Aims: To understand the modification of C4-metabolism under anaerobic glycolysis condition by overexpressing anaplerotic enzymes, which mediating carboxylation of C3 into C4 metabolites, in Escherichia coli. Methods and Results: Anaplerotic NADP-dependent malic enzyme (MaeB), as well as the other anaplerotic enzymes, including phosphoenolpyruvate carboxylase (Ppc), phosphoenolpyruvate carboxykinase (Pck) and NAD-dependent malic enzyme (MaeA), were artificially expressed and their C4 metabolism was compared in E. coli. Increasing MaeB expression enhanced the production of C4 metabolites by 2.4 times compared to the wild-type strain in anaerobic glucose medium with bicarbonate supplementation. In MaeB expression, C4 metabolism by supplementing 10 g l(-1) of NaHCO3 was three times than that by no supplementation, which showed the greatest response to increased CO2 availability among the tested anaplerotic enzyme expressions. Conclusions: The higher C4 metabolism was achieved in E. coli expressing increased levels of the NADPH-dependent MaeB. The greatest increase in the C4 metabolite ratio compared to the other tested enzymes were also found in E. coli with enhanced MaeB expression as CO2 availability increased. Significance and Impact of the Study: The higher C4 metabolites and related biomolecule productions can be accomplished by MaeB overexpression in metabolically engineered E. coli.