Fungi: Pioneers of chemical creativity – Techniques and strategies to uncover fungal chemistry

Rivera-Illanes D., Recabarren-Gajardo G.

Stadler M., Kolarik M.

Over three decades ago, the plant-derived anticancer agent taxol (brand name: paclitaxel) was reported from a fungal endophyte colonizing the producing plant. The hope that this finding could ever result in a sustainable production process has thus far been disappointed. Modern evidence on the evolution of secondary metabolites in plants vs. fungi suggests that this hypothesis (that fungi could produce such complex plant metabolites) is invalid. Still, numerous inconclusive original studies -and in particular, review papers by non-experts in the field-are continuously being published that claim the opposite. The current commentary tries to deal with the topic, taking the findings of –OMICS studies and current state-of-the art mycology into account. This can hopefully help to stop the scientific papermills from further spreading the fake news that fungi were capable of sustainable production of taxol.

Gärditz K.F., Czesnick H.

AbstractTaxol (common name: paclitaxel) is an extremely important component of drugs for the treatment of various cancers. Thirty years after the discovery of its effectiveness, a metabolic precursor of Taxol (10-deacetylbaccatin III) is still primarily extracted from needles of European yew trees. In order to meet the considerable demand, hopes were pinned on the possibilities of biotechnological production from the very beginning. In 1993, as if by chance, Taxol was supposedly discovered in fungi that grow endobiotically in yew trees. This finding aroused hopes of biotechnological use to produce fungal Taxol in large quantities in fermenters. It never came to that. Instead, a confusing flood of publications emerged that claimed to have detected Taxol in more and more eukaryotic and even prokaryotic species. However, researchers never reproduced these rather puzzling results, and they could certainly not be applied on an industrial scale. This paper will show that some of the misguided approaches were apparently based on a seemingly careless handling of sparse evidence and on at least questionable publications. Apparently, the desired gold rush of commercial exploitation was seductive. Scientific skepticism as an indispensable core of good scientific practice was often neglected, and the peer review process has not exerted its corrective effect. Self-critical reflection and more healthy skepticism could help to reduce the risk of such aberrations in drug development. This article uses this case study as a striking example to show what can be learned from the Taxol case in terms of research ethics and the avoidance of questionable research practices.

Hyde K.D., Baldrian P., Chen Y., Thilini Chethana K.W., De Hoog S., Doilom M., de Farias A.R., Gonçalves M.F., Gonkhom D., Gui H., Hilário S., Hu Y., Jayawardena R.S., Khyaju S., Kirk P.M., et. al.

AbstractThe field of mycology has grown from an underappreciated subset of botany, to a valuable, modern scientific discipline. As this field of study has grown, there have been significant contributions to science, technology, and industry, highlighting the value of fungi in the modern era. This paper looks at the current research, along with the existing limitations, and suggests future areas where scientists can focus their efforts, in the field mycology. We show how fungi have become important emerging diseases in medical mycology. We discuss current trends and the potential of fungi in drug and novel compound discovery. We explore the current trends in phylogenomics, its potential, and outcomes and address the question of how phylogenomics can be applied in fungal ecology. In addition, the trends in functional genomics studies of fungi are discussed with their importance in unravelling the intricate mechanisms underlying fungal behaviour, interactions, and adaptations, paving the way for a comprehensive understanding of fungal biology. We look at the current research in building materials, how they can be used as carbon sinks, and how fungi can be used in biocircular economies. The numbers of fungi have always been of great interest and have often been written about and estimates have varied greatly. Thus, we discuss current trends and future research needs in order to obtain more reliable estimates. We address the aspects of machine learning (AI) and how it can be used in mycological research. Plant pathogens are affecting food production systems on a global scale, and as such, we look at the current trends and future research needed in this area, particularly in disease detection. We look at the latest data from High Throughput Sequencing studies and question if we are still gaining new knowledge at the same rate as before. A review of current trends in nanotechnology is provided and its future potential is addressed. The importance of Arbuscular Mycorrhizal Fungi is addressed and future trends are acknowledged. Fungal databases are becoming more and more important, and we therefore provide a review of the current major databases. Edible and medicinal fungi have a huge potential as food and medicines, especially in Asia and their prospects are discussed. Lifestyle changes in fungi (e.g., from endophytes, to pathogens, and/or saprobes) are also extremely important and a current research trend and are therefore addressed in this special issue of Fungal Diversity.

Pfütze S., Charria-Girón E., Schulzke E., Toshe R., Khonsanit A., Franke R., Surup F., Brönstrup M., Stadler M.

AbstractIn this investigation, we explored the diversity of melleolide‐type meroterpenoids produced by Armillaria ostoyae, one of the largest and oldest organisms on Earth, using extracts from liquid and solid fermentation media. The study unveiled three unprecedented dimeric bismelleolides and three novel fatty‐acid‐substituted congeners, along with 11 new and 21 known derivatives. The structures were elucidated by 1D and 2D NMR spectroscopy and HRESI‐MS, and ROESY spectral analysis for relative configurations. Absolute configurations were determined from crystal structures and through ECD spectra comparison. A compound library of melleolide‐type meroterpenoids facilitated metabolomics‐wide associations, revealing production patterns under different culture conditions. The library enabled assessments of antimicrobial and cytotoxic activities, revealing that the Δ2,4 double bond is not crucial for antifungal activity. Cytotoxicity was linked to the presence of an aldehyde at C1, but lost with hydroxylation at C13. Chemoinformatic analyses demonstrated the intricate interplay of chemical modifications on biological properties. This study marks the first systematic exploration of Armillaria spp. meroterpenoid diversity by MS‐based untargeted metabolomics, offering insight into structure–activity relationships through innovative chemoinformatics.

Cox R.J.

Filamentous fungi produce a very wide range of complex and often bioactive metabolites, demonstrating their inherent ability as hosts of complex biosynthetic pathways. Recent advances in molecular sciences related to fungi have afforded the development of new tools that allow the rational total biosynthesis of highly complex specialized metabolites in a single process. Increasingly, these pathways can also be engineered to produce new metabolites. Engineering can be at the level of gene deletion, gene addition, formation of mixed pathways, engineering of scaffold synthases and engineering of tailoring enzymes. Combination of these approaches with hosts that can metabolize low-value waste streams opens the prospect of one-step syntheses from garbage. The total biosynthesis and engineering of complex natural products is now routinely achieved in filamentous fungal host organisms. This technology offers substantial advantages over traditional total chemical synthesis for the production of both known and new specialized metabolites.

Chemutai Sum W., Ebada S.S., Clement Matasyoh J., Stadler M.

Fukaya M., Nagamine S., Ozaki T., Liu Y., Ozeki M., Matsuyama T., Miyamoto K., Kawagishi H., Uchiyama M., Oikawa H., Minami A.

Mushroom terpenoids are biologically and chemically diverse fungal metabolites. Among them, melleolides are representative sesquiterpenoids with a characteristic protoilludane skeleton. In this study, we applied a recently established hot spot knock‐in method to elucidate the biosynthetic pathway leading to 1a‐hydroxymelleolide. The biosynthesis of the sesquiterpene core involves the cytochrome P450 catalyzing stepwise hydroxylation on the D6‐protoilludene framework and a stereochemical inversion process at the C5 position catalyzed by short‐chain dehydrogenase/reductase family proteins. The highlight on the biosynthesis is that the flavoprotein Mld7 catalyzes an oxidation triggered double bond shift accompanying dehydration and acyl group assisted substitution with two different nucleophiles at the C6 position to afford the D7‐protoilludene derivatives such as melleolide and armillarivin. The complex reaction mechanism was proposed by density functional theory (DFT) calculations. Of particularly importance is that product distribution is regulated by the interaction with cell membrane. This proposed biosynthetic pathway provides an opportunity to understand the structural diversification mechanisms of melleolides and protoilludane sesquiterpenes.

Lambert C., Schmidt K., Karger M., Stadler M., Stradal T.E., Rottner K.

The eukaryotic actin cytoskeleton comprises the protein itself in its monomeric and filamentous forms, G- and F-actin, as well as multiple interaction partners (actin-binding proteins, ABPs). This gives rise to a temporally and spatially controlled, dynamic network, eliciting a plethora of motility-associated processes. To interfere with the complex inter- and intracellular interactions the actin cytoskeleton confers, small molecular inhibitors have been used, foremost of all to study the relevance of actin filaments and their turnover for various cellular processes. The most prominent inhibitors act by, e.g., sequestering monomers or by interfering with the polymerization of new filaments and the elongation of existing filaments. Among these inhibitors used as tool compounds are the cytochalasans, fungal secondary metabolites known for decades and exploited for their F-actin polymerization inhibitory capabilities. In spite of their application as tool compounds for decades, comprehensive data are lacking that explain (i) how the structural deviances of the more than 400 cytochalasans described to date influence their bioactivity mechanistically and (ii) how the intricate network of ABPs reacts (or adapts) to cytochalasan binding. This review thus aims to summarize the information available concerning the structural features of cytochalasans and their influence on the described activities on cell morphology and actin cytoskeleton organization in eukaryotic cells.

Niego A.G., Lambert C., Mortimer P., Thongklang N., Rapior S., Grosse M., Schrey H., Charria-Girón E., Walker A., Hyde K.D., Stadler M.

AbstractFungi provide ecological and environmental services to humans, as well as health and nutritional benefits, and are vital to numerous industries. Fermented food and beverage products from fungi are circulating in the market, generating billions of USD. However, the highest potential monetary value of fungi is their role in blue carbon trading because of their ability to sequester large amounts of carbon in the soil. There are no conclusive estimates available on the global monetary value of fungi, primarily because there are limited data for extrapolation. This study outlines the contribution of fungi to the global economy and provides a first attempt at quantifying the global monetary value of fungi. Our estimate of USD 54.57 trillion provides a starting point that can be analysed and improved, highlighting the significance of fungi and providing an appreciation of their value. This paper identifies the different economically valuable products and services provided by fungi. By giving a monetary value to all important fungal products, services, and industrial applications underscores their significance in biodiversity and conservation. Furthermore, if the value of fungi is well established, they will be considered in future policies for effective ecosystem management.

Zeng H., Stadler M., Abraham W., Müsken M., Schrey H.

The two fungal human pathogens, Candida auris and Candida albicans, possess a variety of virulence mechanisms. Among them are the formation of biofilms to protect yeast against harsh conditions through the development of (pseudo)hyphae whilst also facilitating the invasion of host tissues. In recent years, increased rates of antifungal resistance have been associated with C. albicans and C. auris, posing a significant challenge for the effective treatment of fungal infections. In the course of our ongoing search for novel anti-infectives, six selected azaphilones were tested for their cytotoxicity and antimicrobial effects as well as for their inhibitory activity against biofilm and hyphal formation. This study revealed that rubiginosin C, derived from stromata of the ascomycete Hypoxylon rubiginosum, effectively inhibited the formation of biofilms, pseudohyphae, and hyphae in both C. auris and C. albicans without lethal effects. Crystal violet staining assays were utilized to assess the inhibition of biofilm formation, while complementary microscopic techniques, such as confocal laser scanning microscopy, scanning electron microscopy, and optical microscopy, were used to investigate the underlying mechanisms. Rubiginosin C is one of the few substances known to effectively target both biofilm formation and the yeast-to-hyphae transition of C. albicans and C. auris within a concentration range not affecting host cells, making it a promising candidate for therapeutic intervention in the future.

Blin K., Shaw S., Augustijn H.E., Reitz Z.L., Biermann F., Alanjary M., Fetter A., Terlouw B.R., Metcalf W.W., Helfrich E.J., van Wezel G.P., Medema M.H., Weber T.

Abstract Microorganisms produce small bioactive compounds as part of their secondary or specialised metabolism. Often, such metabolites have antimicrobial, anticancer, antifungal, antiviral or other bio-activities and thus play an important role for applications in medicine and agriculture. In the past decade, genome mining has become a widely-used method to explore, access, and analyse the available biodiversity of these compounds. Since 2011, the ‘antibiotics and secondary metabolite analysis shell—antiSMASH’ (https://antismash.secondarymetabolites.org/) has supported researchers in their microbial genome mining tasks, both as a free to use web server and as a standalone tool under an OSI-approved open source licence. It is currently the most widely used tool for detecting and characterising biosynthetic gene clusters (BGCs) in archaea, bacteria, and fungi. Here, we present the updated version 7 of antiSMASH. antiSMASH 7 increases the number of supported cluster types from 71 to 81, as well as containing improvements in the areas of chemical structure prediction, enzymatic assembly-line visualisation and gene cluster regulation.

Pfütze S., Khamsim A., Surup F., Decock C., Matasyoh J.C., Stadler M.

With heimionones A–E (1–5), five new terpenoids were isolated from submerged cultures of Heimiomyces sp. in addition to the previously described compounds hispidin, hypholomin B, and heimiomycins A and B. Planar structures of the metabolites were elucidated by 1D and 2D NMR in addition to HRESIMS data. While ROESY data assigned relative configurations, absolute configurations were determined by the synthesis of MTPA esters of 1, 3, and 5. The [6.3.0] undecane core structure of compounds 3–5 is of the asteriscane-type, however, the scaffold of 1 and 2 with their bicyclo [5.3.0] decane core and germinal methyl substitution is, to our knowledge, unprecedented. Together with several new compounds that were previously isolated from solid cultures of this strain, Heimiomyces sp. showed an exceptionally high chemical diversity of its secondary metabolite profile.

Wongkanoun S., Chainuwong B., Kobmoo N., Roytrakul S., Somrithipol S., Luangsa-ard J., Charria-Girón E., Srikitikulchai P., Stadler M.

Over the past two decades, hypoxylaceous specimens were collected from several sites in Thailand. In this study, we examined their affinity to the genus Pyrenopolyporus using macroscopic and microscopic morphological characters, dereplication of their stromatal secondary metabolites using ultrahigh performance liquid chromatography coupled to diode array detection and ion mobility tandem mass spectrometry (UHPLC-DAD-IM-MS/MS), and molecular phylogenetic analyses. We describe and illustrate five novel species and a new record for the country, present multi-locus phylogenetic analyses that show the distinction between the proposed species, and provide proteomic profiles of the fungi using matrix associated laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) for the first time. Based on our findings, this strategy is useful as a complementary tool to distinguish species between Daldinia and Pyrenopolyporus in a consistent way with the phylogenetic analysis.

Cedeño-Sanchez M., Charria-Girón E., Lambert C., Luangsa-ard J.J., Decock C., Franke R., Brönstrup M., Stadler M.

During a mycological survey of the Democratic Republic of the Congo, a fungal specimen that morphologically resembled the American species Hypoxylon papillatum was encountered. A polyphasic approach including morphological and chemotaxonomic together with a multigene phylogenetic study (ITS, LSU, tub2, and rpb2) of Hypoxylon spp. and representatives of related genera revealed that this strain represents a new species of the Hypoxylaceae. However, the multi-locus phylogenetic inference indicated that the new fungus clustered with H. papillatum in a separate clade from the other species of Hypoxylon. Studies by ultrahigh performance liquid chromatography coupled to diode array detection and ion mobility tandem mass spectrometry (UHPLC-DAD-IM-MS/MS) were carried out on the stromatal extracts. In particular, the MS/MS spectra of the major stromatal metabolites of these species indicated the production of hitherto unreported azaphilone pigments with a similar core scaffold to the cohaerin-type metabolites, which are exclusively found in the Hypoxylaceae. Based on these results, the new genus Parahypoxylon is introduced herein. Aside from P. papillatum, the genus also includes P. ruwenzoriensesp. nov., which clustered together with the type species within a basal clade of the Hypoxylaceae together with its sister genus Durotheca.