Engineering 7β-HSDH_Osp for enhanced catalytic efficiency: Advancing sustainable biosynthesis of ursodeoxycholic acid

Ursodeoxycholic acid (UDCA) as a valuable pharmaceutical agent and is predominantly synthesized via biocatalytic conversion, with 7β-hydroxysteroid dehydrogenase (7β-HSDH) playing a central role in this enzymatic process. To enhance its catalytic efficiency, we performed directed-site saturation mutagenesis on 25 residues within 5 Å of the active site in the 7β-HSDH_Osp mutant T189V/V207M. High-throughput screening identified five improved mutants (V91P, M144H, G146A, A158S, V183I) with specific activities up to 31.57 U·mg^-1, surpassing the wild-type (9.6 U·mg^-1) and T189V/V207M (26.9 U·mg^-1). Iterative saturation mutagenesis on the best variant (T189V/V207M/G146A) yielded a quintuple mutant (T189V/V207M/G146A/V183S/M144L/V91I/A158G) with a 65.18 U·mg^-1 specific activity-a 5.79-fold increase over the wild-type. Kinetic analysis revealed progressive improvements in substrate affinity (K_m) and catalytic efficiency (k_cat/K_m), reaching 1.66-fold that of T189V/V207M. Molecular docking and dynamics simulations demonstrated enhanced substrate binding (ΔG = -13.18 kcal·mol^-1) and increased structural flexibility, facilitating catalysis. The mutant exhibited optimal activity at pH 7.0, with broad pH stability (6.5-10.0). The conversion of 50 g·L^-1 CDCA to UDCA via the "two-step" reaction in "one-pot" achieved a yield of 94.3 %, representing the highest conversion rate reported to date. This study demonstrated the significant potential of the engineered 7β-HSDH_Osp variant as a robust biocatalyst for sustainable industrial-scale UDCA production, offering enhanced process efficiency and improved environmental compatibility.