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RNAi expression tun...
RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae
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- Wang, Guokun, 1988 (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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- Björk, Sara (author)
- KTH,Proteinvetenskap,Science for Life Laboratory, SciLifeLab,Kungliga Tekniska Högskolan (KTH),Royal Institute of Technology (KTH)
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- Huang, Mingtao, 1984 (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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- Liu, Quanli, 1988 (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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- Campbell, Kate, 1987 (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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- Nielsen, Jens B, 1962 (author)
- Chalmers tekniska högskola,Chalmers University of Technology,Danmarks Tekniske Universitet,Technical University of Denmark
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- Jönsson, Håkan, 1979- (author)
- KTH,Nanobioteknologi,Science for Life Laboratory, SciLifeLab,Kungliga Tekniska Högskolan (KTH),Royal Institute of Technology (KTH)
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- Petranovic Nielsen, Dina, 1975 (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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(creator_code:org_t)
- 2019-04-18
- 2019
- English.
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 116:19, s. 9324-9332
- Related links:
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https://www.pnas.org...
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https://doi.org/10.1...
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https://research.cha...
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https://urn.kb.se/re...
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Abstract
Subject headings
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- The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered protein production. Due to the complexity of such cellular machinery, the challenge in generating a superior cell factory is to fully exploit the production potential by finding beneficial targets for optimized strains, which ideally could be used for improved secretion of other proteins. We focused on an approach in the yeast Saccharomyces cerevisiae that allows for attenuation of gene expression, using RNAi combined with high-throughput microfluidic single-cell screening for cells with improved protein secretion. Using direct experimental validation or enrichment analysis-assisted characterization of systematically introduced RNAi perturbations, we could identify targets that improve protein secretion. We found that genes with functions in cellular metabolism (YDC1, AAD4, ADE8, and SDH1), protein modification and degradation (VPS73, KTR2, CNL1, and SSA1), and cell cycle (CDC39), can all impact recombinant protein production when expressed at differentially down-regulated levels. By establishing a workflow that incorporates Cas9-mediated recombineering, we demonstrated how we could tune the expression of the identified gene targets for further improved protein production for specific proteins. Our findings offer a high throughput and semirational platform design, which will improve not only the production of a desired protein but even more importantly, shed additional light on connections between protein production and other cellular processes.
Subject headings
- NATURVETENSKAP -- Biologi -- Cellbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Cell Biology (hsv//eng)
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinsk bioteknologi -- Medicinsk bioteknologi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Medical Biotechnology -- Medical Biotechnology (hsv//eng)
- TEKNIK OCH TEKNOLOGIER -- Industriell bioteknik (hsv//swe)
- ENGINEERING AND TECHNOLOGY -- Industrial Biotechnology (hsv//eng)
- TEKNIK OCH TEKNOLOGIER -- Industriell bioteknik -- Annan industriell bioteknik (hsv//swe)
- ENGINEERING AND TECHNOLOGY -- Industrial Biotechnology -- Other Industrial Biotechnology (hsv//eng)
Keyword
- RNA interference
- Droplet microfluidic screening
- Genome recombineering
- Protein production
- Saccharomyces cerevisiae
Publication and Content Type
- art (subject category)
- ref (subject category)
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Wang, Guokun, 19 ...
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Björk, Sara
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Huang, Mingtao, ...
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Liu, Quanli, 198 ...
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Campbell, Kate, ...
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Nielsen, Jens B, ...
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show more...
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Jönsson, Håkan, ...
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Petranovic Niels ...
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- NATURAL SCIENCES
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NATURAL SCIENCES
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and Biological Scien ...
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and Cell Biology
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- NATURAL SCIENCES
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NATURAL SCIENCES
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and Biological Scien ...
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and Biochemistry and ...
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MEDICAL AND HEAL ...
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ENGINEERING AND ...
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- ENGINEERING AND TECHNOLOGY
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ENGINEERING AND ...
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and Industrial Biote ...
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and Other Industrial ...
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Proceedings of t ...
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Chalmers University of Technology
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Royal Institute of Technology