Fernando, E. Y.

Poly and per fluorinated substances (PFAS) are emerging contaminants of concern that are thought to be involved in causing numerous adverse health effects, such as immunosuppression, increased chance of cancer development, and altered levels of hepatic enzyme levels in humans. However, PFAS are considered highly persistent and resistant to biodegradation given the fact that the C-F bond can have a bond dissociation energy of up to 544 kJ/mol. Though many studies have reported PFAS biodefluorination by bacterial isolates and microbial communities, little is known regarding the molecular foundations for biodefluorination. In this study, we identified a novel defluorinase that is responsible for the biodefluorination of 6:2 fluorotelomer carboxylic acid (6:2 FTCA) in Rhodococcus sp. using the combination of transposome-based insertional mutagenesis and heterologous expression. From a library of 41 Rhodococcus Sp. mutants, 3 individual mutants lost their ability for defluorination when they were exposed to 6:2 FTCA (mutant # 15, 32 and 38 , Supplementary table S-2). The disruption of the genetic locus in all 3 non-defluorinating mutants was identified coding for a putative MhPC superfamily protein. The MhPC superfamily of proteins is known to harbor other proteins such as fluoroacetate dehalogenase (UniProt;Q6NAM1) that are capable of C-F bond cleavage. This identified gene was cloned into the heterologous expression host M. smegmatis MC2-155. After induction, the M. smegmatis MC2-155 transformant exhibited the ability to defluorinate 6:2 FTCA at a rate of 13 micromol/h. In contrast, defluorination was not observed in either abiotic or biotic controls. Further characterization of the novel defluorinase indicated that it could moderately defluorinate the unsaturated PFAS compound 6:2 FTCUA (4.9 micromol/h fluoride) and minimally defluorinate 5:2 sFTOH (1.3 micromol/h fluoride). However, it showed no activity with 5:3 FTCA or sulfonated PFAS compounds such as 6:2 FTS and 8:2 FTS. The wild-type Rhodococcus could defluorinate 6:2 FTCA at a rate of 2.2 micromol/h. The discovery of this MhPC class novel defluorinase in WT Rhodococcus Sp. has substantial value since it is responsible for the critical step that initiates defluorination of PFAS compounds such as 6:2 FTCA.