Engineering the Secretory Pathway for Recombinant Protein Secretion in Saccharomyces cerevisiae

Detta är en avhandling från Chalmers University of Technology

Sammanfattning: Over the past few decades, there has been an increasing demand for biopharmaceutical proteins. Several types of cell factories are used to produce different pharmaceutical proteins. However, manufacturers prefer to use a few favourable biological platforms with low costs, high productivity and proper post-translational modifications to undertake production tasks. The yeast Saccharomyces cerevisiae is a preferred cell factory because it has many advantages. There are several reports on the improvement of recombinant protein production by S. cerevisiae through the rational engineering of different stages of the protein secretion pathway.

Here we engineered protein anterograde trafficking by over-expressing SEC16 to increase the secretory capacity of yeast. We performed bioreactor fermentation to further characterize the engineered strains, and we analysed the reactive oxygen species accumulation, endoplasmic reticulum exit sites, amount of endoplasmic reticulum membranes in different strains, etc. Next, we engineered retrograde trafficking by over-expressing GLO3 and GCS1 to further increase the secretory capacity of yeast based on the strain over-expressing SEC16. Physiological changes in the engineered strains were analysed. We also performed additional experiments to investigate the changes in the amount of endoplasmic reticulum membranes and reactive oxygen species accumulation. We also performed a systems-level analysis of strains with high α-amylase production, which were screened for UV mutations in an earlier study. We identified common regulation patterns and consequently could specify some general rules for efficient protein secretion. Lastly, we report on an efficient yeast secretion platform for biomedical and biotechnological applications. This platform is responsive to secretory disturbances from both chemicals and proteins and is potentially applicable for drug screening and for selecting cell engineering targets for protein production.