# Clade III Synthases Add Cyclic and Linear Terpenoids to the Psilocybe Metabolome

## TL;DR

This study identifies new enzymes in magic mushrooms that produce diverse bioactive compounds, expanding our understanding of their chemical diversity.

## Contribution

The functional characterization of four Psilocybe cubensis terpene synthases reveals new cyclic and linear terpenoids in the Psilocybe metabolome.

## Key findings

- CubB is a (3R,6E)-(−)-nerolidol synthase.
- CubC produces multiple terpenoids including β-caryophyllene and β-farnesene.
- CubD and CubE primarily form sterpurene.

## Abstract

Psilocybe “magic mushrooms” are best known for their indolethylamine psilocybin, yet they encode enzymes for a much more diverse arsenal of small and potentially bioactive molecules. Herein, four Psilocybe cubensis clade III sesquiterpene synthases, CubB‐CubE, whose genes are differently expressed in fruiting bodies compared to vegetative mycelium are reported. CubB‐CubE were functionally characterized in vitro by product formation assays with heterologously produced enzymes and in vivo by transgene expression in Aspergillus niger, followed by extensive gas chromatography‐mass spectrometry analyzes. CubB was identified as a single product (3R,6E)‐(‐)‐nerolidol synthase. CubC is a multiproduct enzyme producing β‐caryophyllene, β‐elemene, α‐humulene, and β‐farnesene. CubD and CubE catalyze (near‐)exclusively sterpurene formation. P. cubensis young fruiting bodies and vegetative mycelium were analyzed for sesquiterpenes, which verified the presence of the CubB product α‐(3R,6E)‐(‐)‐nerolidol. As various Psilocybe species encode highly similar enzymes, this study contributes generally to the as‐yet little‐understood secondary metabolome of the genus.

The Psilocybe cubensis terpene synthases, CubB‐CubE, were investigated. Product formation assays identified CubB as a (3R,6E)‐(‐)‐nerolidol or linalool synthase, whereas CubC is a multiproduct synthase catalyzing the formation of β‐caryophyllene and other linear and cyclic sesquiterpenes/‐terpenoids. CubD and CubE catalyze predominantly sterpurene formation. The results help to explore the true natural product diversity of this iconic mushroom genus.© 2025 WILEY‐VCH GmbH