To enhance succinic acid formation during xylose fermentation in Escherichia coli, overexpression of ATP-forming phosphoenolpyruvate carboxykinase (PEPCK) from Bacillus subtilis 168 in an ldhA, pilB, and ppc deletion strain resulted in a significant increase in cell mass and succinic acid production. However, BA204 displays a low yield of glucose fermentation and sequential glucose-xylose utilization under regulation by the phosphotransferase system (PTS). To improve the capability of glucose fermentation and simultaneously consume sugar mixture for succinic acid production, a pilB, ldhA, ppc, and ptsG deletion strain overexpressing ATP-forming PEPCK, named E. coli BA305, was constructed. As a result, after 120 h fed-batch fermentation of sugarcane bagasse hydrolysate, the dry cell weight and succinic acid concentration in BA305 were 4.58 g L-1 and 39.3 g L-1, respectively. (C) 2013 Elsevier Ltd. All rights reserved.