Gene discovery and virus-induced gene silencing reveal branched pathways to major classes of bioactive diterpenoids in Euphorbia peplus.
Gene discovery and virus-induced gene silencing reveal branched pathways to major classes of bioactive diterpenoids in Euphorbia peplus.
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DOI：
10.1073/pnas.2203890119
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发表时间：
2022-05-24
影响因子：
11.1
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中科院分区：
文献类型：
--
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大戟（Euphorbia peplus）是大戟属（Euphorbia）植物中的一种，含有丰富的麻风树烷和巨大戟烷型二萜类化合物。利用代谢组学指导的转录组学方法对候选基因进行鉴定，我们发现了一个参与生产lathyrane jolkinol E的短链脱氢酶基因。我们建立了一种病毒诱导的基因沉默方法。peplus使我们能够证明卡宾和多环二萜类化合物之间的直接关系，并且乔金醇C在巨大戟烷和麻风树烷的生产中充当关键的分支点中间体。这项工作为在异源宿主系统中工程化生产生物活性二萜类化合物提供了知识和工具，从而使其能够进一步评估和开发。大多数大环和多环大戟科二萜衍生自常见的C20前体casbene。虽然从casbene到lathyrane jolkinol C的生物合成途径的特征在于，其他更复杂的生物活性二萜类化合物的途径仍有待阐明。代谢组学指导的转录组学方法和基因组学方法，导致两个casbene衍生的二萜类基因簇的发现，产生了总共68个候选基因，这些基因在本塞姆氏烟草中瞬时表达，对jolkinol C和其他lathyrane具有活性。我们报告了两个短链淀粉酶/还原酶（SDRs），通过RNA测序鉴定为在大戟胶乳中高度表达。其中之一EpSDR-5是一种C3-酮还原酶，将jolkinol C转化为lathyrane jolkinol E。EpSDR-5的基因功能通过在酿酒酵母中的异源表达得到进一步证实。为了研究EpSDR-5在体内的作用，我们在大肠杆菌中建立了病毒诱导的基因沉默（VIGS）。PEPLUS，导致显着减少jatrophanes和相应增加的巨大戟烷。Casbene合成酶的VIGS导致了Jatrophanes和ingenanes这两种最丰富的E. peplus二萜类。CYP71D365的VIGS具有类似的作用，与先前确定的该基因在jolkinol C途径中的作用一致。这些结果表明，jolkinol C是生成巨大戟烷和麻风树烷途径中的分支点中间体，EpSDR-5负责从jolkinol C生成麻风树烷的第一步。
Euphorbia peplus, a member of the Euphorbia genus, is rich in jatrophane and ingenane diterpenoids. Using a metabolomics-guided transcriptomic approach to gene candidate identification, we have discovered a short-chain dehydrogenase gene involved in the production of the lathyrane jolkinol E. We have developed a virus-induced gene-silencing method in E. peplus that has allowed us to demonstrate the direct relationship between casbene and polycyclic diterpenoids and that jolkinol C acts as a key branch point intermediate in the production of ingenanes and jatrophanes. This work contributes both knowledge and tools for engineering production of bioactive diterpenoids in heterologous host systems, thus enabling their further evaluation and development. Most macro- and polycyclic Euphorbiaceae diterpenoids derive from the common C20 precursor casbene. While the biosynthetic pathway from casbene to the lathyrane jolkinol C is characterized, pathways to other more complex classes of bioactive diterpenoids remain to be elucidated. A metabolomics-guided transcriptomic approach and a genomics approach that led to the discovery of two casbene-derived diterpenoid gene clusters yielded a total of 68 candidate genes that were transiently expressed in Nicotiana benthamiana for activity toward jolkinol C and other lathyranes. We report two short-chain dehydrogenases/reductases (SDRs), identified by RNA sequencing to be highly expressed in Euphorbia peplus latex. One of these, EpSDR-5, is a C3-ketoreductase, converting jolkinol C to the lathyrane jolkinol E. Gene function of EpSDR-5 was further confirmed by heterologous expression in Saccharomyces cerevisiae. To investigate the in vivo role of EpSDR-5, we established virus-induced gene silencing (VIGS) in E. peplus, resulting in a significant reduction in jatrophanes and a corresponding increase in ingenanes. VIGS of Casbene Synthase results in a major reduction in both jatrophanes and ingenanes, the two most abundant classes of E. peplus diterpenoids. VIGS of CYP71D365 had a similar effect, consistent with the previously determined role of this gene in the pathway to jolkinol C. These results point to jolkinol C being a branch point intermediate in the pathways to ingenanes and jatrophanes with EpSDR-5 responsible for the first step from jolkinol C to jatrophane production.
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影响因子：
13.8
作者：
Forestier ECF;Czechowski T;Cording AC;Gilday AD;King AJ;Brown GD;Graham IA
通讯作者：
Graham IA
影响因子：
64.8
作者：
BAILEY, JA;CARTER, GA;WAIN, RL
通讯作者：
WAIN, RL
影响因子：
5.1
作者：
Giner, JL;Berkowitz, JD;Andersson, T
通讯作者：
Andersson, T
影响因子：
6.4
作者：
Callari, Roberta;Fischer, David;Weber, Nora
通讯作者：
Weber, Nora
DOI：
10.1073/pnas.1607504113
发表时间：
2016-08-23
影响因子：
11.1
作者：
Luo, Dan;Callari, Roberta;Hamberger, Bjoern
通讯作者：
Hamberger, Bjoern