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

• Published in: Molecular systems biology 2009, Vol.5 (1), p.309-n/a
• Main Authors: Widmaier, Daniel M, Tullman‐Ercek, Danielle, Mirsky, Ethan A, Hill, Rena, Govindarajan, Sridhar, Minshull, Jeremy, Voigt, Christopher A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Araneae; automated DNA synthesis; Automation; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding sites; biomaterials; Biotechnology; cellular engineering; Cellulase; Circuits; Cytoplasm; Deoxyribonucleic acid; DNA; DNA biosynthesis; EMBO41; Enzymes; Exports; Fibroins - genetics; Fibroins - metabolism; Fibroins - secretion; Gene expression; Genetic engineering; genetic parts; Gram-negative bacteria; Humans; Membrane Proteins - genetics; Membrane Proteins - metabolism; Membranes; Models, Biological; Molecular Sequence Data; Monomers; Outer membranes; Polypeptides; Protein Engineering - methods; Protein Transport; Proteins; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - secretion; Salmonella; Salmonella - genetics; Salmonella - metabolism; Salmonella - physiology; Scholarships & fellowships; Secretion; Sequence Alignment; Signal Transduction; Silk; Spiders - genetics; systems biology; Transcription
• ISSN: 1744-4292; 1744-4292
• DOI: 10.1038/msb.2009.62

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.