Engineering Yeast for the Production of Biologicals
Doctoral thesis, 2022
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.
biopharmaceuticals
genome-scale models.
kinase Gcn2
Recombinant protein production
proteases
Saccharomyces cerevisiae
biologicals
i Konferensrummet 10’an, Kemihuset våning 10 (Forskarhus 1), Kemigården 4, Göteborg
Opponent: Associate Professor Brigitte Gasser, BOKU University of Natural Resources and Life Sciences Vienna, Austria
Author
Veronica Gast
Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology
Gast V., Li F., Domenzain I., Molin M., Siewers V. Improving the production of biologicals in Saccharomyces cerevisiae by overexpressing native target genes predicted by two proteome constrained genome-scale models.
Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules
Microbial Cell Factories,;Vol. 21(2022)
Journal article
A Hypersensitive Genetically Encoded Fluorescent Indicator (roGFP2-Prx1) Enables Continuous Measurement of Intracellular H<inf>2</inf>O<inf>2</inf> during Cell Micro-cultivation
Bio-protocol,;Vol. 12(2022)
Journal article
The Yeast eIF2 Kinase Gcn2 Facilitates H<inf>2</inf>O<inf>2</inf>-Mediated Feedback Inhibition of Both Protein Synthesis and Endoplasmic Reticulum Oxidative Folding during Recombinant Protein Production
Applied and Environmental Microbiology,;Vol. 87(2021)p. e0030121-16
Journal article
The yeast Saccharomyces cerevisiae has been a central part of the development and implementation of biotechnology by human society. We have been using this cell factory for thousands of years to produce some of our all-time favorite products like beer, wine, and bread. Due to the advances in the synthetic biology field over the last decades, S. cerevisiae yeast can nowadays also be implemented as a cell factory to produce biopharmaceutical proteins.
The market for pharmaceutical proteins is developing and increasing rapidly. An essential part of this market and the production of biopharmaceutical proteins, or biologicals, is the use of efficient and reliable production hosts. S. cerevisiae is momentarily one of the most popular hosts to produce biologicals and is currently responsible for 60% of the industrially produced human insulin. S. cerevisiae is known for its robustness under harsh industrial conditions, rapid growth, and simple growth requirements, and has, unlike bacterial hosts, a eukaryotic protein production machinery similar to human cells. The similarity of yeast to other eukaryotes makes it an attractive host to produce more complex human proteins.
In this thesis, I present several strategies to improve the production of biologicals with S. cerevisiae. One strategy was dedicated to engineering the stress response to increased levels of intracellular oxidants caused by protein production. Our engineering strategy improved the production of a model protein 2-fold. Additionally, we identified a mechanism that was before not documented within yeast biology. Secondly, we showed that the removal of native yeast proteases increases the integrity of secreted biologicals. Finally, we implemented the use of state-of-the-art genome-scale models as guides to improve biological production and showed by combining the selected targets that the production of insulin was improved by up to 10-fold. The results summarized within this thesis provide new insight into yeast cellular biology and suggest effective strategies to improve S. cerevisiae as a cell factory for recombinant proteins.
The market for pharmaceutical proteins is developing and increasing rapidly. An essential part of this market and the production of biopharmaceutical proteins, or biologicals, is the use of efficient and reliable production hosts. S. cerevisiae is momentarily one of the most popular hosts to produce biologicals and is currently responsible for 60% of the industrially produced human insulin. S. cerevisiae is known for its robustness under harsh industrial conditions, rapid growth, and simple growth requirements, and has, unlike bacterial hosts, a eukaryotic protein production machinery similar to human cells. The similarity of yeast to other eukaryotes makes it an attractive host to produce more complex human proteins.
In this thesis, I present several strategies to improve the production of biologicals with S. cerevisiae. One strategy was dedicated to engineering the stress response to increased levels of intracellular oxidants caused by protein production. Our engineering strategy improved the production of a model protein 2-fold. Additionally, we identified a mechanism that was before not documented within yeast biology. Secondly, we showed that the removal of native yeast proteases increases the integrity of secreted biologicals. Finally, we implemented the use of state-of-the-art genome-scale models as guides to improve biological production and showed by combining the selected targets that the production of insulin was improved by up to 10-fold. The results summarized within this thesis provide new insight into yeast cellular biology and suggest effective strategies to improve S. cerevisiae as a cell factory for recombinant proteins.
Driving Forces
Sustainable development
Subject Categories (SSIF 2011)
Biochemistry and Molecular Biology
Biological Sciences
Microbiology
Infrastructure
Chalmers Infrastructure for Mass spectrometry
ISBN
978-91-7905-638-4
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5104
Publisher
Chalmers
i Konferensrummet 10’an, Kemihuset våning 10 (Forskarhus 1), Kemigården 4, Göteborg
Opponent: Associate Professor Brigitte Gasser, BOKU University of Natural Resources and Life Sciences Vienna, Austria