Petersen, Lene Maj

Filamentiøse svampe producerer små bioaktive molekyler, betegnet sekundære metabolitter, som spænder bredt i både funktionel og strukturel diversitet. De sekundære metabolitter kan være biologisk aktive mod f.eks. bakterier, insekter, humane celler eller andre svampe. Genomerne for adskillige filamentiøse svampe er på det seneste blevet sekventeret, hvilket har afsløret et stort antal genklustre, der koder for biosynteser, og dermed blotlagt et enormt potentiale af hidtil uopdagede sekundære metabolitter, der potentielt kan udnyttes til medicinske formål.Arbejdet, der præsenteres i denne afhandling, har fokuseret på filamentiøse svampe indenfor den vigtige og spændende slægt Aspergillus. Den indeholder arter der; kontaminerer fødevarer og foder, bruges som modelsystemer til genetiske undersøgelser, er patogene overfor mennesker, og bruges til industrielle applikationer, f.eks. til produktion af både små molekyler og enzymer.Formålet for dette PhD studie har været opdelt i to hovedtemaer:1) Opdagelse og karakterisering af nye metabolitter fra filamentiøse svampe2) Kobling af svampe sekundære metabolitter til gener, samt udredning af biosyntesevejeDen første del af dette studie, opdagelsen af nye sekundære metabolitter, har resulteret i karakterisering af 22 nye sekundære metabolitter fra udvalgte Aspergiller. To forskellige tilgange blev udforsket: Dereplikerings baseret opdagelse samt opdagelse ved induktion af sclerotium dannelse. Resultatet omfatter karakterisering af følgende nye metabolitter: aculene A-D, acucalbistrin A-B, acu-dioxomorpholine, okaramine S, samt epi-10,23- dihydro-24,25-dehydroaflavinin fra A. aculeatus, aspiperidine oxide fra A. indologenus, homomorphosin A-F fra A. homomorphus, sclerotionigrin A-B fra A. sclerotioniger, emindole SC, sclerolizine samt carbonarin I-J fra A. sclerotiicarbonarius. Desuden blev flere kendte metabolitter, rapporteret i de forskellige arter for første gang. Et vigtigtresultat af dette studie, er opdagelsen af, hvordan svampe relaterede strukturer kaldet sclerotier, kan induceres i sorte Aspergiller, ved at kombinere brugen af naturlige substrater og indførelse af et frysningstrin forud for podning. Dette er af generel interesse for opdagelsen af nye bioaktive metabolitter, da sclerotiedannelse, aktiver biosynteseveje, der ellers ikke er udtrykte, og som en direkte konsekvens, ses en stor ændring i metabolitprofilen.Den anden del af PhD studiet, hvor gener kobles til sekundære metabolitter og biosynteseveje udledes, førte til indsigt i det biosyntetiske potentiale i en række Aspergiller. Dette omfattede en sammenligning af den kemiske profil af A. oryzae og A. flavus, der er to nærtbeslægtede arter (99,5% homologi på genomniveau), men som viser en overraskende høj grad af forskelle i deres kemiske profiler. Opklaring af kemiske strukturer for nye stoffer fra A. oryzae, afslørede dog hidtil usete kemiske link mellem de to arter. I to parallelle projekter, i henholdsvist A. niger og A. aculeatus, blev polyketidet 6- methyl-salicylsyre (6-MSA) og de respektive biosynteseveje med udgangspunkt i 6-MSA undersøgt. I A. niger blev 6-MSA omdannet til meroterpenoidet yanuthone D. Via enmultidisciplinær tilgang kunne generne, i et genkluster, ansvarligt for produktionen af yanuthone D identificeres, og biosyntesevejen over 8 trin udledes. Desuden blev ti nye yanuthoner karakteriseret, hvoraf to viste sig ikke at være afledt af 6-MSA. Dog var produktionen af disse to yanuthoner afhængig af flere af enzymerne kodet af det pågældende genkluster ansvarlig for yanuthone D, hvilket, definererede en ny klasse af yanuthoner. A. aculeatus 6-MSA biosyntesevejen varierede. Her blev 6-MSA omdannet til aculiner, hybrider af terpener/ikke-ribosomale peptider/polyketider, for hvilke der blev foreslået en biosyntesevej, baseret på bioinformatiske og strukturkemiske betragtninger. En transskriptionsfaktor tæt på 6-MSA synthasen, blev overudtrykt, hvilket resulterede ikarakterisering af yderligere to stoffer, der dog ikke var afledte fra 6-MSA. I A. nidulans blev to fysisk adskilte gener, bestående af en non-ribosomal peptidsynthetase og en prenyltransferase forudset til at være koblet til den samme biosyntesevej. Dette blev eftervist og forbundet med biosyntesen af tetra-cyclopeptidet nidulanin A, hvilket illustrerede styrken af bioinformatiske værktøjer til at forudsige sammenhænge mellem fysisk adskilte gener, samt strukturer af non-ribosomale peptider. Endelig blev det undersøgt, hvordan hele genklustre fra svampe kunne udtrykkes heterologt i modelorganismen A. nidulans. Dette projekt vil forbedre opdagelse samt karakterisering af gener involveret i biosynteser. Cytochalasin biosyntesevejen fra A. clavatus blev undersøgt og med succes rekonstrueret i A. nidulans.Resultaterne af dette PhD studie bringer ny kemisk viden om den komplekse sekundære metabolisme af vigtige filamentiøse svampe og illustrerer samtidig det enorme potentiale for at opdage nye sekundære metabolitter. Et målrettet samarbejde mellem bioinformatik, molekylærbiologi, analytisk kemi og naturstof kemi er afgørende for fremskridt i både koblingen af svampe metabolitter til gener og udledning af biosynteseveje, samt til opdagelsen af de mange nye metabolitter, der stadig ligger skjult i de filamentiøse svampe.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 beexploited, 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 fungi2) Linking of fungal SMs to genes and elucidation of biosynthetic pathwaysThe 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 themodel 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

Online Research Database In Technology

Linking fungal secondary metabolites and pathways to their genes in Aspergillus

http://orbit.dtu.dk/ws/files/127767218/Linking_fungal_secondary_metabolites.pdf

Linking fungal secondary metabolites and pathways to their genes in Aspergillus

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