Linking fungal secondary metabolites and pathways to their genes in Aspergillus

Filamentous fungi are producers of small bioactive molecules termed secondary metabolites (SMs), which display a wide range of functional and structural diversity. The compounds have several important activities including antifungal, antibacterial, anticancer, antiparasitic, antiinsectan and immunosuppressive activities. The rapid increase in genomes sequences of filamentous fungi has revealed that the biosynthetic potential is enormous, due to the presence of huge numbers of biosynthetic gene clusters. Thus, there is an enormous potential of previously undiscovered SMs waiting to be
exploited, which can potentially be used as pharmaceuticals. The work has focused on filamentous fungi from the important genus Aspergillus. It contains species that are both food and feed contaminants, some that are used for industrial applications for production of small compounds and enzymes, as well as model organisms for genetic studies and human opportunistic pathogens. The aim of this PhD study has been divided into two major topics:
1) Discovery and characterization of novel SMs from filamentous fungi
2) Linking of fungal SMs to genes and elucidation of biosynthetic pathways
The first part of this study, the discovery of novel SMs, has resulted in characterization of 22 novel SMs from selected Aspergilli. Two different approaches were undertaken: Dereplication based discovery and discovery by induction of sclerotium formation. The outcome included characterization of the following novel compounds: aculenes A-D, acucalbistrins A-B, acu-dioxomorpholine, okaramine S, and epi-10,23-dihydro-24,25- dehydroaflavinin from A. aculeatus, aspiperidine oxide from A. indologenus, homomorphosins A-F from A. homomorphus, sclerotionigrins A-B from A.
sclerotioniger, as well as emindole SC, sclerolizine and carbonarins I-J from A. sclerotiicarbonarius. In addition, several known compounds could be reported from the different species for the first time. Furthermore it was discovered how sclerotia formation could be induced in black Aspergilli, using a combination of natural substrates and by introduction of a pre-freezing step prior to inoculation. This is of general interest for the discovery of novel bioactive SMs, since sclerotia formation trigger otherwise silent biosynthetic pathways, resulting in greatly altered metabolic profiles.
The second part of the study, linking genes to SMs and elucidating biosynthetic pathways, led to insights into the biosynthetic potential of a number of Aspergilli. This included comparative metabolic profiling of A. oryzae and A. flavus, two species with an overall high homology of 99.5 % on genome level, but surprisingly high degree of chemical differences. Elucidation of structures from novel A. oryzae metabolites, however, revealed the chemical link between the two species. In two parallel projects, involving A. niger and A. aculeatus respectively, the polyketide 6-methyl salicylic acid (6-MSA), and corresponding biosynthetic pathways, were investigated. In A. niger, 6-MSA was converted into meroterpenoid yanuthone D. The biosynthetic pathway was investigated by a multidisciplinary approach. As a result the cluster responsible for production of yanuthone D was identified and a biosynthetic pathway presented, where 6-MSA is converted into yanuthone D in eight steps. Furthermore a total of ten novel yanuthones were characterized, whereof two did not originate from 6-MSA, but did utilize several of the enzymes encoded by the cluster, defining a new class of yanuthones. In A. aculeatus the 6-MSA pathway was found to differ, with conversion of 6-MSA into the terpenoid/non-ribosomal peptide/polyketide hybrids aculins A-B, for which a biosynthetic pathway has been proposed. By overexpression of a TF located immediately downstream of the 6-MSA synthase gene, two non-6-MSA derived compounds were additionally discovered. In A. nidulans a supercluster consisting of a non-ribosomal peptide synthase and a prenyltransferase was predicted, by full genome gene expression comparison, which ultimately linked to the tetracyclopeptide nidulanin A, illustrating the strength of bioinformatic tools to predict superclusters and structures of NRPs. Finally, it was investigated how entire gene clusters from filamentous fungi could be reconstituted in the
model organism A. nidulans. This project would improve discovery, and ease characterization, of genes. As proof of concept, the cytochalasin gene cluster from A. clavatus was successfully reconstituted in A. nidulans, allowing further biochemical characterization.
The work of this thesis represents a major step forward in extending the chemical knowledge on the complex secondary metabolism of important filamentous fungi and at the same time illustrates the enormous potential for still discovering novel SMs. A focused collaboration between bioinformatics, molecular biology, analytical and natural products chemistry is critical for advances in both the linking of fungal SMs to genes and unraveling the biosynthetic pathways, as well as for the discovery of novel SMs hidden in a treasury of biosynthetic potential of filamentous fungi.