Abstract
<jats:p>The steroid hormone 20-hydroxyecdysone (20E) controls molting and metamorphosis in arthropods, yet it also accumulates in plants, where its biosynthesis has remained unknown. We show that quinoa (Chenopodium quinoa) produces 20E through a seven-gene biosynthetic cluster encoding five cytochrome P450s, a fatty acid hydroxylase-like enzyme (FAH), and a 2-oxoglutarate-dependent dioxygenase (2OGD). Distinct mutant alleles, complementation crosses, and chemical rescue established FAH as essential for pathway entry, whereas natural accessions carrying a large cluster deletion lacked 20E and all detectable intermediates. Heterologous reconstruction in Nicotiana benthamiana demonstrated that the cluster is sufficient to convert lathosterol to 20E through an unusual FAH-catalyzed C6 oxidation and 2OGD-mediated C5 epimerization. Plants and arthropods therefore evolved distinct enzymatic routes to the same molecule. This pathway provides a blueprint for engineering 20E and defining its broader ecological functions.</jats:p>