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dc.contributor.author
Moosmann, Philipp
dc.contributor.supervisor
Piel, Jörn
dc.contributor.supervisor
Aebi, Markus
dc.contributor.supervisor
Kast, Peter
dc.date.accessioned
2018-10-31T15:28:34Z
dc.date.available
2018-10-31T14:10:35Z
dc.date.available
2018-10-31T15:28:34Z
dc.date.issued
2018
dc.identifier.uri
http://hdl.handle.net/20.500.11850/300232
dc.identifier.doi
10.3929/ethz-b-000300232
dc.description.abstract
Natural products, also called secondary or specialised metabolites, are compounds that are produced by cells and are intended to have an effect outside of the producer. These compounds often have potent biological activities, and humankind has been using them as medicines in their crude forms for centuries. Even today, natural products are an important source and inspiration for drug development. An especially rich source of novel natural products are marine sponges. For many compounds isolated from sponges, though, it has been shown by genome sequencing and other methods that they are produced by symbiotic microorganisms. The genes necessary for the production of secondary metabolites are often clustered together in microorganisms, a phenomenon generally called a biosynthetic gene cluster (BGC). This clustering allows for the bioinformatic identification of BGCs, so-called genome mining, and subsequent investigation of natural product biosynthesis. The focus of this thesis lies on cyanobacteria. These fascinating microorganisms are not only exceptional producers of specialised metabolites, they have also been shown to occasionally produce compounds with high similarity to natural products isolated from sponges. The goal of this thesis was to further explore the biochemical potential of cyanobacteria in natural product research. This was done by either identifying new natural products and their biosynthetic gene clusters (chapter 2, 4 and 5), by characterising novel biochemistry and new enzymes involved in natural product biosynthesis (chapter 2, 3 and 5) or by testing the potential of cyanobacteria to heterologously express biosynthetic gene clusters (chapter 5). Chapter 2 focuses on the discovery and structure elucidation of new cytotoxic cyanobacterial meroterpenes termed merosterols. Their structure was determined with a combination of nuclear magnetic resonance spectroscopy (NMR), prediction of 13C chemical shifts, and a comparison of measured and predicted circular dichroism spectra. Additionally, the biosynthetic gene cluster responsible for merosterol production was identified. The substrate of the terpene cyclase was investigated by testing different surrogates with an intrinsic tryptophan fluorescence assay. The terpene cyclase was investigated in vitro and the structure of the formed product was determined by NMR analysis. The molecule consists of a polycyclic sterol-like terpene part annelated to an aromatic benzoic acid. A notable feature is the adopted opposite stereochemistry of the one of common steroids. Known terpene cyclases of the same class consist of two or three domains, and are often membrane bound proteins. The merosterol terpene cyclase on the other hand is soluble and smaller than the known enzymes. Structural 10 modelling proposed a minimalistic monodomain architecture, which was confirmed with an X-ray crystal structure of the enzyme, as further described in chapter 3. In chapter 4 the biosynthetic potential of cyanobacteria is further explored. Analysis of cyanobacterial genomes revealed a gene cluster putatively encoding a nonribosomal peptide synthetase/polyketide synthase hybrid accompanied by seven Fe(II)/α-ketoglutarate-dependent enzymes. Bioinformatic analysis suggested that six of them are halogenases, an unprecedented number of halogenases in a single gene cluster. Mass spectrometry-guided screening of cyanobacterial extracts led to the discovery and structure elucidation of the novel, extensively halogenated nonribosomal peptide/polyketide hybrids termed aranazoles. Chapter 5 is based on previous observations that cyanobacteria can produce similar secondary metabolites as symbionts of marine macroorganisms such as sponges. A putative gene cluster encoding genes for the biosynthesis of an indolocarbazole was identified in the genome of 'Candidatus Entotheonella serta'. It contains a glutathione sulphur transferase, a class of enzymes that has not yet been described in the context of indolocarbazoles. Investigation of the presence of homologous genes in cyanobacterial genomes led to the identification of similar gene clusters in two Fischerella species. Even though no activity was detected for the glutathione sulphur transferase so far, it was confirmed that the gene cluster is indeed responsible for the biosynthesis of indolocarbazoles. Detected products included the already known indolocarbazole arcyriaflavine A and a novel compound with two additional oxygens. This product can be explained by the presence of an additional cytochrome P450 enzyme encoded in the cyanobacterial gene clusters. Oxygenations of indolocarbazoles catalysed by P450s have only been described in the context of environmental DNA libraries. Otherwise only hydroxylations catalysed by FAD-dependent enzymes have been described so far for these compounds. All in all, this thesis provides further evidence that cyanobacteria are not only capable of producing unique novel secondary metabolites, but also have a repertoire of natural products closely resembling compounds, which are produced by symbionts of marine macroorganisms.
en_US
dc.format
application/pdf
en_US
dc.language.iso
en
en_US
dc.publisher
ETH Zurich
en_US
dc.subject
Natural product
en_US
dc.subject
Natural product discovery
en_US
dc.subject
Natural product biosynthesis
en_US
dc.subject
Terpene synthase
en_US
dc.subject
Terpenes
en_US
dc.subject
Meroterpenes
en_US
dc.subject
Non-ribosomal peptidesynthase
en_US
dc.subject
Polyketide
en_US
dc.subject
Polyketide synthase
en_US
dc.subject
halogenated natural products
en_US
dc.subject
Alkaloids
en_US
dc.subject
Indolocarbazole
en_US
dc.title
Strategies for the Identification of Biosynthetic Novelty in the Specialised Metabolite Repertoire of Cyanobacteria
en_US
dc.type
Doctoral Thesis
ethz.size
192 p.
en_US
ethz.code.ddc
DDC - DDC::5 - Science::570 - Life sciences
ethz.identifier.diss
25290
en_US
ethz.publication.place
Zurich
en_US
ethz.publication.status
published
en_US
ethz.leitzahl
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02030 - Dep. Biologie / Dep. of Biology::02520 - Institut für Mikrobiologie / Institute of Microbiology::03980 - Piel, Jörn / Piel, Jörn
en_US
ethz.date.deposited
2018-10-31T14:10:43Z
ethz.source
FORM
ethz.eth
yes
en_US
ethz.availability
Embargoed
en_US
ethz.date.embargoend
2021-10-31
ethz.rosetta.installDate
2018-10-31T15:29:02Z
ethz.rosetta.lastUpdated
2018-10-31T15:29:02Z
ethz.rosetta.exportRequired
true
ethz.rosetta.versionExported
true
ethz.COinS
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