Efficient production of Antrodin C by microparticle-enhanced cultivation of medicinal mushroom Antrodia cinnamomea.

The microparticle-enhanced cultivation (MPEC) was used to enhance the production of Antrodin C by submerged fermentation of medicinal mushroom Antrodia cinnamomea. The crucial factors such as types, sizes, concentrations, and addition time of microparticles were optimized. The mechanism of MPEC on the membrane permeability and fluidity of A. cinnamomea and the expression of key genes in Antrodin C were investigated. When talc (18 µm, 2 g/L) was added into the fermentation liquid at 0 h, the promoting effect on Antrodin C was the best. The maximum yield of Antrodin C was 1615.7 mg/L, which was about 2.98 times of the control (541.7 mg/L). Talc slightly damaged the mycelia of A. cinnamomea, increased the release of intracellular constituents, and enhanced the index of unsaturated fatty acid. In addition, the key genes (IDI, E2.3.3.10, HMGCR, atoB) that might play an important role in the synthesis of the triquine-type sesquiterpene Antrodin C, were upregulated. In conclusion, talc increased the permeability and fluidity of cell membrane, upregulated the key genes and improved the biosynthesis process to enhance the yield of Antrodin C in the submerged fermentation of A. cinnamomea.

Structural sequencing and anti-inflammatory, anti-lung cancer activities of 1,4-α/ß-sulfomalonoglucan in Antrodia cinnamomea.

Antrodia cinnamomea is a precious Polyporaceous fungus with various bioactivities. This study reports the chemical identification and biological activities of sulfomalonoglucan, a sulfated polysaccharide (SPS), from the sodium sulfate enriched medium of the title fungus. The SPS-containing fraction was separated by gel filtration chromatography (GFC) to give the title SPS (denoted as Na10_SPS-F3). By analyzing the evidence for key inter-glycosidic linkages in the 1D and 2D NMR spectroscopic data, one possible repeat unit was proposed as: Na10_SPS-F3 inhibited the secretion of tumor necrosis factor (TNF-α) and interleukin (IL)-6 after lipopolysaccharide (LPS) stimulation in RAW264.7 macrophages. Mechanistically, Na10_SPS-F3 downregulated TGFRII also attenuated the LPS-induced IκB-α degradation. Moreover, Na10_SPS-F3 inhibited lung cancer cell H1975 EGFR/ERK signaling. This is the first paper reporting a 3-O-sulfomalonyl glucan (Na10_SPS-F3) with eight 1,4-ß-Glc moieties connected with ten 1,4-α-Glc moieties from Antrodia cinnamomea and its anti-inflammatory and anti-cancer activities.

Oxidative Stress Induction Is a Rational Strategy to Enhance the Productivity of Antrodia cinnamomea Fermentations for the Antioxidant Secondary Metabolite Antrodin C.

Antioxidant metabolites contribute to alleviating oxidative stress caused by reactive oxygen species (ROS) in microorganisms. We utilized oxidative stressors such as hydrogen peroxide supplementation to increase the yield of the bioactive secondary metabolite antioxidant antrodin C in submerged fermentations of the medicinal mushroom Antrodia cinnamomea. Changes in the superoxide dismutase and catalase activities of the cells indicate that ROS are critical to promote antrodin C biosynthesis, while the ROS production inhibitor diphenyleneiodonium cancels the productivity-enhancing effects of H2O2. Transcriptomic analysis suggests that key enzymes in the mitochondrial electron transport chain are repressed during oxidative stress, leading to ROS accumulation and triggering the biosynthesis of antioxidants such as antrodin C. Accordingly, rotenone, an inhibitor of the electron transport chain complex I, mimics the antrodin C productivity-enhancing effects of H2O2. Delineating the steps connecting oxidative stress with increased antrodin C biosynthesis will facilitate the fine-tuning of strategies for rational fermentation process improvement.

Morphological plasticity in brown-rot fungi: Antrodia is redefined to encompass both poroid and corticioid species.

Most known brown rot-producing species of Polyporales belong to the so-called "Antrodia clade" that largely consists of poroid species. In this study, we use three genetic markers to revise Antrodia s. str., the core group of this clade. We show that a corticioid species with a smooth hymenophore, Phlebia griseoflavescens, belongs to Antrodia s. str. Accordingly, we revise the generic concept of Antrodia s. str. to accommodate this species and two recently described poroid taxa, A. tenerifensis and A. multiformis. In addition, we describe two new poroid species within Antrodia s. str., A. latebrosa from Africa and A. peregrina from East Asia, and provide new documentation for the Southeast Asian species A. parvula based on recent collections from the type location.

Diversity of potentially exploitable pharmacological activities of the highly prized edible medicinal fungus Antrodia camphorata.

Antrodia camphorata, also known as A. cinnamomea, is a precious medicinal basidiomycete fungus endemic to Taiwan. This article summarizes the recent advances in research on the multifarious pharmacological effects of A. camphorata. The mushroom exhibits anticancer activity toward a large variety of cancers including breast, cervical, ovarian, prostate, bladder, colorectal, pancreatic, liver, and lung cancers; melanoma; leukemia; lymphoma; neuroblastoma; and glioblastoma. Other activities encompass antiinflammatory, antiatopic dermatitis, anticachexia, immunoregulatory, antiobesity, antidiabetic, antihyperlipidemic, antiatherosclerotic, antihypertensive, antiplatelet, antioxidative, antiphotodamaging, hepatoprotective, renoprotective, neuroprotective, testis protecting, antiasthmatic, osteogenic, osteoprotective, antiviral, antibacterial, and wound healing activities. This review aims to provide a reference for further development and utilization of this highly prized mushroom.

Comparative Transcriptomic and Proteomic Analyses Reveal a FluG-Mediated Signaling Pathway Relating to Asexual Sporulation of Antrodia camphorata.

Medicinal mushroom Antrodia camphorata sporulate large numbers of arthroconidia in submerged fermentation, which is rarely reported in basidiomycetous fungi. Nevertheless, the molecular mechanisms underlying this asexual sporulation (conidiation) remain unclear. Here, we used comparative transcriptomic and proteomic approaches to elucidate possible signaling pathway relating to the asexual sporulation of A. camphorata. First, 104 differentially expressed proteins and 2586 differential cDNA sequences during the culture process of A. camphorata were identified by 2DE and RNA-seq, respectively. By applying bioinformatics analysis, a total of 67 genes which might play roles in the sporulation were obtained, and 18 of these genes, including fluG, sfgA, SfaD, flbA, flbB, flbC, flbD, nsdD, brlA, abaA, wetA, ganB, fadA, PkaA, veA, velB, vosA, and stuA might be involved in a potential FluG-mediated signaling pathway. Furthermore, the mRNA expression levels of the 18 genes in the proposed FluG-mediated signaling pathway were analyzed by quantitative real-time PCR. In summary, our study helps elucidate the molecular mechanisms underlying the asexual sporulation of A. camphorata, and provides also useful transcripts and proteome for further bioinformatics study of this valuable medicinal mushroom.

Studies in the Antrodia serialis group (Polyporales, Basidiomycota).

Taxonomy and phylogeny of the Antrodia serialis group are revised with morphological, ecological, and geographic data, partial translation elongation factor 1-α (tef1) gene sequences, and nuc rDNA ITS1-5.8S-ITS2-28S sequences. The group contains 13 species found in boreal and temperate zones of the Northern Hemisphere. The species are limited to certain geographic areas within Eurasia and North America. The traditional morphology-based concept of A. serialis covers at least four closely related species: A. serialis s. str. in Eurasia, A. angusta, sp. nov., in East Asia, A. serrata, sp. nov., in the American Northeast, and A. calcitrosa, sp. nov., in the American Northwest. They all are associated mostly with Picea spp. and show small, but stable morphological differences from each other. In addition, A. morganii, comb. nov., inhabiting wood of Populus spp., occurs in North America, and Antrodia alaskana, comb. nov., a large-pored species, macroscopically similar to A. variiformis, is distributed along the Pacific coast of North America. The pine-dwelling A. flavimontis, sp. nov., similar to A. primaeva from Eurasia, is so far known only from the eastern part of the Rocky Mountains (Utah and Wyoming).

Identification of the orsellinic acid synthase PKS63787 for the biosynthesis of antroquinonols in Antrodia cinnamomea.

Antrodia cinnamomea, an endemic basidiomycete used as a health food in Taiwan, is known to synthesize antroquinonols, which were reported to have notable medicinal potential in oncology and immunology. However, the biosynthetic pathway of these compounds is currently unclear. Our previous study showed that a pks63787 knockout mutant of A. cinnamomea (∆pks63787) is deficient in the biosynthesis of several aromatic metabolites. In this study, we pointed by phylogenetic analysis that pks63787 likely encodes an orsellinic acid synthase. Moreover, amendment of the cultural medium with orsellinic acid not only restores the ability of ∆pks63787 to produce its major pigment and other deficient metabolites, e.g., antroquinonols, but also enhances the productivity of several antroquinonols, including two new compounds 2 and 3. These results provide direct evidence that the PKS63787 is involved in the biosynthesis of antroquinonols and confirmed our hypothesis that the 6-methylcyclohexenone moiety was synthesized via the PKS63787-mediated polyketide pathway. In conclusion, PKS63787 might function as orsellinic acid synthase and orsellinic acid is an important precursor indispensable for the biosynthesis of the major pigment and antroquinonols in A. cinnamomea. To facilitate further basic or applied study, a putative biosynthesis pathway map of antroquinonols is proposed.

Differential Gene Expression Network in Terpenoid Synthesis of Antrodia cinnamomea in Mycelia and Fruiting Bodies.

Antodia cinnamomea, a precious brown-rot fungus endemic to Taiwan, has pharmaceutical applications due to its diverse array of metabolites. The terpenoids found in A. cinnamomea contribute to its most important bioactivities. We identified several terpenoid compounds in A. cinnamomea and revealed that their content in mycelium and fruiting body were significantly different. Using next-generation sequencing and an in-house transcriptome database, we identified several terpene synthase (TPS) candidates. After sequence analysis and functional characterization, 10 out of 12 candidates were found to have single or multiple terpene synthesis functions. Most of the terpenoid compounds were found to confer important bioactivities. RT-PCR results showed a positive correlation between terpene synthase expression pattern and terpenoid content. In addition, we identified several modification enzyme candidates that may be involved in the postmodification of terpenoid compounds with a genomic DNA scaffold, and a putative genetic network.

pks63787, a Polyketide Synthase Gene Responsible for the Biosynthesis of Benzenoids in the Medicinal Mushroom Antrodia cinnamomea.

Antrodia cinnamomea, a unique resupinate basidiomycete endemic to Taiwan, has potent medicinal activities. The reddish basidiocarps and mycelia generally exhibit abundant metabolites and higher biological activity. To investigate the pigments of A. cinnamomea, polyketide synthase (PKS) genes were characterized based on its partially deciphered genome and the construction of a fosmid library. Furthermore, a gene disruption platform was established via protoplast transformation and homologous recombination. Of four putative polyketide synthase genes, pks63787 was selected and disrupted in the monokaryotic wild-type (wt) strain f101. Transformant Δpks63787 was deficient in the synthesis of several aromatic metabolites, including five benzenoids and two benzoquinone derivatives. Based on these results, a biosynthetic pathway for benzenoid derivatives was proposed. The pks63787 deletion mutant not only displayed a reduced red phenotype compared to the wt strain but also displayed less 1,1-biphenyl-2-picrylhydrazyl free radical scavenging activity. This finding suggests that PKS63787 is responsible for the biosynthesis of pigments and metabolites related to the antioxidant activity of A. cinnamomea. The present study focuses on the functional characterization of the PKS gene, the fluctuations of its profile of secondary metabolites, and interpretation of the biosynthesis of benzenoids.

Species diversity in the Antrodia crassa group (Polyporales, Basidiomycota).

Antrodia is a polyphyletic genus, comprising brown-rot polypores with annual or short-lived perennial resupinate, dimitic basidiocarps. Here we focus on species that are closely related to Antrodia crassa, and investigate their phylogeny and species delimitation using geographic, ecological, morphological and molecular data (ITS and LSU rDNA, tef1). Phylogenetic analyses distinguished four clades within the monophyletic group of eleven conifer-inhabiting species (five described herein): (1)A. crassa s. str. (boreal Eurasia), Antrodia cincta sp. nova (North America) and Antrodia cretacea sp. nova (holarctic), all three being characterized by inamyloid skeletal hyphae that dissolve quickly in KOH solution; (2) Antrodia ignobilis sp. nova, Antrodia sitchensis and Antrodia sordida from North America, and Antrodia piceata sp. nova (previously considered conspecific with A. sitchensis) from Eurasia, possessing amyloid skeletal hyphae; (3) Antrodia ladiana sp. nova from the southern part of the USA, Antrodia pinea from East Asia, and Antrodia ferox - so far known from subtropical North America, but here reported also from Eurasia. These three species have inamyloid hyphae and narrow basidiospores; (4) the North American Antrodia pini-cubensis, sharing similar morphological characters with A. pinea, forming a separate clade. The habitat data indicate that several species are threatened by intensive forestry.

Characterization of the 2,3-Oxidosqualene Cyclase Gene from Antrodia cinnamomea and Enhancement of Cytotoxic Triterpenoid Compound Production.

Antrodia cinnamomea is a scarce, epiphyte, host-specific, brown-rot fungus that produces diverse bioactive compounds with potent biological activity. Natural wild-type fruiting bodies of A. cinnamomea are rare and highly valued, but their artificial culture poses challenges. Triterpenoids are a group of secondary metabolites that contribute to the bioactivities of A. cinnamomea. 2,3-Oxidosqualene cyclase (OSC) is a key enzyme in triterpenoid biosynthesis, which converts 2,3-oxidosqualene (OS) into polycyclic triterpenoids. In this study, we isolated a 2,3-oxidosqualene cyclase gene from A. cinnamomea with degenerate primers and designated it as AcOSC. The full length AcOSC cDNA was subcloned into a yeast expression vector, and AcOSC activity was confirmed. RT-PCR results showed that AcOSC expression was highest in the wild-type fruiting body and correlated with a higher concentration of triterpenoids. Agrobacterium-mediated gene transformation was conducted to enhance the triterpenoid synthesis capacity of the cultured mycelium. Metabolite profiling was conducted by LC-MS/MS and principal component analysis (PCA). The compositions and contents of metabolites in the AcOSC transgenic lines were different from those in the wild-type mycelium and vector control. The levels of two important triterpenoids, dehydrosulphurenic acid (DSA) and dehydroeburicoic acid (DEA), were increased in A. cinnamomea oxidosqualene cyclase overexpression strains compared to controls. In summary an Agrobacterium-mediated gene transformation procedure was established that successfully increased the level of transgene expression and enhanced the triterpenoid content in cultured A. cinnamomea.

MicroRNA-like small RNAs prediction in the development of Antrodia cinnamomea.

Antrodia cinnamomea, a precious, host-specific brown-rot fungus that has been used as a folk medicine in Taiwan for centuries is known to have diverse bioactive compounds with potent pharmaceutical activity. In this study, different fermentation states of A. cinnamomea (wild-type fruiting bodies and liquid cultured mycelium) were sequenced using the next-generation sequencing (NGS) technique. A 45.58 Mb genome encoding 6,522 predicted genes was obtained. High quality reads were assembled into a total of 13,109 unigenes. Using a previously constructed pipeline to search for microRNAs (miRNAs), we then identified 4 predicted conserved miRNA and 63 novel predicted miRNA-like small RNA (milRNA) candidates. Target prediction revealed several interesting proteins involved in tri-terpenoid synthesis, mating type recognition, chemical or physical sensory protein and transporters predicted to be regulated by the miRNAs and milRNAs.

Genomic and transcriptomic analyses of the medicinal fungus Antrodia cinnamomea for its metabolite biosynthesis and sexual development.

Antrodia cinnamomea, a polyporus mushroom of Taiwan, has long been used as a remedy for cancer, hypertension, and hangover, with an annual market of over $100 million (US) in Taiwan. We obtained a 32.15-Mb genome draft containing 9,254 genes. Genome ontology enrichment and pathway analyses shed light on sexual development and the biosynthesis of sesquiterpenoids, triterpenoids, ergostanes, antroquinonol, and antrocamphin. We identified genes differentially expressed between mycelium and fruiting body and 242 proteins in the mevalonate pathway, terpenoid pathways, cytochrome P450s, and polyketide synthases, which may contribute to the production of medicinal secondary metabolites. Genes of secondary metabolite biosynthetic pathways showed expression enrichment for tissue-specific compounds, including 14-α-demethylase (CYP51F1) in fruiting body for converting lanostane to ergostane triterpenoids, coenzymes Q (COQ) for antroquinonol biosynthesis in mycelium, and polyketide synthase for antrocamphin biosynthesis in fruiting body. Our data will be useful for developing a strategy to increase the production of useful metabolites.

Effect of cultural conditions on antrodin C production by basidiomycete Antrodia camphorata in solid-state fermentation.

Antrodia camphorata is a medicinal fungus and antrodin C is one of the main bioactive components of A. camphorata in the submerged fermentation (SmF). To optimize the culture conditions, the factors influencing the production of antrodin C by A. camphorata under solid-state fermentation (SSF) were investigated in this study. Different solid substrates and external nitrogen sources were tested for their efficiency in producing antrodin C. The response surface methodology was applied to evaluate the influence of several variables, namely, the concentrations of soybean meal, initial moisture content, and inoculum density on antrodin C production in solid-state fermentation. The experimental results show that the optimum fermentation medium for antrodin C production by A. camphorata was composed of 0.578 g soybean meal, 0.05 g Na2 HPO4 , 0.05 g MgSO4 for 100 g rice, with 51.83% initial moisture content, 22 day culture time, 28 °C culture temperature, and 35.54% inoculum density. At optimized conditions, 6,617.36 ± 92.71 mg kg(-1) yield of antrodin C was achieved. Solid-state fermentation is one good cultural method to improve the production of antrodin C by A. camphorata.

What is Antrodia sensu stricto?

The polypore genus Antrodia (Polyporales, Basidiomycota) in the strict sense consists of a small number of species grouped around the type species A. serpens in phylogenetic analyses. This distinct clade (Antrodia sensu stricto in our view) contains species of the Antrodia heteromorpha complex, A. macra coll. and Antrodia mappa (formerly Postia mappa). Nuclear rDNA ITS and tef1 data show that the Antrodia heteromorpha species complex includes four species: A. heteromorpha sensu stricto (mostly on gymnosperms, large pores and spores), A. serpens (on angiosperms in Europe, resupinate, smaller pores but large spores), A. favescens (smaller pores and spores, pileate species in North America, formerly known as Trametes sepium), and A. tanakai (a close kin of A. favescens in Eurasia). Antrodia albida is a synonym of A. heteromorpha sensu stricto. We combine A. mappa, A. favescens and A. tanakai in Antrodia and designate neotypes for A. albida and A. heteromorpha, and an epitype for A. serpens. We also compare the morphologically similar but distantly related A. albidoides and A. mellita, and conclude that A. macrospora and A. subalbidoides are synonyms of A. albidoides.

Cytochrome P450 genes in medicinal mushroom Antrodia cinnamomea T.T. Chang et W.N. Chou (higher Basidiomycetes) are strongly expressed during fruiting body formation.

Medicinal mushroom Antrodia cinnamomea is a higher Basidiomycetes endemic to Taiwan, where it is commonly used as a traditional folk medicine. It is well known for its multiple biologic activities and its potential for commercial development. Here, ten full lengths of cytochrome P450 (CYP) genes (ac-1 to ac-10) from A. cinnamomea were cloned and identified. With the exception of ac-3 and ac-8, which will probably be assigned as new CYP families, these genes had more than 40% amino acid identity and close evolutionary relationships to known CYPs. Among the ten genes, only Ac-7 did not possess a transmembrane domain but had an N-terminal signal peptide, so it was considered a novel extracellular CYP. The ten A. cinnamomea CYPs had different expression profiles in different growth conditions. In general, they were strongly expressed during the formation of fruiting bodies, especially in natural basidiomycetes. The expression of six CYPs of A. cinnamomea (ac-1 to ac-3 and ac-5 to ac-7) were strictly inhibited in the mycelia cell type. It was therefore concluded that these CYPs are most active in the fruiting bodies of A. cinnamomea.

Optimization of fermentation medium for triterpenoid production from Antrodia camphorata ATCC 200183 using artificial intelligence-based techniques.

In this study, alteration in morphology of submergedly cultured Antrodia camphorata ATCC 200183 including arthroconidia, mycelia, external and internal structures of pellets was investigated. Two optimization models namely response surface methodology (RSM) and artificial neural network (ANN) were built to optimize the inoculum size and medium components for intracellular triterpenoid production from A. camphorata. Root mean squares error, R (2), and standard error of prediction given by ANN model were 0.31%, 0.99%, and 0.63%, respectively, while RSM model gave 1.02%, 0.98%, and 2.08%, which indicated that fitness and prediction accuracy of ANN model was higher when compared to RSM model. Furthermore, using genetic algorithm (GA), the input space of ANN model was optimized, and maximum triterpenoid production of 62.84 mg l(-1) was obtained at the GA-optimized concentrations of arthroconidia (1.78 × 105 ml(-1)) and medium components (glucose, 25.25 g l(-1); peptone, 4.48 g l(-1); and soybean flour, 2.74 g l(-1)). The triterpenoid production experimentally obtained using the ANN-GA designed medium was 64.79 ± 2.32 mg l(-1) which was in agreement with the predicted value. The same optimization process may be used to optimize many environmental and genetic factors such as temperature and agitation that can also affect the triterpenoid production from A. camphorata and to improve the production of bioactive metabolites from potent medicinal fungi by changing the fermentation parameters.

[Medium optimization for mycelia production of Antrodia camphorata based on artificial neural network-genetic algorithm].

To illustrate the complex fermentation process of submerged culture of Antrodia camphorata ATCC 200183, we observed the morphology change of this filamentous fungus. Then we used two optimization models namely response surface methodology (RSM) and artificial neural network (ANN) to model the fermentation process of Antrodia camphorata. By genetic algorithm (GA), we optimized the inoculum size and medium components for Antrodia camphorata production. The results show that fitness and prediction accuracy of ANN model was higher when compared to those of RSM model. Using GA, we optimized the input space of ANN model, and obtained maximum biomass of 6.2 g/L at the GA-optimized concentrations of spore (1.76x 10(5) /mL) and medium components (glucose, 29.1 g/L; peptone, 9.3 g/L; and soybean flour, 2.8 g/L). The biomass obtained using the ANN-GA designed medium was (6.1+/-0.2) g/L which was in good agreement with the predicted value. The same optimization process may be used to improve the production of mycelia and bioactive metabolites from potent medicinal fungi by changing the fermentation parameters.

Characterization and heterologous expression of a novel lysophospholipase gene from Antrodia cinnamomea.

AIMS: A novel lysophospholipase (LysoPL) from the basidiomycetous fungi Antrodia cinnamomea named ACLysoPL was cloned, heteroexpressed in Escherichia coli and characterized. METHODS AND RESULTS: The gene encoding ACLysoPL was obtained from expressed sequence tags from A. cinnamomea. The full length of this gene has a 945 -bp open reading frame encoding 314 amino acids with a molecular weight of 35.5 kDa. ACLysoPL contains a lipase consensus sequence (GXSXG) motif and a Ser-His-Asp catalytic triad. A putative peroxisomal targeting signal type 1 was found in the C-terminal. Heterologous expression of ACLysoPL in E. coli showed that the enzyme preferentially hydrolyses long-chain acyl esterases at pH 7 and 30 degrees C. ACLysoPL is a psychrophilic enzyme about 40% of whose maximum activity remained at 4 degrees C. The LysoPL activities with lysophospholipids as substrate were analysed by gas chromatography/mass spectrometry. CONCLUSION: We have identified and characterized a gene named ACLysoPL encoding a protein performing LysoPL and esterase activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first LysoPL of A. cinnamomea identified and characterized at the molecular level.