Inactivation of an Aldehyde/Alcohol Dehydrogenase Gene from Clostridium acetobutylicum ATCC 824

  • Edward M. Green
  • George N. Bennett
Part of the ABAB Symposium book series (ABAB, volume 57/58)


A nonreplicative plasmid containing an internal aad gene fragment has been integrated into the chromosome of Clostridium acetobutylicum ATCC 824. Transformation was accomplished by electroporation with relatively high concentrations of methylated plasmid DNA. Southern hybridization experiments revealed that integration occurred by single crossover homologous recombination inactivating the aad gene. Integrants were relatively stable after 25 generations. Inactivation of the aad gene drastically reduced solvent production. This result suggests that aldehyde/alcohol dehydrogenase (AAD) plays a important role in butanol production.

Index Entries

Clostridium acetobutylicum aldehyde/alcohol dehydrogenase solvent production gene inactivation homologous recombination 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Jones, D. T. and Woods, D. R. (1986) Microbiol. Rev. 50, 484–524.Google Scholar
  2. 2.
    Cary, J. W., Petersen, D. J., Papoutsakis, E. T., and Bennett, G. N. (1990) Appl. Environ. Microbiol. 56, 1576–1583.Google Scholar
  3. 3.
    Nair, R. V., Bennett, G. N., and Papoutsakis, E. T. (1994) J. Bacteriol. 176, 871–885.Google Scholar
  4. 4.
    Fischer, R. J., Helms, J., and Durre, P. (1993) J. Bacteriol. 175, 6959–6969.Google Scholar
  5. 5.
    Petersen, D. J., Cary, J. W., Vanderleyden, J., and Bennett, G. N. (1993) Gene 123, 93–97.CrossRefGoogle Scholar
  6. 6.
    Walter, K. A., Bennett, G. N., and Papoutsakis, E. T. (1992) J. Bacteriol. 174, 7149–7158.Google Scholar
  7. 7.
    Nair, R. V. and Papoutsakis, E. T. (1994) J. Bacteriol. 176, 5843–5846.Google Scholar
  8. 8.
    Perego, M. (1993), in Bacillus subtilis and other Gram-Positive Bacteria, Sonenshein, A. L., Hoch, J. A., and Losick, R., eds., American Society for Microbiology, Washington, DC, pp. 615–624.Google Scholar
  9. 9.
    Leenhouts, K. J., Kok, J., and Venema, G. (1989) Appl. Environ. Microbiol. 55, 394–400.Google Scholar
  10. 10.
    Campbell, A. M. (1962) Adv. Genet. 11, 101–146.CrossRefGoogle Scholar
  11. 11.
    Pigot, P. J., Curtis, C. A. M., and de Lencastre, H. (1984) J. Gen. Microbiol. 130, 2123–2136.Google Scholar
  12. 12.
    Young, M. (1984) J. Gen. Microbiol. 130, 1613–1621.Google Scholar
  13. 13.
    Young, M., Staudenbauer, W. L., and Minton, N. P. (1989) FEMS Microbiol. Rev. 63, 301–325.CrossRefGoogle Scholar
  14. 14.
    Williams, D. R., Young, D. I., and Young, M. (1990) J. Gen. Microbiol. 136, 819–826.Google Scholar
  15. 15.
    Wilkinson, S. R. and Young, M. (1994) Microbiology. 140, 89–95.CrossRefGoogle Scholar
  16. 16.
    Mermelstein, L. D. and Papoutsakis, E. T. (1993) Appl. Environ. Microbiol. 59, 1077–1081.Google Scholar
  17. 17.
    Papoutsakis, E. T. and Bennett, G. N. (1993), in The Clostridia and Biotechnology, Woods, D. R., ed., Butterworth-Heinemann, Stoneham, MA, pp. 157–199.Google Scholar
  18. 18.
    Roos, J. W., McLaughlin, K., and Papoutsakis, E. T. (1985) Biotechnol. Bioeng. 27, 681–694.CrossRefGoogle Scholar
  19. 19.
    Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY.Google Scholar
  20. 20.
    Mermelstein, L. D., Welker, N. E., Bennett, G. N., and Papoutsakis, E. T. (1992) Bio/Technology 10, 190–195.CrossRefGoogle Scholar
  21. 21.
    Welch, R. W., Rudolph, F. B., and Papoutsakis, E. T. (1989) Arch. Biochem. Biophys. 273, 309–318.CrossRefGoogle Scholar
  22. 22.
    Southern, E. (1975) J. Mol. Biol. 98, 503–517.CrossRefGoogle Scholar
  23. 23.
    Noirot, Ph., Petit, M.-A., and Ehrlich, S. D. (1987) J. Mol. Biol. 196, 39–48.CrossRefGoogle Scholar
  24. 24.
    Lee, S. Y., Bennett, G. N., and Papoutsakis, E. T. (1992) Biotech. Lett. 14, 427–432.CrossRefGoogle Scholar
  25. 25.
    Young, M. and Ehrlich, S. D. (1989) J. Bacteriol. 171, 2653–2656.Google Scholar
  26. 26.
    Petersen, D. J., Welch, R. W., Rudolph, F. B., and Bennett, G. B. (1991) J. Bacteriol. 173, 1831–1834.Google Scholar

Copyright information

© Springer Science+Business Media New York 1996

Authors and Affiliations

  • Edward M. Green
    • 1
  • George N. Bennett
    • 1
  1. 1.Department of Biochemistry and Cell BiologyRice UniversityHoustonUSA

Personalised recommendations