Overexpression of ORCA3 and G10H in Catharanthus roseus Plants Regulated Alkaloid Biosynthesis and Metabolism Revealed by NMR-Metabolomics

Abstract

In order to improve the production of the anticancer dimeric indole alkaloids in Catharanthuse roseus, much research has been dedicated to culturing cell lines, hairy roots, and efforts to elucidate the regulation of the monoterpenoid indole alkaloid (MIA) biosynthesis. In this study, the ORCA3 (Octadecanoid-derivative Responsive Catharanthus AP2-domain) gene alone or integrated with the G10H (geraniol 10-hydroxylase) gene were first introduced into C. roseus plants. Transgenic C. roseus plants overexpressing ORCA3 alone (OR lines), or co-overexpressing G10H and ORCA3 (GO lines) were obtained by genetic modification. ORCA3 overexpression induced an increase of AS, TDC, STR and D4H transcripts but did not affect CRMYC2 and G10H transcription. G10H transcripts showed a significant increase under G10H and ORCA3 co-overexpression. ORCA3 and G10H overexpression significantly increased the accumulation of strictosidine, vindoline, catharanthine and ajmalicine but had limited effects on anhydrovinblastine and vinblastine levels. NMR-based metabolomics confirmed the higher accumulation of monomeric indole alkaloids in OR and GO lines. Multivariate data analysis of 1H NMR spectra showed change of amino acid, organic acid, sugar and phenylpropanoid levels in both OR and GO lines compared to the controls. The result indicated that enhancement of MIA biosynthesis by ORCA3 and G10H overexpression might affect other metabolic pathways in the plant metabolism of C. roseus.

The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus

“…The use of TFs can be a powerful tool in such metabolic engineering programs. ORCA3 has been overexpressed in cell suspensions, hairy roots, and plants in attempts to increase MIA production (14,(23)(24)(25). However, because ORCA3 does not activate iridoid genes, MIA accumulation in these lines required either coexpression with G8O or feeding with iridoid precursors.…”

Natural products – modifying metabolite pathways in plants

“…Overexpression of ORCA3 resulted in the enhanced expression of several MIA biosynthetic genes and, consequently, in increased accumulation of alkaloids in C. roseus cell lines [42]. To date, however, the results of transcription factor overexpression in periwinkle hairy roots or cell lines remain inconclusive [43,44]. Concurrently, enhanced expression of AaORA in A. annua resulted in an approximately 50% increase in artemisinin accumulation, and a 35% boost in DHAA (dihydroartemisinic acid) content, respectively [41].…”

Enzyme Inhibitor Studies Reveal Complex Control of Methyl-D-Erythritol 4-Phosphate (MEP) Pathway Enzyme Expression in Catharanthus roseus

“…As yet, little is known about the multiple levels of MEP pathway regulation in C. roseus . Interestingly, overexpression of DXS in C. roseus hairy roots stimulated the accumulation of several MIAs [33], and DXS expression was induced in ORCA3 overexpression C. roseus cell lines (ORCA3: a jasmonate-responsive APETALA2 (AP2)-domain transcription factor activating MIA biosynthesis [34], [35]). Besides, various analogues of the DXR inhibitor fosmidomycin inhibited MIA synthesis in C. roseus cells [36], [37], indicating that MEP pathway flux may impact on MIA biosynthesis.…”