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13:36
Synthetic Spider Silk Production on a Laboratory Scale
Published on: July 18, 2012
Engineering the Salmonella type III secretion system to export spider silk monomers
Daniel M Widmaier1, Danielle Tullman-Ercek, Ethan A Mirsky
1Chemistry and Chemical Biology Graduate Program, University of California--San Francisco, San Francisco, CA 94110, USA.
Molecular Systems Biology
|September 17, 2009
Summary
Researchers engineered the Salmonella Pathogenicity Island 1 type III secretion system (T3SS) for biotechnological protein export. This system efficiently secretes spider silk monomers, offering broad applications in Gram-negative bacteria.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- The type III secretion system (T3SS) is a crucial virulence factor in many Gram-negative pathogens.
- Harnessing T3SS for heterologous protein export offers a powerful tool for biotechnology.
Purpose of the Study:
- To engineer a T3SS-based system for exporting proteins of biotechnological interest in Gram-negative bacteria.
- To demonstrate the system's efficacy using spider silk monomers.
Main Methods:
- Constructed a T3SS export system using Salmonella Pathogenicity Island 1 components.
- Fused target proteins (spider silk monomers) to an N-terminal secretion tag and cognate chaperone.
- Implemented a genetic circuit for inducible transcription linked to active secretion.
- Designed synthetic genes for silk monomers with optimized genetic stability and codon usage.
- Utilized automated DNA synthesis and cloning for system construction.
Main Results:
- Achieved secretion rates up to 1.8 mg l⁻¹ h⁻¹.
- Demonstrated secretion of up to 14% of expressed spider silk monomers (ADF-1, -2, -3).
- Successfully refined the system using a human DH domain as a model protein.
Conclusions:
- Developed a novel, controllable protein secretion system in Gram-negative bacteria using T3SS.
- The engineered system is broadly applicable for producing biotechnologically relevant proteins.
- This work provides new genetic parts for advanced control of protein secretion.
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