Production of Manoyl Oxide, a Precursor to the Medical Active Compound Forskolin, in Synechocystis PCC 6803

Terpenoids, also known as isoprenoids, are a large and diverse family of natural compounds including sterols, carotenoids and hormones, many of which are commercially interesting. Forskolin is a complex C₂₀ diterpenoid that is produced in the root cork cells of the plant Coleus forskohlii, and has been shown to have pharmacological activities. Because of its complex molecular structure, chemical synthesis of forskolin is not economically attractive, and extraction and purification from C. forskohlii plants is laborious and resource intensive.

We have engineered the unicellular cyanobacterium Synechocystis PCC 6803 to produce the forskolin precursor 13R-manoyl oxide (13R-MO) by expressing the two C. forskohlii enzymes CfTPS2 and CfTPS3. These two nonnative genes were integrated into the genome at three different locations with two different promoters, with the top producing strains yielding production titers of 0.25 mg/L/g DCW 13R-MO. To further increase the yield, 13R-MO producing strains were engineered by introduction of selected enzymes from the C. forskohlii non-mevalonate pathway (MEP), which resulted in improving the titer to 0.46 mg/L/g DCW in the highest producing strain. By cultivating the strains at different light intensities, large differences in product accumulation were observed, attributed to changes in promoter activities. Finally, we quantified carotenoid and chlorophyll content in our different strains since those pigments are made or partially made from the same terpenoid precursor as 13R-MO and the results suggests that heterologous expression of 13R-MO influences pigment composition. This work is of interest for future engineering of terpenoid products in cyanobacteria and provides insight into promoter selection.