13C‐NMR, 1H‐NMR and gas‐chromatography mass‐spectrometry studies of the biosynthesis of 13C‐enriched l‐lysine by Brevibacterium flavum

Abstract

The basic metabolic pathways of lysine biosynthesis in Brevibacterium flavum, a strain which excretes excessive amounts of l-lysine, have been followed by using two ¹³C-labeled precursors. ¹³C- and ¹H-NMR spectroscopies in conjunction with gas chromatography mass spectrometry (GC-MS) have revealed the various metabolic pathways leading to l-[¹³C]lysine. Discrete metabolic pathways give rise to distinct labeling patterns. l-Lysine resulting from [1-¹³C]glucose fermentation is relatively specifically labeled: l-[3,5-¹³C]lysine is the main product. Experimental and theoretical approaches based on the ¹³C-enrichment values of intracellular glutamate, a major intermediate metabolite, allowed us to assess the relative contribution of the major metabolic pathways forming lysine. The labeling pattern of glutamate reflects the isotope distribution in 2-oxoglutarate. When [2-¹³C]acetate is used as the sole carbon source in the culture, the energy-producing steps of the Krebs cycle are essential. The higher activity of the Krebs cycle, when endogenous carbohydrates are exhausted from the culture, is indicated by the increased ¹³C enrichment in C-1 of lysine and reveal a high content of isotopomers of four, five and six ¹³C atoms in the lysine molecule, pointing out that the four-carbon intermediates of the cycle are being derived from the glyoxylate shunt pathway. Such a phenomenon does not occur in glucose fermentation. GC-MS analyses of ¹³C enrichments and isotopomer distributions in metabolites and end products are in good agreement with the predicted contribution of each metabolic pathway. This new methodological approach of combined NMR and GC-MS has been demonstrated to be applicable to various other metabolic studies.