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.

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.

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.

1,2,3-Triazine formation mechanism of the fairy chemical 2-azahypoxanthine in the fairy ring-forming fungus Lepista sordida.

2-Azahypoxanthine (AHX) was first isolated from the culture broth of the fungus Lepista sordida as a fairy ring-inducing compound. It has since been found that a large number of plants and mushrooms produce AHX endogenously and that AHX has beneficial effects on plant growth. The AHX molecule has an unusual, nitrogen-rich 1,2,3-triazine moiety of unknown biosynthetic origin. Here, we establish the biosynthetic pathway for AHX formation in L. sordida. Our results reveal that the key nitrogen sources that are responsible for the 1,2,3-triazine formation are reactive nitrogen species (RNS), which are derived from nitric oxide (NO) produced by NO synthase (NOS). Furthermore, RNS are also involved in the biochemical conversion of 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranosyl 5'-monophosphate (AICAR) to AHX-ribotide (AHXR), suggesting that a novel biosynthetic route that produces AHX exists in the fungus. These findings demonstrate a physiological role for NOS in AHX biosynthesis as well as in biosynthesis of other natural products containing a nitrogen-nitrogen bond.

Biotransformation of bisphenol F by white-rot fungus Phanerochaete sordida YK-624 under non-ligninolytic condition.

Environmental bisphenol F (BPF) has a cyclic endocrine disruption effect, seriously threatening animal and human health. It is frequently detected in environmental samples worldwide. For BPF remediation, biological methods are more environmentally friendly than physicochemical methods. White-rot fungi have been increasingly studied due to their potential capability to degrade environmental pollutants. Phanerochaete sordida YK-624 has been shown to degrade BPF by ligninolytic enzymes under ligninolytic conditions. In the present study, degradation of BPF under non-ligninolytic conditions (no production of ligninolytic enzymes) was investigated. Our results showed that BPF could be completely removed after 7-d incubation. A metabolite of BPF, 4,4'-dihydroxybenzophenone (DHBP) was identified by mass spectrometry and nuclear magnetic resonance, and DHBP was further degraded by this fungus to form 4-hydroxyphenyl 4-hydroxybenzoate (HPHB). DHBP and HPHB were the intermediate metabolites of BPF and would be further degraded by P. sordida YK-624. We also found that cytochrome P450s played an important role in BPF degradation. Additionally, transcriptomic analysis further supported the involvement of these enzymes in the action of BPF degradation. Therefore, BPF is transformed to DHBP and then to HPHB likely oxidized by cytochrome P450s in P. sordida YK-624. Furthermore, the toxicological studies demonstrated that the order of endocrine-disrupting activity for BPF and its metabolites was HPHB > BPF > DHBP. KEY POINTS: • White-rot fungus Phanerochaete sordida YK-624 could degrade BPF. • Cytochrome P450s were involved in the BPF degradation. • The order of endocrine disrupting activity was: HPHB > BPF > DHBP.

A new lanostane triterpenoid from the mushroom Hypholoma fasciculare.

A novel compound (1) and 3 known compounds (2-4) were isolated from the fruiting bodies of Hypholoma fasciculare. The structure of 1 was determined by the interpretation of spectroscopic data. Compounds 2-4 were identified by comparing the spectra data of known compounds. In the bioassay examining growth inhibitory activity against phytopathogenic bacteria Clavibacter michiganensis, Burkholderia glumae, and Peptobacterium carotovorum, compounds 1, 2, and 4 showed inhibition effects on C. michiganensis only.

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.

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.

S-Adenosylhomocysteine Analogue of a Fairy Chemical, Imidazole-4-carboxamide, as its Metabolite in Rice and Yeast and Synthetic Investigations of Related Compounds.

During the course of our investigations of fairy chemicals (FCs), we found S-ICAr-H (8a), as a metabolite of imidazole-4-carboxamide (ICA) in rice and yeast (Saccharomyces cerevisiae). In order to determine its absolute configuration, an efficient synthetic method of 8a was developed. This synthetic strategy was applicable to the preparation of analogues of 8a that might be biologically very important, such as S-ICAr-M (9), S-AICAr-H (10), and S-AICAr-M (11).

An efficient heterologous Escherichia coli-based expression system for lectin production from Pleurocybella porrigens.

In this study, we report a more efficient heterologous expression of lectin from Pleurocybella porrigens (PPL) using an Escherichia coli-based expression system. The yield (9.3 mg/L culture broth) of recombinant PPL (rPPL) using this expression system was increased approximately 9-fold compared to our previous study. The rPPL obtained in this study exhibited the same biochemical properties as the native PPL.

Biosynthesis of the Fairy Chemicals, 2-Azahypoxanthine and Imidazole-4-carboxamide, in the Fairy Ring-Forming Fungus Lepista sordida.

Fairy rings resulting from a fungus-plant interaction appear worldwide. 2-Azahypoxanthine (AHX) and imidazole-4-carboxamide (ICA) were first isolated from the culture broth of one of the fairy ring-forming fungi, Lepista sordida. Afterward, a common metabolite of AHX in plants, 2-aza-8-oxohypoxanthine (AOH), was found in AHX-treated rice. The biosynthetic pathway of the three compounds that are named as fairy chemicals (FCs) in plants has been partially elucidated; however, that in mushrooms remains unknown. In this study, it was revealed that the carbon skeletons of AHX and ICA were constructed from Gly in L. sordida mycelia and the fungus metabolized 5-aminoimidazole-4-carboxamide (AICA) to both of the compounds. These results indicated that FCs were biosynthesized by a diversion of the purine metabolic pathway in L. sordida mycelia, similar to that in plants. Furthermore, we showed that recombinant adenine phosphoribosyltransferase (APRT) catalyzed reversible interconversion not only between 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranosyl 5'-monophosphate (AICAR) and AICA but also between ICA-ribotide (ICAR) and ICA. Furthermore, the presence of ICAR in L. sordida mycelia was proven for the first time by LC-MS/MS detection, and this study provided the first report that there was a novel metabolic pathway of ICA in which its ribotide was an intermediate in the fungus.

Plant growth regulators and Axl and immune checkpoint inhibitors from the edible mushroom Leucopaxillus giganteus.

A novel compound, (R)-4-ethoxy-2-hydroxy-4-oxobutanoic acid (1), and six known compounds (2-7) were isolated from the fruiting bodies of the wild edible mushroom Leucopaxillus giganteus. The planar structure of 1 was determined by the interpretation of spectroscopic data analysis. The absolute configuration of 1 was determined by comparing specific rotation of the synthetic compounds. In the plant regulatory assay, the isolated compounds (1-7) and the chemically prepared compounds (8-10) were evaluated their biological activity against the lettuce (Lactuca sativa) growth. Compounds 1 and 3-10 showed the significant regulatory activity of lettuce growth. 1 showed the strongest inhibition activity among the all the compounds tested. In the lung cancer assay, all the compounds were assessed the mRNA expression of Axl and immune checkpoints (PD-L1, PD-L2) in the human A549 alveolar epithelial cell line by RT-PCR. Compounds 1-10 showed significant inhibition activity against Axl and/or immune checkpoint.

Effects of Glucose Concentration on Ethanol Fermentation of White-Rot Fungus Phanerochaete sordida YK-624 Under Aerobic Conditions.

White-rot fungi are microorganisms capable of ethanol fermentation; however, the specific conditions activating ethanol fermentation are unclear in contrast to fermentation by yeasts. In this study, we investigated the conditions favoring ethanol fermentation by the white-rot fungus Phanerochaete sordida YK-624, which is able to produce ethanol from woody material. In aerobic stationary cultivation with various concentrations of glucose (0.8-33 g/l), the fungus produced ethanol in media containing an initial glucose concentration of 2.8 g/l or higher. The amount of glucose consumption, mycelial weight, and ethanol production on the second day of culture increased in a concentration-dependent manner at low glucose concentrations; however, these were saturated at high concentrations. Biomass yields (growth/glucose consumption) were decreased until the initial glucose concentration increased to 6.0 g/l, after which the biomass yields showed constant values at higher concentrations (12-33 g/l). On the other hand, ethanol yields increased with decreasing biomass yields. In short shaking cultivation using mycelial suspension, trace amounts of instantaneous aerobic ethanol production were observed with 1.1 and 2.1 g/l glucose, but the relative gene expression levels of key enzymes at the pyruvate branch point showed no significant differences between ethanol production and non-production conditions. From these experimental results, it appears that the white-rot fungus P. sordida YK-624 produces ethanol due to overflow in sugar metabolism under aerobic conditions, although P. sordida YK-624 prioritizes glucose utilization for respiratory growth.

Cytotoxic compounds against cancer cells from Bombyx mori inoculated with Cordyceps militaris.

Two compounds, 3'-deoxyinosine and cordycepin, were isolated from Bombyx mori inoculated with Cordyceps militaris. In the bioassay examining cytotoxicity against cancer cells, both compounds showed toxicity against A549, PANC-1, and MCF-7 cancer cells.

Plant growth regulators from the fruiting bodies of Tricholoma flavovirens.

A novel indole derivative (1) and three known compounds (2-4) were isolated from the fruiting bodies of Tricholoma flavovirens. Their structures were determined or identified by the interpretation of spectroscopic data. Compounds 1 and 2 promoted root growth of lettuce and inhibited hypocotyl growth at 1 µmol/paper. Compound 3 inhibited hypocotyl and root growth at 100 nmol/paper.

Analysis of ethanol fermentation mechanism of ethanol producing white-rot fungus Phlebia sp. MG-60 by RNA-seq.

BACKGROUND: The white-rot fungus Phlebia sp. MG-60 shows valuable properties such as high ethanol yield from several lignocellulosic materials, although white-rot fungi commonly degrade woody components to CO2 and H2O. In order to identify genes involved in ethanol production by Phlebia sp. MG-60, we compared genes differentially expressed by the ethanol producing fungus Phlebia sp. MG-60 and the model white-rot fungus Phanerochaete chrysosporium under ethanol fermenting and non-fermenting conditions using next-generation sequencing technologies. RESULTS: mRNAs from mycelia of Phlebia sp. MG-60 and P. chrysosporium under fermenting and non-fermenting conditions were sequenced using the MiSeq system. To detect differentially expressed genes, expression levels were measured in fragments per kilobase of exon per million mapped reads (FPKM). Differentially expressed genes were annotated using BLAST searches, Gene Ontology classifications, and KEGG pathway analysis. Functional analyses of differentially expressed genes revealed that genes involved in glucose uptake, glycolysis, and ethanol synthesis were widely upregulated in Phlebia sp. MG-60 under fermenting conditions. CONCLUSIONS: In this study, we provided novel transcriptomic information on Phlebia sp. MG-60, and these RNA-seq data were useful in targeting genes involved in ethanol production for future genetic engineering.

Bioconversion of AHX to AOH by resting cells of Burkholderia contaminans CH-1.

Fairy rings are zones of stimulated grass growth owing to the interaction between a fungus and a plant. We previously reported the discovery of two novel plant-growth regulating compounds related to forming fairy rings, 2-azahypoxanthine (AHX) and 2-aza-8-oxohypoxanthine (AOH). In this study, a bacterial strain CH-1 was isolated from an airborne-contaminated nutrient medium containing AHX. The strain converted AHX to AOH and identified as Burkholderia contaminans based on the gene sequence of its 16S rDNA. The quantitative production of AOH by resting cells of the strain was achieved. Among seven Burkholderia species, two bacteria and two yeasts tested, B. contaminans CH-1 showed the highest rate of conversion of AHX to AOH. By batch system, up to 10.6 mmol AHX was converted to AOH using the resting cells. The yield of this process reached at 91%.

Effects of Homologous Expression of 1,4-Benzoquinone Reductase and Homogentisate 1,2-Dioxygenase Genes on Wood Decay in Hyper-Lignin-Degrading Fungus Phanerochaete sordida YK-624.

We investigated the function of 1,4-benzoquinone reductase (BQR)- and homogentisate 1,2-dioxygenase (HGD)-like genes in wood degradation by Phanerochaete sordida YK-624, which exhibits high ligninolytic activity and selectivity. We determined homologous expression in the genomic and cDNA sequences of BQR- and HGD-like genes in P. sordida YK-624 (PsBQR and PsHGD). Both genes shared high homology (≥90 % amino acid sequence similarity) with the corresponding genes in Phanerochaete chrysosporium. These genes were co-transformed with a reporter gene into an uracil auxotrophic mutant of P. sordida YK-624. The PsBQR and PsHGD co-transformants exhibited lower holocellulolytic activity and higher ligninolytic selectivity than the control transformants. In liquid culture with vanillin, both co-transformants significantly accelerated vanillin degradation. Thus, we suggest that the rapid metabolism of low-molecular weight lignin fragments, due to the homologous expression of BQR- and HGD-like genes, affects quinone redox cycling to produce hydroxyl radicals, thereby decreasing holocellulose degradation and increasing ligninolytic selectivity.

Bioactive sesquiterpene aryl esters from the culture broth of Armillaria sp.

Two new compounds, 10-dehydroxymelleolide D (1) and 13-hydroxymelleolide K (2), along with seven known compounds, 5'-O-methylmelledonal (3), melleolide D (4), 13-hydroxydihydromelleolide (5), melleolide (6), armillarinin (7), armillaridin (8), and armillarikin (9), were isolated from the culture broth of Armillaria sp. Their structures were determined by spectroscopic data analysis. All the compounds inhibited plant growth of lettuce. Melleolide (6) and armillarikin (9) inhibited mycelial growth of Coprinopsis cinerea and/or Flammulina velutipes.

Erinaceolactones A to C, from the culture broth of Hericium erinaceus.

Three novel compounds, erinaceolactones A to C (1-3), and a known compound (4) were isolated from the culture broth of Hericium erinaceus. The planar structures of 1-3 were determined by the interpretation of spectroscopic data. The absolute configuration of 3 was determined by X-ray crystallography. Although compound 4 had been synthesized, it was isolated from a natural source for the first time. In the bioassay examining plant-growth regulatory activity of these compounds (1-4) and other components of the fungus (5-8), compounds 1, 2, and 4-8 suppressed the growth of lettuce.