Production of nitroaryl secondary metabolites by wood-decaying fungi of Phlebia 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 Phlebia (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 Phlebia spp. Cultivation of P. radiata 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 P. radiata 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 Phlebia.丝状真菌（Filamentous fungi）可产生具有多种生化活性的次级代谢产物。对于担子菌门（Basidiomycota）中的木材腐朽真菌而言，其中部分化合物可作为氧化还原活性介质，参与木质纤维素与生物聚合物的生物降解过程。本研究旨在鉴定卧孔菌属（Phlebia，隶属于皱孔菌科Meruliaceae、多孔菌目Polyporales、伞菌纲Agaricomycetes）的白腐真菌所产生的天然芳香族化合物，该属真菌是木材、废弃物及外源性异生物质的高效降解者。研究团队通过以葡萄糖为碳源的限定性低氮液体培养基培养该类真菌，获取天然合成的芳基化合物。对培养上清液进行萃取后，采用超高效液相色谱-质谱联用仪（UPLC-MS，ultra-performance liquid chromatography–mass spectrometry）与核磁共振波谱仪（NMR，nuclear magnetic resonance）开展分析。通过酶活测定、添加15N同位素标记氮源的培养实验以及芳基化合物饲喂实验，评估其生物合成机制。除了已知的次级代谢产物藜芦醇（veratryl alcohol）及其酶促氧化产物藜芦醛（veratraldehyde）之外，研究团队还在卧孔菌属菌株的培养上清液中鉴定出两种硝基芳基衍生物：6-硝基藜芦醇与4-硝基藜芦醚。当以硝酸铵（NH4NO3）培养辐射卧孔菌（P. radiata）分离株2776时，其硝基芳基化合物的产物得率高于添加15N-氯化铵（15NH4Cl）的实验组，这表明硝酸根离子在硝基芳基产物的形成中发挥关键作用。然而，在15N标记的氮源添加实验中，研究人员同样观察到铵离子也参与了氮元素的掺入。尽管木质素过氧化物酶（LiP，lignin peroxidase）的酶活与硝基芳基化合物的出现呈正相关，但在添加细胞外上清液的体外反应体系中，LiP无法将藜芦醇转化为硝基芳基化合物。在化合物饲喂实验中，辐射卧孔菌的培养物中还检测到6-硝基藜芦醇的另一种糖基化衍生物，且硝基藜芦醚可转化为硝基愈创木酚。上述结果表明，在卧孔菌属真菌的单环芳基化合物及其衍生物的生物合成与生物转化过程中，存在胞内与胞外代谢等多条途径。