasmid system only residual similarity in analyzed genes was observed; a few percent of identity. Acknowledgements: This work was financially supported by Polish Ministry of Science and Higher Education. Project N N 312 258138. http://dx.doi.org/10.1016/j.nbt.2014.05.1013 Genes encoding DNA polymerases on linear dsDNA plasmids of Debaryomyces hansenii yeasts share very high homology Xymena Połomska 1,∗ , Cecile Neuveglise 2 , Zbigniew Lazar 3 , Barbara Zarowska 4 1 The Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Life Sciences, France 2 INRA, MICALIS, AgroParisTech, Thiverval-Grignon, France 3 Wrocław University of Environmental and Life Sciences; INRA, MICALIS, AgroParisTech, Thiverval-Grignon, France 4 Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Wrocław, Poland Debaryomyces hansenii yeasts can keep natural plasmids in their cytoplasm occurring in systems with two or three linear dsDNA www.elsevier.com/locate/nbt S219