Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Effect of odhA overexpression and odhA antisense RNA expression on Tween-40-triggered glutamate production by Corynebacterium glutamicum


Recent studies have suggested that a decrease in the specific activity of the 2-oxoglutarate dehydrogenase complex (ODHC) is important for glutamate overproduction by Corynebacterium glutamicum. To further investigate the role of the odhA gene and its product in this process, we constructed the recombinant strains of C. glutamicum in which the expression of the odhA and its product could be controlled by odhA overexpression and odhA antisense RNA expression. We examined changes in glutamate production and ODHC specific activity of the constructed strains during glutamate production triggered by Tween 40 addition. The ODHC specific activity increased with odhA overexpression, resulting in dramatically reduced glutamate production despite Tween 40 addition, indicating that a decrease in the specific activity of ODHC is required for glutamate production induced by Tween 40 addition. However, odhA antisense RNA expression alone did not result in glutamate overproduction in spite of the decrease in ODHC specific activity. Rather, it enhanced glutamate production triggered by Tween 40 addition due to the additional decrease in ODHC specific activity, suggesting that odhA antisense RNA expression is effective in enhancing Tween-40-triggered glutamate overproduction. Our results suggest that a change in ODHC specific activity is critical but is not the only factor responsible for glutamate overproduction by C. glutamicum.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  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 73:1308–1319

  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

  3. Desai RP, Papoutsakis ET (1999) Antisense RNA strategies for the metabolic engineering of Clostridium acetobutylicum. Appl Environ Microbiol 65:936–945

  4. Gutmann M, Hoischen C, Krämer R (1992) Carrier-mediated glutamate secretion by Corynebacterium glutamicum under biotin limitation. Biochim Biophys Acta 1112:115–123

  5. Hoischen C, Krämer R (1989) Evidence for an efflux carrier system involved in the secretion of glutamate by Corynebacterium glutamicum. Arch Microbiol 151:342–347

  6. Ikeda M, Nakagawa S (2003) The Corynebacterium glutamicum genome: features and impacts on biotechnological processes. Appl Microbiol Biotechnol 62:99–109

  7. 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 104:5–25

  8. 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 61:1109–1112

  9. Kinoshita S, Udaka S, Shimono M (1957) Studies on the amino acid fermentation. Part 1. Production of l-glutamic acid by various microorganisms. J Gen Appl Microbiol 3:193–205

  10. Lee LK, Roth CM (2003) Antisense technology in molecular and cellular bioengineering. Curr Opin Biotechnol 14:505–511

  11. Miwa K, Matsui K, Terabe M, Ito K, Ishida M, Takagi H, Nakamori S, Sano K (1985) Construction of novel shuttle vectors and a cosmid vector for the glutamic acid-producing bacteria Brevibacterium lactofermentum and Corynebacterium glutamicum. Gene 39:281–286

  12. Nakamura J, Hirano S, Yamaguchi M, Ito H (2006) Japan Patent 2006-101875

  13. Nakamura J, Hirano S, Ito H, Wachi M (2007) Mutations of the Corynebacterium glutamicum NCgl1221 gene, encoding a mechanosensitive channel homolog, induce l-glutamic acid production. Appl Environ Microbiol 73:4491–4498

  14. Nakayama K, Kitada S, Kinoshita S (1961) Studies on lysine fermentation I. The control mechanism of lysine accumulation by homoserine and threonine. J Gen Appl Microbiol 7:145–154

  15. Nara T, Samejima H, Kinoshita S (1964) Effect of penicillin on amino acid fermentation. Agric Biol Chem 28:120–124

  16. 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 281:12300–12307

  17. Nunheimer TD, Birnbaum J, Ihnen ED, Demain AL (1970) Product inhibition of the fermentative formation of glutamic acid. Appl Microbiol 20:215–217

  18. 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 151:1359–1368

  19. Sato H, Orishimo K, Shirai T, Hirasawa T, Nagahisa K, Shimizu H, Wachi M (2008) Distinct roles of two anaplerotic pathways in glutamate production induced by biotin limitation in Corynebacterium glutamicum. J Biosci Bioeng. 106:51–58

  20. Schultz C, Niebisch A, Gebel L, Bott M (2007) Glutamate production by Corynebacterium glutamicum: dependence on the oxoglutarate dehydrogenase inhibitor protein OdhI and protein kinase PknG. Appl Microbiol Biotechnol 76:691–700

  21. Schwinde JW, Hertz PF, Sahm H, Eikmanns BJ, Guyonvarch A (2001) Lipoamide dehydrogenase from Corynebacterium glutamicum: molecular and physiological analysis of the lpd gene and characterization of the enzyme. Microbiology 147:2223–2231

  22. Shiio I, Nakamori S (1970) Microbial production of l-threonine. Part II. Production by α-amino-β-hydroxyvaleric acid resistant mutants of glutamate producing bacteria. Agric Biol Chem 34:448–456

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

  24. Shiio I, Otsuka SI, Takahashi M (1962) Effect of biotin on the bacterial formation of glutamic acid. Part I. Glutamate formation and cellular permeability of amino acids. J Biochem 51:56–62

  25. 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 25:291–298

  26. Shingu H, Terui G (1971) Studies on process of glutamic acid fermentation at the enzyme level. Part I. On the change of α-ketoglutaric acid dehydrogenase in the course of culture. J Ferment Technol 49:400–405

  27. 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 7:59–69

  28. Shirai T, Matsuzaki K, Kuzumoto M, Nagahisa K, Furusawa C, Shioya S, Shimizu H (2006) Precise metabolic flux analysis of coryneform bacteria by gas chromatography-mass spectrometry and verification by nuclear magnetic resonance. J Biosci Bioeng 102:413–424

  29. Takinami K, Yoshii H, Tsuri H, Okada H (1965) Biochemical effects of fatty acid and its derivatives on l-glutamic acid fermentation. Part III. Biotin-Tween 60 relationship in the accumulation of l-glutamic acid and the growth of Brevibacterium lactofermentum. Agric Biol Chem 29:351–359

  30. Tummala SB, Junne SG, Papoutsakis ET (2003a) Antisense RNA downregulation of coenzyme A transferase combined with alcohol-aldehyde dehydrogenase overexpression leads to predominantly alcohologenic Clostridium acetobutylicum fermentations. J Bacteriol 185:3644–3653

  31. Tummala SB, Welker NE, Papoutsakis ET (2003b) Design of antisense RNA constructs for downregulation of the acetone formation pathway of Clostridium acetobutylicum. J Bacteriol 185:1923–1934

  32. Udaka S (1960) Screening method for microorganisms accumulating metabolites and its use in the isolation of Micrococcus glutamicus. J Bacteriol 79:754–755

  33. 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 142:3347–3354

  34. Vertès AA, Inui M, Kobayashi M, Kurusu Y, Yukawa H (1993) Presence of mrr- and mcr-like restriction systems in coryneform bacteria. Res Microbiol 144:181–185

  35. Wagner EG, Simons RW (1994) Antisense RNA control in bacteria, phages, and plasmids. Annu Rev Microbiol 48:713–742

  36. Westmoreland J, Porter G, Radman M, Resnick MA (1997) Highly mismatched molecules resembling recombination intermediates efficiently transform mismatch repair proficient E. coli. Genetics 145:29–38

  37. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119

  38. Yukawa H, Omumasaba CA, Nonaka H, Kós P, Okai N, Suzuki N, Suda M, Tsuge Y, Watanabe J, Ikeda Y, Vèrtes AA, Inui M (2007) Comparative analysis of the Corynebacterium glutamicum group and complete genome sequence of strain R. Microbiology 153:1042–1058

Download references


This work was supported by Grant-in-Aids for Young Scientists (B) to TH (19780061) and CF (20700270), respectively, from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. This work was also partly supported by the Global COE program and the Special Coordination Funds for Promoting Science and Technology: Yuragi Project of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Author information

Correspondence to Hiroshi Shimizu.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kim, J., Hirasawa, T., Sato, Y. et al. Effect of odhA overexpression and odhA antisense RNA expression on Tween-40-triggered glutamate production by Corynebacterium glutamicum . Appl Microbiol Biotechnol 81, 1097–1106 (2009).

Download citation


  • Corynebacterium glutamicum
  • Glutamate overproduction
  • odhA
  • Antisense RNA
  • Tween 40
  • 2-Oxoglutarate dehydrogenase complex