Psilocybin-producing fungi contain many less-studied metabolites beyond psilocybin, but most research treats them as a single group. By optimizing extraction and analysis methods, the metabolomes of 42 distinct fungal strains across 9 species were examined, revealing broad diversity within and between species. Optimal extraction of fruiting bodies used a 1:20 tissue-to-solvent ratio with 25:75 water:methanol at pH 9 for 1.5 hours, enabling quantification of 8 tryptophan-derived indolamines by HPLC-DAD and identification of putative hydroxypsilocybin by HPLC-MS/MS. A method mimicking in vivo dephosphorylation for in vitro setups was also developed. The work provides a standardized approach for studying these fungi and highlights their pharmaceutical potential.
A biosynthetic pathway for dimethyltryptamine (DMT) was reconstructed in a plant assay, along with the full pathways of five natural psychedelics: psilocin, psilocybin, DMT, bufotenin, and 5-methoxy-DMT. Halogenated analogs of these molecules, which do not occur naturally and may have therapeutic potential for psychiatric conditions, were also engineered. By blending catalytic functions from different organisms and using rational protein design to create mutant enzymes, the production of indolethylamine components in plants became substantially more efficient. This platform enables concurrent biosynthesis and diversification of psychoactive indolethylamines.