Toosendanin, bearing a furan ring, is a limonoid belonging to the group of tetranortriterpenoids. Toosendanin is a phytochemical found in the medicinal plant Melia toosendan Sieb. et Zucc. of the Meliaceae family. Toosendanin and its derivatives demonstrate high insecticidal activity and are important pesticides derived from plants. Despite intensive investigation of limonoids over several decades, the biosynthetic pathway of these triterpenoids is less understood. To identify the key enzymes involved in the toosendanin biosynthetic pathway, we analyzed the contents of toosendanin in various plant tissues and parts and found that the highest level of toosendanin was found in the developing fruit and gradually decreased as the fruit matured. More than 346 116 transcripts were assembled based on 394 million paired-end Illumina reads and 6 million PacBio reads from the pooled RNA samples of fruits, leaves, and young barks. A total of 186 263 genes were predicted. Six 2,3-oxidoqualene cyclase (OSC) genes were identified by analyzing the association between gene expression and metabolite profiles. Functional analyses using the Nicotiana benthamiana transient expression assay showed that MtOSC1 catalyzed 2,3-oxidoqualene to produce a tetracyclic triterpene skeleton, tirucalla-7,24-dien-3β-ol, which is predicted as the precursor for toosendanin biosynthesis. We identified another OSC, MtOSC6, which is a lupeol synthase. Using synthetic biology methods, these identified enzymes could be used to model a biosynthetic pathway to produce large quantities of toosendanin. This article is protected by copyright. All rights reserved.

中文翻译：

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楝楸生物合成途径中 2,3-氧化角鲨烯环化酶的表征

带有呋喃环的川楝素是属于四降三萜类的柠檬苦素。川楝素是一种在药用植物楝树中发现的植物化学物质。等祖克。楝科。川楝素及其衍生物具有很高的杀虫活性，是植物中重要的杀虫剂。尽管几十年来对柠檬苦素进行了深入研究，但人们对这些三萜类化合物的生物合成途径知之甚少。为确定参与川楝素生物合成途径的关键酶，我们分析了植物各组织和部位中川楝素的含量，发现在发育中的果实中，川楝素的含量最高，随着果实的成熟而逐渐降低。基于 3.94 亿个双端 Illumina 读数和 600 万个 PacBio 读数，组装了超过 346 116 个转录本，这些读数来自水果、叶子和幼树皮的汇总 RNA 样本。总共预测了186 263个基因。通过分析基因表达与代谢物谱之间的关联，鉴定了六个 2,3-氧化奎宁环化酶 (OSC) 基因。使用本氏烟草瞬时表达测定的功能分析表明，MtOSC1 催化 2,3-氧化角鲨烯产生四环三萜骨架，tirucalla-7,24-dien-3β-ol，预测其为山楝素生物合成的前体。我们鉴定了另一种 OSC，MtOSC6，它是羽扇豆醇合酶。使用合成生物学方法，这些鉴定出的酶可用于模拟生物合成途径，以产生大量的川楝素。本文受版权保护。版权所有。