Correlative metabologenomics of 110 fungi reveals metabolite-gene cluster pairs.

Natural products research increasingly applies -omics technologies to guide molecular discovery. While the combined analysis of genomic and metabolomic datasets has proved valuable for identifying natural products and their biosynthetic gene clusters (BGCs) in bacteria, this integrated approach lacks application to fungi. Because fungi are hyper-diverse and underexplored for new chemistry and bioactivities, we created a linked genomics-metabolomics dataset for 110 Ascomycetes, and optimized both gene cluster family (GCF) networking parameters and correlation-based scoring for pairing fungal natural products with their BGCs. Using a network of 3,007 GCFs (organized from 7,020 BGCs), we examined 25 known natural products originating from 16 known BGCs and observed statistically significant associations between 21 of these compounds and their validated BGCs. Furthermore, the scalable platform identified the BGC for the pestalamides, demystifying its biogenesis, and revealed more than 200 high-scoring natural product-GCF linkages to direct future discovery.

Mining for a new class of fungal natural products: the evolution, diversity, and distribution of isocyanide synthase biosynthetic gene clusters.

The products of non-canonical isocyanide synthase (ICS) biosynthetic gene clusters (BGCs) mediate pathogenesis, microbial competition, and metal-homeostasis through metal-associated chemistry. We sought to enable research into this class of compounds by characterizing the biosynthetic potential and evolutionary history of these BGCs across the Fungal Kingdom. We amalgamated a pipeline of tools to predict BGCs based on shared promoter motifs and located 3800 ICS BGCs in 3300 genomes, making ICS BGCs the fifth largest class of specialized metabolites compared to canonical classes found by antiSMASH. ICS BGCs are not evenly distributed across fungi, with evidence of gene-family expansions in several Ascomycete families. We show that the ICS dit1/2 gene cluster family (GCF), which was prior only studied in yeast, is present in ∼30% of all Ascomycetes. The dit variety ICS exhibits greater similarity to bacterial ICS than other fungal ICS, suggesting a potential convergence of the ICS backbone domain. The evolutionary origins of the dit GCF in Ascomycota are ancient and these genes are diversifying in some lineages. Our results create a roadmap for future research into ICS BGCs. We developed a website (https://isocyanides.fungi.wisc.edu/) that facilitates the exploration and downloading of all identified fungal ICS BGCs and GCFs.

A Timeline of Biosynthetic Gene Cluster Discovery in Aspergillus fumigatus: From Characterization to Future Perspectives.

In 1999, the first biosynthetic gene cluster (BGC), synthesizing the virulence factor DHN melanin, was characterized in Aspergillus fumigatus. Since then, 19 additional BGCs have been linked to specific secondary metabolites (SMs) in this species. Here, we provide a comprehensive timeline of A. fumigatus BGC discovery and find that initial advances centered around the commonly expressed SMs where chemical structure informed rationale identification of the producing BGC (e.g., gliotoxin, fumigaclavine, fumitremorgin, pseurotin A, helvolic acid, fumiquinazoline). Further advances followed the transcriptional profiling of a ΔlaeA mutant, which aided in the identification of endocrocin, fumagillin, hexadehydroastechrome, trypacidin, and fumisoquin BGCs. These SMs and their precursors are the commonly produced metabolites in most A. fumigatus studies. Characterization of other BGC/SM pairs required additional efforts, such as induction treatments, including co-culture with bacteria (fumicycline/neosartoricin, fumigermin) or growth under copper starvation (fumivaline, fumicicolin). Finally, four BGC/SM pairs were discovered via overexpression technologies, including the use of heterologous hosts (fumicycline/neosartoricin, fumihopaside, sphingofungin, and sartorypyrone). Initial analysis of the two most studied A. fumigatus isolates, Af293 and A1160, suggested that both harbored ca. 34-36 BGCs. However, an examination of 264 available genomes of A. fumigatus shows up to 20 additional BGCs, with some strains showing considerable variations in BGC number and composition. These new BGCs present a new frontier in the future of secondary metabolism characterization in this important species.

Mining for a New Class of Fungal Natural Products: The Evolution, Diversity, and Distribution of Isocyanide Synthase Biosynthetic Gene Clusters.

The products of non-canonical isocyanide synthase (ICS) biosynthetic gene clusters (BGCs) have notable bioactivities that mediate pathogenesis, microbial competition, and metal-homeostasis through metal-associated chemistry. We sought to enable research into this class of compounds by characterizing the biosynthetic potential and evolutionary history of these BGCs across the Fungal Kingdom. We developed the first genome-mining pipeline to identify ICS BGCs, locating 3,800 ICS BGCs in 3,300 genomes. Genes in these clusters share promoter motifs and are maintained in contiguous groupings by natural selection. ICS BGCs are not evenly distributed across fungi, with evidence of gene-family expansions in several Ascomycete families. We show that the ICS dit1 / 2 gene cluster family (GCF), which was thought to only exist in yeast, is present in ∻30% of all Ascomycetes, including many filamentous fungi. The evolutionary history of the dit GCF is marked by deep divergences and phylogenetic incompatibilities that raise questions about convergent evolution and suggest selection or horizontal gene transfers have shaped the evolution of this cluster in some yeast and dimorphic fungi. Our results create a roadmap for future research into ICS BGCs. We developed a website ( www.isocyanides.fungi.wisc.edu ) that facilitates the exploration, filtering, and downloading of all identified fungal ICS BGCs and GCFs.

Copper starvation induces antimicrobial isocyanide integrated into two distinct biosynthetic pathways in fungi.

The genomes of many filamentous fungi, such as Aspergillus spp., include diverse biosynthetic gene clusters of unknown function. We previously showed that low copper levels upregulate a gene cluster that includes crmA, encoding a putative isocyanide synthase. Here we show, using untargeted comparative metabolomics, that CrmA generates a valine-derived isocyanide that contributes to two distinct biosynthetic pathways under copper-limiting conditions. Reaction of the isocyanide with an ergot alkaloid precursor results in carbon-carbon bond formation analogous to Strecker amino-acid synthesis, producing a group of alkaloids we term fumivalines. In addition, valine isocyanide contributes to biosynthesis of a family of acylated sugar alcohols, the fumicicolins, which are related to brassicicolin A, a known isocyanide from Alternaria brassicicola. CrmA homologs are found in a wide range of pathogenic and non-pathogenic fungi, some of which produce fumicicolin and fumivaline. Extracts from A. fumigatus wild type (but not crmA-deleted strains), grown under copper starvation, inhibit growth of diverse bacteria and fungi, and synthetic valine isocyanide shows antibacterial activity. CrmA thus contributes to two biosynthetic pathways downstream of trace-metal sensing.

Comprehensive Guide to Extracting and Expressing Fungal Secondary Metabolites with Aspergillus fumigatus as a Case Study.

Fungal secondary metabolites (SMs) have captured the interest of natural products researchers in academia and industry for decades. In recent years, the high rediscovery rate of previously characterized metabolites is making it increasingly difficult to uncover novel compounds. Additionally, the vast majority of fungal SMs reside in genetically intractable fungi or are silent under normal laboratory conditions in genetically tractable fungi. The fungal natural products community has broadly overcome these barriers by altering the physical growth conditions of the fungus and heterologous/homologous expression of biosynthetic gene cluster regulators or proteins. The protocols described here summarize vital methodologies needed when researching SM production in fungi. We also summarize the growth conditions, genetic backgrounds, and extraction protocols for every published SM in Aspergillus fumigatus, enabling readers to easily replicate the production of previously characterized SMs. Readers will also be equipped with the tools for developing their own strategy for expressing and extracting SMs from their given fungus or a suitable heterologous model system. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Making glycerol stocks from spore suspensions Alternate Protocol 1: Creating glycerol stocks from non-sporulating filamentous fungi Basic Protocol 2: Activating spore-suspension glycerol stocks Basic Protocol 3: Extracting secondary metabolites from Aspergillus spp grown on solid medium Alternate Protocol 2: Extracting secondary metabolites from Aspergillus spp using ethyl acetate Alternate Protocol 3: High-volume metabolite extraction using ethyl acetate Alternate Protocol 4: Extracting secondary metabolites from Aspergillus spp in liquid medium Support Protocol: Creating an overlay culture Basic Protocol 4: Extracting DNA from filamentous fungi Basic Protocol 5: Creating a DNA construct with double-joint PCR Alternate Protocol 5: Creating a DNA construct with yeast recombineering Basic Protocol 6: Transformation of Aspergillus spp Basic Protocol 7: Co-culturing fungi and bacteria for extraction of secondary metabolites.

Secreted Secondary Metabolites Reduce Bacterial Wilt Severity of Tomato in Bacterial-Fungal Co-Infections.

In order to gain a comprehensive understanding of plant disease in natural and agricultural ecosystems, it is essential to examine plant disease in multi-pathogen-host systems. Ralstonia solanacearum and Fusarium oxysporum f. sp. lycopersici are vascular wilt pathogens that can result in heavy yield losses in susceptible hosts such as tomato. Although both pathogens occupy the xylem, the costs of mixed infections on wilt disease are unknown. Here, we characterize the consequences of co-infection with R. solanacearum and F. oxysporum using tomato as the model host. Our results demonstrate that bacterial wilt severity is reduced in co-infections, that bikaverin synthesis by Fusarium contributes to bacterial wilt reduction, and that the arrival time of each microbe at the infection court is important in driving the severity of wilt disease. Further, analysis of the co-infection root secretome identified previously uncharacterized secreted metabolites that reduce R. solanacearum growth in vitro and provide protection to tomato seedlings against bacterial wilt disease. Taken together, these results highlight the need to understand the consequences of mixed infections in plant disease.