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

Abstract

c Wild-type Corynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO 2 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 CO 2 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 of L-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO 2 was estimated by using 13 C-labeled glucose and 13 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 (ϳ5%) regardless of the presence or absence of CO 2 . 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 by in 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 with C. glutamicum without genetic manipulation.

Anaerobic Growth of Corynebacterium glutamicum via Mixed-Acid Fermentation

“…A limited number of reactions are assumed to be relevant for proton extrusion under fermentative conditions in C. glutamicum. (i) The export of lactate (pK a ϭ 3.90) and succinate (pK a1 ϭ 4.16, pK a2 ϭ 5.61) by nongrowing C. glutamicum cells at an extracellular pH of 5.7 and an intracellular pH of 6.3 was reported to be coupled to the export of 1.4 and 2.3 to 2.7 protons, respectively (24), thereby contributing to the establishment of a proton motive force. The driving force for this proton export is the 10-fold-higher intracellular compared to extracellular concentration of lactate and succinate, as measured by 13 C NMR (24).…”

“…(i) The export of lactate (pK a ϭ 3.90) and succinate (pK a1 ϭ 4.16, pK a2 ϭ 5.61) by nongrowing C. glutamicum cells at an extracellular pH of 5.7 and an intracellular pH of 6.3 was reported to be coupled to the export of 1.4 and 2.3 to 2.7 protons, respectively (24), thereby contributing to the establishment of a proton motive force. The driving force for this proton export is the 10-fold-higher intracellular compared to extracellular concentration of lactate and succinate, as measured by 13 C NMR (24). For succinate, the exporter SucE has been identified in C. glutamicum (64,65) but still awaits a detailed biochemical characterization, including the stoichiometry of cotransported ions.…”

“…This difference can be explained by the different conditions used in the experiments. Rados et al (24) used nongrowing cells resuspended to an OD 600 of 100 in a buffer without nitrogen, sulfur, or phosphorus sources, whereas we analyzed growing cells at a starting OD 600 of 1 in a minimal medium supplemented with 15 g/liter tryptone. Therefore, it has to be assumed that in our experiments, CO 2 was used to enhance CO 2 -requiring biosynthetic reactions, in particular acetyl-CoA carboxylation for fatty and mycolic acid synthesis, rather than for increasing pyruvate and PEP carboxylation.…”

“…For the corresponding production processes, aerobically grown cells are packed at high cell density under oxygen-deprived conditions, where they remain metabolically active and produce and excrete the products mentioned above. Analysis of the carbon flux under anaerobic conditions in a 100% argon atmosphere by 13 C nuclear magnetic resonance (NMR) showed that 95% of the glucose is metabolized in glycolysis and 5% in the pentose phosphate pathway (24); 97% of the succinate was formed in the reductive tricarboxylic acid (TCA) cycle and only 3% in the oxidative TCA cycle, with the latter providing reducing equivalents required for the redox balance (24). Replacement of the argon atmosphere with a 100% CO 2 atmosphere increased the succinate yield about 3-fold to ϳ0.9 mol/mol glucose and the acetate yield about 2-fold to ϳ0.3 mol/mol, while the lactate yield was decreased about 3-fold to ϳ0.4 mol/mol (24).…”

“…Analysis of the carbon flux under anaerobic conditions in a 100% argon atmosphere by 13 C nuclear magnetic resonance (NMR) showed that 95% of the glucose is metabolized in glycolysis and 5% in the pentose phosphate pathway (24); 97% of the succinate was formed in the reductive tricarboxylic acid (TCA) cycle and only 3% in the oxidative TCA cycle, with the latter providing reducing equivalents required for the redox balance (24). Replacement of the argon atmosphere with a 100% CO 2 atmosphere increased the succinate yield about 3-fold to ϳ0.9 mol/mol glucose and the acetate yield about 2-fold to ϳ0.3 mol/mol, while the lactate yield was decreased about 3-fold to ϳ0.4 mol/mol (24). Gene expression studies revealed that several genes involved in glycolysis, lactate formation, C 3 carboxylation, and the reductive TCA cycle were upregulated under oxygen deprivation, such as tpi, gapA, pgk, ldhA, ppc, and mdh, whereas genes of the oxidative TCA cycle were downregulated (25).…”