Acknowledgements... 3 Contents... I Figures... VII Tables... IX Abbreviations... X

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1 I Acknowledgements I Figures... VII Tables... IX Abbreviations... X Amino acids... XII Nucleobases... XII 1 Introduction Polyketides and non-ribosomal peptides Polyketide synthases Modular PKS type I Classification of PKS types Modular trans-at PKS Non-ribosomal peptide synthetases Editing domains and domain subtypes in NRPS Classification of NRPS types Hybrid PKS-NRPS The phylum Chloroflexi Chloroflexales Herpetosiphonales Herpetosiphon sp Siphonazole a secondary metabolite from Herpetosiphon Scope of the Study Materials & Methods Materials Chemicals Devices Antibiotics Disposable material Buffers and stock solutions Media Enzymes... 26

2 II Kits and standards Organisms Vectors Clones & DNA-constructs Primers Software and Databases General methods concerning micro-organisms Sterilisation Storage of organisms Cultivation of bacterial organisms Preparation of a crude extract from Herpetosiphon sp Test for antibiotic resistance of Herpetosiphon sp Imaging mass spectrometry (IMS) Transformation of bacterial cells Preparation of chemically competent cells Preparation of electro-competent cells Transformation of chemically competent cells Transformation of electro-competent E. coli cells Generation of protoplasts from Herpetosiphon PEG-mediated transformation of Herpetosiphon protoplasts Glycine treatment of Herpetosiphon cultures Electroporation of Herpetosiphon sp. 060 cells Electroporation of Herpetosiphon protoplasts Electroporation of Herpetosiphon cells after glycine treatment Sonication Bacterial conjugation Molecular biological methods concerning nucleic acids PCR Whole cell PCR Agarose gel electrophoresis Isolation of DNA from agarose gels Isolation of plasmid and fosmid DNA Isolation of genomic DNA DNA precipitation... 42

3 III Restriction digestion Ligation of DNA fragments Dephosphorylation of linear DNA Determination of nucleic acid concentration and purity Sequencing of vector constructs and PCR-products Genome sequencing Screening of a genomic library RNA-isolation and expression analysis Recombination and cloning methods Λ-Red recombination Generation of linear DNA for λ-red recombination Cloning of PCR-products Yeast recombinational cloning Isolation of plasmid DNA from yeast cells Molecular biological methods concerning proteins Heterologous expression of proteins Cell lysis by sonication Purification of recombinant proteins by Ni-NTA-columns Denaturing SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Coomassie-staining Concentration of purified proteins and buffer exchange Determination of protein concentrations after Lambert-Beer O-methyltansferase activity assay High performance liquid chromatrography (HPLC) Mass spectrometry ATP - PP i exchange assay Results Knock-out experiments with Herpetosiphon sp Antibiotic resistance tests Transformation of vegetative Herpetosiphon cells Transformation of Herpetosiphon protoplasts Genome sequencing of Herpetosiphon sp Genome search for the putatative siphonazole gene cluster Annotation of the contigs from genome sequencing... 62

4 IV Other gene clusters discovered from genome sequences Complete elucidation of the putative siphonazole gene cluster Gap closure and alignment of the contigs Positioning of contig Gap closure between contigs 0668 and Positioning of contig Gap closure between contigs 1518 and Gap closure between contigs 1518 and Organisation of the putative siphonazole gene cluster NRPS domains and modules Adenylation domains Peptidyl carrier proteins The condensation domain of sphf Heterocyclisation modules Cyclisation tandem domains Oxidation domains PKS domains and modules The trans-at and oxidoreductase SphA Ketosynthases Acyl carrier proteins Dehydratases Ketoreductases The thioesterase SphJ Non-PKS/NRPS cluster parts SphB - an O-methyltransferase The α/β-hydrolase encoded by sphh SphI an aldolase Domain structure of the putative siphonazole biosynthetic enzymes Classification of carrier proteins in view of the domain structure Construction of the complete cluster from the fosmid library Screening of the genomic library Yeast recombinational cloning Λ-Red mediated recombination Protein expression and purification... 99

5 V Heterologous expression of the O-methyltransferase SphB In vitro methylation of protocatechuic acid by SphB Heterologous expression of the first A domain of SphC ATP PP i exchange assay Heterologous expression of the DAHP synthase SphI Expression profile of siphonazole in Herpetosiphon sp mrna expression of sph genes Imaging mass spectrometry (IMS) Heterologous expression of the complete gene cluster in E. coli Discussion Genetic accessibility of Herpetosiphon sp Elucidation of the putative siphonazole biosynthetic pathway NRPS modules The cyclisation modules The initiation module The glycine incorporating module NRPS-PKS interfaces trans-at PKS modules The oxidoreductase of SphA Elongating and non-elongating modules The cryptic module The mosaic structure of the siphonazole gene cluster Postulated biosynthesis of siphonazole Nature and origin of the starter unit The O-methyltransferase SphB The DAHP synthase SphI Substrate specificity of the A domain of the initiation module ATP-PP i exchange assay with the initiation module A domain Decarboxylation of the terminal acetyl moiety Transcriptional organisation of the gene cluster Spatiotemporal biosynthesis of siphonazole Biological role of siphonazole Complete assembly of the putative siphonazole cluster In silico and genomic library screening

6 VI Assembly by λ Red recombination Assembly by yeast recombinational cloning Heterologous expression of the siphonazole gene cluster Heterologous protein expression Metabolic potential of Herpetosiphon Summary References Appendix Primary sequences of the proteins SphA SphJ SphA SphB SphC SphD SphE SphF SphG SphH SphI SphJ Search results from the NaPDos Database C domains in contigs from the genome sequencing KS domains in contigs from the genome sequencing Primer sequences Gap closure Protein expression Fosmid library screening Yeast recombinational cloning λ-red mediated recombination Sequencing primers mrna screening primers Complete sequence alignment of the DAHP synthase SphI HPLC UV-spectra Chromatograms from ATP-PP i exchange assay Publications