Xenocoumacin biosynthesis in Xenorhabdus nematophila and regulation of secondary metabolism in entomophathogenic bacteria

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Entomopathogenic bacteria of the genera Photorhabdus and Xenorhabdus have been proven to be a rich source for novel natural products. However, their structure and biosynthesis is often still unknown. Therefore the biosynthesis of four natural product classes will be investigated in detail in this project:1. In the biosynthesis of the antibiotic xenocoumacin, the biologically active compound is produced from a inactive precursor via a membrane-bound peptidase. Thus, this mechanism is an example of a novel natural prodrug-activation mechanism that is widespread in secondary metabolism but is not investigated in detail. Within this project the specificity of the peptidase XcnG will be studied using synthetic and simplified prexenocoumacin analogues. Additionally, the biosynthesis of a similarly activated natural product from X. bovienii will be investigated.2. The chemical analysis of several Xenorhabdus strains revealed that strains that produce the cytotoxic phenylethylamides almost always also produce the antibiotic xenorhabdines although both compound classes are structurally different. As this might indicate that the biosyntheses of both compounds are linked, xenorhabdines will be produced heterologously in E. coli and the resulting strain will be analysed for the production of phenylethylamides. Additionally, the responsible enzymes for the phenylethylamide biosynthesis will be characterized biochemically.3. Nematophin is a structurally simple but potent antibiotic whose biosynthesis could not been revealed using classical methods (gene cluster analysis, mutant construction). Therefore chemical biology methods will be applied to identify the enzymes involved in the biosynthesis directly. Acylcarrierproteins will be functionalized in vitro in the proteome, enabling their identification via fluorophores or by mass spectrometry. Once the enzyme(s) involved are identified, the biosynthesis gene cluster and/or the enzyme itself will be analysed.4. The novel rhabdopeptides are the most abundant compound class in Xenorhabdus, which show insecticidal or cytotoxic activity. For this class of compounds it is not known, how and why the proteins involved are used iteratively. Thus, the genes will be heterologously expressed in E. coli and the resulting strains will be analysed for rhabdopeptide production. Additionally, the protein interaction will be studied in vitro. Protein domains responsible for this interaction will be identified and exchanged. Similarly, condensation domains responsible for connecting the different substrates will be exchanged and DNA-fragments encoding methyltransferase domains will be mutagenized. This will result in a full picture of protein-protein interaction responsible for the rhabdopeptide biosynthesis.