13C nuclear magnetic resonance studies of the biosynthesis by Microbacterium ammoniaphilum of L-glutamate selectively enriched with carbon-13

Abstract

13C NMR of isotopically enriched metabolites has been used to study the metabolism of Microbacterium ammoniaphilum, a bacterium which excretes large quantities of L-glutamic acid into the medium. Biosynthesis from 90% [1-13C]glucose results in relatively high specificity of the label, with [2,4-13C2]glutamate as the major product. The predominant biosynthetic pathway for synthesis of glutamate from glucose was determined to be the Embden Meyerhof glycolytic pathway followed by P-enolpyruvate carboxylase and the first third of the Krebs cycle. Different metabolic pathways are associated with different correlations in the enrichment of the carbons, reflected in the spectrum as different 13C-13C scalar multiplet intensities. Hence, intensity and 13C-13C multiplet analysis allows quantitation of the pathways involved. Although blockage of the Krebs cycle at the alpha-ketoglutarate dehydrogenase step is the basis for the accumulation of glutamate, significant Krebs cycle activity was found in glucose grown cells, and extensive Krebs cycle activity in cells metabolizing [1-13C]acetate. In addition to the observation of the expected metabolites, the disaccharide alpha, alpha-trehalose and alpha, beta-glucosylamine were identified from the 13C NMR spectra.