A 3-hydroxyisobutyrate dehydrogenase-encoding gene mmsB has been identified as one of the key genes responsible for the enhanced organic solvent tolerance (OST) of Pseudomonas putida JUCT1. In this study, the OST-related effect of two 3-hydroxyacid dehydrogenase family genes (mmsB and zwf) was investigated in Escherichia coli JM109. It was noted that the growth of E. coli JM109 was severely hampered in 4 % decalin after zwf knockout. Additionally, its complementation resulted in significantly enhanced solvent tolerance compared with its parent strain. Furthermore, E. coli JM109 carrying mmsB showed better OST capacity than that harboring zwf. To construct E. coli strains with an inheritable OST phenotype, mmsB was integrated into the genome of E. coli JM109 by red-mediated recombination. Using E. coli JM109(DE3) (Delta endA::mmsB) as host strain, whole-cell biocatalysis was successfully carried out in an aqueous/butyl acetate biphasic system with a remarkably improved product yield.