The revenue from protein products manufactured by microbial fermentation eclipsed $50 billion in 2015, which includes pharmaceutical products such as proinsulin and enzymes for food production and agriculture. While bacteria are advantageous as protein production hosts because of their tractability and high titer, the difficulty and expense of downstream purification prevents the market from growing. When proteins are expressed intracellularly in bacteria, product recovery typically requires lysis, protein refolding, and multiple purification steps, which must be carefully optimized for each product. A solution to this problem is to export the product from the cytoplasm to the extracellular space. <i>Salmonella enterica</i> natively employ a Type 3 Secretion System (T3SS) for exporting proteins outside the cell, making it a promising target for this purpose. Previously, we have shown that heterologous proteins can be exported through the T3SS at titers up to 400mg/L, but secretion titer is sensitive to many environmental factors which are critical to scale up. Therefore, determining how bioreactor operating conditions impact T3SS secretion titer is necessary for its future use as a protein production platform. We explored how oxygen transfer, pH, glucose feed rate, and growth rate affect secretion titer through cultivation in a sparged stirred tank bioreactor. In this talk, I will present the results of these optimizations, including the identification of a stir speed and air flow rate regime which maximizes titer, and the observation that a slower cellular growth rate increases secretion rate. These results provide insight into the optimal operating conditions to maximize secreted protein, furthering the development of a protein production platform based on the <i>S. enterica</i> T3SS.

Bioreactor engineering for a type 3 secretion system powered protein production process

ACS Fall 2020 Virtual Meeting

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