Glutamate production by Corynebacterium glutamicum: dependence on the oxoglutarate dehydrogenase inhibitor protein OdhI and protein kinase PknG

  • Christian Schultz
  • Axel Niebisch
  • Lena Gebel
  • Michael Bott
Applied Microbial and Cell Physiology

Abstract

We recently showed that the activity of the 2-oxoglutarate dehydrogenase complex (ODHC) in Corynebacterium glutamicum is controlled by a novel regulatory mechanism that involves a 15-kDa protein called OdhI and serine/threonine protein kinase G (PknG). In its unphosphorylated state, OdhI binds to the E1 subunit (OdhA) of ODHC and, thereby, inhibits its activity. Inhibition is relieved by phosphorylation of OdhI at threonine-14 by PknG under conditions requiring high ODHC activity. In this work, evidence is provided that the dephosphorylation of phosphorylated OdhI is catalyzed by a phospho-Ser/Thr protein phosphatase encoded by the gene cg0062, designated ppp. As a decreased ODHC activity is important for glutamate synthesis, we investigated the role of OdhI and PknG for glutamate production under biotin limitation and after addition of Tween-40, penicillin, or ethambutol. A ΔodhI mutant formed only 1–13% of the glutamate synthesized by the wild type. Thus, OdhI is essential for efficient glutamate production. The effect of a pknG deletion on glutamate synthesis was dependent on the induction conditions. Under strong biotin limitation and in the presence of ethambutol, the ΔpknG mutant showed significantly increased glutamate production, offering a new way to improve production strains.

Keywords

Corynebacterium glutamicum Glutamate OdhI 2-Oxoglutarate dehydrogenase Oxoglutarate dehydrogenase inhibitor PknG Serine/threonine protein kinase 

Abbreviations

cdw

cell dry weight

PknG

serine/threonine protein kinase G

ODHC

2-oxoglutarate dehydrogenase complex

OdhI

oxoglutarate dehydrogenase inhibitor protein

TCA cycle

tricarboxylic acid cycle

References

  1. Asakura Y, Kimura E, Usuda Y, Kawahara Y, Matsui K, Osumi T, Nakamatsu T (2007) Altered metabolic flux due to deletion of odhA causes l-glutamate overproduction in Corynebacterium glutamicum. Appl Environ Microbiol 73:1308–1319CrossRefGoogle Scholar
  2. Delaunay S, Gourdon P, Lapujade P, Mailly E, Oriol E, Engasser JM, Lindley ND, Goergen JL (1999) An improved temperature triggered process for glutamate production with Corynebacterium glutamicum. Enzyme Microb Technol 25:762–768CrossRefGoogle Scholar
  3. Duperray F, Jezequel D, Ghazi A, Letellier L, Shechter, E. (1992) Excretion of glutamate from Corynebacterium glutamicum triggered by amine surfactants. Biochim Biophys Acta 1103:250–258CrossRefGoogle Scholar
  4. Eggeling L, Bott M (2005) Handbook of Corynebacterium glutamicum. CRC Press, Boca Raton, FL, USAGoogle Scholar
  5. Eggeling L, Krumbach K, Sahm H (2001) l-glutamate efflux with Corynebacterium glutamicum: why is penicillin treatment or Tween addition doing the same? J Mol Microbiol Biotechnol 3:67–68Google Scholar
  6. Gutmann M, Hoischen C, Krämer R (1992) Carrier-mediated glutamate secretion by Corynebacterium glutamicum under biotin limitation. Biochim Biophys Acta 1112:115–123CrossRefGoogle Scholar
  7. Hanahan D (1985) Techniques for transformation of E. coli. In: Glover DM (eds) DNA-Cloning: a practical approach. IRL-Press, Oxford, pp 109–135Google Scholar
  8. Hermann T (2003) Industrial production of amino acids by coryneform bacteria. J Biotechnol 104:155–172CrossRefGoogle Scholar
  9. Hoischen C, Krämer R (1989) Evidence for an efflux carrier system involved in the secretion of glutamate by Corynebacterium glutamicum. Arch Microbiol 151:342–347CrossRefGoogle Scholar
  10. Hoischen C, Krämer R (1990) Membrane alteration is necessary but not sufficient for effective glutamate secretion in Corynebacterium glutamicum. J Bacteriol 172:3409–3416Google Scholar
  11. Kabus A, Niebisch A, Bott M (2007) Role of cytochrome bd oxidase from Corynebacterium glutamicum in growth and lysine production. Appl Environ Microbiol 73:861–868CrossRefGoogle Scholar
  12. 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, Krämer R, Linke B, McHardy AC, Meyer F, Möckel B, Pfefferle W, Pühler A, Rey DA, Rückert C, Rupp O, Sahm H, Wendisch VF, Wiegräbe 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
  13. Kanzaki T, Isobe K, Okazaki H, Motizuki K, Fukuda H (1967) l-Glutamic acid production. Part I. Selection of an oleic acid-requiring mutant and its properties. Agric Biol Chem 31:1307–1313Google Scholar
  14. Kataoka M, Hashimoto KI, Yoshida M, Nakamatsu T, Horinouchi S, Kawasaki H (2006) Gene expression of Corynebacterium glutamicum in response to the conditions inducing glutamate overproduction. Lett Appl Microbiol 42:471–476CrossRefGoogle Scholar
  15. Kawahara Y, Takahashi-Fuke K, Shimizu E, Nakamatsu T, Nakamori S (1997) Relationship between the glutamate production and the activity of 2-oxoglutarate dehydrogenase in Brevibacterium lactofermentum. Biosci Biotechnol Biochem 61:1109–1112CrossRefGoogle Scholar
  16. Keilhauer C, Eggeling L, Sahm H (1993) Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvBilvNilvC operon. J Bacteriol 175:5595–5603Google Scholar
  17. Kimura E (2002) Triggering mechanism of l-glutamate overproduction by DtsR1 in coryneform bacteria. J Biosci Bioeng 94:545–551Google Scholar
  18. 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 produces l-glutamate in the presence of an excess of biotin. Biochem Biophys Res Commun 234:157–161CrossRefGoogle Scholar
  19. Kinoshita S, Udaka S, Shimono M (1957) Studies on the amino acid fermentation. I. Production of l-glutamic acid by various microorganisms. J Gen Appl Microbiol 3:193–205Google Scholar
  20. Krug A, Wendisch VF, Bott M (2005) Identification of AcnR, a TetR-type repressor of the aconitase gene acn in Corynebacterium glutamicum. J Biol Chem 280:585–595Google Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  22. Liebl W (2005) Corynebacterium taxonomy. In: Eggeling L, Bott M (eds) Handbook of Corynebacterium glutamicum. CRC Press, Boca Raton, pp 9–34Google Scholar
  23. Momose H, Takagi T (1978) Glutamic acid production in biotin-rich media by temperature sensitive mutants of Brevibacterium lactofermentum, a novel fermentation process. Agric Biol Chem 42:1911–1917Google Scholar
  24. Nakao Y, Kikuchi M, Suzuki M, Doi M (1972) Microbial production of l-glutamic acid by glycerol auxotrophs. I. Induction of glycerol auxotrophs and production of l-glutamic acid from n-paraffins. Agric Biol Chem 36:490–496Google Scholar
  25. Nampoothiri KM, Hoischen C, Bathe B, Möckel B, Pfefferle W, Krumbach K, Sahm H, Eggeling L (2002) Expression of genes of lipid synthesis and altered lipid composition modulates l-glutamate efflux of Corynebacterium glutamicum. Appl Microbiol Biotechnol 58:89–96CrossRefGoogle Scholar
  26. Niebisch A, Bott M (2001) Molecular analysis of the cytochrome bc 1aa 3 branch of the Corynebacterium glutamicum respiratory chain containing an unusual diheme cytochrome c 1. Arch Microbiol 175:282–294CrossRefGoogle Scholar
  27. Niebisch A, Kabus A, Schultz C, Weil B, Bott M (2006) Corynebacterial protein kinase G controls 2-oxoglutarate dehydrogenase activity via the phosphorylation status of the OdhI protein. J Biol Chem 281:12300–12307CrossRefGoogle Scholar
  28. Nunheimer TD, Birnbaum J, Ihnen ED, Demain AL (1970) Product inhibition of the fermentative formation of glutamic acid. Appl Microbiol 20:215–217Google Scholar
  29. Okazaki H, Kanzaki T, Doi M, Sumino Y, Fukuda H (1967) l-Glutamic acid fermentation. Part II. The production of l-glutamic acid by an oleic acid-requiring mutant. Agric Biol Chem 31:1314–1317Google Scholar
  30. Radmacher E, Stansen KC, Besra GS, Alderwick LJ, Maughan WN, Hollweg G, Sahm H, Wendisch VF, Eggeling L (2005) Ethambutol, a cell wall inhibitor of Mycobacterium tuberculosis, elicits l-glutamate efflux of Corynebacterium glutamicum. Microbiology 151:1359–1368CrossRefGoogle Scholar
  31. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  32. 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
  33. Shiio I, Ujigawa-Takeda K (1980) Presence and regulation of α-ketoglutarate dehydrogenase complex in a glutamate producing bacterium, Brevibacterium flavum. Agric Biol Chem 44:1897–1904Google Scholar
  34. Shiio I, Otsuka SI, Takahashi M (1962a) Effect of biotin on the bacterial formation of glutamic acid. I. Glutamate formation and cellular permeability of amino acids. J Biochem 51:56–62Google Scholar
  35. Shiio I, Otsuka SI, Katsuya N (1962b) Effect of biotin on the bacterial formation of glutamic acid. II. Metabolism of glucose. J Biochem 52:108–116Google Scholar
  36. Shimizu H, Tanaka H, Nakato A, Nagahisa K, Kimura E, Shioya S (2003) 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 25:291–298Google Scholar
  37. Shingu H, Terui G (1971) Studies on the process of glutamic acid fermentation at the enzyme level: I. On the changes of α-ketoglutaric acid dehydrogenase in the course of culture. J Ferment Technol 49:400–405Google Scholar
  38. Shirai T, Nakato A, Izutani N, Nagahisa K, Shioya S, Kimura E, Kawarabayasi Y, Yamagishi A, Gojobori T, Shimizu H (2005) Comparative study of flux redistribution of metabolic pathway in glutamate production by two coryneform bacteria. Metab Eng 7:59–69CrossRefGoogle Scholar
  39. Takinami K, Yoshii H, Tsuri H, Okada H (1965) Biochemical effects of fatty acid and its derivatives on l-glutamic acid fermentation: III. Biotin–Tween 60 relationship in the accumulation of l-glutamic acid and the growth of Brevibacterium lactofermentum. Agric Biol Chem 29:351–359Google Scholar
  40. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354CrossRefGoogle Scholar
  41. Usuda Y, Tujimoto N, Abe C, Asakura Y, Kimura E, Kawahara Y, Kurahashi O, Matsui H (1996) Molecular cloning of the Corynebacterium glutamicum (‘Brevibacterium lactofermentum’ AJ12036) odhA gene encoding a novel type of 2-oxoglutarate dehydrogenase. Microbiology 142:3347–3354CrossRefGoogle Scholar
  42. Uy D, Delaunay S, Goergen JL, Engasser JM (2005) Dynamics of glutamate synthesis and excretion fluxes in batch and continuous cultures of temperature-triggered Corynebacterium glutamicum. Bioprocess Biosyst Eng 27:153–162CrossRefGoogle Scholar
  43. van der Rest ME, Lange C, Molenaar D (1999) A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA. Appl Microbiol Biotechnol 52:541–545CrossRefGoogle Scholar
  44. Villarino A, Duran R, Wehenkel A, Fernandez P, England P, Brodin P, Cole ST, Zimny-Arndt U, Jungblut PR, Cervenansky C, Alzari PM (2005) Proteomic identification of M. tuberculosis protein kinase substrates: PknB recruits GarA, a FHA domain-containing protein, through activation loop-mediated interactions. J Mol Biol 350:953–963CrossRefGoogle Scholar
  45. Wennerhold J, Bott M (2006) The DtxR regulon of Corynebacterium glutamicum. J Bacteriol 188:2907–2918CrossRefGoogle Scholar
  46. Wennerhold J, Krug A, Bott M (2005) The AraC-type regulator RipA represses aconitase and other iron proteins from Corynebacterium under iron limitation and is itself repressed by DtxR. J Biol Chem 280:40500–40508CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Christian Schultz
    • 1
  • Axel Niebisch
    • 1
  • Lena Gebel
    • 1
  • Michael Bott
    • 1
  1. 1.Institut für Biotechnologie 1Forschungszentrum JülichJülichGermany

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