Cytotoxic activity of cordycepin produced by marine-derived fungus Emericella sp. against HT29 human colon cancer cell lines.

Colorectal cancer accounted for the third most common cancer in the world. The search for new drug candidates that can be used for colorectal cancer treatment from marine-derived fungi, Emericella sp. The present study was performed to isolate the cytotoxic compound from Emericella sp. The isolation method was carried out by using a combination of chromatographic techniques to afford compound 1. The cytotoxic activity and the exosome production property were determined by using proliferation and luciferase assay against HT29 CD63 Nluc cells, respectively. The chemical structure of compound 1 was identified as cordycepin based on spectroscopy methods such as mass spectrometry and nuclear magnetic resonance (1D and 2D NMR) analyses and comparison with authentic spectral data. The biological activity assay showed that cordycepin exhibited cytotoxic activity with an IC50 value of 92.05 µM through proliferation assay, and also inhibited the exosome production by luciferase assay with an IC50 value of 86.47 µM. Cordycepin was isolated from culture broth Emericella sp., exhibiting moderate cytotoxic activity and inhibitory activity of exosome production. Thus, cordycepin is a potential compound to be investigated further for its exosome production inhibition activity for further use as an anticancer lead compound.

Emeridones A-F, a Series of 3,5-Demethylorsellinic Acid-Based Meroterpenoids with Rearranged Skeletons from an Endophytic Fungus Emericella sp. TJ29.

Six new 3,5-demethylorsellinic acid-based meroterpenoids, emeridones A-F (1-6), and eight known analogues (7-14) were isolated from Emericella sp. TJ29. Their structures and absolute configurations were elucidated by comprehensive spectroscopic analyses, single-crystal X-ray diffraction experiments, and electronic circular dichroism calculations. Emeridone A (1) represents the first meroterpenoid featuring a unique rigid 6/6/5/6 tetracyclic carbon ring system with two additional lactone rings. Emeridones B and C (2 and 3) possess a 2,6-dioxabicyclo[2.2.1]heptane and a spiro[bicyclo[3.2.2]nonane-2,1'-cyclohexane] moiety, respectively, and both functionalities were found for the second time in meroterpenoids. These new compounds were evaluated for their cytotoxic activities against five human cancer cells, and compounds 2, 4, and 6 exhibited moderate cytotoxic activities, with IC50 values ranging from 8.19 to 18.80 µM.

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.

Structural and Computational Bases for Dramatic Skeletal Rearrangement in Anditomin Biosynthesis.

AndA, an Fe(II)/α-ketoglutarate (αKG)-dependent enzyme, is the key enzyme that constructs the unique and congested bridged-ring system of anditomin (1), by catalyzing consecutive dehydrogenation and isomerization reactions. Although we previously characterized AndA to some extent, the means by which the enzyme facilitates this drastic structural reconstruction have remained elusive. In this study, we have solved three X-ray crystal structures of AndA, in its apo form and in the complexes with Fe(II), αKG, and two substrates. The crystal structures and mutational experiments identified several key amino acid residues important for the catalysis and provided insight into how AndA controls the reaction. Furthermore, computational calculations validated the proposed reaction mechanism for the bridged-ring formation and also revealed the requirement of a series of conformational changes during the transformation.

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.

Computational Studies on Biosynthetic Carbocation Rearrangements Leading to Quiannulatene: Initial Conformation Regulates Biosynthetic Route, Stereochemistry, and Skeleton Type.

The results of quantum chemical calculations on the mechanism of the carbocation cascade of reactions in the biosynthetic pathways leading to the pentacyclic sesterterpenes quiannulatene and sesterfisherol provide reasonable answers to several persistent mechanistic questions in sesterterpene biosynthesis, including: 1) the reaction pathways of the multicyclic ring system construction and skeletal rearrangements, 2) the mechanism of triquinane skeleton formation, which requires more complicated rearrangements than previously proposed, 3) the stereochemistry of the final carbocation intermediate, and 4) the determining factor of biosynthetic selection for either 5/6/4/6/5 or 5/6/5/5/5 pentacyclic skeleton formation. This in-depth mechanistic study on sesterterpene biosynthesis revealed that the shape of the final product and the type of triquinane skeleton formed are regulated by the stereochemistry and conformation of the common starting material, geranylfarnesyl diphosphate (GFPP).

Bioassay-Guided Isolation of Antibacterial Metabolites from Emericella sp. TJ29.

Bioassay-guided isolation of metabolites from cultures of the plant-derived fungus Emericella sp. TJ29 yielded three new terpene-polyketide hybrid meroterpenoids, emervaridones A-C (1-3), two new polyketides, varioxiranediols A and B (5 and 6), and three known analogues (4, 7, and 8). The structures and absolute configurations of these new compounds were elucidated by spectroscopic analyses, single-crystal X-ray diffraction, Mo2(OAc)4-induced electronic circular dichroism (ECD) data, and ECD calculations. To date, only one compound (4) bearing the emervaridone-type carbocyclic skeleton has been reported. The structures of emervaridones A-C (1-3) are new members of this type of natural product, and 1 features the first example of an α-directional H-7' in this structural category. Compounds 1 and 5 were active against five drug-resistant microbial pathogens [methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-producing E. coli), Pseudomonas aeruginosa, and Klebsiella pneumoniae] with minimum inhibitory concentration (MIC) values in the micrograms per milliliter range. Notably, the inhibitory effect of emervaridone A (1) against ESBL-producing E. coli was comparable to that of the clinically used antibiotic amikacin, with an MIC value of 2 µg/mL. Compounds 1 and 5, both with low toxicities to mammalian cells, were bacteriostatic and bactericidal, respectively. Importantly, these two compounds may provide novel chemical scaffolds for the discovery of antibacterial agents for drug-resistant microbial pathogens.

[Study on metabolites from a sponge-derived fungus].

We have carried out the investigation on a sponge-derived fungus,which was identified as Emericella variecolor from the south sea of China. Two new chemical constituents,(+)-2-acetyl-dihydroterrein (1) and (+)-3-acetyl-dihydroterrein (2),with four known compounds,anditomin (3),andilesin A (4),andilesin C (5) and andilesin B (6),were isolated from this fungus by column chromatography over silica gel, Sephadex LH-20, and ODS. The structures of 1 and 2 were elucidated by spectroscopic methods including NMR,HR-ESI-MS,and CD.

Genome-Based Discovery of an Unprecedented Cyclization Mode in Fungal Sesterterpenoid Biosynthesis.

Sesterterpenoids are a group of terpenoid natural products that are primarily biosynthesized via cyclization of the C25 linear substrate geranylfarnesyl pyrophosphate (GFPP). Although the long carbon chain of GFPP in theory allows for many different cyclization patterns, sesterterpenoids are relatively rare species among terpenoids, suggesting that many intriguing sesterterpenoid scaffolds have been overlooked. Meanwhile, the recent identification of the first sesterterpene synthase has allowed the discovery of new sesterterpenoids by the genome mining approach. In this study, we characterized the unusual fungal sesterterpene synthase EvQS and successfully obtained the sesterterpene quiannulatene (1) with a novel and unique highly congested carbon skeleton, which is further oxidized to quiannulatic acid (2) by the cytochrome P450 Qnn-P450. A mechanistic study of its cyclization from GFPP indicated that the biosynthesis employs an unprecedented cyclization mode, which involves three rounds of hydride shifts and two successive C-C bond migrations to construct the 5-6-5-5-5 fused ring system of 1.

The First Case of Invasive Mixed-Mold Infections Due to Emericella nidulans var. echinulata and Rasamsonia piperina in a Patient with Chronic Granulomatous Disease.

A 16-year-old boy with chronic granulomatous disease presented with pneumonia and rib osteomyelitis. Emericella nidulans var. echinulata was isolated from his sputum. After starting voriconazole, Rasamsonia piperina was isolated from the rib swelling. A combination therapy of voriconazole and micafungin effectively eradicated this invasive mixed-mold infection. In immunocompromised patients, a precise pathogenic diagnosis is clinically useful for administration of an appropriate treatment regimen.

Astellifadiene: Structure Determination by NMR Spectroscopy and Crystalline Sponge Method, and Elucidation of its Biosynthesis.

Genome mining of a terpene synthase gene from Emericella variecolor NBRC 32302 and its functional expression in Aspergillus oryzae led to the production of the new sesterterpene hydrocarbon, astellifadiene (1), having a 6-8-6-5-fused ring system. The structure of 1 was initially investigated by extensive NMR analyses, and was further confirmed by the crystalline sponge method, which established the absolute structure of 1 and demonstrated the usefulness of the method in the structure determination of complex hydrocarbon natural products. Furthermore, the biosynthesis of 1 was proposed on the basis of isotope-incorporation experiments performed both in vivo and in vitro. The cyclization of GFPP involves a protonation-initiated second cyclization sequence, 1,2-alkyl migration, and 1,5-hydride shift to generate the novel scaffold of 1.

An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping.

Di- and sesterterpene synthases produce C20 and C25 isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene (1), a novel tricyclic diterpene hydrocarbon. Intriguingly, in vitro reaction with the enzyme afforded the new macrocyclic sesterterpene 2 as a minor product from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The TC domain thus produces the diterpene 1 and the sesterterpene 2 from GGPP and GFPP, respectively. Notably, a domain swap of the PT domain of EvVS with that of another chimeric sesterterpene synthase, EvSS, successfully resulted in the production of 2 in vivo as well. Cyclization mechanisms for the production of these two compounds are proposed.

Cytochrome P450-catalyzed regio- and stereoselective phenol coupling of fungal natural products.

For almost 100 years, phenoxy radical coupling has been known to proceed in nature. Because of the linkage of their molecular halves (regiochemistry) and the configuration of the biaryl axis (stereochemistry), biaryls are notoriously difficult to synthesize. Whereas the intramolecular enzymatic coupling has been elucidated in detail for several examples, the bimolecular intermolecular coupling could not be assigned to one single enzyme in the biosynthesis of axially chiral biaryls. As these transformations often take place regio- and stereoselectively, enzyme-catalyzed control is reasonable. We now report the identification and expression of fungal cytochrome P450 enzymes that catalyze regio- and stereoselective intermolecular phenol couplings. The cytochrome P450 enzyme KtnC from the kotanin biosynthetic pathway of Aspergillus niger was expressed in Saccharomyces cerevisiae. The recombinant cells catalyzed the coupling of the monomeric coumarin 7-demethylsiderin both regio- and stereoselectively to the 8,8'-dimer P-orlandin, a precursor of kotanin. The sequence information obtained from the kotanin biosynthetic gene cluster was used to identify in silico a similar gene cluster in the genome of Emericella desertorum, a producer of desertorin A, the 6,8'-regioisomer of orlandin. The cytochrome P450 enzyme DesC was also expressed in S. cerevisiae and was found to regio- and stereoselectively catalyze the coupling of 7-demethylsiderin to M-desertorin A. Our results show that fungi use highly specific cytochrome P450 enzymes for regio- and stereoselective phenol coupling. The enzymatic activities of KtnC and DesC are relevant for an understanding of the mechanism of this important biosynthetic step. These results suggest that bimolecular phenoxy radical couplings in nature can be catalyzed by phenol-coupling P450 heme enzymes, which might also apply to the plant kingdom.

Varioxiranols A-G and 19-O-Methyl-22-methoxypre-shamixanthone, PKS and Hybrid PKS-Derived Metabolites from a Sponge-Associated Emericella variecolor Fungus.

Chemical examination of a sponge (Cinachyrella sp.)-associated Emericella variecolor fungus resulted in the isolation of seven new polyketide derivatives, namely, varioxiranols A-G (1-7), and a new hybrid PKS-isoprenoid metabolite, 19-O-methyl-22-methoxypre-shamixanthone (8), together with nine known analogues. Their structures were elucidated on the basis of extensive spectroscopic analyses, including ECD effects, Mosher's method, X-ray diffraction, and chemical conversion for the determination of absolute configurations. Varioxiranols F and G were found for the first time to link a xanthone moiety with a benzyl alcohol via an ether bond, while the dioxolanone group of 5 is unusual in nature. A cell-based lipid-lowering assay revealed that pre-shamixanthone (12) exerted significant inhibition against lipid accumulation in HepG2 cells without cytotoxic effects, accompanying the potent reduction of total cholesterol and triglycerides. Real-time quantitative PCR indicated that pre-shamixanthone (12) mediated the reduction of lipid accumulation related to the down-regulation of the expression of the key lipogenic transcriptional factor SREBP-1c and its downstream genes encoding FAS and ACC.

Varioxiranols I-L, new lactones from a sponge-associated Emericella variecolor fungus.

Chemical examination of the sponge-associated fungus Emericella variecolor resulted in the isolation of four new lactones namely varioxiranols I-L(1-4)with different scaffolds, together with asteltoxin (5) and asteltoxin B (6). The structure elucidation of new compounds was accomplished by spectroscopic analysis, while the absolute configurations were determined by computed circular dichroism (ECD) and induced CD effects. Antitumor activities of these compounds were evaluated against different tumor cell lines, while the result indicated that the new compounds showed moderate cytotoxic activity against a panel of tumor cell lines.

Iterative breakthrough invasive aspergillosis due to TR(34) /L98H azole-resistant Aspergillus fumigatus and Emericella sublata in a single hematopoietic stem cell transplant patient.

We report 3 consecutive episodes of invasive aspergillosis in a single hematopoietic stem cell transplant patient successively attributed to TR(34) /L98H azole-resistant Aspergillus fumigatus and to a first occurrence of invasive Emericella sublata infection. This case illustrates potential selection of resistant molds during antifungal therapy in hematological patient.

New prenylxanthones from the deep-sea derived fungus Emericella sp. SCSIO 05240.

Four new prenylxanthones, emerixanthones A-D (1-4), together with six known analogues (5-10), were isolated from the culture of the deep-sea sediment derived fungus Emericella sp. SCSIO 05240, which was identified on the basis of morphology and ITS sequence analysis. The newstructures were determined by NMR (1H, 13C NMR, HSQC, HMBC, and 1H-1H COSY), MS, CD, and optical rotation analysis. The absolute configuration of prenylxanthone skeleton was also confirmed by the X-ray crystallographic analysis. Compounds 1and 3 showed weak antibacterial activities, and 4 displayed mild antifungal activities against agricultural pathogens.

Emerindanols A and B: Two Bipolyhydroindenol Sesterterpenes with 5/6-6/5 Coupled Ring System Discovered by Genome Mining.

Genome mining of Emericella sp. XL-029 achieved a new type E sesterterpene synthase, EmES, which affored a novel bipolyhydroindenol sesterterpene, emerindanol A. Heterologous coexpression with the upstream P450 oxidase revealed C-4 hydroxylated product, emerindanol B. Notably, emerindanols A and B represented the first sesterterpenes featuring a unique 5/6-6/5 coupled ring system. EmES was postulated to initiate through C1-IV-V pathway and convert the fused ring intermediate into the final coupled ring product through a spiro skeleton.

Unexpected Noremestrin with a Sulfur-Bearing 15-Membered Macrocyclic Lactone from Emericella sp. 1454.

Two previous unreported epipolythiodioxopiperazines of the emestrin family, namely, noremestrin A (1) and secoemestrin E (2), were successfully isolated from the fungal source Emericella sp. 1454. Employing comprehensive spectroscopic techniques, such as high-resolution electrospray ionization mass spectrometry, infrared, and nuclear magnetic resonance (NMR), along with NMR and electronic circular dichroism calculations, the chemical structures of compounds 1 and 2 were elucidated. Particularly noteworthy is the distinctive nature of noremestrin A, representing the inaugural instance of a noremestrin variant incorporating a sulfur-bearing 15-membered macrocyclic lactone moiety. Compounds 1 and 2 exhibited weak cytotoxic activities against the human chronic myelocytic leukemia cell lines MEG-01 and K562.

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.