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Influence of l-isoleucine and pantothenate auxotrophy for l-valine formation in Corynebacterium glutamicum revisited by metabolome analyses

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Abstract

The effect of different amounts of supplemented l-isoleucine and pantothenate has been analysed with the auxotrophic strain Corynebacterium glutamicum ΔilvA ΔpanB, showing that the final biomass concentration of this preliminary l-valine production strain can be controlled by the amount of added l-isoleucine. One gramme cell dry weight is formed from 48 μmol l-isoleucine. Different amounts of available pantothenate affect the intracellular pyruvate concentration. By limiting pantothenate supplementation from 0.8 to 0.1 μM, a 35-fold increase of cytoplasmic pyruvate up to 14.2 mM can be observed, resulting in the increased formation of l-valine, l-alanine and organic acids in the presence of low pantothenate concentrations. These findings can be used to redirect the carbon flux from glycolysis via pyruvate to the TCA cycle towards the desired product l-valine.

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References

  1. Sahm H, Eggeling L, de Graaf AA (2000) Pathway analysis and metabolic engineering in Corynebacterium glutamicum. Biol Chem 381:899–910

    Article  CAS  Google Scholar 

  2. Lee SY, Lee DY, Kim TY (2005) Systems biotechnology for strain improvement. Trends Biotechnol 23:349–358

    Article  CAS  Google Scholar 

  3. Mashego MR, Rumbold K, De Mey M, Vandamme E, Soetaert W, Heijnen JJ (2007) Microbial metabolomics: past, present and future methodologies. Biotechnol Lett 29:1–16

    Article  CAS  Google Scholar 

  4. Oldiges M, Lütz S, Pflug S, Schroer K, Stein N, Wiendahl C (2007) Metabolomics: current state and evolving methodologies and tools. Appl Microbiol Biotechnol 76:495–511

    Article  CAS  Google Scholar 

  5. Wendisch VF, Bott M, Kalinowski J, Oldiges M, Wiechert W (2006) Emerging Corynebacterium glutamicum systems biology. J Biotechnol 124:74–92

    Article  CAS  Google Scholar 

  6. Magnus JB, Hollwedel D, Oldiges M, Takors R (2006) Monitoring and modeling of the reaction dynamics in the valine/leucine synthesis pathway in Corynebacterium glutamicum. Biotechnol Prog 22:1071–1083

    Article  CAS  Google Scholar 

  7. Eggeling L, Pfefferle W, Sahm H (2001) Amino acids. In: Ratledge C, Bjoern K (eds) Basic biotechnology. Cambridge University Press, Cambridge, pp 281–303

    Google Scholar 

  8. Leyval D, Uy D, Delaunay S, Goergen JL, Engasser JM (2003) Characterisation of the enzyme activities involved in the valine biosynthetic pathway in a valine-producing strain of Corynebacterium glutamicum. J Biotechnol 104:241–252

    Article  CAS  Google Scholar 

  9. Hüser AT, Chassagnole C, Lindle ND, Merkamm M, Guyonvarch A, Elisakova V, Patek M, Kalinowski J, Brune I, Pühler A, Tauch A (2005) Rational design of a Corynebacterium glutamicum pantothenate production strain and its characterization by metabolic flux analysis and genome-wide transcriptional profiling. Appl Environ Microbiol 71:3255–3268

    Article  Google Scholar 

  10. Radmacher E, Vaitsikova A, Burger U, Krumbach K, Sahm H, Eggeling L (2002) Linking central metabolism with increased pathway flux: l-valine accumulation by Corynebacterium glutamicum. Appl Environ Microbiol 68:2246–2250

    Article  CAS  Google Scholar 

  11. Leonardi R, Zhang YM, Rock CO, Jackowski S (2005) Coenzyme A: back in action. Prog Lipid Res 44:125–153

    Article  CAS  Google Scholar 

  12. Cocaign-Bousquet M, Lindley ND (1995) Pyruvate overflow and carbon flux within the central metabolic pathways of Corynebacterium glutamicum during growth on lactate. Enzyme Microb Technol 17:260–267

    Article  CAS  Google Scholar 

  13. Elisakova V, Patek M, Holatko J, Nesvera JN, Leyval D, Goergen JL, Delaunay S (2005) Feedback-resistant acetohydroxy acid synthase increases valine production in Corynebacterium glutamicum. Appl Environ Microbiol 71:207–213

    Article  CAS  Google Scholar 

  14. Sambrook J, Russel DW (2001) Molecular cloning, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  15. Brik-Ternbach M, Bollman C, Wandrey C, Takors R (2005) Application of model discriminating experimental design for modeling and development of a fermentative fed-batch l-valine production process. Biotechnol Bioeng 91:356–368

    Article  Google Scholar 

  16. Zelic B, Vasic-Racki D, Wandrey C, Takors R (2004) Modeling of the pyruvate production with Escherichia coli in a fed-batch bioreactor. Bioprocess Biosyst Eng 26:249–258

    Article  CAS  Google Scholar 

  17. Luo B, Groenke K, Takors R, Wandrey C, Oldiges M (2007) Simultaneous determination of multiple intracellular metabolites in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle by liquid chromatography-mass spectrometry. J Chromatogr A 1147:153–164

    Article  CAS  Google Scholar 

  18. de Koning W, van Dam K (1992) A method for the determination of changes of glycolytic metabolites in yeast on a subsecond time scale using extraction at neutral ph. Anal Biochem 204:118–123

    Article  Google Scholar 

  19. Rönsch H, Krämer R, Morbach S (2003) Impact of osmotic stress on volume regulation, cytoplasmic solute composition and lysine production in Corynebacterium glutamicum MH20–22B. J Biotechnol 104:87–97

    Article  Google Scholar 

  20. Ruklisha M, Paegle L, Denina I (2007) l-valine biosynthesis during batch and fed-batch cultivations of Corynebacterium glutamicum: Relationship between changes in bacterial growth rate and intracellular metabolism. Process Biochem 42:634–640

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  22. Vadali RV, Bennett GN, San KY (2004) Cofactor engineering of intracellular CoA/acetyl-CoA and its effect on metabolic flux redistribution in Escherichia coli. Metab Eng 6:133–139

    Article  CAS  Google Scholar 

  23. Begley TP, Kinsland C, Strauss E (2001) The biosynthesis of coenzyme A in bacteria. In: Litwack G, Begley TP (eds) Vitamins and hormones—advances in research and applications, vol 61. Academic Press, San Diego, pp 157–171

  24. Schreiner ME, Fiur D, Holatkoj J, Patek M, Eikmanns BJ (2005) E1 enzyme of the pyruvate dehydrogenase complex in Corynebactetium glutamicum: molecular analysis of the gene and phylogenetic aspects. J Bacteriol 187:6005–6018

    Article  CAS  Google Scholar 

  25. Bott M (2007) Offering surprises: TCA cycle regulation in Corynebacterium glutamicum. Trends Microbiol 15:417–425

    Article  CAS  Google Scholar 

  26. Jackowski S, Rock CO (1986) Consequences of reduced intracellular coenzyme A content in Escherichia coli. J Bacteriol 166:866–871

    CAS  Google Scholar 

  27. Takayama K, Wang C, Besra GS (2005) Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clin Microbiol Rev 18:81–101

    Article  CAS  Google Scholar 

  28. Inui M, Murakami S, Okino S, Kawaguchi H, Vertes AA, Yukawa H (2004) Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J Mol Microbiol Biotechnol 7:182–196

    Article  CAS  Google Scholar 

  29. Tarmy EM, Kaplan NO (1968) Kinetics of Escherichia coli B d-lactate dehydrogenase and evidence for pyruvate-controlled change in conformation. J Biol Chem 243:2587–2596

    CAS  Google Scholar 

  30. Schreiner ME, Eikmanns BJ (2005) Pyruvate:quinone oxidoreductase from Corynebacterium glutamicum: purification and biochemical characterization. J Bacteriol 187:862–871

    Article  CAS  Google Scholar 

  31. Gerstmeir R, Wendisch VF, Schnicke S, Ruan H, Farwick M, Reinscheid D, Eikmanns BJ (2003) Acetate metabolism and its regulation in Corynebacterium glutamicum. J Biotechnol 104:99–122

    Article  CAS  Google Scholar 

  32. Wendisch VF, Spies M, Reinscheid DJ, Schnicke S, Sahm H, Eikmanns BJ (1997) Regulation of acetate metabolism in Corynebacterium glutamicum: transcriptional control of the isocitrate lyase and malate synthase genes. Arch Microbiol 168:262–269

    Article  CAS  Google Scholar 

  33. Richter H, Vlad D, Unden G (2001) Significance of pantothenate for glucose fermentation by Oenococcus oeni and for suppression of the erythritol and acetate production. Arch Microbiol 175:26–31

    Article  CAS  Google Scholar 

  34. An GH, Song KB, Sinskey AJ (1999) Redirection of carbon flux to lysine in a recombinant of Corynebacterium lactofermentum ATCC 21799 by limited supply of pantothenate. J Biosci Bioeng 88:168–172

    Article  CAS  Google Scholar 

  35. Marienhagen J, Kennerknecht N, Sahm H, Eggeling L (2005) Functional analysis of all aminotransferase proteins inferred from the genome sequence of Corynebacterium glutamicum. J Bacteriol 187:7639–7646

    Article  CAS  Google Scholar 

  36. Atkinson DE, Walton GM (1967) Adenosine triphosphate conservation in metabolic regulation—rat liver citrate cleavage enzyme. J Biol Chem 242:3239–3241

    CAS  Google Scholar 

  37. Wittmann C, Krömer JO, Kiefer P, Binz T, Heinzle E (2004) Impact of the cold shock phenomenon on quantification of intracellular metabolites in bacteria. Anal Biochem 327:135–139

    Article  CAS  Google Scholar 

  38. Leder IG (1972) Interrelated effects of cold shock and osmotic pressure on permeability of Escherichia coli membrane to permease accumulated substrates. J Bacteriol 11:211–219

    Google Scholar 

  39. Andersen KB, Meyenburg KV (1977) Charges of nicotinamide adenine-nucleotides and adenylate energy-charge as regulatory parameters of metabolism in Escherichia coli. J Biol Chem 252:4151–4156

    CAS  Google Scholar 

  40. Bolten CJ, Kiefer P, Letisse F, Portais JC, Wittmann C (2007) Sampling for metabolome analysis of microorganisms. Anal Chem 79:3843–3849

    Article  CAS  Google Scholar 

  41. Shiio I, Ujigawa-Takeda K (1980) Presence and regulation of α-ketoglutarate dehydrogenase complex in a glutamate-producing bacterium, Brevibacterium flavum. Agric Biol Chem 44:1897–1904

    CAS  Google Scholar 

  42. Blombach B, Schreiner ME, Holatko J, Bartek T, Oldiges M, Eikmanns BJ (2007) (l)-Valine production with pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum. Appl Environ Microbiol 73:2079–2084

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was financially supported by the Fachagentur Nachwachsende Rohstoffe of the BMVEL (grant 04NR003/22000304) and by Evonik Degussa GmbH. The authors thank Bernhard J. Eikmanns and Bastian Blombach at the University of Ulm as well as Robert Gerstmeir and Andreas Karau from Evonik Degussa GmbH for the fruitful cooperation and the valuable discussion of results. We also thank Lothar Eggeling for providing the used C. glutamicum ΔilvA ΔpanB.

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Correspondence to Marco Oldiges.

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Bartek, T., Makus, P., Klein, B. et al. Influence of l-isoleucine and pantothenate auxotrophy for l-valine formation in Corynebacterium glutamicum revisited by metabolome analyses. Bioprocess Biosyst Eng 31, 217–225 (2008). https://doi.org/10.1007/s00449-008-0202-z

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  • DOI: https://doi.org/10.1007/s00449-008-0202-z

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