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

Kangling Chen,Dongzhi Wei,Baoqi Zhang,Jian Deng,Jinping Lin,Kefei Li

doi:10.1186/s12934-016-0585-5

Kangling Chen, Dongzhi Wei + Show 4 more

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https://doi.org/10.1186/s12934-016-0585-5

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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.ResultsA 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).ConclusionsRecombinant 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0585-5) contains supplementary material, which is available to authorized users.

Highlights

Active alcohols are highly valuable chiral synthon pharmaceuticals and fine chemicals [1, 2]. (R)[3,5-bis(trifluoromethyl) phenyl] ethanol [(R)-3,5-BTPE] is a key intermediate for the synthesis of neurokinin-1 receptor antagonists, such as Aprepitant (Emend) and Fosaprepitant (Ivemend), which are widely used in the Asymmetric reduction of the prochiral ketone 3,5-bis(trifluoromethyl) acetophenone (3,5-BTAP) is an efficient and powerful way to produce highly optically active (R)-3,5-BTPE
The large-scale biocatalytic production of (R)-3,5BTPE remains difficult because asymmetric reduction of 3,5-BTAP to (R)-3,5-BTPE requires a biocatalyst with excellent anti-Prelog stereoselectivity, which is relatively rare in nature
Screening of oxidoreductases A genome mining approach was used to search for carbonyl reductases that might be able to asymmetrically reduce 3,5-BTAP to the corresponding alcohol

Summary

Introduction

Active alcohols are highly valuable chiral synthon pharmaceuticals and fine chemicals [1, 2]. (R)[3,5-bis(trifluoromethyl) phenyl] ethanol [(R)-3,5-BTPE] is a key intermediate for the synthesis of neurokinin-1 receptor antagonists, such as Aprepitant (Emend) and Fosaprepitant (Ivemend), which are widely used in the Asymmetric reduction of the prochiral ketone 3,5-bis(trifluoromethyl) acetophenone (3,5-BTAP) is an efficient and powerful way to produce highly optically active (R)-3,5-BTPE. Only five microbial strains, including Lactobacillus kefir [10], Penicillium expansum [11], Leifsonia xyli [12], Microbacterium oxydans [13], and Trichoderma asperellum ZJPH0801 [14], were reported for their abilities to reduce 3,5-BTAP to (R)-3,5-BTPE enantioselectively with >99% ee These microbial cell-mediated bioreduction methods were successfully performed with conversion rates of 31–95% at relatively low substrate concentrations (less than 200 mM 3,5-BTAP), which restricted their industrial applications. Wang et al reported that the whole cells of Escherichia coli expressing a mutant form of carbonyl reductase from L. xyli HS0904 (LXCAR-S154Y) reduced 1 M (256 g/L) 3,5-BTAP in the presence of 20% (v/v) isopropanol as co-substrate [21] This reaction produced the desired (R)-3,5-BTPE with >99% ee but with an unsatisfactory 82.5% product yield. There is great interest in searching for new carbonyl reductases with high enantioselectivity at high substrate concentrations and improving their application performance

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