Biotransformation and detoxification of tetrabromobisphenol A by white-rot fungus Phanerochaete sordida YK-624.

The bulky phenolic compound tetrabromobisphenol A (TBBPA) is a brominated flame retardant used in a wide range of products; however, it diffuses into the environment, and has been reported to have toxic effects. Although it is well-known that white-rot fungi degrade TBBPA through ligninolytic enzymes, no other metabolic enzymes have yet been identified, and the toxicity of the reaction products and their risks have not yet been examined. We found that the white-rot fungus Phanerochaete sordida YK-624 converted TBBPA to TBBPA-O-ß-D-glucopyranoside when grown under non-ligninolytic-enzyme-producing conditions. The metabolite showed less cytotoxicity and mitochondrial toxicity than TBBPA in neuroblastoma cells. From molecular biological and genetic engineering experiments, two P. sordida glycosyltransferases (PsGT1c and PsGT1e) that catalyze the glycosylation of TBBPA were newly identified; these enzymes showed dramatically different glycosylation activities for TBBPA and bisphenol A. The results of computational analyses indicated that the difference in substrate specificity is likely due to differences in the structure of the substrate-binding pocket. It appears that P. sordida YK-624 takes up TBBPA, and reduces its cytotoxicity via these glycosyltransferases.

Ergosterol and Its Metabolites Induce Ligninolytic Activity in the Lignin-Degrading Fungus Phanerochaete sordida YK-624.

White-rot fungi are the most important group of lignin biodegraders. Phanerochaete sordida YK-624 has higher ligninolytic activity than that of model white-rot fungi. However, the underlying mechanism responsible for lignin degradation by white-rot fungi remains unknown, and the induced compounds isolated from white-rot fungi for lignin degradation have never been studied. In the present study, we tried to screen ligninolytic-inducing compounds produced by P. sordida YK-624. After large-scale incubation of P. sordida YK-624, the culture and mycelium were separated by filtration. After the separation and purification, purified compounds were analyzed by high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance. The sterilized unbleached hardwood kraft pulp was used for the initial evaluation of ligninolytic activity. Ergosterol was isolated and identified and it induced the lignin-degrading activity of this fungus. Moreover, we investigated ergosterol metabolites from P. sordida YK-624, and the ergosterol metabolites ergosta-4,7,22-triene-3,6-dione and ergosta-4,6,8(14),22-tetraen-3-one were identified and then chemically synthesized. These compounds significantly improved the lignin-degrading activity of the fungus. This is the first report on the ligninolytic-inducing compounds produced by white-rot fungi.

Aerobic H2 production related to formate metabolism in white-rot fungi.

Biohydrogen is mainly produced by anaerobic bacteria, anaerobic fungi, and algae under anaerobic conditions. In higher eukaryotes, it is thought that molecular hydrogen (H2) functions as a signaling molecule for physiological processes such as stress responses. Here, it is demonstrated that white-rot fungi produce H2 during wood decay. The white-rot fungus Trametes versicolor produces H2 from wood under aerobic conditions, and H2 production is completely suppressed under hypoxic conditions. Additionally, oxalate and formate supplementation of the wood culture increased the level of H2 evolution. RNA-seq analyses revealed that T. versicolor oxalate production from the TCA/glyoxylate cycle was down-regulated, and conversely, genes encoding oxalate and formate metabolism enzymes were up-regulated. Although the involvement in H2 production of a gene annotated as an iron hydrogenase was uncertain, the results of organic acid supplementation, gene expression, and self-recombination experiments strongly suggest that formate metabolism plays a role in the mechanism of H2 production by this fungus. It is expected that this novel finding of aerobic H2 production from wood biomass by a white-rot fungus will open new fields in biohydrogen research.

Total Biosynthesis of Melleolides from Basidiomycota Fungi: Mechanistic Analysis of the Multifunctional GMC Oxidase Mld7.

Mushroom terpenoids are biologically and chemically diverse fungal metabolites. Among them, melleolides are representative sesquiterpenoids with a characteristic protoilludane skeleton. In this study, we applied a recently established hot spot knock-in method to elucidate the biosynthetic pathway leading to 1α-hydroxymelleolide. The biosynthesis of the sesquiterpene core involves the cytochrome P450 catalyzing stepwise hydroxylation of the Δ6 -protoilludene framework and a stereochemical inversion process at the C5 position catalyzed by short-chain dehydrogenase/reductase family proteins. The highlight of the biosynthesis is that the flavoprotein Mld7 catalyzes an oxidation-triggered double-bond shift accompanying dehydration and acyl-group-assisted substitution with two different nucleophiles at the C6 position to afford the Δ7 -protoilludene derivatives, such as melleolide and armillarivin. The complex reaction mechanism was proposed by DFT calculations. Of particular importance is that product distribution is regulated by interaction with the cell membrane.

"Fruiting Liquid" of Mushroom-Forming Fungi, A Novel Source of Bioactive Compounds - Fruiting-Body Inducer and HIF and Axl Inhibitors.

In general, mushroom-forming fungi secrete liquid on the surface of mycelia just before fruiting-body formation. However, no researchers in mushroom science have paid attention to the liquid until now. We formulated a hypothesis that the liquid plays an important role(s) in the formation of the fruiting body and produces various bioactive compounds and named it the "fruiting liquid (FL)". Four novel compounds (1-4) were isolated from FL of Hypholoma lateritium and Hericium erinaceus. The structures of 1-4 except for their stereochemistry were determined by interpretation of MS and NMR data. The absolute configurations of compounds 1-4 were determined by quantum chemical calculation of the ECD spectrum, by single-crystal X-ray diffraction analyses, or by chemical syntheses. Compounds 1, 3, and 4 induced fruiting body formation of Flammulina velutipes. Compound 4 inhibited the activity of hypoxia-inducible factor, and compounds 2-4 suppressed receptor tyrosine kinase (Axl) expression.

Chemical elucidation of acute encephalopathy by ingestion of angel-wing mushroom (Pleurocybella porrigens) - involvement of three constituents in onset.

The mushroom, Pleurocybella porrigens, is widely consumed in Japan; however, in autumn 2004, acute encephalopathy due to ingestion of the mushroom in a large group of patients was reported in Japan. We have continued working on the mushroom to clarify the mechanisms underlying the acute encephalopathy that occurred due to its consumption. The data collected to date have shown that three compounds, pleurocybelline (PC), a Pleurocybella porrigens lectin (PPL), and pleurocybellaziridine (PA), in the mushroom are potentially responsible for the onset of the disease; PC that exhibit lethal activity in mice and PPL formed a complex, and the complex of the two components exhibited proteolytic activity and disrupted the blood-brain barrier. Although PA was not isolated directly from the mushroom, the existence of this compound in the mushroom was predicted. The compound was chemically synthesized and its endogeneity in the mushroom was demonstrated. Furthermore, PA exhibited toxicity to oligodendrocytes.

Construction of white-rot fungal-bacterial consortia with improved ligninolytic properties and stable bacterial community structure.

It is believed that wood-rot fungi change their wood decay activities due to influences from co-existing bacterial communities; however, it is difficult to elucidate experimentally the interaction mechanisms in fungal-bacterial consortia because the bacterial community structure is quite unstable and readily changes. Indeed, the wood decay properties of fungal-bacterial consortia consisting of a white-rot fungus Phanerochaete sordida YK-624 and a natural bacterial community changed dramatically during several sub-cultivations on wood. Therefore, development of a sub-cultivation method that imparts stability to the bacterial community structure and fungal phenotype was attempted. The adopted method using agar medium enabled maintenance of fungal phenotypes relating to wood decay and the bacterial community even through dozens of repetitive sub-cultures. Some bacterial metabolic pathways identified based on gene predictions were screened as candidates involved in P. sordida-bacterial interactions. In particular, pathways related to prenyl naphthoquinone biosynthesis appeared to be involved in an interaction that promotes higher lignin degradation selectivity by the consortia, as naphthoquinone derivatives induced phenol-oxidizing activity. Based on these results, it is expected that detailed analyses of the relationship between the wood-degrading properties of white-rot fungal-bacterial consortia and bacterial community structures will be feasible using the sub-cultivation method developed in this study.

Role of hypoxanthine-guanine phosphoribosyltransferase in the metabolism of fairy chemicals in rice.

Fairy chemicals (FCs), 2-azahypoxanthine (AHX), imidazole-4-carboxamide (ICA), and 2-aza-8-oxohypoxanthine (AOH), are molecules with many diverse functions in plants. The defined biosynthetic pathway for FCs is a novel purine metabolism in which they are biosynthesized from 5-aminoimidazole-4-carboxamide. Here, we show that one of the purine salvage enzymes, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), recognizes AHX and AOH as substrates. Two novel compounds, AOH ribonucleotide and its ribonucleoside which are the derivatives of AOH, were enzymatically synthesized. The structures were determined by mass spectrometry, 1D and 2D NMR spectroscopy, and X-ray single-crystal diffraction analysis. This report demonstrates the function of HGPRT and the existence of novel purine metabolism associated with the biosynthesis of FCs in rice.

Bioactive Compounds from the Mushroom-Forming Fungus Chlorophyllum molybdites.

A novel compound (1) along with two known compounds (2 and 3) were isolated from the culture broth of Chlorophyllum molybdites, and three known compounds (4-6) were isolated from its fruiting bodies. The planar structure of 1 was determined by the interpretation of spectroscopic data. By comparing the specific rotation of the compound with that of the analog compound, the absolute configuration of 1 was determined to be R. This is the first time that compounds 2-4 were isolated from a mushroom-forming fungus. Compound 2 showed significant inhibition activity against Axl and immune checkpoints (PD-L1, PD-L2). In the bioassay to examine growth inhibitory activity against the phytopathogenic bacteria Peptobacterium carotovorum, Clavibacter michiganensis and Burkholderia glumae, compounds 2 and 3 inhibited the growth of P. carotovorum and C. michiganensis. In the bioassay to examine plant growth regulatory activity, compounds 1-4 showed a significant regulatory activity on lettuce growth.

The role of xanthine dioxygenase in the biosynthetic pathway of 2-aza-8-oxohypoxanthine of Lepista sordida.

2-Azahypoxanthine (AHX) and 2-aza-8-oxohypoxanthine (AOH), discovered as causal substances of fairy rings are known to be endogenous in the fairy ring-forming Lepista sordida. In this study, we showed that xanthine dioxygenase, an a-ketoglutarate-dependent dioxygenase, might catalyze the conversion of AHX to AOH in the fungus. Furthermore, this enzyme is the first reported molybdopterin-independent protein of hypoxanthine metabolism.

Identification of Biosynthetic and Metabolic Genes of 2-Azahypoxanthine in Lepista sordida Based on Transcriptomic Analysis.

2-Azahypoxanthine was isolated from the fairy ring-forming fungus Lepista sordida as a fairy ring-inducing compound. 2-Azahypoxanthine has an unprecedented 1,2,3-triazine moiety, and its biosynthetic pathway is unknown. The biosynthetic genes for 2-azahypoxanthine formation in L. sordida were predicted by a differential gene expression analysis using MiSeq. The results revealed that several genes in the purine and histidine metabolic pathways and the arginine biosynthetic pathway are involved in the biosynthesis of 2-azahypoxanthine. Furthermore, nitric oxide (NO) was produced by recombinant NO synthase 5 (rNOS5), suggesting that NOS5 can be the enzyme involved in the formation of 1,2,3-triazine. The gene encoding hypoxanthine-guanine phosphoribosyltransferase (HGPRT), one of the major phosphoribosyltransferases of purine metabolism, increased when 2-azahypoxanthine content was the highest. Therefore, we hypothesized that HGPRT might catalyze a reversible reaction between 2-azahypoxanthine and 2-azahypoxanthine-ribonucleotide. We proved the endogenous existence of 2-azahypoxanthine-ribonucleotide in L. sordida mycelia by LC-MS/MS for the first time. Furthermore, it was shown that recombinant HGPRT catalyzed reversible interconversion between 2-azahypoxanthine and 2-azahypoxanthine-ribonucleotide. These findings demonstrate that HGPRT can be involved in the biosynthesis of 2-azahypoxanthine via 2-azahypoxanthine-ribonucleotide generated by NOS5.

Possible molecular mechanism for acute encephalopathy by angel-wing mushroom ingestion - Involvement of three constituents in onset.

In Japan in 2004, 59 people who had consumed angel-wing mushroom, Pleurocybella porrigens, experienced acute encephalopathy, and of these 17 died. We purified a lethal protein to mice, pleurocybelline (PC), from P. porrigens. Although PC caused no damage to the brain, PC formed a complex with a lectin (PPL) and showed exo-protease activity, degrading substrates from both N- and C-termini. In addition, the presence of an unstable toxic compound, pleurocybellaziridine (PA), in the mushroom was demonstrated. We hypothesized that the complex and PA are involved in disease development and verified that apoptotic cells in the hippocampus were significantly increased by injection of the mixture of PC, PPL, and PA, indicating that these substances might be involved in acute encephalopathy.

Utilization of Corn Steep Liquor for the Production of Fairy Chemicals by Lepista sordida Mycelia.

There are various potential practical uses of fairy chemicals (FCs) in the fields of agriculture, cosmetics, and medicine; however, the production costs of FCs are very high. To enable the practical use of FCs, more efficient and inexpensive methods of culturing the mycelia of FCs-producing fungi and producing FCs need to be developed. The purpose of the present study was to determine methods of reducing the production costs of FCs and mycelia of the FCs-producing fungus Lepista sordida. We investigated the effects of four food industrial by-products, i.e., corn steep liquor (CSL), rice bran, wheat bran, and Japanese liquor lees, as nutritional additives in the liquid culture medium of the fungus. We found that CSL was more effective than the other tested additives in increasing the production of FCs and mycelia. Medium containing 1% CSL was optimal for increasing the mycelial yield while medium containing 6% CSL was optimal for increasing the production of FCs. The reason for this difference in the optimal CSL concentration was considered to be related to the stress on the mycelia caused by the amount of nutrients in the liquid medium. These results are expected to facilitate the practical use of FCs and the mycelia of FCs-producing fungi.

Axl, Immune Checkpoint Molecules and HIF Inhibitors from the Culture Broth of Lepista luscina.

Two compounds 1 and 2 were isolated from the culture broth of Lepista luscina. This is the first time that compound 1 was isolated from a natural source. The structure of compound 1 was identified via 1D and 2D NMR and HRESIMS data. Compounds 1 and 2 along with 8-nitrotryptanthrin (4) were evaluated for their biological activities using the A549 lung cancer cell line. As a result, 1 and 2 inhibited the expression of Axl and immune checkpoint molecules. In addition, compounds 1, 2 and 4 were tested for HIF inhibitory activity. Compound 2 demonstrated statistically significant HIF inhibitory effects on NIH3T3 cells and 1 and 2 against ARPE19 cells.

The complete mitochondrial genome of the white-rot fungus Phanerochaete sordida YK-624.

The white-rot fungus Phanerochaete sordida (Karsten) Eriksson and Ryvarden 1978 is known for its excellent ligninolytic activity and capability to degrade various recalcitrant organic pollutants. In this study, we determined the complete mitochondrial genome sequence of P. sordida YK-624. The mitochondrial genome is 129,567 bp in length with a GC content of 28.9%, and contains two ribosomal RNA genes, 26 transfer RNA genes, and 50 open reading frames, including 14 conserved proteins. Phylogenetic analysis based on the mitochondrial genome confirmed that P. sordida belongs to the family Phanerochaetaceae in the order Polyporales, and showed the general phylogenetic relationships.

Anti-phytopathogenic-bacterial fatty acids from the mycelia of the edible mushroom Agaricus blazei.

Five compounds including a new compound (1) were isolated from mycelia of a mushroom-forming fungus Agaricus blazei. Compound 2 was isolated from nature for the first time. Their structures were determined by the interpretation of spectroscopic data. In the bioassay examining growth inhibitory activity against phytopathogenic bacteria Clavibacter michiganensis, Burkholderia glumae, and Peptobacterium carotovorum, all the compounds showed inhibition effects on C. michiganensis. Compounds 3 and 4 also showed weak inhibitory activity against growth of B. glumae.

Potential of Fairy Chemicals as Functional Cosmetic Ingredients: Effect of 2-Aza-8-Oxohypoxanthine on Skin Lightness.

Among the "fairy chemicals" involved in forming the natural phenomenon of "fairy rings," we focused on 2-aza-8-oxohypoxanthine (AOH) as a candidate functional cosmetic ingredient. In previous studies, AOH was confirmed to be safe for use on human skin, and no adverse reactions were observed in any of the safety studies. In this study, we report the results of a clinical trial using a lotion containing AOH. Our analysis using the L* value for indices of skin lightness indicated that the AOH application significantly increased the L* value after 8 weeks. Since a previous DNA microarray study using normal human epidermal cells showed that AOH suppressed the expression of a group of genes that induce inflammatory cytokines (prostaglandin E synthase, prostaglandin-endoperoxide synthase 2 [cyclooxygenase-2], and interleukin-18), our results suggest that the AOH-induced suppression of inflammatory factors results in skin lightening.

CmLec4, a lectin from the fungus Cordyceps militaris, controls host infection and fruiting body formation.

Fungi belonging to the Ascomycete genus Cordyceps are endoparasitoids and parasites, mainly of insects and other arthropods. Cordyceps militaris has been used as a therapeutic drug for cancer patients. However, the infection, parasitism, and fruiting body formation mechanisms of this fungus are still unknown. Based on our hypothesis that lectin(s) is involved in the interaction between the C. militaris fungi and insects, we partially purified and characterized a new lectin from C. militaris, designated CmLec4. In addition, we searched for substance(s) in the infected silkworm extracts that could bind to CmLec4, and succeeded in purifying the sex-specific storage protein 2 as a specific binding target. To examine function of the binding protein during the process of parasitism, we investigated the effect of recombinant CmLec4 on silkworms by inoculating the protein into silkworm pupae, and found that it significantly delayed emergence compared to the control. Furthermore, cmlec4 gene knockout strains constructed in this study produced markedly lower amounts of fruiting body than the wild-type strain. All the results revealed that the lectin CmLec4 produced by C. militaris would be involved in the infection into silkworm and fruiting body formation from the host.

The complete mitochondrial genome sequence of the fairy ring-forming fungus Lepista sordida.

Lepista sordida is a fairy ring-forming fungus that belongs to the family Tricholomataceae and is widely distributed in the Northern Hemisphere. Here, we report the complete mitochondrial genome sequence of L. sordida. The mitochondrial genome (57,375 bp) contained 20 protein-coding genes, 2 ribosomal RNA genes, and 26 transfer RNA genes. Phylogenetic analysis based on 14 conserved protein sequences from L. sordida and 15 related basidiomycetes showed that L. sordida was located on the outermost branch of the Tricholomataceae clade. This study is the first to report the complete mitochondrial genome sequence of a fairy ring-forming fungus belonging to the genus Lepista.

Study on Secondary Metabolites of Endophytic Fungus, Aspergillus fumigatus, from Crocus sativus L. Guided byUHPLC-HRMS/MS-Based Molecular Network.

As a traditional Chinese medicine, Crocus sativus Linn has been used for a long time in China. However, the studies on secondary metabolites of its endophytic fungi were not fully sufficient. Thus, the endophytic fungus, Aspergillus fumigatus, collected from the lateral buds of C. sativus, was here investigated. An approach combining UHPLC-HRMS/MS (ultra-high performance liquid chromatography-high resolution mass spectrometry) with molecular network was carried out to construct a molecular network of crude EtOAc extract (CEE) of A. fumigatus, in which 32 chemical compounds were annotated. On the basis of analysis results, a total of 15 known natural compounds were isolated from CEE. Among them, compounds 11 and 12 were isolated for the first time from the genus Aspergillus. Moreover, CEE and compound 7 exhibited moderate inhibitory activity against Erwinia sp. with a MIC value of 100 µg/mL. This study provided a more convenient and rapid approach to investigating the crude extract with complex components of A. fumigatus, which is of great benefit to the further study and utilization of secondary metabolites of the genus Aspergillus.