Discovery of a fungal Tc toxin complex and functional mycoserpin through a novel two-by-two comparative genomics approach.

Introduction: Fungi have been a rich source of pharmaceuticals such as antibiotics, immunosuppressants, and cholesterol-lowering drugs; however, their therapeutic potential remains largely untapped due to difficulties in culturing and elucidating the genetic basis of beneficial traits. Fungi contain 'cryptic' genes that are expressed under certain, and often obscure, growth conditions and can produce complex compounds that are difficult to synthesize economically. Developments in genome sequencing and DNA-synthesis technologies offer new opportunities to produce such compounds using biotechnological techniques, however, accurately identifying useful and novel genes, a prerequisite for such approaches, remains challenging. Methods: We present a novel 'two-by-two' comparative genomics pipeline for comprehensive gene analysis of selected fungal groups, enabling more confident identification of unique genes across the analyzed species. The approach compares gene sets from two strains of the same species with those from two strains of different species or families within a fungal order. Self-clustering orthologs that are unique to strains from the same species provide higher confidence in identifying species-specific proteins and help reduce noise from low-quality genome assemblies and gene prediction errors. Results: We validated our method in a well-studied fungal group, correctly identifying known species-specific genes and gene clusters, and discovering a novel functional myco-serpin and a fungal Tc toxin complex in Discussion: Elucidating the genes underlying beneficial traits in fungi presents significant challenges, largely due to the unique and relatively complex aspects of their lifestyles. The two-by-two approach shows broad potential for fungal genome mining and bioprospecting, demonstrated by our discovery of the first fungal Tc toxin complex and a functional myco-serpin in