Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution

Area of Science:

• Biotechnology
• Enzyme Engineering
• Microfluidics

Background:

• Directed evolution requires high-throughput screening of enzyme variants.
• Miniaturization of assays is crucial for increasing screening capacity.
• Previous methods faced limitations in throughput and sensitivity.

Purpose of the Study:

• To demonstrate a microfluidic platform for ultra-high-throughput screening in directed enzyme evolution.
• To assess the impact of extreme assay miniaturization on signal quality and precision.
• To improve enzyme activity and expression through directed evolution.

Main Methods:

• Utilized a microfluidic platform with water-in-oil droplet compartments for assay miniaturization.
• Screened hydrolytic activities of a promiscuous sulfatase at the single-cell level.
• Sorted 3 × 10^7 monodisperse droplets per round of evolution.

Main Results:

• Achieved a million-fold miniaturization of cell lysate assays.
• Demonstrated high signal quality and quantitative precision at the single-cell level.
• Enriched clones with 6-fold improvements in both enzyme activity and expression.

Conclusions:

• Microfluidic droplet technology enables effective directed evolution by combining high sensitivity and high-throughput screening.
• This approach can rescue and revitalize stalled directed evolution experiments.
• Extreme miniaturization does not compromise assay signal quality, offering precise quantitative readouts.