Abstract 2259 PFAS Are Not Forever: Enzymes and Evolution Rise to the Challenge

Widespread use and new restrictive regulations have focused efforts on environmental remediation of fluorinated compounds known as per- and polyfluorinated chemicals (PFAS). Microbial enzymes offer the most efficient and cost-effective means of PFAS remediation, but some have considered them to be non-biodegradable. They are sometimes called "forever chemicals." In this research, we have identified defluorinating enzymes, engineered microbial systems for defluorination, and evolved higher levels of defluorination. Initial efforts focused on developing a high throughput screen to find and manipulate enzymes catalyzing defluorination. The method was validated initially with fluorinated moieties common in pesticides. A Pseudomonas host was engineered to express a defluorinase, making the cell dependent of non-natural fluorinated growth substrates. The recombinant bacterium was shown to release fluorine as fluoride and exceed 50 mM fluoride concentrations. Fluoride is cytotoxic and thus the ability to carry out extensive degradation of fluorinated compounds is strongly linked to an efficient fluoride stress response. Intracellular fluoride triggers the expression of membrane-bound fluoride export proteins as part of a global stress response. Laboratory evolution is leading to improved rates and extents of defluorination. A comprehensive evolutionary approach is needed to deal with highly fluorinated PFAS chemicals that will impose high levels of fluoride stress on microbial cells. This research was supported by a grant from MnDRIVE Industry and the Environment Program.