Functional analysis of basidiomycete specific chitin synthase genes in the agaricomycete fungus Pleurotus ostreatus.

Chitin is an essential structural component of fungal cell walls composed of transmembrane proteins called chitin synthases (CHSs), which have a large range of reported effects in ascomycetes; however, are poorly understood in agaricomycetes. In this study, evolutionary and molecular genetic analyses of chs genes were conducted using genomic information from nine ascomycete and six basidiomycete species. The results support the existence of seven previously classified chs clades and the discovery of three novel basidiomycete-specific clades (BI-BIII). The agaricomycete fungus Pleurotus ostreatus was observed to have nine putative chs genes, four of which were basidiomycete-specific. Three of these basidiomycete specific genes were disrupted in the P. ostreatus 20b strain (ku80 disruptant) through homologous recombination and transformants were obtained (Δchsb2, Δchsb3, and Δchsb4). Despite numerous transformations Δchsb1 was unobtainable, suggesting disruption of this gene causes a crucial negative effect in P. ostreatus. Disruption of these chsb2-4 genes caused sparser mycelia with rougher surfaces and shorter aerial hyphae. They also caused increased sensitivity to cell wall and membrane stress, thinner cell walls, and overexpression of other chitin and glucan synthases. These genes have distinct roles in the structural formation of aerial hyphae and cell walls, which are important for understanding basidiomycete evolution in filamentous fungi.

Identification of two genes essential for basidiospore formation during the postmeiotic stages in Pleurotus ostreatus.

A sporeless strain is an important breeding target in the mushroom industry. However, basidiospore production in the oyster mushroom Pleurotus ostreatus has been shown to be impaired by single-gene mutations in only two meiosis-related genes, mer3 and msh4. This study proposed a strategy for identifying the genes essential for basidiospore formation after meiotic division to determine new targets for molecular breeding. RNA-seq analysis was performed to identify P. ostreatus genes that are specifically expressed in the gill tissue of fruiting bodies, where basidiospore formation occurs. Transcriptome data during fruiting development of Coprinopsis cinerea, in which the meiotic steps progress synchronously, were then used to identify genes that are active in the postmeiotic stages. Based on these comparative analyses, five P. ostreatus genes were identified. Plasmids containing expression cassettes for hygromycin B-resistance screening, Cas9, and single-guide RNA targeting each gene were introduced into the protoplasts of dikaryotic strain, PC9×#64, to generate dikaryotic gene disruptants. Among the obtained transformants, three dikaryotic pcl1 disruptants and two cro6c disruptants did not produce basidiospores. Microscopic analyses indicated that spore formation was arrested at particular stages in these gene disruptants. These results indicate that these two genes are essential for mature spore formation in this fungus.

Pleurotus ostreatus as a model mushroom in genetics, cell biology, and material sciences.

Pleurotus ostreatus, also known as the oyster mushroom, is a popular edible mushroom cultivated worldwide. This review aims to survey recent progress in the molecular genetics of this fungus and demonstrate its potential as a model mushroom for future research. The development of modern molecular genetic techniques and genome sequencing technologies has resulted in breakthroughs in mushroom science. With efficient transformation protocols and multiple selection markers, a powerful toolbox, including techniques such as gene knockout and genome editing, has been developed, and numerous new findings are accumulating in P. ostreatus. These include molecular mechanisms of wood component degradation, sexual development, protein secretion systems, and cell wall structure. Furthermore, these techniques enable the identification of new horizons in enzymology, biochemistry, cell biology, and material science through protein engineering, fluorescence microscopy, and molecular breeding. KEY POINTS: • Various genetic techniques are available in Pleurotus ostreatus. • P. ostreatus can be used as an alternative model mushroom in genetic analyses. • New frontiers in mushroom science are being developed using the fungus.

Coprinopsis cinerea dioxygenase is an oxygenase forming 10(S)-hydroperoxide of linoleic acid, essential for mushroom alcohol, 1-octen-3-ol, synthesis.

1-Octen-3-ol is a volatile oxylipin found ubiquitously in Basidiomycota and Ascomycota. The biosynthetic pathway forming 1-octen-3-ol from linoleic acid via the linoleic acid 10(S)-hydroperoxide was characterized 40 years ago in mushrooms, yet the enzymes involved are not identified. The dioxygenase 1 and 2 genes (Ccdox1 and Ccdox2) in the mushroom Coprinopsis cinerea contain an N-terminal cyclooxygenase-like heme peroxidase domain and a C-terminal cytochrome P450-related domain. Herein, we show that recombinant CcDOX1 is responsible for dioxygenation of linoleic acid to form the 10(S)-hydroperoxide, the first step in 1-octen-3-ol synthesis, whereas CcDOX2 conceivably forms linoleic acid 8-hydroperoxide. We demonstrate that KO of the Ccdox1 gene suppressed 1-octen-3-ol synthesis, although added linoleic acid 10(S)-hydroperoxide was still efficiently converted. The P450-related domain of CcDOX1 lacks the characteristic Cys heme ligand and the evidence indicates that a second uncharacterized enzyme converts the 10(S)-hydroperoxide to 1-octen-3-ol. Additionally, we determined the gene KO strain (ΔCcdox1) was less attractive to fruit fly larvae, while the feeding behavior of fungus gnats on ΔCcdox1 mycelia showed little difference from that on the mycelia of the WT strain. The proliferation of fungivorous nematodes on ΔCcdox1 mycelia was similar to or slightly worse than that on WT mycelia. Thus, 1-octen-3-ol seems to be an attractive compound involved in emitter-receiver ecological communication in mushrooms.

Experimental evidence that lignin-modifying enzymes are essential for degrading plant cell wall lignin by Pleurotus ostreatus using CRISPR/Cas9.

Lignin-modifying enzymes (LMEs), which include laccases (Lacs), manganese peroxidases (MnPs), versatile peroxidases (VPs), and lignin peroxidases (LiPs), have been considered key factors in lignin degradation by white-rot fungi because they oxidize lignin model compounds and depolymerize synthetic lignin in vitro. However, it remains unclear whether these enzymes are essential/important in the actual degradation of natural lignin in plant cell walls. To address this long-standing issue, we examined the lignin-degrading abilities of multiple mnp/vp/lac mutants of Pleurotus ostreatus. One vp2/vp3/mnp3/mnp6 quadruple-gene mutant was generated from a monokaryotic wild-type strain PC9 using plasmid-based CRISPR/Cas9. Also, two vp2/vp3/mnp2/mnp3/mnp6, two vp2/vp3/mnp3/mnp6/lac2 quintuple-gene mutants, and two vp2/vp3/mnp2/mnp3/mnp6/lac2 sextuple-gene mutants were generated. The lignin-degrading abilities of the sextuple and vp2/vp3/mnp2/mnp3/mnp6 quintuple-gene mutants on the Beech wood sawdust medium reduced drastically, but not so much for those of the vp2/vp3/mnp3/mnp6/lac2 mutants and the quadruple mutant strain. The sextuple-gene mutants also barely degraded lignin in Japanese Cedar wood sawdust and milled rice straw. Thus, this study presented evidence that the LMEs, especially MnPs and VPs, play a crucial role in the degradation of natural lignin by P. ostreatus for the first time.

The lignin-degrading abilities of Gelatoporia subvermispora gat1 and pex1 mutants generated via CRISPR/Cas9.

White-rot fungi efficiently degrade wood lignin; however, the mechanisms involved remain largely unknown. Recently, a forward genetics approach to identify several genes in Pleurotus ostreatus (Agaricales) in which mutations cause defects in wood lignin degradation was used. For example, pex1 encodes a peroxisome biogenesis factor and gat1 encodes a putative Agaricomycetes-specific DNA-binding transcription factor. In this study, we examined the effects of single-gene mutations in pex1 or gat1 on wood lignin degradation in another white-rot fungus, Gelatoporia (Ceriporiopsis) subvermispora (Polyporales), to investigate conserved and derived degradation mechanisms in white-rot fungi. G. subvermispora pex1 and gat1 single-gene mutant strains were generated from a monokaryotic wild-type strain, FP-90031-Sp/1, using plasmid-based CRISPR/Cas9. As in P. ostreatus, Gsgat1 mutants were nearly unable to degrade lignin sourced from beech wood sawdust medium (BWS), while Gspex1 mutants exhibited a delay in lignin degradation. We also found that the transcripts of lignin-modifying enzyme-encoding genes, mnp4, mnp5, mnp6, mnp7, and mnp11, which predominantly accumulate in FP-90031-Sp/1 cultured with BWS, were greatly downregulated in Gsgat1 mutants. Taken together, the results suggest that Gat1 may be a conserved regulator of the ligninolytic system of white-rot fungi and that the contribution of peroxisomes to the ligninolytic system may differ among species.

Physiological function of hydrophobin Vmh3 in lignin degradation by white-rot fungus Pleurotus ostreatus.

Hydrophobins are small-secreted proteins comprising both hydrophobic and hydrophilic parts, that can self-assemble into an amphiphilic film at the air-liquid interface. More than 20 hydrophobin genes have been estimated in the white-rot fungus Pleurotus ostreatus. In our previous studies, three hydrophobin genes were shown to be predominantly expressed under ligninolytic conditions, and only vmh3 was downregulated in both the delignification-deficient mutant Δgat1 and Δhir1 strains. Here, we focused on the function of the hydrophobin Vmh3 to clarify its physiological role in lignin degradation. When the hyphae were observed by transmission electron microscopy, deletion of vmh3 resulted in the disappearance of black aggregates at the interface between the cell wall and outer environment. Deletion of vmh3 resulted in reduced hydrophobicity when 0.2% sodium dodecyl sulfate was dropped onto the mycelial surface. These results suggest that Vmh3 functions on the cell surface and plays a major role in mycelial hydrophobization. Furthermore, the Δvmh3 strain showed a marked delay in lignin degradation on beech wood sawdust medium, while the production of lignin-modifying enzymes was not reduced. This study demonstrated, for the first time, the possible effect of hydrophobin on lignin degradation by a white-rot fungus.

CRISPR/Cas9 using a transient transformation system in Ceriporiopsis subvermispora.

Ceriporiopsis subvermispora is a white-rot fungus with great potential for industrial and biotechnological applications, such as the pretreatment of lignocellulose in biorefineries, as it decomposes the lignin in the plant cell wall without causing severe cellulose degradation. A genetic transformation system was recently developed; however, gene-targeting experiments to disrupt or modify the gene(s) of interest remain challenging, and this is a bottleneck for further molecular genetic studies and breeding of C. subvermispora. Herein, we report efficient clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted gene mutagenesis in this fungus. Two plasmids expressing Cas9 together with a different pyrG-targeting single-guide RNA were separately introduced into the monokaryotic C. subvermispora strain FP-90031-Sp/1, which frequently generated strains that exhibited resistance to 5-fluoroorotic acid and uridine/uracil auxotrophy. Southern blot analyses and genomic polymerase chain reaction followed by DNA sequencing of some mutants revealed that they were pyrG mutants. We also observed that hygromycin resistance of the pyrG mutants was frequently lost after repeated subcultivations, indicating that a maker-free genome editing occurred successfully. It is also suggested that a gene mutation(s) can be introduced via a transient expression of Cas9 and a single-guide RNA; this feature, together with high-frequency gene targeting using the CRISPR/Cas9 system, would be helpful for studies on lignocellulose-degrading systems in C. subvermispora. KEY POINTS: • Efficient plasmid-based CRISPR/Cas9 was established in C. subvermispora. • The mutations can be introduced via a transient expression of Cas9 and sgRNA. • A maker-free CRISPR/Cas9 is established in this fungus.

Overexpressing Pleurotus ostreatus rho1b results in transcriptional upregulation of the putative cellulolytic enzyme-encoding genes observed in ccl1 disruptants.

The transcriptional expression pattern of lignocellulolytic enzyme-encoding genes in white-rot fungi differs depending on the culture conditions. Recently, it was shown that 13 putative cellulolytic enzyme-encoding genes were significantly upregulated in most Pleurotus ostreatus ligninolysis-deficient mutant strains on beech wood sawdust medium. However, the mechanisms by which this transcriptional shift is triggered remain unknown. In this study, we identified one mechanism. Our previous study implied that histone H3 N-dimethylation at lysine 4 level possibly affects the shift; therefore, we analysed the expression pattern in the disruptants of P. ostreatus ccl1, which encodes a putative component of the COMPASS complex mediating the methylation. The results showed upregulation of 5 of the 13 cellulolytic enzyme-encoding genes. We also found that rho1b, encoding a putative GTPase regulating signal transduction pathways, was upregulated in the ccl1 disruptants and ligninolysis-deficient strains. Upregulation of at least three of the five cellulolytic enzyme-encoding genes was observed in rho1b-overexpressing strains but not in ccl1/rho1b double-gene disruptants, during the 20-day culture period. These results suggest that Rho1b may be involved in the upregulation of cellulolytic enzyme-encoding genes observed in the ccl1 disruptants. Furthermore, we suggest that Mpk1b, a putative Agaricomycetes-specific mitogen-activated protein kinase, functions downstream of Rho1b.

Targeted disruption of hir1 alters the transcriptional expression pattern of putative lignocellulolytic genes in the white-rot fungus Pleurotus ostreatus.

Pleurotus ostreatus is frequently used in molecular genetics and genomic studies on white-rot fungi because various molecular genetic tools and relatively well-annotated genome databases are available. To explore the molecular mechanisms underlying wood lignin degradation by P. ostreatus, we performed mutational analysis of a newly isolated mutant UVRM28 that exhibits decreased lignin-degrading ability on the beech wood sawdust medium. We identified that a mutation in the hir1 gene encoding a putative histone chaperone, which probably plays an important role in DNA replication-independent nucleosome assembly, is responsible for the mutant phenotype. The expression pattern of ligninolytic genes was altered in hir1 disruptants. The most highly expressed gene vp2 was significantly inactivated, whereas the expression of vp1 was remarkably upregulated (300-400 fold) at the transcription level. Conversely, many cellulolytic and xylanolytic genes were upregulated in hir1 disruptants. Chromatin immunoprecipitation analysis suggested that the histone modification status was altered in the 5'-upstream regions of some of the up- and down-regulated lignocellulolytic genes in hir1 disruptants compared with that in the 20b strain. Hence, our data provide new insights into the regulatory mechanisms of lignocellulolytic genes in P. ostreatus.

Functional analyses of Pleurotus ostreatus pcc1 and clp1 using CRISPR/Cas9.

Understanding the molecular mechanisms controlling dikaryon formation in Agaricomycetes, which is basically controlled by A and B mating-type loci, contributes to improving mushroom cultivation and breeding. In Coprinopsis cinerea, various mutations in the SRY-type high mobility group protein-encoding gene, pcc1, were shown to activate the A-regulated pathway to induce pseudoclamp (clamp cells without clamp connection) and fruiting body formation in monokaryons. The formation of clamp cells was blocked in AmutBmut strain 326 with clp1-1 mutation in C. cinerea. However, considering the diverse mechanisms of sexual development among Agaricomycetes, it remains unclear whether similar phenotypes are also observed in clp1 or pcc1 mutants in cultivated mushrooms. Therefore, phenotypic analyses of Pleurotus ostreatus pcc1 or clp1 (Popcc1 or Poclp1) mutants generated using CRISPR/Cas9 were performed in this study. Plasmids with Cas9 expression cassette and different single guide RNAs targeting Popcc1 or Poclp1 were individually introduced into a monokaryotic P. ostreatus strain PC9 to obtain the mutants. Unlike in C. cinerea, the pseudoclamp cell was not observed in monokaryotic Popcc1 mutants, but it was observed after crossing two compatible strains with Popcc1 mutations. In Poclp1 mutants, dikaryosis was impaired as clamp cells were not observed after crossing, suggesting that Poclp1 functions may be essential for clamp cell formation, like in C. cinerea. These results provided a clue with respect to conserved and diverse mechanisms underlying sexual development in Agaricomycetes (at least between C. cinerea and P. ostreatus).

Comparative transcriptional analyses of Pleurotus ostreatus mutants on beech wood and rice straw shed light on substrate-biased gene regulation.

Distinct wood degraders occupying their preferred habitats have biased enzyme repertoires that are well fitted to their colonized substrates. Pleurotus ostreatus, commonly found on wood, has evolved its own enzyme-producing traits. In our previous study, transcriptional shifts in several P. ostreatus delignification-defective mutants, including Δhir1 and Δgat1 strains, were analyzed, which revealed the downregulation of ligninolytic genes and the upregulation of cellulolytic and xylanolytic genes when compared to their parental strain 20b on beech wood sawdust medium (BWS). In this study, rice straw (RS) was used as an alternative substrate to examine the transcriptional responses of P. ostreatus to distinct substrates. The vp1 gene and a cupredoxin-encoding gene were significantly upregulated in the 20b strain on RS compared with that on BWS, reflecting their distinct regulation patterns. The overall expression level of genes encoding glucuronidases was also higher on RS than on BWS, showing a good correlation with the substrate composition. Transcriptional alterations in the mutants (Δhir1 or Δgat1 versus 20b strain) on RS were similar to those on BWS, and the extracellular lignocellulose-degrading enzyme activities and lignin-degrading ability of the mutants on RS were consistent with the transcriptional alterations of the corresponding enzyme-encoding genes. However, transcripts of specific genes encoding enzymes belonging to the same CAZyme family exhibited distinct alteration patterns in the mutant strains grown on RS compared to those grown on BWS. These findings provide new insights into the molecular mechanisms underlying the transcriptional regulation of lignocellulolytic genes in P. ostreatus.Key Points• P. ostreatus expressed variable enzymatic repertoire-related genes in response to distinct substrates.• A demand to upregulate the cellulolytic genes seems to be present in ligninolysis-deficient mutants.• The regulation of some specific genes probably driven by the demand is dependent on the substrate.

A 14-bp stretch plays a critical role in regulating gene expression from ß1-tubulin promoters of basidiomycetes.

Cis-acting elements play a vital role in regulation of transcription initiation. Several cis-acting elements have been identified in filamentous fungi; however, the fundamental requirements for basic promoter function in basidiomycetes are obscure. In this study, core elements in ß1-tubulin promoters of basidiomycetes were functionally characterized. Using transient transformation in Ceriporiopsis subvermispora as a promoter assay, we found that a 14-bp region (ß1-tubulin core promoter element, BCE), as well as CT-rich stretch, in the ß1-tubulin promoter of the species played a critical role in the expression of a recombinant hph as a reporter gene. In addition, in silico analysis revealed other members of basidiomycetes also harboured the BCE motif as well as CT-rich stretch in the ß1-tubulin promoter region, suggesting their functional conservation among the species of basidiomycetes. To confirm the function of BCE, we investigated the effects of BCE motif deletion in the Pleurotus ostreatus ß1-tubulin promoter on expression levels of a recombinant luminous shrimp luciferase reporter gene, which was targeted into the Pofcy1 locus. Intriguingly, luciferase activity was abolished when the BCE motif was deleted in the ß1-tubulin promoter, strongly demonstrating its essential function in transcription from this promoter on the chromosome. This study clearly demonstrates the crucial role of the BCE as well as the CT-rich stretch regions in the ß1-tubulin promoter among basidiomycetes and provides new insights into the fundamental mechanism of transcription initiation in this group.

Correction to: A 14-bp stretch plays a critical role in regulating gene expression from ß1-tubulin promoters of basidiomycetes.

The original publication of this paper unfortunately contained three errors in Figs. 2B and 3. In Fig. 2B, the TSS site must be counted as "+ 1" instead of "- 1". And we indicated wrong sequences in Fig. 3: the construct "Control" has a missing one "A" in the BCE sequence, and the reverse direction of BCE sequence in the construct "BCEr" must be "GCGGAGTTTCAATT", not "CGCCTCAAGTTAA". For the reasons stated herein, the authors wish to notify the readers that Figs. 2B and 3 must be interpreted as the followings.

Biosynthesis of lagopodins in mushroom involves a complex network of oxidation reactions.

Use of the ku70-deficient strain of Coprinopsis cinerea enabled confirmation within the native context of the central role the sesquiterpene synthase Cop6 plays in lagopodin biosynthesis. Furthermore, yeast in vivo bioconversion and in vitro assays of two cytochrome P450 monooxygenases Cox1 and Cox2 allowed elucidation of the network of oxidation steps that build structural complexity onto the α-cuprenene framework during the biosynthesis of lagopodins. Three new compounds were identified as intermediates formed by the redox enzymes.

Identification of two mutations that cause defects in the ligninolytic system through an efficient forward genetics in the white-rot agaricomycete Pleurotus ostreatus.

White-rot fungi play an important role in the global carbon cycle because they are the species that almost exclusively biodegrade wood lignin in nature. Lignin peroxidases (LiPs), manganese peroxidases (MnPs) and versatile peroxidases (VPs) are considered key players in the ligninolytic system. Apart from LiPs, MnPs and VPs, however, only few other factors involved in the ligninolytic system have been investigated using molecular genetics, implying the existence of unidentified elements. By combining classical genetic techniques with next-generation sequencing technology, they successfully showed an efficient forward genetics approach to identify mutations causing defects in the ligninolytic system of the white-rot fungus Pleurotus ostreatus. In this study, they identified two genes - chd1 and wtr1 - mutations in which cause an almost complete loss of Mn2+ -dependent peroxidase activity. The chd1 gene encodes a putative chromatin modifier, and wtr1 encodes an agaricomycete-specific protein with a putative DNA-binding domain. The chd1-1 mutation and targeted disruption of wtr1 hamper the ability of P. ostreatus to biodegrade wood lignin. Examination of the effects of the aforementioned mutation and disruption on the expression of certain MnP/VP genes suggests that a complex mechanism underlies the ligninolytic system in P. ostreatus.

Effects of pex1 disruption on wood lignin biodegradation, fruiting development and the utilization of carbon sources in the white-rot Agaricomycete Pleurotus ostreatus and non-wood decaying Coprinopsis cinerea.

Peroxisomes are well-known organelles that are present in most eukaryotic organisms. Mutant phenotypes caused by the malfunction of peroxisomes have been shown in many fungi. However, these have never been investigated in Agaricomycetes, which include white-rot fungi that degrade wood lignin in nature almost exclusively and play an important role in the global carbon cycle. Based on the results of a forward genetics study to identify mutations causing defects in the ligninolytic activity of the white-rot Agaricomycete Pleurotus ostreatus, we report phenotypes of pex1 disruptants in P. ostreatus, which are defective in two major features of white-rot Agaricomycetes: lignin biodegradation and mushroom formation. Pex1 disruption was also shown to cause defects in the hyphal growth of P. ostreatus on certain sawdust and minimum media. We also demonstrated that pex1 is essential for fruiting initiation in the non-wood decaying Agaricomycete Coprinopsis cinerea. However, unlike P. ostreatus, significant defects in hyphal growth on the aforementioned agar medium were not observed in C. cinerea. This result, together with previous C. cinerea genetic studies, suggests that the regulation mechanisms for the utilization of carbon sources are altered during the evolution of Agaricomycetes or Agaricales.

A mutation in the Cc.arp9 gene encoding a putative actin-related protein causes defects in fruiting initiation and asexual development in the agaricomycete Coprinopsis cinerea.

Agaricomycetes exhibit a remarkable morphological differentiation from vegetative mycelia to huge fruiting bodies. To investigate the molecular mechanism underlying the fruiting body development, we have isolated and characterized many Coprinopsis cinerea mutant strains defective in fruiting initiation to date. Dikaryon formation in agaricomycetes, which is followed by fruiting development, is governed by the mating type loci, A and B. Recently, mutations in the Cc.snf5 gene, which encodes a putative component of the chromatin remodeling complex switch/sucrose non-fermentable (SWI/SNF), were shown to cause defects in A-regulated clamp cell morphogenesis, as well as in fruiting initiation. Here, we demonstrate that Cc.arp9, which encodes a putative actin-related protein associated with two chromatin remodeling complexes, SWI/SNF and remodels the structure of chromatin (RSC), is also essential for fruiting initiation. In contrast to Cc.snf5 mutants, Cc.arp9 mutants were not defective in clamp cell formation. The effects of mutations in Cc.arp9 and Cc.snf5 on oidia production and the transcriptional expression levels of clp1 and pcc1, which are under the control of the A gene, were also examined. These indicated that Cc.Snf5 is involved in A-regulated pathways, whereas Cc.Arp9 is not apparently. Cc.arp9/Cc.snf5 double-gene disruptants were generated and their phenotypes were analyzed, which suggested a complicated developmental regulation mechanism mediated by chromatin remodeling.

Combinatorial generation of complexity by redox enzymes in the chaetoglobosin A biosynthesis.

Redox enzymes play a central role in generating structural complexity during natural product biosynthesis. In the postassembly tailoring steps, redox cascades can transform nascent chemical scaffolds into structurally complex final products. Chaetoglobosin A (1) is biosynthesized by a hybrid polyketide synthase-nonribosomal peptide synthetase. It belongs to the chaetoglobosin family of natural products, comprising many analogs having different degrees of oxidation introduced during their biosynthesis. We report here the determination of the complete biosynthetic steps leading to the formation of 1 from prochaetoglobosin I (2). Each oxidation step was elucidated using Chaetomium globosum strains carrying various combinations of deletion of the three redox enzymes, one FAD-dependent monooxygenase, and two cytochrome P450 oxygenases, and in vivo biotransformation of intermediates by heterologous expression of the three genes in Saccharomyces cerevisiae. Five analogs were identified in this study as intermediates formed during oxidization of 2 to 1 by those redox enzymes. Furthermore, a stereochemical course of each oxidation step was clearly revealed with the absolute configurations of five intermediates determined from X-ray crystal structure. This approach allowed us to quickly determine the biosynthetic intermediates and the enzymes responsible for their formation. Moreover, by addressing the redox enzymes, we were able to discover that promiscuity of the redox enzymes allowed the formation of a network of pathways that results in a combinatorial formation of multiple intermediate compounds during the formation of 1 from 2. Our approach should expedite elucidation of pathways for other natural products biosynthesized by many uncharacterized enzymes of this fungus.

Targeted disruption of transcriptional regulators in Chaetomium globosum activates biosynthetic pathways and reveals transcriptional regulator-like behavior of aureonitol.

Postgenomic analysis revealed that many microorganisms carry numerous secondary metabolite biosynthetic genes on their genome. However, activities of those putative genes are not clearly reflected in the metabolic profile of the microorganisms, especially in fungi. A recent genome mining effort is promising in discovering new natural products. However, many fungi and other organisms are not amenable to molecular genetics manipulations, making the study difficult. Here we report successful engineering of Chaetomium globosum, a known producer of various valuable natural products, that allows its genetic manipulation via targeted homologous recombination. This strain permitted us to abolish transcriptional regulators associated with epigenetic silencing of secondary metabolite biosynthetic pathways, leading to the identification of the products generated by different gene clusters and isolation of novel secondary metabolites. We were able to identify six gene clusters that are responsible for the biosynthesis of 11 natural products previously known to be produced by C. globosum, including one cytochalasan and six azaphilone-type compounds. In addition, we isolated two new compounds, mollipilin A and B, that were only recently identified in a related Chaetomium species. Furthermore, our investigation into the mechanism of biosynthesis of those natural products in C. globosum also led to the discovery of a secondary metabolite, aureonitol, that acts like a transcriptional regulator for the biosynthesis of other secondary metabolites. Similar approaches should facilitate exploration of the untapped potential of fungal biosynthetic capability and identification of various unique biological functions that those secondary metabolites possess.