## 5 Rational Approaches to the Biotechnological Production of TIAs
### Elicitor Effects on the Expression of Genes Involved in TIA Metabolism  
As mentioned above, empirical studies on _C. roseus in vitro_ cultures have shown that TIA biosynthesis can be markedly induced by elicitors, however how the elicitors affect these specific metabolic pathways is not truly known [90]. In this way, different authors have studied the relationship between TIA accumulation and gene expression of _G10H_, _SLS_, _TDC_ and _STR_ under elicitation conditions, specifically in treatments with MeJA [80, 118, 119, 120, 121], CDs [80], and fungal homogenates [83, 122, 123] alone or in combination [80]. In fact, the transcript levels of _TDC_ and _STR_ were induced by MeJA treatments [80, 119, 120, 121]. In contrast to these results, the expression levels of _SLS_ and _G10H_ were not increased [80] or increased slightly at short periods of time analyzed and then, these transcripts levels returned to the control levels at long treatment times [118, 119, 120]. This could be due to the fact that the expression profile of transcripts induced by MeJA in _C. roseus_ depends on factors such as the developmental stage and type of the plant material, the cultivar of _C. roseus_ and MeJA concentration used in the experiments. It is also important to note that the expression levels of these transcripts were always well-correlated with the levels of TIAs accumulated in MeJA-treated cells [80].  
The expression of genes encoding enzymes from TIA biosynthesis can also be stimulated by adding biotic elicitors such as fungal preparations. [PERSON] _et al._[121] and [PERSON] _et al._[90] observed that _TDC_ and _STR_ genes in _C. roseus_ cell cultures were strongly up-regulated after treatment with a filtrate  
Figure 2: Gene regulation scheme of transcription factors involved in the TIA biosynthetic pathways. AS, anthranilate synthase; TDC, tryptophan decarboxylase; G10H, geraniol 10-hydroxidase; SLS, secologanin synthase; STR, strictosidine synthase; SGD, strictosidine \(\beta\)-d-glucosidase, T16H, tabersonine 16-hydroxylase, OMT, 16-hydroxadenosine 16-O-methyltransferasease; NMT, N-methyltransferasease; D4H, desacetoxyvindoline 4-hydroxylase; DAT, deacetylvindoline 4-O-acetyltransferase; Prx, peroxidase.  
of _Pythium aphanidermatum_ and yeast extract. Similarly, [PERSON] _et al._[83] also demonstrated that _C. roseus_ cells treated with PB90, displayed a time-dependent increase in catharanthine production involving a high level expression of _TDC_ and _STR_. Similarly, CDs which have oligosaccharidesacharide structure and mimick fungal attack acting as elicitors [123], activated the expression of _G10H_, _SLS_, _TDC_ and _STR_ at all time points analyzed [80].  
On the other hand, the combined effect of elicitors on the production of TIAs is a widely used strategy to increase TIAs, however there is few studies related with the joint action of elicitors on the expression of genes codifying enzymes from TIA biosynthesis. [PERSON] _et al._[80] showed that the joint action of CDs and MeJA induced in _C. roseus_ cell cultures, the highest expression levels of _G10H_, _SLS_, _TDC_ and _STR_, observing a synergistic effect on both gene expression and TIA production. Therefore, it is evident that CDs activated a distinct pathway or played a different role in regulating TIA metabolism of that seen with MeJA in _C. roseus_ cell cultures. Also the joint use of CDs and MeJA induced an important synergistic reprogramming of gene expression in _C. roseus_ suspension cultured cells of _C. roseus_, which is probably the reason for the high accumulation levels of ajmalicine and catharanthine in those conditions. The molecular mechanism underlying the synergy between these two elicitors needs to be explored further.