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Engineering the Secretory Pathway for Recombinant Protein Secretion in Saccharomyces cerevisiae

Jichen Bao (Institutionen för biologi och bioteknik, Systembiologi)
Gothenburg : Chalmers University of Technology, 2018. ISBN: 978-91-7597-598-6.
[Doktorsavhandling]

In the past few decades there has been an increasing demand of biopharmaceutical proteins in the market. Several types of cell factories are applied to produce different pharmaceutical proteins. However, manufacturers prefer to use a few favorable biological platforms to undertake the production tasks with low cost, high productivity and proper post-translational modifications. The yeast Saccharomyces cerevisiae is one of these preferred cell factories as it has many advantages. There are several reports on improvement of recombinant protein production by S. cerevisiae through rational engineering of different stages of the protein secretion pathway.

In the first story of this thesis, we engineered protein anterograde trafficking by over-expression of SEC16 to increase the secretory capacity of yeast. We performed bioreactor fermentation to further characterize the engineered strains, and we analysis the reactive oxygen species accumulation, endoplasmic reticulum exit sites, the amount of endoplasmic reticulum membranes of the strains, etc. In the second story, we engineered the retrograde trafficking by over-expression of GLO3 and GCS1 to further increase the secretory capacity of yeast based on the strain constructed in the first story. Physiological changes in the engineered strains were analyzed. We also performed additional experiments to investigate the changes in the amount of endoplasmic reticulum membranes and reactive oxygen species accumulation. In the third story, we performed a systems level analysis of the high α-amylase production strains, which were screened from UV mutation in the previous study. We identified common regulation patterns and hereby we could specify some general rules for efficient protein secretion. Last, we reported an efficient yeast secretion assay platform for biomedical and biotechnological applications. This platform is responsive to secretory disturbances from both chemicals and proteins and is potentially applicable to drug screening and the selection of cell engineering targets for protein production.

Nyckelord: transcriptomics, protein secretion, SEC16, endoplasmic reticulum, oxidative stress, Saccharomyces cerevisiae, drug screening, GLO3



Denna post skapades 2018-01-08. Senast ändrad 2018-01-09.
CPL Pubid: 500034

 

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Institutioner (Chalmers)

Institutionen för biologi och bioteknik, Systembiologi

Ämnesområden

Biokemi och molekylärbiologi
Annan industriell bioteknik
Medicinsk bioteknologi (med inriktning mot cellbiologi)

Chalmers infrastruktur

Examination

Datum: 2018-02-26
Tid: 10:00
Lokal: Sal KA, Kemihuset (Chalmers, Johanneberg campus)
Opponent: Associate Professor Brigitte Gasser, Institute of Biotechnology, University of Natural Resources and Life Sciences, Austria

Ingår i serie

Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie 4279