Validamycin A is widely used to control Basidiomycetes, which causes sheath blight disease in rice, potatoes, vegetables, and other crops as well as dumping-off disease in vegetable seedlings, cotton, sugar beets, and other plants. In order to improve the content of validamycin A in the commercial products, valG from Streptomyces hygroscopicus was successfully cloned into Escherichia coli BL21(DE3) and was directly employed as the biocatalyst in the biotransformation from validoxylamine A to validamycin A with the existence of D-cellobiose using the free resting cells in the present study. The fermentation medium was optimized through single factor experiment and response surface method. With the optimized medium, which contained lactose 4.7 g/L, yeast extract 49.5 g/L, ammonium chloride 2.7 g/L, potassium phosphate buffer solution 110 mL/L, Ca2+ 0.0352 g/L, the biomass yield and enzyme activity reached 5.5 g/L and 1.49 U/mL, respectively, which were nearly twice higher than those with initial medium. (C) 2012 Elsevier Ltd. All rights reserved,