Current status of secondary metabolite pathways linked to their related biosynthetic gene clusters in Aspergillus section Nigri

Xinhui Wang , Scott A. Jarmusch , Jens C. Frisvad and Thomas O. Larsen *
DTU Bioengineering, Technical University of Denmark, DK-2800, Kgs. Lyngby, Denmark. E-mail: tol@bio.dtu.dk

First published on 19th May 2022

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Xinhui Wang

Xinhui Wang received her Master's degree in Pharmacognosy from Tianjin University, supervised by Prof. Robert P. Borris in 2018, and obtained her PhD in fungal natural product discovery from Technical University of Denmark (DTU) in 2022. She is currently a Postdoc researcher at the DTU with Prof. Thomas O. Larsen. Her research focuses on mass spectrometry analysis and bioguided discovery of fungal natural products and fish killing toxins, including characterization of complex structures of ladder-frame polyethers by advanced NMR techniques.

Scott A. Jarmusch

Scott A. Jarmusch received a B.S. in Biochemistry from the University of North Carolina at Greensboro, a M.A. Ancient History from University of Liverpool and a PhD in Chemistry from the University of Aberdeen under the supervision of Prof Marcel Jaspars and Dr Rainer Ebel. After a one year postdoc at Uppsala University, he is currently a postdoc in the Center for Microbial Secondary metabolites (CeMiSt) at the Technical University of Denmark. His research interests include RiPP ecology, microbial chemical ecology and MS-based metabolomics applied to micro-organisms.

Jens C. Frisvad

Jens C. Frisvad was born in Kgs. Lyngby, Denmark in 1952. He studied chemical engineering at DTU with focus on organic chemistry and mycology. He received his PhD in 1982 and defended his Dr techn. thesis on “Secondary metabolites and species concepts in Penicillium and Aspergillus” in 1998. He became full professor in Industrial Mycology at DTU in 2001. In 1994, he stayed for six months with late Professor Martha Christensen, Dept. of Botany, University of Wyoming and has been collaborating extensively for many years with Dr Robert A. Samson at the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands.

Thomas O. Larsen

Thomas O. Larsen was born in Slagelse, Denmark in 1963. He studied Chemical Engineering at DTU with focus on Organic Chemistry and Microbiology. After a job at Danisco A/S working with aroma synthesis he returned to DTU to study fungal volatile production and received his PhD in 1994. He then joined the Marine Chemistry Group at the University of Copenhagen, headed by Reader Carsten Christophersen where he was trained as a classical natural product chemist, before returning to DTU to take up a permanent position. Currently he holds a position as full Professor in Microbial Natural Product Chemistry at Department of Biotechnology and Biomedicine at DTU.

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1 Introduction

1.1 Chemodiversity within genus Aspergillus

Fungal species within genus Aspergillus are able to produce a large number of secondary metabolites including polyketides, non-ribosomal peptides, terpenoids and numerous hybrids of these aforementioned classes. It has been well documented that the combined profile of fungal secondary metabolites in a given species is very specific and can therefore be used in chemotaxonomy.¹ In line with this there is a high degree of metabolite consistency from isolate-to-isolate within a species even though certain metabolite biosynthetic gene clusters (BGCs) might be silenced in some isolates.² On the other hand individual secondary metabolites may occur in other species, even those phylogenetically and ecologically distantly related to Aspergillus. Consequently, correct identification of species in genus Aspergillus, based partly on chemotaxonomy, has notoriously been challenging, since closely related species often share many phenotypic features including metabolite production. This has led to the majority of the series in Aspergillus being invalidly described or named as clades, where a new valid series classification was introduced by Houbraken et al.³ By using a phylogenetic approach, including data on secondary metabolite production, Aspergillus was subdivided in six subgenera, 27 sections and 75 series, altogether including a list of 446 Aspergillus species.³

To further investigate both inter- and intraspecies genomic variation as well as the chemical potential of species in genus Aspergillus, a large sequencing project including one representative strain of >350 known Aspergillus spp. has been initiated using the Joint Genome Institute (JGI) fungal genome pipeline.^4,5 One of the first major outcomes from this initiative was a comprehensive bioinformatic analysis of all detected BGCs from species in section Nigri.⁶ In line with the above mentioned studies, a major conclusion from this work was that secondary metabolism is unique to individual species. The authors reported a total of 2717 encoding secondary metabolite BGCs from a total of 37 genomes and found that on average, each species contains 8.75 unique BGCs.⁶ Similar to previous studies,^7,8 the authors also identified and linked groups of BGCs, that are functionally closely related and encode the production of the same or very similar secondary metabolites, into 455 biosynthetic gene cluster families.⁶ This large number is in sharp contrast to the relatively few secondary metabolites that have so far been linked to their corresponding BGCs, indicating the overall potential for discovery in Nigri.

1.2 Biosystematics and genomes of Aspergillus subgenus Circumdati section Nigri

Species in Aspergillus section Nigri are among the most important filamentous fungi used in biotechnology and are also common in many natural habitats. Most species in section Nigri are asexual, producing a large number of asexual (mitotic) spores (conidia), however, a few species have been shown to have a sexual state.^7–11 Interestingly, the two mating types needed for sexual reproduction have been found in different Aspergillus section Nigri species.^11,12 A large number of species can produce sclerotia (large, firm, frequently rounded masses of hyphae serving as propagules of new growth or as long-lasting resting structures) under certain conditions,^13–19 and sclerotium production is important as certain indoloterpenes are only produced in the sclerotia, and indoloterpenes may go unnoticed if sclerotia are not formed.⁸ Section Nigri is most closely related to sections Flavi, Candidi, and Circumdati in subgenus Circumdati of Aspergillus according to phylogeny,¹⁹ morphology and secondary metabolite production.^20,21 Notably, section Nigri seems to be an outlier as full genome sequencing indicates that section Nigri is phylogenetically more closely related to subgenus Nidulantes than to subgenus Circumdati.^6,22 Furthermore members of section Nigri differ from these groups of Aspergillus subgenera by having the ubiquinone Q-9 rather than the Q-10 (and/or Q-10 (H2)), as do species in sections Flavi, Candidi, Circumdati and subgenus Nidulantes. Visagie et al. placed biseriate section Nigri species as related to the subgenus Circumdati sections Terrei and Flavipedes, and uniseriate section Nigri species close to subgenus Circumdati section Petersoniorum, leaving section Nigri as a polyphyletic taxon, based on ITS and calmodulin sequences.²³ Here we follow the phylogeny and taxonomy suggested by Houbraken et al.,³ except that we provisionally accept the species A. fijiensis, A. violaceofuscus and A. lacticoffeatus.

The species in the A. niger group (now called Aspergillus subgenus Circumdati section Nigri) have been treated by Mosseray (1934), and Raper and Fennell (1965).^24,25 Similarly, very detailed morphological and physiological taxonomic studies were done by Murakami et al.^26–36 with an emphasis on industrial black Kuro koji strains. Later new taxonomies and phylogenies were suggested, based on morphology, physiology, household gene sequences and secondary metabolite profiles.^{3,6,13,15–17,20,22,37–72}

Aspergillus subgenus Circumdati section Nigri presently consists of 5 formal series: ser. Nigri, ser. Carbonarii, ser. Heteromorphi (biseriate species, i.e. species with both metulae and phialides on the globose conidial Aspergillus head (vesicle)), in addition to ser. Japonici (uniseriate species, i.e. species with phialides only on the globose conidial Aspergillus head (vesicle)) and ser. Homomorphi (biseriate).^3,46 Ser. Nigri includes 14 species; Ser. Carbonarii includes 4 species; series Heteromorphi includes 2 species; series Japonici includes 15 species, and finally ser. Homomorphi only has one member (Table 1). Many other names have been published and frequently used, especially A. acidus, A. aureus var. pallidus, A. awamori, A. coreanus, A. inuii, A. kawachii and A. nakazawai (all synonyms of A. luchuensis) and A. pulverulentus, A. saitoi and A. saitoi var. kagoshimaensis (synonyms of A. tubingensis), and A. aureus, A. batatae, A. miyakoensis, A. usamii and A. usamii var. shirousamii (all synonyms of A. niger).^61,62Aspergillus awamori is a name often used, and was originally a name for a domesticated form of A. niger, later it was regarded as cryptic phylogenetic species in A. niger based on a non-domesticated strain from the plant Welwitschia mirabilis.⁵⁴ This interpretation of A. awamori was abandoned and the name A. welwitschiae given to this taxon, while the domesticated forms were named A. luchuensis, which is the most commonly used Kuro koji fungus.^61,62 The names A. phoenicis and A. ficuum have been rejected in order to conserve the well-known name A. niger.⁷³ The number of accepted species in Aspergillus section Nigri is therefore currently 36. Additionally, many of the species of Aspergillus section Nigri have been genome sequenced (Table 1). Thus there are good opportunities for mining these genomes for BGCs encoding for secondary metabolites.

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Some species in Aspergillus section Nigri can produce mycotoxins, notably ochratoxin A (A. carbonarius, A. lacticoffeatus, A. niger, A. sclerotioniger, A. welwitschiae),^{40,47,90–96} and fumonisins B₂, B₄ and B₆ (A. lacticoffeatus, A. niger and A. welwitschiae).^46,95–100 Secalonic acid, only found in the uniseriate Aspergillus section Nigri species, has also been mentioned as a mycotoxin.¹⁰¹ Oxalic acid is a widespread weak mycotoxin of Aspergillus niger and related species in series Nigri and Carbonarii. It has been linked to kidney failure after growth and in human lungs and transport of calcium oxalate, via the blood to the kidneys.¹⁰² Oxalic acid, citric acid and gluconic acid are produced by many species in section Nigri. These natural products should be regarded as secondary metabolites when they are accumulated, secreted and regulated by the general regulator LaeA, in addition to being taxonomically restricted,^103–105 but as primary metabolites when they are in flux in the TCA cycle (and therefore not secreted and accumulated and furthermore present in all fungi and many other organisms). Altogether, a large number of secondary metabolites have been reported from Aspergillus niger and similar species,^{102,106–109} and preliminary genome scale models for Aspergillus niger metabolic networks have been published.^110,111

2 Secondary metabolites and their biosynthetic pathway from Aspergillus section Nigri

As of June 2021, a total of 176 compounds have been isolated and characterized from the species of Aspergillus section Nigri. Table 2 highlights some metabolites discovered from 2009 to 2021 not included in our previous review paper,¹⁰⁹ and Table 3 highlights the secondary metabolites for which their biosynthetic pathway have been characterized.

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2.1 Non-reduced polyketides

Fungal polyketides are synthesized by multidomain polyketide synthases (PKSs), which can be classified as nonreducing (NR), partially reducing (PR), and highly reducing (HR) PKSs. NR-PKSs synthesize a diverse array of polycyclic compounds that contain several important domains at minimum, including: ACP transacylase (SAT), β-ketoacyl synthase (KS), acyl transferase (AT), product template (PT), and acyl carrier protein (ACP). Additional domains that may be present include: C-methyltransferase (C-MeT), thioesterase (TE), Claisen cyclase (CLC) or reductase (R) domains. This section summarizes the secondary metabolites of NR-PKS origin in Aspergillus section Nigri, including azanigerone A (1), kotanin (2), naphtho-γ-pyrones, TAN-1612 (3), 2,4-dihydroxy-3,5,6-trimethylbenzaldehyde (4), nigerpyrone (5) and funalenone (6) (Table 4 and Fig. 1). The PT domains of NR-PKSs can be categorized phylogenetically into eight major groups (group I–VIII), each of which corresponds to a unique polyketide length or cyclization regioselectivity. The NR-PKS derived secondary metabolites in black aspergilli described here belong to group III, group V and group VII. Group III catalyze a single C2–C7 cyclization and multicyclization and product release is TE/CLC mediated. Group VII also catalyze C2–C7 monocyclization and most contain a C-MeT domain. Furthermore, all NR-PKSs in this group release the polyketide product via a reductive release mechanism (terminal R domain). Group V catalyze C6–C11 cyclization most frequently and C4–C13 positions for the secondary cyclization. Group V NR-PKSs are unique due to the fact they all lack the C-terminal release domain. Alternatively, they use a stand-alone metallo-β-lactamase-type TE (MβL-TE).¹⁵⁵

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2.2 Partially reduced polyketides

In section Nigri, the secondary metabolites of PR-PKSs origin that have so far been linked to their related gene cluster include aculinic acid (48), yanuthone D (49) and ochratoxin A (50) (Table 4). The biosynthetic pathway of 48 further downstream products are described in this section, whereas the biosynthetic pathways of 49 and 50 are shown in the section describing hybrid compounds. PR-PKSs typically possess a ketoreductase (KR) domain, introducing hydroxyl groups at specific sites of the growing polyketide chain thereby setting the structural basis for subsequent cyclization reactions, including lactone formation of otherwise aromatic compounds. In line with this they usually lack dehydratase (DH) or enoyl reductase (ER) domains for further reductive processing as found in the HR-PKSs. These enzymes catalyze the production of small molecules, such as 6-methylsalicylic acid (51, 6-MSA) and mellein.¹⁷⁶

2.3 Highly reduced polyketides

Highly-reducing PKSs (HR-PKSs) possess the complete or almost complete ensemble of β-keto processing domains (Table 4). Fumonisins (fum1) and azanigerone A (1) (azaB) are secondary metabolites produced from section Nigri derived from HR-PKSs. In addition to the minimal PKS domains for all fungal iterative PKSs (ACP, MAT, KS), HR-PKSs also have a ketoreductase (KR) domain that reduces the β-ketone to the corresponding β-alcohol and a dehydratase (DH) domain that subsequently removes the β-alcohol to obtain an enoyl thioester. An enoyl reductase (ER) domain is also found in some HR-PKSs, which further reduces the enoyl thioester to a saturated acyl thioester. Many HR-PKSs also have a C-methyltransferase (CMeT) domain that may methylate the α position of the growing polyketide chain before the β-ketone is reduced.¹⁷⁹

3 Linear and cyclic non-ribosomal peptides

Nonribosomal peptides (NRPs) are produced by dedicated nonribosomal peptide synthetases (NRPSs) that are mainly found in bacteria and fungi.¹⁸⁸ NRPSs are large modular multi-functional enzymes that contain at least three domains: an adenylation (A) domain for selection, activation, and loading of the amino acid, a thiolation (T) domain, or peptidyl carrier protein (PCP) domain, for binding the activated substrate to a 4′-phosphopantetheine cofactor via a thioester bond and a condensation (C) domain for the peptide formation between two amino acids from the adjacent T domains.^188,189

Although the canonical function of C domains is to catalyze the formation of a peptide bond between the growing peptide, the C_T domain (a subset of C domains) can be recruited by fungal NRPS to perform the equivalent reaction of a thioesterase domain. To date, all fungal NRPSs that assemble macrocyclic peptides terminate with a condensation-like C_T domain that catalyzes the cyclization reaction, and thus, is a near universal macro-cyclization strategy used by fungal NRPSs.^188,190 Optional domains include epimerization (E), formylation (F), methylation (M), heterocyclization (Cy), reduction (R), and oxidation (Ox) domains for additional modifications. In this section, aculeacin A (72)/echinocandin B (73) (ecd and hty) and malformins (mlfA) are secondary metabolites produced from section Nigri derived from NRPSs. Additionally, a putative BGC for asperazine biosynthesis was proposed in the A. tubingensis G131 and this cluster shows homology with the fumitremorgin biosynthesis cluster identified in A. fumigatus Af293 and the ditryptophenaline production in A. flavus.^191,192 However, genetic experiments should be performed in the future to confirm the involvement of this gene cluster in biosynthesis of asperazine.

3.1 Aculeacin A

Aculeacin A (72) is an antifungal lipopeptide isolated from A. aculeatus that contains palmitic acid as the lipophilic side chain.¹⁹³ It relates to the echinocandin class, which consists of cyclic non-ribosomal hexapeptides with a lipophilic side chain that exhibits high antifungal bioactivity. Furthermore, 72 includes the same six amino acids as echinocandin B (73), while aculeacin biosynthesis is very homologous to 73 biosynthesis.^194,195 This gene cluster responsible for the synthesis of 73 (ecd and hty) in A. pachycristatus NRRL 11440 (previously named as Emericella rugulosa) was characterized via genome sequencing and bioinformatics analysis and was confirmed by functional knock-out experiments and heterologous expression of selected pathway genes.¹⁴⁰ This gene cluster contains genes encoding for a six-module nonribosomal peptide synthetase EcdA, an acyl-AMP ligase EcdI, and oxygenases EcdG, EcdH, and EcdK. The hty subcluster containing htyA, htyB, htyC, and htyD genes has been shown to be responsible for L-homotyrosine (L-homoTyr) biosynthesis (Fig. 15).¹⁴⁰

The researchers proposed one possible pathway for the biosynthesis of 73 (Fig. 16): linoleoyl-AMP, produced by EcdI from linoleate, is transferred to the initiation T0 (first thiolation domain) of EcdA. The linoleoyl-S-phosphopantetheinyl-T0 is sequentially extended with L-ornithine, L-threonine, L-proline, L-homotyrosine, L-threonine, and 4R-methyl-L-proline to form the linear hexapeptide. Thereafter, the terminal condensation (C_T) performs macrocyclization of the NRPS product and the cyclic scaffold is released from EcdA. In addition, an intriguing structural feature of echinocandins is the incorporation of hydroxylated amino acids. Later research identified five oxygenation events by these three iron dependent enzymes, EcdG, EcdH and EcdK.¹⁹⁵ EcdH, a predicted P450 type hemeprotein monoxygenase, has been shown to iteratively hydroxylate C5 and C4 of L-ornithine after the formation of the cyclic peptide, while the ecdG gene encodes α-ketoglutarate (α-KG)-dependent mononuclear, nonheme iron oxygenase that has been shown to hydroxylate free L-homoTyr to generate 3-hydroxyl-L-homoTyr. Furthermore, another α-KG-dependent nonheme iron oxygenase EcdK is involved in hydroxylating C5 of methyl-proline, to produce 4R-methyl-proline (4-Me-Pro).¹⁹⁵ However, the last echinocandin oxygenation event is still to be characterized.

Moreover, to investigate the evolution of chemical diversity in echinocandin lipopeptide metabolites, the gene cluster responsible for 72 synthesis was characterized via DNA sequences for additional echinocandin-type pathway gene clusters.¹³⁹ In the gene cluster of 72, the hty genes are located immediately upstream of and are separated by, two unknown genes and a cytochrome P450 from the core NRPS gene. Recently, Zhang et al. characterized the α-ketoglutarate/Fe²⁺-dependent proline hydroxylase (HtyE) from A. pachycristatus and A. aculeatus in the respective 73 and 72 BGCs through heterologous expression in Escherichia coli.^196,197 This study showed that both Ap- and Aa-HtyE converted L-proline to trans-4- and trans-3-hydroxyproline but at different ratios.

3.2 Malformins

Malformins are a group of cyclic pentapeptide compounds produced by several species in section Nigri,¹⁹⁸ which exhibit antibacterial activity and anti-cancer properties in mouse and human carcinoma cells.^199,200 Recently, researchers have conducted a workflow of genome analysis to predict the gene cluster responsible for biosynthesis of malformin A2 (74) and C (75) (Fig. 17).¹⁰⁷

Since malformins are pentapeptides that contain a disulphide bond, only one gene cluster (mlf) containing NRPS genes and disulphide bond-associated genes were included as search criteria. This search led to the identification of the likely related gene cluster responsible for biosynthesis of malformin in 18 fungal strains, importantly, including previous reported malformin producing species.^107,109 The NRPS responsible for malformin biosynthesis was confirmed to be mlfA through targeted gene deletion in A. brasiliensis, which led to a total abolishment of 74 and 75 production. Furthermore, a genetic complementation by a constitutively expressed mlfA in the mlfAΔ strain restored 74 and 75 production, to further confirm the function of mlfA. Based on these results, it was hypothesized that some of the individual NRPS adenylation domains are less selective since multiple malformins disappear after deletion of the NRPS.

3.3 Atrofuranic acid (NRPS-like)

Single modular NRPS-like enzymes can be considered as a special group of NRPS, which contain a thioesterase (TE) or reductase (R) domain instead of the peptide-forming C domain. NRPS-like enzymes with A–T–TE architectures recognize and activate α-keto acids by their A domains. The activated substrates are tethered to the 4′-phosphopantetheine arm of the T domain. After the transfer to the TE domain, a second α-keto acid molecule is activated by the A domain and bound to the T domain. The two building blocks are then linked by the TE domain and released as condensation products with different ring systems.²⁰¹ The condensation of aromatic α-keto acids produces a variety of interconnecting core ring structures, which are proposed to be performed by non-reducing NRPS-like enzymes.

When the researchers heterologously expressed atromentin synthetases in A. niger in order to explore the reaction mechanism of the thioesterase domain, they discovered the metabolite atrofuranic acid (76), as well as metabolite X (77), gyroporin (78) and p-hydroxybenzoic acid (decomposed product of atrofuranic acid). However, atromentin synthetases produce atromentin in A. oryzae, which implied that the physiology of A. niger initiates a cross-specificity and cross-chemistry event on the NRPS-like enzyme. When feeding with 2-¹³C-labeled L-tyrosine, which was incorporated into 76 as a precursor, the biosynthesis pathway for metabolites produced in A. niger could be proposed (Fig. 18). Biosynthesis of 78 was suggested to be proceed via an uncharacterized metabolite, 77, proposed to be derived by a Piancatelli-type rearrangement from 76,^202,203 which subsequently by decarboxylation and oxidation leads to formation of 78.

Further exploration showed that this cross-chemistry was not limited to A. niger but also occurred in A. brasiliensis, which contains an intrinsic atromentin synthetase, indicating that modifications of benzoquinone core structures are common to Aspergillus species of the section Nigri. Therefore, characterization of products from NRPS-like enzymes may be hampered by cross-chemistry occurring on metabolite core structures during heterologous expression.¹⁴¹ It appears that using different heterologous expression platforms in parallel to elucidate the true nature of products generated by uncharacterized NRPS-like enzymes is critical.

4 Terpenoids

Despite being an incredibly structurally diverse class of natural products in fungi, all terpenoids are derived from the common five carbon precursor molecules dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), which are produced from acetyl-CoA via the mevalonate pathway. Successive head-to-tail condensation of one to three IPP extender units to DMAPP results in monoterpenes (C10), sesquiterpenes (C15) and diterpenes (C20). Longer chains (C30 and C40) are usually formed by a head-to-head condensation of two sesquiterpenes (C15) or diterpenes (C20) molecules.²⁰⁴ Then these linear hydrocarbons undergo a dephosphorylation and cyclization cascade to produce terpenes. This highly complex reaction is catalyzed by enzymes known as terpene synthases. Two distinct classes of terpene synthases exist, defined according to their substrate activation mechanism. Class I terpene synthases catalyze an ionization-dependent cyclization of substrate, while class II terpene synthases catalyze a protonation-dependent cascade.²⁰⁵ Further biosynthetic pathway enzymes such as cytochrome P450 monooxygenases, oxidoreductases, and different groups of transferases modify this initial terpene scaffold, producing the final terpenoid.²⁰⁵

4.1 Ergosterimide

Ergosterimide was characterized from the culture extract of Aspergillus niger EN-13, an endophytic fungus isolated from the marine brown alga Colpomenia sinuosa.¹⁴² Its synthetic steroidal derivative, 3β-acetoxy-7α,15-ethanocholest-8(14)-ene-1′,2′- dicarboxylic acid anhydride, was formed in the Diels–Alder addition of maleic anhydride to 3β-acetoxy-ethanocholest-7,14-diene under mild condition.²⁰⁶ Based on this information, the researchers proposed that ergosterimide is a natural Diels–Alder adduct of ergosteroid and maleimide, which is probably enzymatically catalyzed. Additionally, this is the first reported cycloadduct of a steroid and maleimide so far.¹⁴² However, more genetic experiments are needed in the future.

4.2 Taxol

Taxol is an extremely effective but rare antitumor agent extracted from Taxus spp. barks. Many taxol-producing endophytic fungi have been reported,²⁰⁷ including A. aculeatinus Tax-6 that was isolated from Taxus chinensis var. mairei.²⁰⁸ In addition, a mutant strain, A. aculeatinus BT-2 was obtained from the original strain, A. aculeatinus Tax-6, using fungicidin as the mutagenic agent, which produces higher quantities of taxol than the A. aculeatinus BT-2.¹⁴³ The biosynthetic pathway of taxol in Taxus spp. has been revealed,²⁰⁹ yet the biosynthesis pathway of taxol formed from endophytic fungi is not clear. One study explored the genes involved in taxol synthesis in A. aculeatinus Tax-6 to understand the molecular mechanisms of producing fungal taxol. The results showed that genes involved in the mevalonate (MVA) pathway and non-mevalonate (MEP) pathway were expressed, including isopentenyl pyrophosphate transferase, geranyl pyrophosphate transferase, and geranylgeranyl pyrophosphate synthetase. In addition, to identify the mechanism of the difference in taxol production, the transcriptomes of the two taxol-producing fungi were compared and the changes in the gene expression between them were explored. Most genes related to the MVA pathway in the mutant strain BT-2 showed upregulation. However, the key downstream genes related to taxol biosynthesis, i.e., taxadiene synthetase (TS) were not found.¹⁴³

5 Hybrid PKS–NRPS

5.1 Pyranonigrins E–J and pyranonigrins A, S

Pyranonigrins are a family of antioxidative compounds isolated from A. niger, which all feature a unique pyrano[2,3-c]pyrrole bicyclic skeleton.¹⁴⁵ The biosynthetic pathway of pyranonigrins E–J (79–84) was elucidated in A. niger, while the pyranonigrins A (85) and S (86) were in Penicillium thymicola (Fig. 19).

The BGC which is related to production of 79 (not to be confused with pyranonigrin E reported in another study²¹⁰) was induced and the metabolites was isolated in A. niger ATCC 1015 as a new PKS–NRPS hybrid secondary metabolite after the pyn gene cluster was activated by the expression of the pathway-specific Zn₂Cys₆ transcriptional regulator pynR under control of the arginase (aga) promoter.¹⁴⁴ Furthermore, cluster activation resulted in induced transcriptions of the pyranonigrin encoding genes pynA, pynB, pynC, pynD, and pynE. PynA encodes a PKS–NRPS hybrid enzyme, PynB encodes a FAD/FMN-containing dehydrogenase, PynC encodes a methyltransferase, PynD encodes a cytochrome P450 oxidase, and PynE encodes an NAD-binding protein.¹⁴⁴

In subsequent research, Yamamoto et al. found more pyranonigrins through activating the pyn cluster in high yields by replacing of the pynR promoter with the robust glaA promoter. Subsequently, the complete pathway of pyranonigrins was identified by targeted gene deletion, heterologous in vivo bioconversion and in vitro assays. Furthermore, this study identified three extra genes, including the protease-like protein PynH that performs an exo-methylene formation, the stand-alone thioesterase PynI that is capable of concomitant tetramic acid formation and chain release from the PKS–NRPS hybrid enzyme PynA, and FAD-containing monooxygenase (FMO) PynG that catalyzes an epoxidation-mediated cyclization to form the dihydro-γ-pyrone moiety.¹⁴⁵

Altogether, the proposed biosynthetic pathway initiates with biosynthesis of the PKS–NRPS product by PynA, followed by release of the intermediate from PynA by PynI, generating 84. Next, PynC catalyzes N-methylation of 84, resulting in 83. Then PynG catalyzes an epoxidation-mediated cyclization to form the dihydro-γ-pyrone moiety, which is followed by PynD-catalyzed alcohol oxidation and enolization to form 82. PynH then catalyzes the formation of 79, which is dimerized to form 80. Alternatively, PynE can catalyze the conversion of 79 to 81, which can be reversed by PynB.^144,171

A very notable discovery in this pathway was PynH, a predicted protease that performs dehydration of a serine side chain to form an exo-methylene in pyranonigrin E. It is highly unusual to find an exo-methylene-forming enzyme that is involved in the modification of a fungal polyketide-nonribosomal peptide hybrid compound. Another important finding is that the stand-alone thioesterase PynI is capable of tetramic acid formation and chain release from the PKS–NRPS hybrid enzyme PynA (Fig. 20).

In addition, the potent antioxidant pyranonigrin A (85), which was first reported in A. niger,^211,212 was found to be biosynthesized by a PKS–NRPS gene cluster pyr in P. thymicola, which is different from the pyn cluster for 79. Its biosynthetic pathway is elucidated in P. thymicola by genome mining and heterologous expression.¹⁴⁶

Basic Local Alignment Search Tool (BLAST) search results revealed a PKS–NRPS (PyrA) from P. thymicola IBT 5891 that exhibits moderate sequence similarity to PynA (50% identity/67% similarity) and An18g00520 (61% identity/75% similarity), both of which are from A. niger A1179.²¹³ In addition, An18g00520 was shown to be responsible for the production of tetramic acid type compound, proposed to be the precursor to 86.²¹⁴ Based on these information, the heterologous expression of pyrA in A. nidulans was carried out, resulting in the production of the product of An18g00520. This indicated the product encoded by pyr cluster shares the same biosynthetic precursor with 86.¹⁴⁶ Subsequent heterologous expression of all the genes involved in the entire pyr gene cluster in A. nidulans led to the production of 85. Based on the findings from both in vivo and in vitro studies, only four genes, pyrA–D, are required for the biosynthesis of 85.¹⁴⁶

Therefore, the biosynthetic pathway of 85 in P. thymicola initiates the biosynthesis of the PKS–NRPS product by the PyrA, followed by product release catalyzed by either a Dieckmann cyclase (DKC) and/or the hydrolase PyrD. Then, FAD-binding monooxygenase PyrC may catalyze epoxidation followed by subsequent ring closure to form the pyrano[2,3-c]pyrrole core, followed by conversion to 86 and finally the α-carbon of the glycine part of 85 is oxidized by cytochrome P450 PyrB (Fig. 21).

5.2 Pyranoviolin A

Pyranoviolin A (87) was discovered from the genome-sequenced fungus A. violaceofuscus CBS 115571 and it is the first pyranonigrin analogue containing the C-3 methoxy group.¹⁴⁷

The BGC pyv of 87 was identified via bioinformatics analysis and comparison to the homologous genes of pyranonigrins (Fig. 22).^144,146,214 This revealed the pyv cluster encodes a PKS–NRPS hybrid PyvA, a cytochrome P450 monooxygenase PyvB, two flavin-dependent monooxygenases (FMOs) PyvC and PyvE, an α/β hydrolase PyvD, a glucose-methanol-choline (GMC) family of oxidoreductase PyvF, and O-methyltransferase PyvH. The PKS–NRPS responsible for 87 was confirmed to be PyvA through targeted gene deletion, which led to complete abolishment of 87 production. Based on the bioinformatic analysis and previous studies on pyranonigrins, the biosynthetic pathway leading to 87 was proposed (Fig. 23).

The pathway initiates by the PKS PyvA to synthesize C10 backbone from one acetyl-Co unit and four malonyl-CoA units, followed by the condensation with the thiolation (T) domain-bound glycine activated by the adenylation (A) domain. Then the product release is catalyzed by Dieckmann condensation (DKC) and/or the α/β hydrolase PyvD, yielding the tetramic acid moiety. Next, the FMO PyvC may catalyze the epoxidation of one of the olefins on the polyketide chain, followed by the epoxide ring-opening and the dihydro-γ-pyrone ring formation. Next, the P450 PyvB maybe catalyze two consecutive reactions, in which the dihydro-γ-pyrone is oxidized to γ-pyrone and C-7 is hydroxylated to yield pyranonigrin F (88) [note that two distinct natural products are individually named “pyranonigrin F”].^145,215 Finally, the O-methyltransferase PyvH, which is specifically found in the 87 pathway, catalyze the methylation for the C-3 hydroxy group to produce 87.

5.3 Ochratoxin A

Ochratoxin A (50) is a potent mycotoxin which can be found in a wide variety of food products, produced by among others the species of A. carbonarius and A. niger in section Nigri. Structurally, 50 comprises a dihydrocoumarin moiety linked via an amide bond to a molecule of L-phenylalanine, derived from the shikimic acid pathway. In ochratoxigenic Penicillium species, the majority of the molecular characteristics of 50 biosynthesis have been identified, which includes genes encoding a PKS (otapksPN), an NRPS (otanpsPN) and other tailoring genes.^216–218 The 50 PKS was recently identified in Penicillium verrucosum, and its involvement in 50 biosynthesis was verified by targeted gene disruption.²¹⁹

In Aspergillus species, a PKS required for 50 biosynthesis was identified in A. ochraceus and partially characterized in Aspergillus westerdijkiae.^220,221 In addition, in A. carbonarius, the AcOTAnrps gene encoding the NRPS responsible for the ligation of the phenylalanine to the polyketide group, was identified by gene disruption.¹⁴⁸ In the same study, the order of reactions in the biosynthetic pathway of 50 in A. carbonarius was clarified demonstrating that the amide bond between the phenylalanine and the polyketide dihydroisocoumarin is catalyzed by the synthetase and that it precedes the chlorination step.¹⁴⁸ Furthermore, a flavin-halogenase in the biosynthesis of 50 in A. carbonarius was identified by gene deletion and confirmed that the chlorination step which converts ochratoxin B (OTB, 89) to 50 represents the final stage of the biosynthetic pathway.

Recently, in order to study the regulatory mechanism of 50 biosynthetic pathway, Wang et al. sequenced and assembled the complete genome of A. ochraceus fc-1 and used that for comparative genomic analyses with five other sequenced ochratoxin A-producing fungi (A. carbonarius, A. niger, A. steynii, A. westerdijkiae, and P. nordicum).¹⁴⁹ By comparing the genomic analysis of these six 50 producers, a conserved ochratoxin A (50) BGC was identified, including a PKS (otaA), an NRPS (otaB), a cytochrome P450 monooxygenase (otaC), a halogenase (otaD), and a basic leucine zipper (bZIP) transcription factor (otaR1), which are all required for 50 biosynthesis. A GAL4-like Zn₂Cys₆ binuclear cluster DNA-binding protein (otaR2) and a FAD-dependent oxidoreductase (otaE) were also identified but they are not essential to the 50 production (Fig. 24). Ochratoxin A (50) biosynthesis begins with the formation of 7-methylmellein catalyzed by OtaA utilizing acetyl-CoA and four malonyl-CoA units, followed by the oxidation to OTβ by OtaC and OtaE. Next, OTβ and L-phenylalanine are combined by the NRPS OtaB to form an amide bond to synthesize 89. Finally, 89 is chlorinated by a halogenase OtaD to give 50 (Fig. 25).

5.4 Acurin A

Acurin A (90) was isolated from an extract of A. aculeatus. The BGC was identified based on bioinformatics, RT-qPCR experiments and gene-deletion analysis and revealed that at least eight genes (acrA-G and acrR) are vital for the biosynthesis of 90.¹⁵⁰

Since the structure of 90 is very similar to that of fusarin C, for which the biosynthesis is partly characterized, it was hypothesized that 90 originated from a polyketide and an amino acid fusion. The candidate clusters were further narrowed down to identify the putative PKS and NRPS genes for the production of 90. Intriguingly, bioinformatics analysis indicated that the scaffold for 90 formation potentially could be synthesized by unlinked PKS and NRPS enzymes. In addition, the researchers also found that uncoupled PKS and NRPS encoding genes are conserved in a fungal clade in section Nigri. Moreover, the PKS didn't appear to have a release domain but instead the predicted NRPS contained a reductase domain for the potential release of 90 backbone. Examination of genes encoding the PKS and NRPS responsible for 90 biosynthesis were identified to be acrA and acrB through gene deletion experiment in A. aculeatus, which led to the abolishment of 90 production. Further bioinformatics analyses showed a lack of an epimerase domain is present in the NRPS indicating L-serine is incorporated into 90. In addition to the PKS and NRPS encoding genes of 90, there are six other surrounding genes (acrC–G) including a regulatory transcription factor (acrR). Next, a functional analysis of the genes involved in the formation of 90 was also performed by targeted gene deletions. This confirmed the involvement of the transcription factor acrR and O-methyltransferase (acrG). However, no accumulation of potential intermediates were found in acrC (hydrolase), acrD (oxidoreductase), acrE (CYP450) and acrF (CYP450) deletion strains, which might be because of the unstable intermediate products or a scenario where more than two enzymes interact with each other during the reactions. Altogether, these results enabled Wolff et al. to propose a pathway for biosynthesis of 90.¹⁵⁰

The pathway starts with the polyketide assembly by the PKS AcrA forming a heptaketide backbone from successive condensations of six malonyl-CoA units (Fig. 26). Then the fusion of an amino-acid moiety is catalyzed by the NRPS AcrB. The functions of AcrC–F are still unclear, however the researchers hypothesized that the PK-NRP hybrid product release from the enzyme by reductive release to set up the formation of the lactam ring by Aldol condensation, followed by a water loss to generate a double bond followed by reduction. Finally, an O-methyltransferase AcrG is needed for the final step in the biosynthesis of 90.

6 Meroterpenoids

6.1 Yanuthones

The yanuthones comprise a group of compounds containing a core structure constituted of an epoxylated six-membered ring with a sesquiterpene chain at C-13 and varied side chains at C-15 and C-16.^171,222 Many yanuthones have shown antifungal bioactivity, and among them yanuthone D (49) displayed the most potent antifungal activity against Candida albicans.¹⁵²

The yan BGC for 49 contains ten genes in the genome of A. niger. The PKS-encoding YanA was heterologously expressed in A. nidulans to confirm its involvement in the biosynthesis of the initial polyketide product 6-MSA (51). The biosynthetic pathway of 49 was further investigated by individually deleting all genes within the yan cluster (Fig. 27) which resulted in the accumulation of numerous yanuthone intermediates and enabled a biosynthetic pathway to be proposed (Fig. 28).

The pathway starts with the biosynthesis of 6-MSA (51) catalyzed by the PKS YanA, followed by decarboxylation by YanB producing m-cresol (57), and hydroxylation by the cytochrome P450 YanC, generating toluquinol (91). The latter two steps for epoxidation and prenylation are still not very clear since no biosynthetic intermediate has been obtained from the targeted gene deletions. It has been proposed that the farnesylation likely precede epoxidation since the C-prenylation reaction generally requires the π-electron of a prenyl acceptor to attack the allylic cation generated on the prenyl donor. Therefore, 91 may also be accepted by YanG, followed by epoxidation by YanD/YanE generating 7-deacetoxyyanuthone (92), as epoxidized 91 loses a π-electron at the prenylation site due to the epoxide formation.^151,223 Next, cytochrome P450 YanH catalyzes the conversion to 22-deacetylyanuthone A (93), which is then converted to yanuthone E (94) by the O-mevalon transferase YanI. Importantly, this is the first time that O-mevalon transferase activity has been molecularly characterized. Finally, the oxidase YanF catalyzes the formation of 49 from 94.¹⁷¹ Deletion of the individual genes in the BGC also led to formation of not only hydroxylated metabolites but also a glycosylated yanuthone.⁵⁰

In addition to 49 and its analogues with a C7 scaffold defined as class I yanuthones, yanuthone X1 and X2 were also discovered, which instead contain a C6-core scaffold oxygenated at C-16 defined as class II yanuthones and derived from an unknown precursor.^108,222

6.2 Aculene A–C

A. aculeatus is an industrially important black filamentous fungus. After the prioritization of the growing conditions and the dereplication by UV- and HRMS-data to identify known compounds, a unique type of norsesquiterpenes (C₁₄), named aculenes A and B (55, 56), were isolated and structurally characterized from A. aculeatus IBT 21030.¹¹⁵55 and 56 are of mixed biosynthetic origin containing the amino acid L-proline moiety but also a nordaucane skeleton that the researchers proposed to be derived from an ent-daucane precursor through demethylation (loss of a methyl group on the C_5n terpene scaffold) (Fig. 31). Lee et al. used gene inactivation, feeding experiments, and heterologous reconstitution approaches in order to understand the demethylation (C-12) process that converts the ent-daucane-containing asperaculane into the nordaucane aculene scaffold.¹⁵³

Because the L-proline moiety in 55 was likely introduced by a nonribosomal peptide synthase (NRPS), the researchers accessed the genome sequence from JGI of A. aculeatus ATCC16872 (=BCRC32190) and scanned for all adenylation domain encoding genes and their neighboring genes.²²⁴ A candidate BGC, ane, was found containing one single-module NRPS (AneB) with adenylation and thiolation domains as well as a Class I terpene cyclase (AneC). Further investigation revealed several genes nearby aneB and aneC with putative activities consistent with the structure of 55, including a phytanoyl-CoA dioxygenase (aneA), three cytochrome P450 monooxygenases (aneD, aneF and aneG) that could perform oxidative modifications on the terpene scaffold, a α,β-hydrolase (aneE) that may transfer or release L-proline from AneB to the sesquiterpene moiety, and a proline iminopeptidase (aneH) (Fig. 29).

To further explore the functions of those enzymes, genetic inactivation on aneC was performed which produced no aculene products and showed no accumulation of any intermediates. Next, intron-free aneC from A. aculeatus was cloned and transformed into Saccharomyces cerevisiae strain BJ5464-NpgA, which showed the production of dauca-4, 7-diene (95) as a sole product. These results confirmed that AneC is a terpene cyclase that produce 95 with an ent-daucane skeleton for the biosynthesis of 55.

Each of the three P450 monooxygenase genes (aneG, aneF and aneD) were then inactivated. The results indicated the involvement of oxidation by AneG, AneD and AneF at C-2, C-10 and C12 respectively. In addition, the researchers hypothesized that the P450s could also play a role in the demethylation process. To further verify the function of the three P450 monooxygenases (ane G, D, F), heterologous co-expression of these enzymes with AneC using a S. cerevisiae strain harboring a single copy of the cytochrome P450 reductase gene from A. terreus (AtCPR) were performed first.²²⁴ They confirmed the role of aneF, aneD, aneG when they coexpressed aneF with aneC, aneC/F/D and aneC/G/F. However, the enzyme involved in the second oxidation at C-2 to generate a ketone functionality from a C-2 hydroxy group and decarboxylation at C-12 was still unknown because of the decreasing amounts of downstream metabolites when coexpressing aneC/F/D/G. This might be because of degradation by endogenous enzymes in S. cerevisiae of the aculenes homologues. Therefore, the coexpression of aneC/F/D/G in A. oryzae was performed, which led to the production of aculene D (96) as a major product together with asperaculane G (97). Thus, AneG performs C-H activation at the C-2 position of asperaculane E (98) and catalyzes a double oxidation to install a C-2 ketone group. The involvement of 97 as pathway intermediate was validated, as the feeding of 97 to ΔaneC restored the production of 55. Overall, the researchers proposed that all the three P450s are necessary for this stepwise demethylation process. AneF oxidizes C-12 to a carboxylate, AneG installs a necessary electro-withdrawing carbonyl group on C-2, while AneD introduces a10-hydroxy group to assist the tautomerization.

Next, the modification enzymes involved in the gene cluster was investigated. Deletion of AneA, which encodes an α-ketoglutarate-dependent dioxygenase, led to the abolishment of the production of 55 and aculene C (99), and accumulation of 56 and 96, and a minor component asperaculane B (100) (shunt product). This suggest that the unsaturation is catalyzed by AneA.

Deletion of aneB and aneE both led to the absence of 55 and 56 and the accumulation of 96 and 100. This result suggests that AneB adenylates L-proline and AneE transfers this activated L-proline derivative to 96 to form 56.

Compared to the downstream pathway products, there are several upstream intermediates containing a 14-hydroxy group. To confirm that the 14-hydroxylated compounds are shunt products, the researchers supplied 96 and 100 to aneC-deleted strain. The former restores the production of aculene but the latter does not, which suggest that the metabolites with 14-hydroxy group are shunt products in the biosynthesis of 55.

Overall, the biosynthetic pathway of 55 initiates with the biosynthesis of 95 by AneC, followed by C-12 carboxylation by AneF to produce asperaculane D (101), and C-10 hydroxylation by AneD, yielding asperaculane E (98). Then AneG installs an electron-withdrawing carbonyl group at the C-2 position, which triggers C-12 decarboxylation to yield the nordaucane skeleton in 96. Next, the unsaturation between C5 and C6 are catalyzed by AneA, yielding 55 and 99. Finally, the adenylation is catalyzed by AneB, followed by transferring the activated L-proline derivatives to 96, yielding 56.

7 Fatty acid derived secondary metabolites

7.1 Alkylcitric acids

Alkylcitric acids comprise a saturated alkyl “tail” and a citrate-derived “head”. Many of these compounds have shown bioactive properties, such as plant root growth promotion (hexylitaconic acid) and anti-fungal (hexylaconitic acid anhydride) bioactivity.^225,226 Their BGC and the transcriptional factor (akcR) have been identified recently in A. niger by examining the functional annotation of BGCs predicted from genomic data (Fig. 30). The transcriptional factor akcR was overexpressed, leading to the production of several alkylcitric acids. In addition, an unlinked tailoring gene (hexylaconitic acid decarboxylase gene, hadA) and akcR gene were overexpressed together in A. niger, leading to the overproduction of alkylcitric acids and several previously unreported alkylcitric acids.¹⁵⁴

The proposed alkylcitrate pathway starts with the production of hexylcitric acid (102) catalyzed by a citrate synthase (akcB) and a fatty acid synthase (akcA and akcD). Next, the dihydroxylation of hexylcitric is catalyzed by 2-methylcitrate dehydratase (akcC) to produce hexylaconitic acid A (103). Finally, a decarboxylation reaction is catalyzed by hexylaconitic acid decarboxylase (hadA), yielding hexylitaconic acid B (104) (Fig. 31).¹⁵⁴

8 Conclusion and future perspectives

In this review we have highlighted and described the nineteen biosynthetic pathways that have so far been characterized from species belonging to the group of black aspergilli, linking the secondary metabolites to their corresponding BGCs. Considering that Vesth et al. recently identified 455 BGC families, across 37 genomes in section Nigri,⁶ this is a surprisingly small number taking the importance of black aspergilli as food contaminants and industrial workhorses for enzyme production into account. First, this means that many of the 176 secondary metabolites from which structures have already been reported from black aspergilli need to be linked to their respective BGCs. Secondly, the many unknown secondary metabolites predicted from these fungi need to be discovered and characterized, including the approximately 8.75 unique clusters found among both uni- and bi-seriate black aspergilli.² With the continued rapid collection of high quality genomic information, the development of new integrated omics technologies and more effective strategies for manipulation and mining of fungal genomes, the number of fully characterized fungal BGCs is expected to increase substantially in the coming years.²²⁷

9 Conflicts of interest

There are no conflicts to declare.

10 Acknowledgements

This work was supported by the Danish National Research Foundation (DNRF137) for the Center for Microbial Secondary Metabolites (CeMiSt), as well as a grant from the Novo Nordic Foundation (NNF18OC0034952) and from the China Scholarship Council (No. 201806250032) and the Department of Biotechnology and Biomedicine at the Technical University of Denmark.

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