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Discovery of Bioactive Natural Products from Actinomycetes by Inactivation and Heterologous Expression of Biosynthetic Gene Clusters

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- 1 - Discovery of Bioactive Natural Products from Actinomycetes by Inactivation and Heterologous Expression of Biosynthetic Gene Clusters Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Albert-Ludwigs-Universität Freiburg im Breisgau Vorgelegt von Jing Zhu Aus Wuhan, China 2019 - 2 - Dekan: Prof. Dr. Oliver Einsle Vorsitzender des Promotionsausschusses: Prof. Dr. Stefan Weber Referent: Prof. Dr. Andreas Bechthold Korreferent: Prof. Dr. Irmgard Merfort Drittprüfer: Prof. Dr. Stefan Günther Datum der mündlichen Prüfung: 15.05.2019 Datum der Promotion: 04.07.2019 - 3 - Acknowledgements I would like to express my greatly gratitude to my supervisor, Prof. Dr. Andreas Bechthold for giving me the opportunity to study at his group in University of Freiburg and for his enthusiasm, encouragement and support over the last 4 years. I also appreciate his patience and good suggestions when we discuss my project. I also want to thank my second supervisor Prof. Dr. Irmgard Merfort for kind reviewing my dissertation and caring of my research, her serious attitude for scientific research infected me. I would like to express my appreciation to Prof. Dr. Stefan Günther for being the examiner of my dissertation defense. I am very grateful to Dr. Thomas Paululat from University of Siegen for reviewing my manuscript and for structures elucidation and NMR data preparation. I also want to thank Dr. Manfred Keller and Christoph Warth for NMR and HR-ESIMS measurement. I would like to thank Prof. Dr. David Zechel from Queen's University for reviewing my manuscript. I would like to thank Dr. Lin Zhang for his great help with protein crystallization and for his technical advices. I am grateful to Dr. Claudia Jessen-Trefzer for her kind help about my projects. I am especially thankful to Dr. Roman Makitrynskyy and Olya Tsypik for their useful discussions and suggestions of my study. I am really thankful to Prof. Dr. Mervyn Bibb from John Innes Centre, Prof. Dr. Andriy Luzhetskyy from Saarland University, Dr. Dennis Klementz from the group of Prof. Dr. Stefan Günther, Dr. Robin Teufel and Ying Duan for their kindly providing experimental materials. I would like to express my greatest gratitude to Phillips Schwarzer and Dimah Wassouf for caring of me and for giving me power working in the lab for the final six months. I would like to thank Ahmad Alali and Dimah Wassouf for sharing their delicious Syrian food to me. I want to thank Miriam Bernhardt for teaching me swimming and Fabienne Gutacker for bringing her energy to me. I wish to express my warmest gratitude to my office colleagues Marcus Essing and Sandra Groß for their daily story and laboratory support. I would like to thank Sandra Groß again for helping me correct the german version of abstract. And I also want to thank Elisabeth Welle for strong technician assistance on HPLC problems and taking care of our lab. I would like to thank Dr. Gabriele Weitnauer for her unselfish help in many ways. I want to thank Judy Wang, Tanja Herbstritt, Sandra Cabrera and Dr. Susanne Elfert for their kind help. - 4 - I am indebted to all my current and previous colleagues in the lab, Dimah Wassouf, Phillips Schwarzer, Ahmad Alali, Miriam Bernhardt, Fabienne Gutacker, Olya Tsypik, Dr. Roman Makitrynskyy, Desiree Nuzzo, Miaomiao Li, Xiaokang Li, Philip Lohner, Dr. Anja Greule, Dr. Suzan Samra, Dr. Denise Deubel, Dr. Astrid Erber, Dr. Stefanie Hackl, Dr. chijian zuo for their kind accompany with me during my PhD study and have fun with me. I would like to acknowledge Chinese Scholarship Council and Andreas for providing scholarship and financial support. Last but not least, Special thanks to my parents and my brother, for all of their support and encouragement throughout my PhD study. I especially would like to gratitude to my husband Songya for his wholehearted support and dedication. I owe all the success of my PhD study to my family. Publication Publication I. Jing Zhu, Songya Zhang, David Zechel, Thomas Paululat, Andreas Bechthold. Rational design of hybrid natural products by genome editing utilizing the promiscuity of an amid synthase. ACS Chemical Biology (submitted) II. Songya Zhang, Dennis Klementz, Jing Zhu, Roman Makitrynskyy, A. R. Ola Pasternak, Stefan Günther, David L. Zechel, Andreas Bechthold. Genome Mining Reveals the Origin of a Bald Phenotype and a Cryptic Nucleocidin Gene Cluster in Streptomyces asterosporus DSM 41452. Journal of Biotechnology, 2019, 292, 23-31. III. Songya Zhang, Jing Zhu, David Zechel, Claudia Jessen-Trefzer, Richard T. Eastman, Thomas Paululat, Andreas Bechthold. Novel WS9326A derivatives and one novel Annimycin derivative with antimalarial activity are produced by S. asterosporus DSM 41452 and its mutant, ChemBioChem, 2018, 19(3), 272-279. IV. Arslan Sarwar, Zakia Latif, Songya Zhang, Jing Zhu, Andreas Bechthold, Biological control of potato common scab with rare Isatropolone C compound produced by Streptomyces sp. A1RT. Front Microbiol, 2018, 9: 1126-118. V. Songya Zhang, Jing Zhu, Tao Liu, Suzan Samra, Huaqi Pan, Jiao Bai, Huiming Hua, Andreas Bechthold. a Novel Glycosylated Polyketide from the Terrestrial Fungus Myrothecium sp. GS-17. Helvetica Chimica Acta, 2016, 99 , 215-219. Poster - 5 - I. Jing Zhu, Xiaohui Yan, Anja Greule, Songya Zhang, Andreas Bechthold. Exploring the Biosynthetic Capability of Ganefromycin by Direct Cloning and Heterologous Expression. Annual Conference 2016 of the Association for General and Applied Microbiology (VAAM), 03/2016, Jena, Germany II. Jing Zhu, Songya Zhang, Andreas Bechthold. Revealing the Hidden “domain skipping” Biosynthetic Mechanism in the Annimycin Polyketide Synthase from S. asterosporus DSM 41452. VAAM workshop 2016: Biology of Bacteria Producing Natural Products, 09/2016, Freiburg, Germany III. Songya Zhang, Jing Zhu, Andreas Bechthold. WS9326A Derivatives from S. asterosporus DSM 41152: Chemical Structure and Biosynthesis. VAAM workshop 2016: Biology of Bacteria Producing Natural Products, 09/2016, Freiburg, Germany IV. Songya Zhang, Jing Zhu, Roman Makitrynskyy, Olga Tsypik, Andreas Bechthold. Connecting Chemotype, Phenotype and Genotype, Revealing the Gene Regulatory Mechanism of Morphological Development and Secondray Metabolism in S. asterosporus DSM 41452. Tag der Forschung der Universität Freiburg 2016, 07/2016, Freiburg, Germany V. Songya Zhang, Jing Zhu, Tao Liu, Suzan Samra, Huiming Hua, Andreas Bechthold. Exploiting and Elucidation of a new Glycosylated Polyketide from Fungus Myrothecium sp., 2016 VAAM Annual Conference, 03/2016, Jena, Germany VI. Songya Zhang, Lin Zhang, Anja Greule, Jing Zhu, Max Cryle, Oliver Einsle, Andreas Bechthold. Structural Characterization of Cytochrome P450 Sas16, mediates the formation of the olefinic bond to generate the dehydrotyrosine formation in WS9326As Biosynthesis. RTG 1976 Symposium 2017: Unique Cofactor-dependent Enzymes in Microbes, 10/2017, Freiburg, Germany VII. Songya Zhang, Dennis Klementz, Mingjian Wang, Jing Zhu, Stefan Günther, Verónica I. Dumit, Andreas Bechthold. Complete genome sequencing and comparative Proteomic Analysis of S. asterosporus DSM 41452 reveals the AdpA regulon in a native non- sporulating Streptomyces species. VAAM workshop 2017: Biology of Bacteria Producing Natural Products, 09/2017, Tübingen, Germany. - 6 - Abstract With the help of modern genomic sequencing and molecular biotechnology, it is still valuable to exploit cryptic bioactive natural products from microorganism. To explore the biosynthetic potential of the strain Streptomyces asterosporus DSM 41452, we disrupted the production of two main secondary metabolites (WS9326A and annimycin) in this wildtype strain by genetic inactivation. Through comparison of secondary metabolic profiles, six new derivatives (SY10, J1, J3, J4, J5 and J6) were detected and isolated from the resultant mutants Streptomyces asterosporus DSM 41452::pUC19Δ3100spec and Streptomyces asterosporus DSM 41452ΔNmet::pUC19Δ3100spec. Based on the NMR and MS data, their chemical structures were elucidated. Interestingly, J5 is a hybrid compound consisting of the 2-(1(Z)- pentenyl) cinnamoyl moiety from WS9326A and the C5N unit from annimycin. J1 is a shunt product in which the olefinic double bond at the α,β-position of the cinnamoyl moiety is reduced. Considering that both compounds containing structural elements of WS9326A and annimycin, we predicted that J1 and J5 are assembled by these two discrete gene clusters of annimycin and WS9326A. About the biosynthetic mechanism of this substituted cinnamoyl moiety, it was hypothesized previously that the final aromatic cinnamoyl moiety is generated via the formation of a corresponding linear polyene followed by isomerization of olefinic bond, 6π- electrocyclization and dehydrogenation in skyllamycin and WS9326A. However, the structural complexity and instability of the intermediate probably hindered the further investigation on this special biosynthetic machinery. The discovery of J1 and J5 provides us a straightforward platform to investigate the biosynthetic mechanism of this special 2-(1(Z)-pentenyl) cinnamoyl moiety. Through bioinformatic analysis and gene mutagenesis, we postulated that 14 genes (sas24- sas37) of sas gene cluster are involved in the biosynthesis of the substituted cinnaomyl moiety. The assembly of the nascent polyketide chain of this cinnamoyl acid is catalyzed by 3-oxoacyl- ACP synthases (Sas30, Sas31, Sas32 and Sas33), ACPs (Sas29 and Sas34) reductase (Sas37) and dehydrase (Sas35 and Sas36). Based on the high similarity
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  • Precursor-Directed Biosynthesis of Azinomycin a and Related Metabolites by Streptomyces Sahachi- Roi

    Precursor-Directed Biosynthesis of Azinomycin a and Related Metabolites by Streptomyces Sahachi- Roi

    ORBIT-OnlineRepository ofBirkbeckInstitutionalTheses Enabling Open Access to Birkbeck’s Research Degree output Precursor-Directed Biosynthesis of Azinomycin A and Related Metabolites by Streptomyces sahachi- roi https://eprints.bbk.ac.uk/id/eprint/40052/ Version: Full Version Citation: Sebbar, Abdel-Ilah (2014) Precursor-Directed Biosynthesis of Azinomycin A and Related Metabolites by Streptomyces sahachiroi. [Thesis] (Unpublished) c 2020 The Author(s) All material available through ORBIT is protected by intellectual property law, including copy- right law. Any use made of the contents should comply with the relevant law. Deposit Guide Contact: email Precursor-Directed Biosynthesis of Azinomycin A and Related Metabolites by Streptomyces sahachiroi Abdel-Ilah Sebbar Department of Biological Sciences School of Science Birkbeck College University of London A thesis submitted to the University of London for the degree of Doctor of Philosophy August 2014 Declaration The work presented in this thesis in entirely on my own, except where I have either acknowledged help from a known person or given a published reference. 15/8/14 Signed: ………………. Date:… ………………... PhD student: Abdel-Ilah Sebbar Department of Biological Sciences School of Science Birkbeck College University of London 15/8/14 Signed: Date: …………………... Supervisor: Dr. Philip Lowden Department of Biological Sciences School of Science Birkbeck College University of London 2 Abstract Azinomycins A and B are bioactive compounds produced by Streptomyces species. These naturally occurring antibiotics exhibit potent in vitro cytotoxic activity, promising in vivo antitumor activity and exert their effect by disruption of DNA replication by the formation of interstrand cross-links. The electrophilic C-10 and C-21 carbons contained within the aziridine and epoxide moieties are known to be responsible for the interstrand cross-links through alkylation of N-7 atoms of guanine bases.