Actinomycetes from neglected areas, Iranian soil, their taxonomy, and secondary metabolites Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades einer Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Shadi Khodamoradi aus Tabriz / Iran 1. Referent: Professor Dr. Michael Steinert 2. Referent: Privatdozent Dr. Joachim Wink eingereicht am: 09.02.2021 mündliche Prüfung (Disputation) am: 16.04.2021 Druckjahr 2021 ii Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Khodamoradi S, Stadler M, Wink J, Surup F. Litoralimycins A and B, New Cytotoxic Thiopeptides from Streptomonospora sp. M2. Marine Drugs. 2020 Jun;18(6):280. Conference Talk Shadi Khodamoradi. Streptomonospora litoralis sp. nov., a halophilic actinomycetes with antibacterial activity from beach sands. 11th International PhD Symposium, Braunschweig, Germany (2018) Posterbeiträge Shadi Khodamoradi, Richard L. Hahnke, Frank Surup, Manfred Rohde, Michael Steinert, Marc Stadler, Joachim Wink. Isolation of a new Streptomonospora-species producing a new peptide antibiotic. The HIPS Symposium, Saarbrücken, Germany (2019). Shadi Khodamoradi. Isolation of new Streptomyces sp. producing the new antibacterial compound from the soil sample collected from Iran/ Tehran/ Alborz Mountain HZI. 12th International PhD Symposium, Braunschweig, Germany (2019) Shadi Khodamoradi. Isolation, characterization, and Antagonistic Properties of novel species of halophilic Streptomonospora.10th International PhD Symposium, Braunschweig, Germany (2017) iii Acknowledgements First of all, I want to give my sincere gratitude to my principal supervisor PD Dr. Joachim Wink for giving me the chance to explore the wonderful science and sharing his knowledge with me. I appreciate his patience and constant support throughout my PhD journey. I would also like to thank Prof. Dr. Marc Stadler for his great support and giving the opportunity to work in his group. Your guidance and teaching have developed my skill in many ways, especially in academic writing. I thank my associated supervisor Prof. Dr. Michael Steinert for his scientific advice and support. I would like to thank Dr. Stephan Hüttel for his kind support, continuous help and encourage during my PhD study. I would like to give special thanks to my collaboration partners Dr. Frank Surup, Dr. Kathrin Mohr, Dr. Kathrin Wittstein, Dr. Steffen Bernecker, Dr Manfred Rohde, Prof. Dr. Peter Kämpfer, Dr. Eike Steinmann, Dr. Christian Rückert, and Christel Kakoschke. My sincere thanks to my coauthors Prof. Dr. Yvonne Mast for her kind help and advices, Dr. Peter Schumann, Dr. Richard Hahnke, and other members of DSMZ, Dr. Meina Neumann-Schaal and Gabriele Pötter. I would like to further acknowledge to our graduate school in the HZI for their astonishing support and organization during my study especially Dr. Sabine Kirchhoff. I am much grateful to Prof. Dr. Ludger Beerhues for taking over the chair during my defense. My deepest thank to Romy Schade and Silke Reinecke for their great technical support. I would like to mention my appreciation to all members of the MWIS group, especially to Kerstin Schober, Esther Surges, Axel Schulz, Kevin Becker, and Sebastian Pfütze for helping me with chemistry part. I extend my iv Acknowledgements special thanks to my colleague Dr. Chandra Risdian for sharing his knowledge and so many advices in the last years. I would also like to express my gratitude to my group members, Stephanie Schulz, Klaus Peter Conrad, Aileen Gollasch, Birte Trunkwalter, Wera Collisi and Jolanta Lulla for the professional technical support. I thank all of my other colleagues, Nasim Safaei, Senlie Octaviana, Rina Andriyani, Hani Pira, Sana Ullah Tareen for the warm environment in our group. Also I thank previous students Dr. Lucky Mulwa and Dr. Omar Messaoudi for their kind scientific advices during my PhD study. Many thanks to all friends for their support and all the happy time we spent together during this journey. Finally, I would like to thank my kind family for their love and support throughout the years, which enabled me to follow my dreams. v List of Contents Table of Contents Acknowledgements ............................................................................................................. iv List of Figures ..................................................................................................................... ix List of Tables ....................................................................................................................... xi List of Supplementary Figures ........................................................................................... xii List of Supplementary Tables ............................................................................................ xiii List of Abbreviations ......................................................................................................... xiv Abstract .............................................................................................................................. xv Abstrakt ............................................................................................................................. xvi 1 Introduction ....................................................................................................................... 1 1.1 Natural products ......................................................................................................... 1 1.2 The Class of Actinobacteria ....................................................................................... 4 1.2.1 Rare Actinobacteria ............................................................................................. 6 1.2.2 Bioactive compounds from rare actinomycetes................................................... 7 1.3 Prokaryotic taxonomy ................................................................................................ 9 1.4 The polyphasic classification in Actinobacteria ....................................................... 10 1.4.1 Phenotypic classification ................................................................................... 10 1.4.1.1 Microscopic and macroscopic characteristics ............................................ 10 1.4.1.2 Physiological characteristics ...................................................................... 11 1.4.2 Chemical classification (chemotaxonomy) ....................................................... 12 1.4.3 Genomic classification ...................................................................................... 13 1.5 Dereplication of compounds from Actinobacteria ................................................... 14 1.6 Previous work in search of novel Actinobacteria and their secondary metabolites . 15 1.7 Study aims ................................................................................................................ 16 5) Taxonomic study of the identified novel Actinobacteria strains. .............................. 16 2. Materials and Methods ................................................................................................... 17 2.1 Materials ....................................................................................................................... 17 2.1.2 Chemicals .......................................................................................................... 24 2.1.3 Equipment ......................................................................................................... 28 2.1.4 Primers............................................................................................................... 31 2.1.5 Kits and Enzymes .............................................................................................. 32 2.1.6 Microorganisms ................................................................................................. 33 2.2 Method ..................................................................................................................... 34 2.2.1 Sampling ............................................................................................................ 34 2.2.2 Isolation ............................................................................................................. 34 vi List of Contents 2.2.3 Cultivation ......................................................................................................... 35 2.2.3.1 Cultivation of Actinobacteria strains .......................................................... 35 2.2.3.2 Cultivation in production media and extraction of secondary metabolite .. 35 2.2.4 Taxonomic classification ................................................................................... 35 2.2.4.1 Extraction, amplification, sequencing of 16S rRNA .................................. 36 2.2.4.2 MALDI-TOF analysis ................................................................................ 37 2.2.4.3 RiboPrinter® analysis ................................................................................ 38 2.2.4.5 DNA-DNA hybridization ........................................................................... 38 2.2.4.6 Whole genome sequencing ......................................................................... 39