Tracing the origins of crmA megasynthase through lichen genomes

The products of isocyanide synthase (ICS) biosynthetic gene clusters (BGCs) have been implicated in microbial interactions, pathogenesis, and metal homeostasis. While several ICS BGCs have been described as mediating metal-associated ecologies, the evolutionary history of these clusters is unexplored among Lecanoromycetes, a clade comprised predominantly of symbiotic, lichen-forming fungi (LFF), which are known to thrive in both metal-contaminated and scarce environments. Analyzing nearly 4,000 fungal genomes, including 90 Lecanoromycetes, we identified a significant 3-fold enrichment of ICS-encoding genes in lichenized fungi compared with non-lichenized counterparts. This expansion includes six distinct clades enriched in LFF. Evolutionary reconstruction uncovered a widespread "split" variant of the copper-responsive (crmA) pathway, where the ICS- and NRPS-like components are encoded on separate genes, contrasting to the canonical "fused" crmA megasynthase. Metabolic characterization and genetic deletions in Fusarium graminearum confirmed that this split architecture is functionally equivalent to the fused form. Our chemical analysis suggests the first evidence of a potential leucine-derived isocyanide metabolite. Phylogenetic reconstruction indicates that the fused crmA arose from a split ancestor whose NRPS-like subdomain evolved from a canonical thioester reductase architecture likely via domain replacement. Redefinition of the crmA pathway to include the split ICS/NRPS-like variant reveals that crmA is one of the most prevalent ICSs in the fungi. We developed a website (https://isocyanides.fungi.wisc.edu/) that facilitates the exploration and downloading of all major results in our study. Our work demonstrates how exploring understudied fungal lineages can define new specialized metabolism lineages and reshape our understanding of the evolution of broadly conserved biosynthetic pathways.