Engineering Alcohol Dehydrogenase for Efficient Catalytic Synthesis of Ethyl (<i>R</i>)-4-Chloro-3-hydroxybutyrate

Ethyl (<i>R</i>)-4-chloro-3-hydroxybutyrate [(<i>R</i>)-CHBE] is an intermediate with high value in medicine and pesticide applications. Alcohol dehydrogenase serves as an excellent biocatalyst during the synthesis of (<i>R</i>)-CHBE. However, the lack of effective engineering methods limits its wider application. In this study, the sequence-modeling-docking-principle (SMDP) method was used to screen enzymes with catalytic activity. Three protein modification strategies were established for the active center, substrate channel, and distal hotspot to enhance the catalytic efficiency of alcohol dehydrogenase LCRIII. Substrate batch replenishment was used to alleviate substrate inhibition. Subsequently, optimal mutant M3 (W151F-S167A-F215Y) was successfully obtained with a specific enzyme activity of 23.00 U/mg and <i>k</i>_cat/<i>K</i>_m of 11.22 (mM^-1·min^-1), which were 4.55- and 3.98-fold higher than those of the wild type, respectively. (<i>R</i>)-CHBE was prepared using M3 and GDH at 298.21 g/L (>99% <i>e.e.</i>). This study provides a promising approach for the protein engineering modification of alcohol dehydrogenase and industrial-scale production of (<i>R</i>)-CHBE.