Batch culture characterization and metabolic flux analysis of succinate-producing Escherichia coli strains.

This study presents an in-depth analysis of the anaerobic metabolic fluxes of various mutant strains of Escherichia coli overexpressing the Lactococcus lactis pyruvate carboxylase (PYC) for the production of succinate. Previously, a metabolic network design that includes an active glyoxylate pathway implemented in vivo increased succinate yield from glucose in an E. coli mutant to 1.6 mol/mol under fully anaerobic conditions. The design consists 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 a lower NADH requirement). Mutant strains previously constructed during the development of high-yield succinate-producing strains were selected for further characterization to understand their metabolic response as a result of several genetic manipulations and to determine the significance of the fermentative and the glyoxylate pathways in the production of succinate. Measured fluxes obtained under batch cultivation conditions were used to estimate intracellular fluxes and identify critical branch point flux split ratios. The comparison of changes in branch point flux split ratios to the glyoxylate pathway and the fermentative pathway at the oxaloacetate (OAA) node as a result of different mutations revealed the sensitivity of succinate yield to these manipulations. The most favorable split ratio to obtain the highest succinate yield was the fractional partition of OAA to glyoxylate of 0.32 and 0.68 to the fermentative pathway obtained in strains SBS550MG (pHL413) and SBS990MG (pHL413). The succinate yields achieved in these two strains were 1.6 and 1.7 mol/mol, respectively. In addition, an active glyoxylate pathway in an ldhA, adhE, ack-pta mutant strain is shown to be responsible for the high succinate yields achieved anaerobically. Furthermore, in vitro activity measurements of seven crucial enzymes involved in the pathways studied and intracellular measurements of key intermediate metabolite pools provided additional insights on the physiological perturbations caused by these mutations. The characterization of these recombinant mutant strains in terms of flux distribution pattern, in vitro enzyme activity and intracellular metabolite pools provides useful information for the rational modification of metabolic fluxes to improve succinate production.

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