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

Succinate production under different concentrations of carbon dioxide (CO2) 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 CO2. As the concentration of CO2 in the gas phase increased from 0% to 50%, the succinate specific productivity increased from 1.9 mg/g h to 225 mg/g h, and the succinate yield increased from 0.04 g/g to 0.75 g/g. Above 50% CO2, succinate production did not increase further. Intracellular fluxes were determined at three different CO2 concentrations (3%, 10%, and 50%) using 13C-label tracing coupled with LC–MS analysis. The fraction of carbon flux into the pentose phosphate pathway increased from 0.04 at 3% CO2 to 0.17 at 50% CO2. Also, the fractional flux through anaplerotic carboxylation at the phosphoenolpyruvate (PEP) node increased slightly from 0.53 at 3% CO2 to 0.63 at 50% CO2. The increased flux into the pentose phosphate pathway is attributed to an increased demand for reduced cofactors with elevated CO2. A four-process explicit model to describe the CO2 transfer and utilization was proposed. The model predicted that at CO2 concentrations below about 30–40% the system becomes limited by gas phase CO2, while at higher CO2 concentrations the system is limited by PEP carboxylase enzyme kinetics.