Orsellinic Acid Research Articles

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.

Biosynthesis of Antroquinonol and 4-Acetylantroquinonol B via a Polyketide Pathway Using Orsellinic Acid as a Ring Precursor in Antrodia cinnamomea.

Antroquinonol (AQ) and 4-acetylantroquinonol B (4-AAQB), isolated from the mycelium of Antrodia cinnamomea, have a similar chemical backbone to coenzyme Q (CoQ). Based on the postulation that biosynthesis of both AQ and 4-AAQB in A. cinnamomea starts from the polyketide pathway, we cultivated this fungus in a culture medium containing [U-13C]oleic acid, and then we analyzed the crude extracts of the mycelium using UHPLC-MS. We found that AQ and 4-AAQB follow similar biosynthetic sequences as CoQ. Obvious [13C2] fragments on the ring backbone were detected in the mass spectrum for [13C2]AQ, [13C2]4-AAQB, and their [13C2] intermediates found in this study. The orsellinic acid, formed from acetyl-CoA and malonyl-CoA via the polyketide pathway, was found to be a novel benzoquinone ring precursor for AQ and 4-AAQB. The identification of endogenously synthesized farnesylated intermediates allows us to postulate the routes of AQ and 4-AAQB biosynthesis in A. cinnamomea.

Biochemical and genetic basis of orsellinic acid biosynthesis and prenylation in a stereaceous basidiomycete

The prenylphenols are a class of natural products that have been frequently isolated from basidiomycetes, e.g., from the genus Stereum (false turkey tail fungi) and other Russulales as well as from ascomycetes. Biosynthetically, these compounds are considered hybrids, as the orsellinic acid moiety is a polyketide and the prenyl side chain originates from the terpene metabolism, although no literature on the genetic and biochemical background of the biosynthesis is available. In a stereaceous basidiomycete, referred to as BY1, a new prenylphenol, now termed cloquetin, was identified and its structure elucidated by mass spectrometry and nuclear magnetic resonance spectroscopy. Genes for two non-reducing polyketide synthases (PKS1 and PKS2) were identified in the BY1 genome, and heterologously expressed in Aspergillus niger. Product formation identified both PKSs as orsellinic acid synthases. A putative prenyltransferase gene (BYPB) found in the BY1 genome was expressed in Escherichia coli. In vitro characterization showed that BYPB activity depends on bivalent cations and that it uses orsellinic acid as acceptor substrate for the transfer of a prenyl group. The two orsellinic acid synthases support the emerging notion that fungi secure individual metabolic steps or entire pathways by redundant enzymes.

A Novel Class of Plant Type III Polyketide Synthase Involved in Orsellinic Acid Biosynthesis from Rhododendron dauricum.

Rhododendron dauricum L. produces daurichromenic acid, the anti-HIV meroterpenoid consisting of sesquiterpene and orsellinic acid (OSA) moieties. To characterize the enzyme responsible for OSA biosynthesis, a cDNA encoding a novel polyketide synthase (PKS), orcinol synthase (ORS), was cloned from young leaves of R. dauricum. The primary structure of ORS shared relatively low identities to those of PKSs from other plants, and the active site of ORS had a unique amino acid composition. The bacterially expressed, recombinant ORS accepted acetyl-CoA as the preferable starter substrate, and produced orcinol as the major reaction product, along with four minor products including OSA. The ORS identified in this study is the first plant PKS that generates acetate-derived aromatic tetraketides, such as orcinol and OSA. Interestingly, OSA production was clearly enhanced in the presence of Cannabis sativa olivetolic acid cyclase, suggesting that the ORS is involved in OSA biosynthesis together with an unidentified cyclase in R. dauricum.

Two new threitol orsellinates from a sea mud-derived fungus, Ascotricha sp. ZJ-M-5

Two new d-threitol orsellinates (1–2) were isolated from the EtOAc extract of a sea mud-derived fungus, Ascotricha sp. ZJ-M-5, cultured in Czapek Dox broth. These two compounds featured in the symmetrical substitution of orsellinic acid or acetic acid, which was established on the basis of 1D and 2D NMR experiments. The characteristic optical rotations enabled the assignment of the absolute configuration.

Discovery of novel xylosides in co-culture of basidiomycetes Trametes versicolor and Ganoderma applanatum by integrated metabolomics and bioinformatics.

Transcriptomic analysis of cultured fungi suggests that many genes for secondary metabolite synthesis are presumably silent under standard laboratory condition. In order to investigate the expression of silent genes in symbiotic systems, 136 fungi-fungi symbiotic systems were built up by co-culturing seventeen basidiomycetes, among which the co-culture of Trametes versicolor and Ganoderma applanatum demonstrated the strongest coloration of confrontation zones. Metabolomics study of this co-culture discovered that sixty-two features were either newly synthesized or highly produced in the co-culture compared with individual cultures. Molecular network analysis highlighted a subnetwork including two novel xylosides (compounds 2 and 3). Compound 2 was further identified as N-(4-methoxyphenyl)formamide 2-O-β-D-xyloside and was revealed to have the potential to enhance the cell viability of human immortalized bronchial epithelial cell line of Beas-2B. Moreover, bioinformatics and transcriptional analysis of T. versicolor revealed a potential candidate gene (GI: 636605689) encoding xylosyltransferases for xylosylation. Additionally, 3-phenyllactic acid and orsellinic acid were detected for the first time in G. applanatum, which may be ascribed to response against T.versicolor stress. In general, the described co-culture platform provides a powerful tool to discover novel metabolites and help gain insights into the mechanism of silent gene activation in fungal defense.

Structural Elucidation and Free Radical Scavenging Activity of a new o-Orsellinic Acid Derivative Isolated from the Lichen Cladonia rappii

Rappiidic acid, a new o-orsellinic acid derivative, was isolated from the lichen Cladonia rappii. Its capability to scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS) was investigated and compared with resveratrol and (+)-usnic acid. Usnic acid at 100 μM was the most efficient ROS scavenger, exhibiting activity 3-fold higher than that of resveratrol. At the same concentration, rappidic acid scavenged 23.1% of ROS formed, demonstrating that this compound is twice as active as resveratrol. Both compounds were shown to be poor RNS scavengers.

Transcriptomic and metabolomic profiling of ionic liquid stimuli unveils enhanced secondary metabolism in Aspergillus nidulans.

BackgroundThe inherent potential of filamentous fungi, especially of Ascomycota, for producing diverse bioactive metabolites remains largely silent under standard laboratory culture conditions. Innumerable strategies have been described to trigger their production, one of the simplest being manipulation of the growth media composition. Supplementing media with ionic liquids surprisingly enhanced the diversity of extracellular metabolites generated by penicillia. This finding led us to evaluate the impact of ionic liquids’ stimuli on the fungal metabolism in Aspergillus nidulans and how it reflects on the biosynthesis of secondary metabolites (SMs).ResultsWhole transcriptional profiling showed that exposure to 0.7 M cholinium chloride or 1-ethyl-3-methylimidazolium chloride dramatically affected expression of genes encoding both primary and secondary metabolism. Both ionic liquids apparently induced stress responses and detoxification mechanisms but response profiles to each stimulus were unique. Primary metabolism was up-regulated by choline, but down-regulated by 1-ethyl-3-methylimidazolium chloride; both stimulated production of acetyl-CoA (key precursor to numerous SMs) and non proteinogenic amino acids (building blocks of bioactive classes of SMs). In total, twenty one of the sixty six described backbone genes underwent up-regulation. Accordingly, differential analysis of the fungal metabolome showed that supplementing growth media with ionic liquids resulted in ca. 40 differentially accumulated ion masses compared to control conditions. In particular, it stimulated production of monodictyphenone and orsellinic acid, otherwise cryptic. Expression levels of genes encoding corresponding polyketide biosynthetic enzymes (i.e. backbone genes) increased compared to control conditions. The corresponding metabolite extracts showed increased cell polarity modulation potential in an ex vivo whole tissue assay (Thelial Live Targeted Epithelia; theLiTE™).ConclusionsIonic liquids, a diverse class of chemicals composed solely of ions, can provide an unexpected means to further resolve the diversity of natural compounds, guiding discovery of fungal metabolites with clinical potential.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2577-6) contains supplementary material, which is available to authorized users.

Optimisation and establishment of separation conditions of organic acids from Usnea longissima Ach. by pH-zone-refining counter-current chromatography: Discussion of the eluotropic sequence

The major bioactive constituents of Usnea longissima Ach. are organic acids. However, few recent literatures involve the preparative separation of these organic acids. In the present study, pH zone-refining counter-current chromatography is used to separate organic acids from crude sample of U. longissima Ach. The crude extract was separated with the two-phase solvent system Pet–EtAc–MeOH–H2O (5:5:3:7, v/v) with 10mM TFA in organic stationary phase and different concentration of the eluter in aqueous mobile phase for the screening of the most suitable separation conditions. From the crude extract (1.2g), 74.0mg of orsellinic acid at 92.7% purity, 55.5mg of 4-O-methylorsellinic acid at 97.7% purity, 353.5mg of evernic acid at 93.8% purity, 102.0mg of barbatic acid at 94.8% purity, 19.4mg of diffractaic acid at 92.2% purity, and 44.9mg of usnic acid at 95.7% purity were obtained using the selected conditions in which the concentration of TFA in stationary phase was 10mM and the concentration of NaOH in mobile phase was 10–20mM. The purities of the separated organic acids were measured by HPLC. And the data of electrospray ionization-liquid chromatography/mass spectrometry (ESI–LC/MS), 1H NMR, and 13C NMR were used for confirming chemical structures.

The Distant Siblings-A Phylogenomic Roadmap Illuminates the Origins of Extant Diversity in Fungal Aromatic Polyketide Biosynthesis.

In recent years, the influx of newly sequenced fungal genomes has enabled sampling of secondary metabolite biosynthesis on an unprecedented scale. However, explanations of extant diversity which take into account both large-scale phylogeny reconstructions and knowledge gained from multiple genome projects are still lacking. We analyzed the evolutionary sources of genetic diversity in aromatic polyketide biosynthesis in over 100 model fungal genomes. By reconciling the history of over 400 nonreducing polyketide synthases (NR-PKSs) with corresponding species history, we demonstrate that extant fungal NR-PKSs are clades of distant siblings, originating from a burst of duplications in early Pezizomycotina and thinned by extensive losses. The capability of higher fungi to biosynthesize the simplest precursor molecule (orsellinic acid) is highlighted as an ancestral trait underlying biosynthesis of aromatic compounds. This base activity was modified during early evolution of filamentous fungi, toward divergent reaction schemes associated with biosynthesis of, for example, aflatoxins and fusarubins (C4–C9 cyclization) or various anthraquinone derivatives (C6–C11 cyclization). The functional plasticity is further shown to have been supplemented by modularization of domain architecture into discrete pieces (conserved splice junctions within product template domain), as well as tight linkage of key accessory enzyme families and divergence in employed transcriptional factors. Although the majority of discord between species and gene history is explained by ancient duplications, this landscape has been altered by more recent duplications, as well as multiple horizontal gene transfers. The 25 detected transfers include previously undescribed events leading to emergence of, for example, fusarubin biosynthesis in Fusarium genus. Both the underlying data and the results of present analysis (including alternative scenarios revealed by sampling multiple reconciliation optima) are maintained as a freely available web-based resource: http://cropnet.pl/metasites/sekmet/nrpks_2014.

Fungal biosynthesis of the bibenzoquinone oosporein to evade insect immunity

Quinones are widely distributed in nature and exhibit diverse biological or pharmacological activities; however, their biosynthetic machineries are largely unknown. The bibenzoquinone oosporein was first identified from the ascomycete insect pathogen Beauveria bassiana>50 y ago. The toxin can also be produced by different plant pathogenic and endophytic fungi with an array of biological activities. Here, we report the oosporein biosynthetic machinery in fungi, a polyketide synthase (PKS) pathway including seven genes for quinone biosynthesis. The PKS oosporein synthase 1 (OpS1) produces orsellinic acid that is hydroxylated to benzenetriol by the hydroxylase OpS4. The intermediate is oxidized either nonenzymatically to 5,5'-dideoxy-oosporein or enzymatically to benzenetetrol by the putative dioxygenase OpS7. The latter is further dimerized to oosporein by the catalase OpS5. The transcription factor OpS3 regulates intrapathway gene expression. Insect bioassays revealed that oosporein is required for fungal virulence and acts by evading host immunity to facilitate fungal multiplication in insects. These results contribute to the known mechanisms of quinone biosynthesis and the understanding of small molecules deployed by fungi that interact with their hosts.

Crystal structure of O-methyltransferase CalO6 from the calicheamicin biosynthetic pathway: a case of challenging structure determination at low resolution.

BackgroundCalicheamicins (CAL) are enedyine natural products with potent antibiotic and cytotoxic activity, used in anticancer therapy. The O-methyltransferase CalO6 is proposed to catalyze methylation of the hydroxyl moiety at the C2 position of the orsellinic acid group of CAL.ResultsCrystals of CalO6 diffracted non-isotropically, with the usable data extending to 3.4 Å. While no single method of crystal structure determination yielded a structure of CalO6, we were able to determine its structure by using molecular replacement-guided single wavelength anomalous dispersion by using diffraction data from native crystals of CalO6 and a highly non-isomorphous mercury derivative. The structure of CalO6 reveals the methyltransferase fold and dimeric organization characteristic of small molecule O-methyltransferases involved in secondary metabolism in bacteria and plants. Uncommonly, CalO6 was crystallized in the absence of S-adenosylmethionine (SAM; the methyl donor) or S-adenosylhomocysteine (SAH; its product).ConclusionsLikely as a consequence of the dynamic nature of CalO6 in the absence of its cofactor, the central region of CalO6, which forms a helical lid-like structure near the active site in CalO6 and similar enzymes, is not observed in the electron density. We propose that this region controls the entry of SAM into and the exit of SAH from the active site of CalO6 and shapes the active site for substrate binding and catalysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12900-015-0040-6) contains supplementary material, which is available to authorized users.

Total Synthesis of the Glycosylated Macrolide Antibiotic Fidaxomicin.

The first enantioselective total synthesis of fidaxomicin, also known as tiacumicin B or lipiarmycin A3, is reported. This novel glycosylated macrolide antibiotic is used in the clinic for the treatment of Clostridium difficile infections. Key features of the synthesis involve a rapid and high-yielding access to the noviose, rhamnose, and orsellinic acid precursors; the first example of a β-selective noviosylation; an effective Suzuki coupling of highly functionalized substrates; and a ring-closing metathesis reaction of a noviosylated dienoate precursor. Careful selection of protecting groups allowed for a complete deprotection yielding totally synthetic fidaxomicin.

A new high-performance heterologous fungal expression system based on regulatory elements from the Aspergillus terreus terrein gene cluster

Recently, the Aspergillus terreus terrein gene cluster was identified and selected for development of a new heterologous expression system. The cluster encodes the specific transcription factor TerR that is indispensable for terrein cluster induction. To identify TerR binding sites, different recombinant versions of the TerR DNA-binding domain were analyzed for specific motif recognition. The high affinity consensus motif TCGGHHWYHCGGH was identified from genes required for terrein production and binding site mutations confirmed their essential contribution to gene expression in A. terreus. A combination of TerR with its terA target promoter was tested as recombinant expression system in the heterologous host Aspergillus niger. TerR mediated target promoter activation was directly dependent on its transcription level. Therefore, terR was expressed under control of the regulatable amylase promoter PamyB and the resulting activation of the terA target promoter was compared with activation levels obtained from direct expression of reporters from the strong gpdA control promoter. Here, the coupled system outcompeted the direct expression system. When the coupled system was used for heterologous polyketide synthase expression high metabolite levels were produced. Additionally, expression of the Aspergillus nidulans polyketide synthase gene orsA revealed lecanoric acid rather than orsellinic acid as major polyketide synthase product. Domain swapping experiments assigned this depside formation from orsellinic acid to the OrsA thioesterase domain. These experiments confirm the suitability of the expression system especially for high-level metabolite production in heterologous hosts.

Asperlones A and B, dinaphthalenone derivatives from a mangrove endophytic fungus Aspergillus sp. 16-5C.

Racemic dinaphthalenone derivatives, (±)-asperlone A (1) and (±)-asperlone B (2), and two new azaphilones, 6″-hydroxy-(R)-mitorubrinic acid (3) and purpurquinone D (4), along with four known compounds, (−)-mitorubrinic acid (5), (−)-mitorubrin (6), purpurquinone A (7) and orsellinic acid (8), were isolated from the cultures of Aspergillus sp. 16-5C. The structures were elucidated using comprehensive spectroscopic methods, including 1D and 2D NMR spectra and the structures of 1 further confirmed by single-crystal X-ray diffraction analysis, while the absolute configuration of 3 and 4 were determined by comparing their optical rotation and CD with those of the literature, respectively. Compounds 1, 2 and 6 exhibited potent inhibitory effects against Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) with IC50 values of 4.24 ± 0.41, 4.32 ± 0.60 and 3.99 ± 0.34 μM, respectively.

A New Orsellinic Acid Derivative from the Fungus Chaetomium megalocarpum

A new orsellinic acid derivative, orsellide A1, together with orsellide A, orsellide D, and orsellinic acid, was isolated from the ethyl acetate extract of the solid-state fermented rice culture of the Chaetomium megalocarpum cib-135 fungus. The structure of orsellide A1 (1) was confirmed to be 2-O-(2,4-dihydroxy-6-methylbenzoyl)-6-deoxy-1-O-ethyl-3-keto-α-D-ribo-hexopyranoside on the basis of spectroscopic analysis.

Ascomycotin A, a new citromycetin analogue produced by Ascomycota sp. Ind19F07 isolated from deep sea sediment

A new citromycetin analogue, ascomycotin A (1), together with eight known compounds, wortmannilactone E (2), orcinol (3), orsellinic acid (4), isosclerone (5), (3R,4S)-( − )-4-hydroxymellein (6), diorcinol (7), chaetocyclinone B (8) and 2,5-dimethoxy-3,6-di(p-methoxypheny1)-1,4-benzoquinone (9), was isolated from the fungal strain Ascomycota sp. Ind19F07, which was isolated from the deep sea sediment of the Indian Ocean. The structures of the compounds were established by spectroscopic data including 1D and 2D NMR and HR-ESI-MS. Compounds (1–9) were evaluated for antibacterial activity.

Qualitative and spatial metabolite profiling of lichens by a LC-MS approach combined with optimised extraction.

Lichens are self-sustaining partnerships comprising fungi as shape-forming partners for their enclosed symbiotic algae. They produce a tremendous diversity of metabolites (1050 metabolites described so far). A comparison of metabolic profiles in nine lichen species belonging to three genera (Lichina, Collema and Roccella) by using an optimised extraction protocol, determination of the fragmentation pathway and the in situ localisation for major compounds in Roccella species. Chemical analysis was performed using a complementary study combining a Taguchi experimental design with qualitative analysis by high-performance liquid chromatography coupled with mass spectrometry techniques. Optimal conditions to obtain the best total extraction yield were determined as follows: mortar grinding to a fine powder, two successive extractions, solid:liquid ratio (2:60) and 700 rpm stirring. Qualitative analysis of the metabolite profiling of these nine species extracted with the optimised method was corroborated using MS and MS/MS approaches. Nine main compounds were identified: 1 β-orcinol, 2 orsellinic acid, 3 putative choline sulphate, 4 roccellic acid, 5 montagnetol, 6 lecanoric acid, 7 erythrin, 8 lepraric acid and 9 acetylportentol, and several other compounds were reported. Identification was performed using the m/z ratio, fragmentation pathway and/or after isolation by NMR analysis. The variation of the metabolite profile in differently organised parts of two Roccella species suggests a specific role of major compounds in developmental stages of this symbiotic association. Metabolic profiles represent specific chemical species and depend on the extraction conditions, the kind of the photobiont partner and the in situ localisation of major compounds.

Fusarium graminearum PKS14 is involved in orsellinic acid and orcinol synthesis

The available genome sequences show that the number of secondary metabolite genes in filamentous fungi vastly exceeds the number of known products. This is also true for the global plant pathogenic fungus Fusarium graminearum, which contains 15 polyketide synthase (PKS) genes, of which only 6 have been linked to products. To help remedy this, we focused on PKS14, which has only been shown to be expressed during plant infections or when cultivated on rice or corn meal (RM) based media. To enhance the production of the resulting product we introduced a constitutive promoter in front of PKS14 and cultivated two of the resulting mutants on RM medium. This led to the production of two compounds, which were only detected in the PKS14 overexpressing mutants and not in the wild type or PKS14 deletion mutants. The two compounds were tentatively identified as orsellinic acid and orcinol by comparing spectroscopic data (mass spectroscopy and chromatography) to authentic standards. NMR analysis of putative orcinol isolated from the PKS14 overexpressing mutant supported our identification. Orcinol and orsellinic acid, not previously detected in Fusarium, have primarily been detected in lichen fungi. Orsellinic acid is hypothesized to be the PKS release product which is transformed to orcinol through decarboxylation. Phylogenetic analyses of PKSs placed PKS14 in a subclade of known OA synthases. Expression analysis by microarray of 55 experiments identified seven genes near PKS14 that were expressed in a similar manner. One of the seven genes encodes a predicted carboxylase, which could be responsible for transforming orsellinic acid to orcinol.

Anthraquinone Derivatives and an Orsellinic Acid Ester from the Marine Alga‐Derived Endophytic Fungus Eurotium cristatum EN‐220

AbstractCultivation of the fungal strain Eurotium cristatum EN‐220, an endophyte obtained from the marine alga Sargassum thunbergii, resulted in the isolation of one new anthraquinone glycoside, 3‐O‐(α‐D‐ribofuranosyl)questinol (1) and one new orsellinic acid ester, cristatumside A (2), together with three known aromatic glycosides, 3‐O‐(α‐D‐ribofuranosyl)questin (3), isotorachrysone 6‐O‐α‐D‐ribofuranoside (4), and asperflavin ribofuranoside (5), as well as three anthraquinone derivatives, asperflavin (6), eurorubrin (7), and (+)‐variecolorquinone A (8). The structures of these compounds were determined by extensive analysis of their spectroscopic data, as well as by their comparison with those in the literature. Each of the isolated compounds was evaluated for its antimicrobial activity and brine shrimp lethality.