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RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae
KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH, Centra, Science for Life Laboratory, SciLifeLab.ORCID-id: 0000-0002-3722-5970
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2019 (Engelska)Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 116, nr 19, s. 9324-9332Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

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.

Ort, förlag, år, upplaga, sidor
National Academy of Sciences , 2019. Vol. 116, nr 19, s. 9324-9332
Nationell ämneskategori
Industriell bioteknik Biokemi och molekylärbiologi Annan industriell bioteknik
Identifikatorer
URN: urn:nbn:se:kth:diva-250438DOI: 10.1073/pnas.1820561116ISI: 000467226400031Scopus ID: 2-s2.0-85065621578OAI: oai:DiVA.org:kth-250438DiVA, id: diva2:1307914
Anmärkning

QC 20190529

Tillgänglig från: 2019-04-29 Skapad: 2019-04-29 Senast uppdaterad: 2019-11-13Bibliografiskt granskad
Ingår i avhandling
1. Droplet microfluidics for screening and sorting of microbial cell factories
Öppna denna publikation i ny flik eller fönster >>Droplet microfluidics for screening and sorting of microbial cell factories
2019 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Cell factories are cells that have been engineered to produce a compound of interest, ranging from biopharmaceuticals to biofuels. With advances in metabolic engineering, the number of cell factory variants to evaluate has increased dramatically, necessitating screening methods with increased throughput. Microfluidic droplets, which can be generated, manipulated and interrogated at very high throughput, are isolated reaction vessels at the single cell scale. Compartmentalization maintains the genotype-phenotype link, making droplet microfluidics suitable for screening of extracellular traits such as secreted products and for screening of microcolonies originating from single cells.

 

In Paper I, we investigated the impact of droplet microfluidic incubation formats on cell culture conditions and found that syringe and semi open incubation resulted in different metabolic profiles. Controlling culture conditions is key to cell factory screening, as product formation is influenced by the state of the cell.

 

In Paper II and III, we used droplet microfluidics to perform screening campaigns of interference based cell factory variant libraries. In Paper II, two S. cerevisiae RNAi libraries were screened based on amylase secretion, and from the sorted fraction genes linked to improved protein secretion could be identified. In paper III, we screened a Synecosystis sp. CRISPRi library based on lactate secretion. The library was sorted at different time points after induction, followed by sequencing to reveal genes enriched by droplet sorting.

 

In Paper IV, we developed a droplet microcolony-based assay for screening intracellular triacylglycerol (TAG) in S. cerevisiae, and showed improved strain separation compared to flow cytometry in a hypothetical sorting scenario. By screening microcolonies compartmentalized in droplets, we combine the throughput of single cell screening methods with the reduced impact of cell-to-cell noise in cell ensemble analysis.

Ort, förlag, år, upplaga, sidor
KTH Royal Institute of Technology, 2019. s. 58
Serie
TRITA-CBH-FOU ; 2019:43
Nyckelord
Droplet microfluidics, Cell factories, High-throughput screening, Cell culture
Nationell ämneskategori
Teknik och teknologier
Forskningsämne
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-259490 (URN)978-91-7873-290-6 (ISBN)
Disputation
2019-10-11, Air & Fire, Tomtebodavägen 23A, Solna, 10:00 (Engelska)
Opponent
Handledare
Anmärkning

QC 2019-09-16

Tillgänglig från: 2019-09-16 Skapad: 2019-09-16 Senast uppdaterad: 2019-09-16Bibliografiskt granskad

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Björk, SaraJönsson, Håkan

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Björk, SaraJönsson, Håkan
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ProteinvetenskapScience for Life Laboratory, SciLifeLabNanobioteknologi
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Proceedings of the National Academy of Sciences of the United States of America
Industriell bioteknikBiokemi och molekylärbiologiAnnan industriell bioteknik

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