Developing a Suite of High-throughput Screens for Use in Engineering Bacterial Protein Secretion

Protein production is a multi-billion dollar industry, with applications in pharmaceuticals, industrial enzyme use (eg in laundry detergent), and biomaterials. Bacteria are receiving renewed interest as protein production hosts because of their fast growth and tractability, but traditional production systems in bacteria are intracellular. Secretion is gaining traction as a strategy for bacterial production, as it enables continuous fermentation and reduces purification costs. To engineer these systems to be competitive with production in other microbes, researchers need high-throughput screens to probe the secretion system and amount of secreted protein. A popular choice is a fluorescent protein, but those derived from Aequorea victoria form a covalent bond upon formation of the chromophore, making secretion via systems that unfold the protein impractical. Other assays are highly dependent on environment, rendering them incompatible with various media additives. We present the development of a suite of fluorescence-based assays that overcome these challenges, based on the de novo designed mini Fluorescence Activating Protein (mFAP). This protein is expressed well, is compatbile with secretion in an unfolded state, and only becomes fluorescent upon addition of an inexpensive small molecule. We show that the assay provides a linear range over three orders of magnitude, and maintains linearity in backgrounds ranging from minimal to rich media. We demonstrate the utility of these screens to characterize and engineer the Salmonella enterica Type III Secretion System (T3SS). We identified variants that secrete non-native proteins at high titer, and engineered an optimized, more robust secreting strain of Salmonella. We are using the engineered strain for the high-titer production of a variety of biochemically challenging heterologous proteins, such as growth factors, antibodies, and toxic antimicrobial peptides. 

Learning Objectives: 

1. Explain the advantage of secreting a bioproduct, in the context of a manufacturing process.

2. Define the term fitness landscape in the context of protein engineering.

3. List and briefly describe the four types of assays commonly used to assess secretion titer.