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

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.

Reversible photocontrol of oxidase activity by inserting a photosensitive domain into the oxidase

“…The gene for AdhP was subcloned into pET-28a(+) via BamHI/XhoI (Thermo Fisher Scientific, Rockford, IL, USA) restriction sites to create an in-frame N-terminal 6*histidine tag. The LOV2 and adhP genes were spliced together using the splicing by overlap extension polymerase chain reaction method (SOE-PCR) (the primer information is listed in Additional file 1: Table S1) (Chen et al 2016). The plasmids were then transformed into Escherichia coli BL21 (DE3)-competent cells.…”

Identification of a newly isolated <i>Sphingomonas</i> sp. LZ1 and its application to biosynthesize chiral alcohols

“…Asymmetric biocatalytic reduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) provides a straightforward approach to produce enantiomerically pure BTPE. In recent years, some microorganisms or isolated enzymes have been reported for their ability to produce (R)-[3,5bis(trifluoromethyl)phenyl] ethanol ((R)-BTPE) from BTAP, such as Leifsonia xyli (Wang et al, 2014), Lactobacillus kefir (Chen et al, 2016), Penicillium expansum (Kurbanoglu et al, 2009), Microbacterium oxydans (Gai et al, 2013), Trichoderma asperellum ZJPH0801 (Li et al, 2013), Chryseobacterium sp. CA49 (Liu et al, 2014), Burkholderia cenocepacia (Yu et al, 2018), Leifsonia sp.…”

Identification of a newly isolated Rhodotorula mucilaginosa NQ1 and its development for the synthesis of bulky carbonyl compounds by whole‐cell bioreduction

“…2014), Lactobacillus kefir (Chen et al . 2016), Penicillium expansum (Kurbanoglu et al . 2009), Microbacterium oxydans (Gai et al .…”