Comparative Genomics Identifies Putative Non-Canonical CYP51F1-Associated Clusters in Mycorrhizal Fungi

Mycorrhizal agaricomycetes represent a critical evolutionary and ecological group of fungi that form mutualistic associations with plant roots, contributing to forest ecosystem functioning and global nutrient cycling. Despite extensive genomic studies of these fungi, cytochrome P450 monooxygenases (CYPs/P450s), key enzymes involved in primary and secondary metabolism, remain underexplored in this lineage. In this study, we performed a comprehensive genome-wide analysis of P450s across ten mycorrhizal agaricomycete species to investigate their diversity, evolution, and potential roles in secondary metabolism. A total of 1031 P450s were identified and classified into 74 families and 269 subfamilies. Notably, five P450 families (CYP5144, CYP512, CYP5340, CYP5037, and CYP63) exhibited significant expansion (blooming), suggesting their potential importance in ecological adaptation. Furthermore, 26 previously unreported P450 families were identified in mycorrhizal agaricomycetes, indicating potential roles in lineage- or symbiosis-specific functions. Comparative analysis revealed substantial differences in P450 repertoire between mycorrhizal agaricomycetes and mycorrhizal pezizomycetes. Mycorrhizal agaricomycetes contained about three times as many P450s per species on average (103 versus 33), despite comparable average numbers of P450 families (25 versus 24) and higher average numbers of subfamilies (45 versus 28). Only five P450 families (CYP51, CYP53, CYP61, CYP505, and CYP512) were shared between the two groups, and these largely represent conserved fungal P450 families rather than mycorrhiza-specific convergent families. Biosynthetic gene cluster analysis identified 25 P450s associated with secondary metabolism and two putative non-canonical CYP51F1-associated BGCs predicted to integrate lipid or sterol metabolism with cellular signaling and redox processes. Overall, this study provides systematic insights into P450 diversity, evolution, and biosynthetic potential in mycorrhizal agaricomycetes.