Fungal Secondary Metabolite Biosynthesis Research Articles

Exploring Culture Media Diversity to Produce Fungal Secondary Metabolites and Cyborg Cells.

Fungi are microorganisms of significant biotechnological importance due to their ability to provide food and produce several value-added secondary metabolites and enzymes. Its products move billions of dollars in the pharmaceutical, cosmetics, and additives sectors. These microorganisms also play a notable role in bionanotechnology, leading to the production of hybrid biological-inorganic materials (such as cyborg cells) and the use of their enzyme complex in the biosynthesis of nanoparticles. In this sense, optimizing the fungal growth process is necessary, with selecting the cultivation medium as one of the essential factors for the microorganism to reach its maximum metabolic expression. The culture medium's composition can also impact the nanomaterial's stability and prevent the incorporation of nanoparticles into fungal cells. Therefore, our main objectives are the following: (1) compile and discuss the most commonly employed culture media for the production of fungal secondary metabolites and the formation of cyborg cells, accompanied by preparation methods; (2) provide a six-step guide to investigating the fungal metabolomic profile and (3) discuss the main procedures of microbial cultivation to produce fungal cyborg cells.

Advances in Chemical Epigenetic Modification Methods in the Study of Fungal Secondary Metabolites

Abstract: The secondary metabolites produced by fungi are an important resource for new drug development, and the biosynthesis of fungal secondary metabolites is closely related to the epigenetic status of the chromosomes in which their gene clusters are located. However, the induction of fungal silencing of gene expression is one of the challenges faced at this stage. Chemical epigenetic modification is a simple and effective method to regulate fungal metabolism by adding chemical epigenetic modifiers to the culture medium to activate silent metabolic pathways in the fungus, resulting in the production of cryptic natural products. This paper reviews the progress of research on increasing the chemical diversity of fungal secondary metabolites using chemical epigenetic modifications, with the aim of providing a reference for the in-depth study of fungal natural products.

Nitric oxide mediates red light-induced perylenequinone production in Shiraia mycelium culture.

Perylenequinones (PQs) from bambusicolous Shiraia fungi serve as excellent photosensitizers for photodynamic therapy. However, the lower yield of PQ production in mycelium cultures is an important bottleneck for their clinical application. Light has long been recognized as a pivotal regulatory signal for fungal secondary metabolite biosynthesis. In this study, we explored the role of nitric oxide (NO) in the growth and PQ biosynthesis in mycelium cultures of Shiraia sp. S9 exposed to red light. The continuous irradiation with red light (627nm, 200lx) suppressed fungal conidiation, promoted hyphal branching, and elicited a notable increase in PQ accumulation. Red light exposure induced NO generation, peaking to 81.7μmol/g FW on day 8 of the culture, with the involvement of nitric oxide synthase (NOS)- or nitrate reductase (NR)-dependent pathways. The application of a NO donor sodium nitroprusside (SNP) restored conidiation of Shiraia sp. S9 under red light and stimulated PQ production, which was mitigated upon the introduction of NO scavenger carboxy-PTIO or soluble guanylate cyclase inhibitor NS-2028. These results showed that red light-induced NO, as a signaling molecule, was involved in the regulation of growth and PQ production in Shiraia sp. S9 through the NO-cGMP-PKG signaling pathway. While mycelial H2O2 content exhibited no significant alternations, a transient increase of intracellular Ca2+ and extracellular ATP (eATP) content was detected upon exposure to red light. The generation of NO was found to be interdependent on cytosolic Ca2+ and eATP concentration. These signal molecules cooperated synergistically to enhance membrane permeability and elevate the transcript levels of PQ biosynthetic genes in Shiraia sp. S9. Notably, the combined treatment of red light with 5μM SNP yielded a synergistic effect, resulting in a substantially higher level of hypocrellin A (HA, 254mg/L), about 3.0-fold over the dark control. Our findings provide valuable insights into the regulation of NO on fungal secondary metabolite biosynthesis and present a promising strategy involving the combined elicitation with SNP for enhanced production of photoactive PQs and other valuable secondary metabolites in fungi.

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.

Fusion Enzymes Involved in Biosynthetic Tailoring Reactions in Fungi.

Enzyme fusion, the fusion of enzymes with different domains to a single protein, has been widely recognized as a promising strategy in the development of biocatalysts. Nature has evolved gene fusion to combine different catalytic enzymes to function as a fusion enzyme, and this strategy is utilized in many natural product biosynthetic pathways. Owing to rapid advances in genome sequencing and biosynthetic pathway characterization, there is increasing interest in fusion enzymes from fungal biosynthetic pathways, particularly those involved in tailoring steps. This concept aims to provide an up-to-date overview of fusion enzymes that catalyze tailoring reactions in the biosynthesis of fungal secondary metabolites. Since fungal fusion enzymes are often associated with novel metabolites, this pioneering work may stimulate the exploration of the structural diversity of fungal natural products through genome mining of the untapped biosynthetic pathways involving fusion enzymes.

Application of Gene Knockout and Heterologous Expression Strategy in Fungal Secondary Metabolites Biosynthesis.

The in-depth study of fungal secondary metabolites (SMs) over the past few years has led to the discovery of a vast number of novel fungal SMs, some of which possess good biological activity. However, because of the limitations of the traditional natural product mining methods, the discovery of new SMs has become increasingly difficult. In recent years, with the rapid development of gene sequencing technology and bioinformatics, new breakthroughs have been made in the study of fungal SMs, and more fungal biosynthetic gene clusters of SMs have been discovered, which shows that the fungi still have a considerable potential to produce SMs. How to study these gene clusters to obtain a large number of unknown SMs has been a research hotspot. With the continuous breakthrough of molecular biology technology, gene manipulation has reached a mature stage. Methods such as gene knockout and heterologous expression techniques have been widely used in the study of fungal SM biosynthesis and have achieved good effects. In this review, the representative studies on the biosynthesis of fungal SMs by gene knockout and heterologous expression under the fungal genome mining in the last three years were summarized. The techniques and methods used in these studies were also briefly discussed. In addition, the prospect of synthetic biology in the future under this research background was proposed.

Dynamic regulation of Monascus azaphilones biosynthesis by the binary MrPigE-MrPigF oxidoreductase system.

Monascus azaphilones (MAs) have been extensively applied as natural food coloring agents. MAs are classified into three categories: yellow MAs (YMAs), orange MAs (OMAs), and red MAs with various biological activities. However, the exact biosynthetic mechanism of OMAs and YMAs are not thoroughly elucidated. Firstly, we identified four DNA-binding residues of transcription factor MrPigB and constructed a multi-site saturation mutagenesis library of MrPigB. Then, comparative metabolite and gene expression of the mutants revealed that two oxidoreductases MrPigE and MrPigF were responsible for the formation of YMAs and OMAs. Finally, the in vitro and in vivo assays demonstrated the opposite roles of MrPigE and MrPigF in conversion of OMAs to YMAs. To our knowledge, this is the first report of a binary oxidoreductase system for dynamic regulation of fungal secondary metabolite biosynthesis. Broadly, our work also demonstrates the transcription factor engineering strategy for elucidating the biosynthetic pathway of secondary metabolite. KEY POINTS: • MrPigE converts orange Monascus azaphilones to yellow Monascus azaphilones • MrPigF oxidizes intermediates to afford orange Monascus azaphilones • MrPigE and MrPigF constitute a binary system in Monascus azaphilones biosynthesis.

Genetic Relationships in the Toxin-Producing Fungal Endophyte, Alternariaoxytropis Using Polyketide Synthase and Non-Ribosomal Peptide Synthase Genes.

The legume Oxytropis sericea hosts a fungal endophyte, Alternaria oxytropis, which produces secondary metabolites (SM), including the toxin swainsonine. Polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) enzymes are associated with biosynthesis of fungal SM. To better understand the origins of the SM, an unannotated genome of A. oxytropis was assessed for protein sequences similar to known PKS and NRPS enzymes of fungi. Contigs exhibiting identity with known genes were analyzed at nucleotide and protein levels using available databases. Software were used to identify PKS and NRPS domains and predict identity and function. Confirmation of sequence for selected gene sequences was accomplished using PCR. Thirteen PKS, 5 NRPS, and 4 PKS-NRPS hybrids were identified and characterized with functions including swainsonine and melanin biosynthesis. Phylogenetic relationships among closest amino acid matches with Alternaria spp. were identified for seven highly conserved PKS and NRPS, including melanin synthesis. Three PKS and NRPS were most closely related to other fungi within the Pleosporaceae family, while five PKS and PKS-NRPS were closely related to fungi in the Pleosporales order. However, seven PKS and PKS-NRPS showed no identity with fungi in the Pleosporales or the class Dothideomycetes, suggesting a different evolutionary origin for those genes.

Biosynthesis of a Tricyclo[6.2.2.02,7 ]dodecane System by a Berberine Bridge Enzyme-Like Aldolase.

The aldol reaction is one of the most fundamental stereocontrolled carbon-carbon bond-forming reactions and is mainly catalyzed by aldolases in nature. Despite the fact that the aldol reaction has been widely proposed to be involved in fungal secondary metabolite biosynthesis, a dedicated aldolase that catalyzes stereoselective aldol reactions has only rarely been reported in fungi. Herein, we activated a cryptic polyketide biosynthetic gene cluster that was upregulated in the fungal wheat pathogen Parastagonospora nodorum during plant infection; this resulted in the production of the phytotoxic stemphyloxin II (1). Through heterologous reconstruction of the biosynthetic pathway and in vitro assay by using cell-free lysate from Aspergillus nidulans, we demonstrated that a berberine bridge enzyme (BBE)-like protein SthB catalyzes an intramolecular aldol reaction to establish the bridged tricyclo[6.2.2.02,7 ]dodecane skeleton in the post-assembly tailoring step. The characterization of SthB as an aldolase enriches the catalytic toolbox of classic reactions and the functional diversities of the BBE superfamily of enzymes.

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.

Investigation of Citrinin and Pigment Biosynthesis Mechanisms in Monascus purpureus by Transcriptomic Analysis.

Monascus purpureus YY-1 is widely used in food colorant production in China. Our previous study clearly illustrated the whole-genome data for YY-1 and provided useful insight into evolutionary research and industrial applications. However, the presence of citrinin, which has nephrotoxic, hepatotoxic, and carcinogenic activities, has attracted attention to the safety of Monascus products. In an effort to reduce the harmful effects of citrinin in Monascus-related products, a random mutant of M. purpureus YY-1 with low citrinin production (designated as “winter”) was obtained in this study. To analyze the biosynthesis and regulation mechanisms of pigment and citrinin, a transcriptomic analysis of the M. purpureus YY-1 and winter strains was performed. Comparative transcriptomic analysis reveals pksCT, the essential gene for citrinin synthesis, showed a low expression level in M. purpureus YY-1 and winter, which suggested there might be isoenzymes in M. purpureus YY-1 that were responsible for the citrinin synthesis during evolution. In addition, changes in transcription factor expression may also influence the network regulating the citrinin synthesis pathway in M. purpureus. Moreover, the yields of pigments produced by the winter mutant were significantly increased. Repressing the central carbon metabolism and improving the acetyl-CoA pool can contribute to a high pigment yield, and enhanced NADPH regeneration can also lead to the metabolic flux of pigment production in M. purpureus. Investigations into the biosynthesis and regulation of citrinin and pigment production in M. purpureus will enhance our knowledge of the mechanisms behind the biosynthesis of fungal secondary metabolites.

The fungal myosin I is essential for Fusarium toxisome formation.

Myosin-I molecular motors are proposed to function as linkers between membranes and the actin cytoskeleton in several cellular processes, but their role in the biosynthesis of fungal secondary metabolites remain elusive. Here, we found that the myosin I of Fusarium graminearum (FgMyo1), the causal agent of Fusarium head blight, plays critical roles in mycotoxin biosynthesis. Inhibition of myosin I by the small molecule phenamacril leads to marked reduction in deoxynivalenol (DON) biosynthesis. FgMyo1 also governs translation of the DON biosynthetic enzyme Tri1 by interacting with the ribosome-associated protein FgAsc1. Disruption of the ATPase activity of FgMyo1 either by the mutation E420K, down-regulation of FgMyo1 expression or deletion of FgAsc1 results in reduced Tri1 translation. The DON biosynthetic enzymes Tri1 and Tri4 are mainly localized to subcellular structures known as toxisomes in response to mycotoxin induction and the FgMyo1-interacting protein, actin, participates in toxisome formation. The actin polymerization disruptor latrunculin A inhibits toxisome assembly. Consistent with this observation, deletion of the actin-associated proteins FgPrk1 and FgEnd3 also results in reduced toxisome formation. Unexpectedly, the FgMyo1-actin cytoskeleton is not involved in biosynthesis of another secondary metabolite tested. Taken together, this study uncovers a novel function of myosin I in regulating mycotoxin biosynthesis in filamentous fungi.

KdmB, a Jumonji Histone H3 Demethylase, Regulates Genome-Wide H3K4 Trimethylation and Is Required for Normal Induction of Secondary Metabolism in Aspergillus nidulans.

Histone posttranslational modifications (HPTMs) are involved in chromatin-based regulation of fungal secondary metabolite biosynthesis (SMB) in which the corresponding genes—usually physically linked in co-regulated clusters—are silenced under optimal physiological conditions (nutrient-rich) but are activated when nutrients are limiting. The exact molecular mechanisms by which HPTMs influence silencing and activation, however, are still to be better understood. Here we show by a combined approach of quantitative mass spectrometry (LC-MS/MS), genome-wide chromatin immunoprecipitation (ChIP-seq) and transcriptional network analysis (RNA-seq) that the core regions of silent A. nidulans SM clusters generally carry low levels of all tested chromatin modifications and that heterochromatic marks flank most of these SM clusters. During secondary metabolism, histone marks typically associated with transcriptional activity such as H3 trimethylated at lysine-4 (H3K4me3) are established in some, but not all gene clusters even upon full activation. KdmB, a Jarid1-family histone H3 lysine demethylase predicted to comprise a BRIGHT domain, a zinc-finger and two PHD domains in addition to the catalytic Jumonji domain, targets and demethylates H3K4me3 in vivo and mediates transcriptional downregulation. Deletion of kdmB leads to increased transcription of about ~1750 genes across nutrient-rich (primary metabolism) and nutrient-limiting (secondary metabolism) conditions. Unexpectedly, an equally high number of genes exhibited reduced expression in the kdmB deletion strain and notably, this group was significantly enriched for genes with known or predicted functions in secondary metabolite biosynthesis. Taken together, this study extends our general knowledge about multi-domain KDM5 histone demethylases and provides new details on the chromatin-level regulation of fungal secondary metabolite production.

Next-generation sequencing approach for connecting secondary metabolites to biosynthetic gene clusters in fungi.

Genomics has revolutionized the research on fungal secondary metabolite (SM) biosynthesis. To elucidate the molecular and enzymatic mechanisms underlying the biosynthesis of a specific SM compound, the important first step is often to find the genes that responsible for its synthesis. The accessibility to fungal genome sequences allows the bypass of the cumbersome traditional library construction and screening approach. The advance in next-generation sequencing (NGS) technologies have further improved the speed and reduced the cost of microbial genome sequencing in the past few years, which has accelerated the research in this field. Here, we will present an example work flow for identifying the gene cluster encoding the biosynthesis of SMs of interest using an NGS approach. We will also review the different strategies that can be employed to pinpoint the targeted gene clusters rapidly by giving several examples stemming from our work.

The Fusarium graminearum genome reveals more secondary metabolite gene clusters and hints of horizontal gene transfer.

Fungal secondary metabolite biosynthesis genes are of major interest due to the pharmacological properties of their products (like mycotoxins and antibiotics). The genome of the plant pathogenic fungus Fusarium graminearum codes for a large number of candidate enzymes involved in secondary metabolite biosynthesis. However, the chemical nature of most enzymatic products of proteins encoded by putative secondary metabolism biosynthetic genes is largely unknown. Based on our analysis we present 67 gene clusters with significant enrichment of predicted secondary metabolism related enzymatic functions. 20 gene clusters with unknown metabolites exhibit strong gene expression correlation in planta and presumably play a role in virulence. Furthermore, the identification of conserved and over-represented putative transcription factor binding sites serves as additional evidence for cluster co-regulation. Orthologous cluster search provided insight into the evolution of secondary metabolism clusters. Some clusters are characteristic for the Fusarium phylum while others show evidence of horizontal gene transfer as orthologs can be found in representatives of the Botrytis or Cochliobolus lineage. The presented candidate clusters provide valuable targets for experimental examination.

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.

Fungal secondary metabolite biosynthesis – a chemical defence strategy against antagonistic animals?

Abstract Analogous to their hypothesized benefits in plants, many fungal secondary metabolites may serve as a chemical shield that fends off fungal feeders or competing saprophagous animals. We review different approaches providing increasing evidence that some secondary metabolites may mediate resistance to antagonistic animals, reducing their negative effects on fungal fitness. Because secondary metabolism is under tight regulatory control, that allows adjustment of secondary metabolite formation to diverse ecological challenges, we argue that natural selection has favoured at least some fungal secondary metabolites and the underlying regulatory machinery through antagonistic animals. Yet, whether animals indeed operate as selective agents contributing to the evolution of secondary metabolites as part of a fungal defence strategy remains elusive. We suggest combining eco-evolutionary concepts and methods with genomic and transgenic tools to close this knowledge gap. We predict an increase in fungal species discovered to be amenable to this functional ecological genomic approach.

Origin and distribution of epipolythiodioxopiperazine (ETP) gene clusters in filamentous ascomycetes

BackgroundGenes responsible for biosynthesis of fungal secondary metabolites are usually tightly clustered in the genome and co-regulated with metabolite production. Epipolythiodioxopiperazines (ETPs) are a class of secondary metabolite toxins produced by disparate ascomycete fungi and implicated in several animal and plant diseases. Gene clusters responsible for their production have previously been defined in only two fungi. Fungal genome sequence data have been surveyed for the presence of putative ETP clusters and cluster data have been generated from several fungal taxa where genome sequences are not available. Phylogenetic analysis of cluster genes has been used to investigate the assembly and heredity of these gene clusters.ResultsPutative ETP gene clusters are present in 14 ascomycete taxa, but absent in numerous other ascomycetes examined. These clusters are discontinuously distributed in ascomycete lineages. Gene content is not absolutely fixed, however, common genes are identified and phylogenies of six of these are separately inferred. In each phylogeny almost all cluster genes form monophyletic clades with non-cluster fungal paralogues being the nearest outgroups. This relatedness of cluster genes suggests that a progenitor ETP gene cluster assembled within an ancestral taxon. Within each of the cluster clades, the cluster genes group together in consistent subclades, however, these relationships do not always reflect the phylogeny of ascomycetes. Micro-synteny of several of the genes within the clusters provides further support for these subclades.ConclusionETP gene clusters appear to have a single origin and have been inherited relatively intact rather than assembling independently in the different ascomycete lineages. This progenitor cluster has given rise to a small number of distinct phylogenetic classes of clusters that are represented in a discontinuous pattern throughout ascomycetes. The disjunct heredity of these clusters is discussed with consideration to multiple instances of independent cluster loss and lateral transfer of gene clusters between lineages.