Improving fluoride tolerance in a Pseudomonas sp via adaptive laboratory evolution

Abstract

Per and poly-fluorinated alkyl and aromatic substances (PFAS) are pervasive across the globe in both use and bioaccumulation. They are known for their chemical stability due to the strength of the C-F bond, which makes biodegradation difficult. A secondary challenge in degrading PFAS is fluoride toxicity. PFAS degradation will always produce fluoride due to the electronegativity of the fluorine atom. In perfluorooctanoic acid (PFOA) and similar PFAS compounds, 12 to 15 fluorine atoms would be released per PFAS molecule. Fluoride is toxic to most bacterial species even at small levels of 10 mM. In response, 85% of bacterial species possess fluoride exporters, either the passive channel CrcB or the H+/F- antiporter CLCF, that confer resistance to the low fluoride levels typically seen in the environment. Resistance to significantly higher fluoride concentrations will be required to host PFAS degradation. In this study, adaptive laboratory evolution (ALE) experiments were conducted to improve both intracellular and extracellular fluoride tolerance in terms of growth yield and lag phase after starting on a relatively stress tolerant Pseudomonas sp. With transfers on 20 mM α-fluorophenylacetic acid, rapid adaption was observed in <15 generations wherein the lag phase went from 16 hours to 2.5 hours and maximum cell density increased by 15%. Sequencing revealed a downward titration of plasmid copy number to be the source of adaption wherein the cells decreased the fluoride level produced at once without diminishing the carbon flux. To improve tolerance to fluoride specifically, the fluoride antiporter CLCF was recombinantly expressed in our strain that natively possessed CrcB via two plasmid systems differing in plasmid copy number, respectively. Both showed improved maximum tolerance up to 300 mM NaF, whereas the wildtype strain showed no growth at 100 mM. ALE was then performed on sequentially increasing NaF levels until 550 mM NaF, above which no growth was observed. Sequencing revealed that the strains adapted by knocking out the nondirectional CrcB channel and elevating CLCF levels by increasing plasmid copy. Five biological replicates involving both CLCF strains showed different mutations in CrcB that modeling indicate to be non-functional. It is hypothesized that because CrcB functions based on the ion gradient, at high extracellular fluoride salt concentrations, the gradient will bring fluoride into the cell. With direct implications for maximum PFAS degradation based on fluoride tolerance, ALE was performed in a strain with both the higher copy CLCF plasmid and a defluorinase reacting with 2-fluoropropionic acid (2-FPA). Incrementally increasing 2-FPA concentrations resulted in strong growth of OD600 2.6 on 300 mM racemic 2-FPA. The defluorinase is stereospecifc to only one enantiomer of 2-FPA, and full degradation of 2-FPA was indicated by the release of 150 mM fluoride. Growth declined after a few transfers until strains ceased growth and 2-FPA degradation. Further studies are necessary to determine the cause. ALE on NaF and 2-FPA, respectively, resulted in novel fluoride tolerance 5.5-fold improved and C-F degradation 3-fold higher than previously reported in the literature.