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.

Artificial amplification of gluconeogenic phosphoenolpyruvate carboxykinase (PCK) under glycolytic conditions enables Escherichia coli to maintain a greater intracellular ATP concentration during its growth phase. To demonstrate the biotechnological benefit of E. coli harboring a high intracellular ATP concentration, we compared the recombinant protein synthesis of a soluble protein (enhanced green fluorescence protein, GFP) with that of a secretory protein (alkaline protease, AP), under control of the T7 promoter in E. coli BL21(DE3) overexpressing PCK. According to the batch fermentations, the strain overexpressing PCK produced more GFP and AP with a lower increase in biomass than the control strain. In a chemostat culture (D = 0.7 h(-1)), the GFP production in the PCK overexpressing strain was 99.0 ± 4.31 mg/g cell, with a biomass of 0.22 g/L, while that of the control strain was 53.5 ± 3.07 mg/g cell, with a biomass of 0.35 g/L. These results indicate that the PCK overexpressing E. coli strain harboring high intracellular levels of ATP can be useful as a protein-synthesizing host. The potential uses of the strain and associated rationale are discussed.

Overexpression of gluconeogenic phosphoenolpyruvate carboxykinase (PCK) under glycolytic conditions enables Escherichia coli to maintain a greater intracellular ATP concentration and, consequently, to up-regulate genes for amino acid and nucleotide biosynthesis. To investigate the effect of a high intracellular ATP concentration on heterologous protein synthesis, we studied the expression of a foreign gene product, enhanced green fluorescence protein (eGFP), under control of the T7 promoter in E. coli BL21(DE3) strain overexpressing PCK. This strain was able to maintain twice as much intracellular ATP and to express two times more foreign protein than the control strain. These results indicate that a high energy-charged cell can be beneficial as a protein-synthesizing cell factory. The potential uses of such a cell factory are discussed.

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