High-throughput screening for industrial enzyme production hosts by droplet microfluidics
2014 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 14, no 4, 806-813 p.Article in journal (Refereed) Published
A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its a-amylase production, close to the theoretical maximum enrichment. Furthermore, a 105 member yeast cell library is screened yielding a clone with a more than 2-fold increase in a-amylase production. The increase in enzyme production results from an improvement of the cellular functions of the production host in contrast to previous droplet-based directed evolution that has focused on improving enzyme protein structure. In the workflow presented, enzyme producing single cells are encapsulated in 20 pL droplets with a fluorogenic reporter substrate. The coupling of a desired phenotype (secreted enzyme concentration) with the genotype (contained in the cell) inside a droplet enables selection of single cells with improved enzyme production capacity by droplet sorting. The platform has a throughput over 300 times higher than that of the current industry standard, an automated microtiter plate screening system. At the same time, reagent consumption for a screening experiment is decreased a million fold, greatly reducing the costs of evolutionary engineering of production strains.
Place, publisher, year, edition, pages
2014. Vol. 14, no 4, 806-813 p.
Saccharomyces-Cerevisiae, Directed Evolution, Microdroplets, Selection, Systems, Assays
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:kth:diva-141316DOI: 10.1039/c3lc51202aISI: 000330046100024ScopusID: 2-s2.0-84893020696OAI: oai:DiVA.org:kth-141316DiVA: diva2:696625
FunderSwedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
QC 201402142014-02-142014-02-132015-01-29Bibliographically approved