Efficient Escherichia coli Platform for Cannabinoid Precursor Olivetolic Acid Biosynthesis from Inexpensive Inputs

Abstract

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 Escherichia coli and may be a valuable reference for the development of microbial cell factories that use hexanoyl-CoA and malonyl-CoA as important intermediates.