Opera Bioscience proposes to develop a platform based on the type III secretion system of Salmonella enterica typhimurium, which transports proteins from the cytoplasm to the extracellular space in a single step, to produce cost effective growth factors and recombinant proteins for therapeutic use. Recombinant proteins underlie all facets of biomedical research and therapeutic development. Advancements in genomics, bioinformatics, and tools for genetic manipulation have led to an exponential growth in demand for recombinant proteins to study disease phenotypes, develop therapeutic candidates, and support cell culture for live cellular therapies. Growth factors are in demand for the latter application, especially because of the recent promise of therapies such as CAR-T cells. Many growth factors are expensive to produce with current technologies, leading to increased manufacturing costs for treatments. A novel protein expression system that lowers costs and simplifies the production process would decrease the cost of therapeutic development and manufacturing and expand access to these life-saving products. Bacterial expression systems are preferred for their low cost, high yield, and genetic tractability, but traditional expression strategies require cell lysis and expensive downstream purification processes to recover the product. Non-native proteins often accumulate in insoluble aggregates, which increases cost and complexity by requiring extensive process optimization to recover a soluble and active product. Bacterial protein secretion combines the advantages of traditional bacterial protein expression with the significant benefit of recovering the product from the cell culture medium without cell lysis. This improvement creates high purity proteins that significantly reduces downstream recovery costs and complexity. Secretion also improves product yield by enabling continuous manufacturing technology, which is not currently used in bacterial expression systems. Opera’s platform produces titers on the order of hundreds of milligrams per liter and generate soluble, active enzymes, antibody fragments, and antimicrobial peptides. In this Phase I proposal, Opera outlines an approach to adapt its platform for commercial use. In Aim 1, Opera describes a strategy to create an optimized, safe production strain by removing unnecessary and pathogenic elements to increase safety and initial protein purity and improve strain performance in suspension culture. In Aim 2, Opera Bioscience will unravel the T3SS response to environmental stimuli by defining an optimal growth environment (dissolved oxygen and shear stress, growth medium composition, and temperature) that maximizes secreted protein titer and yields operating parameters Opera can use to scale the technology. The successful outcome of this proposal will be a streamlined, optimized strain with a process environment that promotes secreted titers of growth factors that match or exceed published values...