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Research
Danielle Tullman-Ercek
Northwestern University
Corresponding Author
ORCiD: https://orcid.org/0000-0001-6734-480X
License:
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Background
Protein secretion in bacteria is an attractive strategy for heterologous protein production because it retains the high titers and tractability of bacterial hosts while simplifying downstream processing. Traditional intracellular production strategies require cell lysis and separation of the protein product from the chemically similar cellular contents, often a multi-step process that can include an expensive refolding step. The type III secretion system of Salmonella enterica transports proteins from the cytoplasm to the extracellular environment in a single step and is thus a promising solution for protein secretion in bacteria. Product titer is sensitive to extracellular environmental conditions, however, and is therefore not robust. We investigated growth medium composition to provide a favorable environment for secretion that produces consistently high secretion titers, advancing the type III secretion system as a heterologous protein production platform.
Results
We investigated the effect of carbon sources, buffers, and salts in a rich nutrient base on secretion titer. Carbon sources alone decreased secretion titer, secretion titer increased with salt concentration, and the combination of a carbon source, buffer, and high salt concentration had a synergistic effect on secretion titer. Transcriptional activity measured by flow cytometry showed that medium composition affected secretion system activity, and prolonged secretion system activation correlated strongly with increased secretion titer. We found that an optimal combination of glycerol, phosphate, and sodium chloride provided at least a fourfold increase in secretion titer for a variety of proteins. Further, the increase in secretion titer provided by the optimized medium was additive with strain enhancements.
Conclusions
We leveraged the sensitivity of the type III secretion system to the extracellular environment to increase heterologous protein secretion titer. Our results suggest that maximizing secretion titer via the type III secretion system is not as simple as maximizing secreted protein expression—one must also optimize secretion system activity. This work advances the type III secretion system as a platform for heterologous protein secretion in bacteria and will form a basis for future engineering efforts.
Keywords
protein secretion, T3SS, recombinant protein, Salmonella, media optimization
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CURRENT STATUS: Published
View the published article at Microbial Cell Factories.
Version 1
Posted 08 Dec, 2020
No community comments so far
Editorial decision: Accept with minor revisions
On 30 Dec, 2020
Review #1 received
Received 29 Dec, 2020
Review #2 received
Received 29 Dec, 2020
Reviewer #2 agreed
On 11 Dec, 2020
Reviewer #1 agreed
On 07 Dec, 2020
Reviewers invited
Invitations sent on 06 Dec, 2020
Editor invited
On 01 Dec, 2020
Editor assigned
On 30 Nov, 2020
Submission checks complete
On 30 Nov, 2020
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On 29 Nov, 2020
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Subject Areas
Applied & Industrial Microbiology
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A new preprint design is coming!
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See the published version of this preprint at Microbial Cell Factories.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Danielle Tullman-Ercek
Northwestern University
Corresponding Author
ORCiD: https://orcid.org/0000-0001-6734-480X
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Microbial Cell Factories.
Version 1
Posted 08 Dec, 2020
No community comments so far
Editorial decision: Accept with minor revisions
On 30 Dec, 2020
Review #1 received
Received 29 Dec, 2020
Review #2 received
Received 29 Dec, 2020
Reviewer #2 agreed
On 11 Dec, 2020
Reviewer #1 agreed
On 07 Dec, 2020
Reviewers invited
Invitations sent on 06 Dec, 2020
Editor invited
On 01 Dec, 2020
Editor assigned
On 30 Nov, 2020
Submission checks complete
On 30 Nov, 2020
First submitted
On 29 Nov, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Applied & Industrial Microbiology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Microbial Cell Factories.
Background
Protein secretion in bacteria is an attractive strategy for heterologous protein production because it retains the high titers and tractability of bacterial hosts while simplifying downstream processing. Traditional intracellular production strategies require cell lysis and separation of the protein product from the chemically similar cellular contents, often a multi-step process that can include an expensive refolding step. The type III secretion system of Salmonella enterica transports proteins from the cytoplasm to the extracellular environment in a single step and is thus a promising solution for protein secretion in bacteria. Product titer is sensitive to extracellular environmental conditions, however, and is therefore not robust. We investigated growth medium composition to provide a favorable environment for secretion that produces consistently high secretion titers, advancing the type III secretion system as a heterologous protein production platform.
Results
We investigated the effect of carbon sources, buffers, and salts in a rich nutrient base on secretion titer. Carbon sources alone decreased secretion titer, secretion titer increased with salt concentration, and the combination of a carbon source, buffer, and high salt concentration had a synergistic effect on secretion titer. Transcriptional activity measured by flow cytometry showed that medium composition affected secretion system activity, and prolonged secretion system activation correlated strongly with increased secretion titer. We found that an optimal combination of glycerol, phosphate, and sodium chloride provided at least a fourfold increase in secretion titer for a variety of proteins. Further, the increase in secretion titer provided by the optimized medium was additive with strain enhancements.
Conclusions
We leveraged the sensitivity of the type III secretion system to the extracellular environment to increase heterologous protein secretion titer. Our results suggest that maximizing secretion titer via the type III secretion system is not as simple as maximizing secreted protein expression—one must also optimize secretion system activity. This work advances the type III secretion system as a platform for heterologous protein secretion in bacteria and will form a basis for future engineering efforts.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
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