Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS), and we show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains, use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterized sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle, making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes toward alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids.

In the native pathway to therapeutic cannabinoid biosynthesis in Cannabis sativa, the three‐step production of a key intermediate, olivetolic acid, is catalysed by the enzymes tetraketide synthase (TKS; linear tetraketide intermediate production in two stages) and olivetolic acid cyclase (OAC; final C2 → C7 aldol condensation). In the absence of OAC, a nonenzymatic C2 → C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol. TKS is a type III polyketide synthase, and the question arises why it is unable to form olivetolic acid directly, but instead forms this unwanted side product. We determined the TKS, CoA complex structure, and performed structurally guided mutagenesis studies to identify potential residues responsible for cyclization pathway discrimination in type III polyketide synthases. Prior studies suggested an ‘aldol switch’ is necessary to allow linear tetraketide intermediate release prior to cyclization, thereby enabling subsequent olivetolic acid production by OAC. However, our studies do not support the presence of a universal or predictable ‘aldol switch’ consensus sequence. Instead, we propose the mode of ordered active site water activation between type III polyketide synthases catalysing different cyclization mechanisms is subtle and homologue‐specific. Our work indicates that subtle structural variations between homologous enzymes can have a major mechanistic impact on the catalytic outcome. This highlights the importance of embedding high‐resolution structural analysis of multiple enzyme homologues with classical site‐directed mutagenesis studies when investigating highly similar enzymes with different mechanistic pathway outcomes. Enzymes TKS, EC 2.3.1.206; OAC, EC 4.4.1.26; chalcone synthase, EC 2.3.1.74; stilbene synthase, EC 2.3.1.95; 2‐PS, EC 2.3.1.-. Accession numbers The atomic coordinates and structure factors for the crystal structure of TKS have been deposited in the Protein Data Bank with accession number http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6GW3.

Cannabinoids are a therapeutically valuable class of secondary metabolites with a vast number of substituents. The native cannabinoid biosynthetic pathway of Cannabis sativa generates cannabigerolic acid, 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 synthetic chemical reactions. 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 a synthetic biology platform for cannabigerol biosynthesis in E. coli cells by employing AtaPT under an optimised microbial system. Our results have therefore set the foundation for sustainable production of well researched and rarer cannabinoids in an E. coli chassis.