Investigating Natural Product Biosynthesis In Uncultivated .
Investigating Natural Product Biosynthesisin Uncultivated Symbiotic Bacteria of theMarine Sponge Theonella swinhoeiDissertationzurErlangung des Doktorgrades (Dr. rer. nat.)derMathematisch-Naturwissenschaftlichen FakultätderRheinischen Friedrich-Wilhelms-Universität Bonnvorgelegt vonAgustinus Robert Uriaaus Tambarana, Zentral Sulawesi, IndonesienUniversität Bonn2012
Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultätder Rheinischen Friedrich-Wilhelms-Universität Bonn.Gutachter:1. Prof. Dr. Jörn Piel2. Prof. Dr. Uwe Deppenmeier3. Prof. Dr. Albert Haas4. Prof. Dr. Gabriele M. KönigTag der Promotion: 05.12.2012Erscheinungsjahr:2013
Dedicated to my parents with love and gratitude
AbstractAbstractMarine sponges are a rich source of bioactive natural products with potentanticancer activities. Currently, the limited availability of most of these substancesprohibits further drug development (Proksch et al., 2002). Highly complex consortiaof bacterial symbionts associated with sponges have been frequently proposed tobe the true producers of many secondary metabolites (Piel, 2004). However, themajority of these complex microbial assemblages are not amenable to cultivation(Amann et al., 1995; Hugenholtz et al., 1998; Friedrich et al., 2001; Webster & Hill,2001), thereby hampering efforts to prove the symbiont hypothesis as well as toaccess their biosynthetic potential. Using metagenomic-based approaches, Piel andcolleagues have previously provided the first genetic evidence for the bacterial originof invertebrate-derived natural products by cloning the entire gene cluster foronnamide/theopederin from the Japanese sponge Theonella swinhoei (Piel et al.,2004a, 2004b).The Japanese sponge Theonella swinhoei WPhoto by J. TanakaThe Japanese sponge Theonella swinhoei YPhoto by Y. NakaoIn this work, we investigated further natural product biosynthetic pathways fromuncultivated symbiotic bacteria using Japanese T. swinhoei as a symbioticassemblage model. The reasons for selecting this sponge are the wide variety ofpharmaceutically important secondary metabolites isolated from this sponge as wellas the high complexity of the associated bacteria (Fusetani & Matsunaga, 1993;Henstchel et al., 2002), which might play an important role in metabolitebiosynthesis. Metagenome mining strategies that we applied and developed in thiswork have led to the cloning of two new biosynthetic pathways from this complexsymbiosis model. Our bioinformatic analysis predicted that one pathway isresponsible for the biosynthesis of misakinolide A, and another one for keramamideH. Interestingly, we found that the first pathway contains additional components thati
Abstractmatch structures of swinholide A and hurghadolide A, potent actin polymerizationinhibitors isolated from other sponges (Carmely & Kashman, 1985; Kitagawa et al.,1990; Doi et al. 1991; Youssef & Mooberry, OOONHn2OHOCH 3NOHOOHOMeOHOSOOHOMeNHBrMisakinolide A (n1 n2 0)Swinholide A (n1 n2 0)Hurghadolide A (n1 1, n2 0)Keramamide HBoth biosynthetic pathways were encoded on two different gene clusters thatexhibited typical bacterial gene features, strongly indicating that the producer ofmisakinolide A and keramamide H is a symbiotic bacterium. Since the screeningsystem used to clone the gene clusters was based on the filamentous fractiondominated by “Candidatus Entotheonella sp.”, a heterothropic delta-proteobacteriumassociated with T. swinhoei, we assumed that misakinolide A and keramamide Hare produced by “Entotheonella sp.” To confirm the taxonomic status of the bacterialproducer, further analysis either by single cell studies or its combination withcomplete genome is currently underway.Subsequent genome sequencing of another member of this as-yet uncultivablecandidate genus from a different chemotype of T. swinhoei led to the identification ofgenes for the biosynthesis of orbiculamide-like structure, which is structurally relatedto keramamides. Therefore, the results in this work provide not only convincing prooffor the microbial origin of marine natural products but also specific taxonomicinformation as well as the potential to sustainable supply of pharmacologicallypotential compounds.“Candidatus Entotheonella sp.”OHNOHORHNNHNHNNHOOONOSONHOrbiculamide-like structureHNOBrNHHOPhoto by T. Moriii
AcknowledgementsAcknowledgementsFirst and foremost I would like to thank my supervisor, Prof. Dr. Jörn Piel for thegreat opportunity he has given to me to learn and perform research within his group.I am grateful for his insightful guidance, suggestions, ideas, conversations, supports,and encouragement. Thank you for editing this dissertation; your important advicesand input significantly enhanced the content.I would like to thank Prof. Dr. Grabriele M. König and Priv.-Doz. Dr. Thomas Kolterfor evaluating my research progress and for providing the DAAD withrecommendation letters that allowed the annual extension of my DAAD scholarship.I would like to acknowledge the committee members Prof. Dr. Uwe Deppenmeier,Prof. Dr. Albert Haas, and Prof. Dr. Grabriele M. König for their critical reading,input, and helpful advice.Many thanks go to all my colleagues in both the Piel and Gulder Groups for theirfriendship, humor, and help that provided a nice and fun environment for me to learnand perform research. My dissertation would not be possible without their support.My special thanks to Dr. Christian Gurgui for his kindness in sharing knowledge,protocols and experimental tricks, as well as for proof-reading my thesis draft. Mysincere appreciation goes to him and his wife for their generosity and help especiallywhen I found difficulties in my life. I am grateful to Silke Reiter and Bikram Pandeyfor their involvement and contributions in my PhD project.I am thankful to Dr. Michael Freeman for sharing his incredible scientific knowledgeand practical experiences, and for proof-reading my thesis draft. Many thanks to Dr.Brandon Morinaka for explaining me reaction mechanisms and proof-reading mythesis draft. I am grateful to Dr. Mike Wilson and Dr. Reiko Ueoka for sharing theirscientific knowledge and having nice discussions. A big thank to Max Helf andStefan Künne for their help especially when I have computer problems. I also wantto thank Katja Jensen and Ursula Steffens for sharing valuable “yeast” protocols.Thanks too to Annette Kampa, Xiaofeng Cai, and Dr. Max Crüsemann for sharingtheir knowledge in protein expressions.iii
AcknowledgementsI am indebted to the past members of the Piel Group: Dr. Katja Fisch, Dr. KathrinReinhardt, Dr. Tu Ahn Nguyen, Dr. Sonia van der Sar, Dr. Jana Moldenhauer, Dr.Marija Avramović, Dr. Mina Eklund, Dr. Thomas Hochmuth, Dr. Katrin Zimmermann,Dr. Holger Niederkrueger, and Daniel Flachshaar. They have been supportive andhelpful. Special thanks go to Dr. Katja Fisch for mentoring and teaching me withvarious laboratory techniques during the first year of my PhD research. I amespecially thankful to Dr. Kathrin Reinhardt, Dr. Katrin Zimmermann and Dr. HolgerNiederkrueger for their help, including matters regarding my visa extension.I would like to thank Prof. S. Matsunaga (University of Tokyo), Dr. T. Mori, Prof. M.Takeyama (Waseda University), Dr. T. Wakimoto (University of Tokyo) for theircollaboration in providing sponge and filamentous bacterial samples used in thiswork. I would like to thank Dr. Jörn Kalinowski and Dr. Christian Rückert (BielefeldUniversity) for their collaboration in genome sequencing. I am thankful to Dr.Vladimir Benes (EMBL-Heidelberg) for the collaboration in fosmid sequencing.I am grateful to Prof. Dr. Inneke F.M. Rumengan (my former academic supervisor)for providing me with the foundation during my undergraduate studies for becominga biotechnologist. Her support and encouragement were paramount in motivatingme to get my scientific carrier started on the right path. I would like to thank Prof. Dr.Maggy Thenawidjaja Suhartono, Prof. Dr. Ekowati Chasanah, and Prof. Dr. Hari EkoIrianto for their encouragement and recommendation for me to pursue doctoralstudies.I am especially thankful to my parents and brothers for their prayers, love andencouragement. I am grateful to Edison Macusi and Dr. Jonattan Lassa for sharingbrotherhood in Bonn. Thanks to friends and colleagues in Research Center forMarine and Fisheries Product Processing and Biotechnology, Jakarta for theirfriendship and support.I would like to thank the German Academic Exchange Service (DAAD) for awardingme a scholarship that has enabled me to carry out doctoral studies in Germany. ThisPhD work was done in the Piel Group at Kekulé Institute of Organic Chemistry andBiochemistry, Rheinischen Friedrich‐Wilhelms‐Universität Bonn. This researchproject was financially supported by BMBF through GenBioCom 03155851 given toProf. Dr. Jörn Piel.iv
ContentsContentsAbstract . i-iiAcknowledgements.iii-ivContents . v-viList of Figures . vii-ixList of Tables . x-xiAbbreviations . xii-xiiiChapter 1 Introduction . 1-441.1Pharmacological Potential of Marine Bacteria . 41.2Microbial Associations with Marine Sponges . 91.3Sponge-derived Metabolites and Symbiosis Hypothesis . 131.4Biosynthetic Insights into Microbial Secondary Metabolites . 251.4.1 Polyketide biosynthesis . 251.4.2 Nonribosomal peptide biosynthesis. 361.4.3 Metagenomic strategies to isolate biosynthetic pathways . 41Chapter 2 Research Goals . 45-47Chapter 3 Results and Discussion . 48-1293.1Metagenomic Survey of Biosynthesis Genes . 483.1.1 PCR cloning of biosynthesis genes from sponge metagenome . 493.1.2 PCR cloning of biosynthesis genes from uncultivated bacteria . 523.2Metagenomic Discovery of a Polyketide Biosynthetic Pathway . 633.2.1 Metagenomic library construction and screening . 633.2.2 Isolation of polyketide biosynthesis genes and preliminary analysis . 723.2.3 Completion of the biosynthetic gene cluster . 793.2.4 Bioinformatic analysis of the entire biosynthetic gene cluster . 813.2.5 Proposed model of the discovered biosynthetic pathway . 883.3Metagenomics Insights into the Biosynthesis of a Nonribosomal Peptide . 983.3.1 Isolation of a nonribosomal biosynthetic pathway . 983.3.2 Analysis of the nonribosomal peptide synthase region . 1033.4Genomics-guided Identification of a Biosynthetic Pathway . 1143.4.1 Purity analysis of uncultured bacterial fraction . 1143.4.2 Genome analysis . 1213.4.3 Proposed scheme of the identified biosynthetic pathway . 1243.5Summary and Outlook . 126Chapter 4 Methodology. 130-163v
Contents4.1Materials . 1304.1.1 Organisms and vectors . 1304.1.2 Chemicals, solutions, and instruments . 1314.2General Molecular Biology and Microbiology Techniques . 1344.2.1 DNA isolation and electrophoresis . 1344.2.2 PCR amplification . 1394.2.3 DNA modifications . 1424.2.4 DNA cloning . 1494.3Molecular Diagnostic of Key Biosynthetic Genes . 1464.3.1 Isolation of metagenomic DNA from sponge . 1474.3.2 PCR amplification of biosynthetic genes . 1484.3.3 Cloning of PCR products . 1504.3.4 Clone analysis and sequencing . 1504.4Construction and Screening of Complex Metagenomic Libraries . 1514.4.1 Isolation of high-molecular-weight metagenomic DNA . 1534.4.2 Size selection and end-repairment of DNA fragments . 1534.4.3 DNA packaging and transfection . 1554.4.4 Amplification and screening of 3-D libraries . 1554.4.5 Chromosome walking, fosmid analysis and sequencing . 1594.5Genome Analysis . 1614.5.1 Genetic diversity analysis. 1624.5.2 Gene cluster identification . 163Supplementary . 164-176References . 177-214Curriculum Vitae . 215-217Selbständigkeitserklärung . 218vi
List of FiguresList of FiguresFigure 1.1.1 Examples of pharmacologically potential compounds from marine bacteria . 4Figure 1.1.2 Pharmacologically important natural products from marine sponges,in which their biosynthetic pathways have been characterized. . 7Figure 1.1.3 Pharmacologically important natural products from marine invertebrates,in which their biosynthetic pathways have been characterized . 8Figure 1.2.1 The sponge body s plan . . .10Figure 1.3.1 Sponge-derived natural products and their analogs approved by FDA/EMAas clinically used agents or in clinical trial . . .14Figure 1.3.2 Macrolides closely related to misakinolide A reported from diverse sources . 16Figure 1.3.3 The antifungal complex cyclic peptide theonellamides F and A-E . . 17Figure 1.3.4 Oxazole-containing cyclic peptides isolated from marine sponges . . 20Figure 1.3.5 Thiazole-containing cyclic peptides isolated from marine sponges . . 21Figure 1.3.6 Ureido bond-containing cytotoxic cyclic peptides . . 22Figure 1.3.7 Onnamide-type compounds from various sources . . 23Figure 1.4.1 General biosynthetic steps of a bacterial modular PKS . . 26Figure 1.4.2 The well-studied AT PKS, deoxyerythronolide B synthase (DEBS) . . 27Figure 1.4.3 Basic mechanisms of initiation by AT domain in type I PKS . . 29Figure 1.4.4 Basic mechanisms of elongation by KS domain in type I PKS . . 31Figure 1.4.5 Proposed mechanisms of modifications by KR and DH domainsin type I PKS . 33Figure 1.4.6 Basic mechanisms of terminations in 6-deoxyerythronolide B (6-dEB) . . 34Figure 1.4.7 Basic mechanisms of trans-AT PKS . . 35Figure 1.4.8 Various strategies of amino acid activation to from peptide bonds . . 38Figure 1.4.9 Bond formation in nonribosomal peptides and hybrids with polyketides . . 40Figure 1.4.10 Some examples of molecules from the metagenomic expression ofbiosynthetic pathways . . 42Figure 1.4.9 Bond formation in nonribosomal peptides and hybrids with polyketides . . 40Figure 2.1Two forms of T. swinhoei chemotypes . . 46Figure 2.2Structures of some bioactive secondary metabolites from T. swinhoei Y . 46Figure 2.3The symbiotic “Candidatus Entotheonella sp.” In the homogenized sponge . 47Figure 3.1.1 Isolation of KS domain-encoding genes from the metagenome ofT. swinhoei chemotype W . 50Figure 3.1.2 Phylogenetic analysis of partial KS amplicons . 51Figure 3.1.3 Compound localization in the Japanese T. swinhoei chemotype W . 52Figure 3.1.4 The proposed biogenesis of some building blocks in theonellamide F . 54Figure 3.1.5 Cloning of KS sequences from the uncultivated filamentous bacterialfraction of the Japanese marine sponge T. swinhoei chemotype white . 55Figure 3.1.6 Detection of bacterial NRPS genes based on A domain sequences . 56vii
List of FiguresFigure 3.1.7 Molecular detection of bacterial NRPS genes based on A domainsequences in the filamentous bacterial fraction of T. swinhoei . 58Figure 3.1.8 Strategy for designing degenerate primers specifically to target the Adomain for L-serine developed in this work . 60Figure 3.1.9 Cloning of the A domain-encoding fragments specific for L-serine. 61Figure 3.1.10 The predicted functional role of the isolated SerA homologs . 62Figure 3.2.1 General
Investigating Natural Product Biosynthesis in Uncultivated Symbiotic Bacteria of the Marine Sponge Theonella swinhoei Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.)
the biosynthesis of chlorophylls (phytyl side-chain) and carotenoids, and differentiates it from the cytosolic ac-etate/mevalonate pathway of sterol biosynthesis. The light-induced accumulation and final functional con-centration of carotenoids and chlorophylls in green leaves is presented. Moreover, the biosynthesis of isoprene and
Isoprenoids Fig. 1 The relationship between alginate biosynthesis and the cellular metabolism in P. fluorescens. a The proteins and metabolites needed for alginate biosynthesis. b A simplified model of the cell's metabolism highlighting the processes identified in the present study as being important for full alginate biosynthesis levels.
the fascinating biochemistry of AG biosynthesis and the rich potential in their pathway engineering. In particular, parallel pathways have been shown to be common and widespread in AG biosynthesis, highlighting nature’s ingenuity in accessing diverse natural products from a limited set of genes.
Cholesterol: Biosynthesis, Functional Diversity, Homeostasis and Regulation by Natural Products 423 oxygenase and sequalene 2, 3 epoxidase enzymes. Sequalene mono oxygenase is the second committed step in cholesterol biosynthesis and lead to the formation squalene 2, 3 epoxide.
Terpenoids are also known as Isoprenoids. Terpenes and terpenoids are the primary constituents of the essential oils of many types of plants and . (ca. 1955) shown that the biosynthesis of terpenes does indeed occur starting from isoprene-like C5 building blocks. Labelling experiments, using 14C-labelled acetic acid, showed early on
Isoprenoids represent the largest class of small molecules on earth,1 so it is not surprising that many of the enzymes that are involved in isoprenoid biosynthesis are drug targets. For example, the most widely prescribed drug, Lipitor, targets cho-lesterol biosynthesis at an early stage; bisphosphonates, such
biosynthesis of steroidal alkaloids in Solanaceae plants. We found that GAME9 is part of an ERF-gene cluster existing in potato and tomato. Transactivation and promoter binding assays as well as transgenic tomato and potato plants revealed that GAME9 controls SGA biosynthesis as well as several upstream mevalonate and cholesterol precursor .
INTRODUCTION TO FIELD MAPPING OF GEOLOGIC STRUCTURES GEOL 429 – Field Geology Department of Earth Sciences Montana State University Dr. David R. Lageson Professor of Structural Geology Source: Schmidt, R.G., 1977, Geologic map of the Craig quadrangle, Lewis and Clark and Cascade Counties, Montana: U.S. Geological Survey GQ-1411, 1:24,000. 2 CONTENTS Topic Page Introduction 3 Deliverables 4 .
