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Study on roles of anaplerotic pathways in glutamate overproduction of Corynebacterium glutamicum by metabolic flux analysis.

Shirai T, Fujimura K, Furusawa C, Nagahisa K, Shioya S, Shimizu H - Microb. Cell Fact. (2007)

Bottom Line: It was clarified by a quantitative 13C MFA that the reaction catalyzed by Pc is most markedly increased, whereas other fluxes of PEPc and PEPck remain constant in the glutamate overproduction induced by Tween 40.This result is consistent with the previous results obtained in a comparative study on the glutamate productions of genetically recombinant Pc- and PEPc-overexpressing strains.The importance of a specific reaction in an anaplerotic pathway was elucidated at a metabolic level by MFA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan. shirai@bio.eng.osaka-u.ac.jp

ABSTRACT

Background: Corynebacterium glutamicum has several anaplerotic pathways (anaplerosis), which are essential for the productions of amino acids, such as lysine and glutamate. It is still not clear how flux changes in anaplerotic pathways happen when glutamate production is induced by triggers, such as biotin depletion and the addition of the detergent material, Tween 40. In this study, we quantitatively analyzed which anaplerotic pathway flux most markedly changes the glutamate overproduction induced by Tween 40 addition.

Results: We performed a metabolic flux analysis (MFA) with [1-13C]- and [U-13C]-labeled glucose in the glutamate production phase of C. glutamicum, based on the analysis of the time courses of 13C incorporation into proteinogenic amino acids by gas chromatography-mass spectrometry (GC-MS). The flux from phosphoenolpyruvate (PEP) to oxaloacetate (Oxa) catalyzed by phosphoenolpyruvate carboxylase (PEPc) was active in the growth phase not producing glutamate, whereas that from pyruvate to Oxa catalyzed by pyruvate carboxylase (Pc) was inactive. In the glutamate overproduction phase induced by the addition of the detergent material Tween 40, the reaction catalyzed by Pc also became active in addition to the reaction catalyzed by PEPc.

Conclusion: It was clarified by a quantitative 13C MFA that the reaction catalyzed by Pc is most markedly increased, whereas other fluxes of PEPc and PEPck remain constant in the glutamate overproduction induced by Tween 40. This result is consistent with the previous results obtained in a comparative study on the glutamate productions of genetically recombinant Pc- and PEPc-overexpressing strains. The importance of a specific reaction in an anaplerotic pathway was elucidated at a metabolic level by MFA.

No MeSH data available.


Metabolic fluxes in C. glutamicum in growth and production phases of two different glutamate production activities. Dotted arrows indicate fluxes for biomass. Left, middle, and right values in boxes indicate fluxes in the growth and production phases, where glutamate fluxes were 20 and 68, respectively. In this study, the fluxes with backward (exchange) reactions, i.e., those in glycolysis, the pentose phosphate pathway, the latter steps of the TCA cycle (succinate → oxaloacetate), and C1 metabolisms, are shown as net values [22]. Abbreviations: Gly, glycine; Ser, serine; Glu, glutamate; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; GAP, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; Pyr, pyruvate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; Xu5P, xylulose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; AcCoA, acetyl-CoA; IsoCit, isocitrate; αKG, 2-oxoglutarate; Suc, succinate; Fum, fumarate; Mal, malate; Oxa, oxaloacetate.
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Figure 3: Metabolic fluxes in C. glutamicum in growth and production phases of two different glutamate production activities. Dotted arrows indicate fluxes for biomass. Left, middle, and right values in boxes indicate fluxes in the growth and production phases, where glutamate fluxes were 20 and 68, respectively. In this study, the fluxes with backward (exchange) reactions, i.e., those in glycolysis, the pentose phosphate pathway, the latter steps of the TCA cycle (succinate → oxaloacetate), and C1 metabolisms, are shown as net values [22]. Abbreviations: Gly, glycine; Ser, serine; Glu, glutamate; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; GAP, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; Pyr, pyruvate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; Xu5P, xylulose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; AcCoA, acetyl-CoA; IsoCit, isocitrate; αKG, 2-oxoglutarate; Suc, succinate; Fum, fumarate; Mal, malate; Oxa, oxaloacetate.

Mentions: The 13C MFA in the growth phase was performed by a previously reported method [26], where 13C-labeled glucose was added at the beginning of fermentation, that is, the GC-MS data in the log growth phase was used for analysis (22 h). The 13C MFAs in the two glutamate production phases were performed using the GC-MS data mentioned in the previous section. The GC-MS data of C. glutamicum in the growth phase and two glutamate production phases are shown in Tables 2, 3, 4, respectively. The measurement of each sample was performed five times, and all data errors (standard deviation divided by mean values) were below 2%. The 13C MFA results in the growth phase and two glutamate production phases are shown in Fig. 3. For the result in the growth phase, cross validation was performed using the NMR data shown in Table 5. For the 13C MFA results in the two production phases, the results based on the 13C labeling patterns of proteinogenic amino acids were verified using the GC-MS data of intermediate metabolites (pyruvate, succinate, and fumarate) because of difficulty in cross validation using the NMR data due to a small number of cells in the production phase, as shown in Tables 6 and 7.


Study on roles of anaplerotic pathways in glutamate overproduction of Corynebacterium glutamicum by metabolic flux analysis.

Shirai T, Fujimura K, Furusawa C, Nagahisa K, Shioya S, Shimizu H - Microb. Cell Fact. (2007)

Metabolic fluxes in C. glutamicum in growth and production phases of two different glutamate production activities. Dotted arrows indicate fluxes for biomass. Left, middle, and right values in boxes indicate fluxes in the growth and production phases, where glutamate fluxes were 20 and 68, respectively. In this study, the fluxes with backward (exchange) reactions, i.e., those in glycolysis, the pentose phosphate pathway, the latter steps of the TCA cycle (succinate → oxaloacetate), and C1 metabolisms, are shown as net values [22]. Abbreviations: Gly, glycine; Ser, serine; Glu, glutamate; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; GAP, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; Pyr, pyruvate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; Xu5P, xylulose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; AcCoA, acetyl-CoA; IsoCit, isocitrate; αKG, 2-oxoglutarate; Suc, succinate; Fum, fumarate; Mal, malate; Oxa, oxaloacetate.
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Related In: Results  -  Collection

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Figure 3: Metabolic fluxes in C. glutamicum in growth and production phases of two different glutamate production activities. Dotted arrows indicate fluxes for biomass. Left, middle, and right values in boxes indicate fluxes in the growth and production phases, where glutamate fluxes were 20 and 68, respectively. In this study, the fluxes with backward (exchange) reactions, i.e., those in glycolysis, the pentose phosphate pathway, the latter steps of the TCA cycle (succinate → oxaloacetate), and C1 metabolisms, are shown as net values [22]. Abbreviations: Gly, glycine; Ser, serine; Glu, glutamate; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; GAP, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; Pyr, pyruvate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; Xu5P, xylulose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; AcCoA, acetyl-CoA; IsoCit, isocitrate; αKG, 2-oxoglutarate; Suc, succinate; Fum, fumarate; Mal, malate; Oxa, oxaloacetate.
Mentions: The 13C MFA in the growth phase was performed by a previously reported method [26], where 13C-labeled glucose was added at the beginning of fermentation, that is, the GC-MS data in the log growth phase was used for analysis (22 h). The 13C MFAs in the two glutamate production phases were performed using the GC-MS data mentioned in the previous section. The GC-MS data of C. glutamicum in the growth phase and two glutamate production phases are shown in Tables 2, 3, 4, respectively. The measurement of each sample was performed five times, and all data errors (standard deviation divided by mean values) were below 2%. The 13C MFA results in the growth phase and two glutamate production phases are shown in Fig. 3. For the result in the growth phase, cross validation was performed using the NMR data shown in Table 5. For the 13C MFA results in the two production phases, the results based on the 13C labeling patterns of proteinogenic amino acids were verified using the GC-MS data of intermediate metabolites (pyruvate, succinate, and fumarate) because of difficulty in cross validation using the NMR data due to a small number of cells in the production phase, as shown in Tables 6 and 7.

Bottom Line: It was clarified by a quantitative 13C MFA that the reaction catalyzed by Pc is most markedly increased, whereas other fluxes of PEPc and PEPck remain constant in the glutamate overproduction induced by Tween 40.This result is consistent with the previous results obtained in a comparative study on the glutamate productions of genetically recombinant Pc- and PEPc-overexpressing strains.The importance of a specific reaction in an anaplerotic pathway was elucidated at a metabolic level by MFA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan. shirai@bio.eng.osaka-u.ac.jp

ABSTRACT

Background: Corynebacterium glutamicum has several anaplerotic pathways (anaplerosis), which are essential for the productions of amino acids, such as lysine and glutamate. It is still not clear how flux changes in anaplerotic pathways happen when glutamate production is induced by triggers, such as biotin depletion and the addition of the detergent material, Tween 40. In this study, we quantitatively analyzed which anaplerotic pathway flux most markedly changes the glutamate overproduction induced by Tween 40 addition.

Results: We performed a metabolic flux analysis (MFA) with [1-13C]- and [U-13C]-labeled glucose in the glutamate production phase of C. glutamicum, based on the analysis of the time courses of 13C incorporation into proteinogenic amino acids by gas chromatography-mass spectrometry (GC-MS). The flux from phosphoenolpyruvate (PEP) to oxaloacetate (Oxa) catalyzed by phosphoenolpyruvate carboxylase (PEPc) was active in the growth phase not producing glutamate, whereas that from pyruvate to Oxa catalyzed by pyruvate carboxylase (Pc) was inactive. In the glutamate overproduction phase induced by the addition of the detergent material Tween 40, the reaction catalyzed by Pc also became active in addition to the reaction catalyzed by PEPc.

Conclusion: It was clarified by a quantitative 13C MFA that the reaction catalyzed by Pc is most markedly increased, whereas other fluxes of PEPc and PEPck remain constant in the glutamate overproduction induced by Tween 40. This result is consistent with the previous results obtained in a comparative study on the glutamate productions of genetically recombinant Pc- and PEPc-overexpressing strains. The importance of a specific reaction in an anaplerotic pathway was elucidated at a metabolic level by MFA.

No MeSH data available.