Improved catalytic efficiency of P450 OleP for converting lithocholic acid into murideoxycholic and ursodeoxycholic acids through semi-rational and rational design

Cytochrome P450 enzymes are promising for synthesizing therapeutically beneficial bile acids murideoxycholic acid (MDCA) and ursodeoxycholic acid (UDCA) from lithocholic acid (LCA). However, the catalytic efficiency of P450 enzymes remains inadequate for large-scale production. Here, we address this limitation by engineering P450 OleP, achieving high activity for the efficient synthesis of MDCA and UDCA. Firstly, an improved high-throughput detection method for UDCA was developed to accelerate the screening process for mutants with improved UDCA synthesis capabilities. Next, the mutant S240A was generated through semi-rational design guided by the crystal structure of the OleP-LCA complex, resulting in an improvement in the MDCA conversion rate from 32.5% to 98.3% using 0.5 g L −1 of LCA as the substrate. Furthermore, the crystal structure of the S240A mutant was resolved and analyzed. Subsequently, the mutant F84Q/S240A/V291G/E89 N was developed through rational design, improving the UDCA conversion rate from 6.5% to 12.1% with 0.5 g L −1 of LCA as the substrate. Finally, molecular dynamics simulations were employed to elucidate the mechanisms underlying the enhanced catalytic activity of the S240A and F84Q/S240A/V291G/E89 N mutants. This study provides optimized P450 enzyme mutants for the synthesis of MDCA and UDCA and offers significant insights into the catalytic mechanisms underlying the production of hydroxylated, LCA-derived high-value-added compounds.