Study On Pyruvic Acid Accumulation By Metabolic Engineered Escherichia Coli

The pyruvate, a key branch point in central carbon metabolism, is an important intermediate metabolite connecting glycolysis with tricarboxylic acid (TCA) cycle in bacteria like Escherichia coli under aerobic condition. Lipoamide dehydrogenase (LPD) coded by lpdA gene is an important composition of pyruvate dehydrogenase complex (PDHc). Under aerobic condition, PDHc of Escherichia coli is the key enzyme which connects EMP pathway with TCA cycle. The inactivation of PDHc would lead to pyruvic acid accumulation in E.coli. In this dissertation, IpdA gene knockout Escherichia coli(Escherichia coli lpdA) were cultured in synthetic medium with different carbon source. And the compositions of medium were optimized to increase the pyruvate productivity by lpdA mutant. Moreover, respiratory chain terminal oxidase gene cyoA knockout mutant and cydB knockout mutant were investigated due to the result that NADH/NAD+ratio were associated with the pyruvate yield in E.coli.The ability of the lpdA mutant to ferment different carbon sources (10g/L) in synthetic medium under aerobic condition was investigated. The lpdA mutant converted glucose, fructose, xylose and mannose to pyruvate with yields of0.884g/g,0.802g/g,0.817g/g and0.808g/g, respectively. And the result also shown that the pyruvate accumulation curve couple with cell growth in glucose, fructose and xylose fermentation.The different effects of1%,5%,10%and20%LB medium as addition to lpdA mutant convert glucose to pyruvate in synthetic medium were studied, respectively. The result showed that10%LB as addition is optimized, and the fermentation time reduced from71h to12h with the increased productivity from0.080g-L-1·h-1to0.525g·L-1·h-1.The experiment shown that lower intracellular NADH/NAD+ratio was benificial for pyruvate producing in E.coli. Moreover, the pyruvate yield but productivity can be assosiated with the NADH/NAD+ratio in lpdA mutant under this condition. This results suggested that rational design cell factory combining energy metabolism with substance metabolism would be more effective than substance metabolism.The E.coli cydB gene knockout mutant shown35%higher biomass yield compared to the E.coli wild type in batch culture under aerobic condition. The enzyme activity shown that TCA related enzymes were activated in cydB mutant. These results suggested that respiratory chain terminal oxidase could be a good target to build genetically engineered bacterium with high productivity.