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Effects of the changes in enzyme activities on metabolic flux redistribution around the 2-oxoglutarate branch in glutamate production by Corynebacterium glutamicum

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Abstract

An experimental method for metabolic control analysis (MCA) was applied to the investigation of a metabolic network of glutamate production by Corynebacterium glutamicum. A metabolic reaction (MR) model was constructed and used for flux distribution analysis (MFA). The flux distribution at a key branch point, 2-oxoglutarate, was investigated in detail. Activities of isocitrate dehydrogenase (ICDH), glutamate dehydrogenase (GDH), and 2-oxoglutarate dehydrogenase complex (ODHC) around this the branch point were changed, using two genetically engineered strains (one with enhanced ICDH activity and the other with enhanced GDH activity) and by controlling environmental conditions (i.e. biotin-deficient conditions). The mole flux distribution was determined by an MR model, and the effects of the changes in the enzyme activities on the mole flux distribution were compared. Even though both GDH and ICDH activities were enhanced, the mole flux distribution was not significantly changed. When the ODHC activity was attenuated, the flux through ODHC decreased, and glutamate production was markedly increased. The flux control coefficients of the above three enzymes for glutamate production were determined based on changes in enzyme activities and the mole flux distributions. It was found that the factor with greatest impact on glutamate production in the metabolic network was obtained by attenuation of ODHC activity.

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Abbreviations

A :

stoichiometric coefficient matrix in MR model

Ck i :

flux control coefficients for enzyme reaction of i on flux of k

Dk i :

deviation index in terms for enzyme reaction of i on flux of k

e i :

specific enzyme activity of reaction i, mol/min per mg protein

er i :

perturbed enzyme activity of reaction i, mol/min per mg protein

Δe i :

large perturbation of enzyme activity of reaction i, mol/min per mg protein

fk i :

flux amplification factor of flux of reaction k by perturbation of enzyme of reaction i

Fk i :

correction factor defined in Eq. (8)

h :

consistency index

J k :

flux of reaction k, mol/h

Jr k :

perturbed flux of reaction k, mol/h

ΔJ k :

large perturbation of flux of reaction k, mol/h

K i :

perturbation constants defined in Eq. (15)

r c :

calculated reaction rate vector

\( {\bar {\varvec{r}}}_{\rm m} \) :

measured reaction rates with the measured error vector

\( {\hat {\varvec{r}}}_{\rm m} \) :

reconciled measured reaction rate vector

r i :

enzyme activity amplification factor of reaction i

\({\hat {\varvec {\delta}}} \) :

estimated error vector using the variance–covariance matrix of measured errors

References

  1. Bailey JE (1991) Towards a science of metabolic engineering. Science 252:1668–1675

    CAS  PubMed  Google Scholar 

  2. Stephanopoulos G, Vallino JJ (1991) Network rigidity and metabolic engineering in metabolite over production Science 252:1675–1681

    Google Scholar 

  3. Vallino JJ, Stephanopoulos G (1993) Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine over production. Biotechnol Bioeng 41:633–646

    Google Scholar 

  4. Marx A, de Graaf AA, Wiechert W, Eggeling L, Sahm H (1996) Determination of the fluxes in the central carbon metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing. Biotechnol Bioeng 49:111–129

    Google Scholar 

  5. Small JR, Kacser H (1993) Response of metabolic systems to large changes in enzyme activities and effectors. 1. The linear treatment of unbranched chains. Europ J Biochem 213:613–624

    CAS  PubMed  Google Scholar 

  6. Stephanopoulos G, Simpson T (1997) Flux amplification in complex metabolic network. Chem Eng Sci 52:2607–2627

    Article  CAS  Google Scholar 

  7. Takiguchi N, Shimizu H, Shioya S (1997) An on-line physiological state recognition system for the lysine fermentation process based on a metabolic reaction model. Biotechnol Bioeng 55:170–181

    Google Scholar 

  8. Kawahara Y, Takahashi-Fuke K, Shimizu E, Nakamatsu E, Nakamori S (1997) Relationship between the glutamate production and the activity of 2-oxoglutarate dehydrogenase in Brevibacterium lactofermentum. Biosci Biotechnol Biochem 61:1109–1112

    CAS  Google Scholar 

  9. Eggeling L, Sham H (1999) L-glutamate and L-lysine: traditional products with impetuous development. Appl Microbiol Biotechnol 52:146–153

    CAS  Google Scholar 

  10. Stephanopoulos G, Aristidou A, Nielsen J (1998) Metabolic engineering. Academic, San Diego

  11. Eikmanns BJ, Rittmann D, Sahm H (1995) Cloning, sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme. J Bacteriol 177:774–782

    CAS  PubMed  Google Scholar 

  12. Bormann ER, Eikmanns EJ, Sahm H (1992) Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamate dehydrogenase. Molecular Microbiol 6:317–326

    CAS  Google Scholar 

  13. Rollin C, Morgant V, Guyonvarch A, Guerquin-Kern JL (1995) 13C-NMR studies of Corynebacterium melassecola. Europ J Biochem 227:488–493

    CAS  PubMed  Google Scholar 

  14. Guillouet S, Lessard PA, Sinskey AJ (2000) Investigating microbial metabolic pathways with NMR: metabolism of amino acids in corynebacteria. In: Barbotin JN, Portais JC (eds) NMR in microbiology: theory and applications. Horizon Scientific, Wymondham, UK, pp 259–281

    Google Scholar 

  15. Tesch M, de Graaf AA, Sahm H (1999) In vivo fluxes in the ammonium-assimilatory pathways in Corynebacterium glutamicum studied by 15 N nuclear magnetic resonance. Appl Environ Microbiol 65:1099–1109

    CAS  PubMed  Google Scholar 

  16. van der Heijden RTJM, Heijnen JJ, Hellinga C, Romein B, Luyben KCAM (1994) Linear constraint relations in biochemical reaction systems. I. Classification of the calculability and the balanceability of the conversion rates. Biotechnol Bioeng 43:3–10

    Google Scholar 

  17. van der Heijden RTJM, Romein B, Heijnen JJ, Hellinga C, Luyben KCAM (1994) Linear constraint relations in biochemical reaction systems. II. Diagnosis and estimation of gross errors. Biotechnol Bioeng 43:11–20

    Google Scholar 

  18. Pissara P, Nielsen J, Bazin M (1996) Pathway kinetics and metabolic control analysis. Bioetchnol Bioeng 51:168–176

    Article  Google Scholar 

  19. Yang C, Hua Q, Shimizu K (1999) Development of a kinetic model for L-lysine biosynthesis in Corynebacterium glutamicum and its application to metabolic control analysis. J Biosci Bioeng 88:393–403

    Article  CAS  Google Scholar 

  20. Ruyter GJ, Postma PW, van Dam K (1991) Control of glucose metabolism by enzyme IIGluc of the phosphoenolpyrvate-dependent phosphotranspherase system in Escherichia coli. J Bacteriol 173:6184–6191

    CAS  PubMed  Google Scholar 

  21. Small JR, Kacser H (1993) Response of metabolic systems to large changes in enzyme activities and effectors. 2. The linear treatment of branched pathways and metabolite concentrations, assessment of general non-linear case. Europ J Biochem 213:625–640

    CAS  PubMed  Google Scholar 

  22. Simpson TW, Shimizu H, Stephanopoulos G (1998) Experimental determination of group flux control coefficients in metabolic networks. Biotechnol Bioeng 58:149–153

    Article  CAS  PubMed  Google Scholar 

  23. Shimizu H, Tanaka H, Nakato A, Nagahisa K, Shioya H (2002) Metabolic control analysis in glutamate synthetic pathway. In: Marten M, Park TH, Nagamune T (eds) ACS Symposium Series, Biological systems engineering, vol 830. Oxford University Press, Oxford, pp 39–52

  24. Petersen S, Mack C, de Graaf A, Riedel C, Eikmanns BJ, Sahm H (2001) Metabolic consequences of altered phosphoenolpyruvate carboxykinase activity in Corynebacterium glutamicum reveal anaplerotic regulation mechanism in vivo. Metabol Eng 3:344–361

    Article  CAS  Google Scholar 

  25. Kimura E, Abe C, Kawahara Y, Nakamatsu T, Tokuda H (1997) A dtsR gene-disrupted mutant of Brevibacterium lactofermentum requires fatty acids for growth and efficiently reduces L-glutamate in the presence of an excess of biotin. Biophys Res Comm 234:157–161

    Article  CAS  PubMed  Google Scholar 

  26. Kimura E, Yagoshi C, Kawahara Y, Ohsumi T, Nakamatsu T, Tokuda H (1999) Glutamate overproduction in Corynebacterium glutamicum triggered by a decrease in the level of a complex comprising DtsR and a biotin-containing subunit. Biosci Biotechnol Biochem 63:1274–1278

    CAS  Google Scholar 

  27. HirasawaT, Wachi M, Nagai K (2000) A mutation in the Corynebacterium glutamicum ltsA gene causes susceptibility to lysozyme, temperature-sensitive growth, and L-glutamate production. J Bacteriol 182:2696–2701

    Article  CAS  PubMed  Google Scholar 

  28. Loos A, Glanemann C, Willis LB, O'Brien XM, Lessard PA, Gerstmeier R, Guillouet S, Sinskey A (2001) Development and validation of Corynebacterium glutamicum DNA microarrays. Appl Environ Microbiol 67:2310–2318

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 2–1 Yamadaoka, Suita, 565–0871, Osaka , Japan

    H. Shimizu & K. Nagahisa

  2. Department of Biotechnology, Graduate School of Engineering, Osaka University, 2–1 Yamadaoka, Suita, 565–0871, Osaka, Japan

    H. Tanaka, A. Nakato & S Shioya

  3. Fermentation and Biotechnology Lab., Ajinomoto Co. Ltd., 1–1 Suzuki Cho, Kawasaki-ku, 210–8681, Kawasaki, Japan

    E. Kimura

Authors
  1. H. Shimizu
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  2. H. Tanaka
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  4. K. Nagahisa
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  6. S Shioya
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Correspondence to S Shioya.

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Shimizu, H., Tanaka, H., Nakato, A. et al. Effects of the changes in enzyme activities on metabolic flux redistribution around the 2-oxoglutarate branch in glutamate production by Corynebacterium glutamicum . Bioprocess Biosyst Eng 25, 291–298 (2003). https://doi.org/10.1007/s00449-002-0307-8

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  • DOI: https://doi.org/10.1007/s00449-002-0307-8

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