Phytocannabinoids are a group of plant-derived metabolites that display a wide range of psychoactive as well as health-promoting effects. The production of pharmaceutically relevant cannabinoids relies on extraction and purification from cannabis (Cannabis sativa) plants yielding the major constituents, Δ⁹-tetrahydrocannabinol and cannabidiol. Heterologous biosynthesis of cannabinoids inNicotiana benthamiana orSaccharomyces cerevisiae may provide cost-efficient and rapid future production platforms to acquire pure and high quantities of both the major and the rare cannabinoids as well as novel derivatives. Here, we used a meta-transcriptomic analysis of cannabis to identify genes for aromatic prenyltransferases of the UbiA superfamily and chalcone isomerase-like (CHIL) proteins. Among the aromatic prenyltransferases,CsaPT4 showed CBGAS activity in bothN. benthamiana andS. cerevisiae. Coexpression of selectedCsaPT pairs and of CHIL proteins encoding genes withCsaPT4 did not affect CBGAS catalytic efficiency. In a screen of different plant UDP-glycosyltransferases,Stevia rebaudiana SrUGT71E1 andOryza sativa OsUGT5 were found to glucosylate olivetolic acid, cannabigerolic acid, and Δ⁹-tetrahydrocannabinolic acid. Metabolic engineering ofN. benthamiana for production of cannabinoids revealed intrinsic glucosylation of olivetolic acid and cannabigerolic acid.S. cerevisiae was engineered to produce olivetolic acid glucoside and cannabigerolic acid glucoside.