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Abstract: Microbial response to toxic fluoride anion has traditionally been studied by adding inorganic fluoride salts to growth media. Fluoride is known to spontaneously transit the membrane as hydrogen fluoride (HF) and manifests significant toxicity in the cytoplasm. The present study investigated how microbes respond to high levels of HF generated directly in the cytoplasm to better understand potential limits of microbial defluorination of organofluorine compounds. For that purpose, Pseudomonas putida ATCC 12633 was engineered to express a defluorinase enzyme from Delftia acidovorans strain B that had high activity in producing growth-supporting alcohols from organofluorinated compounds of xenobiotic origin. Genome annotation predicted the growth of P. putida ATCC 12633 on all possible chiral products from 2-fluoropropionic acid and α-fluorophenylacetic acid when defluroinases are expressed. The defluorinase was shown to have complete enantioselectivity for (S)-fluoro substrates via experimental and computational modeling methods. The bacterium grew to a high turbidity with stoichiometric release of fluoride from the (S)-enantiomers into the medium. The highest yield of fluoride obtained was 50 mM with 2-fluoropropionic acid as the growth substrate. The growth yield was significantly lowered by 41% with α-fluorophenylacetic acid or 2-fluoropropionic acid compared to (S)-mandelic acid or D-lactic acid, respectively. Fluoride stress was also indicated by longer lag phases, slower growth, and cell morphology changes on fluorinated substrates or the cognate alcohols with NaF in the medium. In total, these studies show the potential for engineering bacterial defluorination of non-natural substrates within limits posed by fluoride stress. IMPORTANCE: Society uses thousands of organofluorine compounds, sometimes denoted per- and polyfluoroalkyl substances (PFAS), in hundreds of products, but recent studies have shown some to manifest human and environmental health effects. As a class, they are recalcitrant to biodegradation, partly due to the paucity of fluorinated natural products to which microbes have been exposed. Another limit to PFAS biodegradation is the intracellular toxicity of fluoride anion generated from C-F bond cleavage. The present study identified a broader substrate specificity in an enzyme originally studied for its activity on the natural product fluoroacetate. A recombinant Pseudomonas expressing this enzyme was used here as a model system to better understand the limits and effects of a high level of intracellular fluoride generation. A fluoride stress response has evolved in bacteria and has been described in Pseudomonas spp. The present study is highly relevant to organofluorine compound degradation or engineered biosynthesis in which fluoride anion is a substrate.