Designer Pichia Strains That Enable Difficult-To-Express Biologics Production by Addressing Limitations in Secretory Protein Expression - 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.
