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

Perfluoroalkyl and polyfluoroalkyl substances pose significant environmental threats due to their chemical stability and resistance to biodegradation. The discovery of enzymes with selective defluorination capabilities is currently hindered by the lack of rapid and sensitive methods for detecting fluoride. In this study, we developed a fluoride-specific riboswitch biosensor by coupling a fluoride-responsive riboswitch from <i>Pseudomonas syringae</i> with commonly used colorimetric 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside and fluorogenic (4-methylumbelliferyl β-d-galactopyranoside reporter substrates. This biosensor exhibited high sensitivity, specificity, operational simplicity, and compatibility with high-throughput screening. Using defluorinase RPA1163, we validated the biosensor's performance, where enzymatic defluorination was accurately detected across multiple fluorinated substrates, including fluoroacetate, difluoroacetate, 2-fluoropropionic acid, and 2,3,3,3-tetrafluoropropanoic acid. This versatile and cost-effective biosensor combines chemical and biological detection principles, providing a powerful tool for the high-throughput screening of defluorinases and supporting efforts in the engineering of bioremediation enzymes.