Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots

Diterpenoids are important compounds for plant survival and have beneficial properties for humans. Bioactive abietanic diterpenes are synthesized in roots of Salvia sclarea (e.g. aethiopinone, 1-oxoaethiopinone, salvipisone, and ferruginol), but at a very low level (about 1 % of root dry weight). To enhance the biosynthesis of this interesting class of compounds, heterologous AtDXS (D-xylulose 5-phosphate synthase) or AtDXR (1-deoxy-D-xylulose 5 phosphate reductoisomerase) genes, encoding the up-stream enzymes of the plastidial 2-C-methyl-D-erythritol 4-phosphate (MEP)-dependent terpenoid pathway, were ectopically expressed in S. sclarea hairy roots. Quantitative targeted metabolic analysis (HPLC-DAD) revealed that three independent root lines, expressing different levels of DXS or DXR transcripts and proteins, synthesized a significant higher content of abietanic diterpenes, compared to the control hairy root line transformed with the empty vector. The increase was gene-dependent, since the overexpression of the AtDXR triggered a 4.4-fold increase in aethiopinone, an abietane quinone-type tricyclic diterpene. In addition, aethiopinone was proved to be cytotoxic to different solid tumor cell lines, with the highest effect on human melanoma A375 cell line (IC50 11.4 mu M). Overall these results show that it is possible to boost the metabolic flow towards the synthesis of abietanic diterpenes in S. sclarea hairy roots by overexpressing genes involved in the first steps of the MEP-pathway and provide new insights for the large-scale production of this class of compounds, with potential application in cancer treatment.