Engineering fluoroacetate dehalogenase by growth-based selections to degrade non-natural organofluorides

The widespread use of organofluorides in modern society has inadvertently led to the bioaccumulation of harmful pollutants, most prominently per- and polyfluorinated alkyl substances (PFAS). In principle, tailored biocatalysts able to cleave C—F bonds represent an attractive strategy to combat this (emerging) environmental crisis. However, Nature is largely impartial to C—F bonds, with fluoroacetate dehalogenases (FAcDs) standing out as a notable exception, catalyzing the hydrolysis of single C—F bonds in fluoroacetate at high turnover rates. To expand the substrate scope of FAcDs and harness its catalytic prowess for non-natural organofluorides, we designed and applied a robust growth-based selection strategy for large-scale FAcD engineering. Specifically, we demonstrate that FAcD-catalyzed C—F bond cleavage of (natural and) synthetic organofluorides generates metabolizable carbon sources for bacteria, enabling in vivo enrichment of active FAcD variants. By forcing populations expressing diverse FAcD-libraries to utilize various organofluorides as sole carbon source, we elicited a broad panel of FAcD variants that displayed improved activities and drastically altered substrate profiles. In these efforts, we also identified a previously overlooked inhibition pathway, which largely impedes the conversion of gem-difluoride compounds. Overall, our study presents the first large-scale engineering campaign of FAcDs and introduces an operationally simple selection platform that paves the way toward adapting these enzymes for the sustainable degradation of contaminating organofluorides.