A Fluoride-Specific Riboswitch Biosensor for High-Throughput Enzymatic Defluorination Screening.

Area of Science:

• Environmental Science
• Biotechnology
• Biochemistry

Background:

• Per- and polyfluoroalkyl substances (PFAS) are persistent environmental pollutants due to their chemical stability.
• Detecting fluoride release is essential for identifying enzymes capable of defluorination, but current methods lack sensitivity and speed.
• There is a need for efficient tools to screen for and engineer bioremediation enzymes targeting PFAS.

Purpose of the Study:

• To develop a sensitive and specific biosensor for fluoride detection.
• To enable high-throughput screening of enzymes with defluorination capabilities.
• To support the engineering of novel bioremediation strategies for PFAS.

Main Methods:

• A fluoride-specific riboswitch from *Pseudomonas syringae* was coupled with colorimetric and fluorogenic reporter substrates.
• The biosensor was designed for operational simplicity and compatibility with high-throughput screening.
• Enzymatic defluorination activity was validated using the defluorinase RPA1163 and various fluorinated substrates.

Main Results:

• The developed riboswitch biosensor demonstrated high sensitivity and specificity for fluoride detection.
• The biosensor accurately detected enzymatic defluorination across multiple fluorinated compounds, including fluoroacetate and tetrafluoropropanoic acid.
• The system proved versatile, cost-effective, and suitable for high-throughput screening applications.

Conclusions:

• The fluoride-specific riboswitch biosensor is a powerful tool for identifying and characterizing defluorinase enzymes.
• This biosensor facilitates the screening process for enzymes crucial in bioremediation efforts.
• The technology supports advancements in engineering enzymes for the biodegradation of persistent fluorinated compounds.