From discovery to application: cytochrome P450 enzyme in filamentous fungi for biosynthesis of steroids.

Steroids represent a cornerstone of modern pharmacotherapy, with their physiological functions intrinsically linked to precise hydroxylation patterns on the steroid nucleus. Hydroxylation of inert C-H bonds on the steroid nucleus is critical for their physiological and pharmacological activities. However, conventional chemical hydroxylation often suffers from complex procedures and poor regioselectivity. In contrast, microbial cytochrome P450 enzyme (P450) mediated biocatalysis enables precise, single-step steroid hydroxylation under mild conditions, overcoming the limitations of conventional approaches. Filamentous fungi are particularly notable as prolific hosts for steroid transformation, harboring a vast and underexplored repertoire of P450s with exceptional catalytic diversity. Despite their potential, a comprehensive summary of the identification approaches and application of steroid-hydroxylating P450s from filamentous fungi remains limited. This review provides an in-depth overview of recently identified and engineered P450s from filamentous fungi, emphasizing the integration of multi-omics approaches and computational tools in their discovery. We highlight breakthroughs in protein engineering and metabolic regulation that have enhanced the regio- and stereo-selective capabilities of these enzymes. It establishes a foundation for future investigations into the roles and applications of steroid hydroxylase P450s.