A "Magic Mushroom" Multi-Product Sesquiterpene Synthase.

Psilocybe "magic mushrooms" are chemically well understood for their psychotropic tryptamines. However, the diversity of their other specialized metabolites, in particular terpenoids, has largely remained an open question. Yet, knowledge on the natural product background is critical to understand if other compounds modulate the psychotropic pharmacological effects. CubA, the single clade II sesquiterpene synthase of P. cubensis, was heterologously produced in Escherichia coli and characterized in vitro, complemented by in vivo product formation assays in Aspergillus niger as a heterologous host. Extensive GC-MS analyses proved a function as multi-product synthase and, depending on the reaction conditions, cubebol, ß-copaene, δ-cadinene, and germacrene D were detected as the major products of CubA. In addition, mature P. cubensis carpophores were analysed chromatographically which led to the detection of ß-copaene and δ-cadinene. Enzymes closely related to CubA are encoded in the genomes of various Psilocybe species. Therefore, our results provide insight into the metabolic capacity of the entire genus.

Metabolic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and related tryptamine derivatives.

Psilocybin is a tryptamine-derived psychoactive alkaloid found mainly in the fungal genus Psilocybe, among others, and is the active ingredient in so-called "magic mushrooms". Although its notoriety originates from its psychotropic properties and popular use as a recreational drug, clinical trials have recently recognized psilocybin as a promising candidate for the treatment of various psychological and neurological afflictions. In this work, we demonstrate the de novo biosynthetic production of psilocybin and related tryptamine derivatives in Saccharomyces cerevisiae by expression of a heterologous biosynthesis pathway sourced from Psilocybe cubensis. Additionally, we achieve improved product titers by supplementing the pathway with a novel cytochrome P450 reductase from P. cubensis. Further rational engineering resulted in a final production strain producing 627 ± 140 mg/L of psilocybin and 580 ± 276 mg/L of the dephosphorylated degradation product psilocin in triplicate controlled fed-batch fermentations in minimal synthetic media. Pathway intermediates baeocystin, nor norbaeocystin as well the dephosphorylated baeocystin degradation product norpsilocin were also detected in strains engineered for psilocybin production. We also demonstrate the biosynthetic production of natural tryptamine derivative aeruginascin as well as the production of a new-to-nature tryptamine derivative N-acetyl-4-hydroxytryptamine. These results lay the foundation for the biotechnological production of psilocybin in a controlled environment for pharmaceutical applications, and provide a starting point for the biosynthetic production of other tryptamine derivatives of therapeutic relevance.

Iterative l-Tryptophan Methylation in Psilocybe Evolved by Subdomain Duplication.

Psilocybe mushrooms are best known for their l-tryptophan-derived psychotropic alkaloid psilocybin. Dimethylation of norbaeocystin, the precursor of psilocybin, by the enzyme PsiM is a critical step during the biosynthesis of psilocybin. However, the "magic" mushroom Psilocybe serbica also mono- and dimethylates l-tryptophan, which is incompatible with the specificity of PsiM. Here, a second methyltransferase, TrpM, was identified and functionally characterized. Mono- and dimethylation activity on l-tryptophan was reconstituted in vitro, whereas tryptamine was rejected as a substrate. Therefore, we describe a second l-tryptophan-dependent pathway in Psilocybe that is not part of the biosynthesis of psilocybin. TrpM is unrelated to PsiM but originates from a retained ancient duplication event of a portion of the egtDB gene that encodes an ergothioneine biosynthesis enzyme. During mushroom evolution, this duplicated gene was widely lost but re-evolved sporadically and independently in various genera. We propose a new secondary metabolism evolvability mechanism, in which weakly selected genes are retained through preservation in a widely distributed, conserved pathway.

Bioactive alkaloids produced by fungi. I. Updates on alkaloids from the species of the genera Boletus, Fusarium and psilocybe.

Fungi, in particular, are able in common with the higher plants and bacteria, to produce metabolites, including alkaloids. Alkaloids, along with other metabolites are the most important fungal metabolites from pharmaceutical and industrial point of view. Based on this observation, the authors of this review article have tried to provide an information on the alkaloids produced by the species of genera: Boletus, Fusarium and Psilocybef from 1981-2009. Thus the review would be helpful and provides valuable information for the researchers of the same field.

Biotransformation of ent-kaur-16-en-19-oic acid by Psilocybe cubensis.

Biotranformation of ent-kaur-16-en-19-oic acid (1) using Psilocybe cubensis resulted in hydroxylated products. After two days of incubation, ent-16beta,17-dihydroxy-kauran-19-oic acid (2) was isolated. After further incubation for nine days, two novel metabolites, ent-12alpha,16beta,17-trihydroxy-kauran-19-oic acid (3) and ent-11alpha,16beta,17-trihydroxy-kauran-19-oic acid (4), were obtained. The metabolites were identified by spectroscopic methods and X-ray crystallography. Compounds 1-4 were evaluated for their cytotoxic properties against the human leukaemia K562 cell line; only compound 1 showed moderate activity.