文献类型：期刊文献

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

作者：Ye, Wenjie[1];Xie, Jingwen[2];Gao, Weijie[1];Meng, Yifang[1];Liu, Qinghai[1];Wang, Hualei[1];Wei, Dongzhi[1]

机构：[1]East China Univ Sci & Technol, New World Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Shanghai ZJ Biotech Co Ltd, Shanghai 201114, Peoples R China

年份：2025

卷号：73

期号：36

起止页码：22554

外文期刊名：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY

收录：;EI(收录号：20253719136518);WOS:【SCI-EXPANDED(收录号:WOS:001560615100001)】；

基金：This study was supported by the National Key Research and Development Program of China (Grant Number: 2021YFC2102100)

语种：英文

外文关键词：alcohol dehydrogenase; enzyme engineering; virtual saturation mutagenesis; combinational mutagenesis; (S)-2-chloro-1-(2,4-dichlorophenyl)ethanol

摘要：(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, LkADH(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 LkADH(M1) (LkADH(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, LkADH(M3) (LkADH(M0)-T94 V/E145 K/L147M/M206I), with a 29.1-fold increase in catalytic efficiency compared to LkADH(M0). LkADH(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 gL-1d(-1)) ever reported. Molecular dynamics simulations revealed that the enhanced activity was related to the stabilization of the substrate in LkADH(M3).