Peter J. Facchini

SUMMARY Peyote ( Lophophora williamsii ) is an entheogenic and medicinal cactus native to the Chihuahuan desert. The psychoactive and hallucinogenic properties of peyote are principally attributed to the phenethylamine derivative mescaline. Despite the isolation of mescaline from peyote over 120 years ago, the biosynthetic pathway in the plant has remained undiscovered. Here, we use a transcriptomics and homology‐guided gene discovery strategy to elucidate a near‐complete biosynthetic pathway from l ‐tyrosine to mescaline. We identified a cytochrome P450 that catalyzes the 3‐hydroxylation of l ‐tyrosine to l ‐DOPA, a tyrosine/DOPA decarboxylase yielding dopamine, and four substrate‐specific and regiospecific substituted phenethylamine O ‐methyltransferases. Biochemical assays with recombinant enzymes or functional analyses performed by feeding putative precursors to engineered yeast ( Saccharomyces cerevisiae ) strains expressing candidate peyote biosynthetic genes were used to determine substrate specificity, which served as the basis for pathway elucidation. Additionally, an N ‐methyltransferase displaying broad substrate specificity and leading to the production of N ‐methylated phenethylamine derivatives was identified, which could also function as an early step in the biosynthesis of tetrahydroisoquinoline alkaloids in peyote.