Advertisement

Molecular Biotechnology

, Volume 46, Issue 3, pp 250–257 | Cite as

Enhanced Extracellular Production of Heterologous Proteins in Bacillus subtilis by Deleting the C-terminal Region of the SecA Secretory Machinery

  • Hiroshi Kakeshtia
  • Yasushi Kageyama
  • Katsutoshi Ara
  • Katsuya Ozaki
  • Kouji Nakamura
Research

Abstract

In this study, we examined the effects of modifying the C-terminal region of the SecA protein on the production of heterologous proteins in Bacillus subtilis. SecA was selected as a candidate among the components of the Sec system due to its ability to interact directly with both the precursors and membrane translocases. A phylogenetic comparison demonstrated that the C-terminal region is not well conserved among eubacterial SecA proteins. The deletion of the 61 amino acids at the C-terminal region led to an 83% increase in extracellular alkaliphilic Bacillus sp. thermostable alkaline cellulase (Egl-237) activity. Moreover, the productivity of human interferon α (hIFN-α2b) was increased by 2.2-fold compared to the wild-type SecA, by deletion of these 61 amino acids. We indicated that the deletion of the C-terminal domain (CTD) of SecA enhanced the secretion of two different heterologous protein, Egl-237 and hIFN-α2b. This study provides a useful method to enhance the extracellular production of heterologous proteins in B. subtilis.

Keywords

SecA Bacillus subtilis Protein secretion Heterologous proteins Alkaliphilic Bacillus sp. thermostable alkaline cellulase Human interferon α 

Notes

Acknowledgments

This study is the subproject, ‘Development of a Technology for Creation of a Host Cell’ included within the industrial technology project, ‘Development of a Generic Technology for Production Process Starting Productive Function’ of the Ministry of Economy, Trade and Industry (METI), entrusted by the New Energy and Industrial Technology Development Organization (NEDO), Japan.

References

  1. 1.
    Braun, P., Gerritse, G., van Dijl, J. M., & Quax, W. J. (1999). Improving protein secretion by engineering components of the bacterial translocation machinery. Current Opinion in Biotechnology, 10, 376–381.CrossRefGoogle Scholar
  2. 2.
    Tjalsma, H., Bolhuis, A., Jongbloed, J. D., Bron, S., & van Dijl, J. M. (2000). Signal peptide-dependent protein transport in Bacillus subtilis: A genome-based survey of the secretome. Microbiology and Molecular Biology Reviews, 64, 515–547.CrossRefGoogle Scholar
  3. 3.
    Westers, L., Westers, H., & Quax, W. J. (2004). Bacillus subtilis as cell factory for pharmaceutical proteins: A biotechnological approach to optimize the host organism. Biochimica et Biophysica Acta, 1694, 299–310.Google Scholar
  4. 4.
    Tjalsma, H., Antelmann, H., Jongbloed, J. D., Braun, P. G., Darmon, E., Dorenbos, R., et al. (2004). Proteomics of protein secretion by Bacillus subtilis: Separating the “secrets” of the secretome. Microbiology and Molecular Biology Reviews, 68, 207–233.CrossRefGoogle Scholar
  5. 5.
    Yamane, K., Bunai, K., & Kakeshita, H. (2004). Protein traffic for secretion and related machinery of Bacillus subtilis. Bioscience Biotechnology Biochemistry, 68, 2007–2023.CrossRefGoogle Scholar
  6. 6.
    van Wely, K. H., Swaving, J., Freudl, R., & Driessen, A. J. (2001). Translocation of proteins across the cell envelope of Gram-positive bacteria. FEMS Microbiology Reviews, 25, 437–454.CrossRefGoogle Scholar
  7. 7.
    Harwood, C. R., & Cranenburgh, R. (2008). Bacillus protein secretion: An unfolding story. Trends in Microbiology, 16, 73–79.Google Scholar
  8. 8.
    Hartl, F. U., Lecker, S., Schiebel, E., Hendrick, J. P., & Wickner, W. (1990). The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane. Cell, 63, 269–279.CrossRefGoogle Scholar
  9. 9.
    Fekkes, P., van der Does, C., & Driessen, A. J. (1997). The molecular chaperone SecB is released from the carboxy-terminus of SecA during initiation of precursor protein translocation. The EMBO Journal, 16, 6105–6113.CrossRefGoogle Scholar
  10. 10.
    Kunst, F., Ogasawara, N., Moszer, I., Albertini, A. M., Alloni, G., Azevedo, V., et al. (1997). The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature, 390, 249–256.CrossRefGoogle Scholar
  11. 11.
    van Wely, K. H., Swaving, J., Klein, M., Freudl, R., & Driessen, A. J. (2000). The carboxyl terminus of the Bacillus subtilis SecA is dispensable for protein secretion and viability. Microbiology, 146, 2573–2581.Google Scholar
  12. 12.
    Henner, D. J. (1990). Inducible expression of regulatory genes in Bacillus subtilis. Methods in Enzymology, 185, 223–228.CrossRefGoogle Scholar
  13. 13.
    Morimoto, T., Kadoya, R., Endo, K., Tohata, M., Sawada, K., Liu, S., et al. (2008). Enhanced recombinant protein productivity by genome reduction in Bacillus subtilis. DNA Research, 15, 73–81.CrossRefGoogle Scholar
  14. 14.
    Hakamada, Y., Hatada, Y., Koike, K., Yoshimatsu, T., Kawai, S., Kobayashi, T., et al. (2000). Deduced amino acid sequence and possible catalytic residues of a thermostable, alkaline cellulase from an alkaliphilic bacillus strain. Bioscience Biotechnology Biochemistry, 64, 2281–2289.CrossRefGoogle Scholar
  15. 15.
    Kakeshita, H., Oguro, A., Amikura, R., Nakamura, K., & Yamane, K. (2000). Expression of the ftsY gene, encoding a homologue of the α subunit of mammalian signal recognition particle receptor, is controlled by different promoters in vegetative and sporulating cells of Bacillus subtilis. Microbiology, 146, 2595–2603.Google Scholar
  16. 16.
    Takamatsu, H., Fuma, S., Nakamura, K., Sadaie, Y., Shinkai, A., Matsuyama, S., et al. (1992). In vivo and in vitro characterization of the secA gene product of Bacillus subtilis. Journal of Bacteriology, 174, 4308–4316.Google Scholar
  17. 17.
    Matsuzaki, H., Yamane, K., Yamaguchi, K., Nagata, Y., & Maruo, B. (1974). Hybrid α-amylases produced by transformants of Bacillus subtilis. I. Purification and characterization of extracellular α-amylases produced by the parental strains and transformants. Biochemica et Biophysica Acta, 365, 235–247.Google Scholar
  18. 18.
    Herbort, M., Klein, M., Manting, E. H., Driessen, A. J., & Freudl, R. J. (1999). Temporal expression of the Bacillus subtilis secA gene, encoding a central component of the preprotein translocase. Journal of Bacteriology, 181, 493–500.Google Scholar
  19. 19.
    Ling, L., Xu, Z., Li, W., Shuai, J., Lu, P., & Hu, C. (2007). Protein secretion pathways in Bacillus subtilis: Implication for optimization of heterologous protein secretion. Biotechnology Advances, 25, 1–12.CrossRefGoogle Scholar
  20. 20.
    Li, W., Zhou, X., & Lu, P. (2004). Bottlenecks in the expression and secretion of heterologous proteins in Bacillus subtilis. Research in Microbiology, 155, 605–610.CrossRefGoogle Scholar
  21. 21.
    Kouwen, T. R., Dubois, J. Y., Freudl, R., Quax, W. J., & van Dijl, J. M. (2008). Modulation of thiol-disulfide oxidoreductases for increased production of disulfide-bond-containing proteins in Bacillus subtilis. Applied Environmental Microbiology, 74, 7536–7545.CrossRefGoogle Scholar
  22. 22.
    Kouwen, T. R., Nielsen, A. K., Denham, E. L., Dubois, J. Y., Dorenbos, R., Rasmussen, M. D., et al. (2010). Contributions of the pre- and pro-regions of a Staphylococcus hyicus lipase to secretion of a heterologous protein by Bacillus subtilis. Applied Environmental Microbiology, 76, 659–669.CrossRefGoogle Scholar
  23. 23.
    Brockmeier, U., Caspers, M., Freudl, R., Jockwer, A., Noll, T., & Eggert, T. (2006). Systematic screening of all signal peptides from Bacillus subtilis: A powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria. Journal of Molecular Biology, 362, 393–402.CrossRefGoogle Scholar
  24. 24.
    Bolhuis, A., Tjalsma, H., Smith, H. E., de Jong, A., Meima, R., Venema, G., et al. (1999). Evaluation of bottlenecks in the late stages of protein secretion in Bacillus subtilis. Applied Environmental Microbiology, 65, 2934–2941.Google Scholar
  25. 25.
    Sarvas, M., Harwood, C. R., Bron, S., & van Dijl, J. M. (2004). Post-translocational folding of secretory proteins in Gram-positive bacteria. Biochimica et Biophysica Acta, 1694, 311–327.Google Scholar
  26. 26.
    Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., et al. (2002). Nucleotide control of interdomain interactions in the conformational reaction cycle of SecA. Science, 297, 2018–2026.CrossRefGoogle Scholar
  27. 27.
    Mostertz, J., Scharf, C., Hecker, M., & Homuth, G. (2004). Transcriptome and proteome analysis of Bacillus subtilis gene expression in response to superoxide and peroxide stress. Microbiology, 150, 497–512.CrossRefGoogle Scholar
  28. 28.
    Takase, T., Mizuno, H., & Yamane, K. (1988). NH2-terminal processing of Bacillus subtilis α-amylase. Journal of Biological Chemistry, 263, 11548–11553.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Hiroshi Kakeshtia
    • 1
    • 2
  • Yasushi Kageyama
    • 2
  • Katsutoshi Ara
    • 2
  • Katsuya Ozaki
    • 2
  • Kouji Nakamura
    • 1
  1. 1.Graduate School of Life and Environmental SciencesUniversity of TsukubaTsukubaJapan
  2. 2.Biological Science Laboratories, Kao CorporationIchikai-Machi, Haga-GunJapan

Personalised recommendations