Abstract
The carbon flux distribution in the central metabolism of Corynebacterium glutamicum was studied in batch cultures using [1-13C]- and [6-13C]glucose as substrate during exponential growth as well as during overproduction of l-lysine and l-glutamate. Using the 13C NMR data in conjunction with stoichiometric metabolite balances, molar fluxes were quantified and normalised to the glucose uptake rate, which was set to 100. The normalised molar flux via the hexose monophosphate pathway was 40 during exponential growth, whereas it was only 17 during l-glutamate production. During l-lysine production, the normalised hexose monophosphate pathway flux was elevated to 47. Thus, the carbon flux via this pathway correlated with the NADPH demand for bacterial growth and l-lysine overproduction. The normalised molar flux in the tricarboxylic acid cycle at the level of 2-oxoglutarate dehydrogenase was 100 during exponential growth and 103 during l-lysine secretion. During l-glutamate formation, the normalised flux through the tricarboxylic acid cycle was reduced to 60. In contrast to earlier NMR studies with C. glutamicum, no significant activity of the glyoxylate pathway could be detected. All experiments indicated a strong in vivo flux from oxaloacetate back to phosphoenolpyruvate and/or pyruvate, which might be due to phosphoenolpyruvate carboxykinase activity in C. glutamicum.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Eikmanns BJ, Metzger M, Reinscheid D, Kircher M, Sahm H (1991) Amplification of three threonine biosynthesis genes in Corynebacterium glutamicum and its influence on carbon flux in different strains. Appl Microbiol Biotechnol 34:617–622
Gutmann M, Hoischen C, Krämer R (1992) Carrier-mediated glutamate secretion by Corynebacterium glutamicum under biotin limitation. Biochim Biophys Acta 1112:115–123
Inbar L, Lapidot A (1987) 13C-NMR, 1H-NMR and gaschromatography mass-spectrometry studies of the biosynthesis of 13C-enriched l-lysine by Brevibacterium flavum. Eur J Biochem 162:621–633
Inbar L, Kahana Z, Lapidot A (1985) Natural abundance 13C-nuclear magnetic resonance studies of regulation and overproduction of l-lysine by Brevibacterium flavum. J Biochem (Tokyo) 149:601–607
Ishino S, Shimomura-Nishimuta J, Yamaguchi K, Araki K (1991) 13C-NMR studies of glucose metabolism in l-glutamic acid and l-lysine fermentation by Corynebacterium glutamicum. J Gen Appl Microbiol 37:157–165
Jetten MSM, Sinskey AJ (1993) Characterisation of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum. FEMS Microbiol Lett 111:183–188
Jetten MSM, Pitoc GA, Follettie MT, Sinskey AJ (1994) Regulation of phospho(enol)-pyruvate- and oxaloacetate-converting enzymes in Corynebacterium glutamicum. Appl Microbiol Biotechnol 41:47–52
Kinoshita S (1985) Glutamic acid bacteria. In: Demain AL, Solomon NA (eds) Biology of industrial microorganisms. Benjamin Cummings, London, pp 115–142
Marx A, Sonntag K, Graaf AA de, Wiechert W, Sahm H (1993) Simulation of 13C labelling for NMR studies of the pentose phosphate pathway. Biol Chem Hoppe-Seyler 374:689
Marx A, Graaf AA de, Wiechert, W Eggeling, L Sahm H (1994) Carbon flux in the lysine producing strain Corynebacterium glutamicum MH20–22B: analysis by 13C NMR of amino acids and metabolite balancing. In: Gnaiger E, Gellerich FN, Wysss M (eds) What is controlling life?. Modern trends in bio thermokinetics vol 3. Innsbruck University Press, Innsbruck, Austria, pp 240–243
Marx A, Graaf AA de, Wiechert, W Eggeling, L Sahm H (1995) Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by NMR spectroscopy combined with metabolite balancing. Biotechnol Bioeng
Neidhardt FC, Ingraham JL, Schächter M (1982) Physiology of the bacterial cell. Sinauer, Sunderland, Massachusetts
Ozaki H, Shiio I (1969) Regulation of the TCA and glyoxylate cycles in Brevibacterium flavum. J Biochem (Tokyo) 66:297–311
Peters-Wendisch PG, Eikmanns BJ, Thierbach G, Bachmann B, Sahm H (1993) Phosphoenolpyruvate carboxylase in Corynebacterium glutamicum is dispensable for growth and lysine production. FEMS Microbiol Lett 112:269–274
Reinscheid D, Eikmanns BJ, Sahm H (1994a) Characterisation of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme. J Bacteriol 176:3474–3483
Reinscheid D, Eikmanns BJ, Sahm H (1994b) Malate synthase from Corynebacterium glutamicum: sequence analysis of the gene and biochemical characterization of the enzyme. Microbiologia 140:3099–3108
Rollin C, Morgant V, Guyonvarch A, Guerquin-Kern J-L (1995) 13C-NMR studies of Corynebacterium melassecola metabolic pathways. Eur J Biochem 227:488–493
Schrumpf B, Schwarzer A, Kalinowski J, Pühler A, Eggeling L, Sahm H (1991) A functionally split pathway for lysine synthesis in Corynebacterium glutamicum. J Bacteriol 173:4510–4516
Schrumpf B, Eggeling L, Sahm H (1992) Isolation and prominent characteristics of an l-lysine hyperproducing strain of Corynebacterium glutamicum. Appl Microbiol Biotechnol 37:566–571
Shiio I, Otsuka SI, Katsuya M (1962) Effect of biotin on the bacterial formation of glutamic acid. I. Glutamate formation and the cellular permeability of amino acids. J Biochem (Tokyo) 51:56–62
Sonntag K, Eggeling L, Graaf AA de, Sahm H (1993) Flux partitioning in the split pathway of lysine synthesis in Corynebacterium glutamicum, quantification by 13C and 1H-NMR spectroscopy. Eur J Biochem 213:1325–1331
Tosaka O, Morioka H, Kakinami K (1979) The role of biotin-dependent pyruvate carboxylase in l-lysine production. Agric Biol Chem 43:1513–1519
Vallino JJ, Stephanopoulos G (1990) Flux determination in cellular bioreaction networks: application to lysine fermentation. In: Sikdar SK, Bier M, Todd P (eds) Frontiers in bioprocessing, CRC Press, Boca Raton, Fla, pp 205–219
Vallino JJ, Stephanopoulos G (1993) Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction. Biotechnol Bioeng 41:633–646
Walker TE, Han CH, Kollman VH, London RE, Matwiyoff NA (1982) 13C NMR studies of the biosynthesis by Microbacterium ammoniaphilum of l-glutamate selectively enriched with carbon-13. J Biol Chem 257:1189–1195
Walsh K, Koshland DE Jr (1984) Determination of flux through the branch point of two metabolic cycles. J Biol Chem 259:9646–9654
Yamaguchi K, Ishino S, Araki K, Shirahata K (1986) 13C-NMR studies of lysine fermentation with a Corynebacterium glutamicum mutant. Agric Biol Chem 50:2453–2459
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Sonntag, K., Schwinde, J., de Graaf, A.A. et al. 13C NMR studies of the fluxes in the central metabolism of Corynebacterium glutamicum during growth and overproduction of amino acids in batch cultures. Appl Microbiol Biotechnol 44, 489–495 (1995). https://doi.org/10.1007/BF00169949
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00169949