Cofactor-dependence alteration of 7β-hydroxysteroid dehydrogenase: Enhancing one-pot synthesis efficiency of...

NAD⁺-dependent 7α-hydroxysteroid dehydrogenase (7α-HSDH) and NADPH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH) are involved in the biosynthesis of chenodeoxycholic acid (CDCA) to ursodeoxycholic acid (UDCA). To realize the one-pot synthesis of CDCA to UDCA through NAD⁺-NADH cycling, we aimed to improve the binding ability of Hyphomicrobium sp. 7β-HSDH to NADH. The 7β-HSDH structure was modeled and some potential residues to improve NADH affinity near conserved cofactor binding regions were screened, including Ala22, Gln23, Asn24, Asp44, Leu45, and Asn46. The dominant mutant A22T/Q23E/L45A/N46E significantly enhanced the binding affinity for NADH, resulting in a 44.9-fold increase in its k_cat/K_m value. It increased enzymatic activity by 65.2-fold and catalyzed the synthesis of UDCA at a yield of 77.6 % with 5 g/L 7K-LCA and 12.5 mM NADH. Molecular dynamics simulations indicated increased interactions of mutated 7β-HSDH and the ligand NADH by their spatially reduced binding distance and reaction energy. The modified cofactor-dependence of 7β-HSDH realized efficient one-pot synthesis of CDCA to UDCA through strengthening cofactor-recycling and reducing the use of cofactor, achieving 90.1 % UDCA yield and 54.1 g/L/d spatiotemporal yield when coupled with 7α-HSDH with only 0.5 mM NAD⁺ as coenzyme. This work also supplies a universal cofactor-dependence engineering technique for homologous HSDH enzymes.