Engineering fluoroacetate dehalogenase by growth-based selections to degrade 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 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.