Metabolic engineering of Synechocystis sp. PCC 6803 for olivetolic acid production

Olivetolic acid (OLA), the critical aromatic precursor for plant-derived cannabinoids, conventionally relies on resource-intensive plant extraction or complex chemical synthesis. This study establishes an environmentally friendly biosynthetic platform by genetically engineering the model cyanobacterium Synechocystis sp. PCC 6803. We constructed the heterologous OLA pathway by co-expressing plant-derived tetraketide synthase (TKS), olivetolic acid cyclase (OAC), and acyl-activating enzyme 1 (AAE1). Through cultivation supplemented with sodium hexanoate, successful biosynthesis of OLA was achieved within this photosynthetic host. To overcome metabolic bottlenecks, the pathway was further optimized using the robust Pcpc560 promoter coupled with carbon sink redirection strategy. Disruption of the primary glycogen storage sink (ΔglgC) combined with the overexpression of the anaplerotic malic enzyme gene (maeB) significantly enhanced the specific OLA yield. Notably, while continuous aeration with 5% CO2 drastically accelerated cell growth and prolonged the cultivation period, it simultaneously led to a severe decrease in the specific OLA yield. This work demonstrates the feasibility of utilizing a cyanobacterial chassis for precursor-directed OLA biosynthesis, establishing a foundational blueprint for the sustainable manufacturing of cannabinoid-related metabolites. Initial synthesis of olivetolic acid in Synechocystis sp. PCC 6803. Production titer increased after knocking out the glycogen synthesis pathway. Overexpression assays optimized the metabolic pathway and increased production.