Functional expression of a highly-reducing polyketide synthase of Emericella variecolor IFM42010, an asteltoxin-producing strain, resulted in production of two polyenoic ß-ketolactones with opposite stereochemistry.

The asteltoxin-producing fungus Emericella variecolor IFM42010 possesses 22 highly-reducing polyketide synthase (HR-PKS) genes. Of these, an HR-PKS with a methyltransferase domain but lacking an enoylreductase domain could be involved in the biosynthesis of asteltoxin and related compounds. From six such candidate HR-PKS genes, Ev460pks was analyzed by gene disruption in E. variecolor and heterologous expression in Aspergillus oryzae. The Ev460pks-disrupted strain retained asteltoxin production ability, indicating that Ev460pks is not involved in asteltoxin biosynthesis. The A. oryzae transformant harboring the Ev460pks gene produced compounds 1 and 2, along with several unidentified products possibly decomposed from 2. Spectroscopic analyses revealed that 1 was a 4-methyl-ß-ketolactone with a methylheptatriene side-chain at the C-5 position, and 2 was also a 4-methyl-ß-ketolactone, bearing a dimethyltetradecahexaene side-chain at the same position. The relative configuration at C-4 in compounds 1 and 2 was opposite.

Emerixanthone E, a new xanthone derivative from deep sea fungus Emericella sp SCSIO 05240.

The marine fungus Emericella sp was isolated from the deep sea sediments. The fungus was identified by its morphology and ITS region. A new emerixanthone E (1) together with four (2-5) known emodin derivatives were isolated from the metabolites of the fungus Emericella SCSIO05240. The structures were elucidated on the basis of NMR spectroscopic analysis and mass spectrometry. The biological properties of those compounds (1-5) were explored for antimicrobial, antifungal and antitumor activity.

Enhanced production of tanshinone IIA in endophytic fungi Emericella foeniculicola by genome shuffling.

CONTEXT: Tanshinone IIA, commercially produced from Salvia miltiorrhiza Bunge (C.Y.Wu) (Labiatae), has various biological benefits. Currently, this compound is mainly extracted from plants. However, because of the long growth cycle and the unstable quality of plants, the market demands can barely be satisfied. OBJECTIVE: The genomic shuffling technology is applied to screen the high-yield tanshinone IIA strain, which could be used to replace the plant S. miltiorrhiza for the production of tanshinone IIA. The change in the production of tanshinone IIA is clarified by comparing it with the original strain. MATERIALS AND METHODS: Tanshinone IIA was extracted from Strains cells, which was prepared through 0.5 mL protoplast samples by using hypertonic solution I from two different strains. Then, it was analyzed by high-performance liquid chromatography at 30 °C and UV 270 nm. Total DNA from the strains was extracted for RAPD amplification and electrophoresis to isolate the product. RESULTS: In this study, a high-yield tanshinone IIA strain F-3.4 was screened and the yield of tanshinone IIA was increased by 387.56 ± 0.02 mg/g, 11.07 times higher than that of the original strain TR21. DISCUSSION: This study shows that the genetic basis of high-yield strains is achieved through genome shuffling, which proves that genome shuffling can shorten the breeding cycle and improve the mutagenesis efficiency in obtaining the strains with good traits and it is a useful method for the molecular breeding of industrial strains.

Chemogenomics driven discovery of endogenous polyketide anti-infective compounds from endosymbiotic Emericella variecolor CLB38 and their RNA secondary structure analysis.

In the postgenomic era, a new strategy for chemical dereplication of polyketide anti-infective drugs requires novel genomics and chromatographic strategies. An endosymbiotic fungal strain CLB38 was isolated from the root tissue of Combretum latifolium Blume (Combretaceae) which was collected from the Western Ghats of India. The isolate CLB38 was then identified as Emericella variecolor by its characteristic stellate ascospores culture morphology and molecular analysis of ITS nuclear rDNA and intervening 5.8S rRNA gene sequence. ITS2 RNA secondary structure modeling clearly distinguished fungal endosymbiont E. variecolor CLB38 with other lifestyles in the same monophyletic clade. Ethyl acetate fraction of CLB38 explored a broad spectrum of antimicrobial activity against multidrug resistant pathogens. Biosynthetic PKS type-I gene and chromatographic approach afford two polyketide antimicrobial compounds which identified as evariquinone and isoindolones derivative emerimidine A. MIC of purified compounds against test microorganisms ranged between 3.12 µg/ml and 12.5 µg/ml. This research highlights the utility of E. variecolor CLB38 as an anticipate source for anti-infective polyketide metabolites evariquinone and emerimidine A to combat multidrug resistant microorganisms. Here we demonstrates a chemogenomics strategy via the feasibility of PKS type-I gene and chromatographic approach as a proficient method for the rapid prediction and discovery of new polyketides compounds from fungal endosymbionts.

Diasteltoxins A-C, Asteltoxin-Based Dimers from a Mutant of the Sponge-Associated Emericella variecolor Fungus.

Three novel asteltoxin-bearing dimers namely diasteltoxins A-C (1-3) along with asteltoxin were isolated from a mutated strain of a sponge-derived fungus Emericella variecolor XSA-07-2. Their structures were determined by extensive spectroscopic analyses including the computed electronic circular dichroism (ECD) data for the configurational assignment. The biogenetic formation of the dimers through [2 + 2] cycloaddition of asteltoxin was postulated. Diasteltoxins 1-3 exerted inhibitory effects against the tumor cell lines H1299 and MCF7 and exhibited significant inhibition against thioredoxin reductase (TrxR).

Molecular Basis for Stellatic Acid Biosynthesis: A Genome Mining Approach for Discovery of Sesterterpene Synthases.

The search for a new sesterterpene synthase in the genome of Emericella variecolor, which reportedly produces diverse sesterterpenoids, is described. One gene product (a chimeric protein with prenyltransferase and terpene cyclase domains) led to the synthesis of a novel tricyclic sesterterpene, stellata-2,6,19-triene (1), from DMAPP and IPP, and the hydrocarbon was further transformed into stellatic acid (2) by cytochrome P450 monooxygenase encoded by the gene adjacent to the sesterterpene synthase gene.

The binding of zinc ions to Emericella nidulans endo-ß-1,4-galactanase is essential for crystal formation.

A novel Emericella nidulans endo-ß-1,4-galactanase (EnGAL) demonstrates a strong capacity to generate high levels of very potent prebiotic oligosaccharides from potato pulp, a by-product of the agricultural potato-starch industry. EnGAL belongs to glycoside hydrolase family 53 and shows high (72.5%) sequence identity to an endo-ß-1,4-galactanase from Aspergillus aculeatus. Diffraction data extending to 2.0 Å resolution were collected from a crystal of EnGAL grown from conditions containing 0.2 M zinc acetate. The crystal structure showed a high similarity between EnGAL and other endo-ß-1,4-galactanases belonging to GH53. It also revealed 15 zinc ions bound to the protein, one of which is located in the active site, where it is coordinated by residues Glu136 and Glu246 which comprise the catalytic machinery. The majority of the zinc ions are located on the surface of the enzyme, in some cases with side chains from two different molecules as ligands, thus explaining why the presence of zinc ions was essential for crystallization.

EcdGHK are three tailoring iron oxygenases for amino acid building blocks of the echinocandin scaffold.

The echinocandins are a small group of fungal N-acylated cyclic hexapeptides that are fungicidal for candida strains and fungistatic for aspergilli by targeting cell wall 1,3-ß-glucan synthases. The side chains of all six amino acid building blocks have hydroxyl groups, including the nonproteinogenic 4R,5R-dihydroxy-Orn1, 4R-OH-Pro3, 3S,4S-dihydroxy-homoTyr4, and 3S-OH-4S-Me-Pro6. The echinocandin (ecd) gene cluster contains two predicted nonheme mononuclear iron oxygenase genes (ecdG,K) and one encoding a P450 type heme protein (ecdH). Deletion of the ecdH gene in the producing strain Emericella rugulosa generates an echinocandin scaffold (echinocandin D) lacking both hydroxyl groups on Orn1. Correspondingly, the ΔecdG strain failed to hydroxylate C3 of the homoTyr residue, and purified EcdG hydroxylated free L-homoTyr at C3. The ΔecdK strain failed to generate mature echinocandin unless supplemented with either 4R-Me-Pro or 3S-OH-4S-Me-Pro, indicating blockage of a step upstream of Me-Pro formation. Purified EcdK is a Leu 5-hydroxylase, acting iteratively at C5 to yield γ-Me-Glu-γ-semialdehyde in equilibrium with the cyclic imine product. Evaluation of deshydroxyechinocandin scaffolds in the in vitro anticandidal assays revealed up to a 3-fold loss of potency for the ΔecdG scaffolds, but a 3-fold gain of potency for the ΔecdH scaffold, in line with prior results on deoxyechinocandin homologues.

Invasive pulmonary aspergillosis due to Emericella nidulans var. echinulata, successfully cured by voriconazole and micafungin.

A 78-year-old male who was undergoing prolonged glucocorticoid treatment experienced cough and expectoration for 2 weeks. Galactomannan antigen analysis and a chest computed tomography (CT) scan suggested a diagnosis of invasive pulmonary aspergillosis. DNA sequencing indicated that Emericella nidulans var. echinulata was the causative agent. A combination of voriconazole and micafungin successfully treated the illness.

Identification and characterization of the echinocandin B biosynthetic gene cluster from Emericella rugulosa NRRL 11440.

Echinocandins are a family of fungal lipidated cyclic hexapeptide natural products. Due to their effectiveness as antifungal agents, three semisynthetic derivatives have been developed and approved for treatment of human invasive candidiasis. All six of the amino acid residues are hydroxylated, including 4R,5R-dihydroxy-L-ornithine, 4R-hydroxyl-L-proline, 3S,4S-dihydroxy-L-homotyrosine, and 3S-hydroxyl-4S-methyl-L-proline. We report here the biosynthetic gene cluster of echinocandin B 1 from Emericella rugulosa NRRL 11440 containing genes encoding for a six-module nonribosomal peptide synthetase EcdA, an acyl-AMP ligase EcdI, and oxygenases EcdG, EcdH, and EcdK. We showed EcdI activates linoleate as linoleyl-AMP and installs it on the first thiolation domain of EcdA. We have also established through ATP-PP(i) exchange assay that EcdA loads L-ornithine in the first module. A separate hty gene cluster encodes four enzymes for de novo generation of L-homotyrosine from acetyl-CoA and 4-hydroxyphenyl-pyruvate is found from the sequenced genome. Deletions in the ecdA, and htyA genes validate their essential roles in echinocandin B production. Five predicted iron-centered oxygenase genes, ecdG, ecdH, ecdK, htyE, and htyF, in the two separate ecd and hty clusters are likely to be the tailoring oxygenases for maturation of the nascent NRPS lipohexapeptidolactam product.

Rare and new etiological agents revealed among 178 clinical Aspergillus strains obtained from Czech patients and characterized by molecular sequencing.

A collection of 178 Aspergillus isolates, recovered from Czech patients, mostly from 2007-2011, was subjected to multilocus DNA sequence typing using the ITS region, ß-tubulin, and calmodulin genes. An unusually wide spectrum of etiologic agents that included 36 species of Aspergillus is discussed in the context of recent taxonomic and clinical reports. Invasive aspergillosis (IA), onychomycosis, and otitis externa were the predominant clinical entities. Five cases due to species newly proven as etiologic agents of human mycoses, as well as cases with unique clinical manifestations caused by unusual agents are discussed in more detail. Three species (i.e., A. insulicola, A. westerdijkiae and A. tritici) were identified as the confirmed etiologic agents of non-dermatophytic onychomycosis. Emericella rugulosa was recovered from a premature newborn with a fatal necrotising disseminated infection and is reported for only the second time as the cause of IA. Furthermore, we document the first infection due to A. calidoustus in a patient with chronic granulomatous disease. The infection manifested as a latent brain aspergilloma with an unusual clinical-laboratory finding. In addition to the well-known agents of human mycosis, several rarely isolated or poorly documented species were identified. An undescribed cryptic species related to A. versicolor was found to be common among isolates linked to proven and probable onychomycosis. An isolate representing A. fresenii, or an unnamed sister species, were causal agents of otomycosis. Three well defined, and tentative new species belonging to section Cervini, Candidi and Aspergillus (Eurotium spp.), were associated with cases of probable onychomycosis.

Expression and characterization of an endo-1,4-ß-galactanase from Emericella nidulans in Pichia pastoris for enzymatic design of potentially prebiotic oligosaccharides from potato galactans.

Potato pulp is a high-volume side-stream from industrial potato starch manufacturing. Enzymatically solubilized ß-1,4-galactan-rich potato pulp polysaccharides of molecular weights >100 kDa (SPPP) are highly bifidogenic in human fecal sample fermentations in vitro. The objective of the present study was to use potato ß-1,4-galactan and the SPPP as substrates for enzymatic production of potentially prebiotic compounds of lower and narrower molecular weight. A novel endo-1,4-ß-galactanase from Emericella nidulans (anamorph Aspergillus nidulans), GH family 53, was produced in a recombinant Pichia pastoris strain. The enzyme was purified by Cu(2+) affinity chromatography and its optimal reaction conditions were determined to pH 5 and 49°C via a statistical experimental design. The specific activity of the E. nidulans enzyme expressed in P. pastoris was similar to that of an endo-1,4-ß-galactanase from Aspergillus niger used as benchmark. The E. nidulans enzyme expressed in P. pastoris generated a spectrum poly- and oligo-saccharides which were fractionated by membrane filtration. The potential growth promoting properties of each fraction were evaluated by growth of beneficial gut microbes and pathogenic bacteria. All the galactan- and SPPP-derived products promoted the growth of probiotic strains of Bifidobacterium longum and Lactobacillus acidophilus and generally did not support the propagation of Clostridium perfringens in single culture fermentations. Notably the growth of B. longum was significantly higher (p<0.05) or at least as good on galactan- and SPPP-derived products as fructooligosaccharides (FOS). Except in one case these products did not support the growth of the pathogen Cl. perfringens to any significant extent.

Molecular analysis and anticancer properties of two identified isolates, Fusarium solani and Emericella nidulans isolated from Wady El-Natron soil in Egypt against Caco-2 (ATCC) cell line.

OBJECTIVE: To characterize, identify and investigate the anticancer properties of two new soil fungal isolates, Emericella nidulans and Fusarium solani isolated from Wady El-Natron in Egypt against colon cancer Caco-2 (ATCC) cell line. METHODS: Soil sample was cultured and two strains were chosen for morphological and phenotypical characterization. Partial sequences of the 18s rRNA gene and the internal transcribed spacer region ITS of the two isolates were amplified by PCR. Phylogenetic tree construction and analysis of the resulted multiple sequences from the two fugal isolates were also carried out. In vitro anticancer activity of the two strains was done against colon Caco-2 cancer cell line. Reverse transcription - PCR was carried out to detect level of expression of p53 in Caco-2 cell line. RESULTS: HF.1 displayed morphological and genotypic characteristics most similar to that of Fusarium solani while HF.2 was most similar to Emericella nidulans with high similarity of 99% and 97% respectively. The multiple sequence alignment of the two fungal isolates showed that, the maximum identical conserved domains in the 18s rRNA genes were identified with the nucleotide regions of 51st to 399th base pairs, 88th to 525th base pairs respectively. While those in the ITS genes were identified with the nucleotide regions of 88th to 463rd and 51st to 274th. The two isolates showed IC50 value with (6.24±5.21) and (9.84±0.36) µg/mL) concentrations respectively at 28h. Reverse transcription - PCR indicated that these cells showed high level of expression for p53 mRNA. CONCLUSIONS: The morphology and molecular analysis identified HF.1 and HF.2 to be Fusarium solani and Emericella nidulans; new isolates of anticancer producing fungi from Wady El-Natroon city in Egypt. Treatment with the two isolates caused P53 expression in Caco-2 cell line. These two isolates can be used as an anticancer agents.

Polyphasic characterization of "Aspergillus nidulans var. roseus" ATCC 58397.

Polyphasic characterization of the echinocandin B producer Aspergillus nidulans var. roseus ATCC 58397 strain was carried out to elucidate its taxonomical status. According to its carbon source utilization and secondary metabolite spectrum as well as the partial ß-tubulin, calmodulin, and γ-actin gene sequences, A. nidulans var. roseus belongs to the Emericella rugulosa species. Auxotroph mutants of A. nidulans var. roseus ATCC 58397 and E. rugulosa CBS 171.71 and CBS 133.60 formed stable heterokaryons on minimal medium with several A. nidulans strains, and in the case of A. nidulans var. roseus, even cleistothecia were developed.

The mode of reproduction in natural populations of ascomycetous fungus, Emericella nidulans, from Israel.

The mode of reproduction of the soil ascomycetous fungus Emericella nidulans of Israeli populations was studied using 15 microsatellite (simple sequence repeats or SSR) trinucleotide markers. The study was performed in three canyons: two located in the northern part of Israel (Mount Carmel and western Upper Galilee) and one in the southern Negev desert. In each canyon, E. nidulans strains were isolated from the opposite slopes and (in the desert canyon) the valley bottom. Testing the reproductive structure of the populations indicated the presence of sexuality in the northern population and predominant clonality in the desert population. The predominantly clonal character of the desert population of E. nidulans was explained by the assumption that for relevant multilocus systems of a fungus, only several haplotypes can survive in the rather constant, extremely stressful desert conditions. Additionally, the very low density of E. nidulans populations in the soil of the desert canyon, which reduces the probability of finding a sexual partner, might favour predominant clonality via selfing. Increasing sexuality in E. nidulans populations on the north-facing slopes of the northern canyons may be a result of biotic stress (pressure of competitive fungal species), due to the more mild ecological conditions in these canyons.

Development of rapid and specific molecular discrimination methods for pathogenic emericella species.

Aspergillosis is an important mycosis caused primarily by Aspergillus fumigatus and its relatives. The genus Emericella is a teleomorph related to the Aspergillus section Nidulantes. The typical anamorphic stage species in this genus is Aspergillus nidulans, which is sometimes a significant agent in chronic granulomatous disease (CGD) patients. The mortality rate of osteomyelitis in CGD patients due to A. nidulans ( E. nidulans ) is very high compared to that due to A. fumigatus. Moreover, two Emericella species ( E. nidulans and E. quadrilineata ) from clinical specimens exhibit different sensitivities against several antifungal drugs. In aspergillosis, correct species identification is important for antifungal therapy. We attempted to develop rapid and specific molecular discrimination by polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) methods in the principal pathogenic Emericella species, and succeeded in establishing species-specific primers corresponding to the hydrophobin gene. These primers discriminate E. nidulans and E. quadrilineata rapidly and specifically. These methods and primers make it possible to diagnose etiological agents in aspergillosis quickly and easily.

Four new species of Emericella from the Mediterranean region of Europe.

Four new species of Emericella, E. discophora, E. filifera, E. olivicola and E. stella-maris, are proposed. Their new taxonomic status was determined applying a polyphasic taxonomic approach using phenotypic (morphology and extrolite profiles) and molecular (sequences of ITS, beta-tubulin and calmodulin genes) characters. Ascospores of E. stella-maris and E. olivicola have star-shape equatorial crests, those of E. filifera form long appendages that emerge radially from narrow stellate crests and those of E. discophora produce wide and entire, nonstellate equatorial crests. E. stella-maris originated from leaf litter in Tunisia and E. filifera from raisins in Argentina, and both of them also were found in hypersaline water of a saltern in Slovenia. E. olivicola was isolated from olives in Italy and E. discophora from soil in Spain. All listed species possess distinct extrolite profiles: E. stella-maris produced arugosin E, shamixanthone and the yet unelucidated metabolites glia 1-3; E. filifera produced shamixanthone and varitriols; E. discophora produced sterigmatocystin and versicolorins; E. olivicola produced numerous extrolites such as arugosin E, siderin, shamixanthone, sterigmatocystin, terrein, varitriols and aflatoxin B1, of which the latter was detected only in one of the two strains.

Emericella quadrilineata as cause of invasive aspergillosis.

We noted a cluster of 4 cases of infection or colonization by Emericella spp., identified by sequence-based analysis as E. quadrilineata. Sequence-based analysis of an international collection of 33 Emericella isolates identified 12 as E. nidulans, all 12 of which had previously been identified by morphologic methods as E. nidulans. For 12 isolates classified as E. quadrilineata, only 6 had been previously identified accordingly. E. nidulans was less susceptible than E. quadrilineata to amphotericin B (median MICs 2.5 and 0.5 mg/L, respectively, p<0.05); E. quadrilineata was less susceptible than E. nidulans to caspofungin (median MICs, 1.83 and 0.32 mg/L, respectively, p<0.05). These data indicate that sequence-based identification is more accurate than morphologic examination for identifying Emericella spp. and that correct species demarcation and in vitro susceptibility testing may affect patient management.

Isolation and characterization of nudC from mouse macrophages, a gene implicated in the inflammatory response through the regulation of PAF-AH(I) activity.

We report the characterization of a cDNA induced in mouse macrophages that encodes a 332-amino acid protein with extensive sequence identity with members of the mammalian nudC-like genes. The interaction between mNUDC and the regulatory beta subunit of platelet activating factor acetylhydrolase I (PAF-AH(I)) shown in this article indicates a new function of NUDC. Thus, we show that NUDC increases the catalytic activity of PAF-AH(I) and that this regulatory activity is located in the carboxyl terminal half of the protein which is highly conserved. This suggests a novel function for mammalian nudC-like genes as anti-inflammatory proteins.

Glucose-mediated repression of autolysis and conidiogenesis in Emericella nidulans.

Glucose-mediated repression of autolysis and sporulation was studied in submerged Emericellanidulans (anam. Aspergillus nidulans) cultures. Null mutation of the creA gene, which encodes the major carbon catabolite repressor CreA in E. nidulans, resulted in a hyperautolytic phenotype characterized by increased extracellular hydrolase production and dry cell mass declination. Interestingly, glucose, as well as the glucose antimetabolite 2-deoxy-d-glucose, repressed autolysis and sporulation in both the control and the creA null mutant strains suggesting that these processes were also subjected to CreA-independent carbon regulation. For example, the glucose-mediated, but CreA-independent, repression of the sporulation transcription factor BrlA was likely to contribute to the negative regulation of conidiogenesis by glucose. Although CreA played a prominent role in the regulation of autolysis via the repression of genes encoding important autolytic hydrolases like ChiB chitinase and PrtA protease the age-related production of the chitinase activity was also negatively affected by the down-regulation of brlA expression. However, neither CreA-dependent nor CreA-independent elements of carbon regulation affected the initiation and regulation of cell death in E. nidulans under carbon starvation.