Bioprocess and Biosystems Engineering

, Volume 33, Issue 7, pp 873–883 | Cite as

Studies on substrate utilisation in l-valine-producing Corynebacterium glutamicum strains deficient in pyruvate dehydrogenase complex

  • Tobias Bartek
  • Christiane Rudolf
  • Ulrike Kerßen
  • Bianca Klein
  • Bastian Blombach
  • Siegmund Lang
  • Bernhard J. Eikmanns
  • Marco OldigesEmail author
Original Paper


The pyruvate dehydrogenase complex was deleted to increase precursor availability in Corynebacterium glutamicum strains overproducing l-valine. The resulting auxotrophy is treated by adding acetate in addition glucose for growth, resulting in the puzzling fact of gluconeogenic growth with strongly reduced glucose uptake in the presence of acetate in the medium. This result was proven by intracellular metabolite analysis and labelling experiments. To increase productivity, the SugR protein involved in negative regulation of the phosphotransferase system, was inactivated, resulting in enhanced consumption of glucose. However, the surplus in substrate uptake was not converted to l-valine; instead, the formation of up to 289 μM xylulose was observed for the first time in C. glutamicum. As an alternative to the genetic engineering solution, a straightforward process engineering approach is proposed. Acetate limitation resulted in a more efficient use of acetate as cosubstrate, shown by an increased biomass yield Y X/Ac and improved l-valine formation.


Corynebacterium glutamicum l-Valine Pyruvate dehydrogenase complex Substrate uptake Fermentation process development Xylulose 



This work was financially supported by the Fachagentur Nachwachsende Rohstoffe of the BMVEL—Federal Ministry of Food, Agriculture and Consumer Protection—(grant 04NR003/22000304) and by Evonik Degussa GmbH. The authors wish to thank Verena Engels from IBT 1 of Forschungszentrum Jülich GmbH as well as Robert Gerstmeir and Andreas Karau from Evonik Degussa GmbH for fruitful cooperation and the valuable discussion of results, and Pia Makus for her assistance in performing the experiments.


  1. 1.
    Sugisaki Z (1959) Studies on l-valine fermentation. Part 1- Production of l-valine by Aerobacter Bacteria. J Gen Appl Microbiol 5:138–149CrossRefGoogle Scholar
  2. 2.
    Eggeling L, Pfefferle W, Sahm H (2001) Amino acids. In: Colin R, Bjoern K (eds) Basic biotechnology. Cambridge University Press, Cambridge, New York, pp 281–303Google Scholar
  3. 3.
    Leuchtenberger W, Huthmacher K, Drauz K (2005) Biotechnological production of amino acids and derivatives: current status and prospects. Appl Microbiol Biotechnol 69:1–8CrossRefGoogle Scholar
  4. 4.
    Demain AL, Adrio JL (2008) Contributions of microorganisms to industrial biology. Mol Biotechnol 38:41–55CrossRefGoogle Scholar
  5. 5.
    Bartek T, Makus P, Klein B, Lang S, Oldiges M (2008) Influence of l-isoleucine and pantothenate auxotrophy for l-valine formation in Corynebacterium glutamicum revisited by metabolome analyses. Bioprocess Biosyst Eng 31:217–225CrossRefGoogle Scholar
  6. 6.
    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 68:2246–2250CrossRefGoogle Scholar
  7. 7.
    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 73:2079–2084CrossRefGoogle Scholar
  8. 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 104:241–252CrossRefGoogle Scholar
  9. 9.
    Bartek T, Blombach B, Zönnchen E, Makus P, Lang S, Eikmanns BJ, Oldiges M. Importance of NADPH supply for improved l-valine formation in Corynebacterium glutamicum. Biotechnol Prog. doi: 10.1002/btpr.345
  10. 10.
    Blombach B, Schreiner ME, Bartek T, Oldiges M, Eikmanns BJ (2008) Corynebacterium glutamicum tailored for high-yield l-valine production. Appl Microbiol Biotechnol 79:471–479CrossRefGoogle Scholar
  11. 11.
    Wendisch VF, Bott M, Kalinowski J, Oldiges M, Wiechert W (2006) Emerging Corynebacterium glutamicum systems biology. J Biotechnol 124:74–92CrossRefGoogle Scholar
  12. 12.
    Mori M, Shiio I (1987) Phosphoenolypyruvate—sugar phosphotransferase systems and sugar metabolism in Brevibacterium flavum. Agric Biol Chem 51:2671–2678Google Scholar
  13. 13.
    Yokota A, Lindley ND (2005) Central metabolism: sugar uptake and conversion. In: Bott M, Eggeling L (eds) Handbook of Corynebacterium glutamicum. Taylor and Francis, Boca Raton, pp 215–240Google Scholar
  14. 14.
    Engels V, Wendisch VF (2007) The DeoR-type regulator SugR represses expression of ptsG in Corynebacterium glutamicum. J Bacteriol 189:2955–2966CrossRefGoogle Scholar
  15. 15.
    Gaigalat L, Schlüter J-P, Hartmann M, Mormann S, Tauch A, Pühler A, Kalinowski J (2007) The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Corynebacterium glutamicum. BMC Mol Biol 8:104CrossRefGoogle Scholar
  16. 16.
    Tanaka Y, Teramoto H, Inui M, Yukawa H (2008) Regulation of expression of general components of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) by the global regulator SugR in Corynebacterium glutamicum. Appl Microbiol Biotechnol 78:309–318CrossRefGoogle Scholar
  17. 17.
    Oldiges M, Lütz S, Pflug S, Schroer K, Stein N, Wiendahl C (2007) Metabolomics: current state and evolving methodologies and tools. Appl Microbiol Biotechnol 76:495–511CrossRefGoogle Scholar
  18. 18.
    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 1147:153–164CrossRefGoogle Scholar
  19. 19.
    Villas-Boas SG, Mas S, Akesson M, Smedsgaard J, Nielsen J (2005) Mass spectrometry in metabolome analysis. Mass Spectrom Rev 24:613–646CrossRefGoogle Scholar
  20. 20.
    Schreiner ME, Eikmanns BJ (2005) Pyruvate : quinone oxidoreductase from Corynebacterium glutamicum: purification and biochemical characterization. J Bacteriol 187:862–871CrossRefGoogle Scholar
  21. 21.
    Blombach B, Arndt A, Auchter M, Eikmanns BJ (2009) l-Valine production during growth of pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum in the presence of ethanol or by inactivation of the transcriptional regulator SugR. Appl Environ Microbiol 75:1197–1200CrossRefGoogle Scholar
  22. 22.
    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 91:356–368CrossRefGoogle Scholar
  23. 23.
    Link T, Backstrom M, Graham R, Essers R, Zorner K, Gatgens J, Burchell J, Taylor-Papadimitriou J, Hansson GC, Noll T (2004) Bioprocess development for the production of a recombinant MUC1 fusion protein expressed by CHO-K1 cells in protein-free medium. J Biotechnol 110:51–62CrossRefGoogle Scholar
  24. 24.
    Zelic B, Gostovic S, Vuorilehto K, Vasic-Racki B, Takors R (2004) Process strategies to enhance pyruvate production with recombinant Escherichia coli: from repetitive fed-batch to in situ product recovery with fully integrated electrodialysis. Biotechnol Bioeng 85:638–646CrossRefGoogle Scholar
  25. 25.
    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 204:118–123CrossRefGoogle Scholar
  26. 26.
    Thiele B, Füllner K, Stein N, Oldiges M, Kuhn AJ, Hofmann D (2008) Analysis of amino acids without derivatization in barley extracts by LC–MS–MS. Anal Bioanal Chem. doi: 10.1007/s00216-008-2167-9
  27. 27.
    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 104:87–97CrossRefGoogle Scholar
  28. 28.
    Sambrook J, Russel DW (2001) Molecular cloning. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  29. 29.
    Shimizu K (2004) Metabolic flux analysis based on C-13-labeling experiments and integration of the information with gene and protein expression patterns. In: Recent Progress of biochemical and biomedical engineering in Japan Ii. Springer, Berlin, pp 1–49Google Scholar
  30. 30.
    Strelkov S, von Elstermann M, Schomburg D (2004) Comprehensive analysis of metabolites in Corynebacterium glutamicum by gas chromatography/mass spectrometry. Biol Chem 385:853–861CrossRefGoogle Scholar
  31. 31.
    Wagner C, Sefkow M, Kopka J (2003) Construction and application of a mass spectral and retention time index database generated from plant GC/EI–TOF–MS metabolite profiles. Phytochemistry 62:887–900CrossRefGoogle Scholar
  32. 32.
    Rogatsky E, Jayatillake H, Goswami G, Tomuta V, Stein D (2005) Sensitive LC MS quantitative analysis of carbohydrates by Cs+ attachment. J Am Soc Mass Spectrom 16:1805–1811CrossRefGoogle Scholar
  33. 33.
    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 104:99–122CrossRefGoogle Scholar
  34. 34.
    Wendisch VF, De Graaf AA, Sahm H, Eikmanns BJ (2000) Quantitative determination of metabolic fluxes during coutilization of two carbon sources: comparative analyses with Corynebacterium glutamicum during growth on acetate and/or glucose. J Bacteriol 182:3088–3096CrossRefGoogle Scholar
  35. 35.
    Blombach B, Schreiner ME, Moch M, Oldiges M, Eikmanns BJ (2007) Effect of pyruvate dehydrogenase complex deficiency on L-lysine production with Corynebacterium glutamicum. Appl Microbiol Biotechnol 76:615–623CrossRefGoogle Scholar
  36. 36.
    Gourdon P, Raherimandimby M, Dominguez H, Cocaign-Bousquet M, Lindley ND (2003) Osmotic stress, glucose transport capacity and consequences for glutamate overproduction in Corynebacterium glutamicum. J Biotechnol 104:77–85CrossRefGoogle Scholar
  37. 37.
    Dover LG, Cerdeno-Tarraga AM, Pallen MJ, Parkhill J, Besra GS (2004) Comparative cell wall core biosynthesis in the mycolated pathogens, Mycobacterium tuberculosis and Corynebacterium diphtheriae. FEMS Microbiol Rev 28:225–250CrossRefGoogle Scholar
  38. 38.
    Wolf A, Krämer R, Morbach S (2003) Three pathways for trehalose metabolism in Corynebacterium glutamicum ATCC13032 and their significance in response to osmotic stress. Mol Microbiol 49:1119–1134CrossRefGoogle Scholar
  39. 39.
    Jolkver E, Emer D, Ballan S, Krämer R, Eikmanns BJ, Marin K (2009) Identification and characterization of a bacterial transport system for the uptake of pyruvate, propionate, and acetate in Corynebacterium glutamicum. J Bacteriol 191:940–948CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Tobias Bartek
    • 1
    • 4
  • Christiane Rudolf
    • 1
  • Ulrike Kerßen
    • 1
  • Bianca Klein
    • 1
  • Bastian Blombach
    • 2
  • Siegmund Lang
    • 3
  • Bernhard J. Eikmanns
    • 2
  • Marco Oldiges
    • 1
    Email author
  1. 1.Institute of Biotechnology 2Forschungszentrum JülichJülichGermany
  2. 2.Institute of Microbiology and BiotechnologyUniversity of UlmUlmGermany
  3. 3.Institute for Biochemistry and BiotechnologyBraunschweig University of TechnologyBraunschweigGermany
  4. 4.Lonza BiopharmaceuticalsR&D Microbial Services, Lonza AGVispSwitzerland

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