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[1,3]; Wei, Dongzhi[1]

机构：[1] State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China; [2] Shanghai ZJ Bio-Tech Co.,Ltd., Shanghai, 201114, China; [3] State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China

年份：2024

外文期刊名：SSRN

收录：EI(收录号：20240436962)

语种：英文

外文关键词：Enzyme activity - Synthesis (chemical)

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, LkADHM0 from Lactobacillus kefir, was redesigned for the efficient synthesis of (S)-CPEO 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 LkADHM1 (LkADHM0-E145K/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, LkADHM3 (LkADHM0-T94V/E145K/L147M/M206I), with a 29.1-fold increase in catalytic efficiency compared to LkADHM0. LkADHM3 efficiently reduced up to 600 g/L of the substrate with >99.5% ee, achieving the highest space-time yields ever reported. Molecular dynamics simulations revealed that the enhanced activity was related to stabilization of the substrate in LkADHM3. This study describes an enzyme engineering strategy and provides an effective mutant for synthesizing (S)-CPEO. ? 2024, The Authors. All rights reserved.