Regulation Of Fungal Secondary Metabolism Research Articles

Molecular regulation of fungal secondary metabolism.

Many bioactive secondary metabolites synthesized by fungi have important applications in many fields, such as agriculture, food, medical and others. The biosynthesis of secondary metabolites is a complex process involving a variety of enzymes and transcription factors, which are regulated at different levels. In this review, we describe our current understanding on molecular regulation of fungal secondary metabolite biosynthesis, such as environmental signal regulation, transcriptional regulation and epigenetic regulation. The effects of transcription factors on the secondary metabolites produced by fungi were mainly introduced. It was also discussed that new secondary metabolites could be found in fungi and the production of secondary metabolites could be improved. We also highlight the importance of understanding the molecular regulation mechanisms to activate silent secondary metabolites and uncover their physiological and ecological functions. By comprehensively understanding the regulatory mechanisms involved in secondary metabolite biosynthesis, we can develop strategies to improve the production of these compounds and maximize their potential benefits.

Effects of branched-chain amino acids on Shiraia perylenequinone production in mycelium cultures

BackgroundPerylenequinones from Shiraia fruiting bodies are excellent photosensitizers and widely used for anti-cancer photodynamic therapy (PDT). The lower yield of Shiraia perylenequinones becomes a significant bottleneck for their medical application. Branched-chain amino acids (BCAAs) not only serve as important precursors for protein synthesis, but also are involved in signaling pathway in cell growth and development. However, there are few reports concerning their regulation of fungal secondary metabolism. In present study, the eliciting effects of BCAAs including l-isoleucine (l-Ile), l-leucine (l-Leu) and l-valine (l-Val) on Shiraia perylenequinone production were investigated.ResultsBased on the analysis of the transcriptome and amino acid contents of Shiraia in the production medium, we revealed the involvement of BCAAs in perylenequinone biosynthesis. The fungal conidiation was promoted by l-Val treatment at 1.5 g/L, but inhibited by l-Leu. The spore germination was promoted by both. The production of fungal perylenequinones including hypocrellins A (HA), HC and elsinochromes A-C (EA–EC) was stimulated significantly by l-Val at 1.5 g/L, but sharply suppressed by l-Leu. After l-Val treatment (1.5 g/L) in Shiraia mycelium cultures, HA, one of the main bioactive perylenequinones reached highest production 237.92 mg/L, about 2.12-fold than that of the control. Simultaneously, we found that the expression levels of key genes involved in the central carbon metabolism and in the late steps for perylenequinone biosynthesis were up-regulated significantly by l-Val, but most of them were down-regulated by l-Leu.ConclusionsOur transcriptome analysis demonstrated that BCAA metabolism was involved in Shiraia perylenequinone biosynthesis. Exogenous BCAAs exhibit contrasting effects on Shiraia growth and perylenequinones production. l-Val could promote perylenequinone biosynthesis via not only enhancing the central carbon metabolism for more precursors, but also eliciting perylenequinone biosynthetic gene expressions. This is the first report on the regulation of BCAAs on fungal perylenequinone production. These findings provided a basis for understanding physiological roles of BCAAs and a new avenue for increasing perylenequinone production in Shiraia mycelium cultures.Graphical

Global regulation of fungal secondary metabolism in Trichoderma reesei by the transcription factor Ypr1, as revealed by transcriptome analysis

Trichoderma reesei Rut-C-30 is a well-known robust producer of cellulolytic enzymes, which are used to degrade lignocellulosic biomass for the sustainable production of biofuels and biochemicals. However, studies of its secondary metabolism and regulation remain scarce. Ypr1 was previously described as a regulator of the biosynthesis of the yellow pigment sorbicillin (a bioactive agent with great pharmaceutical interest) in T. reesei and several other fungi. However, the manner in which this regulator affects global gene transcription has not been explored. In this study, we report the effect of Ypr1 on the regulation of both the secondary and primary metabolism of T. reesei Rut-C30. A global gene transcription profile was obtained using a comparative transcriptomic analysis of the wild-type strain T. reesei Rut-C-30 and its ypr1 deletion mutant. The results of this analysis suggest that, in addition to its role in regulating sorbicillin and the major extracellular (hemi)cellulases, Ypr1 also affects the transcription of genes encoding several other secondary metabolites. Although the primary metabolism of T. reesei ∆ypr1 became less active compared with that of T. reesei Rut-C-30, several gene clusters involved in its secondary metabolism were activated, such as the gene clusters for the biosynthesis of specific polyketides and non-ribosomal peptides, together with the “sorbicillinoid–cellulase” super cluster, indicating that specific secondary metabolites and cellulases may be co-regulated in T. reesei Rut-C-30. The results presented in this study may benefit the development of genetic engineering strategies for the production of sorbicillin by T. reesei Rut-C-30, and provide insights for enhancing sorbicillin production in other filamentous fungal producers.

Orchestrated Biosynthesis of the Secondary Metabolite Cocktails Enables the Producing Fungus to Combat Diverse Bacteria.

ABSTRACTFungal secondary metabolites with antibiotic activities can promote fungal adaptation to diverse environments. Besides the global regulator, individual biosynthetic gene clusters (BGCs) usually contain a pathway-specific transcription factor for the tight regulation of fungal secondary metabolism. Here, we report the chemical biology mediated by a supercluster containing three BGCs in the entomopathogenic fungus Metarhizium robertsii. These clusters are jointly controlled by an embedded transcription factor that orchestrates the collective production of four classes of chemicals: ustilaginoidin, indigotide, pseurotin, and hydroxyl-ovalicin. The ustilaginoidin BGC is implicated as a late-acquired cluster in Metarhizium to produce both the bis-naphtho-γ-pyrones and the monomeric naphtho-γ-pyrone glycosides (i.e., indigotides). We found that the biosynthesis of indigotides additionally requires the functions of paired methylglucosylation genes located outside the supercluster. The pseurotin/ovalicin BGCs are blended and mesosyntenically conserved to the intertwined pseurotin/fumagillin BGCs of Aspergillus fumigatus. However, the former have lost a few genes, including a polyketide synthase gene responsible for the production of a pentaene chain used for assembly with ovalicin to form fumagillin, as observed in A. fumigatus. The collective production of chemical cocktails by this supercluster was dispensable for fungal virulence against insects and could enable the fungus to combat different bacteria better than the metabolite(s) produced by an individual BGC could. Thus, our results unveil a novel strategy employed by fungi to manage chemical ecology against diverse bacteria.

Screening of tenuazonic acid production-inducing compounds and identification of NPD938 as a regulator of fungal secondary metabolism.

The control of secondary metabolism in fungi is essential for the regulation of various cellular functions. In this study, we searched the RIKEN Natural Products Depository (NPDepo) chemical library for inducers of tenuazonic acid (TeA) production in the rice blast fungus Pyricularia oryzae and identified NPD938. NPD938 transcriptionally induced TeA production. We explored the mode of action of NPD938 and observed that this compound enhanced TeA production via LAE1, a global regulator of fungal secondary metabolism. NPD938 could also induce production of terpendoles and pyridoxatins in Tolypocladium album RK99-F33. Terpendole production was induced transcriptionally. We identified the pyridoxatin biosynthetic gene cluster among transcriptionally induced secondary metabolite biosynthetic gene clusters. Therefore, NPD938 is useful for the control of fungal secondary metabolism.

Interspecies Genomic Variation and Transcriptional Activeness of Secondary Metabolism-Related Genes in Aspergillus Section Fumigati.

Filamentous fungi produce various bioactive compounds that are biosynthesized by sets of proteins encoded in biosynthesis gene clusters (BGCs). For an unknown reason, many BGCs are transcriptionally silent in laboratory conditions, which has hampered the discovery of novel fungal compounds. The transcriptional reactiveness of fungal secondary metabolism is not fully understood. To gain the comprehensive view, we conducted comparative genomic and transcriptomic analyses of nine closely-related species of Aspergillus section Fumigati (A. fumigatus, A. fumigatiaffinis, A. novofumigatus, A. thermomutatus, A. viridinutans, A. pseudoviridinutans, A. lentulus, A. udagawae, and Neosartorya fischeri). For expanding our knowledge, we newly sequenced genomes of A. viridinutans and A. pseudoviridinutans, and reassembled and reannotated the previously released genomes of A. lentulus and A. udagawae. Between 34 and 84 secondary metabolite (SM) backbone genes were identified in the genomes of these nine respective species, with 8.7-51.2% being unique to the species. A total of 247 SM backbone gene types were identified in the nine fungi. Ten BGCs are shared by all nine species. Transcriptomic analysis using A. fumigatus, A. lentulus, A. udagawae, A. viridinutans, and N. fischeri was conducted to compare expression levels of all SM backbone genes in four different culture conditions; 32-83% of SM backbone genes in these species were not expressed in the tested conditions, which reconfirmed that large part of fungal SM genes are hard to be expressed. The species-unique SM genes of the five species were expressed with lower frequency (18.8% in total) than the SM genes that are conserved in all five species (56%). These results suggest that the expression tendency of BGCs is correlated with their interspecies distribution pattern. Our findings increase understanding of the evolutionary processes associated with the regulation of fungal secondary metabolism.

Mycovirus-Induced Tenuazonic Acid Production in a Rice Blast Fungus Magnaporthe oryzae.

Fungi are a rich source of natural products with biological activities. In this study, we evaluated viral effects on secondary metabolism of the rice blast fungus Magnaporthe oryzae using an isolate of APU10-199A co-infected with three types of mycoviruses: a totivirus, a chrysovirus, and a partitivirus. Comparison of the secondary metabolite profile of APU10-199A with that of the strain lacking the totivirus and chrysovirus showed that a mycotoxin tenuazonic (TeA) acid was produced in a manner dependent on the mycoviruses. Virus reinfection experiments verified that TeA production was dependent on the totivirus. Quantitative reverse transcription PCR and RNA-sequencing analysis indicated the regulatory mechanism underlying viral induction of TeA: the totivirus activates the TeA synthetase gene TAS1 by upregulating the transcription of the gene encoding a Zn(II)2-Cys6-type transcription factor, TAS2. To our knowledge, this is the first report that confirmed mycovirus-associated regulation of secondary metabolism at a transcriptional level by viral reinfection. Because only treatment with dimethyl sulfoxide has been reported to trigger TeA production in this fungus without gene manipulation, our finding highlights the potential of mycoviruses as an epigenomic regulator of fungal secondary metabolism.

Overexpression of an LaeA-like Methyltransferase Upregulates Secondary Metabolite Production in Aspergillus nidulans.

Fungal secondary metabolites (SMs) include medically valuable compounds as well as compounds that are toxic, carcinogenic, and/or contributors to fungal pathogenesis. It is consequently important to understand the regulation of fungal secondary metabolism. McrA is a recently discovered transcription factor that negatively regulates fungal secondary metabolism. Deletion of mcrA (mcrAΔ), the gene encoding McrA, results in upregulation of many SMs and alters the expression of more than 1000 genes. One gene strongly upregulated by the deletion of mcrA is llmG, a putative methyl transferase related to LaeA, a major regulator of secondary metabolism. We artificially upregulated llmG by replacing its promoter with strong constitutive promoters in strains carrying either wild-type mcrA or mcrAΔ. Upregulation of llmG on various media resulted in increased production of the important toxin sterigmatocystin and compounds from at least six major SM pathways. llmG is, thus, a master SM regulator. mcrAΔ generally resulted in greater upregulation of SMs than upregulation of llmG, indicating that the full effects of mcrA on secondary metabolism involve genes in addition to llmG. However, the combination of mcrAΔ and upregulation of llmG generally resulted in greater compound production than mcrAΔ alone (in one case more than 460 times greater than the control). This result indicates that deletion of mcrA and/or upregulation of llmG can likely be combined with other strategies for eliciting SM production to greater levels than can be obtained with any single strategy.

Regulation of subtelomeric fungal secondary metabolite genes by H3K4me3 regulators CclA and KdmB.

Studies on the regulation of fungal secondary metabolism highlight the importance of histone H3K4 methylation regulators Set1, CclA (Ash2) and KdmB (KDM5), but it remains unclear whether these proteins act by direct modulation of H3K4me3 at the target genes. In filamentous fungi, secondary metabolite genes are frequently located near telomeres, a site where H3K4 methylation is thought to have a repressive role. Here we analyzed the role of CclA, KdmB and H3K4me3 in regulating the subtelomeric EAS and LTM cluster genes in Epichloë festucae. Depletion of H3K4me3 correlated with transcriptional activation of these genes in ΔcclA, similarly enrichment of H3K4me3 correlated with transcriptional repression of the genes in ΔkdmB which was accompanied by significant reduction in the levels of the agriculturally undesirable lolitrems. These transcriptional changes could only be explained by the alterations in H3K4me3 and not in the subtelomerically-important marks H3K9me3/K27me3. However, H3K4me3 changes in both mutants were not confined to these regions but occurred genome-wide, and at other subtelomeric loci there were inconsistent correlations between H3K4me3 enrichment and gene repression. Our study suggests that CclA and KdmB are crucial regulators of secondary metabolite genes, but these proteins likely act via means independent to, or in conjunction with the H3K4me3 mark.

Research advances on molecular regulation of filamentous fungal secondary metabolism in China

More than half of the activity secondary metabolites from microorganisms are produced by filamentous fungi. The metabolites that are well known include penicillin, cyclosporine A, lovastatin, and so on. Due to their importance in agriculture, medicine and industry, biosynthesis and regulation of these fungal secondary metabolites have attracted much attention. The biosynthesis of fungal secondary metabolites is a complex process which generally includes multistep enzymatic reactions. Commonly, the process is controlled by elaborate regulations at different levels. A good understanding of the molecular regulation of fungal secondary metabolism will lay a theoretical foundation for improving metabolite production, mining novel compounds and activating the silent gene clusters. In this review, we focus on molecular regulation of filamentous fungal secondary metabolite biosynthesis, especially the research advances of Chinese scientists in recent forty years are mainlyintroduced. Finally, we propose the future challenges and opportunities in this field.

Regulatory Mechanism of Mycotoxin Tenuazonic Acid Production in Pyricularia oryzae.

Tenuazonic acid (TeA) is a mycotoxin produced by the rice blast fungus Pyricularia oryzae and some plant pathogenic fungi. We previously demonstrated that TeA is biosynthesized in P. oryzae by TeA synthetase 1 (TAS1) and that its production is induced by osmo-sensory MAPK-encoding gene (OSM1) deletion or the addition of 1% DMSO to cultures; however, the regulatory mechanisms of TeA production were unknown. Here, we identify a Zn(II)2-Cys6-type transcription factor in the upstream region of TAS1, which is encoded by TAS2 and regulates TeA production. We also find PoLAE1, which is a homologue of LaeA, a regulator of fungal secondary metabolism. Analysis of PoLAE1 deletion and overexpression strains indicate that PoLAE1 drives TeA production. We also demonstrate that two TeA-inducing signals, 1% DMSO addition and OSM1 deletion, were transmitted through PoLAE1. Our results indicate that TeA production is regulated by two specific regulators, TAS2 and PoLAE1, in P. oryzae.

Sirtuin A regulates secondary metabolite production by Aspergillus nidulans.

Late-stage cultures of filamentous fungi under nutrient starvation produce valuable secondary metabolites such as pharmaceuticals and pigments, as well as deleterious mycotoxins, all of which have remarkable structural diversity and wide-spectrum bioactivity. The fungal mechanisms regulating the synthesis of many of these compounds are not fully understood, but sirtuin A (SirA) is a key factor that initiates production of the secondary metabolites, sterigmatocystin and penicillin G, by Aspergillus nidulans. Sirtuin is a ubiquitous NAD+-dependent histone deacetylase that converts euchromatin to heterochromatin and silences gene expression. In this study, we have investigated the transcriptome of a sirA gene disruptant (SirAΔ), and found that SirA concomitantly repressed the expression of gene clusters for synthesizing secondary metabolites and activated that of others. Extracts of SirAΔ cultures grown on solid agar and analyzed by HPLC indicated that SirA represses the production of austinol, dehydroaustinol and sterigmatocystin. These results indicated that SirA is a transcriptional regulator of fungal secondary metabolism.

Nitrogen regulation of fungal secondary metabolism in fungi.

Fungi occupy diverse environments where they are constantly challenged by stressors such as extreme pH, temperature, UV exposure, and nutrient deprivation. Nitrogen is an essential requirement for growth, and the ability to metabolize a wide variety of nitrogen sources enables fungi to colonize different environmental niches and survive nutrient limitations. Favored nitrogen sources, particularly ammonium and glutamine, are used preferentially, while the expression of genes required for the use of various secondary nitrogen sources is subject to a regulatory mechanism called nitrogen metabolite repression. Studies on gene regulation in response to nitrogen availability were carried out first in Saccharomyces cerevisiae, Aspergillus nidulans, and Neurospora crassa. These studies revealed that fungi respond to changes in nitrogen availability with physiological and morphological alterations and activation of differentiation processes. In all fungal species studied, the major GATA transcription factor AreA and its co-repressor Nmr are central players of the nitrogen regulatory network. In addition to growth and development, the quality and quantity of nitrogen also affects the formation of a broad range of secondary metabolites (SMs). Recent studies, mainly on species of the genus Fusarium, revealed that AreA does not only regulate a large set of nitrogen catabolic genes, but can also be involved in regulating production of SMs. Furthermore, several other regulators, e.g., a second GATA transcription factor, AreB, that was proposed to negatively control nitrogen catabolic genes by competing with AreA for binding to GATA elements, was shown to act as activator of some nitrogen-repressed as well as nitrogen-induced SM gene clusters. This review highlights our latest understanding of canonical (AreA-dependent) and non-canonical nitrogen regulation mechanisms by which fungi may regulate biosynthesis of certain SMs in response to nitrogen availability.

Light-Dependent Functions of the Fusarium fujikuroi CryD DASH Cryptochrome in Development and Secondary Metabolism

DASH (Drosophila, Arabidopsis, Synechocystis, human) cryptochromes (cry-DASHs) constitute a subgroup of the photolyase cryptochrome family with diverse light-sensing roles, found in most taxonomical groups. The genome of Fusarium fujikuroi, a phytopathogenic fungus with a rich secondary metabolism, contains a gene encoding a putative cry-DASH, named CryD. The expression of the cryD gene is induced by light in the wild type, but not in mutants of the "white collar" gene wcoA. Targeted ΔcryD mutants show light-dependent phenotypic alterations, including changes in morphology and pigmentation, which disappear upon reintroduction of a wild-type cryD allele. In addition to microconidia, the colonies of the ΔcryD mutants produced under illumination and nitrogen starvation large septated spores called macroconidia, absent in wild-type colonies. The ΔcryD mutants accumulated similar amounts of carotenoids to the control strain under constant illumination, but produced much larger amounts of bikaverin under nitrogen starvation, indicating a repressing role for CryD in this biosynthetic pathway. Additionally, a moderate photoinduction of gibberellin production was exhibited by the wild type but not by the ΔcryD mutants. The phenotypic alterations of the ΔcryD mutants were only noticeable in the light, as expected from the low expression of cryD in the dark, but did not correlate with mRNA levels for structural genes of the bikaverin or gibberellin biosynthetic pathways, suggesting the participation of CryD in posttranscriptional regulatory mechanisms. This is the first report on the participation of a cry-DASH protein in the regulation of fungal secondary metabolism.

糸状菌二次代謝のエピジェネティック制御と天然物探索

ゲノム解読が進むにつれ，糸状菌には，これまで同定された化合物から予測されるよりも，はるかに多くの二次代謝物を生産する能力が秘められていることが明らかになってきた．二次代謝物生合成遺伝子の大半は，通常の培養条件下では休眠状態にあり，われわれは，その能力の一部しか引き出せていない．最近，急速に解明されつつある，エピジェネティクスを介した糸状菌の二次代謝制御は，休眠遺伝子にコードされる新規物質を取得するうえで重要な鍵になることが期待される．本稿では，糸状菌二次代謝制御にかかわるエピジェネティクス現象を概説するとともに，その概念に基づいた天然物探索について紹介する．

Regulation of fungal secondary metabolism

Fungi produce a multitude of low-molecular-mass compounds known as secondary metabolites, which have roles in a range of cellular processes such as transcription, development and intercellular communication. In addition, many of these compounds now have important applications, for instance, as antibiotics or immunosuppressants. Genome mining efforts indicate that the capability of fungi to produce secondary metabolites has been substantially underestimated because many of the fungal secondary metabolite biosynthesis gene clusters are silent under standard cultivation conditions. In this Review, I describe our current understanding of the regulatory elements that modulate the transcription of genes involved in secondary metabolism. I also discuss how an improved knowledge of these regulatory elements will ultimately lead to a better understanding of the physiological and ecological functions of these important compounds and will pave the way for a novel avenue to drug discovery through targeted activation of silent gene clusters.

Analysis of genes early expressed during Aspergillus flavus colonisation of hazelnut

Aflatoxins contamination by Aspergillus flavus is a matter of great concern for oil rich crops among which hazelnuts represent economically important agricultural commodities of Mediterranean countries, mainly used as mixed nuts or as ingredients in the bakery and confectionery industries. Since the biosynthetic pathway of aflatoxin biosynthesis has been elucidated in detail, expression analysis of the genes along the pathway can provide a thorough insight into the molecular mechanisms of toxin production and regulation. In the present work, we carried out a transcriptional analysis of the main genes belonging to aflatoxin biosynthetic cluster of A. flavus, namely the two regulatory genes aflR and aflS and the five structural genes aflD, aflM, aflO, aflP, and aflQ. The analysis was carried out at different stages of fungal growth on two different media: hazelnut agar medium and YES medium. The transcripts of all the genes paralleled the synthesis of aflatoxin and both were detected starting around 36 h in YES medium, and 72 h in hazelnut agar medium. Significantly, the amount of aflatoxin produced was about one order lower in hazelnut agar compared to YES medium. The expression of two genes encoding a lipase and a metalloprotease, potentially involved in lipid and protein catabolism, was also monitored during fungal growth. Noteworthy, the expression of the metalloprotease gene appeared to be specific for the hazelnut medium, whereas the lipase gene was expressed in both media. Finally, we verified the expression profiles of three genes encoding fatty acid dioxygenases/diol synthases involved in the biosynthesis of fungal oxylipins, namely ppoA, ppoB, ppoC. Recent findings have pointed out the importance of fungal oxylipins in fungal growth/mycotoxin production and our results indicated that all the three ppo genes are expressed during A. flavus growth on hazelnut medium. In particular, ppoB appeared to be specifically expressed in this medium. This study reports for the first time on the expression profiles of genes belonging to the biosynthetic cluster and genes potentially involved in the regulation of fungal secondary metabolism during A. flavus colonisation of hazelnuts.