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

• 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.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology (hsv//eng)

TEKNIK OCH TEKNOLOGIER  -- Kemiteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Chemical Engineering (hsv//eng)

Nyckelord

Saccharomyces-Cerevisiae

Directed Evolution

Microdroplets

Selection

Systems

Assays

Publikations- och innehållstyp

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