High-Throughput Absorbance-Activated Droplet Sorting for Engineering Aldehyde Dehydrogenases.

Area of Science:

• Biocatalysis and Enzyme Engineering
• Microfluidics
• Sustainable Chemistry

Background:

• Biocatalyst commercialization is increasing, shifting towards sustainable methods.
• Enzyme engineering requires screening large mutant libraries, a challenge for conventional methods.
• Droplet microfluidics offers high-throughput screening but is limited by fluorescence detection.

Purpose of the Study:

• To develop a novel absorbance-activated droplet sorter for high-throughput enzyme screening.
• To expand the applicability of droplet microfluidics to a wider range of enzymes.
• To enable robust and timely screening of large enzyme mutant libraries.

Main Methods:

• Development of an absorbance-activated droplet sorter for kilohertz-rate sorting without optical monitoring.
• Screening of a 10^5-member aldehyde dehydrogenase library using a NADH-mediated coupled assay.
• Colorimetric detection of WST-1 formazan product for absorbance-based sorting.

Main Results:

• Successful demonstration of an absorbance-activated droplet sorter for high-throughput screening.
• Identification of an aldehyde dehydrogenase variant with a 51% improvement in catalytic efficiency.
• The engineered variant showed increased overall activity across a broad substrate spectrum.

Conclusions:

• The absorbance-activated droplet sorter significantly enhances enzyme evolution capabilities.
• This technology expands the scope of enzymes that can be screened using droplet microfluidics.
• The developed system facilitates the discovery of improved biocatalysts for sustainable chemical processes.