Metabolic Mechanism Of Glutamic Acid Biosynthesis Under Different Dissolved Oxygen Concentrations And Its Metabolic Flux Analysis

In this thesis, we applied multi-scale parameter association analysis and metabolic flux analysis to investigate and analysis Corynebacterium glutamicum's metabolic response to changes of dissolved oxygen concentrations quantitatively. The mechanisms of dissolved oxygen affect Corynebacterium glutamicum's growth and glutamate synthesis capacity were investigated detailly by enzymology, cell redox potential and quantitative metabolic flux analysis.Dissolved oxygen (DO) is an important parameter in aerobic fermentation process. It has a major impact on cell's metabolism and physiology. Glutamate fermentation is a typical aerobic fermentation process, oxygen limitation will reduce the glutamate production and may lead to cell inactivation, quantitative analysis of metabolic flux changes under this condition will contribute to a better understanding of Corynebacterium glutamicum's behaviors in glutamate fermentation process. We understood glutamate fermentation process deeply by multi-scale parameter association analysis and select DO as the starting point to study the mechanism of glutamate overproduction by Corynebacterium glutamicum. Corynebacterium glutamicum was cultivated in three parallel5L fermenters under0%,5%and30%DO concentrations, respectively. The metabolism response of Corynebacterium glutamicum to different DO concentrations was investigated systematically. The results showed that cell growth was affected by dissolved oxygen slightly, but the metabolic flux was quite different under different DO levels. As the DO concentration decreased, the concentrations of citrate and ?-oxoglutarate decreased, the concentrations of organic acids in the reductive arm of the TCA cycle as well as the amount of alanine, valine and phenylalanine were increased. It was also revealed that when the DO concentration was kept at0%, lactate and acetate accumulated largely. By measuring two key enzyme activities and intracellular NADH/NAD+ratio under these three conditions, we found that as the dissolved oxygen concentration decreased, GDH activity decreased, LDH activity increased, intracellular redox potential (NADH/NAD+) increased. All these changes caused metabolic shift towards the reductive arm of the TCA cycle under oxygen limited condition, and which finally led to the decline of the yield of glutamate.1C isotope tracer experiment method was applied to glutamate fermentation process successfully. We cultivated Corynebacterium glutamicum with13C labeled glucose in a300ml mini-reactor. The dissolved oxygen concentrations during production period were controlled at 30%and0%, respectively. The flux ratios of key nodes in the glutamate overproduction pathway were calculated by Fiatflux software under these two conditions. The global metabolic flux was calculated with chemometric method. The results showed that the glucose comsumption rate was lower, PP pathway, anaplerotic pathway and synthesis pathway of lactate, alanine and lysine were enhanced under oxygen limited condition. This indicates that the metabolic flux towards glutamate synthesis pathway was reduced under oxygen limited condition and glutamate production declined.