Engineering of Streptomyces albus J1074 and Streptomyces lividans TK24 for Natural Products Production Dissertation zur Erlangung des Grades des Doktors der Naturwissenschaften der Naturwissenschaftlich-Technischen Fakultät der Universität des Saarlandes Von Yousra Ahmed Saarbrücken 2019 2 Tag des Kolloquiums: 09. August 2019 Dekan: Prof. Dr. rer. nat. Guido Kickelbick Berichterstatter: Prof. Dr. Andriy Luzhetskyy Prof. Dr. Rolf Müller Vorsitz: Prof. Dr. Volkhard Helms Akad. Mitarbeiter: Dr. Stefan Boettcher 3 Diese Arbeit entstand unter der Anleitung von Prof. Dr. Andriy Luzhetskyy in der Fachrichtung Pharmazeutische Biotechnologie der Naturwissenschaftlich-Technischen Fakultät der Universität des Saarlandes von Mai 2013 bis August 2018. 4 Acknowledgement Acknowledgments Firstly, I would like to express my deep sense of gratitude and respectful regards to my supervisor Prof. Dr. Andriy Luzhetskyy for giving me the opportunity to work in his group on these exciting projects. I highly appreciate his invaluable guidance and continuous support throughout my PhD studies. I am also thankful to him for reading the manuscript of this thesis and for his recommendations to improve it. I would like to thank Prof. Dr. Rolf Müller for his insightful comments and suggestions during the committee meetings and for being the second reviewer of this thesis. I am forever grateful to Dr. Yuriy Rebets, my lab supervisor, for his patience, motivation, and for sparing his valuable time whenever I needed. His useful experimental advice, constructive suggestions and scientific inspiration made it possible for me to achieve my goal. I am indebted to him for his help, suggestions and correction of the entire thesis. It has been an absolute pleasure to work with and learn from him. I am also very grateful to all my colleagues in AMEG group for their wonderful collaboration. They supported me impressively and were always willing to help me. Thanks a lot for the nice time we have spent together outside of work. I will cherish these memories forever. I would like to express my deepest gratitude to my parents, Samaa and MhD. Saleh, my sister Souhaila and my brothers: Ossama, Ahmed, Mouwafak and Abderrahman, who always believe in me and encourage me to follow my dreams. Words fail to express my appreciation and respect to them for their moral and emotional support in all aspects of my life. I would like to thank my nieces and nephews that made me laugh from my heart in the hardest moments. To Mouwafak and Abderrahman I am so grateful to have you here beside me and for helping in whatever way you could during all challenging times. Thanks a lot for the very delicious meals and for making sure that I did not have to cook when I was very busy. I am also obliged to my other family members and friends who have supported me along the way. I sincerely express my gratitude from my heart to my beloved husband, Ahmad, for his warm love, continued patience, and endless support during the happy and hard times. He has been a true and great supporter for the last two and half year of my studies, living every single minute of it. I thank God for enlightening my life with his presence. Without him this journey would not have been possible. Yousra Ahmed Saarbrücken, April 01, 2019 5 Publications and Conference Contributions Publications Amin, R., Franz-Wachtel, M., Tiffert, Y., Heberer, M., Meky, M., Ahmed, Y., Matthews, A., Krysenko, S., Jakobi, M., Hinder, M., et al. (2016) Post- translational Serine/Threonine Phosphorylation and Lysine Acetylation: A Novel Regulatory Aspect of the Global Nitrogen Response Regulator GlnR in S. coelicolor M145. Front. Mol. Biosci., 3, 38 Ahmed, Y., Rebets, Y., Tokovenko, B., Brötz, E., Luzhetskyy, A. 2017. Identification of butenolide regulatory system controlling secondary metabolism in Streptomyces albus J1074. Sci Rep 7:9784. Busche, T., Tsolis, K.C., Koepff, J., Rebets Y., Ruckert, C., Hamed, M.B., Bleidt, A., Wiechert, W., Lopatniuk, M., Ahmed, Y. et al. Multi-Omics and Targeted Approaches to Determine the Role of Cellular Proteases in Streptomyces Protein Secretion. Front Microbiol 2018, 9:1174. Rebets, Y., Tsolis, K.C., Guðmundsdóttir, E.E., Koepff, J., Wawiernia, B., Busche, T., Bleidt, A., Horbal, L., Myronovskyi, M., Ahmed, Y., Wiechert, W., Rückert, C., Hamed, M.B., Bilyk, B., Anné, J., Friðjónsson, Ó., Kalinowski, J., Oldiges, M., Economou, A., Luzhetskyy, A. Characterization of Sigma Factor Genes in Streptomyces lividans TK24 Using a Genomic Library-Based Approach for Multiple Gene Deletions. Frontiers in Microbiology 9, doi:10.3389/fmicb.2018.03033 (2018). Ahmed, Y., Rebets, Y., Rodríguez Estévez, M., Luzhetskyy, A. Engineering of Streptomyces lividans for heterologous expression of secondary metabolites gene clusters (manuscript in preparation) Conference Contributions Amin, R., Tiffert, Y., Heberer, M., Ahmed, Y., Krysenko, S., Jakobi, M., Hinder, M., et al. Post-translational Serine/Threonine Phosphorylation and Lysine Acetylation: A Novel Regulatory Aspect of the Global Nitrogen Response Regulator GlnR in S. coelicolor M145, the Annual Meeting of the part "Association for General and Applied Microbiology“ Tuebingen, Germany (VAAM). (Poster) Ahmed, Y., Rebets, Y., Brötz, E. and Luzhetskyy, A. new strategy in awaking silent secondary metabolites biosynthesis gene clusters in Streptomyces albus J1074, VAAM workshop, Freiburg, Germany 2014. (Poster) Ahmed, Y., Rebets, Y., Brötz, E. and Luzhetskyy, A. Regulatory elements silencing the secondary metabolism in Streptomyces albus J1074. 5th HIPS Symposium on Pharmaceutical Sciences Devoted to Infection Research, Saarbruecken, 2015. (Poster) 6 Abstract Abstract Actinobacteria have remarkable chemical potential that is not explored due to low level of corresponding genes expression. In order to uncover this reservoir of natural products we developed a reporter-guided screening strategy combined with transposon mutagenesis. It was used to activate silent polycyclic tetramate macrolactam biosynthesis gene cluster in Streptomyces albus J1074. As result, the mutant with awaken secondary metabolism was obtained. Analysis of this strain led to identification of new regulatory system consisting of transcriptional regulator XNR_3174 and bacterial hormone-like compound butenolide. XNR_3174 and butenolide biosynthesis genes orthologues are present in the genomes of different Streptomyces. The identified regulatory system comprises a new condition-depended cascade controlling secondary metabolism in Actinobacteria. We also developed new host strains for heterologous production of natural products by deleting 11 endogenous secondary metabolite gene clusters from chromosome of S. lividans and introducing up to 2 sites for integration of foreign DNA. When expressing three heterologous gene clusters the generated hosts have shown better performance than the parental strain. S. lividans TK24 was also improved as a host for heterologous protein production by deleting a set of proteases encoding genes. The developed strains represent a step forward to a better panel of organisms for bioprospecting and genome mining of novel natural products. 7 Zusammenfassung Zusammenfassung Actinobakterien besitzen ungeahntes chemisches Potenzial, das aufgrund niedriger Exprimierung entsprechender Gene nicht erforscht werden kann. Um Dieses aufzudecken, wurde eine Kombination aus reportergeführter Screening-Strategie und Transposon Mutagene entwickelt. Die Verwendung dieser Strategie führte zur Aktivierung des polyzyklischen Tetramat- Makrolaktam Gen-Clusters in Streptomyces albus J1074. Der erhaltene Stamm weißt eine aktivierte Produktion von Sekundärmetaboliten auf und Analysen führten zur Identifizierung eines Regulationssystems, bestehend aus dem transkriptionellen Regulator XNR_3174 und dem hormonähnlichen Naturstoff Butenolid. Orthologe von XNR_3174 und der Butenolid Gene findet man in Genomen verschiedener Streptomyceten. Das identifizierte System umfasst die neue „condition-dependent“-Kaskade, die den Sekundärstoffwechsel in Actinobakterien steuert. Zusätzlich entwickelten wir, durch das Entfernen von 11 endogenen Gen-Clustern aus dem Genom von S. lividans und dem Einfügen von zwei DNA Integrationsstellen, neue Wirtsstämme für die heterologe Produktion von Naturstoffen. Bei der heterologen Exprimierung von drei Gen- Clustern zeigten die optimierten Wirte bessere Resultate als der ursprüngliche Stamm. Durch das Entfernen der Proteasegene wurde S. lividans TK24 als Wirt für die Proteinproduktion verbessert. Die entwickelten Stämme vereinfachen die Bioprospektion und die Entdeckung neuer Naturstoffe unter Verwendung des „Genome-Mining“ Ansatzes. 8 Table of Contents Table of Contents Acknowledgment 5 Publications and Conference Contributions 6 Abstract 7 Zusammenfassung 8 Table of Contents 9 1. Introduction 11 1.1 Importance of Natural Products in Medicines Development 11 1.2 Natural Products of Actinobacteria 14 1.3 Activation of Silent Biosynthetic Gene Clusters 15 1.3.1 OSMAC: One Strain Many Compounds 16 1.3.2 Manipulation of Transcriptional and Translational Machineries 18 1.3.3 Manipulation of Global and Pathway-Specific Regulators 19 a) Manipulation of global Regulators 20 b) Manipulation of Pathway-Specific Regulators 21 1.3.4 Gene Cluster Refactoring 23 1.3.5 Reporter-Guided Mutant Selection (RGMS) 25 1.4 Heterologous Expression of Secondary Metabolite Gene Clusters 27 1.4.1 Streptomyces coelicolor 28 1.4.2 Streptomyces avermitilis 29 1.4.3 Streptomyces albus 29 1.4.4 Streptomyces lividans 30 1.5