(269b) Engineering the Type III Secretion Apparatus for Increased Protein Export

Bacterial hosts are attractive for heterologous protein production because they are robust, easy to manipulate, and inexpensive to culture. Protein production traditionally occurs in the cytoplasm; product recovery requires cell lysis and several downstream purification steps, often including refolding. To avoid these challenges, it is desirable to develop a system to export proteins directly to the extracellular space. We use synthetic biology and protein engineering techniques to optimize the Salmonella enterica type III secretion system (T3SS) to export high titers of heterologous protein cargo. The type III secretion apparatus is a multimeric protein needle complex that shuttles proteins from the cytoplasm to the extracellular space through a polymeric needle composed of a single protein, PrgI. Mutational studies in homologous T3SSs indicated that single point mutations in the needle protein alter secretion titer, and we hypothesized that we could replicate this behavior in the S. enterica T3SS. We mapped a set of point mutations from these studies to PrgI and identified a hypersecreting mutant. We are now applying protein engineering techniques to create additional hypersecreting mutants of PrgI and increase bulk secretion titer. Removing bottlenecks in protein export advances development of the T3SS as a general platform for protein production.