## 4 A Hairy Root Platform for the Production of Bioactive Abietane Diterpenes
### Metabolic Engineering to Enrich the Content of Abietane Diterpenes in S. sclarea Hairy Roots  
Plant metabolic engineering is a powerful strategy to modify metabolic routes aimed at increasing the synthesis of a specific bioactive compound, by either genetic engineering of endogenous genes, with the final aim to increase or divert the metabolic flux towards desired/undesired products, respectively. Another application is to generate plants able to synthesize novel compounds through transformation with genes from other plants/organisms [64]. Our previous elicitation studies evidenced a strong correlation between the accumulation of abietane diterpenes and the level of expression of several biosynthetic genes of the MEP-pathway [51]. In the next section, we summarize the different approaches we have used and the results achieved in boosting the synthesis of abietane diterpenes in _S. sclarea_ HRs by: i) tuning the expression of biosynthetic genes that control rate-limiting enzymatic steps (_DXS_, _DXR_, _GGPPS_, _CPPS_ alone or in combination); ii) RNAi silencing of a gene acting at the lateral competitive route of gibberellin (_Ent-CPPS_); iii) overexpressing different transcription factors (_WRKY_s and _MYC_2) involved in the coordinated regulation of several genes of the MEP-pathway. 2.1 Overexpression of _DXS_ and _DXR_ Genes, encoding the First Two Enzymes Acting Up-Stream the MEP-Pathway  
As reported previously, Deoxyxylulose 5-phosphate synthase (DXS), the first enzymatic step of the MEP-pathway, has been often reported as one of the limiting enzymes of the up-stream pathway to GGPP, by influencing the supply of IPP and its isomer DMAPP (extensively and recently reviewed by [65]). Experimental evidence has indicated that also the activity of DXP reducto-isomerase (DXR), the second enzymatic step, is limiting the biosynthesis of several plant isoprenoids [66]. The expression of the _DXS_ and _DXR_ genes has been modified in different plant species to enhance successfully the synthesis of different isoprenoids, although the reported results are not always consistent in different plant species [67; 68; 69]. In our earlier studies, we have demonstrated that constitutive overexpression of the heterologous _A. thaliana AtDXS_ or _AtDXR_ genes is able to enhance the content of bioactive abietane-type diterpenoids in _S. sclarea_ HRs [9]. As reported in Table 2, overexpression of the DXS protein triggered a 2-fold increase in aithopinone content compared to the control HR lines. However, high levels of the exogenous DXS protein caused a negative pleotropic effect on the HR growth, probably due to a general competition with the basal protein synthesis or to the modification of the content of other MEP-derived phytohormones (cytokines, gibberellin or ABA), which might interfere with normal HR growth and development. Interestingly, the overexpression of the _AtDXR_ gene appeared to be slightly more efficient in enhancing the synthesis of abietane-typediterpenes in _S. sclarea_ HRs (a 3-fold increase compared to the control HR line), coupled to no detrimental effects on HR growth [9]. Overexpression both genes in _S. miltiorrhiza_ HRs has improved the accumulation of tanshinones by [68] as well as of other terpenoids in different plant species [69]. In a parallel study, we also tested whether overexpression of _DXS_ and _DXR_ genes of cyanobacterial origin might offer some biotechnological advantages in increasing the synthesis of bioactive diterpenes in _S. sclarea_ HRs. Indeed, the introduction in a plant genome of heterologous genes by genetic transformation might avoid potential gene silencing or co-suppression events, often reported when overexpressing homologous genes, and, at the same time, ensuring a possible greater stability and activity of the two enzymes in a plastidial environment. Orthologous genes, amplified by the genomic DNA of _Syntechocystis_ sp PCC6803, were overexpressed in _S. sclarea_ HRs, by targeting them to the chloroplast by the rbc plastid transit peptide [41].