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

点击翻译按钮获取中文摘要

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.