## 5 Rational Approaches to the Biotechnological Production of TIAs
### Metabolic Engineering to Improve the Production of TIAs
#### 5.2.2 Overexpression of Transcription Factors which Regulate TIA Biosynthesis  
Most of genes codifying enzymes from TIA biosynthetic pathway are tightly regulated by specific transcription factors in a coordinate manner together with developmental and environmental factors. In _C. roseus_, only a few transcription factors have been isolated and characterized, highlighting the Octadecanoid-Responsive Catharanthus AP2/ERF-domain proteins (_ORCA2_ and _ORCA3_), _CrBPF1_, _CrWRKY1_, _CrMYC1_ and _CrMYC2_[73, 78, 79, 80, 81, 82, 83, 84, 86, 87, 88, 89, 90, 91, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127]. These transcription factors respond to jasmonic acid, MeJA, and/or elicitors. Two bHLH (a basic helix-loop-helix) transcription factors, _CrMYC1_ and _CrMYC2_, were isolated and characterized from _C. roseus_. Both _CrMYC1_ and _CrMYC2_ were involved in the response to MeJA while _CrMYC1_ also responded to fungal elicitors [128, 129], suggesting that these transcription factors may be involved in the regulation of TIA biosynthesis in response to these signals. Moreover, _CrMYC2_ activate the expression of two transcription factors, _ORCA2_ and _ORCA3_, which bind to the JERE (a _cis_-acting element involved in jasmonic acid and elicitor responses) element of the _STR_ promoter. However, while _ORCA2_ respond to MeJA and elicitors, _ORCA3_ only respond to jasmonic acid. Likewise, the isolation of _CrBPF1_ transcription factor which is induced by elicitors but not by MeJA, suggest that elicitors induce _STR_ gene expression via jasmonic acid-dependent and independent pathways [130].  
On the other hand, _ORCA3_ overexpression resulted in increasing levels of some genes involved in TIA biosynthetic pathway, specially _TDC_, _STR_ and _D4H_[118,130] which in turn, provokes the accumulation of TIAs [97,99,131]. In fact, the overexpression of _ORCA3_ in hairy root cultures increased the content of catharanthine up to 2.5-fold in comparison with the control cultures [97] (Table 1). Moreover, _ORCA2_ is a transcription factor key in regulating genes encoding the enzymes _STR_, _TDC_ and _SGD_ involved in the first two branches of TIA biosynthetic pathway [98,132]. [PERSON] _et al._[98] also observed that the overexpression of _ORCA2_ in _C. roseus_ hairy root cultures provoked an increase of 2.0-fold in catharanthine (Table 1). Likewise, the combined overexpression of _G10H_ and _ORCA3_ in hairy roots provoked an increase of catharanthine (6.5-fold) [131]. The same overexpression was carried out in plants of _C. roseus_ resulting in an important increase of vindoline, catharanthine, ajmalicine, anhydrovinblastine and vinblastine (Table 1, [99]).  
Other transcription factors involved in the biosynthesis of TIAs is the _WRKY_ family. Overexpression of _CrWRKY1_ in hairy roots increased the expression of genes encoding _AS_, _SLS_, _SDG_ and _TDC_ and repressed _ORCA2_, _ORCA3_, and _CrMYC2_ resulting in an increase of ajmalicine and serpentine (Table 1, [95]).  
Finally, CrMPK3 is also an important component of intracellular signalling pathway triggered by different stresses in _C. roseus_. In fact, the overexpression of CrMPK3 in _C. roseus_ leaves raised both the expression of _ORCA3_ and genes involved in TIA biosynthetic pathway as well as serpentine, vindoline, catharanthine and vincristine levels (Table 1, [96]).