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

photohabdus属和Xenorhabdus属昆虫病原细菌已被证明是新型天然产物的丰富来源。然而，它们的结构和生物合成往往仍然未知。因此，本项目将对四类天然产物的生物合成进行详细的研究：在抗生素异种库马辛的生物合成中，生物活性化合物是由无活性前体通过膜结合肽酶产生的。因此，这种机制是一种新的天然药物前激活机制的一个例子，这种机制在次级代谢中广泛存在，但尚未得到详细研究。在这个项目中，将使用合成和简化的前异种库马辛类似物来研究肽酶XcnG的特异性。此外，我们还将研究一种类似活性的天然产物的生物合成。对几种Xenorhabdus菌株的化学分析表明，产生细胞毒性苯乙胺的菌株几乎总是产生抗生素xenorhabdines，尽管这两类化合物在结构上不同。由于这可能表明这两种化合物的生物合成是联系在一起的，因此将在大肠杆菌中产生异源的外哈伯丁，并将分析产生的菌株以生产苯乙胺。此外，还将对苯乙胺生物合成的酶进行生物化学表征。Nematophin是一种结构简单但有效的抗生素，其生物合成无法用传统的方法（基因聚类分析，突变体构建）揭示。因此，化学生物学方法将被用于直接识别参与生物合成的酶。酰基载体蛋白将在体外蛋白质组中功能化，使其能够通过荧光团或质谱法进行鉴定。一旦所涉及的酶被确定，生物合成基因簇和/或酶本身将被分析。这种新型横纹肌多肽是Xenorhabdus中含量最多的一类化合物，具有杀虫或细胞毒活性。对于这类化合物，目前尚不清楚所涉及的蛋白质如何以及为什么被迭代使用。因此，这些基因将在大肠杆菌中异种表达，并将分析产生的菌株以产生横纹肌多肽。此外，将在体外研究蛋白质相互作用。负责这种相互作用的蛋白质结构域将被识别和交换。类似地，负责连接不同底物的缩合结构域将被交换，编码甲基转移酶结构域的dna片段将被诱变。这将导致对横纹肌多肽生物合成负责的蛋白质-蛋白质相互作用的全面了解。

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.