Engineering Fluoroacetate Dehalogenase by Growth‐Based Selections on 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 by catalyzing the hydrolysis of single C─F bonds in fluoroacetate at high turnover rates. To 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 panel of FAcD variants with improved activities and altered substrate profiles for fluoroacetate, 2-fluoropropionate, and 2,2-difluoroacetate. In these efforts, we also identified a previously overlooked inhibition pathway, which 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 to adapt these enzymes for the sustainable degradation of contaminating organofluorides.