Network Pharmacology Analysis of Liquid-Cultured Armillaria ostoyae Mycelial Metabolites and Their Molecular Mechanism of Action against Gastric Cancer.

Armillaria sp. are traditional edible medicinal mushrooms with various health functions; however, the relationship between their composition and efficacy has not yet been determined. Here, the ethanol extract of liquid-cultured Armillaria ostoyae mycelia (AOME), a pure wild Armillaria sp. strain, was analyzed using UHPLC-QTOF/MS, network pharmacology, and molecular docking techniques. The obtained extract affects various metabolic pathways, such as JAK/STAT and PI3K/AKT. The extract also contains important compounds such as 4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl] benzamide, isoliquiritigenin, and 7-hydroxycoumarin. Moreover, the extract targets key proteins, including EGFR, SCR, and IL6, to suppress the progression of gastric cancer, thereby synergistically inhibiting cancer development. The molecular docking analyses indicated that the main compounds stably bind to the target proteins. The final cell culture experimental data showed that the ethanol extract inhibited MGC-803 gastric cancer cells. In summary, our research revealed the beneficial components of AOME for treating gastric cancer and its associated molecular pathways. However, further research is needed to confirm its effectiveness and safety in gastric cancer patients.

Extraction, purification, structural characteristics and biological properties of the polysaccharides from Armillaria mellea (Vahl) P. Kumm.: A review.

Armillaria mellea (Vahl) P. Kumm. is a well-known homoeopathic plant with medicinal and culinary uses. Modern phytochemical researchers have successfully extracted and purified over 40 types of A. mellea polysaccharides (AMPs) from the fruiting bodies, hyphae and fermentation broth of A. mellea, and some of them have been analyzed and identified by their chemical structures. The impressive biological activity of these polysaccharides has been recognized by scientists worldwide. Many studies show that AMPs have remarkable antioxidant, anti-diabetic, anti-tumor, anti-inflammatory, immunoregulatory, hypolipidemic, thrombectomy, anti-aging, pulmonary protective, hepatic protective, anti-Alzheimer's properties, etc. However, the current understanding of the relationships between their chemical structure and biological activity, toxicological effects and pharmacokinetics remains limited. This article provides a systematic review of the research conducted over the past decades on the extraction and purification methods, structural characteristics, biological activity and mechanism of action of AMPs. The aim is to provide a research base that will benefit the future application of AMPs as therapeutic drugs and functional foods, and also provide insights for the further development of AMPs.

Honey Mushroom, Armillaria mellea (Agaricomycetes) and Its Fermentation Products Target Regulation of OAT1/OAT3 Proteins to Reduce Hyperuricemia in Mice.

BACKGROUND: Disorders of purine metabolism are the main cause of hyperuricemia. Current drugs for the treatment of hyperuricemia usually cause a degree of cardiovascular damage. METHODS: This study aimed to investigate the therapeutic effects of Armillaria mellea fruiting body (AFB), Armillaria rhizomorph (AR) and Armillaria mellea fermentation product (after rhizomorphs removal) (AFP) on hyperuricemic mice. The hyperuricemia mouse model was established by oral administration of potassium oxonate 0.9 g⋅kg-1 and hypoxanthine 0.5 g⋅kg-1 for two weeks. Starting from the third week, the intragastric administration of the intervention drug group was as follows: Allopurinol 0.013 g⋅kg-1, AFB (3.9 and 7.8 g⋅kg-1), AR (3.9 and 7.8 g⋅kg-1), AFP (1.95 and 3.9 g⋅kg-1) once daily for 14 days. RESULTS: Results showed that AFB, AR, and AFP reduced the contents of serum uric acid, serum creatinine, and blood urea nitrogen in hyperuricemic mice and the mechanism of action might be through up-regulation of the expression levels of organic anion transporter 1/organic anion transporter 3 proteins in kidney tissue. AR and AFP both exhibited better uric acid-lowering effects than AFB, which may be due to the higher purine content of AFB. CONCLUSIONS: Armillaria mellea and its fermentation products can treat hyperuricemia by up-regulating OAT1 protein and OAT3 protein, reducing uric acid content in mice.

Critical review of the phytochemical profiles and health-promoting effects of the edible mushroom Armillaria mellea.

Research on the nutritional and medicinal properties of wild edible mushrooms has witnessed a significant surge in recent years. Among these mushrooms, Armillaria mellea (AM) stands out due to its abundant biologically active components. The presence of biological compounds in AM, including carbohydrates, sterols, fatty acids, sesquiterpenes, non-hallucinogenic indole compounds and adenosine derivatives, has been demonstrated in previous studies. Notably, specific bioactive substances isolated from AM, such as armillarikin, have exhibited promising anticancer effects. In vitro studies have elucidated the mechanisms behind these effects, further emphasizing the potential of AM in cancer treatment. Consequently, the objective of this study is to provide a comprehensive overview of the phytochemical profiles of AM while thoroughly investigating its therapeutic benefits. Moreover, this research has uncovered novel and effective treatments, including the utilization of ultrasonic disruption extraction in food processing. These findings highlight the potential of AM as a functional food with possible medical applications. By exploring AM's phytochemical composition and therapeutic effects, this study aims to contribute to a deeper understanding of its potential as a valuable natural resource.

Structural characterization, anti-tumor and immunomodulatory activity of intracellular polysaccharide from Armillaria luteo-virens.

Armillaria luteo-virens (A. luteo-virens) is a kind of edible fungus mainly exists in Qinghai-Tibet of China, but at present only very few studies focus on the bioactivities of its polysaccharides. This study aimed to purify and characterize the structure features of a novel intracellular polysaccharide (ALP-A) derived from A. luteo-virens and explore its potential anti-tumor and immunomodulatory activities. Through systematic separation and purification, we obtained a homogeneous ALP-A with an average molecular weight of 23693Da. Structural analysis indicated that ALP-A was mainly composed of glucose and mannose with a molar ratio of 6.02:1. The repeating unit of ALP-A was →4) -α-D-Glcp-(1→ backbone with α-Glcp-(1→ and α-Manp-(6→ side chains which branched at O-2 position. The anti-tumor assays in vivo suggested that ALP-A could effectively restrain S180 solid tumor growth, protect immune organs and promote the secretion of cytokines (IL2, IL6 and TNF-α) in serum. Besides, in vitro immunomodulatory assays indicated that ALP-A could improve proliferation, phagocytic capacity and raise the level of NO and cytokines in Raw264.7 cells. These results demonstrate that ALP-A which possess potential antitumor and immunomodulatory abilities can be developed as a new functional food.

Characterization of the transcriptional responses of Armillaria gallica 012m to GA3.

Gastrodia elata needs to establish a symbiotic relationship with Armillaria strains to obtain nutrients and energy. However, the signaling cross talk between G. elata and Armillaria strains is still unclear. During our experiment, we found that the vegetative mycelium of Armillaria gallica 012m grew significantly better in the media containing gibberellic acid (GA3) than the blank control group (BK). To explore the response mechanism, we performed an RNA-sequencing experiment to profile the transcriptome changes of A. gallica 012m cultured in the medium with exogenous GA3. The transcriptome-guided differential expression genes (DEGs) analysis of GA3 and BK showed that a total of 1309 genes were differentially expressed, including 361 upregulated genes and 948 downregulated genes. Some of those DEGs correlated with the biological process, including positive regulation of chromosome segregation, mitotic metaphase/anaphase transition, attachment of mitotic spindle microtubules to kinetochore, mitotic cytokinesis, and nuclear division. These analyses explained that GA3 actively promoted the growth of A. gallica to some extent. Further analysis of protein domain features showed that the deduced polypeptide contained 41 candidate genes of GA receptor, and 27 of them were expressed in our samples. We speculate that GA receptors exist in A. gallica 012m. Comparative studies of proteins showed that the postulated GA receptor domains of A. gallica 012m have a higher homologous correlation with fungi than others based on cluster analysis.

Study of anti-fatigue activity of polysaccharide from fruiting bodies of Armillaria gallica.

Fatigue is a common physiological response that is closely related to energy metabolism. Polysaccharides, as excellent dietary supplements, have been proven to have a variety of pharmacological activities. In this study, A 23.007 kDa polysaccharide from Armillaria gallica (AGP) was purified and performed structural characterization, including analysis of homogeneity, molecular weight and monosaccharide composition. Methylation analysis is used to analyze the glycosidic bond composition of AGP. The mouse model of acute fatigue was used to evaluate the anti-fatigue effect of AGP. AGP-treatment improved exercise endurance in mice and reduced fatigue symptoms caused by acute exercise. AGP regulated the levels of adenosine triphosphate, lactic acid, blood urea nitrogen and lactate dehydrogenase, muscle glycogen and liver glycogen of acute fatigue mice. AGP affected the composition of intestinal microbiota, the changes of some intestinal microorganisms are correlated with fatigue and oxidative stress indicators. Meanwhile, AGP reduced oxidative stress levels, increased antioxidant enzyme activity and regulated the AMP-dependent protein kinase/nuclear factor erythroid 2-related factor 2 signaling pathway. AGP exerted an anti-fatigue effect through modulation of oxidative stress, which is related to intestinal microbiota.

Chemical Characterization and Bioactive Properties of the Edible and Medicinal Honey Mushroom Armillaria mellea (Agaricomycetes) from Serbia.

Honey mushroom, Armillaria mellea, is an edible and medicinal lignicolous basidiomycete. In this study, we investigated the chemical composition and bioactive properties of its methanolic and acetonic extracts. The chemical characterization of extracts was done with the HPLC-DAD-MS/MS method. The results showed that potassium was the most abundant mineral; chlorogenic acid was the most abundant polyphenol; malic acid was the most abundant organic acid; and among carbohydrates, the most abundant were sorbitol, glucose, fructose, and saccharose. Antioxidative activity was assessed by DPPH (IC50 of the methanolic extract was 608.32 µg/mL and of the acetonic extract 595.71 µg/mL) and reducing power assays (the results ranged between 0.034 and 0.102 µg/mL). Total phenolic content was determined as gallic acid equivalent (methanolic extract: 4.74 mg GAE/g; acetonic extract: 5.68 mg GAE/g). The microdilution assay was used to evaluate the antimicrobial activity of the extracts, and the results ranged from 1.25 to 20 mg/mL. The antidiabetic effect of the extracts was tested by the α-amylase (the results ranged from 34.90% to 41.98%) and α-glucosidase assays (the results were in the range of 0.55-2.79%). The neuroprotective activity was explored by the acetylcholinesterase inhibition assay (the results were in the range of 1.94-7.76%). The microtetrazolium assay was used to investigate the cytotoxic activity of the extracts (the IC50 values ranged from 212.06 to > 400 µg/mL). Although some results suggest that some activities of the extracts are relatively moderate, the honey mushroom can still be considered an excellent source of food and bioactive compounds with medicinal value.

Research Status and Application Prospects of the Medicinal Mushroom Armillaria mellea.

Armillaria is one of the most common diseases underlying chronic root rot in woody plants. Although there is no particularly effective way to prevent it, soil disinfection is a common effective protective measure. However, Armillaria itself has important medicinal value and is a symbiotic fungus in the cultivation of Gastrodia elata and Polyporus umbellatus. Therefore, researching Armillaria is of great practical significance. In this review, the biological characteristics, cultivation methods, chemical components, food and medicinal value and efficacy of Armillaria were all reviewed, and its development and utilization direction were analyzed and discussed.

Nonribosomal peptide synthetase gene clusters and characteristics of predicted NRPS-dependent siderophore synthetases in Armillaria and other species in the Physalacriaceae.

Fungal secondary metabolites are often pathogenicity or virulence factors synthesized by genes contained in secondary metabolite gene clusters (SMGCs). Nonribosomal polypeptide synthetase (NRPS) clusters are SMGCs which produce peptides such as siderophores, the high affinity ferric iron chelating compounds required for iron uptake under aerobic conditions. Armillaria spp. are mostly facultative necrotrophs of woody plants. NRPS-dependent siderophore synthetase (NDSS) clusters of Armillaria spp. and selected Physalacriaceae were investigated using a comparative genomics approach. Siderophore biosynthesis by strains of selected Armillaria spp. was evaluated using CAS and split-CAS assays. At least one NRPS cluster and other clusters were detected in the genomes studied. No correlation was observed between the number and types of SMGCs and reported pathogenicity of the species studied. The genomes contained one NDSS cluster each. All NDSSs were multi-modular with the domain architecture (ATC)3(TC)2. NDSS clusters of the Armillaria spp. showed a high degree of microsynteny. In the genomes of Desarmillaria spp. and Guyanagaster necrorhizus, NDSS clusters were more syntenic with NDSS clusters of Armillaria spp. than to those of the other Physalacriaceae species studied. Three A-domain orthologous groups were identified in the NDSSs, and atypical Stachelhaus codes were predicted for the A3 orthologous group. In vitro biosynthesis of mainly hydroxamate and some catecholate siderophores was observed. Hence, Armillaria spp. generally contain one highly conserved, NDSS cluster although some interspecific variations in the products of these clusters is expected. Results from this study lays the groundwork for future studies to elucidate the molecular biology of fungal phyto-pathogenicity.

A Combination of Genome Mining with an OSMAC Approach Facilitates the Discovery of and Contributions to the Biosynthesis of Melleolides from the Basidiomycete Armillaria tabescens.

Genome mining revealed that the genomes of basidiomycetes may include a considerable number of biosynthetic gene clusters (BGCs), yet numerous clusters remain unidentified. Herein, we report a combination of genome mining with an OSMAC (one strain, many compounds) approach to characterize the spectrum of melleolides produced by Armillaria tabescens CPCC 401429. Using F1 fermentation medium, the metabolic pathway of the gene cluster mel was successfully upregulated. From the extracts of the wild-type strain, two new melleolides (1 and 2), along with five new orsellinic acid-derived lactams (10-14), were isolated, and their structures were elucidated by LC-HR-ESIMS/MS and 2D-NMR. Several melleolides exhibited moderate anti-carcinoma (A549, NCI-H520, and H1299) effects with IC50 values of 4.0-48.8 µM. RNA-sequencing based transcriptomic profiling broadened our knowledge of the genetic background, regulation, and mechanisms of melleolide biosynthesis. These results may promote downstream metabolic engineering studies of melleolides. Our study demonstrates the approach is effective for discovering new secondary metabolites from Armillaria sp. and will facilitate the mining of the unexploited biosynthetic potential in other basidiomycetes.

Armillaria mellea fermentation liquor ameliorates p-chlorophenylalanine-induced insomnia associated with the modulation of serotonergic system and gut microbiota in rats.

In China, Armillaria mellea (Vahl) P. Kumm. has been used as a folk medicine to treat insomnia for several hundred years. However, the underlying mechanisms involved are currently unknown. In this study, the anti-insomnia efficacy of A. mellea fermentation liquor (AFL) was evaluated in p-chlorophenylalanine-induced insomnia rats by measuring the serotonergic systems and gut microbiota. Our results demonstrate that all doses of AFL significantly reduced locomotor activity and alleviated decreasing weights in insomnia rats. Further, AFL exhibited better sedative effects by reducing sleep latency and increasing sleep duration in pentobarbital-treated rats. AFL treatment also elevated serum glutathione peroxidase and superoxide dismutase levels, while reducing serum interleukin-6, tumor necrosis factor-α, and interleukin-1ß levels. Furthermore, AFL alleviated insomnia by enhancing 5-hydroxytryptamine content and the expression 5-HT1A and 5-HT2A receptor in the hippocampus. Meanwhile, AFL treatment normalized the composition of gut microbiota in insomnia-model rats, while increasing relative abundance of Lachnospiraceae, Ruminococcaceae, and Saccharimonadaceae restores the gut microbial ecosystem altered in insomnia rats. The experiments show that A. mellea alleviated insomnia by modulating serotonergic system and gut microbiota. PRACTICAL APPLICATIONS: Insomnia has become a serious health issue of global concern. As a well-known traditional Chinese medicine, Armillaria mellea has been clinically employed in the treatment of insomnia for centuries in Asia with significant efficacy. In the present study, we firstly reported A. mellea fermentation liquor potentially relieved insomnia rats by alteration of gut microbiota and serotonergic systems and could guide future clinical studies. As a popular edible and medicinal mushroom, A. mellea also can be potentially used in the development and production of novel food products in the future.

Two new sesquiterpene aryl esters from Armillaria gallica 012m.

Two new sesquiterpene aryl esters, armimelleolides A and B (1 and 2), and four known ones, were isolated from the EtOAc extract of Armillaria gallica 012 m by column chromatography on silica gel, reversed-phase C18 silica gel and semi-preparative HPLC. Their structures were elucidated on the basis of spectroscopic methods, including extensive 1 D NMR, 2 D NMR and MS. All these compounds showed potential antitumor activities against at least one of the human cancer cell lines (A549, HCT-116, M231 and W256), with IC50 ranging from 2.57 to 19.94 µM.

Discovery of inhibition of Burkholderia cenocepacia, Pseudomonas aeruginosa and Stenotrophomonas maltophilia by the Brown Rot Basidiomycete Fungus, Postia placenta.

Antimicrobial resistance (AMR) has now emerged as a major global public health problem. Certain bacterial pathogens, particularly Gram negative organisms associated with patients with cystic fibrosis (CF), have become resistant to several classes of antibiotics resulting in pan-resistance, which creates a clinical treatment dilemma. This study wished to explore the production of antibacterial extracellular metabolites from plant pathogenic fungi. Fungal Culture Extracts (FCEs) were prepared from 10 fungi (Armillaria gallica, Clitocybe nebularis, Fusarium coeruleum, Fusarium oxysporum, Fusarium poae, Hymenoscyphus fraxineus, Nectria fuckeliana, Phytophthora infestans, Phytophthora ramorum, Postia placenta), which were tested for activity against the CF pathogens, Pseudomonas aeruginosa (PA) (n=8), Burkholderia cenocepacia (n=2) and Stenotrophomonas maltophilia (n=2). In addition, FCE were assessed for their ability to alter antibiotic susceptibility in PA (n=8), with six antipseudomonal antibiotics (ceftazidime, ciprofloxacin, colistin, meropenem, piperacillin/tazobactam, tobramycin). None of the FCEs showed inhibitory activity to the 12 bacterial isolates tested, with the exception of the FCE from Postia placenta, which showed inhibition against all 12 bacteria. An antagonistic interaction was observed, where a statistically significant decrease in mean zone sizes was noted with Armillaria gallica (p=0.03) and Phytophthora infestans (p=0.03) FCEs and their interaction with the fluoroquinolone antibiotic, ciprofloxacin. Given the increase in clinical morbidity and mortality associated with chronic lung infections with Pseudomonas aeruginosa, Burkholderia cenocepacia and Stenotrophomonas maltophilia, coupled with the difficulty in treating such chronic infection due to overwhelming antimicrobial resistance, any novel substance showing inhibition of these organisms merits further investigation as a potential future antimicrobial agent, with potential clinical therapeutic application.

The selective anti-fungal metabolites from Irpex lacteus and applications in the chemical interaction of Gastrodia elata, Armillaria sp., and endophytes.

The investigation of the metabolites from endophyte Irpex lacteus cultured in host "tian ma" (Gastrodia elata) revealed five new tremulane sesquiterpenes (1-5), and a new tetrahydrofuran derivative (6). Compound 1 was the first tremulane glucoside, and 6 possessed a rare tetrahydropyran-tetrahydrofuran scaffold. Main metabolite (2,3-dihydroxydodacane-4,7-dione, 14) from I. lacteus showed significant selectivity for antifungal activity against phytopathogen and endophytes associated with G. elata rather than against Armillaria sp. providing nutrition for the host G. elata. 14 accounted for 27.4% of isolated compounds from G. elata medium, and 69.3% by co-culturing with Armillaria sp. So the I. lacteus tended to promote the growth of Armillaria sp. in co-culture by producing 2,3-dihydroxydodacane-4,7-dione (14) to selective inhibit the phytopathogen and endophyte existed in host G. elata for the benefit of G. elata-Armillaria symbiosis. And the results were in accord with the real environment of G. elata depending on the nutrition of Armillaria. Some metabolites had anti-fungal activities against phytopathogens of G. elata with MICs ≤8 µg/mL.

The catalytic activity of mycelial fungi towards 7-oxo-DHEA - an endogenous derivative of steroidal hormone dehydroepiandrosterone.

Seventeen species of fungi belonging to thirteen genera were screened for the ability to carry out the transformation of 7-oxo-DHEA (7-oxo-dehydroepiandrosterone). Some strains expressed new patterns of catalytic activity towards the substrate, namely 16ß-hydroxylation (Laetiporus sulphureus AM498), Baeyer-Villiger oxidation of ketone in D-ring to lactone (Fusicoccum amygdali AM258) and esterification of the 3ß-hydroxy group (Spicaria divaricata AM423). The majority of examined strains were able to reduce the 17-oxo group of the substrate to form 3ß,17ß-dihydroxy-androst-5-en-7-one. The highest activity was reached with Armillaria mellea AM296 and Ascosphaera apis AM496 for which complete conversion of the starting material was achieved, and the resulting 17ß-alcohol was the sole reaction product. Two strains of tested fungi were also capable of stereospecific reduction of the conjugated 7-keto group leading to 7ß-hydroxy-DHEA (Inonotus radiatus AM70) or a mixture of 3ß,7α,17ß-trihydroxy-androst-5-ene and 3ß,7ß,17ß-trihydroxy-androst-5-ene (Piptoporus betulinus AM39). The structures of new metabolites were confirmed by MS and NMR analysis. They were also examined for their cholinesterase inhibitory activity in an enzymatic-based assay in vitro test.

Element concentration, daily intake of elements, and health risk indices of wild mushrooms collected from Belgrad Forest and Ilgaz Mountain National Park (Turkey).

The aim of this study was to determine the element content of wild edible and inedible mushroom species (Agaricus campestris, Armillaria ostoyae, Boletus reticulatus, Bondarzewia mesenterica, Bovistella utriformis, Cantharellus cibarius, Marasmius oreades, Megacollybia platyphylla, Meripilus giganteus, Neoboletus erythropus, Panellus stipticus, Phaeotremella foliacea, Pleurotus ostreatus, Podoscypha multizonata, Russula aurea, R. chloroides, R. virescens, T. versicolor, Trametes gibbose, and Trichaptum biforme) collected from the Belgrad Forests and the Ilgaz Mountain National Park. Based on the results of elemental analyses, daily metal intake (DMI) and health risk index (HRI) values of edible mushrooms collected from both localities were also calculated. As, Cd, Cr, Se, P, Hg, Cu, Mn, Fe, Zn, Al, Ca, Mg, and K contents of mushrooms were in the ranges of 0.16-3.45, 0.09-2.4, 0.15-2.34, 0.3-8.13, 0.28-11.44, 14.03-37.81, 3.87-108.57, 6.18-149.77, 11.9-776.1, 5.4-317.4, 7.4-355.2, 15.4-3517.3, 266.0-2500.0, and 628.0-24083.0 mg/kg dry weight, respectively. As a result of the DMI and HRI analyses, Cu concentration of B. utriformis (DMI: 46.53 µg/kg body weight/serving, HRI: 1.16) and Cd concentrations of A. campestris (DMI: 0.49 µg/kg body weight/serving, HRI: 1.36), A. ostoyae (DMI: 1.03 µg/kg body weight/serving, HRI: 2.86), B. utriformis (DMI: 0.52 µg/kg body weight/serving, HRI: 1.44), and P. ostreatus (DMI: 0.45 µg/kg body weight/serving, HRI: 1.24) were found to exceed the legal limits determined by authorities. It was concluded that the species collected from the regions in question should be consumed in a controlled manner.

Study on antidepressant-like effect of protoilludane sesquiterpenoid aromatic esters from Armillaria Mellea.

Armillaria mellea, also known as Hazel mushroom, is a delicious food material and traditional herbal medicine in East Asia. Protoilludane sesquiterpenoid aromatic esters from A. mellea (PSAM) are the main active components with antibacterial and anticancer activities. This study explored the antidepressant-like activities of PSAM and its possible mechanisms of action using the open field test (OFT), tail suspension test (TST) and forced swimming test (FST) in mice for the first time. The results revealed that PSAM (1 mg/kg, i.p.) exhibited markedly antidepressant-like activity, which could be reversed by pretreatment with haloperidol (a non-selective D2 receptor antagonist), bicuculline (a competitive GABA antagonist), NMDA (an agonist at the glutamate site). Meanwhile, PSAM also effectively increased the hippocampus dopamine (DA) and γ-aminobutyric acid (GABA) and decreased the hippocampus glutamate (Glu) levels of mice, indicating that the antidepressant-like effect of PSAM might be mediated by the DAergic, GABAergic and Gluergic systems.

Isolation of a gene cluster from Armillaria gallica for the synthesis of armillyl orsellinate-type sesquiterpenoids.

Melleolides and armillyl orsellinates are protoilludene-type aryl esters that are synthesized exclusively by parasitic fungi of the globally distributed genus Armillaria (Agaricomycetes, Physalacriaceae). Several of these compounds show potent antimicrobial and cytotoxic activities, making them promising leads for the development of new antibiotics or drugs for the treatment of cancer. We recently cloned and characterized the Armillaria gallica gene Pro1 encoding protoilludene synthase, a sesquiterpene cyclase catalyzing the pathway-committing step to all protoilludene-type aryl esters. Fungal enzymes representing secondary metabolic pathways are sometimes encoded by gene clusters, so we hypothesized that the missing steps in the pathway to melleolides and armillyl orsellinates might be identified by cloning the genes surrounding Pro1. Here we report the isolation of an A. gallica gene cluster encoding protoilludene synthase and four cytochrome P450 monooxygenases. Heterologous expression and functional analysis resulted in the identification of protoilludene-8α-hydroxylase, which catalyzes the first committed step in the armillyl orsellinate pathway. This confirms that ∆-6-protoilludene is a precursor for the synthesis of both melleolides and armillyl orsellinates, but the two pathways already branch at the level of the first oxygenation step. Our results provide insight into the synthesis of these valuable natural products and pave the way for their production by metabolic engineering. KEY POINTS: • Protoilludene-type aryl esters are bioactive metabolites produced by Armillaria spp. • The pathway-committing step to these compounds is catalyzed by protoilludene synthase. • We characterized CYP-type enzymes in the cluster and identified novel intermediates.

Do the Main Components of the Sclerotia of Umbrella Polypore Mushroom, Polyporus umbellatus (Agaricomycetes), Correlate with Armillaria Associates?

Polyporus umbellatus is a traditional Chinese medicinal mushroom. The growth of P. umbellatus sclerotia requires the rhizomorphs of Armillaria spp. to supply nutrition. Whether the main components (MC) of sclerotia of P. umbellatus are related to the phylogeny of Armillaria associates or other environmental factors is largely unknown. In this study, we collected 17 sclerotia and soil samples from northeast to southwest China. In total, 17 Armillaria associates were isolated, and sclerotial MC contents and soil characteristics (total N, P, K, and organic matter) were determined. The analysis revealed that the MC content of P. umbellatus did not resemble a Brownian motion process in phylogeny of Armillaria associates, but were significantly influenced by the total N content of the soil. These results provide clear evidence that sclerotia of P. umbellatus associating with phylogenetic related Armillaria associates possess differing MC content. The mechanisms of nutrient exchange in P. umbellatus-Armillaria associations now require further elucidation.