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Droplet microfluidics for screening and sorting of microbial cell factories
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. (Nanobiotechnology)
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2019. , p. 58
Series
TRITA-CBH-FOU ; 2019:43
Keywords [en]
Droplet microfluidics, Cell factories, High-throughput screening, Cell culture
National Category
Engineering and Technology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-259490ISBN: 978-91-7873-290-6 (print)OAI: oai:DiVA.org:kth-259490DiVA, id: diva2:1351561
Public defence
2019-10-11, Air & Fire, Tomtebodavägen 23A, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 2019-09-16

Available from: 2019-09-16 Created: 2019-09-16 Last updated: 2019-09-16Bibliographically approved
List of papers
1. Metabolite profiling of microfluidic cell culture conditions for droplet based screening
Open this publication in new window or tab >>Metabolite profiling of microfluidic cell culture conditions for droplet based screening
2015 (English)In: Biomicrofluidics, ISSN 1932-1058, E-ISSN 1932-1058, Vol. 9, no 4, article id 044128Article in journal (Refereed) Published
Abstract [en]

We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format impacts cell proliferation and metabolite production. The standard syringe incubation of droplets exhibited metabolite profiles similar to oxygen limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format.

Keywords
Biomolecules;Cell culture;Cell proliferation;Cells;Dimensional stability;Glucose;Metabolism;Metabolites;Microfluidics;Yeast
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-173782 (URN)10.1063/1.4929520 (DOI)000360311900030 ()2-s2.0-84940909670 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20150921

QC 20191008

Available from: 2015-09-21 Created: 2015-09-18 Last updated: 2019-10-08Bibliographically approved
2. RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae
Open this publication in new window or tab >>RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae
Show others...
2019 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 116, no 19, p. 9324-9332Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
National Academy of Sciences, 2019
National Category
Industrial Biotechnology Biochemistry and Molecular Biology Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-250438 (URN)10.1073/pnas.1820561116 (DOI)000467226400031 ()2-s2.0-85065621578 (Scopus ID)
Note

QC 20190529

Available from: 2019-04-29 Created: 2019-04-29 Last updated: 2019-09-30
3. Droplet microfluidic screening of a Synechocystis sp. CRISPRi library based on L-lactate production
Open this publication in new window or tab >>Droplet microfluidic screening of a Synechocystis sp. CRISPRi library based on L-lactate production
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-259487 (URN)
Note

QC 20191011

Available from: 2019-09-16 Created: 2019-09-16 Last updated: 2019-10-11Bibliographically approved
4. Droplet microfluidic microcolony analysis of triacylglycerol yields in S. cerevisiae for high throughput screening
Open this publication in new window or tab >>Droplet microfluidic microcolony analysis of triacylglycerol yields in S. cerevisiae for high throughput screening
(English)Manuscript (preprint) (Other academic)
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-259488 (URN)
Available from: 2019-09-16 Created: 2019-09-16 Last updated: 2019-10-18Bibliographically approved

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