Discovery of a novel fungal Tc toxin complex and a functional myco- serpin via a unique two-by-two comparative genomics pipeline

Background 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. Results 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. We validated our method on a well-studied group of fungi, discovering the first functional myco-serpin and an undescribed fungal Tc toxin complex. Using a gene knockout approach, we have implicated both proteins’ roles in the insect host infection process of this entomopathogenic fungal species. Conclusions Elucidating the genes underlying beneficial traits in fungi presents significant challenges, largely due to the unique and relatively complex aspects of their lifestyles. Our two-by-two approach offers broad potential for applications in fungal genome mining and bioprospecting as exemplified in this study by the discovery of the first fungal Tc toxin complex and a functional myco-serpin. We identified genes with high sequence identity to this serpin in other pathogenic fungal strains, including those known to infect humans. Furthermore, the two-by-two approach can be adapted to other organisms with genome architectures similar to fungi.