Abstract: Lipoamide dehydrogenase encoded by lpdA gene is the subunit of pyruvate dehydrogenase complex,α-ketoglutarate dehydrogenase complex and glycine cleavage multi-enzyme systems. In the present study, the comparison of the fermentative properties between lpdA knockout E. coli and the wild type was made with different carbon sources and aeration conditions. Furthermore, the effect of lpdA gene knockout on the metabolism of E. coli was studied based on the measurement of some key enzyme activities and the intracellular metabolite concentrations. The results showed that under aerobic condition, using glucose as a carbon source, the knockout of lpdA gene led to the accumulation of pyruvate, D-lactate and L-glutamate; the repression of the TCA cycle; the activation of the glyoxylate shunt and the glucose-6-phosphate dehydrogenase in the oxidative pentose phosphate pathway. These conclusions were reinforced by the fermentation using acetate or pyruvate as a carbon source under aerobic condition and the fermentation using glucose as a carbon source under microaerobic condition.