• Hybridorubrins A-D, novel azaphilone heterodimers from stromata of Hypoxylon fragiforme and insights into the biosynthetic machinery for azaphilone diversification.

      Becker, Kevin; Pfütze, Sebastian; Kuhnert, Eric; Cox, Russell; Stadler, Marc; Surup, Frank; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley-VCH, 2020-08-04)
      The diversity of azaphilones in stromatal extracts of the fungus Hypoxylon fragiforme was investigated and linked to their biosynthetic machineries using bioinformatics. Nineteen azaphilone-type compounds were isolated and characterized by NMR spectroscopy and mass spectrometry, with their absolute stereoconfigurations assigned using Mosher ester analysis and ECD spectroscopy. Four unprecedented bisazaphilones, named hybridorubrins A-D ( 1 - 4 ), were elucidated, in addition to new fragirubrins F-G ( 5 - 6 ) 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 (BGC) 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 BGC collaborating to produce two families of azaphilones and bisazaphilones derived thereof.
    • Investigating the Function of Cryptic Cytochalasan Cytochrome P450 Monooxygenases Using Combinatorial Biosynthesis.

      Wang, Chongqing; Becker, Kevin; Pfütze, Sebastian; Kuhnert, Eric; Stadler, Marc; Cox, Russell J; Skellam, Elizabeth; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (ACS, 2019-10-23)
      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

      Sir, Esteban B.; Becker, Kevin; Lambert, Christopher; Bills, Gerald F.; Kuhnert, Eric; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Taylor & Francis, 2019-01-01)
    • Ophiocordyceps flavida sp. nov. (Ophiocordycipitaceae), a new species from Thailand associated with Pseudogibellula formicarum (Cordycipitaceae), and their bioactive secondary metabolites

      Mongkolsamrit, Suchada; Noisripoom, Wasana; Pumiputikul, Siraphop; Boonlarppradab, Chollaratt; Samson, Robert A.; Stadler, Marc; Becker, Kevin; Luangsa-Ard, Janet Jennifer; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer Nature, 2021-04-01)
      During a diversity study of entomopathogenic fungi in an agricultural ecosystem, two fungi were collected, isolated, and identified based on molecular phylogenetic analyses of three nuclear loci (LSU, TEF1, and RPB1) combined with morphological data. In this study, one novel species is described, Ophiocordyceps flavida, and a new record of Pseudogibellula formicarum for Thailand. Ophiocordyceps flavida morphologically resembles the Hirsutella anamorph of Ophiocordyceps pruinosa by having a mononematous character of the conidiophores and the same insect host (Hemiptera: Cicadellidae). Pseudogibellula formicarum is found to occur simultaneously with O. flavida, producing white conidiophores on the host. Additionally, secondary metabolites of both fungi were investigated and the major compound in O. flavida was identified as 2-[2-(4-chlorophenyl)ethyl]-2-(1,1-dimethylethyl)-oxirane. Pseudogibellula formicarum from Ghana and Thailand produces 6-methoxy-1H-indole-3-carbonitrile as a main component. These compounds are known from chemical synthesis or as products of biotransformation, respectively. However, they were obtained in our study as genuine fungal metabolites for the first time and may even constitute chemotaxonomic markers for the respective species
    • Phylogenetic and chemotaxonomic studies confirm the affinities of Stromatoneurospora phoenix to the Coprophilous xylariaceae

      Becker, Kevin; Wongkanoun, Sarunyou; Wessel, Anna Charleen; Bills, Gerald F.; Stadler, Marc; Luangsa-ard, J. Jennifer; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-09-01)
      The genus Stromatoneurospora was erected in 1973 by Jong and Davis to accommodate the pyrophilic pyrenomycete Sphaeria phoenix and has traditionally been placed in the family Xylariaceae based on morphological features. However, no living culture of this genus has so far been available in the public domain. Molecular data were restricted to an internal transcribed spacer (ITS) sequence that only confirmed the familial position, and was generated from a strain that is not deposited in a public culture collection. We have recently collected fresh material and were able to culture this fungus from Thailand. The secondary metabolites of this strains were analysed after fermentation in multiple media. The the prominent components of these fermentation were purified, using preparative chromatography. Aside from two new eremophilane sesquiterpenoids named phoenixilanes A-B (1-2), four other components that are known from species of the xylariaceous genera Xylaria and Poronia were identified by spectral methods (nuclear magnetic resonance spectroscopy and high resolution mass spectrometry). Notably, (-)-(R)-6-hydroxy-3-methyl-4-dihydroisocoumarin-5-carboxylic acid (6) has not been reported as a natural product before. Moreover, DNA sequences of Stromatoneurospora phoenix clustered with members of the genera Poronia and Podosordaria in a multi-locus molecular phylogeny. These results confirmed that the genus belongs to the same evolutionary lineage as the coprophilic Xylariaceae. The results also suggest that this lineage has evolved independently from the plant-inhabiting saprotrophs and endophytes that are closely related to the genus Xylaria. These findings are discussed in relation to some theories about the endophytic vs. the pyrophilic/coprophilic fungal life style.
    • Phylogenetic Assignment of the Fungicolous (Ascomycota, Xylariales) and Investigation of its Secondary Metabolites.

      Becker, Kevin; Lambert, Christopher; Wieschhaus, Jörg; Stadler, Marc; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-09-11)
      The ascomycete Hypoxylon invadens was described in 2014 as a fungicolous species growing on a member of its own genus, H.fragiforme, which is considered a rare lifestyle in the Hypoxylaceae. This renders H.invadens an interesting target in our efforts to find new bioactive secondary metabolites from members of the Xylariales. So far, only volatile organic compounds have been reported from H.invadens, but no investigation of non-volatile compounds had been conducted. Furthermore, a phylogenetic assignment following recent trends in fungal taxonomy via a multiple sequence alignment seemed practical. A culture of H.invadens was thus subjected to submerged cultivation to investigate the produced secondary metabolites, followed by isolation via preparative chromatography and subsequent structure elucidation by means of nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). This approach led to the identification of the known flaviolin (1) and 3,3-biflaviolin (2) as the main components, which had never been reported from the order Xylariales before. Assessment of their antimicrobial and cytotoxic effects via a panel of commonly used microorganisms and cell lines in our laboratory did not yield any effects of relevance. Concurrently, genomic DNA from the fungus was used to construct a multigene phylogeny using ribosomal sequence information from the internal transcribed spacer region (ITS), the 28S large subunit of ribosomal DNA (LSU), and proteinogenic nucleotide sequences from the second largest subunit of the DNA-directed RNA polymerase II (RPB2) and β-tubulin (TUB2) genes. A placement in a newly formed clade with H.trugodes was strongly supported in a maximum-likelihood (ML) phylogeny using sequences derived from well characterized strains, but the exact position of said clade remains unclear. Both, the chemical and the phylogenetic results suggest further inquiries into the lifestyle of this unique fungus to get a better understanding of both, its ecological role and function of its produced secondary metabolites hitherto unique to the Xylariales.
    • Recent progress in biodiversity research on the Xylariales and their secondary metabolism.

      Becker, Kevin; Stadler, Marc; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer Nature, 2020-10-23)
      The families Xylariaceae and Hypoxylaceae (Xylariales, Ascomycota) represent one of the most prolific lineages of secondary metabolite producers. Like many other fungal taxa, they exhibit their highest diversity in the tropics. The stromata as well as the mycelial cultures of these fungi (the latter of which are frequently being isolated as endophytes of seed plants) have given rise to the discovery of many unprecedented secondary metabolites. Some of those served as lead compounds for development of pharmaceuticals and agrochemicals. Recently, the endophytic Xylariales have also come in the focus of biological control, since some of their species show strong antagonistic effects against fungal and other pathogens. New compounds, including volatiles as well as nonvolatiles, are steadily being discovered from these ascomycetes, and polythetic taxonomy now allows for elucidation of the life cycle of the endophytes for the first time. Moreover, recently high-quality genome sequences of some strains have become available, which facilitates phylogenomic studies as well as the elucidation of the biosynthetic gene clusters (BGC) as a starting point for synthetic biotechnology approaches. In this review, we summarize recent findings, focusing on the publications of the past 3 years.
    • Three novel species and a new record of Daldinia (Hypoxylaceae) from Thailand

      Wongkanoun, Sarunyou; Becker, Kevin; Boonmee, Kanthawut; Srikitikulchai, Prasert; Boonyuen, Nattawut; Chainuwong, Boonchuai; Luangsa-ard, Jennifer; Stadler, Marc; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer Science and Business Media LLC, 2020-10-08)
      n an investigation of stromatic Xylariales in Thailand, several specimens of Daldinia were discovered. Three novel species (D. flavogranulata, D. phadaengensis, and D. chiangdaoensis) were recognized from a molecular phylogeny based on concatenated ITS, LSU, RPB2, and TUB2 sequence data, combined with morphological characters and secondary metabolite profiles based on high performance liquid chromatography coupled to diode array detection and mass spectrometry (HPLC-MS). The major components detected were cytochalasins (in D. flavogranulata and D. chiangdaoensis) and daldinin type azaphilones (in D. phadaengensis). In addition, D. brachysperma, which had hitherto only been reported from America, was found for the first time in Asia. Its phylogenetic affinities were studied, confirming previous suspicions from morphological comparisons that the species is closely related to D. eschscholtzii and D. bambusicola, both common in Thailand. Daldinia flavogranulata, one of the new taxa, was found to be closely related to the same taxa. The other two novel species, D. phadaengensis and D. chiangdaoensis, share characters with D. korfii and D. kretzschmarioides, respectively.
    • Viridistratins A-C, Antimicrobial and Cytotoxic Benzo[]fluoranthenes from Stromata of (Hypoxylaceae, Ascomycota).

      Becker, Kevin; Wessel, Anna-Charleen; Luangsa-Ard, J Jennifer; Stadler, Marc; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-05-23)
      During the course of our search for novel biologically active metabolites from tropical fungi, we are using chemotaxonomic and taxonomic methodology for the preselection of interesting materials. Recently, three previously undescribed benzo[j]fluoranthenes (1-3) together with the known derivatives truncatones A and C (4, 5) were isolated from the stromata of the recently described species Annulohypoxylon viridistratum collected in Thailand. Their chemical structures were elucidated by means of spectral methods, including nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). The new compounds, for which we propose the trivial names viridistratins A-C, exhibited weak-to-moderate antimicrobial and cytotoxic activities in cell-based assays.