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A Microfluidics-Based Ultrahigh-Throughput Screening Unveils Diverse Ketoreductases Relevant to Pharmaceutical
Laura Blas-Muñoz1,2, Alejandro H Orrego1,2, Michael Hofmeister3
1Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas (UAM-CSIC), Nicolás Cabrera 1, Madrid 28049, Spain.
Analytical Chemistry
|September 16, 2025
Summary
We developed an ultrahigh-throughput screening assay for discovering ketoreductases (KREDs), enabling the identification of novel enzymes with unique capabilities. This advanced method significantly boosts enzyme discovery and evolution efforts.
Area of Science:
- Biocatalysis
- Enzyme Engineering
- Microfluidics
Background:
- Ketoreductases (KREDs) are valuable biocatalysts for producing chiral alcohols.
- Previous KRED discovery assays had limited throughput.
- Thai et al. developed an efficient but low-throughput KRED discovery assay.
Purpose of the Study:
- To develop an ultrahigh-throughput screening assay for KRED discovery.
- To overcome the throughput limitations of existing KRED screening methods.
- To enable the discovery of novel KREDs with unique substrate scopes.
Main Methods:
- Optimized Thai's assay for droplet microfluidics.
- Integrated droplet microfluidics with fluorescence-activated cell sorting (FACS).
- Screened a library of 1.5 million clones using the developed assay.
Main Results:
- Successfully screened 1.5 million clones, demonstrating assay reliability and sensitivity.
- Discovered KREDs with low sequence identity to known enzymes.
- Identified KREDs with previously undescribed substrate scopes, not predictable computationally.
Conclusions:
- The developed assay is the first to perform metagenomic screening for KREDs in microdroplets.
- This ultrahigh-throughput assay is applicable to large KRED libraries for enzyme discovery and evolution.
- The platform enables coupled ultrahigh-throughput screening for other enzyme activities.
