Evaluation of bottlenecks in the late stages of protein secretion in Bacillus subtilis

Albert Bolhuis, Harold Tjalsma, Hilde E. Smith, Anne De Jong, Rob Meima, Gerard Venema, Sierd Bron, Jan Maarten Van Dijl

Research output: Contribution to journalArticlepeer-review

80 Citations (Scopus)

Abstract

Despite a high capacity for secretion of homologous proteins, the secretion of heterologous proteins by Bacillus subtilis is frequently inefficient. In the present studies, we have investigated and compared bottlenecks in the secretion of four heterologous proteins: Bacillus lichenifomis α-amylase (AmyL), Escherichia coli TEM β-lactamase (Bla), human pancreatic α-amylase (HPA), and a lysozyme-specific single-chain antibody. The same expression and secretion signals were used for all four of these proteins. Notably, all identified bottlenecks relate to late stages in secretion, following translocation of the preproteins across the cytoplasmic membrane. These bottlenecks include processing by signal peptidase, passage through the cell wall, and degradation in the wall and growth medium. Strikingly, all translocated HPA was misfolded, its stability depending on the formation of disulfide bonds. This suggests that the disulfide bond oxidoreductases of B. subtilis cannot form the disulfide bonds in HPA correctly. As the secretion bottlenecks differed for each heterologous protein tested, it is anticipated that the efficient secretion of particular groups of heterologous proteins with the same secretion bottlenecks will require the engineering of specifically optimized host strains.

Original languageEnglish
Pages (from-to)2934-2941
Number of pages8
JournalApplied and Environmental Microbiology
Volume65
Issue number7
Early online date1 Jul 1999
Publication statusPublished - 1 Jul 1999

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

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