Clade III Synthases Add Cyclic and Linear Terpenoids to the <i>Psilocybe</i> Metabolome

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.