Recombinant Pseudomonas growing on non-natural fluorinated substrates shows stress but overall tolerance to cytoplasmically released fluoride anion

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 fluoropropionic2-fluoropropionicacid and α-fluorophenylacetic acid when defluroinases are expressed. The defluorinasedefluorinasedefluorinasewas 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 fluorophenylacetic-fluorophenylaceticacid 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 defluorina tion of non-natural substrates within limits posed by fluoride stress.