UC Berkeley

Recombinant proteins comprise billion-dollar industries, from therapeutics to enzymes used as catalysts in industrial processes. As the application space of recombinant proteins expands as a result of advancements in protein design and engineering, so does the demand for production systems that minimize the cost of developing and manufacturing the new protein products. Protein secretion in bacteria has the potential to combine the inexpensive cultivation, genetic tractability, and high productivity of traditional intracellular bacteria expression with the simplified downstream processing provided by eukaryotic systems that secrete the product into the extracellular space. The SPI-1 type III secretion system (T3SS) of Salmonella enterica Typhimurium is an ideal engineering target for heterologous protein secretion in bacteria—it is well-characterized, not essential for cell viability, and has demonstrated the capability to secrete heterologous proteins at titers up to hundreds of milligrams per liter.

This work expands upon initial engineering achievements in adapting the SPI-1 T3SS for heterologous protein production. Chapters 2 and 3 describe design rules for growth media promote high secretion titers. A simple combination of glycolytic carbon source, buffering agent, high osmolarity, and essential nutrients was sufficient to increase secretion titer at least fourfold for multiple secreted proteins. Chapter 4 describes the advantages and disadvantages of several methods to measure secretion titer. Chapter 5 combines the knowledge acquired in Chapters 2-4 to create a library construction and screening workflow to identify hypersecreting variants of the T3SS needle protein, PrgI, via systematic, comprehensive mutagenesis. In addition to identifying many hypersecreting variants, systematic mutagenesis of PrgI revealed design rules for functional needle assembly and high secretion titers. Finally, Chapter 6 outlines strain modifications that increase the utility of S. enterica Typhimurium as a recombinant protein production strain. Deletion of several pathogenic and non-essential genetic loci had a minimal effect on secretion titer, while deletion of the T3SS tip complex increased secretion titer. In combination with an optimized growth medium, genomic modifications that increased expression of the master regulator hilD produced higher secretion titers than observed in Chapter 2 and, importantly, simplified T3SS system expression. Collectively, the work described here advances the SPI-1 T3SS as a heterologous protein production platform and provides insight into the native function of the system.