Rapid discovery of terpene tailoring enzymes for total biosynthesis.

Twenty oxygenated aristolochene congeners were rapidly synthesised by combining genes from four different fungal pathways in the fungal host organism Aspergillus oryzae. Compounds produced in a single step include the natural product hypoxylan A and an epimer of guignaderemophilane C. A new fungal aromatase was discovered that produces phenols by oxidative demethylation.

Total biosynthesis of fungal tetraketide pyrones.

Fungal tetraketide pyrones possess important and potent bioactivities, but their detailed biosynthetic pathways are unknown and synthetic routes to their production are lengthy. Here we investigated the fungal pathways to the multiforisins and compounds related to islandic acid. Heterologous expression experiments yield high titres of these compounds and pathway intermediates. The results both elucidate the pathway and offer a platform for the total biosynthesis of this class of metabolites.

Ten decadal advances in fungal biology leading towards human well-being.

Fungi are an understudied resource possessing huge potential for developing products that can greatly improve human well-being. In the current paper, we highlight some important discoveries and developments in applied mycology and interdisciplinary Life Science research. These examples concern recently introduced drugs for the treatment of infections and neurological diseases; application of -OMICS techniques and genetic tools in medical mycology and the regulation of mycotoxin production; as well as some highlights of mushroom cultivaton in Asia. Examples for new diagnostic tools in medical mycology and the exploitation of new candidates for therapeutic drugs, are also given. In addition, two entries illustrating the latest developments in the use of fungi for biodegradation and fungal biomaterial production are provided. Some other areas where there have been and/or will be significant developments are also included. It is our hope that this paper will help realise the importance of fungi as a potential industrial resource and see the next two decades bring forward many new fungal and fungus-derived products.

A genomic journey in the secondary metabolite diversity of fungal plant and insect pathogens: from functional to population genomics.

Fungal pathogens produce a broad array of secondary metabolites (SMs), which allow the fungus to thrive in its natural habitat and gain competitive advantage. Analysis of the genetically encoded blueprints for SM assembly highlighted that only a small portion of the SMs these fungi are capable of producing are known, and even fewer have been investigated for their natural function. Using molecular tools, a lot of progress has been made recently in identifying the blueprint products and linking them to their ecological purpose such as the peptide virulence factor fusaoctaxin A released by Fusarium graminearum during infection of wheat or the F. oxysporum polyketide bikaverin that provides competitive advantage against bacteria in tomato. In addition, population genomics have given particularly important insights into the species-specific plasticity of the SM blueprint arsenal, showcasing the ongoing evolution and adaptation of fungal pathogens. This approach holds promise in inferring roles in pathogenicity of many more fungal SMs.

Genomic characterization of three marine fungi, including Emericellopsis atlantica sp. nov. with signatures of a generalist lifestyle and marine biomass degradation.

Marine fungi remain poorly covered in global genome sequencing campaigns; the 1000 fungal genomes (1KFG) project attempts to shed light on the diversity, ecology and potential industrial use of overlooked and poorly resolved fungal taxa. This study characterizes the genomes of three marine fungi: Emericellopsis sp. TS7, wood-associated Amylocarpus encephaloides and algae-associated Calycina marina. These species were genome sequenced to study their genomic features, biosynthetic potential and phylogenetic placement using multilocus data. Amylocarpus encephaloides and C. marina were placed in the Helotiaceae and Pezizellaceae (Helotiales), respectively, based on a 15-gene phylogenetic analysis. These two genomes had fewer biosynthetic gene clusters (BGCs) and carbohydrate active enzymes (CAZymes) than Emericellopsis sp. TS7 isolate. Emericellopsis sp. TS7 (Hypocreales, Ascomycota) was isolated from the sponge Stelletta normani. A six-gene phylogenetic analysis placed the isolate in the marine Emericellopsis clade and morphological examination confirmed that the isolate represents a new species, which is described here as E. atlantica. Analysis of its CAZyme repertoire and a culturing experiment on three marine and one terrestrial substrates indicated that E. atlantica is a psychrotrophic generalist fungus that is able to degrade several types of marine biomass. FungiSMASH analysis revealed the presence of 35 BGCs including, eight non-ribosomal peptide synthases (NRPSs), six NRPS-like, six polyketide synthases, nine terpenes and six hybrid, mixed or other clusters. Of these BGCs, only five were homologous with characterized BGCs. The presence of unknown BGCs sets and large CAZyme repertoire set stage for further investigations of E. atlantica. The Pezizellaceae genome and the genome of the monotypic Amylocarpus genus represent the first published genomes of filamentous fungi that are restricted in their occurrence to the marine habitat and form thus a valuable resource for the community that can be used in studying ecological adaptions of fungi using comparative genomics.

Total synthesis and mechanism of action of the antibiotic armeniaspirol A.

Emerging antimicrobial resistance urges the discovery of antibiotics with unexplored, resistance-breaking mechanisms. Armeniaspirols represent a novel class of antibiotics with a unique spiro[4.4]non-8-ene scaffold and potent activities against Gram-positive pathogens. We report a concise total synthesis of (±) armeniaspirol A in six steps with a yield of 20.3% that includes the formation of the spirocycle through a copper-catalyzed radical cross-coupling reaction. In mechanistic biological experiments, armeniaspirol A exerted potent membrane depolarization, accounting for the pH-dependent antibiotic activity. Armeniaspirol A also disrupted the membrane potential and decreased oxygen consumption in mitochondria. In planar lipid bilayers and in unilamellar vesicles, armeniaspirol A transported protons across membranes in a protein-independent manner, demonstrating that armeniaspirol A acted as a protonophore. We provide evidence that this mechanism might account for the antibiotic activity of multiple chloropyrrole-containing natural products isolated from various origins that share a 4-acylphenol moiety coupled to chloropyrrole as a joint pharmacophore. We additionally describe an efflux-mediated mechanism of resistance against armeniaspirols.

Hybridorubrins A-D: Azaphilone Heterodimers from Stromata of Hypoxylon fragiforme and Insights into the Biosynthetic Machinery for Azaphilone Diversification.

The diversity of azaphilones in stromatal extracts of the fungus Hypoxylon fragiforme was investigated and linked to their biosynthetic machineries by using bioinformatics. Nineteen azaphilone-type compounds were isolated and characterized by NMR spectroscopy and mass spectrometry, and their absolute stereoconfigurations were assigned by using Mosher ester analysis and electronic circular dichroism spectroscopy. Four unprecedented bis-azaphilones, named hybridorubrins A-D, were elucidated, in addition to new fragirubrins F and G and various known mitorubrin derivatives. Only the hybridorubrins, which are composed of mitorubrin and fragirubrin moieties, exhibited strong inhibition of Staphylococcus aureus biofilm formation. Analysis of the genome of H. fragiforme revealed the presence of two separate biosynthetic gene clusters (BGCs) hfaza1 and hfaza2 responsible for azaphilone formation. While the hfaza1 BGC likely encodes the assembly of the backbone and addition of fatty acid moieties to yield the (R)-configured series of fragirubrins, the hfaza2 BGC contains the necessary genes to synthesise the widely distributed (S)-mitorubrins. This study is the first example of two distant cross-acting fungal BGCs collaborating to produce two families of azaphilones and bis-azaphilones derived therefrom.

Biosynthesis of oxygenated brasilane terpene glycosides involves a promiscuous N-acetylglucosamine transferase.

Investigation of the metabolome of the ascomycete Annulohypoxylon truncatum led to the identification of novel oxygenated brasilane glycosides and the revision of the stereochemistry of the brasilane A octahydro-1H-indene core scaffold to trans. The bra biosynthetic gene cluster containing five genes (braA-braE) was identified and verified by heterologous expression experiments in Aspergillus oryzae demonstrating that BraC is a multifunctional P450 monooxygenase. In vitro studies of BraB revealed it to be a very rare fungal UDP-GlcNAc dependent N-acetylglucosamine transferase. UDP-glucose is also accepted as a donor, and a broad acceptor substrate tolerance for various primary and secondary alcohols was observed.

Contrasting Effects of Grass - Endophyte Chemotypes on a Tri-Trophic Cascade.

Systemic grass-endophytes of the genus Epichloë symbiotically infect the above-ground plant parts of many grass species, where they produce alkaloids in a grass- and endophyte-specific manner that are toxic or deterrent to herbivores. An increasing number of studies show cascading negative effects of endophyte-derived alkaloids that extend to higher trophic levels, harming beneficial insects, including those that control aphid populations. Lacewings are one of the major biological aphid controls, and are especially resistant to insecticides and pollutants, but their susceptibility to endophyte infection in the food chain has never been studied. Our study found variability in aphid population growth depending on the endophyte-grass chemotype, where aphid population growth was lowest on chemotypes known for producing high amounts of loline alkaloids. We also showed that larval and pupal development and mortality of the Common Green Lacewing (Chrysoperla carnea) was, in a non-choice experiment, not affected by endophyte infection in the food chain. This is a first indication that lacewings might be resistant to endophyte-derived alkaloids and could be robust biocontrol agents when applied together with endophyte-infected grass, possibly replacing chemical pesticides.

The sporothriolides. A new biosynthetic family of fungal secondary metabolites.

The biosynthetic gene cluster of the antifungal metabolite sporothriolide 1 was identified from three producing ascomycetes: Hypomontagnella monticulosa MUCL 54604, H. spongiphila CLL 205 and H. submonticulosa DAOMC 242471. A transformation protocol was established, and genes encoding a fatty acid synthase subunit and a citrate synthase were simultaneously knocked out which led to loss of sporothriolide and sporochartine production. In vitro reactions showed that the sporochartines are derived from non-enzymatic Diels-Alder cycloaddition of 1 and trienylfuranol A 7 during the fermentation and extraction process. Heterologous expression of the spo genes in Aspergillus oryzae then led to the production of intermediates and shunts and delineation of a new fungal biosynthetic pathway originating in fatty acid biosynthesis. Finally, a hydrolase was revealed by in vitro studies likely contributing towards self-resistance of the producer organism.

Investigating the Function of Cryptic Cytochalasan Cytochrome P450 Monooxygenases Using Combinatorial Biosynthesis.

Tailoring enzymes in cytochalasan biosynthesis are relatively promiscuous. Exploiting this property, we deduced the function of four cryptic cytochrome P450 monooxygenases via heterologous expression of six cytochrome P450-encoding genes, originating from Hypoxylon fragiforme and Pyricularia oryzae, in pyrichalasin H ΔP450 strains. Three cryptic cytochrome P450 enzymes (HffD, HffG, and CYP1) restored pyrichalasin H production in mutant strains, while CYP3 catalyzed a site-selective epoxidation leading to the isolation of three novel cytochalasans.

Observations on Texas hypoxylons, including two new Hypoxylon species and widespread environmental isolates of the H. croceum complex identified by a polyphasic approach.

Two new species and a new combination of Hypoxylon from Texas were identified and described based on morphological, multigene phylogenetic (ITS [nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2], 28S [5' 1200 bp of nuc 28S rDNA], RPB2 [partial second largest subunit of the DNA-directed RNA polymerase II], TUB2 [partial ß-tubulin]), and chemotaxonomic data. Hypoxylon olivaceopigmentum is characterized by its pulvinate to glomerate stromata, olivaceous KOH-extractable pigments, equilateral ascospores, and indehiscent perispore. Hypoxylon texense can be distinguished from morphologically similar species by its rust to dark brick KOH-extractable pigments and the high-performance liquid chromatography (HPLC) profile of its stromatal secondary metabolites. Hypoxylon hinnuleum is proposed as the sexual morph of Nodulisporium hinnuleum, featuring dark vinaceous glomerate stromata with dark brick KOH-extractable pigments composed of cohaerin-type azaphilones and smooth equilateral ascospores with indehiscent perispore. Based on these diagnostic characters, H. hinnuleum forms a complex with H. croceum and H. minicroceum. More than 50 ITS sequences with high identity originating from North American and East Asian environmental isolates formed a well-supported clade with the type of N. hinnuleum, demonstrating the widespread distribution of the species complex. In addition, updated descriptions and comprehensive illustrations with detailed information on the diagnostic features of H. fendleri and H. perforatum are provided. The multilocus phylogenetic reconstruction of Hypoxylon supported the status of the new species and broadened the knowledge about intergeneric relationships.

Oryzines A & B, Maleidride Congeners from Aspergillus oryzae and Their Putative Biosynthesis.

Aspergillus oryzae is traditionally used in East Asia for the production of food and brewing. In addition, it has been developed into a suitable host for the heterologous expression of natural product biosynthetic genes and gene clusters, enabling the functional analysis of the encoded enzymes. A. oryzae shares a 99.5% genome homology with Aspergillus flavus, but their secondary metabolomes differ significantly and various compounds unique to A. oryzae have been reported. While using A. oryzae as a host for heterologous expression experiments we discovered two new metabolites in extracts of A. oryzae M-2-3 with an unusual maleidride backbone, which were named oryzine A and B. Their structures were elucidated by high resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) analysis. Their structural relationships with known maleidrides implied involvement of a citrate synthase (CS) and a polyketide (PKS) or fatty acid synthase (FAS) in their biosynthesis. Analysis of the A. oryzae genome revealed a single putative biosynthetic gene cluster (BGC) consistent with the hypothetical biosynthesis of the oryzines. These findings increase knowledge of the chemical potential of A. oryzae and are the first attempt to link a novel product of this fungus with genomic data.

Enfumafungin synthase represents a novel lineage of fungal triterpene cyclases.

Enfumafungin is a glycosylated fernene-type triterpenoid produced by the fungus Hormonema carpetanum. Its potent antifungal activity, mediated by its interaction with ß-1,3-glucan synthase and the fungal cell wall, has led to its development into the semi-synthetic clinical candidate, ibrexafungerp (=SCY-078). We report on the preliminary identification of the enfumafungin biosynthetic gene cluster (BGC) based on genome sequencing, phylogenetic reconstruction, gene disruption, and cDNA sequencing studies. Enfumafungin synthase (efuA) consists of a terpene cyclase domain (TC) fused to a glycosyltransferase (GT) domain and thus represents a novel multifunctional enzyme. Moreover, the TC domain bears a phylogenetic relationship to bacterial squalene-hopene cyclases (SHC) and includes a typical DXDD motif within the active centre suggesting that efuA evolved from SHCs. Phylogenetic reconstruction of the GT domain indicated that this portion of the fusion gene originated from fungal sterol GTs. Eleven genes flanking efuA are putatively involved in the biosynthesis, regulation, transport and self-resistance of enfumafungin and include an acetyltransferase, three P450 monooxygenases, a dehydrogenase, a desaturase and a reductase. A hypothetical scheme for enfumafungin assembly is proposed in which the E-ring is oxidatively cleaved to yield the four-ring system of enfumafungin. EfuA represents the first member of a widespread lineage of fungal SHCs.

The Rickiols: 20-, 22-, and 24-membered Macrolides from the Ascomycete Hypoxylon rickii.

In preceding studies the neotropical ascomycete Hypoxylon rickii turned out to be a prolific source of new secondary metabolites, considering that we had obtained terpenoids with five different scaffolds along with a series of terphenyls. From the mycelial extracts of a 70 L scale fermentation of this strain we additionally isolated nine new macrolides (1-9) by RP-HPLC. The planar structures were elucidated by NMR spectroscopy complemented by HR-ESIMS. The relative configurations were assigned by J-based configuration analyses and confirmed by Kishi's Universal Database. Subsequently, the absolute configurations were assigned by Mosher's method using the shift analysis of a tetra-MTPA derivative. For rickiol A (1) and E (5) we observed transesterification of 20-membered ring structures to 22-membered isomers rickiol A2 (6) and E2 (7), and to 24-membered isomers rickiol A3 (8) and rickiol E3 (9), respectively. Cytotoxic effects and moderate antibiotic activity against Gram-positive bacteria were observed for 1-8 and 1-6 and 8, respectively. The total synthesis of rickiol E3 (9) established easier access to these compounds.

Minutellins A - D, azaphilones from the stromata of Annulohypoxylon minutellum (Xylariaceae).

During the course of our screening for new metabolites with chemotaxonomic importance from stromata of fungi from the family Xylariaceae, we characterized several interesting metabolites in the fungus Annulohypoxylon minutellum. Extraction of the fruiting bodies and purification by preparative HPLC resulted in the isolation of five metabolites. The main compound was identified as the known metabolite hinnulin A (5), while four minor compounds were found to represent previously undescribed azaphilones, named minutellins A - D (1-4). Their planar structures were elucidated using NMR and HRESIMS data; absolute stereochemistry was assigned by CD data and Mosher's method. Compounds 1, 3 and 5 showed cytotoxic effects against murine and human cells. As the production of 1-5 is restricted to a group of closely related Annulohypoxylon species, they serve well as chemotaxonomic marker.

Rickicaryophyllane A, a Caryophyllane from the Ascomyceteous Fungus Hypoxylon rickii and a 10-Norbotryane Congener.

Herein we report the isolation from Hypoxylon rickii of a new sesquiterpenoid (1) with a caryophyllane skeleton. The planar structure of 1 was elucidat ed by NMR and HRMS data as the 1,12-dihydro-l-hydroxyl derivative of caryophyllenol-I, for which we propose the name rickicaryophyllane A. Its relative stereochemistry was assigned with a series of ID NOESY experiments, while the IR,2S,5R,9R absolute configuration was demonstrated by Mosher's analysis. Besides, we isolated 3-(hydroxymethyl)-1,1,3,5-tetramethyl-1,2,3,5,6,7-hexahydro-4H-inden-4-one (2) as a new 10-norbotryane derivative and the known metabolite orcacetophenone (3).

Rickenyls A-E, antioxidative terphenyls from the fungus Hypoxylon rickii (Xylariaceae, Ascomycota).

Our screening efforts for new natural products with interesting bioactivity have revealed the neotropical ascomycete Hypoxylon rickii as a prolific source. We isolated five secondary metabolites with a p-terphenyl backbone from the mycelial extract of a fermentation of this fungus in 70 l scale by using RP-HPLC, which were named rickenyls A-E (1-5). Their structures were elucidated by X-ray crystallography and NMR spectroscopy, complemented by HRESIMS. Two of the compounds contained a quinone core structure in ortho (2) and para-position (5), respectively. We obtained 2 spontaneously and by lead tetraacetate oxidation from 1. All compounds were screened for antimicrobial, antioxidative and cytotoxic activities. Rickenyl A (1) exhibited strong antioxidative effects and moderate cytotoxic activity against various cancer cell lines.

Silphiperfolene-Type Terpenoids and Other Metabolites from Cultures of the Tropical Ascomycete Hypoxylon rickii (Xylariaceae).

A culture isolated from ascospores of Hypoxylon rickii, a xylariaceous ascomycete collected in Martinique, had yielded botryane, noreremophilane and abietane-type terpenoids in a preceding study, but additional metabolites were detected by extensive HPLC-MS analysis in other fractions. Herein we report the further isolation of four new sesquiterpenoids with a silphiperfol-6-ene skeleton from extracts of H. rickii. The planar structures were elucidated by NMR and HRMS data as 13-hydroxysilphiperfol-6-ene (1), 9-hydroxysilphiperfol-6-en-13-oic acid (2), 2-hydroxysilphiperfol-6-en-13-oic acid (3) and 15-hydroxysilphiperfol-6-en-13-oic acid (4). For compounds 2-4 we propose the trivial names rickinic acids A-C. Their stereochemistry was assigned by ROESY correlations as well as by the specific optical rotation. Additionally, the known compounds, botryenanol, dehydrobotrydienol, cyclo(Phe-Pro), cyclo(Pro-Leu), (+)-ramulosin and α-eleostearic acid were isolated. The antimicrobial and cytotoxic activities of the new compounds are also reported.

Botryane, noreudesmane and abietane terpenoids from the ascomycete Hypoxylon rickii.

In the course of our screening for new bioactive natural products, a culture of Hypoxylon rickii, a xylariaceous ascomycete collected from the Caribbean island Martinique, was identified as extraordinary prolific producer of secondary metabolites. Ten metabolites of terpenoid origin were isolated from submerged cultures of this species by preparative HPLC. Their structures were elucidated using spectral techniques including 2D NMR and HRESIMS. Three of the compounds were elucidated as new botryanes (1-3) along with three known ones, i.e. (3aS)-3a,5,5,8-tetramethyl-3,3a,4,5-tetrahydro-1H-cyclopenta[de]isochromen-1-one (4), (3aS,8R)-3a,5,5,8-tetramethyl-3,3a,4,5,7,8-hexahydro-1H-cyclopenta[de]isochromen-1-one (5) and botryenanol (6). Further three new sesquiterpenoids featured a 14-noreudesmane-type skeleton and were named hypoxylan A-C (7-9); the diterpenoid rickitin A (10) contains an abietane-type backbone. Compounds 1, 2, 3, 7, and 10 showed cytotoxic effects against murine cells.