Engineering P450BM3 for highly regio- and stereoselective C15α-hydroxylation of D-ethylgonendione

Steroids are important pharmaceutically active compounds, and cytochrome P450 monooxygenases (CYPs) serve as promising biocatalysts for steroidal drug synthesis. However, their application is often limited by low catalytic efficiency and incomplete understanding of regioselectivity mechanisms, despite their remarkable oxygen activation capabilities. In this study, we engineered P450BM3 to enable high-efficiency C15α-hydroxylation of d -ethylgonendione, producing a key intermediate in the synthesis of gestodene, a widely used progestogen hormonal contraceptive. Initial screening of a laboratory P450 library identified variant HG12 (P450BM3/F87G/A328G/A330W), which exhibited 54.1 % regioselectivity and 45.8 % substrate conversion. Subsequent semi-rational design targeting residues lining in the active pocket and substrate channel, variant HG12M8 (R47W/F87G/M185L/A328G/A330W/T438G) was obtained, achieving 89.5 % regioselectivity and 90.1 % conversion. Molecular dynamics simulations revealed that F87G and M185L mutations alleviate steric hindrance and stabilize substrate binding, while R47W widens the substrate access channel. This work presents a dual-purpose protein engineering strategy to overcome efficiency and selectivity challenges in P450s while providing a robust biocatalyst for sustainable steroidal pharmaceutical production.