Metabolic Investigation and Auxiliary Enzyme Modelization of the Pyrrocidine Pathway Allow Rationalization of Paracyclophane-Decahydrofluorene Formation.

Fungal paracyclophane-decahydrofluorene-containing natural products are complex polycyclic metabolites derived from similar hybrid PKS-NRPS pathways. Herein we studied the biosynthesis of pyrrocidines, one representative of this family, by gene inactivation in the producer Sarocladium zeae coupled to thorough metabolic analysis and molecular modeling of key enzymes. We characterized nine pyrrocidines and analogues as well as in mutants a variety of accumulating metabolites with new structures including rare cis-decalin, cytochalasan, and fused 6/15/5 macrocycles. This diversity highlights the extraordinary plasticity of the pyrrocidine biosynthetic gene cluster. From accumulating metabolites, we delineated the scenario of pyrrocidine biosynthesis. The ring A of the decahydrofluorene is installed by PrcB, a membrane-bound cyclizing isomerase, on a PKS-NRPS-derived pyrrolidone precursor. Docking experiments in PrcB allowed us to characterize the active site suggesting a mechanism triggered by arginine-mediated deprotonation at the terminal methyl of the substrate. Next, two integral membrane proteins, PrcD and PrcE, each predicted as a four-helix bundle, perform hydroxylation of the pyrrolidone ring and paracyclophane formation, respectively. Modelization of PrcE highlights a topological homology with vitamin K oxido-reductase and the presence of a disulfide bond. Our results suggest a previously unsuspected coupling mechanism via a transient loss of aromaticity of tyrosine residue to form the strained paracyclophane motif. Finally, the lipocalin-like protein PrcX drives the exo-cycloaddition yielding ring B and C of the decahydrofluorene to afford pyrrocidine A, which is transformed by a reductase PrcI to form pyrrocidine B. These insights will greatly facilitate the microbial production of pyrrocidine analogues by synthetic biology.

Discovery of Enzyme Inhibitors from Mangrove Sediment Derived Fungus Trichoderma harzianum SCSIO 41051.

One new fatty acid derivative, (2E,4E)-6,7-dihydroxy-2-methylocta-2,4-dienoic acid (1), and 16 known compounds (2-17) were isolated from the mangrove sediment derived fungus Trichoderma harzianum SCSIO 41051. Their structures were established by spectroscopic methods, computational ECD, and Mo2(OAc)4-induced ECD experiment. All the compounds were evaluated for their acetylcholinesterase (AChE) and pancreatic lipase (PL) inhibition. Compounds 9 and 14 exhibited moderate AChE inhibitory activities with IC50 values of 2.49 and 2.92 µM, respectively, which compounds 8 and 9 displayed moderate inhibition on PL with IC50 value of 2.30 and 2.34 µM, respectively.

Ochrolines A-C, three new indole diketopiperazines from cultures of endophytic fungi Bionectria ochroleuca SLJB-2.

Three new indole diketopiperazines, ochrolines A-C (1-3), along with three known compounds (4-6), were isolated and identified from the EtOAc extract of the solid fermentation of Bionectria ochroleuca SLJB-2. Notably, compound 1 featured a natural rarely-occurring caged skeleton with a 6/5/6/7 heterotetracyclic bridged ring system. The structures including absolute configurations of 1-3 were fully accomplished by extensive spectroscopic analyses, DFT GIAO 13C NMR and electronic circular dichroism (ECD) calculations. The plausible biogenetic pathways of these new indole diketopiperazines were also proposed. Moreover, the cytotoxic activity screening revealed that compound 2 exhibited moderate inhibitory effect against A549 with inhibition rate of 57.44% at the concentration of 50 µM and compound 1 exhibited mild inhibitory activities against A549, Hela and MCF-7.

A novel aromatic compound from the fungus Synnemellisia sp. FKR-0921.

The filamentous fungus Synnemellisia sp. strain FKR-0921 was obtained from soil collected on Kume Island, Okinawa. The MeOH extract of FKR-0921 cultured on a solid rice medium yielded a new aromatic compound, synnemellisitriol A (1). The structure, including the absolute configuration, was elucidated by spectroscopic analysis (FT-IR, NMR, and HR-ESI-MS), and the absolute configuration at C-9 of 1 was determined using the modified Mosher's method. Additionally, 1 was evaluated for its biological activities, including metallo-ß-lactamase inhibitory activity, type III secretion system inhibitory activity, antimicrobial activity, antimalarial activity, and cytotoxicity.

Metabolomics-Guided Discovery, Isolation, Structure Elucidation, and Bioactivity of Myropeptins C-E from Myrothecium inundatum.

The saprotrophic filamentous fungus Myrothecium inundatum represents a chemically underexplored ascomycete with a high number of putative biosynthetic gene clusters in its genome. Here, we present new linear lipopeptides from nongenetic gene activation experiments using nutrient and salt variations. Metabolomics studies revealed four myropeptins, and structural analyses by NMR, HRMS, Marfey's analysis, and ECD assessment for their helical properties established their absolute configuration. A myropeptin biosynthetic gene cluster in the genome was identified. The myropeptins exhibit general nonspecific toxicity against all cancer cell lines in the NCI-60 panel, larval zebrafish with EC50 concentrations of 5-30 µM, and pathogenic bacteria and fungi (MICs of 4-32 µg/mL against multidrug-resistant S. aureus and C. auris). In vitro hemolysis, cell viability, and ionophore assays indicate that the myropeptins target mitochondrial and cellular membranes, inducing cell depolarization and cell death. The toxic activity is modulated by the length of the lipid side chain, which provides valuable insight into their structure-activity relationships.

Koshidacins A and B, Antiplasmodial Cyclic Tetrapeptides from the Okinawan Fungus Pochonia boninensis FKR-0564.

Two new antiplasmodial peptides, named koshidacins A (1) and B (2), were discovered from the culture broth of the Okinawan fungus Pochonia boninensis FKR-0564. Their structures, including absolute configurations, were elucidated by a combination of spectroscopic methods and chemical derivatization. Both compounds showed moderate in vitro antiplasmodial activity against Plasmodium falciparum strains, with IC50 values ranging from 17.1 to 0.83 µM. In addition, compound 2 suppressed 41% of malaria parasites in vivo when administered intraperitoneally at a dose of 30 mg/kg/day for 4 days.

Tolypocladamides A-G: Cytotoxic Peptaibols from Tolypocladium inflatum.

Seven new peptaibols named tolypocladamides A-G have been isolated from an extract of the fungus Tolypocladium inflatum, which inhibits the interaction between Raf and oncogenic Ras in a cell-based high-throughput screening assay. Each peptaibol contains 11 amino acid residues, an octanoyl or decanoyl fatty acid chain at the N-terminus, and a leucinol moiety at the C-terminus. The peptaibol sequences were elucidated on the basis of 2D NMR and mass spectral fragmentation analyses. Amino acid configurations were determined by advanced Marfey's analyses. Tolypocladamides A-G caused significant inhibition of Ras/Raf interactions with IC50 values ranging from 0.5 to 5.0 µM in a nanobioluminescence resonance energy transfer (NanoBRET) assay; however, no interactions were observed in a surface plasmon resonance assay for binding of the compounds to wild type or G12D mutant Ras constructs or to the Ras binding domain of Raf. NCI 60 cell line testing was also conducted, and little panel selectivity was observed.

Halovirs I-K, antibacterial and cytotoxic lipopeptaibols from the plant pathogenic fungus Paramyrothecium roridum NRRL 2183.

Three new lipopeptaibols, halovirs I-K (1-3), were isolated from the solid culture of the phytopathogenic fungus Paramyrothecium roridum NRRL 2183. Their planar structures, which consist of a hexapeptide backbone and acyl substitutions at the N- and C-termini, were elucidated by comprehensive analysis of the 1D and 2D NMR spectroscopic data along with the detailed interpretation of the MS/MS fragmentation pattern. Absolute configurations of the amino acid/1,2-amino alcohol residues were determined using the advanced Marfey's method. Bioinformatics analysis of the genome assembly of P. roridum NRRL 2183 revealed a gene cluster that is likely responsible for the biosynthesis of halovirs I-K. Analysis of the module and domain organization of the putative halovir synthetase PrHalA indicated that the assembly of 1-3 proceeds in an unconventional nonlinear fashion. 1 and 2 exhibited potent antibacterial activity against both antibiotic-sensitive and multidrug-resistant Gram-positive pathogens. These lipopeptaibols also displayed significant cytotoxicity toward human lung carcinoma A549, human breast carcinoma MCF-7, and human cervical carcinoma HeLa cells with IC50 values ranging from 1.3 to 3.3 µM.

Sesquiterpene and Sorbicillinoid Glycosides from the Endophytic Fungus Trichoderma longibrachiatum EN-586 Derived from the Marine Red Alga Laurencia obtusa.

An unusual sesquiterpene glycoside trichoacorside A (1) and two novel sorbicillinoid glycosides sorbicillisides A (2) and B (3), together with a known compound sorbicillin (4), were isolated and identified from the culture extract of an endophytic fungus Trichoderma longibrachiatum EN-586, obtained from the marine red alga Laurencia obtusa. Trichoacorside A (1) is the first representative of a glucosamine-coupled acorane-type sesquiterpenoid. Their structures were elucidated based on detailed interpretation of NMR and mass spectroscopic data. The absolute configurations were determined by X-ray crystallographic analysis, chemical derivatization, and DP4+ probability analysis. The antimicrobial activities of compounds 1-4 against several human, aquatic, and plant pathogens were evaluated.

New Sorbicillinoids with Tea Pathogenic Fungus Inhibitory Effect from Marine-Derived Fungus Hypocrea jecorina H8.

Four new dimeric sorbicillinoids (1-3 and 5) and a new monomeric sorbicillinoid (4) as well as six known analogs (6-11) were purified from the fungal strain Hypocrea jecorina H8, which was obtained from mangrove sediment, and showed potent inhibitory activity against the tea pathogenic fungus Pestalotiopsis theae (P. theae). The planar structures of 1-5 were assigned by analyses of their UV, IR, HR-ESI-MS, and NMR spectroscopic data. All the compounds were evaluated for growth inhibition of tea pathogenic fungus P. theae. Compounds 5, 6, 8, 9, and 10 exhibited more potent inhibitory activities compared with the positive control hexaconazole with an ED50 of 24.25 ± 1.57 µg/mL. The ED50 values of compounds 5, 6, 8, 9, and 10 were 9.13 ± 1.25, 2.04 ± 1.24, 18.22 ± 1.29, 1.83 ± 1.37, and 4.68 ± 1.44 µg/mL, respectively. Additionally, the effects of these compounds on zebrafish embryo development were also evaluated. Except for compounds 5 and 8, which imparted toxic effects on zebrafish even at 0.625 µM, the other isolated compounds did not exhibit significant toxicity to zebrafish eggs, embryos, or larvae. Taken together, sorbicillinoid derivatives (6, 9, and 10) from H. jecorina H8 displayed low toxicity and high anti-tea pathogenic fungus potential.

Shikinefragalides A-D, new tricyclic macrolides produced by Stachybotryaceae sp. FKI-9632.

Four new tricyclic macrolides, named shikinefragalides A (1), B (2), C (3) and D (4), were isolated by physicochemical (PC) screening from a static culture material of Stachybotryaceae sp. FKI-9632. Their structures were elucidated as new analogs of colletofragarones by MS and NMR analyses. Compounds 1 and 2 showed weak antimalarial activity and cytotoxicity.

Xanthone glucoside from an insect pathogenic fungus Conoideocrella luteorostrata NBRC106950.

A new compound, 3-O-(4-O-methyl-ß-D-glucopyranosyl) xanthone (1) was isolated from the culture of Conoideocrella luteorostrata NBRC106950. The structure of 1 was mainly determined by 1H, 13C, 2D-NMR and HREIMS spectral analyses. The absolute configuration of 4-O-methylglucopyranosyl moiety was determined by the optical rotation of aqueous layer of hydrolyzed 1 as D-configuration.

Nigirpexin E, a new azaphilone derivative with anti-tobacco mosaic virus activity from soil-derived fungus Trichoderma afroharzianum LTR-2.

A new compound classified as one new azaphilone derivative, nigirpexin E (1), was obtained from the soil-derived fungus Trichoderma afroharzianum LTR-2, together with seven known compounds (2-8). The structures of 1-8 were determined by their HRESIMS, optical rotation, and NMR spectroscopic data. The absolute configuration of nigirpexin E (1) was determined on the basis of comparisons of experimental and theoretically calculated ECD spectra. Compound 3 was firstly isolated from Trichoderma. Bioactivities of the isolated compounds were assayed their anti-tobacco mosaic virus (anti-TMV) activities. The results showed that compound 1 exhibited significant inactivation effect against TMV with an inhibition rate of 67.25% (0.5 mg ml-1), which was higher than that of positive control ribavirin (56.74%). This is the first report of the anti-TMV activity of azaphilone derivatives.

Diversification of ergot alkaloids and heritable fungal symbionts in morning glories.

Heritable microorganisms play critical roles in life cycles of many macro-organisms but their prevalence and functional roles are unknown for most plants. Bioactive ergot alkaloids produced by heritable Periglandula fungi occur in some morning glories (Convolvulaceae), similar to ergot alkaloids in grasses infected with related fungi. Ergot alkaloids have been of longstanding interest given their toxic effects, psychoactive properties, and medical applications. Here we show that ergot alkaloids are concentrated in four morning glory clades exhibiting differences in alkaloid profiles and are more prevalent in species with larger seeds than those with smaller seeds. Further, we found a phylogenetically-independent, positive correlation between seed mass and alkaloid concentrations in symbiotic species. Our findings suggest that heritable symbiosis has diversified among particular clades by vertical transmission through seeds combined with host speciation, and that ergot alkaloids are particularly beneficial to species with larger seeds. Our results are consistent with the defensive symbiosis hypothesis where bioactive ergot alkaloids from Periglandula symbionts protect seeds and seedlings from natural enemies, and provide a framework for exploring microbial chemistry in other plant-microbe interactions.

Luteapyrone, a Novel Æ´-Pyrone Isolated from the Filamentous Fungus Metapochonia lutea.

In the process of screening for new bioactive microbial metabolites we found a novel Æ´-pyrone derivative for which we propose the trivial name luteapyrone, in a recently described microscopic filamentous fungus, Metapochonia lutea BiMM-F96/DF4. The compound was isolated from the culture extract of the fungus grown on modified yeast extract sucrose medium by means of flash chromatography followed by preparative HPLC. The chemical structure was elucidated by NMR and LC-MS. The new compound was found to be non-cytotoxic against three mammalian cell lines (HEK 263, KB-3.1 and Caco-2). Similarly, no antimicrobial activity was observed in tested microorganisms (gram positive and negative bacteria, yeast and fungi).

Targeted Isolation of a Cytotoxic Cyclic Hexadepsipeptide from the Mesophotic Zone Sponge-Associated Fungus Cymostachys sp. NBUF082.

LC-MS/MS-based molecular networking facilitated the targeted isolation of a new cyclic hexadepsipeptide, cymodepsipeptide (1), and two known analogues, RF-2691A (2) and RF-2691B (3), from the fungus Cymostachys sp. NBUF082 that was derived from a mesophotic zone Aaptos sponge collected near Apo Island. The constitution and configuration of 1 was elucidated through 1D and 2D NMR-spectroscopy, high resolution mass-spectrometry, and chemical degradations including Marfey's analysis and chiral HPLC. It was observed that 1 was moderately cytotoxic against CCRF-CEM human acute lymphocytic leukemia cells in vitro with the IC50 value of 9.2 ± 1.1 µM.

Cochlioquinone derivatives produced by coculture of endophytes, Clonostachys rosea and Nectria pseudotrichia.

Three new meroterpenoid derivatives, furanocochlioquinol (1) and furanocochlioquinone (2), as well as nectrianolin D (3), together with two known biogenetically related compounds 4 and 5 were isolated from a mixed culture of two mangrove-derived fungi, Clonostachys rosea B5-2 and Nectria pseudotrichia B69-1. The structures of 1-3 were deduced based on the interpretation of HRMS and NMR data. Compounds 1-5 exhibited cytotoxicity against human promyelocytic leukemia (HL60) cells with IC50 values ranging from 0.47 to 10.16 µM.

Sesquicillin F, a new insecticidal meroterpenoid produced by Mariannaea macrochlamydospora FKI-4735.

A new insecticidal meroterpenoid, named sesquicillin F (1), has been isolated from a culture broth of Mariannaea macrochlamydospora FKI-4735, together with 4-hydroxy-5,6-dimethylpyran-2-one (2). Compounds 1 and 2 were insecticidally active against Halyomorpha halys at 1 ppm.

Indole Alkaloids from a Soil-Derived Clonostachys rosea.

Clonorosins A (1) and B (2), two novel indole alkaloids featuring unprecedented 6/5/6/6/5 and 6/5/5 cores, together with seven known indole-linked 2,5-diketopiperazine alkaloids (3-9), were isolated from the soil-derived fungus Clonostachys rosea YRS-06. The new structures were proposed through HR-MS, NMR, and ECD spectroscopic data. They were established by comparing the calculated NMR, ECD, and specific rotation data with the experimental. To assist in determining the absolute configuration of the chiral carbon in the side chain of 2,5-diketopiperazine derivatives, flexible analogues 3i-3iv were synthesized and analyzed. 1 was active against Fusarium oxysporum with an MIC value of 50 µg/mL. 7 and 8 showed excellent activity against human HeLa and HepG2 cells with IC50 values of 0.12-0.60 µM.

Bisabolane, cadinane, and cyclonerane sesquiterpenes from an algicolous strain of Trichoderma asperelloides.

Ten new bisabolane derivatives, trichobisabolins Q-Z (1-10), one new cadinane derivative, cadin-4-en-11-ol (11), and three new cyclonerane derivatives, cycloner-3-en-7,11-diol (12), isoepicyclonerodiol oxide (13), and norepicyclonerodiol oxide (14), were isolated from the endophytic fungal strain RR-dl-6-11 of Trichoderma asperelloides that was obtained from a marine alga. Their structures along with relative configurations were established mainly by NMR and IR as well as MS techniques, and the absolute configurations of 10 and 11 were assigned by ECD and X-ray diffraction data, respectively. Sesquiterpenes from the fungus T. asperelloides are reported for the first time. It is interesting that half of the bisabolane derivatives are demethylated. Compound 12 represents the first the occurrence of cyclopentenyl-bearing cycloneranes, and 14 seems a cyclopentyl-degrading cyclonerane derivative. Several isolates feature potent inhibition of marine phytoplankton species.