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Molecular metabolic engineering of clinically-used anti-cancer plant products

临床抗癌植物产品的分子代谢工程

基本信息

批准号：
12558075
负责人：
SAITO Kazuki
金额：
$ 7.87万
依托单位：
CHIBA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

Camptothecin derivatives are clinically used anti-tumor alkaloids. Transformed plant cell cultures may be an alternative source of production of camptothecin and related compounds, although they are now producted by extraction from intact plants. We have established a hairy root culture of Ophiorriza pumila (Rubiaceae) incited by Agrobacteriun rhizogenes strain 15834. This hairy root grew well 16-fold times for 5 weeks in liquid culture, and it produced camptothecin as a main alkaloid up to 0.1% per dry weight of cells. Interestingly, not only the hairy root cells contained camptothecin, but the culture medium also accumulated substantial amount of camptothecin. The camptothecin content in the medium was increased by the presence of a polystyrene resin (Diaion HP-20), to which camptothecin was absorbed. Camptothecin was easily recovered from the resin. Our established method is the most feasible and commercially applicable way to produce camptothecin by in vitro cell culture. We have cloned and characterized cDNAs encoding strictosidine synthase (OpSTR ; EC4.3. 3.2) and tryptophan decarboxylase (OpTDC ; EC4.1.1. 28), two key enzymes in the biosynthesis of camptothecin from hairy roots of 0. Pumila. We also idolated the cDNA coding for NADPH : cytochrome P450 reductase (OpCPR ; EC1. 6.2.4) that is presumed to be indirectly involved in camptothecin synthesis. The recombinant OpSTR and OpTDC proteins exhibit STR and TDC activities, respectively, when expressed in Escherichia coli. The tissue-specific and stress-inducible expression patterns of OpSRC and OpTDC were quite similar, unlike those of OpCPR The high expression of OpSTR and OpTDC observed in hairy roots, roots and stems were closely correlated with STR protein accumulation as observed by immunoblot analysis. Plant stress compounds like salicylic acid (SA) repressed expression of OpSTR and OpTDC, suggesting coordinate regulation of these genes for camptothecin biosynthesis.

喜树碱衍生物是临床上常用的抗肿瘤生物碱。转化的植物细胞培养物可能是生产喜树碱和相关化合物的替代来源，尽管它们现在通过从完整植物中提取来生产。以发根农杆菌15834菌株为诱导剂，建立了茜草科蛇莓的毛状根培养体系。这种毛状根在液体培养中生长良好，5周可生长16倍，并产生喜树碱作为主要生物碱，每细胞干重可达0.1%。有趣的是，不仅毛状根细胞中含有喜树碱，而且培养基中也积累了大量的喜树碱。培养基中的喜树碱含量通过存在吸收喜树碱的聚苯乙烯树脂（Diaion HP-20）而增加。喜树碱易于从树脂中回收。我们建立的方法是最可行的和商业化的方法，生产喜树碱的体外细胞培养。我们已经克隆并表征了编码strictosidine合酶（OpSTR ; EC4.3）的cDNA。3.2)和色氨酸脱羧酶（OpTDC ; EC4.1.1. 28），是喜树毛状根生物合成喜树碱的两个关键酶。普米拉我们还将编码NADPH的cDNA：细胞色素P450还原酶（OpCPR ; EC 1. 6.2.4），推测其间接参与喜树碱合成。重组OpSTR和OpTDC蛋白在大肠杆菌中表达时分别具有STR和TDC活性。OpSRC和OpTDC的组织特异性和胁迫诱导表达模式非常相似，与OpCPR不同。免疫印迹分析显示，在毛状根、根和茎中观察到的OpSTR和OpTDC的高表达与STR蛋白积累密切相关。植物胁迫化合物如水杨酸（SA）抑制了OpSTR和OpTDC的表达，表明这些基因对喜树碱生物合成的协调调节。