Biosynthesis of cannabigerol and cannabigerolic acid, the gateways to further cannabinoid production.

Cannabinoids are a therapeutically valuable class of secondary metabolites with a vast number of substituents. Currently over 100 cannabinoids have been isolated form the plant species Cannabis sativa, with many more non-natural analogues being chemically synthesised. Cannabinoids are primarily sourced from the cannabis plant, which has drawbacks including the resource costs of crop cultivation, inconsistent yields, and extraction and purification difficulties. In the native cannabinoid biosynthetic pathway of C. sativa, cannabigerolic acid is the gateway to the synthesis of many of these compounds, being the common substrate to multiple cannabinoid synthases. The bioactive decarboxylated analogue of this compound, cannabigerol, represents an alternate gateway into the cannabinoid space either as a substrate to non-canonical cannabinoid synthase homologues or to chemical reactions. The construction of a synthetic biology production platform to these compounds would open opportunities for sustainable production of well researched and rarer cannabinoids. Herein, we describe the identification and repurposing of the aromatic prenyl transferase, AtaPT, which when coupled with native enzymes of C. sativa can form an E. coli production system for cannabigerolic acid in cell lysates, and cannabigerol in whole cells. Engineering of AtaPT, guided by structural analysis, was performed to enhance its kinetics towards cannabigerolic acid production for subsequent use in a proof-of-concept lysate system. For the first time, we show cannabigerol production in E. coli cells by employing AtaPT under an optimised microbial system. Our results have therefore set the foundation for a cannabinoid production platform in an E. coli chassis.