Metabolic and Genomic Profiling of Actinobacteria Strains: from New Natural Products to Biosynthetic Pathways Dissertation zu Erlangung des Grades des Doktors der Naturwissenschaften der Naturwissenschaftlich-Technischen Fakultät der Universität des Saarlandes von Constanze Paulus Saarbrücken 2018 Tag des Kolloquiums: 01. Februar 2019 Dekan: Prof. Dr. Guido Kickelbick Berichterstatter: Prof. Dr. Andriy Luzhetskyy Prof. Dr. Christian Ducho Vorsitz: Prof. Dr. Anna Hirsch Akad. Mitarbeiter: Dr. Angelika Ullrich 2 Diese Arbeit entstand unter der Anleitung von Prof. Dr. Andriy Luzhetskyy in der Fachrichtung 8.2, Pharmazeutische Biotechnologie der Naturwissenschaftlich-Technischen Fakultät der Universität des Saarlandes von Januar 2015 bis Oktober 2018. 3 Acknowledgements My deepest gratitude goes to my supervisor Prof. Dr. Andriy Luzhetskyy for giving me the opportunity to do my PhD in his group. I would like to thank him for all the interesting and challenging projects I was working on and for the great support and encouragement throughout that time. Furthermore, I would like to express my thanks to my mentor Dr. Yuriy Rebets. I was lucky to have such a good teacher who introduced me into the world of microbiology and who answered every question I had during my PhD. I also owe my special gratitude to my second supervisor Prof. Dr. Christian Ducho. I thank him for his time and the useful advices to my work in the committee meetings. Moreover, I would like to thank Birgit Rosenkränzer who facilitated the entry into the new work environment and laboratory work. I owe my thanks also to Dr. Thomas Paululat, Dr. Suvd Nadmid and Dr. Josef Zapp for supporting me in NMR questions and for all useful discussions regarding structure elucidation problems. I would like to thank of course all my colleagues for the pleasant working atmosphere in the laboratory and all discussions that helped to find solutions during difficult steps within a project. And moreover for having a great time and so many nice and fun activities during the last three years. Mein tiefster Dank gilt meinen Eltern Richard und Ursula Paulus. Dafür, dass sie immer für mich da sind, mich zu jedem Zeitpunkt unterstützen und mir vor allem in schwierigen Zeiten immer Mut zusprechen. Der Dank geht auch an meine drei lieben Geschwister, Julia, Philipp und Catharina Paulus, dafür, dass sie mir immer zur Seit stehen würden und für all die schönen Momente, die wir zusammen verbracht haben trotz der Entfernung. 4 Publications Sun, Y.Q.; Busche, T.; Rückert, C.; Paulus, C.; Rebets, Y.; Novakova, R.; Kalinowski, J.; Luzhetskyy, A.; Kormanec, J.; Sekurova, O.N.; Zotchev, S.B. Development of a biosensor concept to detect the production of cluster-specific secondary metabolites, ACS Synthetic Biology, 2017, 6 (6), 1026-1033. Paulus, C.; Rebets, Y.; Tokovenko, B.; Nadmid, S.; Terekhova, L.P.; Myronovski, M.; Zotchev, S.B.; Rückert, C.; Braig, S.; Zahler, S.; Kalinowski, J.; Luzhetskyy, A. New natural products identified by combined genomics-metabolomics profiling of marine Streptomyces sp. MP 131- 18, Scientific Reports, 2017, 7, 42382. Paulus, C.; Rebets, Y.; Zapp, J.; Rückert, C.; Kalinowski, J.; Luzhetskyy, A. New Alpiniamides from Streptomyces sp. IB2014/011-12 assembled by an unusual hybrid nonribosomal peptide synthetase trans-AT polyketide synthase enzyme, Frontiers in Microbiology, 2018, 9, 1959. Conference contributions Paulus, C.; Rebets, Y.; Tokovenko, B.; Nadmid, S.; Terekhova, L.P.; Myronovski, M.; Zotchev, S.B.; Rückert, C.; Braig, S.; Zahler, S.; Kalinowski, J.; Luzhetskyy, A. New natural products identified by combined genomics-metabolomics profiling of marine Streptomyces sp. MP 131- 18, 7th HIPS Symposium, Saarbrücken, Germany, 2016 (poster) Paulus, C.; Rebets, Y.; Tokovenko, B.; Nadmid, S.; Terekhova, L.P.; Myronovski, M.; Zotchev, S.B.; Rückert, C.; Braig, S.; Zahler, S.; Kalinowski, J.; Luzhetskyy, A. New natural products identified by combined genomics-metabolomics profiling of marine Streptomyces sp. MP 131- 18, VAAM international Workshop, Freiburg, 2016 (poster) 5 Zusammenfassung Marine Aktinomyceten stellen eine beliebte Quelle neuer Naturstoffe dar, welche unter Umständen Anwendung als therapeutische Arzneimittel finden. Daher steht die Untersuchung neuer Aktinomyceten Stämme im Fokus der Naturstoffforschung. Die dargelegte Arbeit handelt von der Isolierung und strukturellen Charakterisierung neuer Naturstoffe, welche von Streptomyceten produziert wurden. Desweiteren, wird die Konstruktion eines neuen Biosensors dargestellt, welcher die Produktion von Sekundärmetaboliten detektieren soll. Es wurden zwei neue spiroindimicine E und F aus der Streptomyces sp. MP131-18 isoliert und die chemische Struktur mittels NMR bestätigt. Das Genom von MP131-18 wurde sequenziert und der Gencluster, welcher verantwortlich ist für die Synthese der Bisindole-Substanzen identifiziert und den Lynamicinen/Spiroindmicinen zugeordnet. Weiterhin, wurden vier neue Alpiniamide B-D aus dem Stamm Streptomyces sp. IB 2014/11-12 isoliert. Das sequenzierte Genom ermöglichte die Identifizierung des Genclusters, welcher für die Alpiniamide Produktion verantwortlich ist. Die vorgeschlagene Biosyntheseroute wurde bestätigt durch Fütterungsexperimente und Gendeletierungen in einem Hostorganismus. Abschließend wurde ein Beitrag zur Konstruktion eines Repressor-basierender Biosensors geleistet, welcher die Aktivierung von zuvor inaktiven Gencluster in Streptomyceten detektieren soll. Dieses Konzept wurde erfolgreich an dem aktivierten Coelimycin Gencluster angewendet. 6 Abstract Marine actinomycetes are known to be a promising source for new natural products with putative application as therapeutic agents. Thus, the exploitation of novel discovered actinomycetes strains remains in the focus of NP research. The presented thesis deals on the one hand with the isolation and structural characterization of new NPs produced by streptomycetes species. On the other hand, the construction of a novel biosensor concept for the detection of secondary metabolite production is discussed. New spiroindimicins E and F were isolated from the Streptomyces sp. MP131-18 and structures were confirmed by NMR. Additionally, two new lagunapyrones D and E were identified by characteristic MS/MS fragmentation. The genome of MP131-18 has been sequenced and the gene cluster, responsible for the synthesis of bisindole compounds has been identified and connected to lynamicins/spiroindimicin production. Furthermore, four new alpiniamides B-D have been isolated from the Streptomyces sp. IB 2014/11-12. The sequenced genome enabled the identification of the gene cluster responsible for alpiniamide production. The predicted biosynthetic pathway was confirmed by feeding experiments and gene deletions in a heterologous host. Finally, a contribution to the construction of a repressor-based biosensor, which detects the products of awakened silent gene clusters in streptomycetes, has been made. This concept was successfully applied to the activated coelimycin gene cluster. 7 Table of Contents Acknowledgments ...................................................................................................................... 4 Publications and contributions to conferences ........................................................................... 5 German and English abstract ...................................................................................................... 6 1. Introduction .......................................................................................................................... 9 1.1 Impact of Natural Products in Drug Development ................................................... 9 1.2 Natural Products from Actinobacteria .................................................................... 11 1.3 Dereplication .......................................................................................................... 14 1.4 Genomic Profiling of Actinomycetes ..................................................................... 16 1.4.1 Bacterial Genome Sequencing and Identification of Biosynthetic Gene Cluster (BGC) ................................................................................................... 16 1.4.2 Enzyme Machinery for Secondary Metabolite Production ..................... 18 1.5 Isolation and Characterization of Secondary Metabolites ...................................... 27 1.6.1 Extraction and Purification ...................................................................... 27 1.6.2 Structure Elucidation and Assignment of Stereochemistry ..................... 27 1.6.2.1 Nuclear Magnetic Resonance Spectroscopy (NMR) ................ 27 1.6.2.2 Determination of Stereochemistry ............................................ 28 1.6 Outline of the Work ................................................................................................ 32 References .................................................................................................................... 34 2. Publications ......................................................................................................................... 42 New natural products identified by combined genomics-metabolomics profiling of marine Streptomyces sp. MP 131-18 ....................................................................................................... I New alpiniamides from Streptomyces sp. IB 2014/011-12 assembled by an unusual hybrid nonribosomal peptide synthetase trans-AT polyketide synthase