The ORCA2 transcription factor plays a key role in regulation of the terpenoid indole alkaloid pathway

Abstract

BackgroundThe terpenoid indole alkaloid (TIA) pathway leads to the production of pharmaceutically important drugs, such as the anticancer compounds vinblastine and vincristine. Unfortunately, these drugs are produced in trace amounts, causing them to be very costly. To increase production of these drugs, an improved understanding of the TIA regulatory pathway is needed. Towards this end, transgenic Catharanthus roseus hairy roots that overexpress the ORCA2 TIA transcriptional activator were generated and characterized.ResultsTranscriptional profiling experiments revealed that overexpression of ORCA2 results in altered expression of key genes from the indole and terpenoid pathways, which produce precursors for the TIA pathway, and from the TIA pathway itself. In addition, metabolite-profiling experiments revealed that overexpression of ORCA2 significantly affects the levels of several TIA metabolites. ORCA2 overexpression also causes significant increases in transcript levels of several TIA regulators, including TIA transcriptional repressors.ConclusionsResults presented here indicate that ORCA2 plays a critical role in regulation of TIA metabolism. ORCA2 regulates expression of key genes from both feeder pathways, as well as the genes (STR and SGD) encoding the enzymes that catalyze the first two steps in TIA biosynthesis. ORCA2 may play an especially important role in regulation of the downstream branches of the TIA pathway, as it regulates four out of five genes characterized from this part of the pathway. Regulation of TIA transcriptional repressors by ORCA2 may provide a mechanism whereby increases in TIA metabolite levels in response to external stimuli are transient and limited in magnitude.

Perspectives of the Metabolic Engineering of Terpenoid Indole Alkaloids in Catharanthus roseus Hairy Roots

“…Yeast one-hybrid screening with a Str promoter indicated that ORCA2 activates the Str promoter and its expression is rapidly inducible with JA and fungal elicitation; in contrast, ORCA1 is expressed constitutively and is not involved in JAand fungal elicitor-induced Str gene expression [31]. Recently, the overexpression of ORCA2 in C. roseus hairy roots enhanced the transcript levels of AS, TDC, G10H, STR, D4H, T16H, and PRX1 genes [37]. In addition to the three ORCAs, another transcription factor, the C. roseus box P-binding factor (CrBPF-1) can also bind the Str promoter but at a different position.…”

“…C. roseus G10H [50] Ajmalicine:, lochnericine:, tabersonine: Arabidopsis DXS ? feedback-insensitive Arabidopsis ASa [50] Lochnericine:, hörhammericine:, tabersonine: C. roseus T16H [49] Strictosidine;, catharanthine;, lochnericine:, hörhammericine;, tabersonine; C. roseus DAT [51] Hörhammericine: C. roseus ORCA3 [35] Lochnericine;, hörhammericine;, tabersonine; C. roseus ORCA2 [37] Strictosidine;, secologanin;, serpentine:, tabersonine;; C. roseus WRKY1 [43] Serpentine::, catharanthine;, tabersonine; C. roseus WRKY2 [44] Serpentine:, tabersonine::, vindoline:::…”

“…Although many transcriptional regulators have been identified, metabolic engineering with these regulators is still limited. So far, only ORCA2, ORCA3, CrWRKY1, and CrWRKY2 have been overexpressed separately in C. roseus hairy roots to study their effect on the regulators, genes, and metabolites in the TIA metabolic pathway [35,37,43,44]. JA treatment and the overexpression of ORCA3 in C. roseus hairy roots under a glucocorticoid-inducible promoter were investigated for their effect on TIA metabolite levels and gene expression [35].…”

“…ORCA2 was overexpressed under an ethanol-inducible promoter in C. roseus hairy roots, and the expression levels of key genes involved in the TIA pathway, as well as the important metabolites, were examined [37]. Compared with the uninduced ORCA2 line, the induced ORCA2 line produced more serpentine, 16-hydroxytabersonine, and 19-hydroxytabersonine and less secologanin, strictosidine, tabersonine, and hörhammericine.…”

CrMYC1 transcription factor overexpression promotes the production of low abundance terpenoid indole alkaloids in Catharanthus roseus

“…Nevertheless, determining an increase in the yield or function of the enzymes in the TIA pathway through overexpression of CrMYC1 cannot confirm that they are the main targets of this transcription factor because CRMYC1 may indirectly enhance the function of some TIA enzymes by targeting another one or more regulatory genes. Similarly, overexpression of ORCA2 transcription factor in Catharanthus roseus hairy root resulted in a significant increase in catharanthine content but the amount of vinblastine and vincristine was below the detectable level (Li et al, 2013). However, there are some limitations for detailed investigation since the catharanthine production pathway is not recognized genetically and enzymatically.…”