(Abstract) Xylariales (Ascomycota) is a fungal order comprising, inter alia, the large families Hypoxylaceae and Xylariaceae, known as particularly prolific producers of (bioactive) natural products in mycelial cultures and stromata. Recent genome analyses of model fungi revealed a large discrepancy between numbers of predicted biosynthetic gene clusters (BGCs) and known secondary metabolite (SM) classes. Finding the right triggers to induce these “silent” BGCs is therefore expected to substantially expand the known chemodiversity in Xylariales. In the overarching project of this work, 14 high-quality genome sequences from members of Xylariales were obtained, allowing for in-depth studies of their biosynthetic machineries. This work is dedicated to investigating the SMs of Xylariales and establishing the link to the underlying BGCs. A coordinated screening of mycelial cultures of eleven species with available genome data was conducted to define the limitations of a “classical” approach to induce silent BGCs. Moreover, stromata of the widespread European species Hypoxylon fragiforme and H. rubiginosum were investigated for novel azaphilone SMs and their biosynthesis studied using the generated high-quality genomes. In parallel, three rare, unstudied species of Xylariales were investigated in a classical screening approach and characterised for novel SMs. The coordinated screening approach yielded a valuable HPLC−MS dataset that can be used to link BGCs to analytical data. However, the approach was found to induce an unexpectedly low number of clusters. This proved that even elaborate screenings, which go beyond common approaches in natural product research, are unable to activate the majority of silent BGCs. Biosynthetic methods such as heterologous expression are able to overcome this challenge, but were beyond the scope of this work. Therefore, the biosynthesis has been investigated by genome mining on the constitutively-produced azaphilone pigments from H. fragiforme and H. rubiginosum stromata. Isolation efforts yielded 17 novel azaphilones with varying bioactivities, of which the fragirubrins and heterodimeric hybridorubrins constitute novel subclasses. Genome data of H. fragiforme revealed two distantly-located BGCs to collaboratively produce the known azaphilone diversity. In H. rubiginosum, three BGCs were found to produce a single class of SMs, which is unprecedented in fungi. In parallel, mycelial cultures of the fungicolous H. invadens produced known flaviolin naphthalenes, while two novel sesquiterpenoids and a number of chemotaxonomic marker compounds were obtained from the pyrophilic Stromatoneurospora phoenix. Stromata of Annulohypoxylon viridistratum yielded three novel benzo[ j ]fluoranthenes, which showed antimicrobial and cytotoxic activities and are chemotaxonomic markers. Summary 11 To conclude, this work revealed the intricate machinery of azaphilone biosynthesis in H. fragiforme and H. rubiginosum and characterised unprecedented azaphilone congeners. It was also found that even elaborate screening approaches are limited in the chemical diversity they can deliver. What is more, the overarching project of this work revealed hundreds of unassignable BGCs in the genome data of only 14 species from Xylariales. Thus, this work demonstrates the need for future characterisation of SM biosynthesis, as well as the chemical ecology of selected species of the Xylariales.
(Table Of Contents) Abbreviations 2 List of figures 4 List of tables 5 List of publications 6 Contribution to publications 7 Summary 10 1. Introduction 12 1.1. The fungal order Xylariales 12 1.2. Chemical diversity and bioactivity of secondary metabolites from Xylariales 13 1.3. Biosynthesis research on secondary metabolites from Xylariales 22 1.4. Aims of the thesis 27 2. Materials and methods 30 2.1. Medium-scale screening for secondary metabolites 30 2.1.1. Fungal material and cultivation 30 2.1.2. Harvest of cultures and preparation of crude extracts 32 2.1.3. HPLC−DAD/MS analysis for dereplication and identification of novel secondary metabolites 33 2.1.4. Agar-diffusion assay for evaluation of antimicrobial activity 33 2.2. Scale-up of selected fungal cultures 34 2.3. Preparative extraction and isolation of secondary metabolites 35 2.4. Structure elucidation of pure compounds 36 3. Results and discussion 38 3.1. Secondary metabolites from species of Xylariales and correlation to biosynthetic gene clusters 38 3.1.1. Medium-scale screening for secondary metabolites [II] 38 3.1.2. Chemistry, bioactivity, and biosynthesis of azaphilones from stromata of Hypoxylon fragiforme and H. rubiginosum [I, VI, VII] 43 3.2. Secondary metabolites from rare and ecologically interesting species of Xylariales 56 3.2.1. Secondary metabolites from mycelial cultures of the fungicolous Hypoxylon invadens [III] 56 3.2.2. Secondary metabolites from mycelial cultures of the pyrophilic Stromatoneurospora phoenix [IV] 58 3.2.3. Stromatal constituents of Annulohypoxylon viridistratum [V] 60 4. Conclusions and outlook 62 5. References 70 6. Appendices: publications of this dissertation 86 I. Identification of fungal fossils and novel azaphilone pigments in ancient carbonised specimens of Hypoxylon fragiforme from forest soils of Châtillon-sur-Seine (Burgundy) 88 II. Investigating the function of cryptic cytochalasan cytochrome P450 monooxygenases using combinatorial biosynthesis 102 III. Phylogenetic assignment of the fungicolous Hypoxylon invadens (Ascomycota, Xylariales) and investigation of its secondary metabolites 108 IV. Phylogenetic and chemotaxonomic studies confirm the affinities of Stromatoneurospora phoenix to the coprophilous Xylariaceae 124 V. Viridistratins A−C, antimicrobial and cytotoxic benzo[j]fluoranthenes from stromata of Annulohypoxylon viridistratum (Hypoxylaceae, Ascomycota) 146 VI. Hybridorubrins A−D, novel azaphilone heterodimers from stromata of Hypoxylon fragiforme and insights into the biosynthetic machinery for azaphilone diversification 158 VII. Azaphilone pigments from Hypoxylon rubiginosum and H. texense: absolute configuration, bioactivity, and biosynthesis 172 VIII. Recent progress in biodiversity research on the Xylariales and their secondary metabolism 182
Ascomycetes, azaphilones, bioactivity, fungi, Hypoxylaceae, naphthalenes, natural products, secondary metabolites, Xylariaceae, Xylariales