Untersuchungen zur Biosynthese von Indolalkaloiden aus den Basidiomyceten der Gattung Psilocybe

This doctoral thesis characterizes the biosynthesis of indole alkaloids in Psilocybe fungi, including the first description of psilocybin production and the simultaneous discovery of β-carbolines. The genome of Psilocybe cyanescens was sequenced, revealing a psilocybin biosynthesis cluster that was expressed heterologously in E. coli and A. niger. A continuous in vitro synthesis was developed by linking tryptophan synthase from P. cubensis with the psilocybin pathway, enabling biocatalytic production of psilocybin analogues, including 6-methylpsilocybin. The methyltransferase TrpM was used to characterize l-Hypaphorin biosynthesis without interfering with psilocybin methylation. β-carbolines were identified in Psilocybe fruiting bodies and mycelia, suggesting possible synergistic effects on psilocybin's psychoactivity via monoamine oxidase inhibition.

Study at a glance

Abstract

The goal of this doctoral thesis was the close examination of the biosynthesis of different indole alkaloides produced by different species of the fungal genus Psilocybe. This included the first characterization of the biosynthesis of psilocybin as the main psychotropic agent and the first discovery of the simultaneous production of β-carbolines. In the first step, the genome of P. cyanescens was sequenced and analyzed for characteristic genes and clusters or modifying enzymes. After the detection of the psilocybin biosynthesis cluster in Psilocybe, the cluster was expressed heterologous in E. coli and A. niger and an in vitro characterization of the psilocybin biosynthesis was done. Because of the raised pharmaceutical interest as well as some collaboration with the company Promega and the non-profit organization Usona, an expanded biocatalytic route for the in vitro production of psilocybin was established. The solution was the development of a continuous in vitro synthesis through a connection of the tryptophan synthase (TS) out of the basidiomycete P. cubensis with the biosynthesis route of psilocybin. Additionally, the substrate specifity of the TS were tested against different other halogenated indoles and for the first time different analogues of psilocybin could be produced biocatalytically with the same set of enzymes (Blei, Baldeweg et al. 2018). The generation of another derivate the 6-methylpsilocybin succeeded through an exchange with the TS of S. enterica (Fricke, Sherwood et al. 2019). Furthermore, the biosynthesis of the indole alkaloid l-Hypaphorin was characterized using the methyltransferase TrpM. Surprisingly no interference occured between the substrates of TrpM and the methyltransferase from the psilocybin biosynthesis PsiM because of strict substrate specifics. Supplemental to the already addressed indole alkaloides my work could identify further natural products out of the β-carboline class in the fruiting bodies and mycelia of different Psilocybe species. This was a big discovery in regards to the possible synergistic effects this could have on the psychoactive effect of Psilocybin through an inhibition of the monoaminoxidase enzyme (Blei, Dörner et al. 2019).