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

Abstract

The C3-C4 metabolite interconversion at the anaplerotic node in many microorganisms involves a complex set of reactions. C3 carboxylation to oxaloacetate can originate from phosphoenolpyruvate and pyruvate, and at the same time multiple C4-decarboxylating enzymes may be present. The functions of such parallel reactions are not yet fully understood. Using a (13)C 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 (13)C-labeled glucose isotopomer mixture as the main carbon source and (13)C-labeled lactate as a cosubstrate. 58 isotopomers as well as 15 positional labels of biomass compounds were quantifled. Applying a generally applicable mathematical model to include metabolite mass and carbon labeling balances, it is shown that pyruvate carboxylase contributed 91 ± 7% to C3 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 C4 metabolites for biosynthesis.