Substrate Engineering Enabling Fluorescence Droplet Entrapment for IVC-FACS-Based Ultrahigh-Throughput Screening

Area of Science:

• Biotechnology
• Molecular Biology
• Biochemistry

Background:

• In vitro compartmentalization-based fluorescence-activated cell sorting (IVC-FACS) is crucial for enzyme directed evolution.
• Reporter diffusion from droplets in IVC-FACS decouples genotype from phenotype, limiting screening efficiency.

Purpose of the Study:

• To develop a novel strategy, fluorescence droplet entrapment (FDE), to overcome reporter diffusion limitations in IVC-FACS.
• To engineer FDE substrates for detecting various enzyme activities, including phosphotriesterases, carboxylesterases, and glycosidases.

Main Methods:

• Designed substrates with specific polarity for oil-phase passage, enzymatic reaction, and generation of oil-impermeable fluorescent products.
• Employed two distinct substrate engineering strategies for FDE development.
• Conducted model screening assays using phosphotriesterase-active cells and large excesses of inactive cells.

Main Results:

• Achieved a 900-fold enrichment efficiency in model screening assays, the highest reported for IVC-FACS.
• Demonstrated that FDE tightly controls enzymatic reactions, minimizes droplet cross-talk, and reduces background fluorescence.
• Successfully detected phosphotriesterase, carboxylesterase, and glycosidase activities using engineered FDE substrates.

Conclusions:

• Fluorescence droplet entrapment (FDE) is a highly effective strategy for improving IVC-FACS performance.
• FDE enables precise control over enzymatic reactions and enhances screening sensitivity.
• This method holds significant potential for screening enzymes, antibodies, and other proteins via IVC-FACS.