Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming

Abstract

Transcriptional activation of genes belonging to the plastidial MEP-derived isoprenoid pathway by elicitation with methyl jasmonate and coronatine enhanced the content of bioactive abietane diterpenes in Salvia sclarea hairy roots. We have shown that aethiopinone, an abietane diterpene synthesized in Salvia sclarea roots is cytotoxic and induces apoptosis in human melanoma cells. To develop a production platform for this compound and other abietane diterpenes, hairy root technology was combined with the elicitation of methyl jasmonate (MeJA) or the phytotoxin coronatine (Cor). Both MeJA and Cor induced a significant accumulation of aethiopinone, but prolonged exposure to MeJA irremediably caused inhibition of hairy root growth, which was unaffected by Cor treatment. Considering together the fold increase in aethiopinone content and the final hairy root biomass, the best combination was a Cor treatment for 28 days, which allowed to obtain up to 105.34 ± 2.30 mg L of this compound to be obtained, corresponding to a 24-fold increase above the basal content in untreated hairy roots. MeJA or Cor elicitation also enhanced the synthesis of other bioactive abietane-quinone diterpenes. The elicitor-dependent steering effect was due to a coordinated transcriptional activation of several biosynthetic genes belonging to the plastidial MEP-derived isoprenoid pathway. High correlations between aethiopinone content and MeJA or Cor-elicited level of gene transcripts were found for DXS2 (r = 0.99), DXR (r = 0.99), and GGPPS (r = 0.98), encoding enzymes acting upstream of GGPP, the common precursor of diterpenes and other plastidial-derived terpenes, as well as CPPS (r = 0.99), encoding the enzyme involved in the first cyclization steps leading to copalyl-diphosphate, the precursor of abietane-like diterpenes. These results point to these genes as possible targets of metabolic engineering approaches to establish a more efficient production platform for such promising anti-proliferative plant-derived compounds.

High Yield of Bioactive Abietane Diterpenes in Salvia sclarea Hairy Roots by Overexpressing Cyanobacterial DXS or DXR Genes

“…In conclusion, the overexpression of cyanobacterial DXS and DXR genes proved to boost the accumulation of abietane diterpenes in S. sclarea hairy roots and, to our knowledge, this is the first example of a significant enhancement of the content of abietane diterpenes achieved in a medicinal plant by overexpressing cyanobacterial genes. A significant increase in this class of compounds in S. sclarea hairy roots was also obtained by elicitation with MJ by Kuzma et al [36] and by our group [21]. In both cases, the MJ-induced accumulation in abietane diterpenes was higher than that triggered by overexpressing individually the plant and the bacterial DXS or DXR genes, corroborating the notion that simultaneous overexpression of multiple biosynthetic genes is often necessary to efficiently increase the accumulation of valuable plant secondary metabolites.…”

“…We have also demonstrated that S. sclarea hairy roots overexpressing the Arabidopsis DXS and DXR genes produced a 2-fold and about 4-fold higher content of abietane-type diterpenes, respectively, especially aethiopinone [8]. In addition, the expression of the genes encoding these two enzymes acting upstream of the MEP pathway is highly correlated to an enhanced accumulation of abietane diterpenes in S. sclarea hairy roots elicited with MJ or coronatine [21] as well as in hairy roots overexpressing the WRKY or MYC2 transcription factor [22]. In trying to further optimize the conditions to produce an increased content of aethiopinone from S. sclarea hairy roots, cyanobacterial orthologous genes encoding these two first enzymes of the MEPderived isoprenoid pathway were ectopically expressed in S. scla- rea hairy roots.…”

“…In both cases, the MJ-induced accumulation in abietane diterpenes was higher than that triggered by overexpressing individually the plant and the bacterial DXS or DXR genes, corroborating the notion that simultaneous overexpression of multiple biosynthetic genes is often necessary to efficiently increase the accumulation of valuable plant secondary metabolites. It can be envisaged that simultaneous overexpression of the bacterial DXR gene in combination with other biosynthetic genes encoding limiting enzymatic steps of this metabolic pathway, identified in our previous studies [21,22], might positively further affect the accumulation of abietane diterpenes in S. sclarea hairy roots.…”

“…(e.g., Salvia aethiopis and others) have been shown to be cytotoxic against different tumor lines [4][5][6][7][8], but their low amount has hampered the possibility of accurately investigating the underlying mechanisms of its antiproliferative activity. In an effort to understand the contribution of different biosynthetic genes to a higher accumulation of bioactive abietane diterpenes in S. sclarea hairy roots, we targeted this metabolic pathway by different and complementary approaches, including the transcriptional reprograming of multiple genes by MJ elicitation [21] or by overexpressing MYC and WRKY transcription factors [22]. From both studies, it was clearly evident that the expression level of the DXS and DXR genes were highly correlated with an enhanced content of abietane diterpenes in S. sclarea hairy roots, confirming our previous data obtained by overexpressing the Arabidopsis thaliana DXS1 and DXR genes [8].…”

Natural Cytotoxic Diterpenoids, a Potential Source of Drug Leads for Melanoma Therapy

“…This approach depends on a reliable source for the core skeleton, and diterpenes are notoriously minor compounds in most cases. Cell culture in bioreactors may afford a more reliable and sustainable supply such in the case of the bioactive abietane diterpenes from Salvia sclarea (Lamiaceae) which synthesis in vitro can be by elicited transcriptional reprogramming of their hairy roots [106]. Total synthesis is always complicated and usually the last resort but nevertheless possible and always with the advantage of giving a total control on the quality, purity and supply of the drug.…”