WO2006034811A2 - Use of laccases as reporter genes - Google Patents

Use of laccases as reporter genes Download PDF

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Publication number
WO2006034811A2
WO2006034811A2 PCT/EP2005/010239 EP2005010239W WO2006034811A2 WO 2006034811 A2 WO2006034811 A2 WO 2006034811A2 EP 2005010239 W EP2005010239 W EP 2005010239W WO 2006034811 A2 WO2006034811 A2 WO 2006034811A2
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dna
laccase
sequence
candida
expression
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PCT/EP2005/010239
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German (de)
French (fr)
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WO2006034811A3 (en
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Marvin Karos
Thomas Subkowski
Hans-Georg Lemaire
Claus Bollschweiler
Bernhard Nüßlein
Reinhard Fischer
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Basf Aktiengesellschaft
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests

Definitions

  • the invention relates to the use of laccases from Aspergillus nidulans as reporter systems in methods for the qualitative and quantitative determination of gene expression, in particular for the qualitative and quantitative determination of promoter strengths in processes for the localization of proteins in a cell, and to methods for Selection of Certain Recombinant Cells and in Methods for Heterologous Gene Expression.
  • Laccases (EC 1.10.3.2) are blue multi-hybrid enzymes produced by fungi and plants. They oxidize a number of aromatic compounds such as mono-, di- and polyphenols, aromatic amines and methoxy-phenols. During the oxidation, electrons are removed from the substrate, "collected” in the enzyme, and then four electrons each are transferred to oxygen and water is formed. (Messerschmidt & Huber, 1990).
  • Fungi usually contain several laccases, which presumably have different biological functions.
  • Basidiomycin Coprinus cinereus eight lac- cases were described, while in the Ascomycete ' n A. nidulans six laccases occur (Wagner, 2003, Hoegger et al., 2004).
  • the exact biological role of fungal lacquases is largely unknown. They usually have an N-terminal secretion signal and are secreted.
  • the function of basidiomycete laccases in lignin degradation is best studied (Crestini et al., 2003). In other fungi, a function is assumed in the melanin biosynthesis or in the degradation of phenolic substances (Hermann et al., 1983, Fernandez-Larrea & Stahl, 1996).
  • Reporter genes such as beta-galactosidase or glucuronidase are widely used in molecular biology and biotechnology. However, they have the disadvantage that the substrates must be introduced into the cells. In addition, many fungi possess endogenous enzymes which lead to high basal enzyme activities.
  • the present invention relates, in a first embodiment, to a method for the determination of regulatory properties of a genetic element comprising the steps of: (a) introducing a DNA molecule having a DNA I sequence whose regulatory
  • the invention in a second embodiment, relates to a
  • a method of localizing a desired protein in a cell compartment comprising the steps of:
  • the invention relates to a method for determining expression of a desired gene in a cell comprising the steps of:
  • the invention relates to a
  • a method of expressing a desired gene in a cell comprising the steps of: (a) introducing a DNA molecule having a DNA II sequence coding for an Aspergillus nidulans laccase and having the C-terminal one thereof End of another DNA-I sequence which codes for the desired gene product is linked so that the expression takes place as a fusion protein of laccase and the desired gene product, (b) cultivating the cells obtained under (a) under conditions which The expression of the desired gene allows (c) isolation of the fusion protein from laccase and desired gene product.
  • laccase is used as reporter gene or reporter protein for heterologously expressed proteins. Many industrially important proteins are produced in filamentous fungi. An attempt is made to secrete the proteins in order to facilitate the purification. Laccases can be used to secrete non-secreted proteins. A C-terminal fusion of a desired protein with laccase leads to the secretion of the protein and, due to the laccase activity, allows the ready detection of the secreted protein. By introducing a protease cleavage between the two proteins, the desired protein can be released.
  • Suitable cells for the abovementioned processes are vegetable, for example carrot roots, animal and human, for example HeIa or HEK293, and in particular fungal cells.
  • the processes can be carried out particularly well in cells of the following types: Alternaria alternata.Clathrospora diplospora.Alternaria brassicae, Alternaria brassicico- Ia 1 Alternaria raphani, Pleospora herbarum, Pyrenophora trichostoma Cochliobolus sativus, Cochliobolus heterostrophus, Setosphaeria monoceras Setosphaeria rostrata, Pleospora betae, Leptosphaeria maculans, Leptosphaeria miscroscopica, Ophiobolus herpotrichus, Cucurbitaria elongata. Phoma sp.
  • Leptosphaeria Doliolum Cucurbitaria Berberidis, Paraphaeosphaeria agavensis Paraphaeosphaeria nolinae, Paraphaeosphaeria obtusispora, Paraphaeosphaeria con- glomerata, Paraphaeosphaeria quadriseptat, Paraphaeosphaeria filamento- sa.Melanomma Sanguinarium, Paraphaeosphaeria glauco-punct.Septoria nodo- rum.Phoma herbarum Pleospora rudis.Helminthosporium solani.Helminthosporium velutinum, Leptosphaeria bicolor, Paraphaeosphaeria michotii, Paraphaeosphaeria pileatea.Cucurbidothis pityophila.Bimuria novae-zelandiae.Letendraea helminthico-la.Kirschsteiniothelia
  • Phialophora sp. Coniosporium perforans. Coniosporium apollinis. Coniosporium per- forans.Glyphium elatum.Euascomycetes sp., Rhynchostoma minutum.Ceramothyrium camiolicum, Caliciopsis pineam.Lasioderma serricorne yeast-like.Halosarpheia marina.Halosarpheia viscosa.Phaeonectriella lignicola
  • Ophorus Dictyonema pavonia.Tulostoma macrocepha- la. Pluteus petasatus.Lentinula lateritia.Lentinula lateritia.Cyathus striatus.Calvatia gi- gantea.Lycoperdon sp.Agaricus bisporus, Amanita musca ria (fly agaric), Leucopaxillus albissimus.Tricholoma matsutake.Entoloma strictius.Stropharia rugosoannulata Basidiomycete symbiont of Apte.Calathella mangrovei.Nia vibrissa.Halocyphina villo- sa.Cyphellopsis anomala.Physalacria maipoensis.Favolaschia intermedia subsp.
  • Lentinellus omphalodes Lentinellus omphalodes. Lentinellus omphalodes. Lentinellus ursinus. Lentinellus ursinus, Clavicorona pyxi- data. Clavicorona pyxidata.
  • Syncephalastrum racemosum U.
  • Mucor ramannianus Blastocladiella emersonii, Entomophthora muscae. Entomophthora schizophora. Eryniopsis ptycopterae. Entomophaga aulicae, Pandora neoaphidis. Strongwellsea castrans. Zoophthora culisetae.
  • Conidiobolus lamprauges Conidiobolus thromboides, Conidiobolus coronatus.Chytridiomycetes sp., Smittium simulii.Smittium commune.Smittium simulii.Smittium cylindrospo- rum.Smittium imitatum.Smittium imitatum.Smittium tronadorium.Smittium orthocla-dii.Smittium tipulidarum.Smittium dipterorum.Smittium megazygosporum.Smittium cf. dipterorum.Smittium cf.
  • Candida versatilis. Candida otoi, Candida silvanorum., Candida entomophila., Candida sorbophila., Candida uringiensis.Candida antillancae, Candida tepae., Candida musae., Candida ishiwadae., Candida ncudensis, Candida castrensis., Candida vanderwaltii ., Metschnikowia australis., Candida fructus.Metschnikowia pulcherrima., Candida bertae., Wangiella dermatitidis.Candida ga lac ta., Candida salmanticensis., Candida parapsilosis.Candida petrohuensis., Candida polleni., Candida blankii., Ochrolechia parella, Candida rhagii., Candida homilentoma, Candida fennica., Bulgaria inquinans ,, Sporidiobolus sp.
  • Wickerhamiella domercqiea Zygosaccharomyces rouxii.Metschnikowia gruessii., Metschnikowia reukau- fii., Metschnikowia bicuspidata., Metschnikowia krissii., Candida anatomiae., Arxula terrestris.Candida agrestis., Glomerobolus gelineus.Candida vinaria.Blastocladiella emerso- nii.Candida lactis-condensi.Metschnikowia zobellii.Candida bondarzewiae.Candida sequanensis.Candida edax.Candida ernobii, Candida karawaiewii.Saccharomyces cerevisiae.Barrmaelia oxyacanthae.Arxula adeninivorans.
  • the DNA I I sequence is a DNA sequence which is suitable for a Polypeptide according to SEQ ID NO: 2,4,6 or 8 or a DNA sequence which codes for a polypeptide which has 80% identity to SEQ ID NO: 2,4,6 or 8, comprises.
  • DNA-II sequences which code for a polypeptide having laccase activity, the 85, 90, 92, 94, 95, 96, 97, 98, 99% identity to SEQ ID NO: 2, 4, 6 or 8 owns.
  • identity between two polypeptide sequences is determined by means of the GCG software developed by the University of Wisconsin by means of the program GAP using the settings for gap as proposed by the program as default values.
  • Laccase activity is the ability to oxidize phenolic substances, as described, for example, in Machuca et al., 1998, at page 218 (Determination of laccase type PO activity).
  • DNA molecules into cells is carried out by familiar cloning and transfection methods known to the person skilled in the art, such as, for example, co-precipitation, protoplast fusion, electroporation, retroviral transfection and the like, in order to express said nucleic acids in the respective expression system. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Ed., Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd ed., Colard Spring Harbor Laboratory, Col. Spring Harbor Laboratory Press, Col. Spring Harbor, NY, 1989.
  • DNA into cells can also be by homologous recombination.
  • the DNA is usually introduced into a corresponding vector for homologous recombination, as described, for example, by Thomas, K. R. and Capecchi, M.R. (1987) Cell 51: 503.
  • the cells are cultured according to the host organism in a manner known to those skilled in the art.
  • Microorganisms are generally contained in a liquid medium which contains a carbon source mostly in the form of sugars, a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese, magnesium salts and optionally vitamins.
  • a carbon source mostly in the form of sugars
  • a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate
  • trace elements such as iron, manganese, magnesium salts and optionally vitamins.
  • the pH of the nutrient fluid can be kept at a fixed value, that is regulated during the cultivation or not.
  • the cultivation can be batch-wise, semi-batchwise or continuous.
  • Nutrients can be presented at the beginning of the fermentation or fed semi-continuously or continuously. Selecting cells which express a Laccase from Aspergillus nidulans ge done by laccase catalyzed conversion of a substrate, which is then present as an easily identifiable product, usually as a dye. Such stained cells or colonies which convert the substrate in agar in their environment can easily be differentiated from the non-stained cells or colonies and subsequently separated.
  • Particularly suitable substrates are the compounds ABTS (2,2'-azino-bis- (3-ethylbenzthiazoline-6-sulfonate), DMA (3,5-dimethylaniline) and ADBP (4-amino-2,6-dibromophenol) In the following, compounds are also termed "test substrates".
  • test substrates can be converted by laccases in the presence of oxygen to a blue dye, allowing for rapid recognition of laccase activity.
  • the color change can be detected directly with the eye or measured photometrically, in particular for quantification.
  • An advantage in some embodiments of the methods of the invention is the fact that some A. nidulans laccases are secreted from the cell into the medium. This makes it possible to detect the laccase outside the cell, for example directly on the agar plate, without having to take up the test substrates in the cell.
  • a further preferred embodiment of the method according to the invention consists in that the individual laccases have different specificities with respect to the test substrates.
  • the individual laccases have different specificities with respect to the test substrates.
  • differential investigations with, for example, two laccases, wherein the first laccase converts the test substrate 1 and not the test substrate 2 and the second laccase converts the test substrate 2 and not the test substrate 1.
  • the substrate specificity of the laccases IccB, IccC, and IccD given in the table below allows the selection of suitable laccases and test substrates for such a differential method.
  • Suitable laccases for the processes according to the invention are the laccases naturally occurring in Aspergillus nidulans, including polymorphisms, alleles and sequence variants, and the laccases derivable therefrom, insofar as they still have the essential biological properties of the naturally occurring lac- cases, in particular the substrate specificity over those above mentioned test substrates.
  • laccases are the laccases IccA, IccB, IccC and IccD from A. nidulans, whose DNA and polypeptide sequences are listed in the Sequence Listing.
  • SEQ ID NO: 1 IccA DNA Sequence SEQ ID NO: 2 IccA Polypeptide Sequence SEQ ID NO: 3 IccB DNA Sequence SEQ ID NO: 4 IccB Polypeptide Sequence SEQ ID NO: 5 IccC DNA Sequence 1 SEQ ID NO: 6 IccC Polypeptide Sequence SEQ ID NO: 7 IccD DNA sequence SEQ ID NO: 8 IccD polypeptide sequence
  • a particularly advantageous embodiment of the process according to the invention results from the fact that the A. nidulans laccases have different specificities compared to the test substrates.
  • the following table shows the substrate specificities:
  • laccases IccB, IccC and IccD were amplified by PCR and expressed under the control of the alcohol-inducible promoter, alcA, and the xy / A promoter in A. nidulans. (Waring et al., 1989, Zadra et al., 2000). To test the enzyme activity, the strains were cultivated on threonine-containing or xylose-containing agar plates and the laccase-catalyzed oxidation of ABTS (2,2'-azino-bis- (3-ethylbenzthiazoline-6-sulfonate) was investigated.
  • ABTS 2,2'-azino-bis- (3-ethylbenzthiazoline-6-sulfonate
  • Laccases as reporter genes for the determination of promoter activities
  • laccase C can be fused to a promoter - analogous to the fusion with the alcA or xylA promoter - and thereby its activity can be determined.
  • a fungus can be cultured under different conditions (temperature, pH, medium composition) and thus the conditions of promoter activity can be determined.
  • the fungus can be cultured on agar plates and the activity assessed visually or spectroscopically.
  • the fungus can be grown in liquid culture and the laccase activity in the culture supernatant can be quantified.
  • Laccase B and D can be used in accordance with the other two substrates ein ⁇ .
  • Laccases for the localization of proteins The functionality of a predicted secretion signal can be tested by a translational fusion of an N-terminal region of a protein to be tested with a laccase in which the secretion signal has been deleted.
  • Laccases as a reporter gene for genetic screens
  • LccC can be fused with DNA fragments.
  • the generated library can be transformed into a fungal strain and positive, i. Laccase-secreting strains can be isolated by the blue color. This approach can also be carried out in microtiter plates, so that the "readout" by robots can take place. Thus the method is also suitable for high-throughput experiments.
  • Laccases may also serve to isolate regulators of defined promoters.
  • a specific promoter is fused with the laccase gene and introduced into a fungal strain.
  • the strain which then shows expression of the laccase according to the promoter activity, can be mutagenized to isolate mutants with altered regulation. Thereby, e.g. Strains are produced with enhanced promoter activity. If the same promoter in the strain is used to express a desired gene, the expression of this gene can be increased. Mutants with reduced PM motor strength can also be isolated. This effect may be due to the fact that positive regulators have been mutated. These mutants can be used to generate the corresponding regulators, e.g. by isolation with a gene bank.
  • Laccases IccB, IccC or IccD as reporter genes for the quantification of promoter activities
  • the open reading frame of the three laccases in which the secretion signals were deleted, can be fused with promoters and transformed into fungi. These constructs lead to the production of intracellular laccase.
  • the enzyme activity can be exactly quantified after cell disruption. Because the three laccases are different Reacting substrates, several promoter activities can be quantified simultaneously. This system is similar to the simultaneous use of beta-galactosidase and gluturonidase.
  • Fig. 2 Expression of LccC and LccD in RMS011.
  • the laccase genes were introduced into RMS011 under the control of the xylA promoter and the colonies were cultured on agar plates with glucose (A) or glycerol (B 1 C) for 2 days at 37 ° C.
  • the medium was spiked with ABTS.
  • the cultures in (C) were overlaid with ADBP and DMA after growth.
  • Fig. 3 Expression of LccC under the control of the xylA promoter.
  • the / ccC gene was introduced into RMS011 under the control of the xylA promoter.
  • the strains express the gene to different extents, which can be seen in the different dyeing intensity.
  • Fig. 4 Quantification of LccC activity.
  • the / ccC gene was introduced into RMS011 under the control of the alcA promoter. Liquid medium was seeded with spores and cultured with glucose on substrate for 20 h. Subsequently, the hyphae were harvested and induced in medium with 100 mM ethanol. After different times, equal amounts of mycelium were removed and incubated for 2 h in Laccase assay buffer (137 mM di-sodium hydrogen phosphate, 26 mM citric acid, pH 6.0) at 4 ° C., the enzyme from the hyphae solved.
  • Laccase assay buffer 137 mM di-sodium hydrogen phosphate, 26 mM citric acid, pH 6.0
  • laccase activity was measured: to 900 ⁇ l of laccase assay buffer was added 100 ⁇ l of ABTS (50 mM in H 2 O). The reaction was photometrically followed by addition of enzyme started at 405 nm. Reaction temperature was at 37 ° C. Samples 1-3 are independent cultures derived from independent precultures. The labels a and b result from the duplicate determination of the samples.
  • laccase I Aspergillus nidulans yA (laccase I) gene. Nucl. Acid Res. 18, 3415. Crestini, C, Jurasek, L. & Argyropoulos, D.S. (2003). On the mechanism of the lac-case mediator System in the oxidation of lignin. Chemistry 9, 5371-5378.

Abstract

Method for determining the regulatory properties of a genetic element, which comprises the following steps: a) introducing a DNA molecule having a DNA-I sequence whose regulatory properties are to be determined into a cell, said DNA-I sequence being linked with an additional DNA-II sequence encoding a laccase from Aspergillus nidulans in such a manner that the regulatory elements of the DNA-I sequence control expression of the laccase, b) cultivating the cells obtained in step (a) under conditions allowing expression of the laccase, c) associating the conditions selected under (b) with the regulatory properties of the DNA-I sequence.

Description

Verwendung von Laccasen als ReportergeneUse of laccases as reporter genes
Beschreibungdescription
Die Erfindung betrifft die Verwendung von Laccasen aus Aspergillus nidulans als Re¬ portersysteme in Verfahren zur qualitativen und quantitativen Bestimmung von Genex¬ pression, insbesondere zur qualitativen und quantitativen Bestimmung von Promotor¬ stärken, in Verfahen zur Lokalisation von Proteinen in einer Zelle, sowie Verfahren zur Selektion bestimmter rekombinanter Zellen sowie in Verfahren zur heterologen Genex- pression.The invention relates to the use of laccases from Aspergillus nidulans as reporter systems in methods for the qualitative and quantitative determination of gene expression, in particular for the qualitative and quantitative determination of promoter strengths in processes for the localization of proteins in a cell, and to methods for Selection of Certain Recombinant Cells and in Methods for Heterologous Gene Expression.
Stand der TechnikState of the art
Laccasen (EC 1.10.3.2) sind blaue Multi-Kupferenzyme, die von Pilzen und Pflanzen produziert werden. Sie oxidieren eine Reihe aromatischer Verbindungen wie Mono-, Di, und Polyphenole, aromatische Amine und Methoxy-phenole. Bei der Oxidation werden dem Substrat Elektronen entzogen, im Enzym "gesammelt" und dann jeweils vier Elek¬ tronen auf Sauerstoff übertragen und Wasser gebildet. (Messerschmidt & Huber, 1990).Laccases (EC 1.10.3.2) are blue multi-hybrid enzymes produced by fungi and plants. They oxidize a number of aromatic compounds such as mono-, di- and polyphenols, aromatic amines and methoxy-phenols. During the oxidation, electrons are removed from the substrate, "collected" in the enzyme, and then four electrons each are transferred to oxygen and water is formed. (Messerschmidt & Huber, 1990).
Pilze enthalten in der Regel mehrere Laccasen, die vermutlich unterschiedliche biolo¬ gische Funktionen ausüben. Im Basidiomycenten Coprinus cinereus wurden acht Lac¬ casen beschrieben, während im Ascomycete'n A. nidulans sechs Laccasen vorkommen (Wagner, 2003; Hoegger et al., 2004). Die genaue biologische Rolle der pilzlichen Lac- casen ist weitgehend unbekannt. Sie besitzen meistens ein N-terminales Sekretionssi¬ gnal und werden sekretiert. Am besten ist die Funktion der Basidiomycetenlaccasen im Ligninabbau untersucht (Crestini et al., 2003). In anderen Pilzen wird eine Funktion bei der Melaninbiosynthese angenommen oder beim Abbau von phenolischen Substanzen (Hermann et al., 1983; Fernändez-Larrea & Stahl, 1996).Fungi usually contain several laccases, which presumably have different biological functions. In the basidiomycin Coprinus cinereus eight lac- cases were described, while in the Ascomycete ' n A. nidulans six laccases occur (Wagner, 2003, Hoegger et al., 2004). The exact biological role of fungal lacquases is largely unknown. They usually have an N-terminal secretion signal and are secreted. The function of basidiomycete laccases in lignin degradation is best studied (Crestini et al., 2003). In other fungi, a function is assumed in the melanin biosynthesis or in the degradation of phenolic substances (Hermann et al., 1983, Fernandez-Larrea & Stahl, 1996).
Reportergene wie beta-Galaktosidase oder Glucuronidase werden vielfältig in der Mo¬ lekularbiologie und Biotechnologie eingesetzt. Sie haben allerdings den Nachteil, dass die Substrate in die Zellen eingebracht werden müssen. Ausserdem besitzen viele Pil¬ ze endogene Enzyme, die zu hohen basalen Enzymaktivitäten führen.Reporter genes such as beta-galactosidase or glucuronidase are widely used in molecular biology and biotechnology. However, they have the disadvantage that the substrates must be introduced into the cells. In addition, many fungi possess endogenous enzymes which lead to high basal enzyme activities.
Weitere häufig verwendete Reportergene stellen die fluoreszierenden Proteine (Green Fluorescent Protein GFP, und Yellow Fluorescent Protein , zur Ausbildung der Fluo¬ reszenz und werden vielfältig zur Lokalisierung von Proteinen aber auch zur MessungOther frequently used reporter genes provide the fluorescent proteins (Green Fluorescent Protein GFP, and Yellow Fluorescent Protein, for the formation of Fluo¬ rescence and are versatile for the localization of proteins but also for measurement
[3 Fig.] von Promotorstärken verwendet (US 5,491,084). Allerding s können sie nicht gut für genetische Screens eingesetzt werden, da das Fluoreszenzsignal nur relativ unemp¬ findlich auf Agarplatten ausgelesen werden kann.[3 Fig.] of promoter strengths (US 5,491,084). However, they can not be used well for genetic screens since the fluorescence signal can only be read out on agar plates with relative insensitivity.
Aufgabenstellungtask
Es bestand daher die Aufgabe, neue Verfahren für genetische Screeningverfahren wie Nachweis der Expression eines bestimmten Gens, Messung der Expressionsstärke eines genetischen Regulationssignals, Nachweis der Lokalisation eines Proteins in der Zelle (Zellkompartimenten), bereitzustellen, die nicht die o.g. die o.g. Restriktionen und Nachteile aufweisen.It was therefore an object to provide new methods for genetic screening methods such as detection of the expression of a particular gene, measurement of the expression level of a genetic regulatory signal, detection of the localization of a protein in the cell (cell compartments), not the o.g. the o.g. Have restrictions and disadvantages.
Beschreibung der ErfindungDescription of the invention
Die vorliegende Erfindung betrifft in einer ersten Ausführungsform ein Verfahren zur Bestimmung von regulatorischen Eigenschaften eines genetischen Ele¬ mentes, das die Schritte umfasst: (a) Einführen eines DNA-Moleküls mit einer DNA-I-Sequenz, deren regulatorischeThe present invention relates, in a first embodiment, to a method for the determination of regulatory properties of a genetic element comprising the steps of: (a) introducing a DNA molecule having a DNA I sequence whose regulatory
Eigenschaften bestimmt werden sollen, in eine Zelle, wobei die DNA-I-Sequenz mit einer weiteren DNA-I I-Sequenz , die für eine Laccase aus Aspergillus nidu- lans codiert, so verknüpft ist, dass die regulatorischen Elemente der DNA-I- Sequenz die Expression der Laccase kontrollieren, (b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die die Expression der Laccase erlauben,Properties are to be determined in a cell, the DNA I sequence being linked to a further DNA I 1 sequence which codes for an Aspergillus nidulans laccase in such a way that the regulatory elements of the DNA Control the expression of the laccase, (b) cultivating the cells obtained under (a) under conditions which allow expression of the laccase,
(c) Zuordnen der unter (b) gewählten Bedingungen zu den regulatorischen Eigen¬ schaften der DNA-I-Sequenz.(c) assigning the conditions selected under (b) to the regulatory properties of the DNA I sequence.
In einer zweiten Ausführungsform betrifft die Erfindung einIn a second embodiment, the invention relates to a
Verfahren zur Lokalisation eines gewünschten Proteins in einem Zellkompartiment, das die Schritte umfasst:A method of localizing a desired protein in a cell compartment comprising the steps of:
(a) Einführen eines DNA-Moleküls, das eine DNA Sequenz besitzt, die für das ge- wünschte Protein codiert, und die so mit einer weiteren DNA-Il-Sequenz ver¬ knüpft ist, die für eine Laccase aus Aspergillus nidulans codiert, das das Prote¬ in, das von dem DNA-Molekül codiert wird, eine Fusion aus dem gewünschten Protein mit der Laccase darstellt, in die Zellen,(a) introducing a DNA molecule which has a DNA sequence which codes for the desired protein and which is thus linked to a further DNA II sequence which codes for a laccase from Aspergillus nidulans, the the protein encoded by the DNA molecule is a fusion of the desired protein with the laccase into the cells,
(b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die die Expression des fusionierten Proteins erlauben,(b) culturing the cells obtained under (a) under conditions which allow the expression of the fused protein,
(c) Nachweis der Lokalisation der Laccase in einem Zlellkompartiment und damit der Lokalisation des gewünschten Proteins in diesem Zellkompartiment, insbe- sondere durch Zellfraktionierung und anschliessende Bestimmung der Lacca- seaktivität in den verschiedenen Fraktionen.(c) detection of the localization of the laccase in a cell compartment and thus the localization of the desired protein in this cell compartment, in particular in particular by cell fractionation and subsequent determination of laccase activity in the various fractions.
In einer dritten Ausführungsform betrifft die Erfindung ein Verfahren zur Bestimmung der Expression eines gewünschten Gens in einer Zelle, das die Schritte umfasst:In a third embodiment, the invention relates to a method for determining expression of a desired gene in a cell comprising the steps of:
(a) Einführen eines DNA-Moleküls, das eine DNA Sequenz des gewünschten Gens besitzt, in eine Zelle, wobei die DNA-Sequenz mit einer weiteren DNA-II- Sequenz, die für eine Laccase aus Aspergillus nidulans codiert, so verknüpft ist, dass die regulatorischen Elemente des gewünschten Gens die Expression der Laccase kontrollieren,(A) introducing a DNA molecule which has a DNA sequence of the desired gene into a cell, wherein the DNA sequence is linked to a further DNA II sequence coding for an Aspergillus nidulans laccase such that the regulatory elements of the desired gene control the expression of the laccase,
(b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die d ie Expression des gewünschten Gens erlauben,(b) cultivating the cells obtained under (a) under conditions permitting the expression of the desired gene,
(c) Nachweis der Expression der Laccase in der Zelle und damit Nachweis der Ex- pression des gewünschten Gens.(c) Detection of the expression of the laccase in the cell and thus proof of the expression of the desired gene.
In einer vierten Ausführungsform betrifft die Erfindung einIn a fourth embodiment, the invention relates to a
Verfahren zur Expression eines gewünschten Gens in einer Zelle, das die Schritte um¬ fasst: (a) Einführen eines DNA-Moleküls, das eine DNA-II- Sequenz, die für eine Laccase aus Aspergillus nidulans codiert, besitzt und mit deren C-terminalem Ende eine weitere DNA-I-Sequenz, die für das gewünschte Genprodukt codiert, so verknüft ist, das die Expression als Fusionsprotein aus Laccase u nd gewünsch¬ tem Genprodukt erfolgt, (b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die d ie Expression des gewünschten Gens erlauben, (c) Isolierung des Fusionsproteins aus Laccase und gewünschtem G enprodukt.A method of expressing a desired gene in a cell comprising the steps of: (a) introducing a DNA molecule having a DNA II sequence coding for an Aspergillus nidulans laccase and having the C-terminal one thereof End of another DNA-I sequence which codes for the desired gene product is linked so that the expression takes place as a fusion protein of laccase and the desired gene product, (b) cultivating the cells obtained under (a) under conditions which The expression of the desired gene allows (c) isolation of the fusion protein from laccase and desired gene product.
Hierbei wird Laccase als Reportergen bzw. Reporterprotein für heterolog exprimierte Proteine verwendet. Viele industriell bedeutende Proteine werden in filamentösen Pil¬ zen produziert. Dabei wird versucht, die Proteine zu sekretieren, um die Λufreinigung zu erleichtern. Laccasen können verwendet werden, um nicht-sekretierte Proteine zu sekretieren. Eine C-terminale Fusion eines gewünschten Proteins mit Laccase führt zur Sekretion des Proteins und ermöglicht, aufgrund der Laccaseaktivität, den leichten Nachweis des sekretierten Proteins. Durch Einführung einer Proteaseschnittstelle zwi¬ schen den beiden Proteinen kann das gewünschte Protein freigesetzt werden.Here, laccase is used as reporter gene or reporter protein for heterologously expressed proteins. Many industrially important proteins are produced in filamentous fungi. An attempt is made to secrete the proteins in order to facilitate the purification. Laccases can be used to secrete non-secreted proteins. A C-terminal fusion of a desired protein with laccase leads to the secretion of the protein and, due to the laccase activity, allows the ready detection of the secreted protein. By introducing a protease cleavage between the two proteins, the desired protein can be released.
Geignete Zellen für die o.g. Verfahren sind pflanzliche, beispielsweise Karottenwurzeln, tierische und menschliche, beispielsweise HeIa oder HEK293, und insbesondere pilzli¬ che Zellen. Besonders gut lassen sich die Verfahren in Zellen der folgenden Arten aus¬ führen: Alternaria alternata.Clathrospora diplospora.Alternaria brassicae,Alternaria brassicico- Ia1 Alternaria raphani, Pleospora herbarum, Pyrenophora trichostoma Cochliobolus sativus, Cochliobolus heterostrophus, Setosphaeria monoceras Setosphaeria rostrata, Pleospora betae, Leptosphaeria maculans, Leptosphaeria mi- croscopica, Ophiobolus herpotrichus, Cucurbitaria elongata.Phoma sp.Suitable cells for the abovementioned processes are vegetable, for example carrot roots, animal and human, for example HeIa or HEK293, and in particular fungal cells. The processes can be carried out particularly well in cells of the following types: Alternaria alternata.Clathrospora diplospora.Alternaria brassicae, Alternaria brassicico- Ia 1 Alternaria raphani, Pleospora herbarum, Pyrenophora trichostoma Cochliobolus sativus, Cochliobolus heterostrophus, Setosphaeria monoceras Setosphaeria rostrata, Pleospora betae, Leptosphaeria maculans, Leptosphaeria miscroscopica, Ophiobolus herpotrichus, Cucurbitaria elongata. Phoma sp.
Leptosphaeria doliolum, Cucurbitaria berberidis, Paraphaeosphaeria agavensis Paraphaeosphaeria nolinae, Paraphaeosphaeria obtusispora, Paraphaeosphaeria con- glomerata,Paraphaeosphaeria quadriseptat,Paraphaeosphaeria filamento- sa.Melanomma sanguinarium, Paraphaeosphaeria glauco-punct.Septoria nodo- rum.Phoma herbarum, Pleospora rudis.Helminthosporium solani.Helminthosporium velutinum, Leptosphaeria bicolor, Paraphaeosphaeria michotii, Paraphaeosphaeria pil- leata.Cucurbidothis pityophila.Bimuria novae-zelandiae.Letendraea helminthico- la.Kirschsteiniothelia elaterascu.Helicascus kanaloanus.Herpotrichia diffu¬ sa, Mycosphaerella mycopappi.Herpotrichia juniperi.Byssothecium circi- nans.Lophiostoma crenatum.Trematosphaeria heterospora,Kirschsteiniothelia mariti- ma.Sporomia lignicola.Westerdykella cylindrica.Westerdykella dispersa, Delitschia didyma.Dendryphiopsis atra,Kirschsteiniothelia aethiops, Sarcinomyces pe- tricola.Phaeosclera dematioides.Sarcinomyces crustaceus,Scytalidium dimidia- tum.Scytalidium hyalinum.Scytalidium dimidiatum.Botryosphaeria ribis.Capnobotryella sp.,Capnobotryella renispora.Coccodinium bartschii.Hortaea werneckii.Phaeotheca fissurella.Acrospermum compressum .Acrospermum ramineum,Phaeotrichum benja- minii.Aureobasidium pullulans.Discosphaerina fagi.Dothidea hippophaeos.Dothidea insculpta.Stylodothis puccinioides.Dothidea ribesia, Delphinella strobiligena.Myriangium duriaei.Myriangium duriaei,Piedraia hortae.Raciborskiomyces longiseto- sum.Capnodium citri.Euascomycetes sp.,Thecotheus holmskjoldii.Lasallia rossi- ca.Baeomyces rufus,Anamylopsora pulcherrima,Placopsis gelida.Trapelia involu- ta.Trapelia placodioides.Pertusaria trachythallina.Ochrolechia parella.Coccotrema cu- curbitula.Lepolichen coccophorus.Diploschistes rampoddensis.Diploschistes thunber- gianus.Graphis scripta.Conotrema populorum.Gyalecta ulmi,Dibaeis baeomyces, Thamnolia vermicularis.Stenocybe pullatula.Sphinctrina turbinata,Cladonia sulphuri- na,Cladonia bellidiflora,Cladonia subcervicomis.Metus conglomeratus.Pilophorus robu- stus.Pilophorus acicularis.Pilophorus cereolus.Pilophorus strumaticus.Cladia retipo- ra.Stereocaulon paschale.Stereocaulon taeniarum.Stereocaulon vesuvia- num.Stereocaulon ramulosum.Lecidella meiococca.Squamarina lentigera.Lecanora intumescens.Alectoria sarmentosa.Letharia columbiana.Vulpicida pinastri.Cetraria slandica,Cornicularia normoerica.Pleurosticta acetabulum.Xanthoparmelia consper- sa.Evemia prunastri.Parmelia saxatilis.Hypogymnia physodes.Bunodophoron scrobicu- latum.Leifidium tenerum.Sphaerophorus globosus.Sphaerophorus stereocauloi- des.Neophyllis melacarpa.Austropeltum glareosum.Lecania cyrtella.Psora decipi- ens.Cliostomum griffithii.Toninia sedifolia,Bacidia rosella.Caloplaca flavorubes- cens.Xanthoria elegans.Xanthoria parietina.Tephromela atra.Thelomma mammo- sum.Texosporium sancti-jacobi,Cyphelium inquinans.Buellia disciformis.Physcia aipo- lia.Cyphelium tigillare.Lecanora dispersa, Leprocaulon sp.,Lecidea fuscoatra.Emericella nidulans.Aspergillus ustus.Aspergillus versicolor.Aspergillus oryzae.Aspergillus parasi- ticus.Aspergillus tamarii,Aspergillus flavus .Aspergillus sojae.Aspergillus nomi- us.Aspergillus avenaceus.Eurotium herbariorum.Eurotium rubrum.Aspergillus restric- tus.Aspergillus clavatus.Aspergillus fumigatus.Neosartorya fischeri.Aspergillus ter- reus.Fennellia flavipes.Aspergillus wentii.Chaetosartorya cremea.Aspergillus ochra- ceus.Aspergillus awamori.Aspergillus niger .Aspergillus candidus.Aspergillus cervi- nus.Penicillium allii.Penicillium expansum,Penicillium commune.Penicillium freii.Penicilium notatum.Eupenicillium crustaceum.Geosmithia namyslowskii.Penicillium namyslowskii.Eupenicillium javanicum.Chromocleista malachitea.Monascus purpu- reus,Hamigera avellanea,Merimbla ingelheimense.Hamigera striata, Hemicarpenteles omatus.Penicilliopsis clavariiformis.Aspergillus zonatus.Aspergillus spar- sus.Chromocleista cinnabarina.Chromocleista cinnabarina.Talaromyces fla- vus.Talaromyces bacillisporus.Talaromyces luteus.Talaromyces luteus.Elaphomyces leveillei.Elaphomyces maculatus.Trichocoma paradoxa.Geosmithia cylindrospo- ra.Talaromyces eburneus.Talaromyces emersonii.Byssochlamys nivea, Therm oascus crustaceus.Malbranchea dendritica.Malbranchea filamentosa.Auxarthron zuffia- num.Renispora flavissima.Malbranchea albolutea.Coccidioides immitis .Uncinocarpus reesii.Coccidioides immitis, Onygena equina.Pseudoamauroascus australien- si.Castanedomyces australiensis.Trichophyton rubrum, Malbranchea gypsea.Ascosphaera apis 2,Ascosphaera apis.Eremascus albus.Ajellomyces capsula- tus.Histoplasma capsulatum Eukaryo.Histoplasma capsulatum, Paracoccidioides brasi- liensis.Lacazia loboi.Lacazia loboi.Exophiala dermatidis,Graphium calicioides.Capronia pilosella.Cladophialophora boppii.Cladophialophora arxii.Fonsecaea pedro- soi,Cladophialophora devriesii.Phialophora verrucosa, Capronia mansonii.Exophiala dermatitidis.Püllularia prototropha.Exophiala dermatitidis.Sarcinomyces phaeomurifor- mis.Capronia coronata.Capronia epimyces.Rhinocladiella phaeophora.Ramichloridium anceps.Capronia fungicola, Capronia pilosella.Exophiala jeanselmei.Phaeococcomyces exophialae,Phaeoannellomyces elegans,Nadsoniella nigra, Exophiala sp.,Rhinocladiella atrovirens, Capronia dactylotricha, Capronia pulcherrima.Capronia acutiseta.Cladophialophora sp.,Ceramothyrium linnaeae, Exophiala manso- nii.Phialophora sp.,Coniosporium perforans.Coniosporium apollinis.Coniosporium per- forans.Glyphium elatum.Euascomycetes sp.,Rhynchostoma minutum.Ceramothyrium camiolicum,Caliciopsis pineam.Lasioderma serricorne yeast-like.Halosarpheia mari- na.Halosarpheia viscosa.Phaeonectriella lignicolaLeptosphaeria Doliolum, Cucurbitaria Berberidis, Paraphaeosphaeria agavensis Paraphaeosphaeria nolinae, Paraphaeosphaeria obtusispora, Paraphaeosphaeria con- glomerata, Paraphaeosphaeria quadriseptat, Paraphaeosphaeria filamento- sa.Melanomma Sanguinarium, Paraphaeosphaeria glauco-punct.Septoria nodo- rum.Phoma herbarum Pleospora rudis.Helminthosporium solani.Helminthosporium velutinum, Leptosphaeria bicolor, Paraphaeosphaeria michotii, Paraphaeosphaeria pileatea.Cucurbidothis pityophila.Bimuria novae-zelandiae.Letendraea helminthico-la.Kirschsteiniothelia elaterascu.Helicascus kanaloanus.Herpotrichia diffusa, Mycosphaerella mycopappi.Herpotrichia juniperi.Byssothecium circinans.Lophiostoma crenatum Trematosphaeria heterospora, Kirschsteiniothelia maritima, Sporomia lignicola, Westerdykella cylindrica, Westerdykella dispersa, Delichia didyma Dendryphiopsis atra, Kirschsteiniothelia aethiops, Sarcinomyces per- tricola, Phaeosclera dematioides, Sarcinomyces crustaceus, Scytalidium dimi dytum.Scytalidium hyalinum.Scytalidium dimidiatum.Botryosphaeria ribis.Capnobotryella sp., Capnobotryella renispora.Coccodinium bartschii.Hortaea werneckii.Phaeotheca fissurella.Acrospermum compressum .Acrospermum ramineum, Phaeotrichum benja- minii.Aureobasidium pullulans.Discosphaerina fagi.Dothidea hippophaeos.Dothidea insculpta.Stylodothis puccinioides.Dothidea ribesia, Delphinella strobiligena.Myriangium duriaei.Myriangium duriaei, Piedraia hortae.Raciborskiomyces longisetosum.Capnodium citri.Euascomycetes sp., Thecotheus holmskjoldii.Lasallia rossi ca.Baeomyces rufus, Anamylopsora pulcherrima, Placopsis gelida.Trapelia involute.Trapelia placodioides.Pertusaria trachythallina.Ochrolechia parella.Coccotrema cucurbitula.Lepolichen coccophorus.Diploschistes rampoddensis.Diploschistes thunbergianus.Graphis scripta.Conotrema populorum.Gyalecta ulmi, Dibaeis baeomyces, Thamnolia vermicularis.Stenocybe pullatula.Sphinctrina turbinata, Cladonia sulphuria, Cladonia bellidiflora, Clado nia subcervicomis.Metus conglomeratus.Pilophorus robustus.Pilophorus acicularis.Pilophorus cereolus.Pilophorus strumaticus.Cladia retypoara.Stereocaulon paschale.Stereocaulon taeniarum.Stereocaulon vesuvia num.Stereocaulon ramulosum.Lecidella meiococca.Squamarina lentigera.Lecanora intumescens.Alectoria sarmentosa .Letharia columbiana.Vulpicida pinastri.Cetraria slandica, Cornicularia normoerica.Pleurosticta acetabulum.Xanthoparmelia conspersa.Evemia prunastri.Parmelia saxatilis.Hypogymnia physodes.Bunodophoron scrobicu- latum.Leifidium tenerum.Sphaerophorus globosus.Sphaerophorus stereocauloides.Neophyllis melacarpa.Austropeltum glareosum.Lecania cyrtella.Psora decipi- ens.Cliostomum griffithii.Toninia sedifolia, Bacidia rosella.Caloplaca flavorubes- cens.Xanthoria elegans.Xanthoria parietina.Tephromela atra. Thelomma mammo- sum.Texosporium sancti-jacobi, Cyphelium inquinans.Buellia disciformis.Physcia afipia.Cyphelium tigillare.Lecanora dispersa, Leprocaulon sp., Lecidea fuscoatra.Emericella nidulans.Aspergillus ustus.Aspergillus versicolor.Aspergillus oryzae.Aspergillus parasiticus Aspergillus tamarii, Aspergillus flavus, Aspergillus sojae, Aspergillus nomius, Aspergillus avenaceus, Herotium herbariorum, Eurotum rubrum, Aspergillus restricus, Aspergillus clavatus, Aspergillus fumigatus, Neosartorya fischeri, Aspergillus terreus, Fenellia flavipes, Aspergillus wentii, Chaetosartorya Aspergillus awamori.Aspergillus niger. Aspergillus candidus.Aspergillus cervinus.Penici Àllium allii.Penicillium expansum, Penicillium commune.Penicillium freii.Penicilium notatum.Eupenicillium crustaceum.Geosmithia namyslowskii.Penicillium namyslowskii.Eupenicillium javanicum.Chromocleista malachitea.Monascus purpu- Reus, Hamigera avellanea, Merimbla ingelheimense.Hamigera striata, Hemicarpenteles omatus.Penicilliopsis clavariiformis. Aspergillus zonatus.Aspergillus sparus.Chromocleista cinnabarina.Chromocleista cinnabarina.Talaromyces flavus.Talaromyces bacillisporus.Talaromyces luteus.Talaromyces luteus.Elaphomyces leveillei.Elaphomyces maculatus.Trichocoma paradoxa.Geosmithia cylindrospora.Talaromyces eburneus.Talaromyces emersonii.Byssochlamys nivea , Thermoascus crustaceus.Malbranchea dendritica.Malbranchea filamentosa.Auxarthron zuffia- num.Renispora flavissima.Malbranchea albolutea.Coccidioides immitis .Uncinocarpus reesii.Coccidioides immitis, Onygena equina.Pseudoamauroascus australia- si.Castanedomyces australiensis.Trichophyton rubrum, Malbranchea gypsea.As cosphaera apis 2, Ascosphaera apis.Eremascus albus.Ajellomyces capsula- tus.Histoplasma capsulatum Eukaryo.Histoplasma capsulatum, Paracoccidioides brasilieniensis.Lacazia loboi.Lacazia loboi.Exophiala dermatidis, Graphium calicioides.Capronia pilosella.Cladophialophora boppii.Cladophialophora arxii.Fonsecaea pedro - soi, Cladophialophora devriesii.Phialophora verrucosa, Capronia mansonii.Exophiala dermatitidis.Püllularia prototropha.Exophiala dermatitidis.Sarcinomyces phaeomurifor- mis.Capronia coronata.Capronia epimyces.Rhinocladiella phaeophora.Ramichloridium anceps.Capronia fungicola, Capronia pilosella.Exophiala jeanselmei.Phaeococcomyces exophialae, Phaeoannellomyces elegans, Nadsoniella nigra, Exophiala sp., Rhinocladiella atrovirens, Capronia dactylotricha, Capronia pulcherrima.Capronia acutiseta.Cladophialophora sp., Ceramothyrium linnaeae, Exophiala mansoiiii. Phialophora sp., Coniosporium perforans. Coniosporium apollinis. Coniosporium per- forans.Glyphium elatum.Euascomycetes sp., Rhynchostoma minutum.Ceramothyrium camiolicum, Caliciopsis pineam.Lasioderma serricorne yeast-like.Halosarpheia marina.Halosarpheia viscosa.Phaeonectriella lignicola
.Halosarpheia lotica.Halosarpheia retorquens.Nais inomata.Halosarpheia heteroguttu- lata.Halosarpheia cincinnatula.Halosarpheia fibrosa.Halosarpheia spartinae.Halosarpheia lotica.Halosarpheia retorquens.Nais inomata.Halosarpheia heteroguttu- lata.Halosarpheia cincinnatula.Halosarpheia fibrosa.Halosarpheia spartinae
.Graphium putredinis.Pseudallescheria boydii.Pseudallescheria ellipsoidea.Microascus cirrosus.Graphium putredinis.Graphium tectonae.Graphium putredinis.Petriella setife- ra.Lomentospora prolificans.Ceratocystis fimbriata.Graphium penicillioides.Glomerella cingulata.Verticillium longisporum.Verticillium dahliae.Volutella colletotrichoi- des.Plectosphaerella cucumerina.Cordyceps yakusimensis.Cordyceps sobolife- ra.Cordyceps capitata,Cordyceps cochlidiicola.Cordyceps prolifica.Cordyceps inegoen- sis,Tolypocladium inflatum.Cordyceps paradoxa.Cordyceps japonica.Cordyceps hete- ropoda.Hamiltonaphis styraci yeast-li.Cordyceps ramosopulvinata,Paecilomyces sp.,Cordyceps kanzashiana.Trichoderma koningii.Trichoderma viride.Taphrina defor- mans.Hypocrea lutea, Hypomyces chrysospermus.Volutella ciliata, Volutella cilia- ta.Chaetopsina fulva,Gibberella pulicaris.Gibberella moniliformis,Botryotinia fuckelia- na,Botryotinia convoluta.Mycoarachis inversa.Cordyceps pruinosa.Cordyceps takao- montana.Botrytis cinerea,Cordyceps broπgniartii.Cordyceps militaris,Paecilomyces tenuipes.Bionectria ochroleuca.Nectria ochroleuca.Nectria aureofulva.Nectria cinnaba- rina,Geosmithia putterillii.Geosmithia lavendula.Lulworthia fucicola.Chaetomium ela- tum.Neurospora crassa.Sordaria firmicola.Podospora anserina.Ascovaginospora stelli- pala.Phyllachora graminis.Kionochaeta ramifera.Kionochaeta spissa.Kionochaeta spis- sa.Kionochaeta ivoriensis.Meliola juddiana.Meliola niessleana.Neurospora cras- sa.Ophioceras leptosporum.Magnaporthe grisea.Pseudohalonectria falca- ta.Cryphonectria cubensis.Cryphonectria havanensis,Endothia gyrosa.Leucostoma persoonii.Ophiostoma penicillatum.Pesotum fragrans.Ophiostoma bicolor.Ophiostoma piceae.Ophiostoma floccosum .Ophiostoma piliferum.Ophiostoma ulmi.Ophiostoma schenckii.Ophiostoma stenoceras.Ophiostoma cucullatum.Ophiostoma europhioi- des.Ophiostoma ainoae.Papulosa amerospora.Ascolacicola austriaca.Rhamphoria delicatula.sordariomycete sp.,Poronia punctata, Rosellinia necatrix.Xylaria sp.,Xylaria polymorpha.Muscodor sp.,Barrmaelia oxyacanthae,Hypoxylon fragifor- me.Monographella nivalis, Pestalosphaeria hansenii.Pestalosphaeria sp.,Hyponectria buxi.Monilinia fructicola.Monilinia laxa,Sclerotinia sclerotiorum.dark septate endophyte DS16b,Euascomycetes sp.,Neobulgaria premnophila, Blumeria graminis,Phyllactinia guttata.Spathularia flavida.Cyttaria darwinii.Geomyces pannorum var. panno- ru.Thelebolus stercoreus.Ascozonus woolhopensis.Selenaspora guernisacii.lnermisia aggregata.Pyronema domesticum.Otidea onotica.Glaziella aurantiaca.Cephaliophora tropica.Cephaliophora irregularis.Scutellospora castanea.Geopyxis carbonaria Sarcoscypha austriaca,Pseudopithyella minuscula.Kompsoscypha chudei Pithya cupressina.Nanoscypha tetraspora.Phillipsia domingensis.Wynnea sp.,Cookeina tricholoma.Microstoma floccosum.Wolfina aurantiopsis.Strobiloscypha keliae.Desmazierella acicola.Neournula pouchetii.Chorioactis geaster.Donadinia sp.,Ga!iella rufa.Plectania rhytidia.Sarcosoma mexicanum.Pseudoplectania nigrel- la,Tuber borchii , Tuber gibbosum.Choiromyces meandriformis.Choiromyces veno- sus.Tuber excavatum.Tuber magnatum ,Tuber panniferum.Dingleya verruco- sa.Reddellomyces donkii.Labyrinthomyces varius.Helvella lacunosa.Wynnella silvico- lor.Helvella terrestris.Balsamia vulgaris, Barssia oregonensis.Balsamia magnata, Underwoodia columnaris.Morchella elata 2,Morchella esculenta,Verpa conica.Disciotis venosa.Fischerula subcauüs.Leucangium carthusianum.Verpa bohemi- ca.Pseudorhizina califomica,Gyromitra esculenta 2,Gyromitra melaleucoides.Discina macrospora.Gyromitra montana.Hydnotrya cerebriformis,Rhizina undula- ta.Pachyphloeus citrinus.Cazia flexiascus.Terfezia arenaria.Peziza echinospo- ra.Tβrfezia terfezioides.Orbilia fimicola.Orbilia auricolor.Dactylella oxyspo- ra.Cephaliophora longispora.Cephaliophora muscicola.Arxiozyma tellu- ris.Saccharomyces transvaalensis.Saccharomyces dairensis.Saccharomyces servazzii, Saccharomyces unisporus.Zygosaccharomyces bailii,Zygosaccharomyces len- tus,Zygosaccharomyces bisporus,Zygosaccharomyces melIis,Zygosaccharomyces rouxii.Zygosaccharomyces rouxii 2,Torulaspora globosa.Zygosaccharomyces florenti- nus, Candida colliculosa.Torulaspora delbrueckii.Torulaspora pretorien- sis.Zygosaccharomyces microellipso.Zygosaccharomyces mrakiijorulaspora del- brueckii.Saccharomyces kudriavzevii.Saccharomyces mikatae, Saccharomyces cerevi- siae .Saccharomyces cariocanus.Saccharomyces bayanus, Saccharomyces cerevisi- ae,Saccharomyces paradoxus, Saccharomyces pastorianus.Kluyveromyces polyspo- rus.Kluyveromyces yarrowii, Saccharomyces dairensis 2,Kluyveromyces africa- nus.Kluyveromyces africanus.Saccharomyces rosinii.Kluyveromyces lodde- rae.Saccharomyces spencerorum, Candida holmii.Saccharomyces barnet- tii, Saccharomyces exiguus, Saccharomyces exiguus.Kazachstania vitico- Ia, Saccharomyces castellii, Candida glabrata,,Kluyveromyces delphen- sis, Saccharomyces kluyveri.Eremothecium gossypii.Holleya sinecauda.Kluyveromyces thermotolerans,Kluyveromyces waltii,Zygosaccharomyces cidri,Zygosaccharomyces fermentati.Hanseniaspora uvarum.Saccharomycodes iudwigii.Kluyveromyces marxia- nus.Kluyveromyces wickerhamii.Kliiyveromyces dobzhanskii.Kluyveromyces lactis Kluyveromyces nonfermentati, Kluyveromyces aestuarii,Tetrapisispora nanseien- sis.Tetrapisispora arboricola, Kluyveromyces phaffϊi.Tetrapisispora iriomoten- sis, Kluyveromyces blattae.Phaffomyces opuntiae.Phaffomyces opuntiae.Phaffomyces antillensis.Phaffomyces thermotolerans,Candida insectalens, Candida silvatica.Pichia membranifaciens.lssatchenkia orientalis.Brettanomyces anomalus 2,Dekkera anoma- la.Brettanomyces anomalus, Brettanomyces bruxellensis,Dekkera bruxellensis.Dekkera custersiana.Dekkera naardenensis.Williopsis salicorniae.Candida haemulonii, Candida tsuchiyae.Candida oregonensis.Candida intermedia, Candida pseudointerme- dia, Candida intermedia, Candida akabanensis.Candida melibiosica,Clavispora lusitani- ae, Candida torresii, Candida agrestis.Metschnikowia bicuspidata.Metschnikowia reu- kaufii.Metschnikowia pulcherrima.Candida mogii.Williopsis californica.Williopsis praten¬ sis, Starmera amethionina var. pach, Starmera caribaea.Williopsis saturnus.Candida utilis.Pichia anomala.Williopsis mucosa.Candida albicans,Candida dublinien- sis, Candida maltosa, Candida viswanathii, Candida lodderae, Candida tropicalis, Candida sojae.Candida parapsilosis.Lodderomyces elongisporus,Candida fungicola, Candida sagamina, Candida fukazawae.Candida mesenterica, Candida suecica, Candida rugo- sa.Candida savonica, Candida tanzawaensis.Candida austromarina, Candida sa- ke,Candida kruisii, Candida insectamans.Candida lyxosophila, Candida glaebo- sa, Candida pseudoglaebosa, Candida saitoana, Candida fluviatilis, Candida palmiole- ophila, Candida palmioleophila.Candida shehatae var. insectos.Candida shehatae var. ,shehatae,Candida shehatae var. lignosa.Candida coipomoensis.Candida ergasten- sis.Candida chilensis, Candida glucosophila.Debaryomyces hansenii.Debaryomyces castellii.Debaryomyces udenii, Candida psychrophila.Debaryomyces hansenii var. fab.Candida fermenticarens.Candida oleophila.Candida zeylanoides.Candida boletico- la,Candida schatavii, Candida ralunensis.Candida krissii.Candida laureliae.Candida santamariae var. membr.Candida santamariae var. santa, Candida beechii, Candida sophiae-reginae.Candida natalensis.Candida quercitrusa, Candida fragi, Candida xesto- bii.Candida fukuyamaensis.Taphrina farlowii.Pichia guilliermondii.Pichia mexica- na, Candida conglobata, Candida insectorum, Candida aaseri, Candida butyri, Candida dendronema.Candida naeodendra, Candida tenuis.Candida diddensiae.Candida friedri- chii, Candida membranifaciens.Candida buinensis.Candida atlantica.Candida at- mosphaerica.Pichia triangularis.Candida multigemmis.Pichia farinosa.Candida cylin- dracea.Endomyces fibuliger.Saccharomycopsis fibuligera.Saccharomycopsis capsula- ris.PachysoIen tannophilus,Endomyces geotrichum.Galactomyces geotri- chum.Galactomyces citri-aurantii,Galactomyces reessii,,Dipodascus albi- dus,,Dipodascus albidus,Dipodascus geniculatus.Dipodascus australiensis.Dipodascus armillariae.Dipodascus macrosporus.Geotrichum klebahnii.Dipodascus aggrega- tus.Geotrichum eriense.Geotrichum fermentans.Candida apicola.Candida bom- bi.Starmerella bombicola, Candida geochares.Candida spandovensis.Zygoascus helle- nicus, Candida valdiviana.Arxula terrestris, Candida chiropterorum, Candida drimy- dis.Dipodascopsis uninucleata.Waltomyces lipofer.Neolecta vitelli- na,Schizosaccharomyces pombe,Schizosaccharomyces japonicus, Pneumocystis cari¬ nii, Pneumocystis carinii.Taphrina mirabilis.Taphrina nana.Taphrina pruni.Taphrina ul- mi,Taphrina communis.Taphrina flavorubra.Taphrina deformans 2,Taphrina pruni- subcordatae.Taphrina populina.Taphrina wiesneri.Taphrina carnea,Taphrina virgini- ca.Taphrina letifera.Taphrina robinsoniana,Protomyces inouyei.Protomyces lactu- cae,Protomyces macrosporus.Protomyces pachydermus.Saitoella complica¬ ta, Leucoagaricus gongy!ophorus,Dictyonema pavonia.Tulostoma macrocepha- la.Pluteus petasatus.Lentinula lateritia.Lentinula lateritia.Cyathus striatus.Calvatia gi- gantea.Lycoperdon sp.Agaricus bisporus,Amanita muscaria (fly agaric),Leucopaxillus albissimus.Tricholoma matsutake.Entoloma strictius.Stropharia rugosoannulata Basidiomycete symbiont of Apte.Calathella mangrovei.Nia vibrissa.Halocyphina villo- sa.Cyphellopsis anomala.Physalacria maipoensis.Favolaschia intermedia subsp. .Athelia bombacina.Cortinarius iodes.lnocybe geophylla.Lepiota procera (parasol mushr.Limnoperdon incarnatum.Panellus serotinus.Panellus stipticus.Hygrophorus sordidus.Humidicutis marginata.Typhula phacorrhiza.Crucibulum laeve.Laccaria ame- thystina.Laccaria pumila.Coprinus cinereus.Omphalina ericetorum.Pleurotus ostrea- tus.Fistulina hepatica.Fistulina hepatica.Schizophyllum commune.Pleurotus tuberregi- um.ScIeroderma citrina.Xerocomus chrysenteron.Boletus satanas.Paxillus panuoides Peniophora nuda,Peniophora nuda,Scytinostroma aiutum,Echinodontium tinctori- um.Echinodontium tinctorium.Russula compacta.Russula compacta.Lentinellus ompha- lodes.Lentinellus omphalodes.Lentinellus ursinus.Lentinellus ursinus,Clavicorona pyxi- data.Clavicorona pyxidata.Auriscalpium vuIgare,Auriscalpium vuIgare,Hericium coral- loides.Hericium erinaceum.Hericium ramosum.Hericium ramosum.Stereum gausapa- tum.Stereum ostrea.Stereum hirsutum.Gloeocystidiellum leucoxantha.Aleurodiscus botryosus.Stereum annosum.Bc-ndarzewia berkeleyi.Albatrellus skamani- us.Heterobasidion annosum.Laxitextum bicolor.Trichaptum fusco-violaceum.Graphium putredinis.Pseudallescheria boydii.Pseudallescheria ellipsoidea.Microascus cirrosus.Graphium putredinis.Graphium tectonae.Graphium putredinis.Petriella setifeira.Lomentospora prolificans.Ceratocystis fimbriata.Graphium penicillioides.Glomerella cingulata.Verticillium longisporum.Verticillium dahliae.Volutella colletotrichoi- des. Plectosphaerella cucumerina.Cordyceps yakusimensis.Cordyceps soboliferra.Cordyceps capitata, Cordyceps cochlidiicola.Cordyceps prolifica.Cordyceps inegosensis, Tolypocladium inflatum.Cordyceps paradoxa.Cordyceps japonica.Cordyceps heteropoda.Hamiltonaphis styraci yeast-li.Cordyceps ramosopulvinata, Paecilomyces sp ., Cordyceps kanzashiana.Trichoderma koningii.Trichoderma viride.Taphrina defor- mans.Hypocrea lutea, Hypomyces chrysospermus.Volutella ciliata, Volutella cilia- ta.Chaetopsina fulva, Gibberella pulicaris.Gibberella moniliformis, Botryotinia fuckelia-na, Botryotinia convoluta.Mycoarachis inversa. Cordyceps pruinosa.Cordyceps takao- montana.Botr ytis cinerea, Cordyceps broπgniartii.Cordyceps militaris, Paecilomyces tenuipes.Bionectria ochroleuca.Nectria ochroleuca.Nectria aureofulva.Nectria cinnabarina, Geosmithia putterillii.Geosmithia lavendula.Lulworthia fucicola.Chaetomium elatum.Neurospora crassa.Sordaria firmicola.Podospora anserina.Ascovaginospora stellipala.Phyllachora graminis.Kionochaeta ramifera.Kionochaeta spissa.Kionochaeta spissa.Kionochaeta ivoriensis.Meliola juddiana.Meliola niessleana.Neurospora crassa.Ophioceras leptosporum.Magnaporthe grisea.Pseudohalonectria falcata.Cryphonectria cubensis.Cryphonectria havanensis, Endothia gyrosa.Leucostoma persoonii.Ophiostoma penicillatum.Pesotum fragrans.Ophiostoma bicolor.Ophiostoma piceae.Ophiostoma floccosum .Ophiostoma piliferum.Ophiostoma ulmi.Ophiostoma schenckii.Ophiostoma stenoceras.Ophiostoma cucullatum.Ophiostoma europhioi- des.Ophiostoma ainoae.Papulosa amerospora.Ascolacicola austriaca.Rhamphoria delicatula.sordariomycete sp., Poronia punctata, Rosell inia necatrix.Xylaria sp., Xylaria polymorpha.Muscodor sp., Barrmaelia oxyacanthae, Hypoxylon fragiforme.Monographella nivalis, Pestalosphaeria hansenii.Pestalosphaeria sp., Hyponectria buxi.Monilinia fructicola.Monilinia laxa, Sclerotinia sclerotiorum.dark septate endophyte DS16b, Euascomycetes sp., Neobulgaria premnophila, Blumeria graminis, Phyllactinia guttata.Spathularia flavida.Cyttaria darwinii.Geomyces pannorum var. panno- ru.Thelebolus stercoreus.Ascozonus woolhopensis.Selenaspora guernisacii.lnermisia aggregata.Pyronema domesticum.Otidea onotica.Glaziella aurantiaca.Cephaliophora tropica.Cephaliophora irregularis.Scutellospora castanea.Geopyxis carbonaria Sarcoscypha austriaca, Pseudopithyella minuscula.Kompsoscypha chudei Pithya cupressina.Nanoscypha tetraspora.Phillipsia domingensis.Wynnea sp., Cookeina tricholoma.Microstoma floccosum.Wolfina aurantiopsis.Strobiloscypha keliae.Desmazierella acicola.Neournula pouchetii.Chorioactis geaster.Donadinia sp., Ga! iella rufa.Plectania rhytidia.Sarcosoma mexicanum.Pseudoplectania nigrella, Tuber borchii, Tuber gibbosum.Choiromyces meandriformis.Choiromyces venus sus.Tuber excavatum Magnolia, Tuber panniferum.Dingleya verrucosa.Reddellomyces donkii.Labyrinthomyces varius.Helvella lacunosa.Wynnella silvico- lor.Helvella terrestris.Balsamia vulgaris, Barssia oregonensis.Balsamia magnata, Underwoodia columnaris.Morchella elata 2, Morchella esculenta, Verpa conica .Disciotis venosa.Fischerula subcauüs.Leucangium carthusianum.Verpa bohemic a.Pseudorhizina califomica, Gyromitra esculenta 2, Gyromitra melaleucoides.Discina macrospora.Gyromitra montana.Hydnotrya cerebriformis, Rhizina undula ta.Pachyphloeus citrinus.Cazia flexiascus.Terfezia arenaria.Peziza echinosporo.Tβrfezia terfezioides.Orbilia fimicola.Orbilia auricolor.Dactylella oxysporo.Cephaliophora longispora.Cephaliophora muscicola.Arxiozyma telluris.Saccharomyces transvaalensis.Saccharomyces dairensis.Saccharomyces servazzii, Saccharomyces unisporus.Zygosaccharomyces bailii, Zygosaccharomyces gentus, Zygosaccharomyces bisporus, Zygosaccharomyces melis, Zygosaccharomyces rouxii.Zygosaccharomyces rouxii 2, Torulaspora globosa Zygosaccharomyces florentine, Candida colliculosa, Torulaspora delbrueckii, Torulaspora pretori- sis, Zygosaccharomyces microellipso, Zygosaccharomyces mrakiijorulaspora delbrueckii, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces cerevisiae, Saccharomyces cariocanus, Saccharomyces bayanus, Saccharomyces cerevisiae, Sac charomyces paradoxus, Saccharomyces pastorianus, Klyyveromyces polysporus, Klyyveromyces yarrowii, Saccharomyces dairensis 2, Kluyveromyces africa- nus, Klyyveromyces africanus, Saccharomyces rosinii, Klyyveromyces lodosterae, Saccharomyces spencerorum, Candida holmii, Saccharomyces barnettii, Saccharomyces exiguus, Saccharomyces exiguus Kazakhstania vitico-Ia, Saccharomyces castellii, Candida glabrata, Kluyveromyces delpsis, Saccharomyces kluyveri.Eremothecium gossypii.Holleya sinecauda.Kluyveromyces thermotolerans, Kluyveromyces waltii, Zygosaccharomyces cidri, Zygosaccharomyces fermentati.Hanseniaspora uvarum.Saccharomycodes iudwigii.Kluyveromyces marxianus. Kluyveromyces wickerhamii.Kliiyveromyces dobzhanskii.Kluyveromyces lactis Kluyveromyces nonfermentati, Kluyveromyces aestuarii, Tetrapisispora nanseien- sis.Tetrapisispora arboricola, Kluyveromyces phaffϊi.Tetrapisispora iriomoten- sis, Kluyveromyces blattae.Phaffomyces opuntiae.Phaffomyces opuntiae.Phaffomyces antillensis.Phaffomyces thermotolerans, Candida insectalens, Candida silvatica.Pichia membranifaciens.lssatchenkia orientalis.Brettanomyces Anomalous 2, Dekkera anomalan, Bbrettanomyces anomalis, Brettanomyces bruxellensis, Dekkera bruxellensis Dekkera custersiana Dekkera naardenensis, Williopsis salicorniae, Candida haemulonii, Candida tsuchiyae, Candida oregonensis, Candida intermedia, Candida pseudointermedia, Candida intermedia, Candida akabanensis, Candida melibiosica, Clavispora lusitani- ae, Candida torresii, Candida agrestis.Metschnikowia bicuspidata.Metschnikowia reu- kaufii.Metschnikowia pulcherrima.Candida mogii.Williopsis californica.Williopsis praten¬ sis, Starmera amethionina var. pach, Starmera caribaea.Williopsis saturnus.Candida utilis. Pichia anomala.Williopsis mucosa.Candida albicans, Candida dubliniensis, Candida maltosa, Candida viswanathii, Candida lodderae, Candida tropicalis, Candida sojae.Candida parapsilosis.Lodderomyces elongisporus, Candida fungicola, Candida sagamina, Candida fukazawae.Candida mesenterica, Candida suecica, Candida rugosa.Candida savonica, Candida tanzawaensis.Candida austromarina, Candida sare ke, Candida kruisii, Candida insectamans.Candida lyxosophila, Candida glaebosa, Candida pseudoglaebosa, Candida saitoana, Candida fluviatilis, Candida palmiole- ophila, Candida palmioleophila.Candida shehatae var. insectos.Candida shehatae var., shehatae, Candida shehatae var. lignosa.Candida coipomoensis.Candida ergasten- sis.Candida chilensis, Candida glucosophila.Debaryomyces hansenii.Debaryomyces castellii.Debaryomyces udenii, Candida psychrophila.Debaryomyces hansenii var. fab. Candida fermenticarens.Candida oleophila.Candida zeylanoides.Candida boleticola, Candida schatavii, Candida ralunensis.Candi da krissii.Candida laureliae.Candida santamariae var. membr.Candida santamariae var. santa, Candida beechii, Candida sophiae-reginae.Candida natalensis.Candida quercitrusa, Candida fragi, Candida xesto- bii.Candida fukuyamaensis.Taphrina farlowii.Pichia guilliermondii.Pichia mexica-na, Candida conglobata, Candida insectorum, Candida aaseri, Candida butyri, Candida dendronema, Candida naeodendra, Candida tenuis, Candida diddensiae, Candida friedrichii, Candida membranifacien, Candida buinensis, Candida atlantica, Candida atmosphaerica, Pichia triangularis. Candida multigemmis.Pichia farinosa.Candida cylin- dracea.Endomyces fibuliger.Saccharomycopsis fibuligera.Saccharomycopsis capsula- ris.PachysoIen tannophilus, Endomyces geotrichum.Galactomyces geotrichum.Galactomyces citri-aurantii, Galactomyces reessii, Dipodascus albidus, Dipodascus albidus , Dipodascus geniculatus.Dipodascus australiensis.Dipodascus armillariae.Dipodascus macrosporus.Geotrichum klebahnii.Dipodascus aggrega- Geotrichum eriense.Geotrichum fermentans.Candida apicola.Candida bombi.Starmerella bombicola, Candida geochares.Candida spandovensis.Zygoascus hellenicus, Candida valdiviana.Arxula terrestris, Candida chiropterorum, Candida drimydis.Dipodascopsis uninucleata.Waltomyces lipofer. Neolecta vitellina, Schizosaccharomyces pombe, Schizosaccharomyces japonicus, Pneumocystis cari nii, Pneumocystis carinii.Taphrina mirabilis.Taphrina nana.Taphrina pruni.Taphrina ulmi, Taphrina communis.Taphrina flavorubra.Taphrina deformans 2, Taphrina pruni- subcordatae.Taphrina populina .Taphrina wiesneri.Taphrina carnea, Taphrina virgini- ca.Taphrina letifera.Taphrina robinsoniana, Protomyces inouyei.Protomyces lactu- cae, Protomyces macrosporus.Protomyces pachydermus.Saitoella complica¬ ta, Leucoagaricus gongy! Ophorus, Dictyonema pavonia.Tulostoma macrocepha- la. Pluteus petasatus.Lentinula lateritia.Lentinula lateritia.Cyathus striatus.Calvatia gi- gantea.Lycoperdon sp.Agaricus bisporus, Amanita musca ria (fly agaric), Leucopaxillus albissimus.Tricholoma matsutake.Entoloma strictius.Stropharia rugosoannulata Basidiomycete symbiont of Apte.Calathella mangrovei.Nia vibrissa.Halocyphina villo- sa.Cyphellopsis anomala.Physalacria maipoensis.Favolaschia intermedia subsp. .Athelia bombacina.Cortinarius iodes.lnocybe geophylla.Lepiota procera (parasol mushr.Limnoperdon incarnatum.Panellus serotinus.Panellus stipticus.Hygrophorus sordidus.Humidicutis marginata.Typhula phacorrhiza.Crucibulum laeve.Laccaria amethystina.Laccaria pumila.Coprinus cinereus.Omphalina ericetorum .Pleurotus ostrea- tus.Fistulina hepatica.Fistulina hepatica.Schizophyllum commune.Pleurotus tuberregi- um.ScIeroderma citrina.Xerocomus chrysenteron.Boletus satanas.Paxillus panuoides Peniophora nuda, Peniophora nuda, Scytinostroma aiutum, Echinodontium tinctori- um.Echinodontium tinctorium.Russula compacta .Russula compacta. Lentinellus omphalodes. Lentinellus omphalodes. Lentinellus ursinus. Lentinellus ursinus, Clavicorona pyxi- data. Clavicorona pyxidata. Auriscalpium vuigare, Auriscalpium vuigare, Hericium coralloides, Hericium erinaceum, Hericium ramosum, Hericium ramosum, Stereum gausapatum Stereum ostrea.Stereum hirsutum.Gloeocystidiellum leucoxantha.Aleurodiscus botryosus.Stereum annosum. Bc-ndarzewia berkeleyi.Albatrellus skamani- us.Heterobasidion annosum.Laxitextum bicolor.Trichaptum fusco-violaceum
Trichaptum biforme.TTrichaptum abietinu,Trichaptum abietinum.Trichaptum larici- num.Basidioradulum radula.Hyphodontia alutaria.lnonotus hispidus.Phellinus igniari- us.Coltricia perennis.Schizopora paradoxa.Fomitopsis pinicola,Daedalea querci- na.Antrodia carbonica.Polyporus squamosus.Trametes suaveolens,Fomes fomentari- us.Lentinus tigrinus.Ganoderma austräte, Dentocorticium sulphurellum.Poria co- cos.Cantharellus tubaeformis,Phaeo!us schweinitzii.Laetiporus sulphureus.Sparassis spathulata.Bjerkandera adusta.Oxyporus latemarginatusjrichaptum abietinum,Phlebia radiata.Phanerochaete chrysosporium.Rhizoctonia zeae,Tretopileus sphaerophorus Ceriporia purpurea.Termitomyces cartilagtneus, Partus rudis.Steccherinum rhois.Meripilus giganteus,Albatrellus syringae.Spongipellis unicolor.Hydnellum sp.,Thelephora sp.,Gloeophyllum sepiarium.Geastrum saccatum.Sphaerobolus stella- tus.Gomphus floccosus.Pseudocolus fusiformis.Ramaria stricta.Clavariadelphus pistil- laris.Multiclavula mucida,Clavulina cristata.Hydπum repandum.Thanatephorus cucume- ris.Thanatephorus praticola.Auricularia auricula.Auricularia polytricha.Botryobasidium isabe!linum,Botryobasidium subcoronatum.Oxyporus sp.,Bullera grandispora.Mrakia frigida.Mrakia psychrophilia.Cystofilobasidium capitatum.Leucosporidium lari- marini.Tilletiopsis cremea.THIetiopsis lilacina.Tilletiopsis washingtonensis.Exobasidium vaccinii,Sympodiomycopsis paphiopedilijilletiopsis pallescens.Tilletia cariesjilletiaria anomala.Tilletiopsis fulvescens.Tületiopsis minor, Tilletiopsis albescens,Graphiola cy- lindrica.Exobasidium rhododendri.Exobasidium vaccinii.Dacrymyces chrysosper- mus.Dacrymyces stillatus.Calocera Cornea, Heterotextus alpinus.Trichosporon caseo- rum.Trichosporon lactis.Trichosporon cutaneum,Asterotremella parasiti- ca.Cryptococcus humicolus.Cryptococcus albidus.Filobasidium floriforme.Holtermannia corniformis.Tremella foliacea.Bullera schimicola.Bullera waltii.Bullera huiaensis.Bullera boninensis.Bullera mrakii.Bullera coprosmaensis.Bullera oryzae.BuIlera derxii.Bullera sinensis.Bullera armeniaca.Dioszegia crocea.Bullera variabilis, Filobasidiella neofor- mans.Tsuchiaea wfngfieldii, Bullera dendrophila,Bu!lera miyagiana.Bullera globospo- ra.Tremella globospora.Kockovaella imperatae.Kockovaella sacchari.Kockovaella thai- landica.Kockovaella phaffii.Kockovaella phaffii.Fellomyces fuzhouensis.Kockovaella machilophila.Fellomyces distylii.Fellomyces ogasawarensis.Kockovaella schi- mae.Fellomyces penicillatus.Fellomyces polyborus.Fellomyces horovitzi- ae.Sterigmatosporidium polymorphu.Tremella moriformis, Bullera hannae.Bullera pen- niseticola, Bullera unica.Bulleromyces albus, Bullera pseudoalba.fungus sp.,Sirobasidium magnum.Fibulobasidium inconspicuum.Taphrina California, Taphrina maculans.Bensingtonia yamatoana.SporoboIomyces inositophilus.Sporobolomyces singularis.Sporobolomyces falcatus.Sporobolomyces tsugae.Microbotryum viola- ceum.Leucosporidium scottii.Bensingtonia intermedia.Sporidiobolus pararo- seus,Sporidiobolus pararoseus.Sporobolomyces roseus.Sporidiobolus johnso- nii.Sporidiobolus johnsonii.Sporidiobolus salmonicolor.Rhodotorula glutinis.Rhodotorula mucilaginosa.Rhodosporidium toruloides.Rhodotorula glutinis.Rhodosporidium fluvia- le.Sporidiobolus ruineniae.Mixia osmundae.Sporobolomyces sasicola.Sporobolomyces xanthus.Kurtzmanomyces nectairei.Bensingtonia sp.,Sporobolomyces ru- ber.Sterigmatomyces halophilus.Bensingtonia ingoldii.Bensingtonia mu- sae,Bensingtonia miscanthi.Bensingtonia subrosea.Kondoa ma!vinella,Bensingtonia yuccicola.Bensingtonia phylladus.Bensingtonia ciliata,Bensingtonia naganoen- sis.Sporobolomyces subbrunneus,Cronartium ribicola.Peridermium harknes- seü.Hyalopsora polypodii.Gymnoconia nitens.Nyssopsora echinata.Helicobasidium purpureurn.Helicobasidium corticioides.Helicogloea variabilisTrichaptum biforme.TTrichaptum abietinu, Trichaptum abietinum.Trichaptum laricium num.Basidioradulum radula.Hyphodontia alutaria.lnonotus hispidus.Phellinus igniari- us.Coltricia perennis.Schizopora paradoxa.Fomitopsis pinicola, Daedalea quercina.Antrodia carbonica.Polyporus squamosus.Trametes suaveolens Fomes fomentari- us.Lentinus tigrinus.Ganoderma spp., Dentocorticium sulphurellum.Poria coco.Cantharellus tubaeformis, Phaeo! Us schweinitzii.Laetiporus sulphureus.Sparassis spathulata.Bjerkandera adusta.Oxyporus latemarginatusjrichaptum abietinum, Phlebia radiata.Phanerochaete chrysosporium.Rhizoctonia zeae, Tretopileus Sphaerophorus Ceriporia purpurea.Termitomyces cartilagtneus, parturition rudis.Steccherinum rhois.Meripilus giganteus, Albatrellus syringae.Spongipellis unicolor.Hydnellum sp., Thelephora sp. Gloeophyllum sepiarium.Geastrum saccatum.Sphaerobolus stellatus.Gomphus floccosus.Pseudocolus fusiformis.Ramaria stricta.Clavariadelphus pistillaris.Multiclavula mucida, Clavulina cristata.Hydπum repandum.Thanatephorus cucumeris.Thanatephorus praticola.Auricularia auricula.Auricularia polytricha.Botryobasidium isabe! linum, Botryobasidium subcoronatum.Oxyporus sp., Bullera grandispora.Mrakia frigida.Mrakia psychrophilia.Cystofilobasidium capitatum.Leucosporidium larimini.Tilletiopsis cremea.THIetiopsis lilacina.Tilletiopsis washingtonensis.Exobasidium vaccinii, Sympodiomycopsis paphiopedilijilletiopsis pallescens.Tilletia cariesjilletiaria anomala.Tillet iopsis fulvescens.Tületiopsis minor, Tilletiopsis albescens, Graphiola cyindrica.Exobasidium rhododendri.Exobasidium vaccinii.Dacrymyces chrysospermus.Dacrymyces stillatus.Calocera cornea, Heterotextus alpinus.Trichosporon caseo- rum.Trichosporon lactis.Trichosporon cutaneum, Asterotremella parasiti- ca. Cryptococcus humicolus.Cryptococcus albidus.Filobasidium floriforme.Holtermannia corniformis.Tremella foliacea.Bullera schimicola.Bullera waltii.Bullera huiaensis.Bullera boninensis.Bullera mrakii.Bullera coprosmaensis.Bullera oryzae.BuIlera derxii.Bullera sinensis.Bullera armeniaca.Dioszegia crocea.Bullera variabilis , Filobasidiella neoformans.Tsuchiaea wfngfieldii, Bullera dendrophila, Buerlera miyagiana.Bullera globospora.Tremella globospora.Kockovaella imperatae.Kockovaella sacchari.Kockovaella thai- landica.Kockovaella phaffii.Kockovaella phaffii.Fellomyces fuzhouensis.Kockovaella machilophila.Fellomyces distylii .Fellomyces ogasawarensis.Kockovaella schimae.Fellomyces penic illatus.Fellomyces polyborus.Fellomyces horovitzi- ae.Sterigmatosporidium polymorphu.Tremella moriformis, Bullera hannae.Bullera peniseticola, Bullera unica.Bulleromyces albus, Bullera pseudoalba.fungus sp., Sirobasidium magnum.Fibulobasidium inconspicuum.Taphrina California, Taphrina maculans.Bensingtonia yamatoana.SporoboIomyces inositophilus.Sporobolomyces singularis.Sporobolomyces falcatus.Sporobolomyces tsugae.Microbotryum viola ceum.Leucosporidium scottii.Bensingtonia intermedia.Sporidiobolus pararo- seus, Sporidiobolus pararoseus.Sporobolomyces roseus.Sporidiobolus johnsonii.Sporidiobolus johnsonii.Sporidiobolus salmonicolor.Rhodotorula glutinis. Rhodotorula mucilaginosa.Rhodosporidium toruloides.Rhodotorula glutinis.Rhodosporidium fluvia- le.Sporidiobolus ruineniae.Mixia osmundae.Sporobolomyces sasicola.Sporobolomyces xanthus.Kurtzmanomyces nectairei.Bensingtonia sp., Sporobolomyces ruber.Sterigmatomyces halophilus.Bensingtonia ingoldii.Bensingtonia mu sae, Bensingtonia miscanthi.Bensingtonia subrosea.Kondoa ma! vinella, Bensingtonia yuccicola.Bensingtonia phylladus.Bensingtonia ciliata, Bensingtonia naganoen- sis.Sporobolomyces subbrunneus, Cronartium ribicola.Peridermium harknes- seü.Hyalopsora polypodii.Gymnoconia nitens.Nyssopsora echinata.Helicobasidium purpureurn. Helicobasidium corticioides.Helicogloea variabilis
Sporobolomyces kluyveri-nielii .Sporobolomyces phyllomatis, Sporobolomyces salici- nus.Rhodotorula minuta, Sporobolomyces foliicola, Sporobolomyces oryzico- Ia, Sporobolomyces vermiculatus.Erythrobasidium hasegawianum, Sporobolomyces yunnanensis, Sporobolomyces elongatus.Rhodotorula lactosa.Archaeospora leptoti- cha.Paraglomus brasilianum.Glomus verruculosum.Glomus intraradices.Glomus proli- ferum.Glornus etunicatum.Gigaspora margarita.Scutellospora projectura- ta.Scutellospora castanea.Scutellospora cerradensis.Entrophospora sp.,Glomus versi- forme.Mucor circinelloides f. lusita.Mucor racemosus.Cepedea virguloidea.Mucor ra- mosissimus.Ellisomyces anomalus.Backusella ctenidia.Circinomucor circinelloi- des,Mucor racemosus, Opalina ranarum, Parasitella parasitica.Chaetocladium brefel- dii.Chaetocladium jonesii.Pilaira anomala.Thamnidium elegans.Helicostylum ele- gans.Pirella circinans.Mucor mucedo.Mucor hiemalis f. hiemalis.Rhizomucor variabi- lis.Zygorhynchus heterogamus.Actinomucor elegans.Dicranophora fulva.Pilobolus um- bonatus.Backusella circina.Mucor recurvus var.,indicus,Blakeslea trispora.Poitrasia circinans.Choanephora cucurbitarum, Gilbertella persicaria.Hyphomucor assamen- sis.Rhizopus microsporus var. micr.Rhizopus microsporus var. rhiz,Rhizopus azy- gospoms.Rhizopus microsporus var. chin.Rhizopus microsporus var. olig.Amylomyces rouxii.Rhizopus arrhizus.Sporodiniella umbellata.Syzygites megalocarpus.Rhizopus stolonifer.Benjaminiella poitrasii.Cokeromyces recurvatus,Mycotypha africa- na.Mycotypha microspora.Kirkomyces cordense.Mucor amphibiorum.Mucor indi- cus.Apophysomyces elegans.Saksenaea vasiformis.Radiomyces spectabi- lis.Phycomyces blakesleeanus.Spinellus fusiger.Circinella umbellata.Phascolomyces articulosus.Zychaea mexicana.Fennellomyces linderi.Thamnostylum piriforme.Absidia corymbifera.Absidia blakesleeana,Dichotomocladium elegans.Rhizomucor mie- hei.Rhizomucor pusillus.Thermomucor indicae-seudaticae.Protomycocladus faisalaba- densi.Absidia glauca.Chlamydoabsidia padenii.Absidia coerulea.Absidia re- pens.Halteromyces radiatus,Cunninghamella bertholletiae.Cunninghamella poly- morpha.Cunninghamella elegans.Cunninghamella echinulata.Gongronella butle- ri.Hesseltinella vesiculosa.Syncephalastrum monosporum var.Syncephalastrum race- mosum (U.),Mucor ramannianus.Blastocladiella emersonii,Entomophthora mus- cae.Entomophthora schizophora.Eryniopsis ptycopterae.Entomophaga auli- cae,Pandora neoaphidis.Strongwellsea castrans.Zoophthora culisetae.Conidiobolus lamprauges,Conidiobolus thromboides,Conidiobolus coronatus.Chytridiomycetes sp.,Smittium simulii.Smittium commune.Smittium simulii.Smittium cylindrospo- rum.Smittium imitatum.Smittium imitatum.Smittium tronadorium.Smittium orthocla- dii.Smittium tipulidarum.Smittium dipterorum.Smittium megazygosporum.Smittium cf. dipterorum.Smittium cf. phytotelmatum.Smittium phytotelmatum.Furculomyces boome- rangus.Smittium angustum.Furculomyces boomerangus,Smittium morbosum.Smittium culicis.Smittium simulatum.Smittium culicis.Smittium culicisoides,Smittium fecun- dum.Smittium annulaturn.Smittium coloradense,Smittium mucronatum.Smittium cauda- tum.Genistelloides hibernus.Capniomyces stellatus.Smittium culisetae.Smittium culise- tae.Smittium culisetae.Coemansia reversa.Spirodactylon aureum.Coemansia brazilien- sis.Kickxella alabastrina, Linderina pennispora.Dipsacomyces acuminospo- rus.Martensiomyces pterosporus,Spiromyces aspiralis.Spiromyces minu- tus.Basidiobolus haptosporus.Basidiobolus ranarum.Chytridium confer- vae.Neocallimastix sp.,Neocallimastix frontalis 2,Dissophora decumbens.Mortierella multidivaricata.Echinosporangium transversale, Mortierella verticillata.Mortierella chla- mydospora.Mortierella wolfii.Mortierella polycephala,Monoblepharella elonga- ta.Monoblepharella mexicana.Monoblepharis hypogyna.Monoblepharis insig- nis.Hyaloraphidium curvatum.Spizellomyces acuminatus.Filobasidiella neoformans 3, Candida floricola., Candida santjacobensis.dark septate.Metschnikowia aga- ves,Candida etchellsii., Pneumocystis carinii.Candida cantarellii., Candida stella- ta.,Nadsoniella nigra.Candida paludigena., Candida versatilis., Candida otoi, Candida silvanorum., Candida entomophila., Candida sorbophila., Candida uringiensis.Candida antillancae., Candida tepae., Candida musae., Candida ishiwadae., Candida ncuden- sis, Candida castrensis., Candida vanderwaltii.,Metschnikowia australis., Candida fruc- tus.Metschnikowia pulcherrima., Candida bertae.,Wangiella dermatitidis.Candida galac- ta., Candida salmanticensis., Candida parapsilosis.Candida petrohuensis.,Candida po- puli.,Candida blankii.,Ochrolechia parella,Candida rhagii., Candida homilento- ma., Candida fennica.,Bulgaria inquinans,,Sporidiobolus sp.,Wickerhamiella domercqi- ae.,Zygosaccharomyces rouxii.Metschnikowia gruessii.,Metschnikowia reukau- fii.,Metschnikowia bicuspidata.,Metschnikowia krissii., Candida anatomiae.,Arxula terre- stris.Candida agrestis.,Glomerobolus gelineus.Candida vinaria.Blastocladiella emerso- nii.Candida lactis-condensi.Metschnikowia zobellii.Candida bondarzewiae.Candida sequanensis.Candida edax.Candida ernobii, Candida karawaiewii.Saccharomyces ce- revisiae.Barrmaelia oxyacanthae.Arxula adeninivorans.Sporobolomyces kluyveri nielii .Sporobolomyces phyllomatis, Sporobolomyces salici- nus.Rhodotorula minuta, Sporobolomyces foliicola, Sporobolomyces oryzico- Ia, Sporobolomyces vermiculatus.Erythrobasidium hasegawianum, Sporobolomyces yunnanensis, Sporobolomyces elongatus.Rhodotorula lactosa.Archaeospora leptoti- cha.Paraglomus brasilianum.Glomus verruculosum. Glomus intraradices.Glomus proliferum.Glornus etunicatum.Gigaspora margarita.Scutellospora projecturata.Scutellospora castanea.Scutellospora cerradensis.Entrophospora sp., Glomus versi- forme.Mucor circinelloides f. lusita.Mucor racemosus.Cepedea virguloidea.Mucor ramosissimus.Ellisomyces anomalus.Backusella ctenidia.Circinomucor circinelloides, Mucor racemosus, Opalina ranarum, Parasitella parasitica.Chaetocladium brefeldi.Chaetocladium jonesii.Pilaira anomala.Thamnidium elegans.Helicostylum ele- gans.Pirella circinans.Mucor mucedo.Mucor hiemalis f. hiemalis.Rhizomucor variabilis.Zygorhynchus heterogamus.Actinomucor elegans.Dicranophora fulva.Pilobolus umbonatus.Backusella circina.Mucor recurvus var., indicus, Blakeslea trispora.Poitrasia circinans.Choanephora cucurbitarum, Gilbertella persicaria.Hyphomucor assamernis.Rhizopus microsporus var. micr.Rhizopus microsporus var. rhiz, Rhizopus azygospoms.Rhizopus microsporus var. chin.Rhizopus microsporus var. olig.Amylomyces rouxii.Rhizopus arrhizus.Sporodiniella umbellata.Syzygites megalocarpus.Rhizopus stolonifer.Benjaminiella poitrasii.Cokeromyces recurvatus, Mycotypha africa - mycotypha microspora.Kirkomyces cordense.Mucor amphibiorum.Mucor indicus.Apophysomyces elegans.Saksenaea vasiformis.Radiomyces spectabilis.Phycomyces blakesleeanus.Spinellus fusiger.Circinella umbellata.Phascolomyces articulosus.Cychea mexicana.Fennellomyces linderi.Thamnostylum piriforme.Absidia corymbifera.Absidia blakesleeana, Dichotomocladium elegans.Rhizomucor mie- hei.Rhizomucor pusillus.Ther momucor indicae-seudaticae.Protomycocladus faisalaba- densi.Absidia glauca.Chlamydoabsidia padenii.Absidia coerulea.Absidia re- pens.Halteromyces radiatus, Cunninghamella bertholletiae.Cunninghamella polymorpha.Cunninghamella elegans.Cunninghamella echinulata.Gongronella butleri.Hesseltinella vesiculosa.Syncephalastrum monosporum var. Syncephalastrum racemosum (U.), Mucor ramannianus. Blastocladiella emersonii, Entomophthora muscae. Entomophthora schizophora. Eryniopsis ptycopterae. Entomophaga aulicae, Pandora neoaphidis. Strongwellsea castrans. Zoophthora culisetae. Conidiobolus lamprauges, Conidiobolus thromboides, Conidiobolus coronatus.Chytridiomycetes sp., Smittium simulii.Smittium commune.Smittium simulii.Smittium cylindrospo- rum.Smittium imitatum.Smittium imitatum.Smittium tronadorium.Smittium orthocla-dii.Smittium tipulidarum.Smittium dipterorum.Smittium megazygosporum.Smittium cf. dipterorum.Smittium cf. phytotelmatum.Smittium phytotelmatum.Furculomyces boomerangus.Smittium angustum.Furculomyces boomerangus, Sorbet morbosum.Smittium culicis.Smittium simulatum.Smittium culicis.Smittium culicisoides, Sodium fecun- dum.Smittium annulaturn.Smittium coloradense, Smittium mucronatum.Smittium caudatum.Genistelloides hibernus.Capniomyces stellatus.Smittium culisetae.Smittium culise- tae.Smittium culisetae.Coemansia reversa.Spirodactylon aureum.Coemansia braziliensis.Kickxella alabastrina, Linderina pennispora.Dipsacomyces acuminosporo.Martensiomyces pterosporus, Spiromyces aspiralis.Spiromyces minutus.Basidiobolus haptosporus.Basidiobolus ranarum.Chy tridium confervae.Neocallimastix sp., Neocallimastix frontalis 2, Dissophora decumbens.Mortierella multidivaricata.Echinosporangium transversal, Mortierella verticillata.Mortierella chlamydospora.Mortierella wolfii.Mortierella polycephala, Monoblepharella elongata.Monoblepharella mexicana.Monoblepharis hypogyna.Monoblepharis nis.Hyaloraphidium curvatum.Spizellomyces acuminatus.Filobasidiella neoformans 3, Candida floricola., Candida santjacobensis.dark septate.Metschnikowia agavees, Candida etchellsii., Pneumocystis carinii.Candida cantarellii., Candida stellata., Nadsoniella nigra.Candida paludigena. , Candida versatilis., Candida otoi, Candida silvanorum., Candida entomophila., Candida sorbophila., Candida uringiensis.Candida antillancae, Candida tepae., Candida musae., Candida ishiwadae., Candida ncudensis, Candida castrensis., Candida vanderwaltii ., Metschnikowia australis., Candida fructus.Metschnikowia pulcherrima., Candida bertae., Wangiella dermatitidis.Candida ga lac ta., Candida salmanticensis., Candida parapsilosis.Candida petrohuensis., Candida polleni., Candida blankii., Ochrolechia parella, Candida rhagii., Candida homilentoma, Candida fennica., Bulgaria inquinans ,, Sporidiobolus sp. , Wickerhamiella domercqiea, Zygosaccharomyces rouxii.Metschnikowia gruessii., Metschnikowia reukau- fii., Metschnikowia bicuspidata., Metschnikowia krissii., Candida anatomiae., Arxula terrestris.Candida agrestis., Glomerobolus gelineus.Candida vinaria.Blastocladiella emerso- nii.Candida lactis-condensi.Metschnikowia zobellii.Candida bondarzewiae.Candida sequanensis.Candida edax.Candida ernobii, Candida karawaiewii.Saccharomyces cerevisiae.Barrmaelia oxyacanthae.Arxula adeninivorans.
Besonders geeignete Ausführungsformen der erfindungsgemässen Verfahren sind dadurch gekennzeichnet, dass die DNA-I I-Sequenz eine DNA-Sequenz ist, die für ein Polypeptid gemäss SEQ ID NO: 2,4,6 oder 8 codiert oder eine DNA-Sequenz ist, die für ein Polypeptid codiert, das 80% Identität zu SEQ ID NO: 2,4,6 oder 8 besitzt, um- fasst.Particularly suitable embodiments of the methods according to the invention are characterized in that the DNA I I sequence is a DNA sequence which is suitable for a Polypeptide according to SEQ ID NO: 2,4,6 or 8 or a DNA sequence which codes for a polypeptide which has 80% identity to SEQ ID NO: 2,4,6 or 8, comprises.
Besonders bevorzugt sind solche DNA-Il-Sequenzen, die für ein Polypeptid mit Lacca- seaktivität codieren, das 85, 90, 92, 94, 95, 96, 97, 98, 99 % Identität zu SEQ ID NO: 2,4,6 oder 8 besitzt. Die Identität zwischen zwei Polypeptidsequenzen wird dabei an¬ hand der GCG Software, die von der University of Wisconsin entwickelt worden ist, mittels des Programms GAP unter Verwendung der vom Programm als Standardwerte vorgeschlagenen Einstellungen für gap penalty, ermittelt.Particular preference is given to those DNA-II sequences which code for a polypeptide having laccase activity, the 85, 90, 92, 94, 95, 96, 97, 98, 99% identity to SEQ ID NO: 2, 4, 6 or 8 owns. In this case, the identity between two polypeptide sequences is determined by means of the GCG software developed by the University of Wisconsin by means of the program GAP using the settings for gap as proposed by the program as default values.
Als Laccase-Aktivität wird die Fähigkeit bezeichnet, phenolische Substanzen zu oxidie- ren wie sie beispielsweise in Machuca et al.,1998 , auf Seite 218 (Determination of laccase type PO activity) beschrieben wird.Laccase activity is the ability to oxidize phenolic substances, as described, for example, in Machuca et al., 1998, at page 218 (Determination of laccase type PO activity).
Das Einführen von DNA-Molekülen in Zellen geschieht durch dem Fachmann bekannte geläufige Klonierungs- und Transfektionsmethoden, wie beispielsweise Co- Präzipitation, Protoplastenfusion, Elektroporation, retrovirale Transfektion und derglei¬ chen, verwendet, um die genannten Nukleinsäuren im jeweiligen Expressionssystem zur Expression zu bringen. Geeignete Systeme werden beispielsweise in Current Pro- tocols in Molecular Biology, F. Ausubel et al., Hrsg., Wiley Interscience, New York 1997, oder Sambrook et al. Molecular Cloning: A Laboratory Manual. 2. Aufl., CoId Spring Harbor Laboratory, CoId Spring Harbor Laboratory Press, CoId Spring Harbor, NY, 1989 beschrieben.Introduction of DNA molecules into cells is carried out by familiar cloning and transfection methods known to the person skilled in the art, such as, for example, co-precipitation, protoplast fusion, electroporation, retroviral transfection and the like, in order to express said nucleic acids in the respective expression system. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Ed., Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd ed., Colard Spring Harbor Laboratory, Col. Spring Harbor Laboratory Press, Col. Spring Harbor, NY, 1989.
Weiterhin kann das Einführen von DNA in Zellen auch durch homologe Rekombination erfolgen. Dazu wird die DNA üblicherweise in einen entsprechenden Vektor zur homo¬ logen Rekombination eingebracht, wie er beispielsweise aus Thomas, K. R. und Ca- pecchi, M.R. (1987) Cell 51 :503 bekannt ist.Furthermore, the introduction of DNA into cells can also be by homologous recombination. For this purpose, the DNA is usually introduced into a corresponding vector for homologous recombination, as described, for example, by Thomas, K. R. and Capecchi, M.R. (1987) Cell 51: 503.
Das Züchten der Zellen geschieht je nach Wirtsorganismus in dem Fachmann bekann¬ ter Weise. Mikroorganismen werden in der Regel in einem flüssigen Medium, das eine Kohlenstoffquelle meist in Form von Zuckern, eine Stickstoffquelle meist in Form von organischen Stickstoffquellen wie Hefeextrakt oder Salzen wie Ammoniumsulfat, Spu- renelemente wie Eisen-, Mangan-, Magnesiumsalze und gegebenenfalls Vitamine enthält, bei Temperaturen zwischen 0 0C und 100 CC, bevorzugt zwischen 10 0C bis 60 0C unter Sauerstoffbegasung angezogen. Dabei kann der pH der Nährflüssigkeit auf einen festen Wert gehalten werden, das heißt während der Anzucht reguliert werden oder nicht. Die Anzucht kann „batch"-weise, „semi batch"-weise oder kontinuierlich er- folgen. Nährstoffe können zu beginn der Fermentation vorgelegt oder semikontinuier¬ lich oder kontinuierlich nachgefüttert werden. Auswählen von Zellen, die eine Laccase aus Aspergillus nidulans exprimieren ge¬ schieht durch mittels des Laccase katalysierten Umsatz eines Substrats, das danach als leicht identifizierbares Produkt, üblicherweise als Farbstoff vorliegt. Solche gefärb- ten Zellen bzw. Kolonien, die in ihrer Umgebung das Substrat im Agar umsetzen, kön¬ nen leicht von den nicht-gefärbten Zellen bzw. Kolon ien unterschieden und anschlie- ssend separiert werden.The cells are cultured according to the host organism in a manner known to those skilled in the art. Microorganisms are generally contained in a liquid medium which contains a carbon source mostly in the form of sugars, a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese, magnesium salts and optionally vitamins. at temperatures between 0 0 C and 100 C C, preferably between 10 0 C to 60 0 C attracted under oxygen fumigation. In this case, the pH of the nutrient fluid can be kept at a fixed value, that is regulated during the cultivation or not. The cultivation can be batch-wise, semi-batchwise or continuous. Nutrients can be presented at the beginning of the fermentation or fed semi-continuously or continuously. Selecting cells which express a Laccase from Aspergillus nidulans ge done by laccase catalyzed conversion of a substrate, which is then present as an easily identifiable product, usually as a dye. Such stained cells or colonies which convert the substrate in agar in their environment can easily be differentiated from the non-stained cells or colonies and subsequently separated.
Besonders geeignete Substrate sind die Verbindungen ABTS (2,2'-azino-bis-(3- ethylbenzthiazolin-6-sulfonate), DMA (3,5-dimethylaniline) und ADBP (4-amino-2,6- dibromophenol). Diese Verbindungen werden im folg enden auch mit dem Begriff „Test¬ substrate" bezeichnet.Particularly suitable substrates are the compounds ABTS (2,2'-azino-bis- (3-ethylbenzthiazoline-6-sulfonate), DMA (3,5-dimethylaniline) and ADBP (4-amino-2,6-dibromophenol) In the following, compounds are also termed "test substrates".
Diese Testsubstrate können von Laccasen in Gegenwart von Sauerstoff zu einem blauen Farbstoff umgesetzt werden, wodurch eine schnelle Erkennung von Laccase- Aktivität möglich wird. Der Farbumschlag kann direkt mit dem Auge erkannt werden oder - insbesondere zur Quantifizierung - photometrϊsch gemessen werden.These test substrates can be converted by laccases in the presence of oxygen to a blue dye, allowing for rapid recognition of laccase activity. The color change can be detected directly with the eye or measured photometrically, in particular for quantification.
Ein Vorteil bei einigen Ausführungsformen der erfindungsgemässen Verfahren ist die Tatsache, dass einige Laccasen aus A. nidulans aus der Zelle ins Medium sekretiert werden. Damit ist es möglich, die Laccase ausserhalb der Zelle, beispielsweise direkt auf der Agarplatte nachzuweisen, ohne dass die Testsubstrate in die Zelle aufgenom¬ men werden müssen.An advantage in some embodiments of the methods of the invention is the fact that some A. nidulans laccases are secreted from the cell into the medium. This makes it possible to detect the laccase outside the cell, for example directly on the agar plate, without having to take up the test substrates in the cell.
Eine weitere bevorzugte Ausführungsform der erfindu ngsgemässen Verfahren besteht darin, dass die einzelnen Laccasen verschiedene Spezifitäten gegenüber den Test¬ substraten aufweisen. Somit wird es möglich, differenzielle Untersuchungen mit bei¬ spielsweise zwei Laccasen durchzuführen, wobei die erste Laccase das Testsubstrat 1 und nicht das Testsubstrat 2 umsetzt und die zweite Laccase das Testsubstrat 2 und nicht das Testsubstrat 1 umsetzt. Die in der untenstehenden Tabelle angegebene Substratspezifität der Laccasen IccB, IccC, und IccD erlaubt die Auswahl geeigneter Laccasen und Testsubstrate für ein solches differentielles Verfahren.A further preferred embodiment of the method according to the invention consists in that the individual laccases have different specificities with respect to the test substrates. Thus, it becomes possible to carry out differential investigations with, for example, two laccases, wherein the first laccase converts the test substrate 1 and not the test substrate 2 and the second laccase converts the test substrate 2 and not the test substrate 1. The substrate specificity of the laccases IccB, IccC, and IccD given in the table below allows the selection of suitable laccases and test substrates for such a differential method.
Für die erfindungsgemässen Verfahren geeignete Laccasen sind die in Aspergillus nidulans natürlicherweise vorkommenden Laccasen einschliesslich von Polymorphis¬ men, Allelen und Sequenzvarianten, sowie die davon ableitbaren Laccasen, soweit sie noch die wesentlichen biologischen Eigenschaften der natürlich vorkommenden Lac¬ casen, insbesondere die Substratspezifität gegenüber den oben genannten Testsub- straten, aufweisen.Suitable laccases for the processes according to the invention are the laccases naturally occurring in Aspergillus nidulans, including polymorphisms, alleles and sequence variants, and the laccases derivable therefrom, insofar as they still have the essential biological properties of the naturally occurring lac- cases, in particular the substrate specificity over those above mentioned test substrates.
Besonders geeignete Laccasen sind die Laccasen IccA, IccB, IccC und IccD aus A. nidulans, deren DNA- und Polypeptidsequenzen im Sequenzprotokoll aufgeführt sind. SEQ ID NO: 1 IccA DNA-Sequenz SEQ ID NO: 2 IccA Polypeptidsequenz SEQ ID NO: 3 IccB DNA-Sequenz SEQ ID NO: 4 IccB Polypeptidsequenz SEQ ID NO: 5 IccC DNA-Sequenz 1 SEQ ID NO: 6 IccC Polypeptidsequenz SEQ ID NO: 7 IccD DNA-Sequenz SEQ ID NO: 8 IccD PolypeptidsequenzParticularly suitable laccases are the laccases IccA, IccB, IccC and IccD from A. nidulans, whose DNA and polypeptide sequences are listed in the Sequence Listing. SEQ ID NO: 1 IccA DNA Sequence SEQ ID NO: 2 IccA Polypeptide Sequence SEQ ID NO: 3 IccB DNA Sequence SEQ ID NO: 4 IccB Polypeptide Sequence SEQ ID NO: 5 IccC DNA Sequence 1 SEQ ID NO: 6 IccC Polypeptide Sequence SEQ ID NO: 7 IccD DNA sequence SEQ ID NO: 8 IccD polypeptide sequence
Eine besonders vorteilhafte Ausgestaltung der erfindungsgemässen Verfahren ergibt sich dadurch, dass die Laccasen aus A. nidulans verschiedene Spezifitäten gegenüber den Testsubstraten besitzen. Die folgende Tabelle gibt die Substratspezifitäten wieder:A particularly advantageous embodiment of the process according to the invention results from the fact that the A. nidulans laccases have different specificities compared to the test substrates. The following table shows the substrate specificities:
Figure imgf000017_0001
Figure imgf000017_0001
+ : setzt das genannte Substrat mit Sauerstoff zu einem blauen Farbstoff um - : keine Umsetzung mit Sauerstoff zu einer farbigen Verbindung+: converts said substrate with oxygen to a blue dye -: no reaction with oxygen to form a colored compound
Weiter geeignete Farbstoffe zum Laccasenachweis sind die von Dias et al., 2003 und Johannes & Majcherezyk, 2000 beschriebenen.Further suitable dyes for laccase detection are those described by Dias et al., 2003 and Johannes & Majcherezyk, 2000.
Experimenteller TeilExperimental part
Isolierung von Laccasen aus Aspergillus nidulansIsolation of Laccases from Aspergillus nidulans
Das Genom von A. nidulans wurde auf das Vorkommen von Laccasen untersucht und neben YA und TiIA vier neue Laccasen, IccA, IccB, IccC und Icc-D, entdeckt . Die Se¬ quenz der gefundenen Laccasen ist im Sequenzprotokoll dargestellt.The genome of A. nidulans was tested for the presence of laccases and in addition to YA and TiIA four new laccases, IccA, IccB, IccC and Icc-D, were discovered. The sequence of the laccases found is shown in the sequence listing.
Die Laccasen IccB, IccC und IccD wurden mittels PCR amplifiziert und unter der Kon¬ trolle des durch Alkohole induzierbaren Promotors, alcA, sowie den xy/A Promotor, in A. nidulans exprimiert. (Waring et al. 1989, Zadra et al. 2000). Zum Test der Enzymaktivität wurden die Stämme auf threoninhaltigen oder xylosehal- tigen Agarplatten kultiviert und die Laccase katalysierte Oxidation von ABTS (2,2'- azino-bis-(3-ethylbenzthiazolin-6-sulfonate) untersucht.The laccases IccB, IccC and IccD were amplified by PCR and expressed under the control of the alcohol-inducible promoter, alcA, and the xy / A promoter in A. nidulans. (Waring et al., 1989, Zadra et al., 2000). To test the enzyme activity, the strains were cultivated on threonine-containing or xylose-containing agar plates and the laccase-catalyzed oxidation of ABTS (2,2'-azino-bis- (3-ethylbenzthiazoline-6-sulfonate) was investigated.
Im Falle von LccC kam es zu einem Farbumschlag (Abb. 2). Bei Wachstum mit Gluco- se als Kohlenstoffquelle kam es im Falle der Expression unter der Kontrolle des alcA Promotor zu keiner Blaufärbung. Unter diesen Bedingungen wird der alcA Promotor reprimiert. Demgegenüber zeigte die Laccase D keine Blaufärbung. Da ein anderer Laccasetest andere Substanzen als Substrat verwendet, haben wir diesen alternativen Versuchsansatz gewählt (Hermann, et al., 1983). Dazu wurden die Kulturen mit den beiden Substraten DMA (3,5-dimethylaniline) und ADBP (4-amino-2,6-dibromophenoI) überschichtet und inkubiert. Diese Substrate wurden zu einem blauen Farbstoff umge¬ setzt. LccC reagierte nicht mit diesen Substanzen (Abb. 2). Im Fall der Expression der Laccasen unter der Kontrolle des xylA Promotors kam es bei LccC zu einer Blaufär- bung, wenn die Kolonien auf Xylose-haltigen Platten inkubiert wurden. Analog zu der Expression unter Kontrolle des alcA-Promotors wurde keine Blaufärbung auf Glucose>- haltigen Nährmedien detektiert, da unter diesen Bedingungen der xylA-Promotor eben¬ falls reprimiert ist. Die Blaufärbung des entsprechenden Konstruktes mit LccD konnte ebenfalls nur durch Inkubation mit den oben beschriebenen Farbstoffen erreicht wer- den. Die Färbung trat nur auf, wenn die Kolonien auf Xylose-haltigen Medien wuchsen, auf Glucose trat auch hier keine Färbung auf.In the case of LccC, a color change occurred (Figure 2). When grown with glucose as the carbon source, no blue staining occurred in the case of expression under the control of the αcA promoter. Under these conditions, the alcA promoter is repressed. In contrast, laccase D showed no blue color. Since another laccase test uses substances other than substrate, we chose this alternative approach (Hermann, et al., 1983). For this, the cultures were overlaid with the two substrates DMA (3,5-dimethylaniline) and ADBP (4-amino-2,6-dibromophenol) and incubated. These substrates were converted to a blue dye. LccC did not react with these substances (Figure 2). In the case of expression of the laccases under the control of the xylA promoter, LccC became blue-tinged when the colonies were incubated on xylose-containing plates. Analogous to the expression under control of the alcA promoter, no blue staining was detected on glucose-containing nutrient media, since under these conditions the xylA promoter is also repressed. The blue staining of the corresponding construct with LccD could also be achieved only by incubation with the above-described dyes. The staining occurred only when the colonies grew on xylose-containing media, on glucose also no coloration occurred here.
Laccasen als Reportergene zur Bestimmung von PromotoraktivitätenLaccases as reporter genes for the determination of promoter activities
Der offene Leserahmen von Laccase C kann mit einem Promotor fusioniert werden - analog zu der Fusion mit dem alcA oder xylA Promotor - und dadurch dessen Aktivität bestimmt werden. Ein Pilz kann unter verschiedenen Bedingungen (Temperatur, pH, Mediumzusammensetzung) kultiviert werden und damit die Bedingungen der Promo¬ toraktivität ermittelt werden. Der Pilz kann auf Agarplatten kutliviert werden und die Aktivität visuell oder spektroskopisch beurteilt werden. Ausserdem kann der Pilz in Flüssigkultur gezüchtet und die Laccaseaktivität im Kulturüberstand quantifiziert wer¬ den. Laccase B und D können entsprechend mit den beiden anderen Substraten ein¬ gesetzt werden.The open reading frame of laccase C can be fused to a promoter - analogous to the fusion with the alcA or xylA promoter - and thereby its activity can be determined. A fungus can be cultured under different conditions (temperature, pH, medium composition) and thus the conditions of promoter activity can be determined. The fungus can be cultured on agar plates and the activity assessed visually or spectroscopically. In addition, the fungus can be grown in liquid culture and the laccase activity in the culture supernatant can be quantified. Laccase B and D can be used in accordance with the other two substrates ein¬.
Laccasen zur Lokalisierung von Proteinen Durch eine translationale Fusion eines N-terminalen Bereichs eines zu testenden Pro¬ teins mit einer Laccase, in der das Sekretionssignal deletiert wurde, kann die Funktio¬ nalität eines vorhergesagten Sekretionssignals getestet werden.Laccases for the localization of proteins The functionality of a predicted secretion signal can be tested by a translational fusion of an N-terminal region of a protein to be tested with a laccase in which the secretion signal has been deleted.
Durch eine translationale Fusion eines zu testenden Proteins mit der Laccase (mit deletiertem Sekretionssignal) kann die subzelluläre Lokalisierung des Proteins untersucht werden. Zellorganellen können fraktioniert werden und die Laccaseaktivität in den verschiedenen Fraktionen getestet werden.Translational fusion of a protein to be tested with the laccase (with deleted secretion signal) allows the subcellular localization of the protein to be investigated. Cell organelles can be fractionated and the laccase activity in the different fractions tested.
Laccasen als Reportergen für genetische ScreensLaccases as a reporter gene for genetic screens
Zur Isolierung von Promotoren, die auf bestimmte Umweltparameter (Temperatur, pH, Substrate) reagieren, kann LccC mit DNA-Fragmenten fusioniert werden. Die erstellte Genbank kann in einen Pilzstamm transformiert werden und positive, d.h. Laccase- sekretierende Stämme können anhand der Blaufärbung isoliert werden. Dieser Ansatz kann auch in Mikrotiterplatten durchgeführt werden, so dass der "Readout" durch Ro¬ boter erfolgen kann. Damit ist die Methode auch für Hoch-Durchsatz-Experimente ge¬ eignet.To isolate promoters that react to certain environmental parameters (temperature, pH, substrates), LccC can be fused with DNA fragments. The generated library can be transformed into a fungal strain and positive, i. Laccase-secreting strains can be isolated by the blue color. This approach can also be carried out in microtiter plates, so that the "readout" by robots can take place. Thus the method is also suitable for high-throughput experiments.
Laccasen können auch zur Isolierung von Regulatoren definierter Promotoren dienen. Dazu wird ein bestimmter Promotor mit dem Laccasegen fusioniert und in einen Pilz¬ stamm eingebracht. Der Stamm, der dann Expression der Laccase entsprechend der Promotoraktivität zeigt, kann mutagenisiert werden, um Mutanten mit einer veränderten Regulation zu isolieren. Dadurch können z.B. Stämme mit verstärkter Promotoraktivität hergestellt werden. Wenn der gleiche Promotor in dem Stamm zur Expression eines gewünschten Gens genutzt wird, kann so die Expression dieses Gens gesteigert wer¬ den. Es können auch Mutanten mit verringerter Prommotorstärke isoliert werden. Die¬ ser Effekt kann darauf beruhen, dass positive Regulatoren mutiert wurden. Diese Mut¬ anten können genutzt werden, um die entsprechenden Regulatoren z.B. durch Kom- plementation mit einer Genbank zu isolieren.Laccases may also serve to isolate regulators of defined promoters. For this purpose, a specific promoter is fused with the laccase gene and introduced into a fungal strain. The strain, which then shows expression of the laccase according to the promoter activity, can be mutagenized to isolate mutants with altered regulation. Thereby, e.g. Strains are produced with enhanced promoter activity. If the same promoter in the strain is used to express a desired gene, the expression of this gene can be increased. Mutants with reduced PM motor strength can also be isolated. This effect may be due to the fact that positive regulators have been mutated. These mutants can be used to generate the corresponding regulators, e.g. by isolation with a gene bank.
Laccasen IccB, IccC oder IccD als Reportergene zur Quantifizierung von Promotorakti¬ vitätenLaccases IccB, IccC or IccD as reporter genes for the quantification of promoter activities
Der offene Leserahmen der drei Laccasen, in dem die Sekretionssignale deletiert wur- den, können mit Promotoren fusioniert und in Pilze transformiert werden. Diese Kon- strukte führen zur Produktion von intrazellulärer Laccase. Die Enzymaktivität kann nach Zellaufschluss exakt quantifiziert werden. Da die drei Laccasen unterschiedliche Substrate umsetzen, können mehrere Promotoraktivitäten gleichzeitig quantifiziert wer¬ den. Dieses System ähnelt dem simultanen Einsatz von beta-Galaktosidase und GIu- curonidase. The open reading frame of the three laccases, in which the secretion signals were deleted, can be fused with promoters and transformed into fungi. These constructs lead to the production of intracellular laccase. The enzyme activity can be exactly quantified after cell disruption. Because the three laccases are different Reacting substrates, several promoter activities can be quantified simultaneously. This system is similar to the simultaneous use of beta-galactosidase and gluturonidase.
Verzeichnis der AbbildungenList of pictures
Abb. 2: Expression von LccC und LccD in RMS011. Die Laccasegene wurden unter der Kontrolle des xylA Promotors in RMS011 eingebracht und die Kolonien auf Agar- platten mit Glucose (A) oder Glycerin (B1 C) für 2 Tage bei 37°C kultiviert. Das Medium wurde mit ABTS versetzt. Die Kulturen in (C) wurden nach Wachstum mit ADBP und DMA überschichtet.Fig. 2: Expression of LccC and LccD in RMS011. The laccase genes were introduced into RMS011 under the control of the xylA promoter and the colonies were cultured on agar plates with glucose (A) or glycerol (B 1 C) for 2 days at 37 ° C. The medium was spiked with ABTS. The cultures in (C) were overlaid with ADBP and DMA after growth.
Abb. 3: Expression von LccC unter der Kontrolle des xylA Promotors. Das /ccC-Gen wurde unter der Kontrolle des xylA Promotors in RMS011 eingebracht. Je nach Kopi¬ enzahl exprimieren die Stämme das Gen unterschiedlich stark, was an der unter¬ schiedlichen Färbeintensität zu sehen ist.Fig. 3: Expression of LccC under the control of the xylA promoter. The / ccC gene was introduced into RMS011 under the control of the xylA promoter. Depending on the copy number, the strains express the gene to different extents, which can be seen in the different dyeing intensity.
Abb. 4: Quantifizierung der LccC-Aktivität. Das /ccC-Gen wurde unter der Kontrolle des alcA Promotors in RMS011 eingebracht. Flüssigmedium wurde mit Sporen be¬ impft und mit Glucose auf Substrat für 20 h kultiviert. Anschließend wurden die Hyphen geerntet und in Medium mit 100 mM Ethanol induziert. Nach verschiedenen Zeitpunk¬ ten wurden gleiche Myzelmengen entnommen, und durch Inkubieren für 2 h in Lacca- se-Assay Puffer (137 mM Di-Natrium-hydrogenphosphat, 26 mM Citronensäure, pH 6,0) bei 4°C das Enzym aus den Hyphen gelöst. Anschließend wurde die Laccase- Aktivität gemessen: zu 900 μl Laccase-Assay-Puffer wurden 100 μl ABTS (50 mM in H2O) gegeben. Die Reaktion wurde durch Zugabe von Enzym gestartet bei 405 nm photometrisch verfolgt. Reaktionstemperatur war bei 37°C. Bei den Proben 1-3 handelt es sich um unabhängige Kulturen, die unabhängigen Vorkulturen enstammen. Die Kennzeichnung a und b resultiert aus der Doppelbestimmung der Proben.Fig. 4: Quantification of LccC activity. The / ccC gene was introduced into RMS011 under the control of the alcA promoter. Liquid medium was seeded with spores and cultured with glucose on substrate for 20 h. Subsequently, the hyphae were harvested and induced in medium with 100 mM ethanol. After different times, equal amounts of mycelium were removed and incubated for 2 h in Laccase assay buffer (137 mM di-sodium hydrogen phosphate, 26 mM citric acid, pH 6.0) at 4 ° C., the enzyme from the hyphae solved. Subsequently, the laccase activity was measured: to 900 μl of laccase assay buffer was added 100 μl of ABTS (50 mM in H 2 O). The reaction was photometrically followed by addition of enzyme started at 405 nm. Reaction temperature was at 37 ° C. Samples 1-3 are independent cultures derived from independent precultures. The labels a and b result from the duplicate determination of the samples.
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Claims

Patentansprüche claims
1. Verfahren zur Bestimmung von regulatorischen Eigenschaften eines genetischen Elementes, das die Schritte umfasst: (a) Einführen eines DNA-Moleküls mit einer DNA-I-Sequenz, deren regulatorischeA method of determining regulatory properties of a genetic element comprising the steps of: (a) introducing a DNA molecule having a DNA I sequence whose regulatory
Eigenschaften bestimmt werden sollen, in eine Zelle, wobei die DNA-I-Sequenz mit einer weiteren DNA-Il-Sequenz , die für eine Laccase aus Aspergillus nidu- lans codiert, so verknüpft ist, dass die regulatorischen Elemente der DNA-I- Sequenz die Expression der Laccase kontrollieren, (b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die die Expression der Laccase erlauben,Properties are to be determined in a cell, wherein the DNA-I sequence is linked to a further DNA-II sequence which codes for an Aspergillus nidulans Laccase so that the regulatory elements of the DNA-I sequence control the expression of the laccase, (b) cultivating the cells obtained under (a) under conditions which allow the expression of the laccase,
(c) Zuordnen der unter (b) gewählten Bedingungen zu den regulatorischen Eigen¬ schaften der DNA-I-Sequenz.(c) assigning the conditions selected under (b) to the regulatory properties of the DNA I sequence.
2. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, dass die Zellen ausge¬ wählt sind aus der Gruppe von pilzlichen, pflanzlichen und tierischen Zellen.2. The method according to claim 1, characterized in that the cells are ausge¬ selected from the group of fungal, plant and animal cells.
3. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, dass es sich bei dem ge- netischen Element um einen Promotor handelt.3. The method according to claim 1, characterized in that it is the genetic element is a promoter.
4. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, dass es sich um einen regulierbaren Promotor handelt.4. The method according to claim 1, characterized in that it is a regulatable promoter.
5. Verfahren zur Lokalisation eines gewünschten Proteins in einem Zellkomparti- ment, das die Schritte umfasst:5. A method of localizing a desired protein in a cell compartment comprising the steps of:
(a) Einführen eines DNA-Moleküls, das eine DNA Sequenz besitzt, die für das gewünschte Protein codiert, und die so mit einer weiteren DNA-Il-Sequenz verknüpft ist, die für eine Laccase aus Aspergillus nidulans codiert, das das Protein, das von dem DNA-Molekül codiert wird, eine Fusion aus dem ge¬ wünschten Protein mit der Laccase darstellt, in die Zellen,(a) introducing a DNA molecule having a DNA sequence coding for the desired protein and thus linked to another DNA II sequence coding for an Aspergillus nidulans laccase containing the protein, the is encoded by the DNA molecule, represents a fusion of the desired protein with the laccase into the cells,
(b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die die Expres¬ sion des fusionierten Proteins erlauben,(b) cultivating the cells obtained under (a) under conditions which allow the expression of the fused protein,
(c) Nachweis der Lokalisation der Laccase in einem Zellkompartiment und damit der Lokalisation des gewünschten Proteins in diesem Zellkompartiment.(c) Detection of the localization of the laccase in a cell compartment and thus the localization of the desired protein in this cell compartment.
6. Verfahren nach Anspruch 5, dadurch gekennzeichnet, dass die Zellkompartimen- te fraktioniert werden und die Laccaseaktivität in den einzelnen Fraktionen be¬ stimmt wird. 6. Process according to claim 5, characterized in that the cell compartments are fractionated and the laccase activity in the individual fractions is determined.
7. Verfahren zur Bestimmung der Expression eines gewünschten Gens in einer Zelle, das die Schritte umfasst:7. A method for determining expression of a desired gene in a cell comprising the steps of:
(a) Einführen eines DNA-Moleküls, das eine DNA Sequenz des gewünschten Gens besitzt, in eine Zelle, wobei die DNA-Sequenz mit einer weiteren DNA-I I-Sequenz, die für eine Laccase aus Aspergillus nidulans codiert, so verknüpft ist, dass die regulatorischen Elemente des gewünschten Gens die Expression der Laccase kontrollieren,(a) introducing into a cell a DNA molecule having a DNA sequence of the desired gene, wherein the DNA sequence is linked to another DNA I 1 sequence coding for an Aspergillus nidulans laccase, that the regulatory elements of the desired gene control the expression of the laccase,
(b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die die Expres¬ sion des gewünschten Gens erlauben, (c) Nachweis der Expression der Laccase in der Zelle und damit Nachweis der(b) culturing the cells obtained under (a) under conditions which allow the expression of the desired gene, (c) detecting the expression of the laccase in the cell and thus detecting the
Expression des gewünschten Gens.Expression of the desired gene.
8. Verfahren zur Expression eines gewünschten Proteins als Fusionsprotein in ei- ner Zelle, oder als sekretiertes Fusionsprotein, das die Schritte umfasst:8. A method for expressing a desired protein as a fusion protein in a cell, or as a secreted fusion protein comprising the steps:
(a) Einführen eines DNA-Moleküls, das eine DNA-II- Sequenz, die für eine Lac¬ case aus Aspergillus nidulans codiert, besitzt und mit deren C-terminalem Ende eine weitere DNA-I-Sequenz, die für das gewünschte Genprodukt co¬ diert, so verknüft ist, das die Expression als Fusionsprotein aus Laccase und gewünschtem Genprodukt erfolgt,(A) introducing a DNA molecule which has a DNA II sequence coding for a Lac¬ case from Aspergillus nidulans, and with the C-terminal end of a further DNA I sequence which for the desired gene product co ¬ diert, is linked so that the expression takes place as a fusion protein from laccase and desired gene product,
(b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die die Expres¬ sion des gewünschten Gens erlauben,(b) cultivating the cells obtained under (a) under conditions which allow the expression of the desired gene,
(c) Isolierung des Fusionsproteins aus Laccase und gewünschtem Genprodukt.(c) isolating the fusion protein from laccase and desired gene product.
9. Verfahren nach Anspruch 8, dadurch gekennzeichnet, dass als Zelle eine pilzli¬ che Zelle verwendet wird.9. The method according to claim 8, characterized in that a mushroom cell is used as the cell.
10. Verfahren nach Anspruch 8, dadurch gekennzeichnet, dass das Fusionsprotein sekretiert wird, wobei als Sekretionssequenz die endogene Leadersequenz der verwendeten Laccase eingesetzt wird.10. The method according to claim 8, characterized in that the fusion protein is secreted, wherein the endogenous leader sequence of the laccase used is used as secretion sequence.
11. Verfahren nach Anspruch 8, dadurch gekennzeichnet, dass die Expression des Gens für das gewünschte Fusionsprotein unter der Transkriptionskontrolle des alcA oder des xylA Promotors aus A. nidulans erfolgt.11. The method according to claim 8, characterized in that the expression of the gene for the desired fusion protein under the transcriptional control of the alcA or the xylA promoter from A. nidulans occurs.
12. Verfahren nach einem der Ansprüche 1,5,7,8, dadurch gekennzeichnet, dass die DNA-Il-Sequenz eine DNA-Sequenz ist, die für ein Polypeptid gemäss SEQ ID NO: 2,4,6 oderδ codiert oder eine DNA-Sequenz ist, die für ein Polypeptid co- diert, das 80% Identität zu SEQ ID NO: 2,4,6 oder 8 besitzt, umfasst. 12. The method according to any one of claims 1,5,7,8, characterized in that the DNA II sequence is a DNA sequence which codes for a polypeptide according to SEQ ID NO: 2,4,6 or δ or a DNA Sequence which encodes a polypeptide having 80% identity to SEQ ID NO: 2, 4, 6 or 8.
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CN110184244A (en) * 2019-06-20 2019-08-30 福清市火麒麟食用菌技术开发有限公司 A kind of Sparassis crispa produces the preparation method of laccase

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