The three tricarboxylate synthase activities of Corynebacterium glutamicum and increase of l-lysine synthesis

  • Eva Radmacher
  • Lothar EggelingEmail author
Biotechnologically Relevant Enzymes and Proteins


Corynebacterium glutamicum owns a citrate synthase and two methylcitrate synthases. Characterization of the isolated enzymes showed that the two methylcitrate synthases have comparable catalytic efficiency, k cat/K m, as the citrate synthase with acetyl-CoA as substrate, although these enzymes are only synthesized during growth on propionate-containing media. Thus, the methylcitrate synthases have a relaxed substrate specifity, as also demonstrated by their activity with butyryl-CoA, whereas the citrate synthase does not accept acyl donors other than acetyl-CoA. A double mutant deleted of the citrate synthase gene gltA and one of the methylcitrate synthase genes, prpC1, was made unable to grow on glucose. From this mutant, a collection of suppressor mutants could be isolated which were demonstrated to have regained citrate synthase activity due to the relaxed specificity of the methylcitrate synthase PrpC2. Molecular characterization of these mutants showed that the regulator PrpR (Cg0800) located downstream of prpC1 is mutated with mutations likely to effect the secondary structure of the regulator, thus, resulting in expression of prpC2. This expression results in a citrate synthase activity, which is lower than that due to gltA in the original strain and results in increased l-lysine accumulation.


Citrate synthase Methylcitrate synthase MerR-type regulator Regulator mutations l-lysine production 



This paper is dedicated to Hermann Sahm on the occasion of his 65th birthday. Thanks are due to him for his untiring and extremely successful efforts at turning C. glutamicum into a research subject. I (LE) would like to thank him personally for his confidence in me over a period of several decades, permitting me the freedom to choose and implement research projects of the most varied nature. This work is part of BMBF project 0313704.


  1. Ayed A, Krutchinsky AN, Ens W, Standing KG, Duckworth HW (1988) Quantitative evaluation of protein–protein and ligand–protein equilibria of a large allosteric enzyme by electrospray ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 12:339–344CrossRefGoogle Scholar
  2. Bensadoun A, Weinstein D (1976) Assay of proteins in the presence of interfering materials. Anal Biochem 70:241–250CrossRefGoogle Scholar
  3. Brock M, Darley D, Textor S, Buckel W (2001) 2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans: characterization and comparison of both enzymes. Eur J Biochem 268:3577–3586CrossRefGoogle Scholar
  4. Brune I, Brinkrolf K, Kalinowski J, Pühler A, Tauch A (2005) The individual and common repertoire of DNA-binding transcriptional regulators of Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium diphtheriae and Corynebacterium jeikeium deduced from the complete genome sequences. BMC Genomics 6(1):86 DOI  10.1186/1471-2164-6-86
  5. Claes AW, Pühler A, Kalinowski J (2002) Identification of two prpDBC gene clusters in Corynebacterium glutamicum and their involvement in propionate degradation via the 2-methylcitrate cycle. J Bacteriol 184:2728–2739CrossRefGoogle Scholar
  6. de Graaf AA, Eggeling L, Sahm H (2001) Metabolic engineering for L-lysine production by Corynebacterium glutamicum. Adv Biochem Eng Biotechnol 73:9–29Google Scholar
  7. Eggeling L, Reyes O (2005) Experiments. In: Eggeling L, Bott M (eds) Handbook of Corynebacterium glutamicum. CRC, Taylor and Francis, Roca Baton, Florida, USA, pp 535–565Google Scholar
  8. Eikmanns BJ, Thum-Schmitz N, Eggeling L, Lüdtke KU, Sahm H (1994) Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase. Microbiology 140:1817–1828Google Scholar
  9. Ewering C, Bramer CO, Bruland N, Bethke A, Steinbüchel A (2006) Occurrence and expression of tricarboxylate synthases in Ralstonia eutropha. Appl Microbiol Biotechnol 71:80–89CrossRefGoogle Scholar
  10. Georgi T, Rittmann D, Wendisch VF (2005) Lysine and glutamate production by Corynebacterium glutamicum on glucose, fructose and sucrose: roles of malic enzyme and fructose-1,6-bisphosphatase. Metab Eng 7:291–301CrossRefGoogle Scholar
  11. Gerike U, Hough DW, Russell NJ, Dyall-Smith ML, Danson MJ (1998) Citrate synthase and 2-methylcitrate synthase: structural, functional and evolutionary relationships. Microbiology 144:929–935CrossRefGoogle Scholar
  12. Gerike U, Danson MJ, Hough DW (2001) Cold-active citrate synthase: mutagenesis of active-site residues. Protein Eng 14:655–661CrossRefGoogle Scholar
  13. Heldwein EE, Brennan RG (2001) Crystal structure of the transcription activator BmrR bound to DNA and a drug. Nature 409:378–382CrossRefGoogle Scholar
  14. Hüser AT, Becker A, Brune I, Dondrup M, Kalinowski J, Plassmeier J, Pühler A, Wiegrabe I, Tauch A (2003) Development of a Corynebacterium glutamicum DNA microarray and validation by genome-wide expression profiling during growth with propionate as carbon source. J Biotechnol 106:269–286CrossRefGoogle Scholar
  15. Kalinowski J, Bathe B, Bartels D, Bischoff N, Bott M, Burkovski A, Dusch N, Eggeling L, Eikmanns BJ, Gaigalat L, Goesmann A, Hartmann M, Huthmacher K, Kramer R, Linke B, McHardy AC, Meyer F, Mockel B, Pfefferle W, Pühler A, Rey DA, Rückert C, Rupp O, Sahm H, Wendisch VF, Wiegrabe I, Tauch A (2003) The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of l-aspartate-derived amino acids and vitamins. J Biotechnol 104:5–25CrossRefGoogle Scholar
  16. Karlsson EN, Abou-Hachem M, Holst O, Danson MJ, Hough DW (2002) Rhodothermus marinus: a thermophilic bacterium producing dimeric and hexameric citrate synthase isoenzymes. Extremophiles 6:51–56CrossRefGoogle Scholar
  17. 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 49:1–129CrossRefGoogle Scholar
  18. Marx A, Striegel K, de Graaf AA, Sahm H, Eggeling L (1997) Response of the central metabolism of Corynebacterium glutamicum to different flux burdens. Biotechnol Bioeng 56:168–180CrossRefGoogle Scholar
  19. Marx A, Eikmanns BJ, Sahm H, de Graaf AA, Eggeling L (1999) Response of the central metabolism in Corynebacterium glutamicum to the use of an NADH-dependent glutamate dehydrogenase. Metab Eng 1:35–48CrossRefGoogle Scholar
  20. Marx A, Hans S, Mockel B, Bathe B, de Graaf AA, McCormack AC, Stapleton C, Burke K, O’Donohue M, Dunican LK (2003) Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum. J Biotechnol 104:185–197CrossRefGoogle Scholar
  21. Mitsuhashi S, Hayashi M, Ohnishi J, Ikeda M (2006) Disruption of malate:quinone oxidoreductase increases l-lysine production by Corynebacterium glutamicum. Biosci Biotechnol Biochem 70:2803–2806CrossRefGoogle Scholar
  22. Ohnishi J, Hayashi M, Mitsuhashi S, Ikeda M (2003) Efficient 40 degrees C fermentation of l-lysine by a new Corynebacterium glutamicum mutant developed by genome breeding. Appl Microbiol Biotechnol 62:69–75CrossRefGoogle Scholar
  23. Petersen S, de Graaf AA, Eggeling L, Mollney M, Wiechert W, Sahm H (2000) In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum. J Biol Chem 275:35932–35941CrossRefGoogle Scholar
  24. Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73CrossRefGoogle Scholar
  25. Shiio I, Ozaki H, Ujigawa K (1977) Regulation of citrate synthase in Brevibacterium flavum, a glutamate-producing bacterium. J Biochem (Tokyo) 82:395–405Google Scholar
  26. Shiio I, Ozaki H, Ujigawa-Takeda K (1982) Production of aspartic acid and lysine by citrate synthase mutants of Brevibacterium flavum. Agric Biol Chem 46:101–107Google Scholar
  27. Shiio I, Shin-ichi I, Kazue K (1993a) Isolation and Properties of α-ketobutyrate-resistant lysine-producing mutants from Brevibacterium flavum. Biosci Biotechnol Biochem 57:51–55CrossRefGoogle Scholar
  28. Shiio I, Sugimoto S, Kawamura K (1993b) Isolation and properties of α-ketobutyrate-resistant lysine-producing mutants from Brevibacterium flavum. Biosci Biotechnol Biochem 57:51–55Google Scholar
  29. Tong EK, Duckworth HW (1975) The quaternary structure of citrate synthase from Escherichia coli K12. Biochemistry 14:235–241CrossRefGoogle Scholar
  30. Wiegand G, Remington SJ (1986) Citrate synthase: structure, control, and mechanism. Annu Rev Biophys Biophys Chem 15:97–117CrossRefGoogle Scholar
  31. Wittmann C, Heinzle E (2002) Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria. Appl Environ Microbiol 68:5843–5859CrossRefGoogle Scholar
  32. Wittman C, de Graaf AA (2005) Metabolix flux analysis in Corynebacterium glutamicum. In: Eggeling L, Bott M (eds) Handbook of Corynebacterium glutamicum. CRC, Taylor and Francis, Roca Baton, Florida, USA, pp 277–303Google Scholar
  33. Yokota A, Shiio I (1988) Effects of reduced citrate synthase activity and feedback-resistant phosphoenolpyruvate carboxylase on lysine productivities of Brevibacterium flavurn mutants. Agric Biol Chem 52:455–463Google Scholar

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© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Institute for BiotechnologyResearch Centre JuelichJuelichGermany

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