A growth-based screening method to detect defluorinating enzymes

Organofluorines are persistent, widespread contaminants, yet enzymes that cleave carbon-fluorine (C-F) bonds remain scarce, creating demand for methods to discover or engineer defluorinating enzymes. However, screening enzymes that break C-F bonds is difficult because most defluorination assays test one clone or enzyme at a time. To address this problem, we developed a growth-based screening method in Escherichia coli that links fluoride production to tetracycline resistance. We constructed a selection plasmid, pRS-tet, by placing tetA under the control of a fluoride-responsive riboswitch so that fluoride allows tetA expression and cell growth under tetracycline selection. BL21(DE3) E. coli carrying pRS-tet were co-transformed with plasmids encoding either active RPA1163 fluoroacetate dehalogenase (FAcD) or its D114N variant (ΔFAcD). Active FAcD produced higher mean fluoride concentrations from ethyl fluoroacetate (EFA) and ethyl difluoroacetate (EDFA) than ΔFAcD and cell-free controls. Exogenous fluoride increased colony formation up to 5 mM in both strains, whereas growth declined at 10 mM. Under tetracycline selection, active FAcD colony counts increased with EFA, difluoroacetate, and EDFA concentrations (p < 0.01), while ΔFAcD did not exhibit the same dose response. These results show that fluoride released during defluorination can activate tetA expression and support cell growth. Because the selection responds directly to fluoride, it could be adapted to screen enzyme or environmental gene libraries against other fluorinated compounds, including recalcitrant per- and polyfluoroalkyl substances (PFAS).