文献类型：期刊文献

英文题名：Engineered Imine Reductase for Larotrectinib Intermediate Manufacture

作者：Chen, Qi[1];Li, Bo-Bo[1];Zhang, Lilan[2];Chen, Xin-Ru[1];Zhu, Xin-Xin[1];Chen, Fei-Fei[1];Shi, Min[2];Chen, Chun-Chi[2];Yang, Yu[2];Guo, Rey-Ting[2];Liu, Weidong[3];Xu, Jian-He[1];Zheng, Gao-Wei[1]

机构：[1]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Hubei Univ, State Key Lab Biocatalysis & Enzyme Engn, Hubei Collaborat Innovat Ctr Green Transformat Bio, Sch Life Sci,Hubei Hongshan Lab,Hubei Key Lab In, Wuhan 430062, Peoples R China;[3]Chinese Acad Sci, Tianjin Inst Ind Biotechnol, Tianjin 300308, Peoples R China

年份：2022

卷号：12

期号：23

起止页码：14795

外文期刊名：ACS CATALYSIS

收录：;EI(收录号：20224813174934);WOS:【SCI-EXPANDED(收录号:WOS:000890331800001)】；

基金：The work was financially supported by the National Key Research and Development Program of China (2019YFA0905000, 2021YFC2102800, and 2021YFC2102300) , the National Natural Science Foundation of China (21878085, 31971380, 21472045, and 21871085) , and Natural Science Foundation Innovative Group Project of Hubei Province (2020CFA011) .

语种：英文

外文关键词：asymmetric reduction; biocatalysis; directed evolution; imine reductase; larotrectinib

摘要：Imine reductases (IREDs) are increasingly identified and characterized for the reduction of imines and reductive amination of ketones. However, their practical application is still limited due to the low activity and poor stability of native enzymes. Herein, we developed an engineered IRED through three rounds of evolution from wild-type Streptomyces clavuligerus. The specific activity of the engineered enzyme, ScIRED-R3-V4, was increased >100-fold, and stability was increased >270-fold. Using the more active and stable ScIRED-R3-V4, 80 g L-1 cyclic imine 2-(2,5-difluorophenyl)-pyrroline was completely reduced, producing kilogram quantities of a key chiral intermediate of larotrectinib in 82.5% yield, with >99.5% enantiomeric excess and a space-time yield of 352 g L-1 day-1. In addition, crystal structures of enzyme-substrate complexes and molecular dynamics simulations were conducted to gain insight into the molecular mechanism for improved enzyme performance. The significantly improved process demonstrated the potential of the engineered IRED in industrial applications.