Lochnericine is a major monoterpene indole alkaloid (MIA) in the roots of Madagascar periwinkle (Catharanthus roseus). Lochnericine is derived from the stereoselective C6,C7-epoxidation of tabersonine and can be metabolized further to generate other complex MIAs. While the enzymes responsible for its downstream modifications have been characterized, those involved in lochnericine biosynthesis remain unknown. By combining gene correlation studies, functional assays, and transient gene inactivation, we identified two highly conserved P450s that efficiently catalyze the epoxidation of tabersonine: tabersonine 6,7-epoxidase isoforms 1 and 2 (TEX1 and TEX2). Both proteins are quite divergent from the previously characterized tabersonine 2,3-epoxidase and are more closely related to tabersonine 16-hydroxylase, involved in vindoline biosynthesis in leaves. Biochemical characterization of TEX1/2 revealed their strict substrate specificity for tabersonine and their inability to epoxidize 19-hydroxytabersonine, indicating that they catalyze the first step in the pathway leading to hörhammericine production. TEX1 and TEX2 displayed complementary expression profiles, with TEX1 expressed mainly in roots and TEX2 in aerial organs. Our results suggest that TEX1 and TEX2 originated from a gene duplication event and later acquired divergent, organ-specific regulatory elements for lochnericine biosynthesis throughout the plant, as supported by the presence of lochnericine in flowers. Finally, through the sequential expression of TEX1 and up to four other MIA biosynthetic genes in yeast, we reconstituted the 19-acetylhörhammericine biosynthetic pathway and produced tailor-made MIAs by mixing enzymatic modules that are naturally spatially separated in the plant. These results lay the groundwork for the metabolic engineering of tabersonine/lochnericine derivatives of pharmaceutical interest.

中文翻译：

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两种 Tabersonine 6,7-环氧酶在长春花中启动 Lochnericine 衍生的生物碱生物合成。

Lochnericine 是马达加斯加长春花 (Catharanthus roseus) 根中的主要单萜吲哚生物碱 (MIA)。Lochnericine 衍生自塔博宁的立体选择性 C6,C7 环氧化作用，可以进一步代谢以生成其他复杂的 MIA。虽然负责其下游修饰的酶已被表征，但那些参与洛克尼碱生物合成的酶仍然未知。通过结合基因相关性研究、功能分析和瞬时基因失活，我们确定了两个高度保守的 P450，它们有效地催化了泰伯宁的环氧化：泰伯宁 6,7-环氧化酶亚型 1 和 2（TEX1 和 TEX2）。这两种蛋白质与先前表征的塔伯宁 2,3-环氧化酶完全不同，并且与塔伯宁 16-羟化酶更密切相关，参与叶片中文多林的生物合成。TEX1/2 的生化特征揭示了它们对 tabersonine 的严格底物特异性以及它们无法环氧化 19-羟基 tabersonine，表明它们催化了导致 hörhammericine 产生的途径的第一步。TEX1 和 TEX2 显示出互补的表达谱，其中 TEX1 主要在根中表达，而 TEX2 在气管器官中表达。我们的结果表明 TEX1 和 TEX2 起源于基因复制事件，后来为整个植物的 lochnericine 生物合成获得了不同的器官特异性调控元件，这得到了花中 lochnericine 的支持。最后，通过 TEX1 和最多四个其他 MIA 生物合成基因在酵母中的顺序表达，我们重建了 19-乙酰化鼠李素生物合成途径，并通过混合在植物中自然空间分离的酶模块来生产定制的 MIA。这些结果为具有药用价值的 tabersonine/lochnericine 衍生物的代谢工程奠定了基础。