13C-NMR studies of Corynebacterium melassecola metabolic pathways

Abstract

Coryneform bacteria are widely used to produce amino acids, in particularly glutamic acid, by fermentation. To study the metabolic fate of glucose as the carbon source, we developed a method to analyze intracellular extracts by NMR and HPLC. The intracellular metabolites represent the metabolic state of the cells. Glutamic acid was the major metabolic intermediate found in the extracts and its 13C isotopic enrichment reflected that of pyruvic acid. Thus, it was possible to determine the respective contributions of the two major glucose catabolic pathways during the exponential growth phase; glycolysis (55%) and the pentose phosphate pathway (45%). Absolute glutamate 13C enrichments resulting from the incorporation of [1-13C]glucose were determined to quantify the contribution of several metabolic pathways such as anaplerotic pathways (61%; phosphoenolpyruvate carboxylase, pyruvate carboxylase, malic enzyme), a single turn (32%) or multiple turns of the Krebs cycle and the glyoxylate shunt, to oxaloacetate synthesis. A previously described model was adapted to C. melassecola for these calculations. The Krebs cycle was active, whereas the glyoxylate shunt was inactive in exponentially growing cells of C. melassecola with glucose as the sole carbon source. The contributions of anaplerotic enzymes and pyruvate dehydrogenase to replenishing the Krebs' cycle were determined to be 38% and 62%, respectively.