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 (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 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.
Petersen
S
S
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
de Graaf
A A
AA
Eggeling
L
L
Möllney
M
M
Wiechert
W
W
Sahm
H
H
D000596:Amino Acids / Q000378:metabolism; D000641:Ammonia / Q000378:metabolism; D018533:Biomass; D019149:Bioreactors; D002244:Carbon / Q000378:metabolism; D002247:Carbon Isotopes; D002952:Citric Acid Cycle / Q000502:physiology*; D003352:Corynebacterium / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006019:Glycolysis / Q000502:physiology*; D006038:Glyoxylates / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D010071:Oxaloacetates / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism
D000596:Amino Acids; D002247:Carbon Isotopes; D006038:Glyoxylates; D010071:Oxaloacetates; D019344:Lactic Acid; D010728:Phosphoenolpyruvate; D002244:Carbon; D000641:Ammonia; D019289:Pyruvic Acid; D005947:Glucose