Cavity engineering-driven switching of cofactor specificity in a carbonyl reductase for the synthesis of chiral aryl β-Hydroxy α-amino acid.

Carbonyl reductases (CRs) are important enzymes with a broad spectrum of industrial applications. However, the CRs reported for use in the asymmetric synthesis of chiral aryl β-hydroxy α-amino acids are exclusively specific to NADPH, posing challenges for cost-effective NADH regeneration. In this study, we focused on the carbonyl reductase (M30) from Exiguobacterium sp. F42 and successfully altered its cofactor preference to favor the more economical NADH using a computer-aided design approach. Through protein engineering, we developed an enhanced mutant M36, S10A/Y15R/E16A/K19L/A32D/R33I, which increased the cofactor specificity of M30 by over 1000-fold. This best combinatorial mutant enabled the highly efficient synthesis of the chloramphenicol intermediate (2S,3R)-5b, achieving 99 % conversion from 50 g/L of substrate 5a with excellent stereoselectivity (ee > 99.9 %, de > 99.9 %), hereby significantly enhancing its industrial application potential. Molecular dynamics (MD) simulations provided atomic-level insights into the mechanistic basis for the altered cofactor affinity and revealed the individual contribution of key mutations. This work establishes an efficient approach for engineering cofactor specificity in CRs and underscores the potential of cavity engineering in advancing biocatalytic applications.