Engineering Yeast for the Production of Biologicals

Abstract: The market for biopharmaceutical proteins, or biologicals, has been expanding rapidly over the last decades and its value was estimated in 2020 to exceed 300 billion US dollars. Efficient cell factories that produce the biologicals fulfill an essential role within this industry. Around 20% of the current biologicals are produced in the yeast species Saccharomyces cerevisiae. In this thesis, I focus on the engineering of S. cerevisiae as a cell factory for biologicals; Affibody molecules, filgrastim, adalimumab, and insulin precursor. The first strategy focuses on the role of the eIF2α kinase Gcn2 in S. cerevisiae. Upon removal of the kinase Gcn2 we showed effectiveness to improve the production of the model protein α-amylase and performed initial experiments on the influence of the removal of the kinase Gcn2 on the production of adalimumab. Our results indicate a novel role of the eIF2α kinase Gcn2 in S. cerevisiae.  Secondly, I focused on the removal of vacuolar proteases from S. cerevisiae. The proteolytic degradation of recombinant proteins by yeast is a known phenomenon that reduces production yield. I identified and removed the specific proteases that degrade the synthetic biologicals, Affibody molecules, which resulted in the production of intact and functional Affibody molecules and I concluded the study with a high production experiment. Additionally, I removed the severe degradation phenotype of a previously engineered S. cerevisiae strain and implemented that strain for the production of filgrastim and adalimumab. As a final strategy, I used two proteome constrained genome-scale models of S. cerevisiae as engineering guides. One model, ecYeast8, suggested overexpression targets that combined into one strain improved the titers of filgrastim, adalimumab, and insulin precursor. The other model pcSecYeast proved effective to improve insulin precursor and resulted in a 10-fold increase of final insulin precursor concentration. The results presented in this thesis will contribute to the improvement of S. cerevisiae as a production host for biologicals and other recombinant proteins.