文献类型：期刊文献

英文题名：Carbonyl reductase identification and development of whole-cell biotransformation for highly efficient synthesis of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol

作者：Chen, Kangling[1];Li, Kefei[1];Deng, Jian[1];Zhang, Baoqi[1];Lin, Jinping[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

年份：2016

卷号：15

外文期刊名：MICROBIAL CELL FACTORIES

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000387640700001)】；

基金：This work was financially supported by the National Key Basic Research Development Program of China ("973" Program, No. 2012CB721003), the Natural Science Foundation of China (No. 21276084) and Shanghai Natural Science Foundation (No. 15ZR1408600).

语种：英文

外文关键词：(R)-[3,5-bis(trifluoromethyl)phenyl] ethanol; 3,5-bis(trifluoromethyl) acetophenone; Fusion-protein expression; Carbonyl reductase; Glucose dehydrogenase

摘要：Background: (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol [(R)-3,5-BTPE] is a valuable chiral intermediate for Aprepitant (Emend) and Fosaprepitant (Ivemend). Biocatalyzed asymmetric reduction is a preferred approach to synthesize highly optically active (R)-3,5-BTPE. However, the product concentration and productivity of reported (R)-3,5-BTPE synthetic processes remain unsatisfied. Results: A NADPH-dependent carbonyl reductase from Lactobacillus kefir (LkCR) was discovered by genome mining for reduction of 3,5-bis(trifluoromethyl) acetophenone (3,5-BTAP) into (R)-3,5-BTPE with excellent enantioselectivity. In order to synthesize (R)-3,5-BTPE efficiently, LkCR was coexpressed with glucose dehydrogenase from Bacillus subtilis (BsGDH) for NADPH regeneration in Escherichia coli BL21 (DE3) cells, and the optimal recombinant strain produced 250.3 g/L (R)-3,5-BTPE with 99.9% ee but an unsatisfied productivity of 5.21 g/(L h). Then, four different linker peptides were used for the fusion expression of LkCR and BsGDH in E. coli to regulate catalytic efficiency of the enzymes and improved NADPH-recycling efficiency. Using the best strain (E. coli/pET-BsGDH-ER/K(10 nm)-LkCR), up to 297.3 g/L (R)-3,5-BTPE with enantiopurity > 99.9% ee was produced via reduction of as much as 1.2 M of substrate with a 96.7% yield and productivity of 29.7 g/(L h). Conclusions: Recombinant E. coli/pET-BsGDH-ER/K(10 nm)-LkCR was developed for the bioreduction of 3,5-BTAP to (R)-3,5-BTPE, offered the best results in terms of high product concentration and productivity, demonstrating its great potential in industrial manufacturing of (R)-3,5-BTPE.