Biosynthetic and toxic potential of ergot: Biological activity of ergot alkaloids and genome mining for the identification further potential toxic secondary metabolites

紫色锁头菌是一种植物病原真菌，以产生有毒的麦角生物碱而闻名。除此之外，我们对其他次生代谢物的产生知之甚少。在第一个资助期，研究人员对紫荆的两个次生代谢物基因簇进行了详细的鉴定。第一个以非还原性聚酮合成酶为关键酶的簇（PKS4）被确定为麦角色素基因簇，负责色素的产生。第二个鉴定的以非核糖体肽合成酶为关键酶（NRPS3）的簇与编码二酮哌嗪（ETP）毒素的簇具有同源性，其特征是二酮哌嗪环具有内部二硫化物桥。出乎意料的是，没有检测到具有典型ETP结构的分子。相反，发现了几个具有不寻常的氮硫键的中间产物。细螺旋体ETP簇细胞色素P450氧化酶的过表达揭示了具有典型ETP结构的新化合物clapurines。本研究的目标是：(1)利用基因组挖掘方法和生物合成终产物的结构解析，详细表征C. purpurea中迄今未知功能的新的次级代谢物基因簇；(2)阐明新发现的clapurines形成的详细机制，以及具有异常N-S片段的etp样基因簇的代谢物。(3)研究C. purpurea所有新的次生代谢物的细胞毒性，因为这些代谢物可能作为食品污染物；(4)表征迄今为止未知的应激条件下产生的色素，并阐明其生物合成途径。

Claviceps purpurea is a plant pathogenic fungus which is well known for the production of toxic ergot alkaloids. Besides this not much is known about the production of other secondary metabolites. In the first funding period two secondary metabolite gene clusters were characterized in detail in C. purpurea. The first cluster with a nonreducing polyketide synthase as key enzyme (PKS4) was identified as the ergochrome gene cluster responsible for the production of pigments. The second identified cluster with a nonribosomal peptide synthetase as key enzyme (NRPS3) shows homology to clusters encoding epipolythiodiketopiperazine (ETP) toxins characterized by a diketopiperazine ring with an internal disulfide-bridge. Unexpectedly, no molecules with the typical ETP structures could be detected. Instead, several intermediates with an unusual nitrogen-sulfur bond were identified. The overexpression of a cytochrome P450 oxidase from the ETP cluster in Leptospaeria revealed new compounds, named clapurines, with the typical ETP structure.Goal of this proposal is (1) the detailed characterization of further new secondary metabolite gene clusters with so far unknown function in C. purpurea using a genome mining approach and the structure elucidation of the biosynthetic endproducts, (2) to elucidate the detailed mechanism for the formation of the newly identified clapurines as well as the metabolites of the ETP-like gene cluster with the unusual N-S moiety, (3) to study the cytotoxicity of all new secondary metabolites of C. purpurea as these metabolites might play a role as food contaminants and (4) to characterize so far unknown pigments produced also under stress conditions and to elucidate their biosynthetic pathway.