Ambigols A-C and tjipanazole D: bioinformatic analysis of their putative biosynthetic gene clusters [Elektronische Ressource] / Mustafa El Omari
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Ambigols A-C and tjipanazole D: bioinformatic analysis of their putative biosynthetic gene clusters [Elektronische Ressource] / Mustafa El Omari

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Ambigols A-C and tjipanazole D: bioinformatic analysis of their putative biosynthetic gene clusters Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Mustafa El Omari aus Münster, Deutschland Bonn 2011 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Gutachterin : Prof. Dr. G. M. König 2. Gutachter : Priv.-Doz. Dr. habil. W. Knöss Tag der Promotion : 25.07.2011 Erscheinungsjahr : 2011 Publications I Publications Research papers Wagner C, El Omari M, Konig GM. 2009. Biohalogenation: nature's way to synthesize halogenated metabolites. J.Nat Prod 72: 540-553. Erol Ö, Schäberle TF, Schmitz A, Rachid S, Gurgui C, El Omari M, et al., 2010. Biosynthesis of the myxobacterial antibiotic corallopyronin A. Chembiochem 11: 1253-1265. Research presentations El Omari M & König GM. Halogenases in the cyanobacterium Fischerella ambigua. Poster presentation. Annual conference of the Deutsche Pharmazeutische Gesellschaft (DPHG), Bonn, Germany, October 8-11, 2008. El Omari M, Wagner C, König GM. Halogenases in the cyanobacterium Fischerella ambigua.

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Published 01 January 2011
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Ambigols A-C and tjipanazole D: bioinformatic analysis of
their putative biosynthetic gene clusters







Dissertation
zur
Erlangung des Doktorgrades (Dr. rer. nat.)
der
Mathematisch-Naturwissenschaftlichen Fakultät
der
Rheinischen Friedrich-Wilhelms-Universität Bonn









vorgelegt von
Mustafa El Omari
aus
Münster, Deutschland

Bonn 2011


































Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät
der Rheinischen Friedrich-Wilhelms-Universität Bonn























1. Gutachterin : Prof. Dr. G. M. König
2. Gutachter : Priv.-Doz. Dr. habil. W. Knöss


Tag der Promotion : 25.07.2011


Erscheinungsjahr : 2011



Publications

I Publications

Research papers
Wagner C, El Omari M, Konig GM. 2009. Biohalogenation: nature's way to
synthesize halogenated metabolites. J.Nat Prod 72: 540-553.
Erol Ö, Schäberle TF, Schmitz A, Rachid S, Gurgui C, El Omari M, et al., 2010.
Biosynthesis of the myxobacterial antibiotic corallopyronin A. Chembiochem 11:
1253-1265.

Research presentations
El Omari M & König GM. Halogenases in the cyanobacterium Fischerella ambigua.
Poster presentation. Annual conference of the Deutsche Pharmazeutische
Gesellschaft (DPHG), Bonn, Germany, October 8-11, 2008.
El Omari M, Wagner C, König GM. Halogenases in the cyanobacterium Fischerella
ambigua. Poster presentation. 6th European Conference on Marine Natural Products
(ECMNP), Porto, Portugal, July 19-23, 2009.
El Omari M & König GM. New insights into the biosynthesis of ambigols A-C and
tjipanazole D. Poster presentation. Annual Conference of the Association for General
and Applied Microbiology (VAAM), Karlsruhe, Germany, April 3-6, 2011.










Acknowledgements
II Acknowledgements
My sincere gratitude goes to Prof. Dr. G.M. König for giving me the opportunity to
deal with a highly comprehensive topic involving various branches of biosynthesis
and very interesting methods of molecular biology. I would like to thank her for
supervising my project with expert guidance, encouragement and inspiring ideas as
well as for careful proof-reading of my work. I would further like to appreciate her trust
in me to take on further responsibility as assistant appointee for biological safety.
I would like to express my gratitude to Priv.-Doz. Dr. habil. W. Knöss for accepting
the co-examination of this project.
My appreciation also goes to Prof. Dr. G. Bendas and Prof. Dr. E.A. Galinski for
agreeing to participate in my examination committee.
This study involved many different and difficult tasks and problems to be solved. This
was extremely facilitated by the nice work atmosphere and the support of members
of our group. I would especially thank:
Dr. C. Wagner for introducing me to this project and for guiding me on cultivation of
the cyanobacterial strain and DNA-extraction. I also thank her for providing partial
halogenase sequences to me for genomic library screening.
Ö. Erol for familiarising me with the basic molecular biological laboratory work and
always being a helpful and advising companion and special friend during the whole
study. Furthermore, I would like to thank her for help with the construction of the
genomic library and her good ideas concerning problems of the laboratory work.
Sarah Bouhired for organising the 454 sequencing of the Fischerella ambigua strain
and explaining the CLUSEAN software.
Ms. E. Neu and Ms. E. Eguereva for technical and administrative assistance during
the course of the project. Special thanks go to Ms. E. Neu and Ms. E. Eguereva for
support with the cultivation of the cyanobacterial strain and ambigol-isolation.
S. Kehraus for being always helpful concerning general laboratory questions and for
his humurous comments.
Acknowledgements
M. Elsebai for being always a helpful and funny fellow and good friend during this
study.
Further, my thanks go to Prof. J. Piel for good advice on biosynthesis questions
concerning my project in our collaborative group seminars.
I also would like to address special thanks to Dr. H. Groß for enriching my experience
in teaching students and the nice and familiar atmosphere during the plant anatomy
courses. Furthermore, I acknowledge his precious ideas in discussions.
My appreciation goes to Ms. E. Gassen and Dr. M. Koch for help with administrative
issues and to Mr. T. Kögler for technical support.
Finally, I express my cordial gratitude to my family, especially to my parents Samir
and Irmgard El Omari for their permanent encouragement and trust in me. I would
like to thank my whole family for their extensive support during the course of this
project.

Table of content
III Table of content
1. Introduction .................................................................................................. 1
1.1 Cyanobacterial secondary metabolites .......................... 1
1.2 Halogenated secondary metabolites from cyanobacteria .............................. 3
1.3 Chlorinated secondary metabolites in Fischerella ambigua ........................... 8
1.3.1 Ambigols and related structures ..................................................................... 8
1.3.2 Tjipanazole D and related structures ........................... 12
1.4 Proposed building blocks and reactions in ambigol and tjipanazole D
biosynthesis ................................................................................................. 16
1.4.1 Flavin-dependent halogenases .... 17
1.4.2 Chorismate and halogenases in the biosynthetic process ........................... 25
1.4.3 AMP-dependent enzymes in the biosynthetic process ................................. 27
1.4.4 Cytochrome P450-dependent enzymes in biosynthesis .............................. 29
1.4.5 Starter C domains in NRPS ......................................................................... 33
2. Intent of the presented study .... 35
3. Materials and methods .............................................................................. 36
3.1 Materials ...................................... 36
3.1.1 Chemicals and other materials..................................................................... 36
3.1.2 Enzymes ...... 38
3.1.3 Molecular weight markers ............................................................................ 41
3.1.4 Molecular biological kits ............... 41
3.1.5 Bacterial strains ........................................................................................... 42
3.1.6 Vectors ......................................... 42
3.1.7 Fosmids ....................................................................... 44
3.1.8 Phages ......... 46
3.1.9 Oligonucleotides .......................................................................................... 46
3.1.10 Water ........................................................................................................... 48
3.1.11 Culture Media............................... 49 Table of content

3.1.12 Buffers and solutions ................................................................................... 49
3.1.13 Software and Databases .............. 52
3.2 Cultivation of cyanobacterial organism ........................................................ 53
3.2.1 Origin of cyanobacterial Fischerella ambigua .............. 53
3.2.2 Small-scale cultivation for DNA isolation ...................... 53
3.2.3 Large-scale cultivation for analytical purposes ............................................ 53
3.3 Obtaining axenic cyanobacterial cultures ..................... 53
3.3.1 Phototaxis experiments ................................................ 54
3.3.2 UV treatment of cyanobacterial colonies ...................................................... 54
3.3.3 Cyanobacterial resistance to lysozyme ........................ 54
3.3.4 Antibiotic selection with ampicillin ................................................................ 54
3.4 Molecular biological methods ....... 55
3.4.1 Sterilization of solutions and equipment ....................................................... 55
3.4.2 Isolation of chromosomal DNA from F. ambigua.......... 55
3.4.3 Isolation of genomic DNA from the associated Pseudomonas sp. ............... 57
3.4.4 DNA precipitation ......................................................................................... 57
3.4.5 Determination of nucleic acid concentration and purity of DNA ................... 58
3.4.6 16S rDNA analysis ....................... 58
3.4.7 Agarose gel electrophoresis ........................................................................ 59
3.4.8 Recovery of DNA from agarose gels ............................ 59
3.4.9 Polymerase chain reaction ........... 59
3.4.10 Restriction digestion ..................................................................................... 63
3.4.11 Preparation of cells competent for DNA-transformation ............................... 64
3.4.12 Transformation of host strains...................................................................... 64
3.4.13 Plasmid isolation from transformed E. coli ................... 65
3.4.14 Methods for the establishment of a genomic library ..... 65
3.4.15 Genome sequencing using the 454 sequencing procedure ......................... 72

Table of content
4. Results ........................................................................................................ 74
4.1 Morphological and molecular characteristics of F. ambigua ........................ 74
4.2 Identification and removal of associated bacteria ........ 76
4.2.1 16S rDNA analysis of associated bacteria ................................................... 76
4.2.2 Axenic cultures ............................................................. 77
4.3 Construction and screening of a fosmid library ............................................ 79
4.3.1 DNA extraction from filamentous cyanobacteria .......... 79
4.3.1.1 Pretreatment of cyanobacterial cells for filament breakage ......................... 79
4.3.1.2 Removal of heterotrophic bacteria to obtain axenic cyanobacterial DNA ..... 80
4.3.1.3 Comparison of two methods for DNA extraction .......................................... 80
4.3.2 PCR for phenolic halogenase genes ............................................................ 81
4.3.3 Fosmid library production ............................................. 82
4.3.4 Screening of the fosmid library for phenolic halogenase genes ................... 85
4.4 Sequence analysis of fosmid E8 .................................................................. 86
4.4.1 High yield fosmid isolation from clone E8 and restriction digestion .............. 86
4.4.2 End sequencing of fosmid E8 ...... 87
4.4.3 Subcloning of fosmid E8 .............................................................................. 88
4.4.4 Complete sequencing of fosmid E8 ............................................................. 90
4.5 Screening for fosmids overlapping with E8 .................. 91
4.6 Whole genome sequencing of F. ambigua ................................................... 91
4.7 Elucidation of the putative ambigol biosynthetic gene cluster ...................... 92
4.7.1 Searching the genome assembly for sequences of the E8 fosmid............... 92
4.7.2 Overall sequence analysis of contig 00522 .................................................. 92
4.7.3 Orf21 shows homology to regulative proteins .............. 99
4.7.4 Ab1, an FADH -dependent halogenase ..................................................... 100 2
4.7.5 Two adjacent CYP 450 enzymes ............................... 101
4.7.6 Orf22 belongs to the polyketide_cyc2 superfamily ..................................... 104
4.7.7 Ab4, a putative chorismate lyase ................................ 104