Abstract
Proteins comprise a multibillion-dollar industry in enzymes and therapeutics, but bacterial protein production can be costly and inefficient. Proteins of interest (POIs) must be extracted from lysed cells and inclusion bodies, purified, and resolubilized, which adds significant time and cost to the protein-manufacturing process. The Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS) has been engineered to address these problems by secreting soluble, active proteins directly into the culture media, reducing the number of purification steps. However, the current best practices method of T3SS pathway activation is not ideal for industrial scaleup. Previously, the T3SS was activated by plasmid-based overexpression of the T3SS transcriptional regulator, hilA, which requires the addition of a small molecule inducer (IPTG) to the culture media. IPTG adds significant cost to production and plasmid-based expression is subject to instability in large-scale fermentation. Here, we modulate the upstream transcriptional regulator, hilD, to activate the T3SS via three distinct methods. In doing so, we develop a toolbox of T3SS activation methods and construct constitutively active T3SS strains capable of secreting a range of heterologous proteins at titers comparable to plasmid-based hilA overexpression. We also explore how each activation method in our toolbox impacts the SPI-1 regulatory cascade and discover an epistatic relationship between T3SS regulators, hilE and the hilD 3′ untranslated region (hilD 3′UTR). Together, these findings further our goal of making an industrially competitive protein production strain that reduces the challenges associated with plasmid induction and maintenance.
Key points
• Characterized 3 new type III secretion system (T3SS) activation methods for heterologous protein secretion, including 2 constitutive activation methods.
• Eliminated the need for a second plasmid and a small molecule inducer to activate the system, making it more suitable for industrial production.
• Discovered new regulatory insights into the SPI-1 T3SS, including an epistatic relationship between regulators hilE and the hilD 3′ untranslated region.
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Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
We want to acknowledge and thank everyone in the Tullman-Ercek lab who supported us and made publication of this paper possible. Special thanks to Dr. Carolyn Mills, Dr. Samuel Leach, Nolan Kennedy, and Dr. Svetlana Ikonomova for their invaluable feedback throughout the writing of this manuscript. We would also like to thank Dr. Kevin J. Metcalf for the original construction of Pprg GFPmut2.
Funding
This study was funded by the National Institutes of Health Training Grant (T32 GM008382) awarded to JML, the National Science Foundation Graduate Research Fellowship awarded to LAB, the National Science Scholarship from the Agency of Science, Technology and Research, Singapore awarded to HTW, and the National Science Foundation (award number BBE-1706125) awarded to DTE.
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JML, LB, HTW, and DTE conceived and designed research. JML and HTW conducted experiments and analyzed data. JML and LB wrote the manuscript. All authors read and approved the manuscript.
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DTE and JML have a financial interest in Opera Bioscience, which is commercializing bacterial protein production and secretion. DTE, LAB, and HTW are co-inventors on a US patent application related to this work. JML’s and DTE’s conflicts of interest are reviewed and managed by Northwestern University in accordance with their conflict-of-interest policies.
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Liang, J.M., Burdette, L.A., Wong, H.T. et al. Construction of a constitutively active type III secretion system for heterologous protein secretion. Appl Microbiol Biotechnol 107, 1785–1800 (2023). https://doi.org/10.1007/s00253-023-12411-9
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DOI: https://doi.org/10.1007/s00253-023-12411-9