Identification of new bisabosqual-type meroterpenoids reveals non-enzymatic conversion of bisabosquals into seco-bisabosquals.

Bisabosqual-type meroterpenoids are fungi-derived polyketide-terpenoid hybrids bearing a 2,3,3a,3a1,9,9a-hexahydro-1H-benzofuro[4,3,2-cde]chromene skeleton (6/6/6/5 ring system) or its seco-C-ring structure, and exhibit diverse bioactivities. Their unique structural architecture and impressive biological activities have led to considerable interest in discovering new analogues. However, to date, only nine analogues have been identified. Herein, we reported the isolation and identification of six new bisabosqual-type meroterpenoids stachybisbins C-H (1-6), together with one known compound bisabosqual C (7), from Stachybotrys bisbyi PYH05-7. Intriguingly, we found that 7, which contains the intact tetracyclic skeleton, can be non-enzymatically converted into its seco derivative stachybisbin I (8), unveiling the biosynthetic relationship between bisabosquals and seco-bisabosquals. Moreover, based on CRISPR/Cas9-mediated gene disruption, we revealed that the three-gene cluster responsible for the formation of LL-Z1272ß is associated with the biosynthesis of bisabosqual-type meroterpenoids, and then proposed a plausible route to 1-8.

Trichoderpyrone, a Unique Polyketide Hybrid with a Cyclopentenone-Pyrone Skeleton from the Plant Endophytic Fungus Trichoderma gamsii.

Trichoderpyrone (1), a unique polyketide with a cyclopentenone-pyrone hybrid skeleton, was isolated from the plant endophytic fungus Trichoderma gamsii. The structure of 1 was determined by detailed analysis of NMR data together with comparison of chemical shift values of similar fragments. The relative and absolute configurations were established by NOESY correlations and CD experiment. Trichoderpyrone (1) displayed weak cytotoxic activities against A549, HepG2, and HeLa cancer cell lines. 1 might originate from a hybrid biosynthetic pathway through two nonreduced (NR) polyketide megasynthetases.

Mitochondrial genome of the nematode endoparasitic fungus Hirsutella vermicola reveals a high level of synteny in the family Ophiocordycipitaceae.

Ophiocordycipitaceae is a diverse fungal family comprising multiple ecologically, economically, medicinally, and culturally important fungal species; however, only four species of the family have available mitochondrial genomes (mitogenomes). In this study, the complete mitogenome of the nematode endoparasitic fungus Hirsutella vermicola in Ophiocordycipitaceae was sequenced, and a comparative mitogenomic analysis of Ophiocordycipitaceae was performed. We found that the 53,793-bp circular mitogenome of H. vermicola, except for standard fungal mitochondrial genes, harbors seven introns acquired possibly through lateral transfer from other fungi and three free-standing open reading frames (ORFs) coding for hypothetical proteins. Phylogenetic analysis based on concatenated mitochondrial protein sequences confirmed its placement in Ophiocordycipitaceae. Comparison on five mitogenomes of Ophiocordycipitaceae revealed great variation on their sizes, from 35.2 kb in Tolypocladium ophioglossoides to 157.5 kb in Ophiocordyceps sinensis, mainly due to variable numbers of introns (from 7 to 54) as well as variable lengths of intergenic regions. The five mitogenomes, however, are highly syntenic to each other in terms of gene order, the presence of an intronic ORF encoding ribosomal protein S3 within rnl, and the nad2/nad3 joining pattern. Our study is the first report of the mitogenome of H. vermicola and has facilitated the understanding of mitogenome evolution of Ophiocordycipitaceae.

Complete mitochondrial genome of the endophytic fungus Pestalotiopsis fici: features and evolution.

Endophytic fungi (EF) live within plants and have profound impacts on plant communities. They are astonishingly diverse but poorly studied at the genome level. Herein, we assembled the mitochondrial genome (mitogenome) of the EF Pestalotiopsis fici, annotated and compared it with those of other relatives to better understand the evolution of the EF lineage. Except for standard fungal mitochondrial genes, the 69,529-bp circular mitogenome of P. fici harbors 18 introns acquired possibly through lateral transfer from other fungi and nine free-standing open reading frames with some scarcely seen in fungal mitogenomes. BLAST analysis detected no obvious duplication events of large fragments between mitochondrial and nuclear genomes of the fungus. Transcription analyses validated the expression of all mitochondrial genes, while most genes showed higher expression on rice than in two other media. The mitogenome of P. fici is highly syntenic with the Xylariales species Annulohypoxylon stygium and the endophyte Epichloe festucae var. lolii, but lacks synteny with another endophyte Penicillium polonicum. This study reports the first mitogenome of Pestalotiopsis and the third published mitogenome from an EF and provides insights into the evolution of the EF lineage.

New antibacterial isocoumarin glycosides from a wetland soil derived fungal strain Metarhizium anisopliae.

Eight new isocoumarin glycosides (1-8) were obtained from the solid culture of the wetland soil-derived fungus Metarhizium anisopliae (No. DTH12-10). Their chemical structures were elucidated by analyses of HR ESI-TOF MS, (1)H, (13)C NMR, (1)H-(1)H COSY, HSQC, and HMBC spectra. The absolute configurations were determined by single crystal X-ray diffraction, circular dichroism (CD) spectrum, and chemical derivatization methods. In addition, inhibition of the biofilm formation and the secretion of virulence factor of the new isocoumarin glycosides against Pseudomonas aeruginosa strain PAOA (clinical isolates) were evaluated. The result revealed that compound 1 showed antibacterial activity comparable with (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone (BF).

The intronic minisatellite OsMin1 within a serine protease gene in the Chinese caterpillar fungus Ophiocordyceps sinensis.

Repetitive DNA sequences make up a significant portion of all genomes and may occur in intergenic, regulatory, coding, or even intronic regions. Partial sequences of a serine protease gene csp1 was previously used as a population genetic marker of the Chinese caterpillar fungus Ophiocordyceps sinensis, but its first intron region was excluded due to ambiguous alignment. Here in this study, we report the presence of a minisatellite OsMin1 within this intron, where a 20(19)-bp repeat motif is duplicated two to six times in different isolates. Fourteen intron alleles and 13 OsMin1 alleles were identified among 125 O. sinensis samples distributed broadly on the Tibetan Plateau. Two OsMin1 alleles were prevalent, corresponding to either two or five repeats of the core sequence motif. OsMin1 appears to be a single locus marker in the O. sinensis genome, but its origin is undetermined. Abundant recombination signals were detected between upstream and downstream flanking regions of OsMin1, suggesting that OsMin1 mutate by unequal crossing over. Geographic distribution, fungal phylogeny, and host insect phylogeny all significantly affected intron distribution patterns but with the greatest influence noted for fungal genotypes and the least for geography. As far as we know, OsMin1 is the first minisatellite found in O. sinensis and the second found in fungal introns. OsMin1 may be useful in designing an efficient protocol to discriminate authentic O. sinensis from counterfeits.

Three Pairs of New Isopentenyl Dibenzo[b,e]oxepinone Enantiomers from Talaromyces flavus, a Wetland Soil-Derived Fungus.

Three pairs of new isopentenyl dibenzo[b,e]oxepinone enantiomers, (+)-(5S)-arugosin K (1a), (-)-(5R)-arugosin K (1b), (+)-(5S)-arugosin L (2a), (-)-(5R)-arugosin L (2b), (+)-(5S)-arugosin M (3a), (-)-(5R)-arugosin M (3b), and a new isopentenyl dibenzo[b,e]oxepinone, arugosin N (4), were isolated from a wetland soil-derived fungus Talaromyces flavus, along with two known biosynthetically-related compounds 5 and 6. Among them, arugosin N (4) and 1,6,10-trihydroxy-8-methyl-2-(3-methyl-2-butenyl)-dibenz[b,e]oxepin-11(6H)-one (CAS: 160585-91-1, 5) were obtained as the tautomeric mixtures. The structures of isolated compounds were determined by detailed spectroscopic analysis. In addition, the absolute configurations of these three pairs of new enantiomers were determined by quantum chemical ECD calculations.

[Two new polyesters from wetland soil-derived fungus Talaromyces flavus].

Two new polyesters, talapolyesters G-H (1-2) were isolated from the wetland soil-derived fungus Talaromyces flavus BYD07-13, and their structures were determined by NMR and MS spectroscopic data. The absolute configurations of the residues were determined by alkaline hydrolysis. The cytotoxicity against five tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW480) of 1-2 was examined.

New isocoumarins from a cold-adapted fungal strain mucor sp. and their developmental toxicity to zebrafish embryos.

Three new isocoumarin derivatives, mucorisocoumarins A-C (1-3, resp.), together with seven known compounds, 4-10, were isolated from the cold-adapted fungal strain Mucor sp. (No. XJ07027-5). The structures of the new compounds were identified by detailed IR, MS, and 1D- and 2D-NMR analyses. It was noteworthy that compounds 1, 2, 4, and 5 were successfully resolved by chiral HPLC, indicating that 1-7 should exist as enantiomers. In an embryonic developmental toxicity assay using a zebrafish model, compound 3 produced developmental abnormalities in the zebrafish embryos. This is the first report of isocoumarins with developmental toxicity to zebrafish embryos.

Talaflavuterpenoid A, a new nardosinane-type sesquiterpene from Talaromyces flavus.

Talaflavuterpenoid A (1), a new nardosinane-type sesquiterpene, was isolated from the wetland soil-derived fungus Talaromyces flavus BYD07-13, and its structure was elucidated on the basis of HR-MS, NMR, and X-ray diffraction analysis. The absolute configuration of 1 was established by comparing the experimental electronic circular dichroism (ECD) spectrum with the calculated ECD spectra. Its cytotoxic effects on five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW480), and antimicrobial activity against Escherichia coli, Staphylococcus aureus, Candida albicans, and Aspergillus niger were evaluated. This is the first report of the presence of nardosinane-type sesquiterpene in Talaromyces sp.

Two new coumarins from Talaromyces flavus.

Two new coumarins, talacoumarins A (1) and B (2), were isolated from the ethyl acetate extract of the wetland soil-derived fungus Talaromyces flavus BYD07-13. Their structures were elucidated by spectroscopic data (NMR, MS) analyses. The absolute configuration of C-12 in 1 was assigned using the modified Mosher's method, whereas that of C-12 in 2 was deduced via the circular dichroism data of its corresponding [Rh2(OCOCF3)4] complex. Compounds 1 and 2 were evaluated for their anti-Aß42 aggregation, cytotoxic, and antimicrobial activities. The results showed that the two compounds had moderate anti-Aß42 aggregation activity, and this is the first report on the Aß42 inhibitory aggregation activity of coumarins.

On the reliability of DNA sequences of Ophiocordyceps sinensis in public databases.

Some DNA sequences in the International Nucleotide Sequence Databases (INSD) are erroneously annotated, which has lead to misleading conclusions in publications. Ophiocordyceps sinensis (syn. Cordyceps sinensis) is a fungus endemic to the Tibetan Plateau, and more than 100 populations covering almost its distribution area have been examined by us over recent years. In this study, using the data from authentic materials, we have evaluated the reliability of nucleotide sequences annotated as O. sinensis in the INSD. As of October 15, 2012, the INSD contained 874 records annotated as O. sinensis, including 555 records representing nuclear ribosomal DNA (63.5 %), 197 representing protein-coding genes (22.5 %), 92 representing random markers with unknown functions (10.5 %), and 30 representing microsatellite loci (3.5 %). Our analysis indicated that 39 of the 397 internal transcribed spacer entries, 27 of the 105 small subunit entries, and five of the 53 large subunit entries were incorrectly annotated as belonging to O. sinensis. For protein-coding sequences, all records of serine protease genes, the mating-type gene MAT1-2-1, the DNA lyase gene, the two largest subunits of RNA polymerase II, and elongation factor-1α gene were correct, while 14 of the 73 ß-tubulin entries were indeterminate. Genetic diversity analyses using those sequences correctly identified as O. sinensis revealed significant genetic differentiation in the fungus although the extent of genetic differentiation varied with the gene. The relationship between O. sinensis and some other related fungal taxa is also discussed.

Three new glucosides from a cold-adapted fungal strain Mucor sp.

Two new naphthaline glucosides, 2-hydroxy-3-methoxy-8-methyl-1-O-ß-d-glucopyranosylnaphthaline (1) and 1-hydroxy-3-methoxy-8-methyl-2-O-ß-d-glucopyranosylnaphthaline (2), together with one new isocoumarin glucoside, 3-(3,3-dichloro-2-hydroxylpropyl)-6-methoxy-8-O-ß-d-glucopyranosyl-1H-isochromen-1-one (3), were isolated from a cold-adapted fungal strain Mucor sp. (No. XJ07027-5). Their structures were characterized by detailed analyses of IR, MS, 1D- and 2D-NMR spectra. Among them, 2 showed moderate cytotoxic activity against five tumor cells (A-549, HL-60, MCF-7, SMMC-7721, and SW480).

Determining novel molecular markers in the Chinese caterpillar fungus Ophiocordyceps sinensis by screening a shotgun genomic library.

The Chinese caterpillar fungus Ophiocordyceps sinensis, endemic to alpine regions on the Tibetan Plateau, is one of the most valuable medicinal fungi in the world. Genetic differentiation within this fungus was observed; however, due to lack of highly efficient molecular markers, the overall genetic structure of this fungus has not been clarified. In this study, a shotgun genomic library of O. sinensis was constructed, and >181,848 nt were analyzed from >250 random clones. Primers from 33 sequenced fragments were then designed to amplify O. sinensis samples collected from widely separated regions on the Tibetan Plateau. Ten of the 33 fragments had no amplification or poor sequencing quality from all or certain samples. Sequence variations of the remaining 23 fragments among different samples were investigated in detail. Three fragments (OSRC14, OSRC19, and OSRC32) were the most variable with 7-43 single-nucleotide polymorphism (SNP) sites, representing the SNP frequency of 1.2-6.7 % per nucleotide site. These three fragments have the potential to be useful molecular markers for studying the population genetics of O. sinensis. These results also showed that constructing and screening a shotgun genomic library was an efficient approach to identify novel molecular markers from non-model organisms.

Strategy for efficient cloning of biosynthetic gene clusters from fungi.

Filamentous fungi are excellent sources for the production of a group of bioactive small molecules which are often called secondary metabolites (SMs). The advanced genome sequencing technology combined with bioinformatics analysis reveals a large number of unexplored biosynthetic gene clusters (BGCs) in the fungal genomes. To unlock this fungal SM treasure, many approaches including heterologous expression are being developed and efficient cloning of the BGCs is a crucial step to do this. Here, we present an efficient strategy for the direct cloning of fungal BGCs. This strategy consisted of Splicing by Overlapping Extension (SOE)-PCR and yeast assembly in vivo. By testing 14 BGCs DNA fragments ranging from 7 kb to 52 kb, the average positive rate was over 80%. The maximal insertion size for fungal BGC assembly was 52 kb. Those constructs could be used conveniently for the heterologous expression leading to the discovery of novel natural products. Thus, our results provide an efficient and quick method for the low cost direct cloning of fungal BGCs.

Formation and instability of aerobic granules under high organic loading conditions.

The cultivation and instability of aerobic granular sludge in a sequencing batch reactor under high loading conditions were investigated. Compact bacteria-dominated aerobic granules with a mean diameter of 1 mm were formed at a chemical oxygen demand (COD) loading rate of 6.0 kg m(-3) d(-1) within 30 d. However, the compact bacteria-dominated aerobic granules were not stable and transited to large-sized filamentous ones gradually. With the formation of bacteria-dominated granules, the hydrophobicity and specific gravity of the sludge increased. When the granules were transited to filamentous ones, the hydrophobicity and specific gravity decreased. Both granules had a high COD removal efficiency, excellent settling ability and showed a clear, regular round-shaped outline. After the filamentous granules reached a diameter of 16 mm, due to the mass transfer limitation and the possible presence of anaerobes in the inner part of the granules, they began to disintegrate and be washed out of the reactor, follow by failure of the reactor.

The complete mitochondrial genome of the nematode endoparasitic fungus Hirsutella minnesotensis.

In this study, the complete mitochondrial genome of a nematode fungal pathogen, Hirsutella minnesotensis, is sequenced. The circular mitogenome of H. minnesotensis is 52,245 bp in length and consists of 2 rRNA, 25 tRNA and 14 standard protein-coding genes of the oxidative phosphorylation system as well as four additional free-standing ORFs encoding for an endonuclease or a hypothetical protein. Seven genes (rnl, cob, cox1, nad3, nad4, nad4L and nad5) are invaded by group I or unclassified introns, and these introns carry ORFs of ribosomal protein S3 and GIY-YIG/LAGLIDADG endonucleases or hypothetical proteins. Evidence of intron degeneration is significant for the nad4L intron and cox1-i1 due to unexpected stop codons and/or frame shifting. Phylogenetic analysis based on concatenated protein sequences confirms H. minnesotensis as a member of the fungal order Hypocreales. In this study, we present the complete mitogenome sequence of H. minnesotensis, which is the first complete mitogenome of the family Ophiocordycipitaceae.

The complete mitochondrial genome of the cold-adapted fungus Pseudogymnoascus pannorum syn. Geomyces pannorum.

In this study, the complete mitochondrial genome of a cold-adapted fungus, Pseudogymnoascus pannorum (=Geomyces pannorum), was sequenced. Its mitochondrial genome is 26,918 bp in length and consists of 13 standard protein-coding genes, 2 ribosomal RNA subunits and 27 transfer RNAs. The atp9 gene is absent from the mitochondrial DNA of P. pannorum. The mere intron present in the mitochondrial genome of P. pannorum is found within the rnl gene, and this group-IA intron carries an intronic ORF encoding for ribosomal protein S3. Phylogenetic analysis based on concatenated protein sequences support P. pannorum as a Leotiomycetes species, but its taxonomic resolution at the order level needs to be refined when additional mitochondrial genome data are available.

Same data, different structures: diastereoisomers with substantially identical NMR data from nature.

Fusarins G1/G2 (1/2) and G3/G4 (3/4), two sets of interesting examples of diastereoisomers with substantially identical NMR data, were discovered from natural products. The reason was discussed and the generally applicable determinant conditions were proposed. The minimum interval for stereoclusters to be entirely segregated was also discussed.

Orbilia querci sp. nov. and its knob-forming nematophagous anamorph.

Orbilia querci, a new nematode-trapping fungus, was found on rotten wood of Quercus sp. in Huai-rou County, Beijing, China. It is characterized by having a tear-shaped spore body in the cylindrical ascospore. Pure culture was obtained from the ascospores. Conidiophores were simple or occasionally branched, bearing a single conidium on the tip. Conidia were spindle-shaped, mostly with 3-septa. Nematodes were captured by means of adhesive stalked knobs. The adhesive knobs were produced frequently on nutritional agar plates even in the absence of challenging nematodes. Its anamorph is placed in Dactylellina and named as D. querci. The sequence divergence of the ITS1 region between the fungus and the other knob-forming species tested was 23.8-33.4%, supporting O. querci as a distinct species. This is the first report of the connection between a knob-forming nematophagous hyphomycete and an Orbilia teleomorph.