(4S)-3-[(5S)-5-(4-Fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one ((S)-ET-5) is an important chiral intermediate in the synthesis of chiral side chain of ezetimibe. Recombinant Escherichia coli expressing carbonyl reductase (CBR) was successfully constructed in this study. The total E. coli biomass and the specific activity of recombinant CBR in 5 L fermenter culture were 10.9 g DCW L (1) and 14900.3 U g (1) DCW, respectively. The dual-enzyme coupled biocatalytic process in an aqueous-organic biphasic solvent system was first constructed using p-xylene as the optimal organic phase under optimized reaction conditions, and 150 g L (1) (4S)-3-[5-(4-fluorophenyl)-1,5-dioxophentyl]-4-phenyl-1, 3-oxazolidin-2-one (ET-4) was successfully converted to (S)-ET-5 with a conversion of 99.1% and diastereomeric excess of 99% after 24-h, which are the highest values reported to date for the production of (S)-ET-5. (C) 2016 Elsevier Ltd. All rights reserved.