I. Overview Title: Engineering a secretion-based, high-titer protein production process in bacteria Principal Investigator: Danielle Tullman-Ercek Recombinant protein production is a growing, multi-billion dollar industry that supplies enzymes, such as those used in detergents, textiles, and catalysts for chemical production. Bacteria are highly desired production hosts for such proteins because of their great potential to speed production, increase yields, and reduce costs. However, use of bacteria for this purpose is currently slowed by a state of the art in which proteins are expressed within cells to yield large aggregates known as refractile or inclusion bodies. These aggregates require intensive processing to achieve a pure, soluble, active enzyme. Secreting the protein products directly out of the cell is a promising alternative as this would minimize purification and refolding, and in turn reduce costs and increase production efficiency. This work proposes to engineer the type III secretion system of Salmonella enterica for the high-titer production of non-native proteins because it is easily manipulated, well-characterized, and not required for cellular survival. This proposal’s objectives are to 1) increase the protein titers achieved with bacterial production and secretion, 2) optimize control over the secretion process, and 3) develop a suite of molecular biology tools for the high-throughput production of a broad set of proteins. II. Intellectual merit Controlled transport of proteins from the cytosol to the extracellular space is a challenge that nature has overcome in several ways, but that scientists have yet to fully explore. Transforming the type III secretion system to enable efficient production of heterologous proteins will improve understanding of the mechanisms governing this process. Such information is critical not only to molecular biologists as they construct a detailed picture of cellular function, but also to synthetic biologists that wish to engineer organisms with new levels of complexity and extracellular interaction. This work will also directly produce new vectors and strains for heterologous protein production at both the research and industrial scale. III. Broader impacts The proposed advances in protein production will substantially decrease costs and increase the speed at which new proteins can be developed and brought to market. Moreover, new proteins that are traditionally difficult to produce in high quantities, such as the unique and useful proteins found in spider silk, could be manufactured at industrially-relevant scales using a secretion-based system. Moreover, this work will result in the training of students at all levels for biotechnology careers. The advances will be related to other researchers via publications and conference talks, and to the public via museums, community talks, and science festivals. Keywords: recombinant protein production, bacterial protein transport, type III secretion, toolkit, cellular regulatory mechanisms, synthetic biology
|Effective start/end date||7/1/17 → 6/30/21|
- National Science Foundation (CBET-1706125)
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