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

嗜线虫致病杆菌的异香豆素生物合成及昆虫病原菌次生代谢的调节

• 批准号: 59922156
• 负责人: Professor Dr. Helge Björn Bode
• 金额: --
• 依托单位: Abteilung Naturstoffe in Organismischen Interaktionen
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2008
• 资助国家: 德国
• 起止时间: 2007-12-31 至 2012-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/59922156?language=en
• 关键词: Xenocoumacin; biosynthesis; Xenorhabdus; nematophila; regulation

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.

光杆状菌属和黄杆状菌属的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通常仍然未知。因此，本项目将详细研究四类天然产物的生物合成：1。在抗生素异香豆素的生物合成中，生物活性化合物通过膜结合肽酶由无活性前体产生。因此，这种机制是一种新的天然前药活化机制的一个例子，这种机制在次级代谢中广泛存在，但尚未详细研究。在该项目中，将使用合成和简化的prexenocoumacin类似物研究肽酶XcnG的特异性。此外，从X.将对bovienii进行调查。对几种黄杆菌菌株的化学分析表明，产生细胞毒性苯乙酰胺的菌株几乎总是也产生抗生素黄杆菌，尽管两种化合物类别在结构上不同。由于这可能表明两种化合物的生物合成是相互联系的，因此在E.大肠杆菌，并分析所得菌株的苯乙胺生产情况。此外，负责苯乙酰胺生物合成的酶将进行生物化学表征。杀线虫素是一种结构简单但有效的抗生素，其生物合成不能使用经典方法（基因簇分析，突变体构建）来揭示。因此，将应用化学生物学方法来鉴定直接参与生物合成的酶。酰基载体蛋白将在蛋白质组中进行体外功能化，使其能够通过荧光团或质谱法进行识别。一旦所涉及的酶被鉴定，生物合成基因簇和/或酶本身将被分析。新的杆状肽是杆状菌中含量最丰富的一类化合物，具有杀虫或细胞毒活性。对于这类化合物，尚不清楚所涉及的蛋白质如何以及为什么被反复使用。因此，这些基因将在E.大肠杆菌，并分析所得菌株的横纹肽生产。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。这将导致负责横纹肽生物合成的蛋白质-蛋白质相互作用的全貌。