Engineering High-Titer Heterologous Protein Secretion in Bacteria

Secretion is emerging as a useful strategy for exporting proteins of biotechnological interest from bacteria.  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 (1).  We engineered a super-secreting strain of Salmonella for the high-titer production of a variety of biochemically challenging heterologous proteins, such as degradation-prone biopolymer proteins, antibodies, and toxic antimicrobial peptides.  We achieve titers on the order of 100 mg/ml for a variety of proteins – a 100-fold improvement on wild type levels – at relative purity. To design the super-secreting strain, we exploited a native T3SS protein, SipD.  SipD is one of the first three proteins to be secreted through the assembled T3SS.  These “translocon” proteins form a complex at the tip of the T3SS needle, instigating secretion of native proteins into mammalian cells in the first stage of an infection through a poorly understood signal transduction event (2).  Our experiments suggest that SipD also acts as an intracellular regulator in a tightly controlled secretion hierarchy.  Interestingly, these two roles have opposite effects on protein secretion, and occur on different sides of the cell envelope: the regulatory activity of SipD serves to inhibit secretion of heterologous proteins, while exogenous addition of SipD to T3SS-expressing cultures increases secretion. Using a combination of rationally mutated and truncated variants of SipD, we demonstrate that different domains of the protein are involved in each function and pinpoint the key features responsible for conferring the titer increases. This study serves as an example of how a complicated multi-protein apparatus can be deconstructed and rationally engineered, enabling a new route to industrial protein production.

References

(1)   K Metcalf, C Finnerty, A Azam, E Valdivia, D Tullman-Ercek, Applied and Environmental Microbiology (2014).

(2)   M Lara-Tejero and JE Galán, Infection and Immunity (2009).