| Summary: | <i>Escherichia coli</i> has been engineered for L-malate production via aerobic cultivation. However, the maximum yield obtained through this mode is inferior to that of anaerobic fermentation due to massive amounts of CO<sub>2</sub> emissions. Here, we aim to address this issue by reducing CO<sub>2</sub> emissions of recombinant <i>E. coli</i> during aerobic L-malate production. Our findings indicated that NADH oxidation and ATP-synthesis-related genes were down-regulated with 2 g/L of YE during aerobic cultivations of <i>E. coli</i> E23, as compared to 5 g/L of YE. Then, E23 was engineered via the knockout of <i>nuoA</i> and the introduction of the nonoxidative glycolysis (NOG) pathway, resulting in a reduction of NAD<sup>+</sup> and ATP supplies. The results demonstrate that E23 (<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mi>Δ</mi></semantics></math></inline-formula><i>nuoA</i>, NOG) exhibited decreased CO<sub>2</sub> emissions, and it produced 21.3 g/L of L-malate from glucose aerobically with the improved yield of 0.43 g/g. This study suggests that a restricted NAD<sup>+</sup> and ATP supply can prompt <i>E. coli</i> to engage in incomplete oxidization of glucose, leading to the accumulation of metabolites instead of utilizing them in cellular respiration.
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