GPCR-FEX: A Fluoride-Based Selection System for Rapid GPCR Screening and Engineering.

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biochemistry

Background:

• Directed evolution of proteins is crucial for improving function but limited by screening capacity.
• Coupling protein function to cell viability accelerates the identification of desirable variants.
• G-protein coupled receptors (GPCRs) are vital cellular sensors with broad applications.

Purpose of the Study:

• To introduce GPCR-FEX, a stringent selection platform for engineering GPCRs in yeast.
• To enhance the screening of large GPCR libraries by coupling signaling to cellular fluoride tolerance.
• To enable rapid and cost-effective identification of improved GPCR variants.

Main Methods:

• Developed GPCR-FEX by linking GPCR signaling to a fluoride ion exporter (FEX)-GFP fusion gene and cellular fluoride tolerance in yeast.
• Utilized yeast promoters (P_FUS1 or P_FIG1) activated by native GPCRs (Ste2p or Ste3p) upon agonist binding.
• Incorporated a C-terminal degron to FEX1p to amplify the growth difference between agonist-treated and untreated cells.
• Employed deep sequencing to quantify population dynamics during selection experiments.

Main Results:

• The GPCR-FEX platform effectively depletes inactive GPCR variants, enabling high-throughput screening.
• Demonstrated rapid enrichment of a previously engineered Ste2p receptor mutant over wild-type Ste2p in a model library.
• Showcased the enhanced dynamic range of cell growth achieved by adding a degron to FEX1p.

Conclusions:

• GPCR-FEX provides a powerful mechanism for the rapid engineering of GPCRs.
• This platform significantly expands the accessible sequence space for GPCR discovery.
• GPCR-FEX is a valuable tool for synthetic biology applications requiring engineered cellular sensors.