(160ar) Directed Genomic Engineering of Pichia Pastoris Cell Wall to Enhance Protein Secretion

There is a growing interest in the use of alternative hosts to industrially produce various proteins, including biopharmaceuticals, vaccines, and food components. Yeasts and fungi (including K. phaffii, S. cerevisiae, T. reesei, H. polymorpha and Y. lipolytica) are particularly well-suited for large-scale production because of their rapid growth rates, ability to secrete proteins into culture medium, and general overall robustness in bioprocesses. The cell walls of these organisms contribute significantly to their structural durability and ability to grow at high densities in fermenters. Despite these advantages, the relative porosity of the cell wall may have a negative impact on the ability of these organisms to secrete large and complex proteins. We hypothesized that the cell wall could be modified to enhance protein secretion while maintaining its integrity. If successful, modifications could increase the volumetric productivity of these organisms to realize production of low-cost, high-volume biopharmaceuticals like those used to treat infectious diseases such as COVID-19.

To test our hypothesis, we knocked out multiple genes related to cell wall structure in Komagataella phaffii (Pichia pastoris) using CRISPR/Cas9 directed gene editing. We evaluated the ability of these genetically engineered strains to produce and secrete several recombinant proteins, including a monoclonal antibody. This study demonstrates how advanced gene editing technologies can be used to modify complex biological mechanisms like protein secretion.