Aerobic GenX Defluorination by a Wastewater Cladosporium halotolerans Strain with a Genomically Expanded Haloacid Dehalogenase Repertoire

Per- and polyfluoroalkyl substances (PFAS) such as GenX (HFPO-DA) are aerobically recalcitrant contaminants for which biological treatment options remain scarce; the best-characterized microbial degraders require strictly anaerobic conditions and external cofactors. We isolated Cladosporium halotolerans strain CsHGX-1 from activated sludge at a municipal wastewater treatment plant using GenX as the sole carbon source. Whole-genome sequencing (32.4 Mb; 11,201 genes) revealed a PFAS-degradation gene repertoire substantially expanded relative to congeneric Cladosporium species, including 26 dehalogenases (four type-II haloacid dehalogenases, HADs), 141 cytochrome P450s, and 558 esterases/hydrolases. Under aerobic conditions with GenX (50 mg L-1) as the sole carbon source, strain CsHGX-1 removed 47.6 {+/-} 1.7% of GenX within 48 h, accompanied by fluoride release (0.079 {+/-} 0.018 mM) that was absent in abiotic controls, confirming genuine C-F bond cleavage. Time-resolved RNA sequencing (0, 6, 24, 48 h; n = 6 biological replicates) revealed a phase-structured transcriptional program: oxidative genes, including cytochrome P450s, peaked first (6 h; up to 25.4-fold), hydroxylation and reactive-oxygen-species-management genes peaked next (24 h; up to 33.5-fold), and the three type-II HAD genes peaked last (48 h; up to 50.9-fold), coincident with fluoride accumulation. A parallel resazurin metabolic assay over 5 days confirmed sustained catabolic activity in GenX-exposed cultures relative to controls (1.37-1.52-fold; p [≤] 0.003). These findings identify strain CsHGX-1 as, to our knowledge, the first Ascomycete fungus for which genomic and transcriptomic evidence links oxidative activation to haloacid-dehalogenase-mediated defluorination of an aerobically recalcitrant PFAS, extending the known diversity of fungal PFAS degraders beyond Basidiomycota white-rot taxa. IMPORTANCEGenX is a PFAS "replacement" chemical that the U.S. Environmental Protection Agency added to its list of hazardous constituents in 2024, yet no aerobic biological treatment exists for it: every well-characterized microbial degrader requires oxygen-free conditions and added cofactors. We show that a fungus recovered from ordinary wastewater sludge breaks down GenX while using oxygen, the same conditions already used in conventional treatment plants, with no nutrient or reductant supplementation. Genome sequencing showed why this strain is unusual: it carries far more dehalogenase and cytochrome P450 genes than its close relatives. Time-course RNA sequencing showed these genes switch on in a defined order, oxidation first, then carbon-fluorine bond cleavage, matching the appearance of free fluoride in the culture. This links genome content to a functional outcome in an Ascomycete fungus, suggesting aerobic fungal defluorinators may already be present, unrecognized, in engineered wastewater systems.