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ABSTRACT Protein production strategies in bacteria are often limited due to the need for cell lysis and complicated purification schemes. To avoid these challenges, researchers have developed bacterial strains capable of secreting heterologous protein products outside the cell, but secretion titers often remain too low for commercial applicability. Improved understanding of the link between secretion system structure and its secretory abilities can help overcome the barrier to engineering higher secretion titers. Here, we investigated this link with the PrgI protein, the monomer of the secretory channel of the type 3 secretion system (T3SS) ofSalmonella enterica. Despite detailed knowledge of the PrgI needle’s assembly and structure, little is known about how its structure influences its secretory capabilities. To study this, we recently constructed a comprehensive codon mutagenesis library of the PrgI protein utilizing a novel one-pot recombineering approach. We then screened this library for functional T3SS assembly and secretion titer by measuring the secretion of alkaline phosphatase using a high-throughput activity assay. This allowed us to construct a first-of-its-kind secretion fitness landscape to characterize the PrgI needle’s mutability at each position as well as the mutations which lead to enhanced T3SS secretion. We discovered new design rules for building a functional T3SS as well as identified hypersecreting mutants. This work can be used to increase understanding of the T3SS’s assembly and identify further targets for engineering. This work also provides a blueprint for future efforts to engineer other complex protein assemblies through the construction of fitness landscapes.IMPORTANCEProtein secretion offers a simplified alternative method for protein purification from bacterial hosts. However, the current state-of-the-art methods for protein secretion in bacteria are still hindered by low yields relative to traditional protein purification strategies. Engineers are now seeking strategies to enhance protein secretion titers from bacterial hosts, often through genetic manipulations. In this study, we demonstrate that protein engineering strategies focused on altering the secretion apparatus can be a fruitful avenue toward this goal. Specifically, this study focuses on how changes to the PrgI needle protein from the type 3 secretion system fromSalmonella entericacan impact secretion titer. We demonstrate that this complex is amenable to comprehensive mutagenesis studies and that this can yield both PrgI variants with increased secretory capabilities and insight into the normal functioning of the type 3 secretion system. 

Abstract 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 Typhimurium 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 T3SS regulation is integrated with essential cellular functions. Instead of attempting to untangle a complex web of regulatory input, we took an “outside-in” approach to elucidate the effect of growth medium components on secretion titer. Results We dissected the individual and combined effects 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.