Contributions of the pre- and pro-regions of a Staphylococcus hyicus lipase to secretion of a heterologous protein by Bacillus subtilis

Thijs R H M Kouwen, Allan K Nielsen, Emma L Denham, Jean-Yves F Dubois, Ronald Dorenbos, Michael D Rasmussen, Wim J Quax, Roland Freudl, Jan Maarten van Dijl

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Abstract

Bacillus subtilis is a well-established cell factory for efficient secretion of many biotechnologically relevant enzymes that are naturally produced by it or related organisms. However, the use of B. subtilis as a host for production of heterologous secretory proteins can be complicated by problems related to inefficient translocation of the foreign proteins across the plasma membrane or to inefficient release of the exported proteins from the cell surface into the surrounding medium. Therefore, there is a clear need for tools that allow more efficient membrane targeting, translocation, and release during the production of these proteins. In the present study, we investigated the contributions of the pre (pre(lip)) and pro (pro(lip)) sequences of a Staphylococcus hyicus lipase to secretion of a heterologous protein, the alkaline phosphatase PhoA of Escherichia coli, by B. subtilis. The results indicate that the presence of the pro(lip)-peptide, in combination with the lipase signal peptide (pre(lip)), contributes significantly to the efficient secretion of PhoA by B. subtilis and that pre(lip) directs PhoA secretion more efficiently than the authentic signal peptide of PhoA. Genome-wide transcriptional analyses of the host cell responses indicate that, under the conditions tested, no known secretion or membrane-cell wall stress responses were provoked by the production of PhoA with any of the pre- and pro-region sequences used. Our data underscore the view that the pre-pro signals of the S. hyicus lipase are very useful tools for secretion of heterologous proteins in B. subtilis.

Original languageEnglish
Pages (from-to)659-669
Number of pages11
JournalApplied and Environmental Microbiology
Volume76
Issue number3
Early online date27 Jan 2010
DOIs
Publication statusPublished - Feb 2010

Keywords

  • ATP-Binding Cassette Transporters/genetics
  • Adenosine Triphosphatases/genetics
  • Alkaline Phosphatase/genetics
  • Amino Acid Sequence
  • Bacillus subtilis/genetics
  • Bacterial Proteins/chemistry
  • Biotechnology/methods
  • Cell Membrane/genetics
  • Cloning, Molecular
  • Endopeptidase Clp/genetics
  • Escherichia coli Proteins/genetics
  • Lipase/chemistry
  • Protein Folding
  • Protein Sorting Signals/genetics
  • Protein Transport/genetics
  • Recombinant Fusion Proteins/genetics
  • Serine Endopeptidases/genetics
  • Staphylococcus/enzymology
  • alpha-Amylases/metabolism

Cite this

Kouwen, T. R. H. M., Nielsen, A. K., Denham, E. L., Dubois, J-Y. F., Dorenbos, R., Rasmussen, M. D., Quax, W. J., Freudl, R., & van Dijl, J. M. (2010). Contributions of the pre- and pro-regions of a Staphylococcus hyicus lipase to secretion of a heterologous protein by Bacillus subtilis. Applied and Environmental Microbiology, 76(3), 659-669. https://doi.org/10.1128/AEM.01671-09