Department or Program


Primary Wellesley Thesis Advisor

Nolan Flynn

Additional Advisor(s)

J. Chris Love (MIT)


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. Given the advantages of using P. pastoris over mammalian cells such as Chinese hamster ovary cells (CHO), which are the most commonly used host in industry, the development of an improved medium for P. pastoris expressing heterologous therapeutic proteins is highly desirable. 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. Analysis of the RNA-seq data provided areas of potential further experimentation. 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.

Available for download on Tuesday, April 25, 2023