Carbon Flux Analysis by 13 C Nuclear Magnetic Resonance To Determine the Effect of CO 2 on Anaerobic Succinate Production by Corynebacterium glutamicum

Wild-typeCorynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO2 on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO2 caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense ofl -lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO2 was estimated by using13 C-labeled glucose and13 C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (∼5%) regardless of the presence or absence of CO2 . Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured byin vivo NMR, and the stoichiometry (H+ :organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate withC. glutamicum without genetic manipulation.