We have engineered a strain of Salmonella that can manufacture heterologous proteins of interest and secrete them outside of the cell. Stable expression of these non-native proteins is achieved by using DNA synthesis to build genes that are simultaneously optimized for genetic stability and expression in eubacteria. The resulting proteins are exported from the cell using the type III secretion system encoded on Salmonella Pathogeneity Island 1 (SPI-1), which is naturally utilized for the secretion of virulence proteins. To exploit the SPI-1 secretion process, a synthetic genetic system was constructed that interfaces with native SPI-1 regulation, such that heterologous protein expression only occurs when the secretion machinery is assembled and fully functional. Thus, expression is avoided until the cell is capable of exporting the product. Furthermore, we developed fluorescent reporter fusions to the various promoters of the SPI-1 regulatory circuit. We first verified the natural dynamics of the network as reported in the literature and then used the reporters to study various perturbations to the system, with the ultimate goal of increasing heterologous protein secretion levels. The resulting hybrid natural-synthetic system produces significant yields and a novel route to secreted recombinant proteins of biotechnological interest.

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