Amaurones A-K: Polyketides from the Fish Gut-Derived Fungus Amauroascus sp. CMB-F713.

Using a molecular networking guided strategy, chemical analysis of the Australian mullet fish gastrointestinal tract-derived fungus Amauroascus sp. CMB-F713 yielded a family of polyketide pyrones, amaurones A-I (1-9), featuring an unprecedented carbon skeleton. Structures were assigned to 1-9 by detailed spectroscopic analysis (including X-ray analysis of 1), biosynthetic considerations, and chemical interconversions. For example, the orthoacetate 5 was unstable when stored dry at room temperature, transforming to the monoacetates 2 and 3, while mild heating (40 °C) prompted quantitative conversion of 3 to 2, via an intramolecular trans-acetylation. Likewise, during handling, the monoacetate 1 was prone to intramolecular trans-acetylation, leading to an equilibrium mixture with the isomeric monoacetate amaurone J (10), confirmed when partial hydrolysis of the diacetate 2 yielded the monoacetates 1 and 10 and the triol amaurone K (11).

α-Glucosidase and Protein Tyrosine Phosphatase 1B Inhibitors from Malbranchea circinata.

During a search for new α-glucosidase and protein tyrosine phosphatase 1B inhibitors from fungal sources, eight new secondary metabolites, including two anthranilic acid-derived peptides (1 and 2), four glycosylated anthraquinones (3-6), 4-isoprenylravenelin (7), and a dimer of 5,8-dihydroxy-4-methoxy-α-tetralone (8), along with four known compounds (9-12), were isolated from solid rice-based cultures of Malbranchea circinata. The structural elucidation of these metabolites was performed using 1D and 2D NMR techniques and DFT-calculated chemical shifts. Compounds 1-3, 9, and 10 showed inhibitory activity to yeast α-glucosidase (αGHY), with IC50 values ranging from 57.4 to 261.3 µM (IC50 acarbose = 585.8 µM). The effect of 10 (10.0 mg/kg) was corroborated in vivo using a sucrose tolerance test in normoglucemic mice. The most active compounds against PTP-1B were 8-10, with IC50 values from 10.9 to 15.3 µM (IC50 ursolic acid = 27.8 µM). Docking analysis of the active compounds into the crystal structures of αGHY and PTP-1B predicted that all compounds bind to the catalytic domains of the enzymes. Together, these results showed that M. circinata is a potential source of antidiabetic drug leads.

Specific Xylan Activity Revealed for AA9 Lytic Polysaccharide Monooxygenases of the Thermophilic Fungus Malbranchea cinnamomea by Functional Characterization.

The thermophilic biomass-degrader Malbranchea cinnamomea exhibits poor growth on cellulose but excellent growth on hemicelluloses as the sole carbon source. This is surprising considering that its genome encodes eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), enzymes known for their high potential in accelerating cellulose depolymerization. We characterized four of the eight (M. cinnamomea AA9s) McAA9s, namely, McAA9A, McAA9B, McAA9F, and McAA9H, to gain a deeper understanding about their roles in the fungus. The characterized McAA9s were active on hemicelluloses, including xylan, glucomannan, and xyloglucan, and furthermore, in accordance with transcriptomics data, differed in substrate specificity. Of the McAA9s, McAA9H is unique, as it preferentially cleaves residual xylan in phosphoric acid-swollen cellulose (PASC). Moreover, when exposed to cellulose-xylan blends, McAA9H shows a preference for xylan and for releasing (oxidized) xylooligosaccharides. The cellulose dependence of the xylan activity suggests that a flat conformation, with rigidity similar to that of cellulose microfibrils, is a prerequisite for productive interaction between xylan and the catalytic surface of the LPMO. McAA9H showed a similar trend on xyloglucan, underpinning the suggestion that LPMO activity on hemicelluloses strongly depends on the polymers' physicochemical context and conformation. Our results support the notion that LPMO multiplicity in fungal genomes relates to the large variety of copolymeric polysaccharide arrangements occurring in the plant cell wall.IMPORTANCE The Malbranchea cinnamomea LPMOs (McAA9s) showed activity on a broad range of soluble and insoluble substrates, suggesting their involvement in various steps of biomass degradation besides cellulose decomposition. Our results indicate that the fungal AA9 family is more diverse than originally thought and able to degrade almost any kind of plant cell wall polysaccharide. The discovery of an AA9 that preferentially cleaves xylan enhances our understanding of the physiological roles of LPMOs and enables the use of xylan-specific LPMOs in future applications.

Secondary Metabolites of Onygenales Fungi Exemplified by Aioliomyces pyridodomos.

Fungi from the order Onygenales include human pathogens. Although secondary metabolites are critical for pathogenic interactions, relatively little is known about Onygenales compounds. Here, we use chemical and genetic methods on Aioliomyces pyridodomos, the first representative of a candidate new family within Onygenales. We isolated 14 new bioactive metabolites, nine of which are first disclosed here. Thirty-two specialized metabolite biosynthetic gene clusters (BGCs) were identified. BGCs were correlated to some of the new compounds by heterologous expression of biosynthetic genes. Some of the compounds were found after one year of fermentation. By comparing BGCs from A. pyridodomos with those from 68 previously sequenced Onygenales fungi, we delineate a large biosynthetic potential. Most of these biosynthetic pathways are specific to Onygenales fungi and have not been found elsewhere. Family level specificity and conservation of biosynthetic gene content are evident within Onygenales. Identification of these compounds may be important to understanding pathogenic interactions.

Spiromastilactones: A new class of influenza virus inhibitors from deep-sea fungus.

A new class of phenolic lactones with trivial names of spiromastilactones A-M (1-13) was isolated from a deep-sea derived Spiromastix sp. fungus. Their structures featured by various chlorination at aromatic rings were determined on the basis of extensive spectroscopic analyses. An antiviral assay revealed that most of the tested compounds exert inhibitory activity against WSN influenza virus with low cytotoxicity, while the structure-activity relationships were discussed. Spiromastilactone D (4), a 5'-chloro-2'-methoxy substituted analogue, displayed the most potent to inhibit a panel of influenza A and B viruses in addition to drug-resistant clinical isolates. Mechanistic investigation resulted in that compound 4 bonded to hemagglutinin protein (HA), potentially at the spherical head, and disrupted the HA-sialic acid receptor interaction, that is essential for the attachment and entry of all influenza viruses. In addition, compound 4 also showed inhibitory effect toward viral genome replication via targeting viral RNP complex. The synergistic effects of 4 on both viral entry and replication assumed it to be a promising lead for the development of a new influenza inhibitor.

Utilizing DART mass spectrometry to pinpoint halogenated metabolites from a marine invertebrate-derived fungus.

Prenylated indole alkaloids are a diverse group of fungal secondary metabolites and represent an important biosynthetic class. In this study we have identified new halogenated prenyl-indole alkaloids from an invertebrate-derived Malbranchea graminicola strain. Using direct analysis in real time (DART) mass spectrometry, these compounds were initially detected from hyphae of the fungus grown on agar plates, without the need for any organic extraction. Subsequently, the metabolites were isolated from liquid culture in artificial seawater. The structures of two novel chlorinated metabolites, named (-)-spiromalbramide and (+)-isomalbrancheamide B, provide additional insights into the assembly of the malbrancheamide compound family. Remarkably, two new brominated analogues, (+)-malbrancheamide C and (+)-isomalbrancheamide C, were produced by enriching the growth medium with bromine salts.

Premalbrancheamide: synthesis, isotopic labeling, biosynthetic incorporation, and detection in cultures of Malbranchea aurantiaca.

An advanced metabolite, named premalbrancheamide, involved in the biosynthesis of malbrancheamide (1) and malbrancheamide B (2) has been synthesized in double (13)C-labeled form and was incorporated into the indole alkaloid 2 by Malbranchea aurantiaca. In addition, premalbrancheamide has been detected as a natural metabolite in cultures of M. aurantiaca. The biosynthetic implications of these experiments are discussed.

The effect of halide salts on the production of Gymnoascus reessii polyenylpyrroles.

Addition of NaCl to the solid-phase fermentation of an Australian isolate of Gymnoascus reessii resulted in enhanced production of chloropolyenylpyrroles, while the addition of NaBr suppressed chloropolyenylpyrrole production in favor of bromo and dechloro analogues. Access to a wider selection of polyenylpyrroles provided scope for SAR comparisons on this rare class of cytotoxic natural products, with the bromo- and dechloropolyenylpyrroles displaying significantly reduced cytotoxicity. These results suggest that the chloro substituent is a critical element in the pharmacophore for this rare class of natural product.

4-benzyl-3-phenyl-5H-furan-2-one, a vasodilator isolated from Malbranchea filamentosa IFM 41300.

Screening of Malbranchea filamentosa IFM 41300 for bioactive compounds led to the identification of 4-benzyl-3-phenyl-5H-furan-2-one (1) as a vasodilator and erythroglaucin (2). The structure of 1 was established on the basis of spectroscopic and chemical investigations. Compound 1 inhibited Ca2+-induced vasocintraction in aortic rings pretreated with high K+ (60mM) or norepinephrine. Finally, compound 1 did not exhibit activity against human pathogenic microorganisms.

alpha-galf I -->6-alpha-mannopyranoside side chains in Paracoccidioides brasiliensis cell wall are shared by members of the Onygenales, but not by galactomannans of other fungal genera.

The water-soluble polysaccharide fraction of the cell wall alkali extract (F1SS) from the mycelial phase of the dimorphic fungus Paracoccidioides brasiliensis is compared with F1SS polysaccharides obtained from the Onygenalean mycelial fungi Ascocalvatia alveolata, Onygena equina and Aphanoascus terreus. These polymers were exclusively composed of mannose and galactose. Data from methylation and NMR analyses reveal that F1SS polysaccharides from the four fungi contain the same residues although in different proportions: [-->2,6)-alpha-D-Manp-(1 -->]; [2)-alpha-D-Manp-(1 -->]; [ -->6)-alpha-D-Manp-(1 -->]; and [alpha-D-Galf-(1 -->]. In P. brasiliensis, the repeating unit of the polysaccharide consists of a backbone of [(1 -->6)-alpha-D-Manp] substituted at the 0-2 position by the disaccharide [alpha-D-Galf-(1 -->6)-alpha-D-Manp-(1 -->], while the remaining 0-2 positions are substituted by single residues of mannose or short chains of (1 -->2)-mannose. The other species had a lower proportion of galactofuranose-containing side chains and higher proportion of mannose-containing side chains. The similarities found among the F1SS polysaccharides from P. brasiliensis and the Onygenalean A. alveolata, A. terreus and O. equina, reveal the close relatedness of all these fungi, show differences with polysaccharides from other fungal genera and agree with the molecular evidence provided in the scientific literature for the placement of P. brasiliensis within the Onygenales.

Facile synthesis of a comb-like mannohexaose: a trimer of the disaccharide repeating unit of the cell-wall mannans of Aphanoascus mephitatus and related species.

An efficient method for the preparation of a comb-like mannohexaose having alpha-(1-->6) and alpha-(1-->2) linkages has been described using 6-O-acetyl-2-O-benzoyl-3,4-di-O-benzyl-alpha-D-mannopyranosyl trichloroacetimidate as the key glycosyl donor in an 'inverse Schmidt' procedure.