Biochemical and genetic basis of indole alkaloid formation in the basidiomycete Psilocybe cyanescens

丝状真菌被认为是生物活性天然产物的多产者，其中包括具有巨大药用和商业价值的化合物。先前的努力主要强调研究i）子囊菌产物和ii）其骨架由多模块酶（聚酮酶、非核糖体肽合成酶）、萜烯环化酶组装或由核糖体制备的此类分子。担子菌分子，特别是那些不需要上述酶活性的分子，仍然很少被研究。因此，我们对真菌生物活性小分子组装背后的生物合成化学的理解存在偏差。拟议的研究解决了这种偏见，并侧重于一个很少探索的代谢途径，不基于上述标准的生物合成酶。本研究的目的是从酶和遗传水平上阐明蓝盖菇中吲哚生物碱的代谢。裸盖菇属的物种被通俗地称为所谓的神奇蘑菇，因为它们的主要代谢物（裸盖菇素和间接作为前药的裸盖菇素）具有精神生物活性。然而，这些化合物重新吸引了药学关注，因此再次成为临床研究的对象。拟议的工作有助于了解不寻常的天然产物化学，特别是吲哚羟基化在位置4和磷酸酯的形成。裸盖菇素独特地结合了这些结构特征。其他生物活性产品，包括蜘蛛毒液，对植物有毒的细菌化合物和植物代谢物，也显示出这些特征，但单独。除了了解裸盖菇素的生物合成，这项工作，因此预计将有试点性质的天然产物的研究一般。此外，该项目为未来的真菌生理学研究和法医应用奠定了基础。关于方法学，其工作流程依赖于两条平行但相互补充的研究路线，包括i）从真菌中纯化天然酶的生物化学方法，以及ii）候选基因的异源表达，然后表征酶活性。

Filamentous fungi have been recognized as prolific producers of bioactive natural products, among them compounds of great pharmaceutical and commercial value. Prior efforts primarily emphasized research i) on ascomycete products and ii) on such molecules whose backbone is assembled by multimodular enzymes (polyketide synthases, non-ribosomal peptide synthetases), terpene cyclases, or that are made ribosomally. Basidiomycete molecules, and in particular those that do not require the above enzymatic activities, remained very little investigated. Therefore, a bias exists in our understanding of the biosynthetic chemistry behind fungal bioactive small molecule assembly. The proposed research addresses this bias and focuses on one of the rarely explored metabolic pathways that are not based on the above standard biosynthesis enzymes. The objective of this project is to elucidate the indole alkaloid metabolism in the mushroom Psilocybe cyanescens at the enzymatic and genetic level. Species of the genus Psilocybe are colloquially referred to as so-called magic mushrooms as their major metabolites (psilocin and indirectly psilocybin as prodrug) possess psychotropic bioactivity. However, these compounds re-attracted pharmaceutical attention and, thus, are again object of clinical studies. The proposed work serves to understand unusual natural product chemistry, in particular indole hydroxylation at position 4 and phosphoester formation. Psilocybin uniquely combines these structural features. Other bioactive products, including spider venoms, bacterial compounds toxic to plants, and plant metabolites, show these features as well, but individually. Beyond the understanding of psilocybin biosynthesis, this work is therefore expected to have pilot character for natural product research in general. Further, the project lays the foundation for future research on fungal physiology and for forensic applications. Regarding the methodology, its workflow relies on two parallel - yet mutually complementing - lines of research that include i) a biochemical approach to purify native enzymes from the fungus, and ii) on heterologous expression of candidate genes, followed by characterization of the enzymatic activities.