Completion of the seven-step pathway from tabersonine to the anticancer drug precursor vindoline and its assembly in yeast.
Completion of the seven-step pathway from tabersonine to the anticancer drug precursor vindoline and its assembly in yeast.
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DOI：
10.1073/pnas.1501821112
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发表时间：
2015-05-12
影响因子：
11.1
通讯作者：
De Luca V
中科院分区：
文献类型：
--
作者：
Qu Y;Easson ML;Froese J;Simionescu R;Hudlicky T;De Luca V
生物信息学和病毒诱导基因沉默(VIGS)引导的基因发现和生化酶分析相结合的方法表明，他柏松碱3-加氧酶(T3O)和他柏松碱3-还原酶(T3R)需要生成3-羟基-16-甲氧基-2，3-二氢他伯松宁，这是由他伯松碱合成抗癌药物前体长春花碱的中间产物。在没有T3R的情况下，T3O将Tabersonine转化为一系列T3R不能再使用的副产物，这表明了一种协同反应机制。在酵母中进行七基因途径工程展示了一个具有工业化生产抗癌药物前体长春花碱潜力的原型平台。长春花碱和长春新碱相关的抗肿瘤物质仅见于夹竹桃科植物长春花科植物长春花属植物，并继续广泛用于癌症化疗。尽管需求量很大，但这些有价值的化合物只在玫瑰花叶中积累了微量。长春花碱和长春新碱由单萜类吲哚生物碱(MIA)前体长春花碱和长春新碱缩合而成。虽然长春花碱的生物合成特性还不是很好，但从MIA前体丹参素生物合成长春花碱在分子和生化水平上已有很好的了解。本研究利用病毒诱导的基因沉默(VIGS)技术确定细胞色素P450[细胞色素P450[细胞色素71D1V2；丹参素3-加氧酶(T3O)]和乙醇脱氢酶[ADHL1；丹参碱3-还原酶(T3R)]作为候选基因，分别参与丹参碱或16-甲氧基丹参素转化为3-羟基-2，3-二氢丹参素或3-羟基-16-甲氧基-2，3-二氢丹参素，它们分别是长春花碱和长春花碱途径的中间产物。重组酶的生化分析证实，产物的形成只能通过T3O和T3R的偶联作用，因为T3O的反应产物是T3R不作为底物的环氧化物。T3O和T3R转录本在玫瑰花卉数据库中被鉴定，这些转录本代表了在叶表皮中优先表达的基因，表明随后的反应产物从叶表皮运输到特化的叶肉细胞、异成体细胞和乳管细胞，完成这些MIA的生物合成。有了这两个基因，完整的七个基因途径在酵母中被改造成从他伯松碱生产长春花碱。
Bioinformatics and virus-induced gene silencing (VIGS)-guided gene discovery combined with biochemical enzyme assays show that tabersonine 3-oxygenase (T3O) and tabersonine 3-reductase (T3R) are required to form 3-hydroxy-16-methoxy-2,3-dihydrotabersonine, an intermediate in the formation of anticancer drug precursor vindoline from tabersonine. In the absence of T3R, tabersonine is converted by T3O to a series of byproducts that can no longer be used by T3R, suggesting a concerted reaction mechanism. Engineering the seven-gene pathway in yeast demonstrated a prototype platform of high potential for industrial production of the anticancer drug precursor vindoline. Antitumor substances related to vinblastine and vincristine are exclusively found in the Catharanthus roseus (Madagascar periwinkle), a member of the Apocynaceae plant family, and continue to be extensively used in cancer chemotherapy. Although in high demand, these valuable compounds only accumulate in trace amounts in C. roseus leaves. Vinblastine and vincristine are condensed from the monoterpenoid indole alkaloid (MIA) precursors catharanthine and vindoline. Although catharanthine biosynthesis remains poorly characterized, the biosynthesis of vindoline from the MIA precursor tabersonine is well understood at the molecular and biochemical levels. This study uses virus-induced gene silencing (VIGS) to identify a cytochrome P450 [CYP71D1V2; tabersonine 3-oxygenase (T3O)] and an alcohol dehydrogenase [ADHL1; tabersonine 3-reductase (T3R)] as candidate genes involved in the conversion of tabersonine or 16-methoxytabersonine to 3-hydroxy-2,3-dihydrotabersonine or 3-hydroxy-16-methoxy-2,3-dihydrotabersonine, which are intermediates in the vindorosine and vindoline pathways, respectively. Biochemical assays with recombinant enzymes confirm that product formation is only possible by the coupled action of T3O and T3R, as the reaction product of T3O is an epoxide that is not used as a substrate by T3R. The T3O and T3R transcripts were identified in a C. roseus database representing genes preferentially expressed in leaf epidermis and suggest that the subsequent reaction products are transported from the leaf epidermis to specialized leaf mesophyll idioblast and laticifer cells to complete the biosynthesis of these MIAs. With these two genes, the complete seven-gene pathway was engineered in yeast to produce vindoline from tabersonine.
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影响因子：
11.6
作者：
St-Pierre, B;Vazquez-Flota, FA;De Luca, V
通讯作者：
De Luca, V
DOI：
10.1073/pnas.1307504110
发表时间：
2013-09-24
影响因子：
11.1
作者：
Yu, Fang;De Luca, Vincenzo
通讯作者：
De Luca, Vincenzo
影响因子：
4.9
作者：
Kellner, Franziska;Geu-Flores, Fernando;O'Connor, Sarah E.
通讯作者：
O'Connor, Sarah E.
影响因子：
7.4
作者：
Besseau, Sebastien;Kellner, Franziska;Courdavault, Vincent
通讯作者：
Courdavault, Vincent
影响因子：
7.2
作者：
Murata, J;De Luca, V
通讯作者：
De Luca, V