Welcome to IMC 2018 International Mycological Congress
Discovery of new bioactive secondary metabolites from medicinal mushrooms: Identification, biosynthesis and bioactivity evaluation.
- H. Liu
The Tibetan plateau is well known for its diversity ecological system and extreme environmental condition, and harbors rich medicinal fungi, such as Ganoderma sp., Ophiocordyceps sinensis, Tricholoma matsutake. With the help of chemistry and bioassay-guided separation technology, over 900 natural products, including 460 new compounds (36 new skeletons), have been isolated from the medicinal fungi collected in the Tibetan region. The diterpene eryngiolide A and sesquiterpene dimer Sterhirsutin A were selected as “hot-off-the-press” by Nat. Prod. Rep. Bioactivity screening revealed 460 compounds possessing antivirus, anticancer, antibacterial, and antiplasmodial activities. More importantly, two diterpenes (cyathin R and Q) with in vivo anti-tumor activity, one diterpene with in vivo anti-inflammatory activity, one sesterterpene with in vivo anti-virus effect, and one meroditerpene with in vivo anti-diabetic and anti-obesity activity were obtained. In the field of the biosynthesis of diterpene, we reported the diterpene cyclase responsible for the synthesis of cyathane skeleton in the mushroom of Hericium erinaceus. Furthermore, a new group of diterpene cyclases in the superfamily of UbiA proteins were identified.
Reading fungal genomes to discover and engineer antifungal natural products
- G. Bills
Natural products from fungi have been critical to the development of antifungal agents in use in the clinic and as agrochemicals, including griseofulvin, echinocandins, and strobilurins. Other outstanding preclinical and clinical antifungal leads (sordarins, enfumafungin, aureobasidin, ASP2397) have originated from fungi. Fungi continue to be a promising source of low molecular weight molecules that regulate fungal growth, interact with essential fungal proteins, or modulate the activities of key fungal pathways. Genome sequencing and bioinformatic prediction of secondary metabolite-encoding genes now enable searches for relatives of antifungal-encoding biosynthetic gene clusters that can lead to discovery of even more antifungal metabolites. But can this prospective approach guide discovery of new antifungals belonging to unrecognized structural classes without the aid of bioactivity-directed data? We will relate our experiences in dissecting the distribution of gene clusters of some major classes of antifungal agents produced by fungi. Although genome mining approaches can reveal the potential chemical diversity encoded by sets of related gene clusters, the approach remains limited by the small number of available fungal genomes. Therefore, tracking down historical records and the strains responsible for producing reported antifungals, e.g., echinocandins, has been critical for completing the genetic map of biosynthetic families and for guiding genome mining studies. An expanded focus on the target pathogens for whole-cell screening can also widen the search for new antifungal metabolites. Most of our historical knowledge regarding antifungal activity of fungal metabolites has been based on data from whole-cells assays of Candida albicans and Aspergillus spp., and to a lesser extent, on data from some major crop pathogens. Other major pathogens, like Cryptococcus species, were typically tested for antifungal susceptibility only when C. albicans lead compounds were evaluated for thier antifungal spectrum. We contend that Cryptococcus-centric screening of fungi for antifungal natural products offers an outstanding opportunity for the discovery of new antifungal therapies. In addition to using non-traditional pathogen as targets for discovery of antifungals, prospects can be complemented by targeting fungi with complex secondary metabolism but that have rarely been included in screening programs to date.
Exploiting the secondary metabolome of tropical Basidiomycota
- C. Chepkirui
- T. Cheng
- Z. Rupcic
- K. Yuyama
- L. Wanga
- W. Sum
- C. Decock
- J. Matasyoh
- A. Wolf-Rainer
- M. Stadler
The phylum Basidiomycota comprises the mushroom-forming fungi and various other organisms that represent a considerable part of the global biodiversity. Recent molecular ecology studies have revealed an unprecedented, huge diversity of fungi in different habitats, including soil, plants and invertebrate animals. Most of these organisms remain unknown to Science, and have never been cultured and studied for potential beneficial traits such as the production of antibiotics and other useful secondary metabolites. During the course or our search for new anti-infective agents from nature to combat the newly arising multi-resistant human pathogens, we embark on extensive exploitation of tropical fungi whose riches remain largely untapped for new bioactive metabolites. Our approach which has resulted in a rather high discovery rate of novel metabolites, is based on a combination of extensive field work and classical mycological know-how together with sophisticated methods of analytical chemistry and biotechnological process development. Numerous novel compounds with interesting biological activities, which have been discovered from new and hitherto untapped species from Thailand and Kenya will be presented.
Molecular genetic studies of alkaloid biosynthesis genes in Epichloë coenophiala, a bioprotective symbiont of the forage grass, tall fescue
- S. Florea
- D. Panaccione
- C. Schardl
Epichloë species are fungal endophytes of grasses that confer significant defense against vertebrate and invertebrate herbivores, attributed in part to their production of up to four known classes of alkaloids: aminopyrrolizidines such as lolines, pyrrolopyrazines such as peramine, indole-diterpenes such as lolitrems, and ergot alkaloids such as ergovaline. Epichloë coenophiala is a common seed-borne symbiont (endophyte) of tall fescue, a popular grass for pastures and forage due to its high productivity, stand longevity, stress tolerance and pest resistance. The endophyte is a significant contributor to these characteristics, produces anti-insect lolines and peramine, but also ergovaline, which is toxic to livestock. Our focus has been to manipulate the ergot alkaloid genes in the endophyte, with the aim of improving forage quality of tall fescue. Epichloë coenophiala is a triploid interspecific hybrid with two homeologous ergot alkaloid gene clusters, designated EAS1 and EAS2. Its genome sequence revealed that EAS1 is near a telomere, and that EAS2 has an lpsB2 pseudogene due to a frame shift mutation. We developed a method to knock off chromosome ends without stable introduction of foreign genes, and we eliminated EAS1. The genome sequence of two independent mutants confirmed the elimination of EAS1 and absence of any foreign genes. Once reintroduced into tall fescue, these eas1 deletion mutants produced the ergot alkaloid intermediate, chanoclavine I, plus high levels of the spur product, ergotryptamine, but lacked ergovaline. Interestingly, intermediates such as agroclavine and lysergic acid were undetected, and this ergot alkaloid profile recapitulated that of some naturally occurring grass-Epichloë symbiota. Complementation of the eas1 deletion mutant with a functional lpsB gene restored ergovaline production, but still gave high levels of ergotryptamine. Comparison of the in symbio gene expression data between wild type, the knockoff and complemented strains was highly variable, although two of the genes required for conversion of chanoclavine I to agroclavine were poorly expressed in the eas1 deletion mutant and lpsB complemented strains. Additional gene complementation studies are now underway to further decipher roles and regulation of the pathway genes. In addition, loline-biosynthesis genes have been introduced into the non loline alkaloid producer, Epichloë hybrida, and analysis of plants with those transformants have been generated and will be analyzed to investigate the biosynthesis of this important class of anti-insect alkaloids.
Secondary metabolites from fungal cultures: The role of mycologists in multidisciplinary teams
- H. Raja
- N. Oberlies
Fungi comprise a hyperdiverse group of organisms capable of producing notable secondary metabolites such as Penicillin, cyclosporin A, and the cholesterol lowering class of statins. Approximately, 24, 000 fungal secondary metabolites have been reported in the literature; however, we have only scratched the surface with respect to novel bioactive compound discovery from fungi. In this presentation, I will discuss findings from two projects, which highlight our work on secondary metabolites from fungal cultures. In the first part, I will discuss how isolation and identification of fungal secondary metabolites was used for discovering a quorum-sensing inhibitor in a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA), which is an antibiotic-resistant pathogen causing global health threat. In the second part, I will discuss how mass spectrometry mapping of secondary metabolite biosynthesis in situ can be used to probe a series of ecological questions about fungi that may be lost through traditional natural products chemistry extraction protocols. Together, our results highlight the importance of interdisciplinary nature of our research on fungal secondary metabolites and its contribution to both basic and applied sciences.
Bio-prospection of microfungi from Thailand
- M. Hernández-Restrepo
- Z. Rupcic
- P. Crous
- J. Luangsa-Ard
- M. Stadler
Fungi are regarded as prolific sources of novel secondary metabolites with prominent and selective biological activities that can serve as basis for the development of new antimicrobials, agrochemical pesticides and other useful compounds. In particular the mycobiota of tropical countries are still widely unexplored and can yield a plethora of novel chemical entities. Furthermore, fungi represent a rich source of nematicidal compounds, which are natural antagonists of nematode parasites and thus offer novel biocontrol strategies. During the course of the “GOLDEN MYCOLOGICAL TRIANGLE” project, on the functional biodiversity of the mycobiota inhabiting rainforests in Thailand, we recovered several interesting microfungi from plant debris. Plant samples were transported to the lab in plastic or paper bags and treated in moist chambers. Pure cultures were obtained by single spore cultures in WA and transferred to OA and PCA agar plates. Morphological features were obtained from fungi growing on OA or SNA supplemented with fragments of autoclaved pine needles, incubated at 25 °C under continuous near-ultraviolet light to promote sporulation. DNA was extracted from cultures growing in MEA. ITS barcodes and LSU sequences were generated for all species. Additional molecular markers i.e. rpb2 and tef sequences were generated for selected strains. A preliminary maximum likelihood phylogenetic analysis of the LSU sequences placed all fungal taxa in Ascomycota, except for one isolate that was related to the Basidiomycota. Among the Ascomycota the isolates were distributed in four classes; mainly in Sordariomycetes (57.5%) and Dothideomycetes (32%) and to a lesser extent in the Eurotiomycetes (7.5%) and Leotiomycetes (3%). Familial, generic and species level identification for some of the microfungal strains still remain unknown, and they appear to represent new taxa. Researchers at HZI selected 18 strains for additional studies on biological activity, screening of nematode trapping fungi and secondary metabolites. So far, Dactylaria hyalotunicata, Petrakiopsis sp. nov. and Sirothecium sp. nov. represent three new lineages in Sordariomycetes. Furthermore, some isolates represent a new genus in Sulcatisporaceae introduced as Pseudobambusicola, and other isolates represent a potentially new genus related with Exophiala in Eurotiomycetes. Other new species include Anteaglonium sp. nov., Brachiosphaera sp. nov. and Teichospora sp. nov. in Dothideomycetes; Campylocarpon sp. nov., Halorosellinia sp. nov., Hydea sp. nov. and Kionochaeta sp. nov. in Sordariomycetes. Until now, Pseudobambusicola thailandica gen et. sp. nov. revealed strong antagonistic activity against nematodes (Caenorhabditis elegans). Six novel and two known compounds were isolated. Compounds 4 and 8 showed strong nematicidal activity, while compounds 1 and 8 also inhibited growth of the pathogenic basiodiomycete Phellinus tremulae in a plate diffusion assay.