13C NMR studies of the fluxes in the central metabolism of Corynebacterium glutamicum during growth and overproduction of amino acids in batch cultures

Sonntag, K.; Schwinde, J.; Graaf, Albert A. de; Marx, Achim; Eikmanns, Bernhard J.; Wiechert, Wolfgang; Sahm, Hermann

doi:10.1007/bf00169949

Cited by 59 publications

(24 citation statements)

References 22 publications

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“…For the quantitation of carbon fluxes in the central metabolism of C. glutamicum, NMR spectroscopic and metabolite balancing data were combined in a nonlinear least-squares fitting procedure as described previously (26,27,28,45). C. glutamicum was cultured on labeled carbon sources, and the 13 C-labeling patterns of amino acids purified from exponentially growing cells were determined.…”

Section: Methodsmentioning

confidence: 99%

Quantitative Determination of Metabolic Fluxes during Coutilization of Two Carbon Sources: Comparative Analyses with Corynebacterium glutamicum during Growth on Acetate and/or Glucose

et al. 2000

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Growth of Corynebacterium glutamicum on mixtures of the carbon sources glucose and acetate is shown to be distinct from growth on either substrate alone. The organism showed nondiauxic growth on media containing acetate-glucose mixtures and simultaneously metabolized these substrates. Compared to those for growth on acetate or glucose alone, the consumption rates of the individual substrates were reduced during acetateglucose cometabolism, resulting in similar total carbon consumption rates for the three conditions. By 13 Clabeling experiments with subsequent nuclear magnetic resonance analyses in combination with metabolite balancing, the in vivo activities for pathways or single enzymes in the central metabolism of C. glutamicum were quantified for growth on acetate, on glucose, and on both carbon sources. ) on glucose plus acetate also. Consistent with the predictions deduced from the metabolic flux analyses, a glyoxylate cycle-deficient mutant of C. glutamicum, constructed by targeted deletion of the isocitrate lyase and malate synthase genes, exhibited impaired growth on acetate-glucose mixtures.In their natural environments microorganisms often encounter situations when not a single carbon source but mixtures of carbon and energy sources are present. Under such conditions, bacteria often utilize one carbon source preferentially, with the further carbon source(s) being consumed only, when the preferred one is exhausted. As already shown by Monod (29), the preferred carbon source in general supports the best growth rate and/or growth yield, and the successive utilization of the substrates is often represented by a biphasic growth behavior (29). The classical example of this phenomenon is the diauxic growth of Escherichia coli on glucose plus lactose, and the study of the underlying principles initiated the era of research on gene regulation. On the other hand, by analysis of growth and carbon source consumption, it was shown that, e.g., Leuconostoc oenos cometabolizes glucose with citrate or fructose (38). Also, E. coli cometabolizes hexoses under carbon limitation conditions (reviewed in reference 20). Several other bacteria use two carbon sources in parallel (reviewed in reference 16); among these is Corynebacterium glutamicum, a gram-positive bacterium known for its ability to excrete amino acids. C. glutamicum grows aerobically on a variety of carbohydrates and organic acids as carbon sources (23). The organism cometabolizes glucose and fructose, glucose and lactate, and glucose and pyruvate (6, 7), whereas it shows diauxic growth on glucose-glutamate mixtures (21). The carbon sources glucose and acetate have been shown to serve as substrates for amino acid production by C. glutamicum (17). There is considerable knowledge about the enzymes and genes involved in acetate and glucose metabolism as well as their regulation (35,36,52,53), whereas neither growth on acetate-glucose mixtures nor metabolite fluxes during growth on acetate have been studied in detail.The utilization of acetate involves its uptake ...

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Section: Methodsmentioning

confidence: 99%

Quantitative Determination of Metabolic Fluxes during Coutilization of Two Carbon Sources: Comparative Analyses with Corynebacterium glutamicum during Growth on Acetate and/or Glucose

et al. 2000

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“…Approaches based on the labeling of free intracellular amino acids have proven useful in previous studies of metabolic fluxes of C. glutamicum in batch cultures. Nuclear magnetic resonance (NMR)-measured labeling of free intracellular glutamate was included in a 13 C flux analysis of C. glutamicum grown on fructose (5), whereas free intracellular alanine and glutamate were analyzed by NMR for a flux estimation during the growth and overproduction of amino acids of C. glutamicum in batch cultures (34). Admittedly, the fluxes estimated for the present work and for previous studies may to a certain extent be influenced by process dynamics, but they are still very valuable for investigations of metabolic physiology in batch cultures.…”

Section: Vol 186 2004 In-depth Profiling Of Lysine-producing C Glumentioning

confidence: 99%

“…For proteome analysis of C. glutamicum, twodimensional gel electrophoresis was recently used to identify different proteins (11,12,13,31) and to study the influence of nitrogen starvation on the proteome (32). For the quantification of metabolic fluxes (the fluxome), comprehensive approaches combining 13 C tracer experiments, metabolite balancing, and isotopomer modeling have been developed (16,42,43,48) and applied to C. glutamicum, involving, e.g., comparative fluxome analysis during growth, glutamate, and lysine production (21,34), during lysine production in batch cultures (45), of different mutants of a lysine-producing strain genealogy (47), during growth on acetate and/or glucose (40), and during lysine production on different carbon sources (17).…”

mentioning

confidence: 99%

In-Depth Profiling of Lysine-Producing Corynebacterium glutamicum by Combined Analysis of the Transcriptome, Metabolome, and Fluxome

Krömer

Sorgenfrei²,

Klopprogge³

et al. 2004

J Bacteriol

195

122

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An in-depth analysis of the intracellular metabolite concentrations, metabolic fluxes, and gene expression (metabolome, fluxome, and transcriptome, respectively) of lysine-producing Corynebacterium glutamicum ATCC 13287 was performed at different stages of batch culture and revealed distinct phases of growth and lysine production. For this purpose, 13 C flux analysis with gas chromatography-mass spectrometry-labeling measurement of free intracellular amino acids, metabolite balancing, and isotopomer modeling were combined with expression profiling via DNA microarrays and with intracellular metabolite quantification. The phase shift from growth to lysine production was accompanied by a decrease in glucose uptake flux, the redirection of flux from the tricarboxylic acid (TCA) cycle towards anaplerotic carboxylation and lysine biosynthesis, transient dynamics of intracellular metabolite pools, such as an increase of lysine up to 40 mM prior to its excretion, and complex changes in the expression of genes for central metabolism. The integrated approach was valuable for the identification of correlations between gene expression and in vivo activity for numerous enzymes. The glucose uptake flux closely corresponded to the expression of glucose phosphotransferase genes. A correlation between flux and expression was also observed for glucose-6-phosphate dehydrogenase, transaldolase, and transketolase and for most TCA cycle genes. In contrast, cytoplasmic malate dehydrogenase expression increased despite a reduction of the TCA cycle flux, probably related to its contribution to NADH regeneration under conditions of reduced growth. Most genes for lysine biosynthesis showed a constant expression level, despite a marked change of the metabolic flux, indicating that they are strongly regulated at the metabolic level. Glyoxylate cycle genes were continuously expressed, but the pathway exhibited in vivo activity only in the later stage. The most pronounced changes in gene expression during cultivation were found for enzymes at entry points into glycolysis, the pentose phosphate pathway, the TCA cycle, and lysine biosynthesis, indicating that these might be of special importance for transcriptional control in C. glutamicum.

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“…In C. glutamicum, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate 13 C flux analysis: [112] (white circles), [125] (gray circles), [126] (black circles), [127] (white squares), [111] (gray squares), [111] (black squares), [128] (white triangles), [116] (gray triangles), [26] (black triangles), [129] (white diamonds), [115] (gray diamonds), [120] (black diamonds), [76] (white hexagons), and [118] (gray hexagons). The flux reversibility at the pyruvate node is defined as the ratio of the back flux to the anaplerotic net flux [130] 36 C. Wittmann dehydrogenase, and malic enzyme catalyze NADPH generating reactions, whereas NADPH consuming reactions comprise growth with a stoichiometric demand of 16.4 mmol NADPH (g biomass) À1 [34] and overproduction.…”

Section: Metabolic Fluxes Of Nadph Metabolismmentioning

confidence: 99%

“…Lysine production flux (%) Fig. 6 Metabolic flux analysis linking lysine production with NADPH metabolism involving the pentose phosphate pathway (a) and isocitrate dehydrogenase (b) in different C. glutamicum strains investigated under various cultivation conditions by 13 C flux analysis: [125] (white circles), [129] (gray circles), [126] (black circles), [127] (white squares), [112] (gray squares), [126] (black squares), [128] (white triangles), [116] (gray triangles), [26,76] (black triangles), [115] (white diamonds), and [120] (gray diamonds)…”

Section: Metabolic Fluxes Of Nadph Metabolismmentioning

confidence: 99%

Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

Wittmann

2010

Biosystems Engineering I

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The Gram-positive soil bacterium Corynebacterium glutamicum was discovered as a natural overproducer of glutamate about 50 years ago. Linked to the steadily increasing economical importance of this microorganism for production of glutamate and other amino acids, the quest for efficient production strains has been an intense area of research during the past few decades. Efficient production strains were created by applying classical mutagenesis and selection and especially metabolic engineering strategies with the advent of recombinant DNA technology. Hereby experimental and computational approaches have provided fascinating insights into the metabolism of this microorganism and directed strain engineering. Today, C. glutamicum is applied to the industrial production of more than 2 million tons of amino acids per year. The huge achievements in recent years, including the sequencing of the complete genome and efficient post genomic approaches, now provide the basis for a new, fascinating era of research -analysis of metabolic and regulatory properties of C. glutamicum on a global scale towards novel and superior bioprocesses.

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13C NMR studies of the fluxes in the central metabolism of Corynebacterium glutamicum during growth and overproduction of amino acids in batch cultures

Cited by 59 publications

References 22 publications

Quantitative Determination of Metabolic Fluxes during Coutilization of Two Carbon Sources: Comparative Analyses with Corynebacterium glutamicum during Growth on Acetate and/or Glucose

Quantitative Determination of Metabolic Fluxes during Coutilization of Two Carbon Sources: Comparative Analyses with Corynebacterium glutamicum during Growth on Acetate and/or Glucose

In-Depth Profiling of Lysine-Producing Corynebacterium glutamicum by Combined Analysis of the Transcriptome, Metabolome, and Fluxome

Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

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