Fungal secondary metabolism: regulation, function and drug discovery

Abstract

One of the exciting movements in microbial sciences has been a refocusing and revitalization of efforts to mine the fungal secondary metabolome. The magnitude of biosynthetic gene clusters (BGCs) in a single filamentous fungal genome combined with the historic number of sequenced genomes suggests that the secondary metabolite wealth of filamentous fungi is largely untapped. Mining algorithms and scalable expression platforms have greatly expanded access to the chemical repertoire of fungal-derived secondary metabolites. In this Review, I discuss new insights into the transcriptional and epigenetic regulation of BGCs and the ecological roles of fungal secondary metabolites in warfare, defence and development. I also explore avenues for the identification of new fungal metabolites and the challenges in harvesting fungal-derived secondary metabolites.

Fig. 1 |. The typical building blocks of secondary metabolites and a schematic overview of a biosynthetic gene cluster.

a | Most secondary metabolites can be grouped into three chemical categories: polyketides derived from acylCoAs, terpenes derived from acyl-CoAs and small peptides derived from amino acids. Hybrid molecules (polyketide– terpene, non-ribosomal peptide–polyketides and polyketide–fatty acid) are not shown. Fatty acid synthases (not shown) can occasionally contribute to the biosynthesis of secondary metabolites (for example, aflatoxin and sterigmatocystin are polyketide–fatty acid hybrids). b | Biosynthetic gene clusters (BGCs) are minimally composed of a chemically defining synthase and/or synthetase (polyketide synthase, terpene synthase and/or cyclase, non-ribosomal synthetase and isocyanide synthase) that use primary metabolites to form carbon backbones that are further modified by tailoring enzymes (for example, methyltransferases, p450 monooxygenases, hydroxylases and epimerases). Some BGCs contain cluster-specific transcription factors that typically positively regulate the other genes within the BGC; genes that encode proteins that mitigate the toxic property of the BGC secondary metabolite; and incongruous genes with hypothetical functions that are not obviously involved in the production of secondary metabolites or protection from the encoded metabolite. DMAPP, dimethylallyl diphosphate; DMAT, dimethylallyl tryptophan; GGPP, geranylgeranyl diphosphate; GPP, geranyl diphosphate. Part a adapted with permission from reF., Springer Nature Limited.

Fig. 2 |. Regulation of the sterigmatocystin biosynthetic gene cluster.

The Aspergillus nidulans sterigmatocystin biosynthetic gene cluster (BGC) is one of the most thoroughly studied BGCs at the regulatory level. The pathway-specific regulatory transcription factor, AflR, and its partner, AflS, are induced by specific proteins (for example, RsmA, a basic leucine zipper transcription factor) and are epigenetically regulated by the Velvet complex and chromatin modifiers, including the histone 3 demethylase KdmB, the histone 4 acetylase EsaA, the histone deacetylases RdpA and SirA and the histone reader SntB. Environmental factors such as light and interactions with other microorganisms or insects also affect the induction of the sterigmatocystin BGC. For example, fungus–bacteria interactions induce the cluster through the histone acetyltransferase GcnE, a member of the histone acetyltransferase SAGA–ADA (Spt–Ada–Gcn5–acetyltransferase–ADA) complex, whereas white light can repress expression of some BGC-encoded genes. A schematic of the sterigmatocystin BGC details the structure and encoded genes. Adapted with permission from reF., Springer Nature Limited.

Fig. 3 |. The ecological roles of secondary metabolites.

a | Many fungi produce polyketide-derived melanins, a natural pigment that protects spores from damaging ultraviolet (UV) radiation. b | The bacterium Ralstonia solanacearum secretes the lipopeptide ralsolamycin that induces chlamydospore formation in fungi and expression of the bikaverin gene cluster in Fusarium spp.^,. Bikaverin reduces bacterial entry and growth. Both the bikaverin biosynthetic gene cluster (BGC) and ralsolamycin response (that is, protection from bacterial ingress) have been transferred to some Botrytis species. c | For fungi to protect themselves from their own BGC-encoded antifungal secondary metabolites, they have evolved various self-protection strategies, including duplicated, resistant copies of target proteins within a BGC. The diagram of a fungal cell shows the cellular processes that are targeted by six secondary metabolites containing in-cluster resistant genes. Lovastatin interferes with ergosterol biosynthesis and thus cell membrane integrity by inhibiting 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (not shown), echinocandin targets cell wall synthesis by inhibiting β−1,3-d-glucan synthase (not shown), fellutamide is a proteasome inhibitor targeting the proteasome subunit C5 (REF.), aspterric acid interferes with protein synthesis by targeting the branched chain amino acid synthesis enzyme dihydroxy-acid dehydratase (not shown), fumagillin inhibits RNA synthesis by targeting methionine aminopeptidase (not shown) and mycophenolic acid interferes with purine synthesis by inhibiting inosine-5ʹ-monophosphate dehydrogenase (not shown). d | Secondary metabolites can affect developmental processes in fungi. Deletion of a polyketide synthase in the fungus Sordaria macrospora inhibits perithecial formation, whereas its overexpression results in malformed fruiting bodies that lack the usual perithecial neck.

Fig. 4 |. Integration of genome mining with fungal biology yields valuable secondary metabolites.

Biosynthetic gene clusters (BGCs) can be expressed in either heterologous hosts (typically yeast and Aspergillus spp.) or endogenous filamentous hosts (middle circle). Key input features to select BGCs of interest start with bioinformatic mining of sequenced genomes to eliminate replication and identify uniquegenes. Expression cassettes can be used for gene overexpression, gene deletion, yeast recombineering and fungal artificial chromosome (FAC) construction. Inducers that can activate cryptic BGCs include abiotic stress, epigenetic chemicals, nutrients and co-cultures. Outputs include new drugs that may overlap with endogenous function of the fungus, including warfare and developmental signals. DHN, 1,8-dihydroxynaphthalene; KD, knockdown; OE, overexpression; TF, transcription factor.

Fig. 5 |. Chromosomal position of Aspergillus fumigatus biosynthetic gene clusters and their known or predicted products.

The predicted product nidulanin is based on a near-identical copy of the Aspergillus nidulans nidulanin A biosynthetic gene cluster (BGC) in Aspergillus fumigatus. Details of other clusters can be found in REFS^,. DHN, 1,8-dihydroxynaphthalene; ICS, isocyanide synthase; NRPS, non-ribosomal synthetase; PKS, polyketide synthase. Adapted with permission from reF., Annual Reviews.