Assessing the Limitations to Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Root Cultures Through Gene Expression Profiling and Precursor Feeding
Assessing the Limitations to Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Root Cultures Through Gene Expression Profiling and Precursor Feeding
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DOI：
10.1002/btpr.204
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发表时间：
2009-09-01
影响因子：
2.9
通讯作者：
Lee-Parsons, Carolyn W. T.
中科院分区：
文献类型：
--
作者：
Goklany, Sheba;Loring, Ralph H.;Lee-Parsons, Carolyn W. T.
药用上重要的萜类吲哚生物碱（TIAs）的产生部分受转录水平调控。在这项研究中，通过基因表达谱和前体摄食来评估玫瑰花毛状根培养物合成TIA的局限性。分别采用定量RT-PCR和HPLC检测TIA通路关键基因（G10h、Tdc、Str和Sgd）的转录水平和与TIA通路相关的代谢物（色胺、马头草苷、龙骨草苷、strictosidine、ajmalineine、蛇pentine和tabersonine）的水平。在第21天用茉莉酸甲酯（250 μ M MeJA）诱导的培养中，24 h后，G10h、Tdc、Str和Sgd的表达分别增加了9.1倍、3.1倍、6.7倍和8.3倍。基因表达上调后，5天后，strictosidine、ajmalineine和tabersonine的表达分别增加了160倍、440倍和420%。前体马甲素、色胺或其组合分别饲喂于非诱导和meja诱导的培养物中，以补充上述研究。在未诱导或meja诱导的培养物中，前体喂养均未显著提高TIA的产量。在非诱导细胞中，马鞭草苷和色胺的下游步骤受到限制（SLS， STR或SGD），因为马鞭草苷或色胺在前体喂养的细胞中积累。在meja诱导的培养中，随着Str和Sgd基因的表达和TIA产量的增加，这些瓶颈得到了部分克服。然而，在meja诱导的培养物中，前体喂养的secologanin积累，表明在meja诱导的条件下，STR可能是有限的。(c) 2009年美国化学工程师学会生物技术。掠夺。生物工程学报，25:1289-1296,2009
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 mu M 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. (c) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1289-1296, 2009