Identification of key enzymes responsible for protolimonoid biosynthesis in plants: Opening the door to azadirachtin production
Identification of key enzymes responsible for protolimonoid biosynthesis in plants: Opening the door to azadirachtin production
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DOI：
10.1073/pnas.1906083116
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发表时间：
2019-08-20
影响因子：
11.1
通讯作者：
Osbourn, Anne
中科院分区：
文献类型：
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作者：
Hodgson, Hannah;De La Pena, Ricardo;Osbourn, Anne
limestones是由属于楝科（桃花心木）和芸香科（柑橘）家族的植物制成的天然产物。众所周知，它们具有杀虫活性，导致柑橘类水果苦味，以及潜在的药物特性。最著名的柠檬酸类杀虫剂是印楝素，由印楝树（印楝）产生。尽管在过去的半个世纪里对柠檬苦素类化合物进行了深入的研究，但其生物合成途径仍然是未知的。鲎被分类为四降三萜，因为原型26-碳类柠檬酸骨架被假定为通过4个碳的损失与相关的呋喃环形成，由30-碳三萜骨架通过至今未知的机制形成。在这里，我们已经挖掘了3种不同的柠檬苦素生产物种的基因组和转录组序列资源（A。Indica，Melia azedarach，和Citrus sinensis）中，以阐明柠檬苦素类化合物生物合成的早期步骤。我们确定了氧化角鲨烯环化酶能够产生潜在的30-碳三萜支架前体tirucalla-7，24-二烯-3 β-醇从3个物种。我们进一步鉴定了M. Azedarach（MaCYP 71 CD 2和MaCYP 71 BQ 5）和C. Sinensis（CsCYP 71 CD 1和CsCYP 71 BQ 4），其能够对替鲁卡拉-7，24-二烯-3 β-醇进行3次氧化，导致自发的半缩醛环形成和原乳香醇的产生。我们的工作报告了来自不同植物物种的原酮生物合成酶的特征，并支持两种植物家族之间途径保守的概念。它进一步为工程改造具有增强的抗虫性的作物植物和通过在异源宿主中表达产生用于制药和其他应用的高价值柠檬苦素类化合物铺平了道路。
Limonoids are natural products made by plants belonging to the Meliaceae (Mahogany) and Rutaceae (Citrus) families. They are well known for their insecticidal activity, contribution to bitterness in citrus fruits, and potential pharmaceutical properties. The best known limonoid insecticide is azadirachtin, produced by the neem tree (Azadirachta indica). Despite intensive investigation of limonoids over the last half century, the route of limonoid biosynthesis remains unknown. Limonoids are classified as tetranortriterpenes because the prototypical 26-carbon limonoid scaffold is postulated to be formed from a 30-carbon triterpene scaffold by loss of 4 carbons with associated furan ring formation, by an as yet unknown mechanism. Here we have mined genome and transcriptome sequence resources for 3 diverse limonoid-producing species (A. indica, Melia azedarach, and Citrus sinensis) to elucidate the early steps in limonoid biosynthesis. We identify an oxidosqualene cyclase able to produce the potential 30-carbon triterpene scaffold precursor tirucalla-7,24-dien-3 beta-ol from each of the 3 species. We further identify coexpressed cytochrome P450 enzymes from M. azedarach (MaCYP71CD2 and MaCYP71BQ5) and C. sinensis (CsCYP71CD1 and CsCYP71BQ4) that are capable of 3 oxidations of tirucalla-7,24-dien-3 beta-ol, resulting in spontaneous hemiacetal ring formation and the production of the protolimonoid melianol. Ourwork reports the characterization of protolimonoid biosynthetic enzymes from different plant species and supports the notion of pathway conservation between both plant families. It further paves theway for engineering crop plants with enhanced insect resistance and producing high-value limonoids for pharmaceutical and other applications by expression in heterologous hosts.