Abstract
Monoterpenoid indole alkaloids are a large (∼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.
Highlights
Monoterpenoid indole alkaloids are a large (ϳ3000 members) and structurally diverse class of metabolites restricted to a limited number of plant families in the order Gentianales
Candidates for 10-hydroxylation and 10-O-methylation were identified based on sequence homology to tabersonine 16-hydroxylase (T16H; P98183) and 16-hydroxytabersonine O-methyltransferase (16OMT; B0EXJ8) from C. roseus in a transcriptome of T. iboga
Because ibogaine is currently sourced from plant material, we are interested in simultaneously contributing to improvements in the ibogaine supply chain and developing new synthetic biology applications for gaining access to novel small molecule therapeutics based on the ibogaine scaffold
Summary
Of the disparate stereochemical ibogaine, including a potentially unique cyclization step, and the late stages of ibogaine biosynthesis that are characterized by a net decarboxylation at the 16-position, 10-hydroxylation, and 10-O-methylation of the predicted coronaridine precursor (Fig. 2). Considering that the commercial sources of ibogaine are primarily plant-based and that therapeutic use is contributing to overharvesting, we were keen on discovering ibogaine biosynthetic genes that could bolster current production strategies or lead to the creation of alternative production platforms through modern synthetic biology approaches This would have the added benefit of assisting with iboga conservation efforts, a primary concern among members of the Bwiti community. We report the first transcriptome of T. iboga, which we used to identify a cytochrome P450 (P450), named ibogamine-10-hydroxylase (I10H), and an O-methyltransferase (OMT), named noribogaine-10-O-methyltransferase (N10OMT), that catalyze the penultimate 10-hydroxylation and final 10-O-methylation steps in ibogaine biosynthesis, respectively This is the first report of enzymes involved in ibogaine biosynthesis, and the hydroxylation at the 10-position of ibogamine and coronaridine by I10H is the first example of a regiospecific hydroxylation of the iboga alkaloid scaffold. 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 development of ibogaine as a treatment for addiction
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