Enhancing Alcohol Dehydrogenase Activity for the Efficient Synthesis of (S)-2-Chloro-1-(2,4-dichlorophenyl)ethanol Using Computer-Aided Combinational Mutagenesis

( S )-2-chloro-1-(2,4-dichlorophenyl)ethanol (( S )-CPEO) is an important chiral precursor of the antifungal drug luriconazole. In this study, a mutant alcohol dehydrogenase, Lk ADH M0 from Lactobacillus kefir, was redesigned for the efficient synthesis of ( S )-CPEO by using virtual saturation mutagenesis to assess beneficial site combinations. Five poorly conserved sites in the active pocket of the enzyme were identified via multiple sequence alignment with enzymes exhibiting high activity toward acetophenone derivatives. To stimulate potential synergies while minimizing the screening effort, the five hotspots were randomly paired to generate ten libraries for virtual saturation mutagenesis, with four demonstrating promising libraries that were experimentally constructed and screened. Subsequently, an enhanced double mutant Lk ADH M1 ( Lk ADH M0 -E145 K/M206I) was obtained, which showed a 5.4-fold improvement in activity and was used as a new template to iterate the remaining three sites, leading to the creation of three additional combinatorial libraries. This resulted in the final mutant, Lk ADH M3 ( Lk ADH M0 -T94 V/E145 K/L147M/M206I), with a 29.1-fold increase in catalytic efficiency compared to Lk ADH M0 . Lk ADH M3 efficiently reduced up to 600 g/L of substrate 2-chloro-1-(2,4-dichlorophenyl)ethanone with >99.5% ee, achieving the highest space-time yields (654 g·L –1 ·d –1 ) ever reported. Molecular dynamics simulations revealed that the enhanced activity was related to the stabilization of the substrate in Lk ADH M3 .