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ABSTRACT A major factor limiting the biodegradation of organofluorine compounds has been highlighted as fluoride anion toxicity produced by defluorinating enzymes. Here, two highly active defluorinases with different activities were constitutively expressed inPseudomonas putidaATCC 12633 to examine adaption to fluoride stress. Each strain was grown on α‐fluorophenylacetic acid as the sole carbon source via defluorination to mandelic acid, and each showed immediate fluoride release and delayed growth. Adaptive evolution was performed for each recombinant strain by serial transfer. Both strains adapted to show a much shorter lag and a higher growth yield. The observed adaptation occurred rapidly and reproducibly, within 50 generations each time. After adaption, growth with 50–70 mM α‐fluorophenylacetic acid was significantly faster with more fluoride release than a preadapted culture due to larger cell populations. Genomic sequencing of both pre‐ and postadapted strain pairs revealed decreases in the defluorinase gene content. With both defluorinases, adaption produced a 56%–57% decrease in the plasmid copy number. Additionally, during adaption of the strain expressing the faster defluorinase, two plasmids were present: the original and a derivative in which the defluorinase gene was deleted. An examination of the enzyme rates in the pathway suggested that the defluorinase rate was concurrently optimised for pathway flux and minimising fluoride toxicity. The rapid alteration of plasmid copy number and mutation was consistent with other studies on microbial responses to stresses such as antibiotics. The data presented here support the idea that fluoride stress is significant during the biodegradation of organofluorine compounds and suggest engineered strains will be under strong selective pressure to decrease fluoride stress. 

Per- and polyfluorinated chemicals (PFAS) are of rising concern due to environmental persistence and emerging evidence of health risks to humans. Environmental persistence is largely attributed to a failure of microbes to degrade PFAS. PFAS recalcitrance has been proposed to result from chemistry, specifically C-F bond strength, or biology, largely negative selection from fluoride toxicity. Given natural evolution has many hurdles, this review advocates for a strategy of laboratory engineering and evolution. Enzymes identified to participate in defluorination reactions have been discovered in all Enzyme Commission classes, providing a palette for metabolic engineering. In vivo PFAS biodegradation will require multiple types of reactions and powerful fluoride mitigation mechanisms to act in concert. The necessary steps are to: (1) engineer bacteria that survive very high, unnatural levels of fluoride, (2) design, evolve, and screen for enzymes that cleave C–F bonds in a broader array of substrates, and (3) create overall physiological conditions that make for positive selective pressure with PFAS substrates.