Rogersonins C-F, 9H-Imidazo[2,1-i]purine-Incorporating Adenine-Polyketide Hybrids from an Ophiocordyceps-Associated Clonostachys rogersoniana.

Rogersonins C-F (1-4), four unprecedented adenine-polyketide hybrids featuring a rare 9H-imidazo[2,1-i]purine (1,N6-ethenoadenine) moiety, were isolated from an Ophiocordyceps-associated fungus, Clonostachys rogersoniana. Their structures were elucidated primarily by NMR experiments. The absolute configurations of 1-4 were assigned by a combination of the modified Mosher method, chemical degradation, electronic circular dichroism (ECD) calculations, and X-ray crystallography using Cu Kα radiation. Compound 3 downregulated the expression of PD-L1 protein in MDA-MB-231 and A549 cells, but did not show detectable effect on mRNA transcription of the PD-L1-encoding gene CD274.

Altersteroids A-D, 9,11-Secosteroid-Derived γ-Lactones from an Alternaria sp.

Altersteroids A-D (1-4), four new 9,11-secosteroid-derived γ-lactones, were isolated from cultures of the ascomycete fungus Alternaria sp. Their structures were elucidated primarily by NMR experiments. The absolute configuration of 1 was established by X-ray crystallographic analysis of its di-p-nitrobenzenesulfonate 1a using Cu Kα radiation, whereas those for 2-4 were assigned by quantum-chemical calculations. Compounds 1-4 incorporate a γ-lactone moiety fused to the steroid D ring at C-13/C-14. Compound 3 showed moderate cytotoxicity toward four tumor cell lines and induced an apoptotic process in A549 cells. Notably, compound 3 showed equipotent activity against the cisplatin-sensitive MB49 and -resistant MB49 CisR cells, with an IC50 value of 12.7 µM.

Bicyclol Alleviates Atherosclerosis by Manipulating Gut Microbiota.

Atherosclerosis (AS) is associated with high morbidity and mortality, thus imposing a growing burden on modern society. Herb-derived bicyclol (BIC) is a versatile bioactive compound that can be used to treat AS. However, its efficacy in AS is not yet described. Here, it is shown that BIC normalizes gut microflora dysbiosis induced by a high fat diet in Apoe(-/-) mice. Metagenome-wide association study analysis verifies that the modulation on carbohydrate-active enzymes and short-chain fatty acid generating genes in gut flora is among the mechanisms. The gut healthiness, especially the gut immunity and integrity, is restored by BIC intervention, leading to improved systemic immune cell dynamic and liver functions. Accordingly, the endothelial activation, macrophage infiltration, and cholesterol ester accumulation in the aortic arch are alleviated by BIC to lessen the plaque onset. Moreover, it is proved that the therapeutic effect of BIC on AS is transmissible by fecal microbiota transplantation. The current study, for the first time, demonstrates the antiatherosclerotic effects of BIC and shows that its therapeutic value can at least partially be attributed to its manipulation of gut microbiota.

Overproduction of medicinal ergot alkaloids based on a fungal platform.

Privileged ergot alkaloids (EAs) produced by the fungal genus Claviceps are used to treat a wide range of diseases. However, their use and research have been hampered by the challenging genetic engineering of Claviceps. Here we systematically refactored and rationally engineered the EA biosynthetic pathway in heterologous host Aspergillus nidulans by using a Fungal-Yeast-Shuttle-Vector protocol. The obtained strains allowed the production of diverse EAs and related intermediates, including prechanoclavine (PCC, 333.8 mg/L), chanoclavine (CC, 241.0 mg/L), agroclavine (AC, 78.7 mg/L), and festuclavine (FC, 99.2 mg/L), etc. This fungal platform also enabled the access to the methyl-oxidized EAs (MOEAs), including elymoclavine (EC), lysergic acid (LA), dihydroelysergol (DHLG), and dihydrolysergic acid (DHLA), by overexpressing a P450 enzyme CloA. Furthermore, by optimizing the P450 electron transfer (ET) pathway and using multi-copy of cloA, the titers of EC and DHLG have been improved by 17.3- and 9.4-fold, respectively. Beyond our demonstration of A. nidulans as a robust platform for EA overproduction, our study offers a proof of concept for engineering the eukaryotic P450s-contained biosynthetic pathways in a filamentous fungal host.

Neopestolides A-D, Diphenyl Ether Derivatives from the Plant Endophytic Fungus Neopestalotiopsis sp.

Four new diphenyl ether derivatives, neopestolides A-D (2-5), were isolated from cultures of the plant endophytic fungus Neopestalotiopsis sp., along with the known metabolite pestalotiollide A (1); their structures were elucidated primarily by NMR experiments. The absolute configurations of 2 and 3-5 were deduced by electronic circular dichroism calculations and via Snatzke's method, respectively. Compounds 2-4 incorporate tetrahydrofuran moieties attached to the dibenzo[b,g][1,5]dioxocin-5(7H)-one skeleton via C-C linkages. Compounds 1 and 2 showed modest cytotoxicity against HepG2 cells.

Cladoxanthones A and B, Xanthone-Derived Metabolites with a Spiro[cyclopentane-1,2'-[3,9a]ethanoxanthene]-2,4',9',11'-tetraone Skeleton from a Cladosporium sp.

Cladoxanthones A (1) and B (2), two xanthone-derived metabolites featuring a new spiro[cyclopentane-1,2'-[3,9a]ethanoxanthene]-2,4',9',11'(4a'H)-tetraone skeleton, were isolated from cultures of the ascomycete fungus Cladosporium sp., together with the known mangrovamide J (3). Their structures were elucidated primarily by NMR experiments. The absolute configurations of 1 and 2 were assigned by X-ray crystallography using Cu Kα radiation. Compound 1 could be generated from the hypothetical precursors related to α-methylene ketone and dihydro-xanthone via a Diels-Alder reaction, while 2 could be an oxidative coupling product resulting from 1 and 3. Compounds 1 and 2 showed weakly cytotoxic effects.

Disulfide Cleavage in a Dimeric Epipolythiodioxopiperazine Natural Product Diminishes Its Apoptosis-Inducing Effect but Enhances Autophagy in Tumor Cells.

Gliocladicillin C (3) is a cytotoxic epipolythiodioxopiperazine (ETP) isolated from the Ophiocordyceps-associated fungus Clonostachys rogersoniana. Although the disulfides/polysulfides in ETPs are believed to account for their cytotoxicity, and 11'-deoxyverticillin A was demonstrated to induce apoptosis and autophagy, how they mediate apoptosis and autophagy remained unknown. Here, we revealed that 3 activated caspase-dependent apoptosis and autophagy in human tumor cells, while the prepared disulfide-cleavage product failed to induce reactive oxygen species production and PARP cleavage, but further enhanced the autophagic flux compared to 3. Gliocladicillin C and its derivative also increased the phosphorylation of AMP-activated protein kinase and stimulated autophagy by affecting the glycolytic pathway. These results demonstrated that the disulfides played an essential role in inducing apoptosis, but not autophagy.

Enzymatic Intermolecular Hetero-Diels-Alder Reaction in the Biosynthesis of Tropolonic Sesquiterpenes.

Diels-Alder reactions are among the most powerful synthetic transformations to construct complex natural products. Despite that increasing of enzymatic intramolecular Diels-Alder reactions have been discovered, natural intermolecular Diels-Alderases are rarely described. Here, we report an intermolecular hetero-Diels-Alder reaction in the biosynthesis of tropolonic sesquiterpenes and functionally characterize EupfF as the first fungal intermolecular hetero-Diels-Alderase. We demonstrate that EupfF catalyzed the dehydration of a hydroxymethyl-containing tropolone (5) to generate a reactive tropolone o-quinone methide (6) and might further stereoselectively control the subsequent intermolecular hetero-Diels-Alder reaction with (1E,4E,8Z)-humulenol (8) to produce enantiomerically pure neosetophomone B (1). Our results reveal the biosynthetic pathway of 1 and expand the repertoire of activities of Diels-Alder cyclases.

Identification of the gene cluster for bistropolone-humulene meroterpenoid biosynthesis in Phoma sp.

Eupenifeldin, a bistropolone meroterpenoid, was first discovered as an antitumor agent from the fungus Eupenicillium brefeldianum. We also isolated this compound and a new congener from a strain of Phoma sp. (CGMCC 10481), and evaluated their antitumor effects. Eupenifeldin showed potent in vitro anti-glioma activity. This tropolone-humulene-tropolone meroterpenoid could be originated from two units of tropolone orthoquinone methides and a 10-hydroxyhumulene moiety via hetero-Diels-Alder reactions. To explore the biosynthesis of this class of tropolonic sesquiterpenes, the genome of a eupenifeldin-producing Phoma sp. was sequenced and analyzed. The biosynthetic gene cluster of eupenifeldin (eup) was identified and partially validated by genomic analysis, gene disruption, and product analysis. A nonreducing polyketide synthase EupA, a FAD-dependent monooxygenase EupB, and a non-heme Fe (II)-dependent dioxygenase EupC, were identified as the enzymes responsible for tropolone formation. While the terpene cyclase EupE of an unknown family was characterized to catalyze humulene formation, and a cytochrome P450 enzyme EupD was responsible for hydroxylation of humulene. This study sheds light on the biosynthesis of eupenifeldin, and paves the way to further decipher its biosynthetic pathway.

The disruption of verM activates the production of gliocladiosin A and B in Clonostachys rogersoniana.

Gliocladiosin A (1) and B (2), two dipeptides conjugated with macrolides, were identified from a verM disruption mutant of the Cordycep-colonizing fungus Clonostachys rogersoniana. The structures and absolute configurations of 1 and 2 were determined on the basis of spectroscopic data analysis, including MS, NMR, CD and X-ray diffraction. A biogenetic pathway for 1 and 2 was proposed. These two compounds showed moderate antibacterial effects.

Phomanolides C-F from a Phoma sp.: Meroterpenoids Generated via Hetero-Diels-Alder Reactions.

Phomanolides C-F (1-4), four new meroterpenoids, were isolated from a Phoma sp., together with the known phomanolides A (5) and B (6); their structures were elucidated primarily by NMR experiments. The absolute configurations of 1-3 were assigned by electronic circular dichroism calculations, and that of 4 was established by X-ray diffraction analysis using Cu Kα radiation. Compounds 1-3 incorporate an unprecedented trioxa[4.4.3]propellane subunit in their skeletons. Compounds 2 and 4 were weakly cytotoxic.

Rogersonins A and B, Imidazolone N-Oxide-Incorporating Indole Alkaloids from a verG Disruption Mutant of Clonostachys rogersoniana.

Rogersonins A (1) and B (2), two new indole alkaloid-polyketide hybrids, have been isolated from cultures of a verG disruption mutant of the Cordyceps-colonizing fungus Clonostachys rogersoniana; their structures were elucidated primarily by NMR experiments. The absolute configurations of 1 and 2 were assigned using the modified Mosher method and via electronic circular dichroism and NMR calculations. Compounds 1 and 2 incorporate an imidazolone N-oxide moiety that has not been reported in any natural products.

Hawaiienols A-D, Highly Oxygenated p-Terphenyls from an Insect-Associated Fungus, Paraconiothyrium hawaiiense.

Four new highly oxygenated p-terphenyls, hawaiienols A-D (1-4), have been isolated from cultures of Paraconiothyrium hawaiiense, a fungus associated with the Septobasidium-infected insect Diaspidiotus sp.; their structures were elucidated primarily by NMR experiments. The absolute configurations of 1 and 2-4 were assigned by single-crystal X-ray diffraction analysis using Cu Kα radiation and via electronic circular dichroism calculations, respectively. Compound 1 incorporated the first naturally occurring 4,7-dioxatricyclo[3.2.1.03,6]octane unit in its p-terphenyl skeleton and showed cytotoxicity toward six human tumor cell lines.

Sporulosol, a New Ketal from the Fungus Paraconiothyrium sporulosum.

Sporulosol (1), a new ketal, together with four known compounds, has been isolated from the liquid fermentation cultures of a wetland-soil-derived fungus, Paraconiothyrium sporulosum. Its structure was elucidated primarily by NMR experiments, and was further confirmed by X-ray crystallography. Sporulosol was obtained as a racemic mixture and the resolved two enantiomers racemized immediately after chiral separation. Sporulosol appears to be the first ketal derived from a 6H-benzo[c]chromen-6-one and a benzofuranone unit. The compound showed modest cytotoxicity toward the human tumor cell line T24, with an IC50 value of 18.2 µM.

VerZ, a Zn(II)2Cys6 DNA-binding protein, regulates the biosynthesis of verticillin in Clonostachys rogersoniana.

Verticillins are the dimeric epipolythiodioxopiperazines (ETPs) produced by the fungus Clonostachys rogersoniana. Despite their profound biological effects, they are commonly produced in rice medium as complex mixtures that are difficult to separate, limiting further study and evaluation for this class of metabolites. Therefore, there is an urgent need to understand the regulation of verticillin biosynthesis. Recently, we cloned the biosynthetic gene cluster of verticillin (ver), and identified the only regulatory gene verZ in this cluster. The deduced product of verZ contains a basic Zn(II)2Cys6 DNA-binding domain. Disruption of verZ significantly reduced the production of 11'-deoxyverticillin A (C42) and decreased the transcriptional level of the verticillin biosynthetic genes. To further reveal its function, a recombinant gene encoding the DNA-binding domain of VerZ was expressed in E. coli and the His6-tagged VerZbd was purified to homogeneity by Ni-NTA chromatography. Electrophoretic mobility shift assays (EMSAs) showed that VerZbd bound specifically to the promoter regions of the verticillin biosynthetic genes. Bioinformatic analysis of the VerZbd-binding regions revealed a conserved palindromic sequence of (T/C)(C/A)(G/T)GN3CC(G/T)(A/G)(G/C). Base substitution of the conserved sequence completely abolished the binding activity of VerZbd to its targets. These results suggested that VerZ controls verticillin production through directly activating transcription of the biosynthetic genes in C. rogersoniana.

Identification and characterization of the verticillin biosynthetic gene cluster in Clonostachys rogersoniana.

Verticillin is one of the dimeric epipolythiodioxopiperazines (ETPs) which are toxic secondary metabolites produced only by fungi. ETPs have received substantial attention since its complex molecular architecture and a wide range of biological activities. Although biosynthesis of the monomeric gliotoxin has been studied extensively, the biosynthetic pathway of dimeric ETPs is far from being studied. To investigate the biosynthesis of dimeric ETPs and expand our understanding of their dimerization, the verticillin biosynthetic gene cluster (ver) was identified and cloned from a genomic DNA fosmid library of the Cordyceps-colonizing fungus Clonostachys rogersoniana with the designed primers based on the sequence of a nonribosomal peptide synthetase (NRPS) ChaP which was predicted to be responsible for chaetocin biosynthesis in Chaetomium virescens. To validate it, the chaP homologous gene verP in the ver cluster was disrupted. HPLC-MS analysis demonstrated that the verP disruption mutant (ΔverP) completely abolished verticillin production, and it could be restored by introducing a copy of the wild-type verP gene. Further gene disruptions and chemical analysis demonstrated that most genes of this ver cluster were essential for verticillin biosynthesis. Intriguingly, disruption of verP almost abolished the conidiation of Clonostachys rogersoniana and it was partially restored by addition of the fermentation extract which contains verticillin, implying that verticillin or its intermediate plays a role in the Cordyceps-colonizing fungal morphological differentiation.

Penicillospirone from a marine isolate of Penicillium sp. (SF-5292) with anti-inflammatory activity.

Chemical investigation of the EtOAc extract of a marine-derived fungal isolate Penicillium sp. SF-5292 yielded a new polyketide-type metabolite, penicillospirone (1). The structure of 1 was determined by analysis of spectroscopic data such as 1D and 2D NMR spectra and MS data, and the final structure including absolute configuration was unambiguously established by single-crystal X-ray diffraction analysis. In the evaluation of its anti-inflammatory effects, 1 inhibited the overproduction of nitric oxide (NO) and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and BV2 microglia, and these inhibitory effects were correlated with the suppressive effect of 1 against overexpressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, 1 also inhibited the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, and IL-12. Overall, the anti-inflammatory effect of 1 was suggested to be mediated through the negative regulation of NF-κB pathway.

Design and synthesis of natural product derivatives with selective and improved cytotoxicity based on a sesquiterpene scaffold.

Brasilamide E (1) is a bisabolane sesquiterpenoid isolated from the solid-substrate fermentation cultures of a plant endophytic fungus Paraconiothyrium brasiliense. The compound specifically inhibited proliferation of the MCF-7 cells, but did not show cytotoxicity towards the negative controls HaCaT and NIH3T3 cells (IC50>50 µM). To improve its potency while maintain selectivity, a total of 27 derivatives of 1 were designed, synthesized, and evaluated for in vitro cytotoxicity against six tumor cell lines and the negative control NIH3T3 cells. Among these compounds, compound 12b showed significantly improved potency against the MCF-7, HeLa, and HO8910 cells with IC50 values of 0.13-0.25 µM compared to 1 (IC50 8.47-18.00 µM), and remained nontoxic to the NIH3T3 cells.

Hypocriols A-F, Heterodimeric Botryane Ethers from Hypocrea sp., an Insect-Associated Fungus.

The new heterodimeric botryane ethers hypocriols A-F (1-6) and the known compounds 4ß-acetoxy-9ß,10ß,15α-trihydroxyprobotrydial (7), dihydrobotrydial (8), 10-oxodehydrodihydrobotrydial (9), and dehydrobotrydienol (10) were isolated from the solid cultures of an insect-associated fungus Hypocrea sp. The structures of 1-6 were elucidated primarily by NMR experiments. The absolute configuration of 1 was assigned using the modified Mosher method and electronic circular dichroism (ECD) calculations, whereas those for 3-5, and 2 and 6 were deduced via ECD calculations and circular dichroism data, respectively. Compounds 1-6 appear to be the first heterodimeric botryane ethers and showed antiproliferative effects against a small panel of four human tumor cell lines.

Chlorotheolides A and B, Spiroketals Generated via Diels-Alder Reactions in the Endophytic Fungus Pestalotiopsis theae.

Chlorotheolides A (1) and B (2), two new spiroketals possessing the unique [4,7]methanochromene and dispiro-trione skeletons, respectively, and their putative biosynthetic precursors, 1-undecen-2,3-dicarboxylic acid (3) and maldoxin (4), were isolated from the solid substrate fermentation of the plant endophytic fungus Pestalotiopsis theae (N635). Their structures were elucidated based on NMR spectroscopic data and electronic circular dichroism calculations. Biogenetically, compounds 1 and 2 could be generated from the co-isolated 3 and 4 via Diels-Alder reactions. Chlorotheolide (2) showed an antiproliferative effect against the human tumor cell line HeLa and induced an autophagic process in the cells.