Genome Breeding of an Amino Acid-Producing Corynebacterium glutamicum Mutant

  • Masato Ikeda
  • Junko Ohnishi
  • Satoshi Mitsuhashi
Part of the Methods in Biotechnology book series (MIBT, volume 18)


The classical strain breeding method based on random mutation and selection cannot avoid introducing detrimental or unnecessary mutations into the genome. A methodology that overcomes the limitations of the classical method is “genome breeding.” In this approach, biotechno-logically useful mutations identified through the genome analysis of classical mutants are systematically introduced into the wild-type genome in a pinpointed manner, thus allowing creation of a defined mutant that carries only useful mutations. This methodology was applied to generate an efficient L-lysine-producing mutant of Corynebacterium glutamicum. Introduction of the Val-59<-Ala mutation in the homoserine dehydrogenase gene and the Thr-311<-Ile mutation in the aspartokinase gene into the wild-type strain by allelic replacement resulted in accumulation of 8 and 55 g/L of L-lysine, respectively. The two mutations were then reconstituted on the wild-type genome, which led to a synergistic effect on production and accumulation of 75 g/L of L-lysine in a relatively short period of cultivation. The procedure and the impact of this methodology are described.

Key Words

Genome breeding Corynebacterium glutamicum amino acid production L-lysine homoserine dehydrogenase aspartokinase 


  1. 1.
    Ikeda, M. (2003) Amino acid production processes, in Microbial Production of L-Amino Acids (Faurie, R. and Thommel, J., eds.), Advances in Biochemical Engineering and Biotechnology Vol. 79. Springer-Verlag, Berlin, pp. 1–35.Google Scholar
  2. 2.
    Ikeda, M. and Nakagawa, S. (2003) The Corynebacterium glutamicum genome: features and impacts on biotechnological processes. Appl. Microbiol. Biotechnol. 62, 99–109.PubMedCrossRefGoogle Scholar
  3. 3.
    Ohnishi, J., Mitsuhashi, S., Hayashi, M., et al. (2002) A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant. Appl. Microbiol. Biotechnol. 58, 217–223.PubMedCrossRefGoogle Scholar
  4. 4.
    Ohnishi, J., Hayashi, M., Mitsuhashi, S., and Ikeda, M. (2003) Efficient 40°C fermentation of L-lysine by a new Corynebacterium glutamicum mutant developed by genome breeding. Appl. Microbiol. Biotechnol. 62, 69–75.PubMedCrossRefGoogle Scholar
  5. 5.
    Schweizer, H. P. (1992) Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol. Microbiol. 6, 1195–1204.PubMedCrossRefGoogle Scholar
  6. 6.
    Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  7. 7.
    Takeshita, S., Sato, M., Toba, M., Masahashi, W., and Hashimoto-Gotoh, T. (1987) High-copy-number and low-copy-number plasmid vectors for lacZ α-complemen-tation and chloramphenicol-or kanamycin-resistance selection. Gene 61, 63–74.PubMedCrossRefGoogle Scholar
  8. 8.
    Rest, M. E. van der, Lange, C., and Molenaar, D. (1999) A heat shock following electroporation of Corynebacterium glutamicum with xenogenein plasmid DNA. Appl. Microbiol. Biotechnol. 52, 541–545.PubMedCrossRefGoogle Scholar
  9. 9.
    Jäger, W., Schäfer, A., Pühler, A., Labes, G., and Wohlleben, W. (1992) Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the Gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans. J. Bacteriol. 174, 5462–5465.PubMedGoogle Scholar
  10. 10.
    Kwok, S., Chang, S.-Y., Sinskey, A. J., and Wang, A. (1995) Design and use of mismatched and degenerate primers, in PCR Primer: A Laboratory Manual (Dieffenbach, C. W. and Dveksler, G. S., eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 143–155.Google Scholar
  11. 11.
    Hill, D. W., Walters, F. H., Wilson, T. D., and Stuart, J. D. (1979) High performance liquid chromatographic determination of amino acids in the picomole range. Anal. Chem. 51, 1338–1341.PubMedCrossRefGoogle Scholar
  12. 12.
    Schwarzer, A. and Pühler, A. (1991) Manipulation of Corynebacterium glutamicum by gene disruption and replacement. Bio/Technology 9, 84–87.PubMedCrossRefGoogle Scholar
  13. 13.
    Reyes, O., Guyonvarch, A., Bonamy, C., Salti, V., David, F., and Leblon, G. (1991) &quote;Integron&quote;-bearing vectors: a method suitable for stable chromosomal integration in highly restrictive Corynebacteria. Gene 107, 61–68.PubMedCrossRefGoogle Scholar
  14. 14.
    Vertès, A. A., Hatakeyama, K., Inui, M., Kobayashi, M., Kurusu, Y., and Yukawa, H. (1993) Replacement recombination in Coryneform bacteria: high efficiency integration requirement for non-methylated plasmid DNA. Biosci. Biotechnol. Biochem. 57, 2036–2038.CrossRefGoogle Scholar
  15. 15.
    Kimura, E., Abe, C., Kawahara, Y., Nakamatsu, T., and 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, 157–161.PubMedCrossRefGoogle Scholar
  16. 16.
    Ikeda, M. and Katsumata, R. (1998) A novel system with positive selection for the chromosomal integration of replicative plasmid DNA in Corynebacterium glutamicum. Microbiology 144, 1863–1868.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Masato Ikeda
    • 1
  • Junko Ohnishi
    • 2
  • Satoshi Mitsuhashi
    • 2
  1. 1.Department of Bioscience and Biotechnology, Faculty of AgricultureShinshu UniversityNaganoJapan
  2. 2.Tokyo Research LaboratoriesKyowa Hakko Kogyo Co., Ltd.TokyoJapan

Personalised recommendations