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

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.