To reduce dependence on the expensive cofactor and effectively biotransform ethyl 4-chloro-3-oxobutanoate, L-glutamine and glycine were found to enhance the content of intracellular NADH and the reductase activity. Adding the mixture of 200 mM of L-glutamine and 500 mM of glycine to the reaction media, a 1.67-fold of reductase activity was increased over the control without the addition of the two compounds. Moreover, beta-cyclodextrin (0.4 mol beta-cyclodextrin/mol ethyl 4-chloro-3-oxobutanoate) was also added into this reaction media, and the biocatalytic activity of the whole-cell biocatalyst of Escherichia coli CCZU-K14 was increased by 1.34-fold than that without beta-cyclodextrin. In this beta-cyclodextrin-water media containing L-glutamine (200 mM) plus glycine (500 mM), ethyl (S)-4-chloro-3-hydroxybutanoate (>99% ee) could be obtained from 3000 mM ethyl 4-chloro-3-oxobutanoate in the yield of 98.0% after 8 h. All the positive features demonstrate the potential applicability of the bioprocess for the large-scale production of ethyl (S)-4-chloro-3-hydroxybutanoate. (c) 2015 Elsevier Ltd. All rights reserved.