Engineering of a hydroxysteroid dehydrogenase with simultaneous enhancement in activity and thermostability for efficient biosynthesis of ursodeoxycholic acid

Hydroxysteroid dehydrogenases have emerged as indispensable tools in the synthesis of steroids, bile acids, and other steroid derivatives for the pharmaceutical and chemical industries. In this study, a novel approach was developed to simultaneously improve the stability and activity of a hydroxysteroid dehydrogenase by combining B-factor analysis and computer-aided prediction. This semi-rational method was demonstrated to be highly effective for enzyme engineering. In addition, NAD kinase was introduced to convert NAD⁺ to NADP⁺ for effective coenzyme regeneration in the whole-cell multienzyme-catalyzed system. This strategy reduces the significant economic costs associated with externally supplemented cofactors in NADP-dependent biosynthetic pathways.