title: High-throughput screening for industrial enzyme production hosts by droplet microfluidics.
abstract: 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 α-amylase production, close to the theoretical maximum enrichment. Furthermore, a 10(5) member yeast cell library is screened yielding a clone with a more than 2-fold increase in α-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.
authors.affiliation: Division of Proteomics and Nanobiotechnology, Science for Life Laboratory, Royal Institute of Technology (KTH), Sweden. helenea@biotech.kth.se.
authors.forename: Staffan L
authors.initials: SL
authors.lastname: Sjostrom
authors.forename: Yunpeng
authors.initials: Y
authors.lastname: Bai
authors.forename: Mingtao
authors.initials: M
authors.lastname: Huang
authors.forename: Zihe
authors.initials: Z
authors.lastname: Liu
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
authors.forename: Haakan N
authors.initials: HN
authors.lastname: Joensson
authors.forename: Helene
authors.initials: H
authors.lastname: Andersson Svahn
mesh_terms: D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D007221:Industry; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D015003:Yeasts / Q000235:genetics; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000516:alpha-Amylases