UC Berkeley

Type III secretion systems (T3SS) are complex, membrane-embedded macromolecular machines found in Gram-negative bacteria. These bacteria utilize T3SSs to facilitate microbe-host interactions by translocating specific proteins out of their cytosol in a single step. This provides an interesting system for engineering the selective translocation of proteins out of the bacterial cell. Secretion strategies have been useful in exporting proteins of biotechnological interest in systems such as yeast, insect cells and mammalian cells. Bacteria fermentations are faster and cheaper than other systems.

In our lab, we use the Salmonella Pathogenicity Island 1 (SPI-1) T3SS in Salmonella enterica serovar Typhimurium for heterologous protein export. As the Salmonella SPI-1 T3SS is not an essential metabolic pathway, we can repurpose it for heterologous protein secretion without affecting cell viability under laboratory conditions. The availability of numerous genetic tools for S. enterica makes it an attractive platform for engineering purposes. Different heterologous proteins of interest can be directed to the T3SS using known native secretion signals. We were able to achieve high secretion titer greater than 400mg/mL through extensive engineering efforts.

My work focuses on the contribution of cellular factors to heterologous secretion by the SPI-1 T3SS through the use of comparative genomics and transcriptomics. Despite a deep understanding of the regulation of SPI-1 and its regulatory inputs, this complex circuit has yet to be successfully expressed heterologously in another host such as E. coli. This suggests that SPI-1 relies on cellular factors specific to S. enterica, despite the high genetic conservation of homologs between S. enterica and E. coli. The difference in the molecular makeup of the cellular environment could be a result of differential regulation of genes or genes unique to each species. Through my work presented here, I identified numerous cellular factors important for SPI-1 activation and engineered strains capable of high secretion of heterologous proteins. I also showed how insights from systems biology can be used to guide precise strain engineering and process engineering.