13C-metabolic flux analysis of Actinobacillus succinogenes fermentative metabolism at different NaHCO3 and H2 concentrations

Actinobacillus succinogenes naturally produces high concentrations of succinate, a potential intermediary feedstock for bulk chemical productions. A. succinogenes responds to high CO2 and H2 concentrations by producing more succinate and by producing less formate, acetate, and ethanol. To determine how intermediary fluxes in A. succinogenes respond to CO2 and H2 perturbations, 13C-metabolic flux analysis was performed in batch cultures at two different NaHCO3 concentrations, with and without H2, using a substrate mixture of [1-13C]glucose, [U-13C]glucose, and unlabeled NaHCO3. The resulting amino acid, organic acid, and glycogen isotopomers were analyzed by gas chromatography–mass spectrometry and NMR. In all conditions, exchange flux was observed through malic enzyme and/or oxaloacetate decarboxylase. The presence of an exchange flux between oxaloacetate, malate, and pyruvate indicates that, in addition to phosphoenolpyruvate, oxaloacetate, and malate, pyruvate is a fourth node for flux distribution between succinate and alternative fermentation products. High NaHCO3 concentrations decreased the amount of flux shunted by C4-decarboxylating activities from the succinate-producing C4 pathway to the formate-, acetate-, and ethanol-producing C3 pathway. In addition, pyruvate carboxylating flux increased in response to high NaHCO3 concentrations. C3-pathway dehydrogenase fluxes increased or decreased appropriately in response to the different redox demands imposed by the different NaHCO3 and H2 concentrations. Overall, these metabolic flux changes allowed A. succinogenes to maintain a constant growth rate and biomass yield in all conditions. These results are discussed with respect to A. succinogenes’ physiology and to metabolic engineering strategies to increase the flux to succinate.