title: Assessing the limitations to terpenoid indole alkaloid biosynthesis in Catharanthus roseus hairy root cultures through gene expression profiling and precursor feeding.
abstract: The production of pharmaceutically important terpenoid indole alkaloids (TIAs) from Catharanthus roseus is partly regulated at the transcriptional level. In this study, limitations in TIA biosynthesis from C. roseus hairy root cultures were assessed through gene expression profiling and precursor feeding. The transcript levels of key TIA pathway genes (G10h, Tdc, Str, and Sgd) and metabolite levels associated with the TIA pathway (tryptamine, loganin, secologanin, strictosidine, ajmalicine, serpentine, and tabersonine) were monitored using quantitative RT-PCR and HPLC, respectively. In cultures elicited with methyl jasmonate (250 microM MeJA on day 21), G10h, Tdc, Str, and Sgd expression increased by 9.1, 3.1, 6.7, and 8.3-fold, respectively, after 24 h. Up-regulation of gene expression was followed by a 160, 440, and 420% increase in strictosidine, ajmalicine, and tabersonine levels, respectively, after 5 days. Precursors loganin, tryptamine, or their combination were fed to noninduced and MeJA-induced cultures to complement the above studies. TIA production was not significantly enhanced in either noninduced or MeJA-induced cultures with precursor feeding. In noninduced cells, steps downstream of loganin and tryptamine were limiting (SLS, STR, or SGD) because either loganin or tryptamine accumulated in the cells with precursor feeding. These bottlenecks were partly overcome in MeJA-induced cultures as the expression of Str and Sgd genes and TIA production increased. However, secologanin accumulated in MeJA-induced cultures with precursor feeding, suggesting that STR was likely limiting under MeJA-induced conditions.
authors.affiliation: Dept. of Chemical Engineering, Northeastern University, Boston, MA 02115, USA.
authors.forename: Sheba
authors.initials: S
authors.lastname: Goklany
authors.forename: Ralph H
authors.initials: RH
authors.lastname: Loring
authors.forename: James
authors.initials: J
authors.lastname: Glick
authors.forename: Carolyn W T
authors.initials: CW
authors.lastname: Lee-Parsons
mesh_terms: D030008:Catharanthus / Q000235:genetics* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D020869:Gene Expression Profiling / Q000379:methods*; D039823:Iridoids / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D018517:Plant Roots / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D046948:Secologanin Tryptamine Alkaloids / Q000378:metabolism*; D046509:Tissue Culture Techniques; D014363:Tryptamines / Q000378:metabolism
chemical_list: D039823:Iridoids; D012333:RNA, Messenger; D018749:RNA, Plant; D046948:Secologanin Tryptamine Alkaloids; D014363:Tryptamines; C030820:tryptamine; C059516:loganin