ABSTRACT Codomax has developed the Epi-MAX platform, an integrated omics and computational-based strain and gene engineering technology that leverages cellular stress responses to enhance protein production and enable the efficient expression of difficult-to-express proteins. Many therapeutic proteins are difficult to express in high yields due to mRNA that are inefficiently translated or suboptimal polypeptide assembly into functional protein. Achieving efficient protein production is a pain point for customers in the biomanufacturing industry, as poor expressing and costly proteins may be abandoned due to their lack of commercial viability. Codomax’s innovation is grounded in our team’s discovery that cells adapt to stress, including the cumulative stress of protein over- production during biomanufacturing, by reprogramming their tRNA pools to enhance translation of mRNAs with distinct codon usage patterns. The Epi-MAX platform leverages this translational reprogramming mechanism to increase recombinant protein expression in cells by matching codon usage in the target gene to the stress- reprogrammed tRNAs. To date, the Epi-MAX platform has yielded up to 25-fold boosts in production of difficult- to-express proteins in Pichia pastoris. Despite the utility of Pichia as one of the major hosts for industrial protein manufacturing, protein production is plagued by intracellular aggregation and faulty transport through the secretory pathway. We now propose to extend the Epi-MAX platform to increase stress translation of endogenous Pichia chaperones that prevent intracellular protein aggregation and facilitate transport of secreted recombinant proteins. Overexpression of the chaperones Kar2, calnexin, and protein disulfide isomerase (PDI) has been demonstrated to significantly increase secreted recombinant protein yields for a range of enzymes and therapeutic proteins produced in Pichia. However, chaperone over-expression may create additional stressors that impede cellular productivity, as exemplified by reports of reduced recombinant protein secretion following Kar2 overexpression. Thus, the goal of this project is to address limitations in secretory protein production in Pichia to boost recombinant protein production yields by increasing the levels of chaperones via enhanced stress translation of their mRNAs. We will apply the Epi-MAX platform to Pichia expressing human interleukin-2 (IL-2), as a model recombinant biologic, to optimize the IL-2 gene and the genes for Kar2, PDI, and calnexin to enhance translation of their mRNAs (Aim 1). CRISPR-Cas9 tools will be used to replace the native genes with the stress codon-engineered chaperone genes. To prevent exhaustion of the cell’s tRNA pool from competition for the limited tRNA pool, we will identify and over-express the tRNAs responsible for decoding the engineered optimal stress codons (Aim 2). Limited proteolysis-coupled mass spectrometry will be used to assess intracellular protein aggregation and proper folding. Successful completion of this Phase I project will demonstrate the Epi-MAX platform’s applicability to address protein assembly bottlenecks and generate designer Pichia strains, chaperone gene sequences, and CRISPR-Cas9 reagents for enabling efficient difficult-to-express biologics production.

NARRATIVE Intracellular protein aggregation and retention of misfolded proteins in the endoplasmic reticulum is a universal issue plaguing secretory recombinant protein production in Pichia pastoris across all sectors of the biomanufacturing industry. Codomax has developed the Epi-MAX platform, a strain and gene engineering technology that leverages cellular stress responses to enhance protein production and enable the efficient expression of difficult-to-express biologics. Here we propose to use the Epi-MAX platform to boost the efficiency of secreted recombinant therapeutic proteins from Pichia by enhancing stress mRNA translation of three secretory pathway chaperones required to prevent intracellular aggregation and promote assembly and secretion of the polypeptides into functional recombinant protein.