Identification and recombinant expression of a novel defluorinase from Rhodococcus jostii RHA1, for defluorination and biotransformation of the PFAS compound 6:2 fluorotelomer carboxylic acid

Abstract

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, a novel defluorinase was identified, that is responsible for the biodefluorination of 6:2 fluorotelomer carboxylic acid (6:2 FTCA) in R.jostii RHA1 using the combination of transposome-based insertional mutagenesis and heterologous expression. From a library of 417 R.jostii RHA1. mutants, 3 individual mutants lost their ability for defluorination when they were exposed to 6:2 FTCA (mutant # 15, 32 and 38 – Table S2). 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 µmol/h (V_max = 80.9 µmol/min and K_m = 5.04 mM in Michaelis-Menten models). 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 µmol/h fluoride) and minimally defluorinate 5:2 sFTOH (1.3 µmol/h fluoride). The novel defluorinase indicated a maximal specific activity of 12.9 ± 1.9 µmol F/hr/g protein, against its primary PFAS substrate, 6:2 FTCA. 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 µmol/h. The discovery of this MhPC class novel defluorinase in WT R.jostii RHA1. has substantial value since it is responsible for the critical step that initiates defluorination of PFAS compounds such as 6:2 FTCA.