Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity.

A novel in vivo method of producing succinate has been developed. A genetically engineered Escherichia coli strain has been constructed to meet the NADH requirement and carbon demand to produce high quantities and yield of succinate by strategically implementing metabolic pathway alterations. Currently, the maximum theoretical succinate yield under strictly anaerobic conditions through the fermentative succinate biosynthesis pathway is limited to one mole per mole of glucose due to NADH limitation. The implemented strategic design involves the construction of a dual succinate synthesis route, which diverts required quantities of NADH through the traditional fermentative pathway and maximizes the carbon converted to succinate by balancing the carbon flux through the fermentative pathway and the glyoxylate pathway (which has less NADH requirement). The synthesis of succinate uses a combination of the two pathways to balance the NADH. Consequently, experimental results indicated that these combined pathways gave the most efficient conversion of glucose to succinate with the highest yield using only 1.25 moles of NADH per mole of succinate in contrast to the sole fermentative pathway, which uses 2 moles of NADH per mole of succinate. A recombinant E. coli strain, SBS550MG, was created by deactivating adhE, ldhA and ack-pta from the central metabolic pathway and by activating the glyoxylate pathway through the inactivation of iclR, which encodes a transcriptional repressor protein of the glyoxylate bypass. The inactivation of these genes in SBS550MG increased the succinate yield from glucose to about 1.6 mol/mol with an average anaerobic productivity rate of 10 mM/h (approximately 0.64 mM/h-OD600). This strain is capable of fermenting high concentrations of glucose in less than 24 h. Additional derepression of the glyxoylate pathway by inactivation of arcA, leading to a strain designated as SBS660MG, did not significantly increase the succinate yield and it decreased glucose consumption by 80%. It was also observed that an adhE, ldhA and ack-pta mutant designated as SBS990MG, was able to achieve a high succinate yield similar to SBS550MG when expressing a Bacillus subtilis NADH-insensitive citrate synthase from a plasmid.