In this study, an engineered strain Escherichia coli MLB (ldhA(-) pflB(-)) was constructed for production of succinate from glycerol. The succinate yield was 0.37 mol/mol in anaerobic culture, however, the growth and glycerol consumption rates were very slow, resulting in a low succinate level. Two-stage fermentation was performed in flasks, and the succinate yield reached 0.93 mol/mol, but the succinate titer was still low. Hence, overexpression of malate dehydrogenase, malic enzyme, phosphoenolpyruvate (PEP) carboxylase and PEP carboxykinase (PCK) from E. coli, and pyruvate carboxylase from Corynebacterium glutamicum in MLB was investigated for improving succinate production. Overexpression of PCK resulted in remarkable enhancement of glycerol consumption and succinate production. In flask experiments, the succinate concentration reached 118.1 mM, and in a 1.5-L bioreactor the succinate concentration further increased to 360.2 mM. The highest succinate yield achieved 0.93 mol/mol, which was 93% of the theoretical yield, in the anaerobic stage. (C) 2016 Elsevier Ltd. All rights reserved.