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

Award Number: R41TR005278
ORGANIZATION: NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 07/01/2024
PERIOD OF PERFORMANCE END DATE: 06/30/2025

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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.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $0 )
2025	2024	CODOMAX INC.	17 BRIDEN ST STE 220	WORCESTER	MA	01605	WORCESTER	USA	National Center for Advancing Translational Sciences	000	1	9/9/2025	NEW	$0
														Subtotal = $0

Issue Date FY: 2024 ( Subtotal = $350,000 )
2024	2024	CODOMAX INC.	17 BRIDEN ST STE 220	WORCESTER	MA	01605	WORCESTER	USA	National Center for Advancing Translational Sciences	000	1	6/29/2024	NEW	$350,000
														Subtotal = $350,000

Grand Total All Awards = $350,000

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

Award Number: R41TR005278

ORGANIZATION: NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 07/01/2024

PERIOD OF PERFORMANCE END DATE: 06/30/2025

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