Enhanced Regioselectivity and Catalytic Activity on C7Hydroxylation of Lithocholic Acid to Produce Ursodeoxycholic Acid by Monooxygenase CYP107D1 Based on Semi‐Rational Design

ABSTRACT Ursodeoxycholic acid (UDCA), a natural bile acid, is widely utilized in treating hepatobiliary disorders due to its ability to reduce bile concentration and protect liver function. While the CYP107D1 (OleP) triple‐mutant F84Q/S240A/V291G is the first identified bacterial monooxygenase capable of directly converting lithocholic acid (LCA) to UDCA via C7β‐hydroxylation, its industrial application has been hindered by low catalytic efficiency. The study implemented a semi‐rational design strategy combined with a cell‐free enzyme catalysis method, utilizing the OleP triple mutant as a template, to screen for mutants with enhanced 7β‐hydroxylation activity. The superior quadruple‐mutant G294A/N236H/F321W/V297A was engineered through iterative combinatorial mutagenesis, which catalyzed the production of about 0.68 mM UDCA from 1 mM substrate LCA. This variant exhibited a 50% increase in catalytic activity and a 40% improvement in regioselectivity compared to the template OleP triple‐mutant. Molecular docking and kinetic simulations further demonstrated that the quadruple mutant stabilized the enzyme‐substrate complex through optimized binding interactions, thereby enhancing catalytic proficiency. Our findings elucidate critical structural determinants governing C7‐hydroxylation of LCA.