Metabolic Engineering Of Central Carbon Metabolism Of Riboflavin-producing Bacillus Subtilis

In order to reduce acetate formation and increase carbon yield, A series of genetic modified central carbon metabolism of riboflavin-production Bacillus subtilis were constructed based on the principles of metabolic engineering. The riboflavin producing capabilities of these strains were studied systematically and the major findings are:In this thesis, a simple metabolic network of riboflavin-producing B.subtilis was constructed. The theoretical flux distribution of maximum riboflavin production was computed through flux balance analysis. Acid metabolites such as acetate, lactate and pyruvate are not formated when maximum riboflavin production in B.subtilis is obtained. After comparisons with the metabolic flux analysis (MFA) results computed from the wet experiments data of riboflavin-producing B.subtilis, the potential improvement targets for further genetic modification were identified and selected.Riboflavin-producing B.subtilis pta mutant was constrcuted. The lower rates of cell growth and glucose consumption were found in the pta-disrupted mutant while the rate of acetate formation was reduced. Moreover, pyruvate accumulation was found in pta mutant by metabolic analysis. In contrast, the strain has the ablity to redirect pyruvate toward the synthesis of a nonacidic end production, acetoin, when the expression of the gene encoding acetolactate synthase was increased in the pta-disrupted mutant, and cell growth of pta mutant was restored. The riboflavin yield of B. subtilis achieved 54.4mg/l in basic medium containing 0.5% glucose , which was 85.7% enhancement compared to B. subtilis RH33::[pRB63]_n. These resultes from metabolic flux analysis showed that genetic modified central carbon metabolism of riboflavin-production B.subtilis could not significantly affect the flux distribution of key points in metabolic network, but carbon flux through the riboflavin biosynthesis pathway was improved by these changes.The effects of gdh gene amplification on riboflavin biosynthesis of strain B.subtilis RH33::[pRB63]_n was studied in this thesis. The results showed that riboflavin yield achieved 37.2 mg/L when the expression of gdh gene was obtained, which was 29.7% enhancement compared to the parental strain. Moreover, the formation rate of acid metabolites was reduced. These indicated that the increased carbon flow through the gluconate bypass could improve riboflavin yield. Riboflavin-producing B.subtilis pgi mutant was constructed by genetic modified phosphoglucose isomerase. Comparesions with the pgi mutant and the parental strain is different in physiological and fermentation products yields. The mutant showed more slowly specific growth rate, lower glucose consumption. The lower rates of cell growth and glucose consumption could be restored when yeast extract was added into the basic fermentation medium containing 0.5% glucose, and riboflavin yield achieved 35.9mg/L, which was 25.1% enhancement compared to the parental strain.Metabolic control analysis of 6-phosphate glucose branch point showed that carbon flux through HMP pathway could be increased by reducing carbon flux in EMP pathway.