In the biopharmaceutical industry, media for host organisms have been optimized with nutrients like amino acids to maximize recombinant protein production. Because amino acids serve as the building blocks of proteins, as well as intermediates in various metabolic pathways, they are posited to relieve much of the metabolic burden experienced by organisms producing heterologous proteins. Both complex amino acid mixtures and defined amino acids have shown to improve protein titers in various organisms. While there have been some studies on media optimization with amino acids for yeast, few systematic studies of amino acid supplementation have been reported for Pichia pastoris, a widely-used organism for producing biological therapeutics. Commercially available defined media often lacks nutrients, including specific amino acids found in complex media. However, defined media has the potential to simplify downstream protein purification and quality control, and is therefore of great interest. Here, we present a study of the impact of adding specific amino acids on recombinant protein titers. To uncover a general formulation, we studied three P. pastoris strains, each expressing a different heterologous protein. Across all three strains, some amino acids increased protein production, while some amino acids decreased protein production relative to controls lacking amino acids. Given its ability to uncover metabolic pathways associated with a particular phenotypic state, RNA sequencing was performed on a subset of the media conditions to further understand the biological benefits of adding amino acids to the media. Metabolic intermediates of relevant pathways associated with highly productive fermentations, containing either upregulated or downregulated genes depending on the pathway, were further mined for additional gains in heterologous protein expression in Pichia. We believe that this method of amino acid optimization has general application and could be applied to other organisms of interest for improved recombinant protein production.

Optimizing amino acids in defined media for Pichia pastoris recombinant protein expression

255th National Meeting (2018)

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