Carbonyl reductase identification and development of whole-cell biotransformation for highly efficient synthesis of (R)-[3, 5-bis(trifluoromethyl)phenyl] ethanol. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Chen, Kangling; Li, Kefei; Deng, Jian; Zhang, Baoqi; Lin, Jinping; Wei, Dongzhi
• Is Part Of: Microbial cell factories. Volume 15:Issue 1(2016)
• Page Start: 1
• Page End: 11
• Publication Date: 2016-12
• Subjects: (R)-[3, 5-bis(trifluoromethyl)phenyl] ethanol -- 3, 5-bis(trifluoromethyl) acetophenone -- Fusion-protein expression -- Carbonyl reductase -- Glucose dehydrogenase; Microbial biotechnology -- Periodicals; Recombinant proteins -- Synthesis -- Periodicals; 660.62
• DOI: 10.1186/s12934-016-0585-5
• Languages: English
• ISSNs: 1475-2859

Abstract

Abstract 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 fromLactobacillus kefir (Lk CR) 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, Lk CR was coexpressed with glucose dehydrogenase fromBacillus subtilis (Bs GDH) for NADPH regeneration inEscherichia 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 ofLk CR andBs GDH inE. coli to regulate catalytic efficiency of the enzymes and improved NADPH-recycling efficiency. Using the best strain (E. coli /pET-Bs GDH-ER/K(10 nm)-Lk CR), 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 RecombinantE. coli /pET-Bs GDH-ER/K(10 nm)-Lk CR 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.