Upscale production of ethyl (S)-4-chloro-3-hydroxybutanoate by using carbonyl reductase coupled with glucose dehydrogenase in aqueous-organic solvent system.

Abstract

(S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) is an important chiral intermediate to synthesize the side chain of cholesterol-lowering drug atorvastatin. To biosynthesize the (S)-CHBE, a recombinant Escherichia coli harboring the carbonyl reductase and glucose dehydrogenase was successfully constructed. The recombinant E. coli was cultured in a 500-L fermentor; after induction and expression, the enzyme activity and cell biomass were increased to 23,661.65U/L and 13.90g DCW/L which was 3.24 and 2.60-folds compared with those in the 50L fermentor. The biocatalytic process for the synthesis of (S)-CHBE in an aqueous-organic solvent system was constructed and optimized with a substrate fed-batch strategy. The ethyl 4-chloro-3-oxobutanoate concentration reached to 1.7M, and the (S)-CHBE with yield of 97.2% and enantiomeric excess (e.e.) of 99% was obtained after 4-h reaction in a 50-L reactor. In this study, the space-time yield and space-time yield per gram of biomass (dry cell weight, DCW) were 413.17mM/h and 27.55mM/h/g DCWfor(S)-CHBE production, respectively, which were the highest values as compared to previous reports. Finally, (S)-CHBE was extracted from the reaction mixture with 82% of yield and 95% of purity. This study paved the foundation for the upscale production of (S)-CHBE by biocatalysis method.