Asymmetric Bioreduction of Ethyl 4-Chloroacetoacetate into Ethyl 4-Chloro-3-hydroxybutyrate by Recombinant Escherichia coli CgCR in Ethyl Acetate-Betaine:Lactic Acid-Water

Abstract

Objective: Optically active (R)-ethyl 4-chloro-3-hydroxybutyrate ((R)-CHBE) is a useful chiral building block for the synthesis of pharmaceuticals. Recently, there has been great interest in the synthesis of (R)-CHBE via the highly stereoselective bioreduction of ethyl 4-chloro-3-oxobutanoate (COBE) under mild conditions. Methods: A highly efficient bioreduction process for transforming COBE into (R)-CHBE was developed in a biocompatible organic solvent–deep eutectic solvent–water reaction medium. Results: Recombinant Escherichia coli containing carbonyl reductase (CgCR) and glucose dehydrogenase (GDH) was successfully constructed and characterized. In addition, the feasibility of the asymmetric bioreduction of COBE to (R)-CHBE was verified in an organic solvent–deep eutectic solvent–water (ethyl acetate-betaine/lactic acid-water) system. At pH 7.0 and 30 °C, the kinetic constants Km and kcat of COBE were 20.9 mM and 56.1 s−1, respectively. A high (R)-CHBE yield (≥90%) was achieved by catalyzing COBE (1000 mM) in 12 h with E. coli CgCR cells in the presence of Ni2+ (7 mM) and glucose (3.5 mM glucose/mM COBE) in an ethyl acetate-betaine/lactic acid-H2O (50/7/43, v/v/v) system. The effects of organic solvents and DESs on whole-cell permeability were analyzed. Conclusions: An efficient bioreduction system was constructed for biologically transforming COBE to (R)-CHBE via whole-cell biocatalysis, and the established bioprocess has potential application in future.