Efficient Platform for Cannabinoid Precursor Olivetolic Acid Biosynthesis from Inexpensive Inputs.

Olivetolic acid (OLA), an initial precursor of cannabinoids, is catalyzed by type III polyketide synthase, which has a wide range of pharmacological activities, such as antimicrobial and cytotoxic effects. Here, we applied systematic metabolic engineering to develop a multienzyme cascade system to produce OLA via two low-cost inputs. The polyketide synthase (OLS) and cyclase enzymes (OAC), along with the best combination of hexanoyl-CoA and malonyl-CoA synthetases (AEE3 and MatB), were first introduced into the biocatalytic system to increase the supply of hexanoyl-CoA and malonyl-CoA as starting and extender units. To drive the catalysis smoothly, an ATP regeneration system and a CoA-sufficient supply system were incorporated into the biocatalysts to provide enough cofactors. Furthermore, malonyl-CoA flux was redirected to OLA biosynthesis through delicate control of the fatty acid biosynthesis (FAB) pathway via promoter engineering. Collectively, these strategies have led us to produce OLA at a titer of 102.1 mg/L with a productivity of 25.5 mg/L/h by using malonate and hexanoate as direct substrates. Our biocatalytic system provides an effective platform for the production of the cannabinoid precursor OLA in and may be a valuable reference for the development of microbial cell factories that use hexanoyl-CoA and malonyl-CoA as important intermediates.