Carbonyl reductase identification and development of whole-cell biotransformation for highly efficient synthesis of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol.
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.
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China.
Kangling
K
Chen
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China.
Kefei
K
Li
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China.
Jian
J
Deng
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China.
Baoqi
B
Zhang
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China. jplin@ecust.edu.cn.
Jinping
J
Lin
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China.
Dongzhi
D
Wei
D000449:Aldehyde Reductase / Q000235:genetics / Q000378:metabolism*; D000074408:Aldo-Keto Reductases; D001711:Biotransformation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D042843:Glucose 1-Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010626:Phenylethyl Alcohol / Q000031:analogs & derivatives / Q000378:metabolism
C000616616:1-(3,5-bis(trifluoromethyl)phenyl)ethanol; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; D042843:Glucose 1-Dehydrogenase; D010626:Phenylethyl Alcohol
(R)-[3,5-bis(trifluoromethyl)phenyl] ethanol; 3,5-bis(trifluoromethyl) acetophenone; Carbonyl reductase; Fusion-protein expression; Glucose dehydrogenase