High-throughput screening for industrial enzyme production hosts by droplet microfluidics

• Published in: Lab on a chip Vol. 14; no. 4; p. 806
• Main Authors: Sjostrom, Staffan L, Bai, Yunpeng, Huang, Mingtao, Liu, Zihe, Nielsen, Jens, Joensson, Haakan N, Andersson Svahn, Helene
• Format: Journal Article
• Language: English
• Published: England 01-01-2014
• Subjects: alpha-Amylases - biosynthesis; alpha-Amylases - genetics; Bacillus - enzymology; Bacillus - genetics; Genetic Engineering - methods; Industry; Microfluidic Analytical Techniques - methods; Yeasts - genetics
• ISSN: 1473-0189
• DOI: 10.1039/c3lc51202a

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.