| This thesis is a study of metabolic engineering to design and engineer novel metabolic networks with improved metabolic processes that can increase product yield and enhance cellular properties. The succinate synthesis pathways in E. coli were chosen as the model system for network design and optimization. The ultimate goal is to create succinate production systems in E. coli that not only achieve the maximum theoretical yield of succinate, but also are highly efficient and robust.; Different carbon sources with different oxidation states and transport systems, such as sorbitol and xylose, were used to address the requirements of cofactor NADH and precursor PEP in order to improve succinate synthesis.; Phosphoenolpyruvate carboxylase and pyruvate carboxylase were coexpressed to drive the carbon flux toward succinate. Competing pathways of succinate synthesis, the lactate and acetate pathways, were also inactivated to increase more carbon flux toward succinate. The intracellular acetyl-CoA pool was increased by overexpressing pantothenate kinase to enhance the activity of PEPC and PYC in order to improve succinate production.; Novel metabolic networks were designed and constructed to enable E. coli to produce succinate as a product under complete aerobic conditions. Since this is naturally not possible, extensive pathway manipulations had to be carried out.; The potential to produce succinate aerobically in E. coli would offer great advantages over anaerobic fermentation in terms of higher biomass generation, faster carbon throughput and product formation. After a series of pathway reconstructions, several aerobic succinate production systems were finally developed that could achieve the maximum theoretical succinate yield predicted by pathway modeling and simulation.; Fed batch reactor experiments were carried out for the most efficient succinate production system under aerobic conditions and the results demonstrated that it has a tremendously high capacity for succinate production. This system not only sustained fast productivity and maximal yield, it also produced succinate at a level never imagined feasible under aerobic conditions. Examination of the metabolite profiles, enzyme activities, and gene expression profiles showed that the metabolic processes of the most efficient aerobic succinate production system were more robust than the other systems. |
