4.4.2. Metabolic engineering
Metabolic engineering is another strategy for enhancing the biosynthesis of secondary metabolites of economic importance by C. roseus . TIA biosynthetic pathway can be accelerated at any stage of synthesis, tryptamine synthesis (Shikimate pathway), secologanin synthesis (terpenoid pathway), and terpenoid synthesis. Several cloned genes have been used to enhance the production of TIAs including Anthranilate synthase α (ASα), Tryptophan decarboxylase (TDC),1-deoxy-D-xylulose 5-phosphate synthase (DXS), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), Strictosidine synthase(STR), Strictosidine β-D-glucosidase (SGD),Octadecanoid-responsive Catharanthus (ORCA1,ORCA2,ORCA3) and zinc finger protein family from C. roseus (ZCT1, ZCT2, ZCT3) (Liu et al., 2007). The STRpromoter in C. roseus contains three cis-regulatory sequences. The BA element (BA region) in the STR gene promoter also acts as a functional unit in elicitor and jasmonate-responsive gene expression (Hughes et al., 2004). By interacting with TDC and STR promoters, the ORCA proteins consequently activate gene expression. ORCA1 contains a single AP2-domain on the N-terminal part of the protein, whereas the ORCA2 cDNA encoded an AP2-domain protein on the C-terminal part of an ORCA2 protein (Pauw et al., 2004).
Genetic manipulation of genes involved in the metabolic pathways of these compounds can lead to an increase in the yield of TIAs in C. roseus . Overexpression of 1-deoxy-D-xylulosesynthase (DXS ) or geraniol-10-hydroxylase (G10H ) in a hairy root line of C. roseus produced a significant increase in metabolite production (Peebles et al., 2011). Terpenoid biosynthesis involves transactivation of several genes by specific transcription factors such as jasmonate-regulated basic helix-loop-helix (bHLH ) iridoid synthesis 1 (BIS1 ) transcription factor controls the expression of genes lead to the synthesis of iridoid organic acid. Overexpression of BIS1 in hairy root lines ofC. roseus upregulated the tabersonine (0.18 mg/ gm DW), which is an essential precursor in the vindoline synthetic pathway for the production of vincristine and vinblastine (Van Moerkercke et al., 2015). Expression studies of ORCA3 showed no change in TIAs synthesis. However, co-overexpression of pathway regulator ORCA3 and pathway gene strictosidine glucosidase (SGD ) remarkably increased alkaloid pool by 47% in C. roseus hairy root lines (Sun and Peebles, 2016). Genetic transformation experiments on C. roseus using hypocotyls as explants effectively increases anticancer compound synthesis.
A 2.4-fold increase in vindoline accumulation in transgenic C. roseus compared to control was evident by the overexpression of deacetylvindoline-4-O-acetyltransferase (DAT ) in the TIA pathway (Wang et al., 2012). Transcriptomic studies of the TIA pathway in C. roseus indicated that anthranilate synthase (AS), a rate-limiting enzyme, regulates the transcription of various genes involved in the TIA pathway (Sun et al., 2016) (Table 3 ).