Gene hunting and semi-rational design of carbonyl reductase from Kosakonia radicincitans for highly efficient synthesis of the key chiral intermediate of Telotristat ethyl

Abstract

Carbonyl reductase exhibits significant potential in the asymmetric production of chiral alcohols. (αR)-4-Chloro-2-(3-methyl-1H-pyrazol-1-yl)-α-(trifluoromethyl)benzenemethanol ((R)-CMPPFO) is a critical precursor for the synthesis of Telotristat ethyl, an oral drug for the treatment of diarrhea in carcinoid syndrome. Herein, a novel carbonyl reductase KrSDR5 from Kosakonia radicincitans was obtained using gene hunting strategy, capable of asymmetrically reducing the precursor ketone 1-[4‑chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanone (CMPPFA) to (R)-CMPPFO with strict R-stereoselectivity (>99.9 % ee). Further, semi-rational design was adopted to acquire a positive mutant KrSDR5T91V/V141M/I159V, with assistance from a comparative analysis of enzyme-substrate binding mode in molecular dynamics (MD) simulations. This variant displayed a 12.4-fold increase in kcat/Km towards CMPPFA compared to the wild-type (WT) KrSDR5. Insights were gained on the high enantioselectivity and the enhancement of enzyme catalytic activity of the mutant through MD simulations. Using the whole-cells of KrSDR5T91V/V141M/I159V as biocatalyst, the asymmetric synthesis of (R)-CMPPFO was achieved within 20 h at 500 mM CMPPFA concentration, resulting in a 95.0 % yield with >99.9 % ee, and a highest space-time yield (STY) of 165.7 g·L-1·d-1 compared with previous reports. This study provides a robust biocatalyst for highly efficient production of the key precursor (R)-CMPPFO for Telotristat ethyl, highlighting its potential in the biosynthesis of pharmaceutical intermediates.