7β-Hydroxysteroid dehydrogenase (7β-HSDH) catalyzes the reversible reaction between 7-ketolithocholic acid (7K-LCA) and ursodeoxycholic acid (UDCA). However, its much lower forward reaction activity led to the unsatisfactory UDCA production. Here, by autodocking 7K-LCA and UDCA into the structure of Hyphomicrobium sp. 7β-hydroxysteroid dehydrogenase (Hs7β-HSDH) respectively, several key amino acids in the substrate/product channel were identified for virtual mutagenesis. After three-round screening, a dominant mutant F152L/W101N was obtained, which increased forward reaction activity by 3.2-fold and decreased reverse reaction activity by 3.6-fold under optimal conditions: pH 7.5 and 30 °C. Compared to the wild-type, the mutant significantly improved the binding affinity (Km) and kcat/Km by 3.2-fold and 4.3-fold towards 7K-LCA. Moreover, glucose dehydrogenase-based cofactor regeneration system was integrated into the Hs7β-HSDH-mediated UDCA synthesis pathway. The enzyme-coupled system achieved a yield of 92.8 % with 1 mM NADH, and it maintained an average yield of 89.2 % with a theoretical space-time yield of 171 g/L/d UDCA even after four batches. This work semi-rationally designs 7β-HSDH affinity with the substrate and product, and rebalance the forward and reverse reaction activity to effectively improve UDCA production, which supplies a good strategy for the efficient preparation of target product in the reversible reaction.
Keywords: 7β-hydroxysteroid dehydrogenase; Forward reaction activity; Semi-rational engineering.
Copyright © 2024. Published by Elsevier B.V.