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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.


Comparison of fluxes in anaplerotic pathways in different phases. Black, white, zebra, and dotted bars indicate the reactions catalyzed by Pc, PEPc, PEPck, and malic enzymes, respectively. Phases 1 and 2 indicate the fermentations with the glutamate production fluxes of 20 and 68, respectively. Flux values of the anaplerosis in three phases (growth, Phase 1, and Phase 2) were as follows: 0, 10, 56 by Pc, 37, 41, 39 by PEPc, 4, 0, 6 by PEPck, and 13, 18, 18 by malic enzymes, respectively. Error bars indicate those with fluxes induced by the errors of GC-MS data, which were obtained from 20 sets of artificial GC-MS data with a variance due to the observed experimental errors (below 2% in this study), as previously described [25].
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Figure 4: Comparison of fluxes in anaplerotic pathways in different phases. Black, white, zebra, and dotted bars indicate the reactions catalyzed by Pc, PEPc, PEPck, and malic enzymes, respectively. Phases 1 and 2 indicate the fermentations with the glutamate production fluxes of 20 and 68, respectively. Flux values of the anaplerosis in three phases (growth, Phase 1, and Phase 2) were as follows: 0, 10, 56 by Pc, 37, 41, 39 by PEPc, 4, 0, 6 by PEPck, and 13, 18, 18 by malic enzymes, respectively. Error bars indicate those with fluxes induced by the errors of GC-MS data, which were obtained from 20 sets of artificial GC-MS data with a variance due to the observed experimental errors (below 2% in this study), as previously described [25].

Mentions: We determined which reaction in the anaplerotic pathways of C. glutamicum has the greatest correlation with the amount of glutamate produced by the quantitative analysis of the 13C MFA results. As shown in Fig. 4, reactions from PEP to oxaloacetate catalyzed by PEPc and from malate to pyruvate catalyzed by malic enzymes were mainly active in the growth phase. The flux catalyzed by PEPc, which showed a certain extent value (around 40) in the growth phase, was not significantly changed, even though the condition of fermentation was changed from cell growth to glutamate production by Tween 40 addition. The flux of the reaction from pyruvate to oxaloacetate catalyzed by Pc was almost zero in the growth phase. As glutamate production increased with the amount of Tween 40 added, the flux of this reaction catalyzed by Pc increased. The fluxes of the other anaplerotic pathways from oxaloacetate to PEP catalyzed by PEPck and glyoxylate shunt were almost zero in all cases.


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)

Comparison of fluxes in anaplerotic pathways in different phases. Black, white, zebra, and dotted bars indicate the reactions catalyzed by Pc, PEPc, PEPck, and malic enzymes, respectively. Phases 1 and 2 indicate the fermentations with the glutamate production fluxes of 20 and 68, respectively. Flux values of the anaplerosis in three phases (growth, Phase 1, and Phase 2) were as follows: 0, 10, 56 by Pc, 37, 41, 39 by PEPc, 4, 0, 6 by PEPck, and 13, 18, 18 by malic enzymes, respectively. Error bars indicate those with fluxes induced by the errors of GC-MS data, which were obtained from 20 sets of artificial GC-MS data with a variance due to the observed experimental errors (below 2% in this study), as previously described [25].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919393&req=5

Figure 4: Comparison of fluxes in anaplerotic pathways in different phases. Black, white, zebra, and dotted bars indicate the reactions catalyzed by Pc, PEPc, PEPck, and malic enzymes, respectively. Phases 1 and 2 indicate the fermentations with the glutamate production fluxes of 20 and 68, respectively. Flux values of the anaplerosis in three phases (growth, Phase 1, and Phase 2) were as follows: 0, 10, 56 by Pc, 37, 41, 39 by PEPc, 4, 0, 6 by PEPck, and 13, 18, 18 by malic enzymes, respectively. Error bars indicate those with fluxes induced by the errors of GC-MS data, which were obtained from 20 sets of artificial GC-MS data with a variance due to the observed experimental errors (below 2% in this study), as previously described [25].
Mentions: We determined which reaction in the anaplerotic pathways of C. glutamicum has the greatest correlation with the amount of glutamate produced by the quantitative analysis of the 13C MFA results. As shown in Fig. 4, reactions from PEP to oxaloacetate catalyzed by PEPc and from malate to pyruvate catalyzed by malic enzymes were mainly active in the growth phase. The flux catalyzed by PEPc, which showed a certain extent value (around 40) in the growth phase, was not significantly changed, even though the condition of fermentation was changed from cell growth to glutamate production by Tween 40 addition. The flux of the reaction from pyruvate to oxaloacetate catalyzed by Pc was almost zero in the growth phase. As glutamate production increased with the amount of Tween 40 added, the flux of this reaction catalyzed by Pc increased. The fluxes of the other anaplerotic pathways from oxaloacetate to PEP catalyzed by PEPck and glyoxylate shunt were almost zero in all cases.

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.