Salidroside is a bioactive tyrosine-derived phenolic natural product found in medicinal plants under the Rhodiola genus.In addition to their anti-fatigue and anti-anoxia roles in traditional medicine,Rhodiola total extract and salidroside have also displayed medicinal properties as anti-cardiovascular diseases and anticancer agents.The resulting surge in global demand of Rhodiola plants and salidroside has driven some species close to extinction.Here,we report the full elucidation of the Rhodiola salidroside biosynthetic pathway utilizing the first comprehensive transcriptomics and metabolomics datasets for Rhodiola rosea.Unlike the previously proposed pathway involving separate decarboxylation and deamination enzymatic steps from tyrosine to the key intermediate 4-hydroxyphenylacetaldehyde (4-HPAA),Rhodiola contains a pyridoxal phosphate-dependent 4-HPAA synthase that directly converts tyrosine to 4-HPAA.We further identified genes encoding the subsequent 4-HPAA reductase and tyrosol:UDP-glucose 8-O-glucosyltransferase,respectively,to complete salidroside biosynthesis in Rhodiola.We show that heterologous production of salidroside can be achieved in the yeast Saccharomyces cerevisiae as well as the plant Nicotiana benthamiana through transgenic expression of Rhodiola salidroside biosynthetic genes.This study provides new tools for engineering sustainable production of salidroside in heterologous hosts.