Latent Functions and Applications of Cytochrome P450 Monooxygenases from Thamnidium elegans: A Novel Biocatalyst for 14α-Hydroxylation of Testosterone

[Image: see text] Cytochrome P450 monooxygenases (P450s) are ubiquitous enzymes with high availability and diversity in nature. Fungi provide a diverse and complex array of P450s, and these enzymes play essential roles in various secondary metabolic processes. Besides the physiological impacts of P450s on fungal life, their versatile functions are attractive for use in advanced applications of the biotechnology sector. Herein, we report gene identification and functional characterization of P450s from the zygomycetous fungus Thamnidium elegans (TeCYPs). We identified 48 TeCYP genes, including two putative pseudogenes, from the whole-genome sequence of T. elegans. Furthermore, we constructed a functional library of TeCYPs and heterologously expressed 46 TeCYPs in Saccharomyces cerevisiae. Recombinants of S. cerevisiae were then used as whole-cell biocatalysts for bioconversion of various compounds. Catalytic potentials of various TeCYPs were demonstrated through a functionomic survey to convert a series of compounds, including steroidal substrates. Notably, CYP5312A4 was found to be highly active against testosterone. Based on nuclear magnetic resonance analysis, enzymatic conversion of testosterone to 14α-hydroxytestosterone by CYP5312A4 was demonstrated. This is the first report to identify a novel fungal P450 that catalyzes the 14α-hydroxylation of testosterone. In addition, we explored the latent potentials of TeCYPs using various substrates. This study provides a platform to further study the potential use of TeCYPs as catalysts in pharmaceutical and agricultural industries and biotechnology.