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 (https://isocyanides.fungi.wisc.edu/) that facilitates the exploration, filtering, and downloading of all identified fungal ICS BGCs and GCFs.

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.