Engineering yeast for improved recombinant protein production
Doktorsavhandling, 2023

Recombinant proteins are broadly used from basic research to therapeutic development and include industrial enzymes and pharmaceutical proteins. The increasing demand for improved production and enhanced quality of recombinant proteins requires robust biotech-based strategies to overcome the limitations of protein extraction from natural sources. A variety of cell factories are therefore established for the large-scale production of recombinant proteins of interest. In comparison to other expression systems, the budding yeast Saccharomyces cerevisiae is an attractive production platform due to its high tolerance to harsh fermentation conditions, and importantly its capability to perform eukaryotic post-translational modifications and to secrete the biologically active product to the extracellular medium. Thus, many strategies have been applied to engineer this organism for increasing its recombinant protein secretory capacity and productivity.

The major aim of this thesis work was to study and develop efficient yeast platforms for the production of different heterologous proteins for medical or industrial use through diverse engineering strategies. The first part of this work explored in depth a line of previously evolved yeast strains with improved protein secretory capacity. The universal applicability of the evolved strains was evaluated to produce different antibody fragments, but it was concluded that this secretion platform was not suitable for all types of pharmaceutical proteins tested. Furthermore, by re-introducing all 42 protein-sequence-altering mutations identified in the evolved strains into the parental strain using the CRISPR/Cas9 technology, 14 targets were shown to be beneficial for protein production and 11 out of these 14 beneficial targets were newly identified to be related to recombinant protein production. The second part of this work focused on investigating novel targets related to the cellular stress response and the protein secretory process to rationally optimize S. cerevisiae. Furthermore, screening for suppressors of amyloid-β cytotoxicity in a yeast Alzheimer’s disease model revealed a number of gene targets that reduced oxidative stress and improved production of recombinant proteins. Additionally, a proteome-constrained genome-scale protein secretory model of S. cerevisiae (pcSecYeast) was constructed to simulate the secretion of various recombinant proteins and predict system-level engineering targets for increasing protein production. In summary, the work presented in this thesis provides different efficient strategies to develop yeast platforms for the high-level production of valuable industrial or pharmaceutical proteins, and also provides general guidelines for designing other cell platforms for efficient protein production. Integrated application of various engineering approaches will make meaningful advancements in the field of recombinant protein production in the future.

point mutation

genome-scale modeling

recombinant protein

omics analysis


protein secretion

Saccharomyces cerevisiae

Hall KE, Chemistry building, Kemigården 4, Chalmers
Opponent: Prof. Paola Branduardi, Department of Biotechnology and Biosciences, University of Milano-Bicocca, Italy


Yanyan Wang

Chalmers, Life sciences, Systembiologi

We use proteins in a wide range of our everyday products, such as pharmaceutical proteins and industrial enzymes. However, producing these proteins on an industrial scale can be challenging due to high costs, long processing times, and low productivities. To address these challenges, researchers are exploring and developing efficient and reliable organisms to produce the proteins – so called production hosts. One of the most promising candidates is Saccharomyces cerevisiae, commonly known as baker’s yeast, which has been used for thousands of years by humans to create some of our favourite food products, such as bread, beer, and wine. S. cerevisiae is also known for its ease of culture, rapid growth, and high robustness to harsh fermentation conditions. Moreover, its protein production process is very similar to that of higher eukaryotic organisms, making it a very attractive cell factory to produce human proteins.

In this thesis, I used different strategies for enhancing the protein production capacity of S. cerevisiae. I investigated the mechanism behind the increased productivity of mutant yeast strains for producing antibody fragments, which are valuable pharmaceutical products, at different extents. I also explored the altered genes from these mutant yeast strains that are responsible for the high protein production by introducing point mutations into the starting strain. Additionally, I applied rational engineering techniques to identify novel targets for optimizing S. cerevisiae to produce valuable proteins. These techniques included a state-of-the-art genome-scale model and screening for genes that reduce the toxic effects of amyloid-β protein, a main component found in the brains of people with Alzheimer's disease.

In conclusion, this thesis provides valuable insights and strategies for improving S. cerevisiae as a cell factory for the production of industrial or pharmaceutical proteins, and in addition, will aid in the future design of other cell factories for efficient protein production.


Industriell bioteknik

Biologiska vetenskaper



Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5245



Hall KE, Chemistry building, Kemigården 4, Chalmers

Opponent: Prof. Paola Branduardi, Department of Biotechnology and Biosciences, University of Milano-Bicocca, Italy

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