## 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
#### 5.2.2 GGPP Availability Limits the Accumulation of Abietane Diterpenes in _S. sclarea_ HRs  
GGPP is the universal isoprenoid precursor and it has been postulated that modifying its distribution among the different downstream metabolic branches of the plastidial MEP-pathway might direct the metabolic flux towards a higher biosynthesis of a targeted end product [70]. We proved that this is also true for an enhanced abietane diterpene accumulation in _S. sclarea_ HRs, by chemically inhibiting the competitive gibberell (GA) route with the 2-chloroethyl-N,N,N-trimethyl-ammonium chloride (CCC), a known inhibitor of entCPPS, the first enzymatic step, that from GGPP leads to gibberellins (GA) biosynthesis, or by RNAi-mediated silencing of the _ent-CPPS_ gene (Figure 3). The block of this competitive metabolic pathway enhanced the content of all analyzed diterpenes (carnosic acid, ferruginol, 1-oxo-ferruginol, salvipsione, aethiopinone, and 1-oxo-aethiopinone) in CCC-treated HRs compared to the control HRs, with the most relevant increase in the content of aethiopinone (a 7-fold increase). RNAi-mediated silencing of the _entCPPS_ gene confirmed that blocking this lateral GA route from GGPP is an efficient strategy to increase the abietane diterpene content in _S. sclarea_ HRs [40] (Table 2). These data indirectly demonstrated that the GGPP pool is limiting in the metabolic route to abietane diterpenes, as already suggested by the significant correlation between the expression level of the _GGPPS_ gene and the abietane diterpene content we have found in _S. sclarea_-elicited HRs [51]. These findings also pointed to the GGPPS enzyme as a potential target for increasing the biosynthesis of abietane diterpenes in _S. sclarea_ HRs. By assuming that the IPP and DMAPP, the immediate GGPP precursors, would not be limiting, the cDNA full-length _SsGGPPS_ gene, including its own plastid transit peptide, was constitutively overexpressed in _S. sclarea_ HRs. Higher transcript levels of the _SsGGPPS_ gene significantly increased the content of aethiopinone (7.80 \(\pm\) 0.71 mg g\({}^{-1}\) dw), salvipsione (5.57 \(\pm\) 0.78 mg g\({}^{-1}\) dw), and ferruginol (8.51 \(\pm\) 0.35 mg g\({}^{-1}\) dw), which was approximately 8, 7, and 28 times, respectively, higher than that in the control HR line (0.99 \(\pm\) 0.08 mg g\({}^{-1}\) dw). Altogether, these results indicate indirectly that the GGPP pool in _S. sclarea_ HRs might limit the biosynthesis of abietane diterpenes, and that GGPPS overexpression is an efficient strategy to increase the overall content of this class of compounds in _S. sclarea_ HRs [40]. 2.3. CPP Is a Precursor of Abietane Diterpenes in _S. sclarea_ and Overexpression of _SsCPPS_ Also Enhances Their Content  
The biosynthetic pathway of the abietane diterpenes synthesized in _S. sclarea_ roots is poorly understood, although their chemical structures were established more than two decades ago [38, 71]. For instance, little is known on the first GGPP cyclization step operated by diterpene synthases (diTPSs) in _S. sclarea_ roots to yield abietane diterpenes. It is well documented that in conifers/gymnosperms, the formation of diterpenes requires an initial double cyclization of GGPP into (+)-copalyl diphosphate [(+)-CPP], operated by the class II active site of the abietadiene synthase, a bifunctional diTPS. The (+)-CPP intermediate is then modified by the class I diTPS active site and is subjected to a second cyclization enzymatic step, followed by structural rearrangements via intermediate carbocations [54]. In Lamiaceae species, it has been reported that the synthesis of labadane-related diterpenes also proceeds through the cyclization of GGPP to CPP, catalyzed by the copalyl diphosphate synthase (CPPS), a class II diTPS [72]. The following enzymatic reaction in the biosynthesis of this group of diterpenes is due to the action of a kaurene synthase-like enzyme, the miltiradiene synthase (MiS), which, through a spontaneous oxidation reaction, transforms CPP to miltiradiene.