Abstract
The bacterial type III secretion system (T3SS) is an important target for enabling high-titer production of proteins of biotechnological interest as well as for synthetic biology applications that rely on protein delivery to host cells. The T3SS forms a membrane-embedded needle complex that is capped by the translocon proteins and extends into the extracellular space. The needle tip complex in Salmonella enterica consists of three translocon proteins: SipB, SipC, and SipD. It is known that knocking out sipD disrupts T3SS regulation to cause constitutive secretion of native proteins. However, we discovered that complementation of SipD in trans via exogenous addition to T3SS-expressing cultures further improves heterologous protein secretion titers, suggesting a previously unknown but important role for this protein. Building on this knowledge, we have engineered a hyper-secreting strain of S. enterica for a greater than 100-fold improvement in the production of a variety of biotechnologically valuable heterologous proteins that are challenging to produce, such as toxic antimicrobial peptides and proteolysis-prone biopolymer proteins. We determined that transcription by several T3SS promoters is upregulated with the addition of SipD, that the N-terminal domain of SipD is sufficient to observe the increased secretion phenotype, and that the effect is post-transcriptional and post-translational. These results lend support to the use of bacterial secretion as a powerful protein production strategy, and the hypothesis that translocon proteins contribute to type III secretion regulation.
Original language | English (US) |
---|---|
Pages (from-to) | 1006-1015 |
Number of pages | 10 |
Journal | ACS synthetic biology |
Volume | 6 |
Issue number | 6 |
DOIs | |
State | Published - Jun 16 2017 |
Funding
The authors thank Jeff Glasgow, Sergey Boyarskiy, Emily Hartman Caroline Ajo-Franklin, Brittney Nguyen, Bill Burkholder and all the members of the Tullman-Ercek lab at UC Berkeley for valuable discussions and Anthony T. Iavarone for help with mass spectrometry of SipD. The QB3/Chemistry Mass Spectrometry Facility at the University of California Berkeley receives support from the National Institutes of Health (grant number 1S10OD020062-01). A.A.G. gratefully acknowledges the support of graduate fellowships from the NSF and Sandia National Laboratories. H.T.W. gratefully acknowledges the support of graduate fellowship from the A∗STAR, Singapore. The authors would also like to thank the Hammond lab for the use of their equipment.
Keywords
- Salmonella
- gene networks
- protein engineering
- translocon
- type III secretion
- virulence mechanisms
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology (miscellaneous)
- Biomedical Engineering