Engineering the Salmonella type III secretion system to export spider silk monomers

Daniel M. Widmaier, Danielle Tullman-Ercek, Ethan A. Mirsky, Rena Hill, Sridhar Govindarajan, Jeremy Minshull, Christopher A. Voigt

Research output: Contribution to journalArticlepeer-review

99 Scopus citations

Abstract

The type III secretion system (T3SS) exports proteins from the cytoplasm, through both the inner and outer membranes, to the external environment. Here, a system is constructed to harness the T3SS encoded within Salmonella Pathogeneity Island 1 to export proteins of biotechnological interest. The system is composed of an operon containing the target protein fused to an N-terminal secretion tag and its cognate chaperone. Transcription is controlled by a genetic circuit that only turns on when the cell is actively secreting protein. The system is refined using a small human protein (DH domain) and demonstrated by exporting three silk monomers (ADF-1, -2, and -3), representative of different types of spider silk. Synthetic genes encoding silk monomers were designed to enhance genetic stability and codon usage, constructed by automated DNA synthesis, and cloned into the secretion control system. Secretion rates up to 1.8 mg l 1 h 1 are demonstrated with up to 14% of expressed protein secreted. This work introduces new parts to control protein secretion in Gram-negative bacteria, which will be broadly applicable to problems in biotechnology.

Original languageEnglish (US)
Article number309
JournalMolecular Systems Biology
Volume5
DOIs
StatePublished - Jan 20 2009

Keywords

  • Automated DNA synthesis
  • Biomaterials
  • Cellular engineering
  • Genetic parts
  • Systems biology

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)
  • Agricultural and Biological Sciences(all)
  • Applied Mathematics

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