Semi-rational engineering of carbonyl reductase YueD for efficient biosynthesis of halogenated alcohols with in situ cofactor regeneration

Abstract Optically active 1-phenylethanol and its derivatives are versatile chiral precursors for many pharmaceuticals. The increasing market demand of enantiopure alcohols calls for exploration of more robust biocatalysts capable of delivering high conversion rates at high substrate concentrations. The carbonyl reductase YueD from Bacillus subtilis was engineered for improved reduction of halogenated acetophenones. Based on in silico docking and Alanine screening, mutant Val181Ala with 97% conversion of the model substrate 3-bromoacetophenone was obtained. Further molecular simulation analysis suggested expansion of the catalytic pocket and altered substrate orientation to provide a structural basis for the improved activity upon Val181 mutation. To eliminate the requirement of expensive exogenous NADPH and thus afford economical synthesis of enantiopure alcohol products, a cofactor regeneration system based on glucose dehydrogenase was introduced. Since the product of glucose oxidation seemed to inhibit the activity of YueD, xylose was used instead as the co-substrate, which efficiently regenerated NADPH in situ, delivering ( S ) or ( R ) alcohols with >99% ee at high conversion rates of 85–99% in the asymmetric reduction of halogenated acetophenones (500 mM). These data demonstrate the industrial potential of the YueD mutant Val181Ala in biosynthesis of valuable chiral alcohols.