With the advent of recombinant DNA technology, proteins can now be made large-scale as therapeutics to treat diseases, enzymes for consumer goods, and even biomaterials like spider silk. Unfortunately, industrial-scale protein production can be costly and inefficient as expressed proteins aggregate and form inclusion bodies. Extensive purification is required to extract, resolubilize, and purify the protein-of-interest (POI) from the production organism. We solve this issue by engineering the Salmonella Pathogenicity Island-1 Type Three Secretion System (SPI-1 T3SS) to secrete POIs directly into the culture media, simplifying purification. The SPI-1 T3SS is natively used by Salmonella to invade and infect host organisms and its expression is governed by several different transcriptional regulators. Activation of the T3SS can be induced by synthetically controlling or modifying these regulators. Here, we will present how varying activation mechanisms influence (1) SPI-1 T3SS gene expression, (2) assembly of the T3SS needle apparatuses, and (3) functional output in the form of protein secretion titer. This expands our understanding of the steps between T3SS activation and invasion. This work enabled us to better engineer the T3SS for heterologous protein secretion and contributed toward the construction of a strain with a constitutively active T3SS. This new strain does not require added inducer to the culture media which reduces the cost of protein production.

Development of a bacterial strain for producing and secreting protein

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.