(81a) Engineering a Bacterial System for the Production of Biologics Leads to Discovery of Two New Functions of a Pathogenesis-Related Protein

Secretion is emerging as a useful strategy for exporting proteins from bacteria for the production of biologics. The type III secretion system (T3SS) in Salmonella enterica is an ideal path to protein export because it is non-essential for bacterial metabolism and allows for target proteins to cross both bacterial membranes in one step, via characteristic needle-like protein structures. We engineered a super-secreting strain of Salmonella for the high-titer production of a variety of biochemically challenging heterologous proteins, including enzymes, antibody fragments, and antimicrobial peptides. We achieve titers of over 100 mg/ml for a variety of proteins â?? a 100-fold improvement on wild type levels â?? at relative purity. To design the super-secreting strain, we investigated SipD, a structural T3SS protein. SipD is one of the first three proteins to be secreted through the assembled T3SS. These â??transloconâ? proteins form a complex at the tip of the T3SS needle that interacts with a mammalian host cell. We find that exogenous addition of SipD results in high secretion titers even in the absence of a host cell. Moreover, our results indicate that SipD also acts as an intracellular regulator in a tightly controlled secretion hierarchy, inhibiting protein secretion. Interestingly, these two roles have opposite effects on protein secretion, and occur on different sides of the cell envelope. Furthermore, evidence suggests that different domains of the protein are involved in each function. We propose a physical and chemical mechanism for the dual role of SipD. This study serves as an example of how a complicated multi-protein apparatus can be rationally engineered for biotechnological protein production while also revealing how it functions in its native role of bacterial pathogenesis.