Engineering Fluoroacetate Dehalogenase by Growth‐Based Selections on Non‐Natural Organofluorides

ABSTRACT 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.