Data from: Cytochrome P450 and O-methyltransferase catalyze the final steps in the biosynthesis of the anti-addictive alkaloid ibogaine from Tabernanthe iboga

Monoterpenoid indole alkaloids (MIAs) are a large (ca. 3000 members) and structurally diverse class of metabolites restricted to a limited number of plant families in the order Gentianales. Tabernanthe iboga or iboga (Apocynaceae) is native to western equatorial Africa and has been used in traditional medicine for centuries. Howard Lotsof is credited with bringing iboga to the attention of Western medicine through his accidental discovery that iboga can alleviate opioid withdrawal symptoms. Since this observation, iboga has been investigated for its use in the general management of addiction. We were interested in elucidating ibogaine biosynthesis to understand the unique reaction steps en route to ibogaine. Furthermore, because ibogaine is currently sourced from plant material, these studies may help improve the ibogaine supply chain through synthetic biology approaches. Here we used next-generation sequencing to generate the first iboga transcriptome, and leveraged homology guided gene discovery to identify the penultimate hydroxylase and final O-methyltransferase steps in ibogaine biosynthesis, herein named ibogamine 10-hydroxylase (I10H) and noribogaine 10-O-methyltransferase (N10OMT). Heterologous expression in Saccharomyces cerevisiae (I10H) or Escherichia coli (N10OMT) and incubation with putative precursors, along with HPLC–MS analysis,
confirmed the predicted activities of both enzymes. Moreover, high expression levels of their transcripts were detected in ibogaine-accumulating plant tissues. These discoveries coupled with our publicly available iboga transcriptome will contribute to additional gene discovery efforts and could lead to the stabilization of the global ibogaine supply chain and to the development of ibogaine as a treatment for addiction.

单萜吲哚生物碱（Monoterpenoid indole alkaloids, MIAs）是一类规模庞大（约含3000个成员）且结构多样的代谢物类群，仅局限于龙胆目（Gentianales）下的少数植物科属。依波加木（Tabernanthe iboga，俗称伊博格iboga）隶属于夹竹桃科（Apocynaceae），原生分布于西非赤道地区，数个世纪以来被应用于传统医药领域。霍华德·洛茨夫（Howard Lotsof）因偶然发现伊博格可缓解阿片类药物戒断症状，从而将伊博格带入西方医学界的视野。自此之后，伊博格被广泛研究用于成瘾的综合管理。我们的研究旨在阐明伊博加因（ibogaine）的生物合成途径，以解析其合成过程中独特的反应步骤。此外，由于当前伊博加因仍依赖植物材料提取，相关研究或可通过合成生物学手段优化伊博加因的供应链。本研究借助下一代测序技术构建了首个伊博格转录组，并通过同源导向的基因挖掘策略，鉴定出伊博加因生物合成途径中的倒数第二步羟化酶与终末O-甲基转移酶，本文将其分别命名为伊博加明10-羟化酶（ibogamine 10-hydroxylase, I10H）与去甲伊博加明10-O-甲基转移酶（noribogaine 10-O-methyltransferase, N10OMT）。通过在酿酒酵母（Saccharomyces cerevisiae，用于表达I10H）或大肠杆菌（Escherichia coli，用于表达N10OMT）中进行异源表达，并与推定前体共孵育，结合高效液相色谱-质谱（High Performance Liquid Chromatography-Mass Spectrometry, HPLC-MS）分析，证实了两种酶的预测催化活性。此外，在积累伊博加因的植物组织中检测到了这两个基因的高转录水平表达。上述发现结合我们公开可获取的伊博格转录组数据，将为后续的基因挖掘工作提供助力，并有望推动全球伊博加因供应链的稳定化，以及将伊博加因开发为成瘾治疗药物的进程。