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Exploring the Biosynthetic Potential of Cystobacter Fuscus

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Exploring the Biosynthetic Potential of Cystobacter Fuscus Exploring the Biosynthetic Potential of Cystobacter fuscus – Characterization of New Structures and Studies on their Biosynthesis Dissertation zur Erlangung des Grades des Doktors der Naturwissenschaften der Naturwissenschaftlich-Technischen Fakultät III Chemie, Pharmazie, Bio- und Werkstoffwissenschaften der Universität des Saarlandes von Lena Etzbach Saarbrücken 2015 Tag des Kolloquiums: 17.04.2015 Dekan: Prof. Dr. Dirk Bähre Berichterstatter: Prof. Dr. Rolf Müller Prof. Dr. Rolf Hartmann Vorsitz: Prof. Dr. Uli Kazmaier Akad. Mitarbeiter: Dr. Martin Frotscher Diese Arbeit entstand unter der Anleitung von Prof. Dr. Rolf Müller in der Fachrichtung 8.2, Pharmazeutische Biotechnologie der Naturwissenschaftlich- Technischen Fakultät III der Universität des Saarlandes von August 2011 bis Februar 2015. “Twenty years from now you will be more disappointed by the things you didn’t do than by the ones you did. So throw off the bowlines, sail away from the safe harbor. Catch the trade winds in your sails. Explore. Dream. Discover.” Marc Twain Danksagung An erster Stelle möchte ich meinem Doktorvater Prof. Dr. Rolf Müller für sein mir entgegengebrachtes Vertrauen und den Freiraum für mein Projekt danken, für die Bereitstellung der außergewöhnlichen instrumentellen Ausstattung sowie für die vielen Ratschläge und die gute Zusammenarbeit. Auch meinem Co-Betreuer Prof. Dr. Rolf W. Hartmann möchte ich für die Übernahme dieser Aufgabe danken. A big thank you goes to Dr. Alberto Plaza who was a great mentor and friend in the last years. He always encouraged me to discover new opportunities and to follow my own ways. Bedanken möchte ich mich auch bei der ganzen Arbeitsgruppe für die gute Zusammenarbeit. Bei Dr. Sascha Baumann für die spontane Übernahme der Betreuung im letzten Jahr meiner Arbeit. Bei Katrin Jungmann und Hilda Sucipto für das Beantworten meiner vielen Fragen rund um die Molekularbiologie sowie bei Dr. Thomas Hoffmann, der mir von Anfang an immer mit Rat und Tat zur Seite stand. Außerdem bei meinen anderen „supergeilen Kollegen“, die ein wichtiger Grund dafür waren, dass ich jeden Morgen gerne zur Arbeit gegangen bin, auch wenn es in der Forschung auf und ab ging. Danke für jedes TGIF-Bier, bei dem Erfolge gefeiert und Niederlagen vergessen wurden! Ein besonderes Anliegen ist es mir, mich bei meinen Eltern Sylvia und Karl-Heinz sowie bei meiner Schwester Britta für die langjährige Unterstützung zu bedanken. Ohne euch wäre ich nicht da, wo ich heute bin. Zu guter Letzt möchte ich mich bei meinem Verlobten Björn Keller bedanken, ohne den diese Promotion nicht möglich gewesen wäre. Dafür, dass er mir in den letzten Jahren immer den Rücken frei gehalten hat und sich unzählige Probevorträge von mir angehört hat. Danke für das Verständnis, die unbezahlbare Unterstützung und auch für das Korrekturlesen meiner Dissertation. Summary Natural products provide an important basis for the drug development in particular to fight the increasing resistance of microorganisms against existing drugs. Myxobacteria, gram negative ubiquitous soil bacteria belonging to the class of γ-proteobacteria, have proven an impressive capacity for the production of chemically intriguing natural products. In a chemical screening approach, Cystobacter fuscus MCy9118 was used to isolate new natural products based on chemical and structural features where two new compound families were identified and characterized. The cystomanamides are characterized by hydroxylated amino acids, an unusual branched chain fatty acid residue as well as N-glycosylation. The macyranones are unusual peptides belonging to the class of peptidic α’,β’-epoxyketones. Macyranone A exhibits potent and selective activity against Trypanosoma brucei rhodesiense, the parasitic agent causing the African sleeping sickness. The target was identified as 20S proteasome by enzymatic assays. A co-crystal structure of macyranone A with the yeast 20S proteasome revealed the binding mode underlying the compound-target interaction. Macyranone A represents the successful isolation of a new natural product with identified mode of action, which can be useful for the drug development process against the African sleeping sickness. The biosynthetic gene clusters for both compound families were identified as PKS / NRPS hybrids by in silico analysis and confirmed by gene deletion. Zusammenfassung Naturstoffe bilden eine wichtige Grundlage zur Entwicklung neuer Wirkstoffe für die Arzneimitteltherapie, vor allem im Kampf gegen Antibiotikaresistenzen bei Pilzen, Viren, Parasiten und Bakterien. Myxobakterien, gramnegative ubiquitäre Bodenbakterien, haben eine beeindruckende Kapazität zur Produktion von chemisch faszinierenden Naturstoffen unter Beweis gestellt. Das Myxobakterium Cystobacter fuscus MCy9118 wurde in einem chemischen Screening auf die produzierten Naturstoffe hin untersucht und dabei konnten zwei neue Substanzfamilien charakterisiert werden. Die Cystomanamide weisen als besondere Strukturelemente hydroxylierte Aminosäuren, eine verzweigte Fettsäurekette sowie eine N-Glykosylierung auf. Bei der zweiten Substanzgruppe handelt es sich um die Macyranone, sehr ungewöhnliche Verbindungen aus der Gruppe der peptidischen α’,β’-Epoxyketone. Macyranone A zeigt biologische Aktivität gegen Trypanosoma brucei rhodesiense, dem parasitären Erreger der Afrikanischen Schlafkrankheit. Das 20S Proteasom konnte als Target identifiziert und durch eine Cokristallisation des Macyranone A mit dem 20S Proteasom der Hefe bestätigt werden. Damit konnte ein Naturstoff isoliert werden, der mit bekanntem Wirkmechanismus als Ansatzpunkt für die Entwicklung neuer Wirkstoffe gegen die Schlafkrankheit genutzt werden kann. Die Biosynthesegencluster beider Substanzklassen wurden durch in silico Analyse als PKS-NRPS Hybrid identifiziert und durch den Knockout von beteiligten Genen bestätigt. Vorveröffentlichungen der Dissertation Teile dieser Arbeit wurden vorab mit Genehmigung der Naturwissenschaftlich- Technischen Fakultät III, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen veröffentlicht oder sind derzeit in Vorbereitung zur Veröffentlichung: Publikationen Etzbach L., Plaza A., Garcia R., Baumann S., Müller R. (2014) Cystomanamides: Structure and Biosynthetic Pathway of a Family of Glycosylated Lipopeptides from Myxobacteria Org. Lett., 16 (9), 2414-2417. Etzbach L., Plaza A., Dubiella C., Groll M., Kaiser M., Müller R. (2015) Macyranones: Structure, Biosynthesis and Binding Mode of an Antiparasitic Epoxyketone that Targets the 20S Proteasome, submitted Tagungsbeiträge Etzbach, L., Plaza A., Garcia R., Harmrolfs K., Müller R. (2012) New Natural products from Myxobacteria of the Recently Discovered Genus „Aetherobacter“ (Poster) International Congress on Natural Products Research, NY, USA Etzbach, L., Plaza A., Garcia R., Harmrolfs K., Müller R. (2012) New Natural products from a Myxobacterium of the Recently Discovered Genus „Aetherobacter“ (Poster) VAAM International Workshop 2012, Braunschweig, Germany Etzbach, L., Plaza A., Garcia R., Müller R. (2013) Macyranine A: An Unusual Peptide from the Myxobacterial Genus Cystobacter Discovered by LC-SPE-NMR (Poster) 1st European Conference on Natural Products, Frankfurt am Main, Germany Etzbach, L., Plaza A., Garcia R., Baumann S., Müller R. (2014) Cystomanamides: Structure and Biosynthetic Pathway of a Family of Glycosylated Lipopeptides from the Myxobacterial Genus Cystobacter (Poster) GRC Marine Natural Products, Ventura, CA, USA Etzbach, L., Plaza A., Garcia R., Baumann S., Müller R. (2014) Cystomanamides: A Family of Novel Glycosylated Lipopeptides from Myxobacteria (Vortrag) Summer Symposium of the Interdisciplinary Graduate School of Natural Products Research, Saarbrücken, Germany Etzbach, L., Plaza A., Garcia R., Baumann S., Müller R. (2014) Cystomanamides: Structure and Biosynthetic Pathway of a Novel Family of Glycosylated Lipopeptides from Myxobacteria (Vortrag) VAAM Workshop Biology of Natural Products-Producing Microorganisms, Dresden, Germany Weitere Publikationen Fu C., Etzbach L., Bauer A., Brönstrup M., Froidbise A., Hammann P., Herrmann J., Mondesert G., Kurz M., Schiell M., Schummer D., Toti L., Wink J., Müller R. (2015) Biosynthetic Studies of Telomycin Reveal Unprecedented Lipopeptides with Enhanced Activity, submitted Table of Contents Danksagung ...................................................................................................................... 12 Summary ........................................................................................................................... 12 Zusammenfassung .......................................................................................................... 12 Vorveröffentlichungen der Dissertation .................................................................... 12 1 Introduction .............................................................................................................. 12 1.1 Importance of Natural Products for Drug Discovery ........................................................ 12 1.2 Discovery of Natural Products from Myxobacteria ........................................................... 13 1.2.1 Polyketide synthases (PKS) ............................................................................................... 15 1.2.2 Nonribosomal peptide synthetases (NRPS) .................................................................... 18 1.2.3 Mixed PKS/NRPS hybrids ...............................................................................................
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