A short‐chain carbonyl reductase mutant is an efficient catalyst in the production of (R)‐1,3‐butanediol

R‐1,3‐butanediol (R‐1,3‐BDO) is an important chiral intermediate of penem and carbapenem synthesis. Among the different synthesis methods to obtain pure enantiomer R‐1,3‐BDO, oxidation–reduction cascades catalysed by enzymes are promising strategies for its production. Dehydrogenases have been used for the reduction step, but the enantio‐selectivity is not high enough for further organic synthesis efforts. Here, a short‐chain carbonyl reductase (LnRCR) was evaluated for the reduction step and developed via protein engineering. After docking result analysis with the substrate 4‐hydroxy‐2‐butanone (4H2B), residues were selected for virtual mutagenesis, their substrate‐binding energies were compared, and four sites were selected for saturation mutagenesis. High‐throughput screening helped identify a Ser154Lys mutant which increased the catalytic efficiency by 115% compared to the parent enzyme. Computer‐aided simulations indicated that after single residue replacement, movements in two flexible areas (VTDPAF and SVGFANK) facilitated the volumetric compression of the 4H2B‐binding pocket. The number of hydrogen bonds between the stabilized 4H2B‐binding pocket of the mutant enzyme and substrate was higher (from four to six) than the wild‐type enzyme, while the substrate‐binding energy was decreased (from −17.0 kJ/mol to −29.1 kJ/mol). Consequently, the catalytic efficiency increased by approximately 115% and enantio‐selectivity increased from 95% to 99%. Our findings indicate that compact and stable substrate‐binding pockets are critical for enzyme catalysis. Lastly, the utilization of a microbe expressing the Ser154Lys mutant enzyme was proven to be a robust process to conduct the oxidation–reduction cascade at larger scales.