Metabolic engineering of cofactors (NADH/NAD+) in Escherichia coli.

摘要

Metabolic engineering studies have generally focused on manipulating enzyme levels. However, cofactors play a major role in the production of fermentation products. This thesis provides the first systematic study of cofactor manipulations for the NADH/NAD+ cofactor pair and establishes these manipulations as an additional tool for metabolic engineering. This work investigates external and genetic means of increasing the availability of NADH and the total levels of NADH/+) and examines the effect of these manipulations on the distribution of metabolites in Escherichia coli. These strategies include feeding carbon sources with different oxidation states, overexpressing an enzyme that can regenerate NADH, overexpressing an enzyme in the NAD salvage pathway (NAPRTase; pncB), and eliminating NADH competing pathways.; Feeding a more reduced carbon source (sorbitol) or regenerating NADH by overexpression of a heterologous NAD+-dependent FDH increased the NADH availability and provoked a significant change in the final metabolite distribution both under anaerobic and aerobic conditions. Anaerobically, the production of reduced metabolites was favored, as evidenced by a dramatic increase in the ethanol to acetate ratio (Et/Ac) and a shift towards ethanol as the major fermentation product. Aerobically, the increased availability of NADH induced a shift to fermentation even in the presence of oxygen. The NADH regeneration system doubled the maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed. This system also allows the uncoupling of NADH generation from carbon source oxidation by formate addition.; Overexpression of the pncB gene in chemostat experiments increased the total NAD levels, decreased the NADH/NAD+ ratio, and did not significantly redistribute the metabolic fluxes. However, under anaerobic tube conditions, this manipulation decreased lactate production and increased the Et/Ac ratio by 2-fold, suggesting that the higher NAD levels increase the rate of NADH-dependent pathways (ethanol). Chemostat results from all manipulations studied imply that NADH availability rather than the NADH/NAD+ ratio dictates the metabolic flux distribution.; This work also investigates the effect of these cofactor manipulations on the production of a model chemical that requires NADH, 1,2-propanediol. Results showed that our current 1,2-PD pathway is not limited by the availability of NADH.