Identification of novel bioactive natural products from species of Xylariales V2

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.