Effect of CO2 on succinate production in dual-phase Escherichia coli fermentations

Succinate production under different concentrations of carbon dioxide (CO(2)) was studied in Escherichia coli AFP111, which contains mutations in pyruvate formate lyase (pfl), lactate dehydrogenase (ldhA) and the phosphotransferase system glucosephosphotransferase enzyme II (ptsG). A series of two-phase fermentations were conducted in which an aerobic cell growth phase was followed by an anaerobic succinate production phase using several constant concentrations of CO(2). As the concentration of CO(2) in the gas phase increased from 0% to 50%, the succinate specific productivity increased from 1.9 mg/gh to 225 mg/gh, and the succinate yield increased from 0.04 g/g to 0.75 g/g. Above 50% CO(2), succinate production did not increase further. Intracellular fluxes were determined at three different CO(2) concentrations (3%, 10%, and 50%) using (13)C-label tracing coupled with LC-MS analysis. The fraction of carbon flux into the pentose phosphate pathway increased from 0.04 at 3% CO(2) to 0.17 at 50% CO(2). Also, the fractional flux through anaplerotic carboxylation at the phosphoenolpyruvate (PEP) node increased slightly from 0.53 at 3% CO(2) to 0.63 at 50% CO(2). The increased flux into the pentose phosphate pathway is attributed to an increased demand for reduced cofactors with elevated CO(2). A four-process explicit model to describe the CO(2) transfer and utilization was proposed. The model predicted that at CO(2) concentrations below about 30-40% the system becomes limited by gas phase CO(2), while at higher CO(2) concentrations the system is limited by PEP carboxylase enzyme kinetics.