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

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Abstract

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 µM). 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.

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Abbreviations

AT:

Arabidopsis thaliana

DXS:

d-xylulose 5-phosphate synthase

DXR:

1-Deoxy-d-xylulose 5 phosphate reductoisomerase

MEP:

2-C-methyl-D-erythritol 4-phosphate

CAMV:

Cauliflower mosaic virus

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Acknowledgments

This work was supported by funds provided to AL by the Research Project of National Interest (PRIN 2005), the Italian Ministry of University of Education (MIUR) and by a Short-Term Scientific Mission to MCV by COST Action FA1006 “Plant Metabolic Engineering for High Value Products”. We would like to acknowledge Prof. M.H. Walter, Plant Biochemical Institute, Halle, Germany, for the gift of ZmDXR antibody.

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Correspondence to Mariacarmela Vaccaro.

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Supplementary material 1 (JPEG 296 kb)

11240_2014_514_MOESM2_ESM.jpg

Supplementary Fig. 1 Stable insertion of the heterologous DXS or DXR genes into the genome of ten independent lines of Salvia sclarea transgenic hairy roots for each gene. PCR amplification from genomic DNA, using specific primers of A. thaliana DXS (a) or A. thaliana DXR (b). Genomic DNA was used for amplifying virD gene to confirm absence of contaminant bacteria. PC, positive control (vector carrying the respective exogenous genes), EV, empty vector (hairy root generate from empty vector transformation), NS, negative control without DNA sample (JPEG 55 kb)

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Vaccaro, M., Malafronte, N., Alfieri, M. et al. Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots. Plant Cell Tiss Organ Cult 119, 65–77 (2014). https://doi.org/10.1007/s11240-014-0514-4

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