In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum

The C-3-C-4 metabolite interconversion at the anaplerotic node in many microorganisms involves a complex set of reactions. C-3, carboxylation to oxaloacetate can originate from phosphoenolpyruvate and pyruvate, and at the Same time multiple C-4-decarboxylating enzymes may be present. The functions of such parallel reactions are not yet fully understood. Using a C-13 NMR-based strategy, we here quantify the individual fluxes at the anaplerotic node of Corynebacterium glutamicum, which is an example of a bacterium possessing multiple carboxylation and decarboxylation reactions. C. glutamicum was grown with a C-13-labeled glucose isotopomer mixture as the main carbon source and 13C-labeled lactate as a cosubstrate. 58 isotopomers as well as 15 positional labels of biomass compounds were quantified. Applying a generally applicable mathematical model to include metabolite mass and carbon labeling balances, it is shown that pyruvate carboxylase contributed 91 +/- 7% to C-3 carboxylation. The total in vivo carboxylation rate of 1.28 +/- 0.14 mmol/g dry weight/h exceeds the demand of carboxylated metabolites for biosyntheses 3-fold. Excess oxaloacetate was recycled to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase. This shows that the reactions at the anaplerotic node might serve additional purposes other than only providing C-4 metabolites for biosynthesis.