Production of nitroaryl secondary metabolites by wood-decaying fungi of <i>Phlebia</i> spp.

Filamentous fungi produce secondary metabolites with multiple biochemical activities. For wood-decaying fungi of Basidiomycota, some of these compounds may act as redox-active mediators involved in biodegradation of lignocelluloses and biopolymers. Our aim was to identify natural aromatic compounds produced by white rot fungi of the genus <i>Phlebia</i> (Meruliaceae, Polyporales, Agaricomycetes), which comprises efficient decomposers of wood, wastes, and xenobiotics. Naturally produced aryl compounds were obtained by cultivating the fungi on a defined low-nitrogen liquid medium with glucose as carbon source. Culture supernatants were extracted and analyzed with UPLC-MS (ultra-performance liquid chromatography-mass spectrometry) and NMR (nuclear magnetic resonance). Enzyme assays, cultivation with ¹⁵N isotope-labeled nitrogen supplement, and aryl compound-feeding experiments were performed to assess biosynthesis mechanisms. Together with the well-known secondary metabolite veratryl alcohol and its enzymatic oxidation product veratraldehyde, we identified two nitroaryl derivatives, 6-nitroveratryl alcohol and 4-nitroveratrole, accumulating in culture supernatants of <i>Phlebia</i> spp. Cultivation of <i>P. radiata</i> isolate 2776 with NH₄NO₃ caused higher product yield of the nitroaryl compounds than ¹⁵NH₄Cl supplementation, suggesting a role of nitrate ions in formation of nitroaryl products. With ¹⁵N-labeled supplementation, however, incorporation of nitrogen also from ammonium ions was observed. Although lignin peroxidase (LiP) enzyme activities correlated with appearance of nitroaryl compounds, their formation from veratryl alcohol by LiP was not accomplished in vitro in reaction mixtures with extracellular supernatants. In compound-feeding experiments, additional glycosylated derivative of 6-nitroveratryl alcohol was detected in <i>P. radiata</i> cultures, and nitroguaiacol was formed from nitroveratrole. These results indicate multiple pathways including both intra- and extracellular metabolism in biosynthesis and bioconversion of monoaromatic aryl compounds and their derivatives in fungi of <i>Phlebia</i>.