WFRF:(Liu Zihe 1984)
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Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast
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- Huang, Mingtao, 1984 (författare)
- Chalmers tekniska högskola,Chalmers University of Technology
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- Bai, Yunpeng (författare)
- KTH,Proteomik och nanobioteknologi,Science for Life Laboratory, SciLifeLab,East China University of Science and Technology, China
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- Sjöström, Staffan L. (författare)
- KTH,Proteomik och nanobioteknologi,Science for Life Laboratory, SciLifeLab
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- (creator_code:org_t)
- 2015-08-10
- 2015
- Engelska.
- Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 112:34, s. E4689-E4696
- Tidskriftsartikel (refereegranskat)
Abstract
Ämnesord
Stäng
- There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant a-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.
Ämnesord
- NATURVETENSKAP -- Biologi -- Bioinformatik och systembiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Bioinformatics and Systems Biology (hsv//eng)
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
Nyckelord
- droplet microfluidics
- protein secretion
- systems biology
- yeast cell factories
- random mutagenesis
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