Formatted Title
PFAS Are Not Forever: Existing Enzymes and New Evolution Show Success
Background/Objectives
Widespread use and new restrictive regulations have focused efforts on environmental remediation of fluorinated compounds, particularly per- and polyfluorinated chemicals (PFAS). When available, microbial enzymes are often the most efficient and cost-effective means of chemical remediation. The biodegradation of most PFAS chemicals is rare or unknown, leading some to call them “forever chemicals.” Here, we describe methods for identifying defluorinating enzymes, re-engineering biological systems for defluorination, and overcoming roadblocks to high level defluorination in microbial systems.
Approach/Activities
Initial efforts focused on developing high throughput methods to rapidly screen enzymes and microbes for biodegradative activity with fluorinated compounds. Fluorine is the most electronegative element and C-F bond cleavage via any mechanism releases fluoride. A rapid and sensitive microwell screen for fluoride allowed us to analyze thousands of enzymes and bacteria daily. Initial successes were demonstrated with fluorinated moieties common in fluorinated pesticides. A microbial system was engineered to catalyze defluorination to support growth, making the cell addicted to the fluorinated substrates. Levels of fluoride release exceeding 50 mM have been attained in this way. Fluoride is cytotoxic due to coordination to calcium and magnesium in essential enzymes, including ATP synthetases. We have shown that the ability to carry out extensive degradation of fluorinated compounds is strongly linked to effective fluoride stress response mechanisms. A major protective mechanism is carried out by membrane-bound fluoride export proteins. Using systems optimized for this purpose, bacteria have been subjected to laboratory evolution to improve rates and extent of fluorinated-compound biodegradation.
Results/Lessons Learned
While PFAS compounds are persistent, mechanisms exist to biodegrade these chemicals if attention is paid to the hurdles that cells must overcome to degrade the chemicals and deal with fluoride toxicity emanating from the defluorination reactions. First, rapid methods must be instituted to determine successful biodegradation. This gives one the ability to combinatorially screen millions of substrate-catalysts pairs. Once high levels of biodegradation are achieved, fluoride toxicity imposes significant limitations on the process. This requires pairing of enzymes with highly protective fluoride stress response mechanisms. Screening is effective, but selection for growth on fluorinated compounds provides a way to achieve rapid degradation and cell protection at the same time. Genome sequencing of improved strains provides new insights into how PFAS biodegradation activities can efficiently evolve over time.