Engineering yeast for improved recombinant protein production

Doctoral thesis, 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.