Heterologous Production of the Structurally Complex Diterpenoid Forskolin in Synechocystis sp. PCC. 6803

Photosynthetic organisms such as cyanobacteria have the potential for the sustainable production of complex organic molecules due to their ability to use light as an energy source to fix CO₂ and assimilate inorganic nutrients. Over the past decade, large efforts have been put into the metabolic engineering of cyanobacteria to produce various compounds such as alcohols, isoprenoids, biopolymers, and recombinant proteins. Forskolin is a structurally complex labdane-type diterpenoid with eight chiral carbon atoms and is naturally produced in the root cork of the plant Plectranthus barbatus. Forskolin is a potent cAMP activator indicated as a pharmaceutical for a variety of diseases. In the plant, forskolin biosynthesis from geranylgeranyl diphosphate involves six enzymes: two terpene synthases, three cytochrome P450s, and a single acetyltransferase. In this work, we express all six biosynthetic genes from Plectranthus barbatus in Synechocystis sp. PCC. 6803 and demonstrate heterologous production of this complex diterpenoid in a phototroph cyanobacterium. Forskolin titers reached 25.0 ± 4.4 µg/L and the forskolin was entirely secreted into the media. The forskolin-producing Synechocystis strain and empty vector control were cultivated in a photobioreactor for 8 days. Both strains showed similar chlorophyll a contents, and the forskolin-producing strain reached a slightly higher OD₇₃₀ than the control. Forskolin began accumulating in the supernatant after 4 days and increased over time. These results indicate that forskolin production did not negatively impact cell growth.