Development of an engineered ketoreductase with improved activity, stereoselectivity and relieved substrate inhibition for enantioselective synthesis of a key (R)-α-lipoic acid precursor

Abstract

Methyl (R)-8-chloro-6-hydroxyoctanoate ((R)-MCHO, (R)-2a) is a key precursor that can be utilized in the production of (R)-α-lipoic acid ((R)-LA) of pharmaceutical and dietary significance. Owing to its high atom economy, maximum theoretical yield of 100%, and environmentally friendliness, ketoreductase (KRED)-catalyzed stereoselective reduction of methyl 8-chloro-6-oxooctanoate (MCOO, 1a), an ε-keto ester, represents an efficient yet challenging approach to the synthesis of (R)-2a. We report herein the development of a versatile KRED, referred to as SsCRL211H/V127A/L135I, through enzyme screening followed by structure-guided protein engineering, which was assisted by comparative analysis of enzyme-/substrate- binding modes in prereaction-state and free-state molecular dynamics (MD) simulations. This enzyme variant displayed 2.8-fold increased kcat (16.77 s−1) towards 1a relative to the wild-type SsCR, improved stereoselectivity (98.0% ee), and no substrate inhibition. Insights were gained on the origin of the enhancement of enzyme's catalytic activity and stereoselectivity, by conducting complete deconvolution experiments and MD simulations. In the presence of 60 g∙L−1 of wet E. coli cells coexpressing SsCRL211H/V127A/L135I and glucose dehydrogenase (GDH), and 0.05 mM NADP+, complete reduction of 260 g∙L−1 of 1a was achieved furnishing (R)-2a with 98.0% ee with a space-time yield of 391 g∙L−1∙d−1, thereby showcasing the promising potential of this ketoreductase in the asymmetric synthesis of (R)-2a.