AbstractCannabinoids are a therapeutically valuable class of secondary metabolites with a vast number of substituents. The native cannabinoid biosynthetic pathway ofCannabis sativagenerates cannabigerolic acid (CBGA), the common substrate to multiple cannabinoid synthases. The bioactive decarboxylated analog of this compound, cannabigerol (CBG), represents an alternate gateway into the cannabinoid space as a substrate either to non-canonical cannabinoid synthase homologs or to synthetic chemical reactions. Herein, we describe the identification and repurposing of aromatic prenyltransferase (AtaPT), which when coupled with native enzymes ofC. sativacan form anEscherichia coliproduction system for CBGA in cell lysates and CBG in whole cells. Engineering of AtaPT, guided by structural analysis, was performed to enhance its kinetics toward CBGA production for subsequent use in a proof-of-concept lysate system. For the first time, we show a synthetic biology platform for CBG biosynthesis inE. colicells by employing AtaPT under an optimized microbial system. Our results have therefore set the foundation for sustainable production of well-researched and rarer cannabinoids in anE. colichassis.Graphical Abstract