Catnip or catmint (Nepeta spp.) is a flowering plant in the mint family (Lamiaceae) famed for its ability to attract cats. This phenomenon is caused by the compound nepetalactone, a volatile iridoid that also repels insects. Iridoids are present in many Lamiaceae species but were lost in the ancestor of the Nepetoideae, the subfamily containing Nepeta. Using comparative genomics, ancestral sequence reconstructions, and phylogenetic analyses, we probed the re-emergence of iridoid biosynthesis in Nepeta. The results of these investigations revealed mechanisms for the loss and subsequent re-evolution of iridoid biosynthesis in the Nepeta lineage. We present evidence for a chronology of events that led to the formation of nepetalactone biosynthesis and its metabolic gene cluster. This study provides insights into the interplay between enzyme and genome evolution in the origins, loss, and re-emergence of plant chemical diversity.

Terpene synthases typically form complex molecular scaffolds by concerted activation and cyclization of linear starting materials in a single enzyme active site. Here we show that iridoid synthase, an atypical reductive terpene synthase, catalyzes the activation of its substrate 8-oxogeranial into a reactive enol intermediate, but does not catalyze the subsequent cyclization into nepetalactol. This discovery led us to identify a class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) from catmint (Nepeta mussinii) that capture this reactive intermediate and catalyze the stereoselective cyclisation into distinct nepetalactol stereoisomers. Subsequent oxidation of nepetalactols by NEPS1 provides nepetalactones, metabolites that are well known for both insect-repellent activity and euphoric effect in cats. Structural characterization of the NEPS3 cyclase reveals that it binds to NAD+ yet does not utilize it chemically for a non-oxidoreductive formal [4 + 2] cyclization. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis.

The carbon skeleton of ecologically and pharmacologically important iridoid monoterpenes is formed in a reductive cyclization reaction unrelated to canonical terpene cyclization. Here we report the crystal structure of the recently discovered iridoid cyclase (Catharanthus roseus) bound to a mechanism-inspired inhibitor that illuminates substrate binding and catalytic function of the enzyme. Key features that distinguish iridoid synthase from its close homologue, progesterone 5β-reductase, are highlighted.

The evolution of chemical complexity has been a major driver of plant diversification, with novel compounds serving as key innovations. The species-rich mint family (Lamiaceae) produces an enormous variety of compounds that act as attractants and defense molecules in nature and are used widely by humans as flavor additives, fragrances, and anti-herbivory agents. To elucidate the mechanisms by which such diversity evolved, we combined leaf transcriptome data from 48 Lamiaceae species and four outgroups with a robust phylogeny and chemical analyses of three terpenoid classes (monoterpenes, sesquiterpenes, and iridoids) that share and compete for precursors. Our integrated chemical-genomic-phylogenetic approach revealed that: (1) gene family expansion rather than increased enzyme promiscuity of terpene synthases is correlated with mono- and sesquiterpene diversity; (2) differential expression of core genes within the iridoid biosynthetic pathway is associated with iridoid presence/absence; (3) generally, production of iridoids and canonical monoterpenes appears to be inversely correlated; and (4) iridoid biosynthesis is significantly associated with expression of geraniol synthase, which diverts metabolic flux away from canonical monoterpenes, suggesting that competition for common precursors can be a central control point in specialized metabolism. These results suggest that multiple mechanisms contributed to the evolution of chemodiversity in this economically important family.

(−)-Ibogaine and (−)-voacangine are plant derived psychoactives that show promise as treatments for opioid addiction. However, these compounds are produced by hard to source plants, making these chemicals difficult for broad-scale use. Here we report the complete biosynthesis of (−)-voacangine, and de-esterified voacangine, which is converted to (−)-ibogaine by heating, enabling biocatalytic production of these compounds. Notably, (−)-ibogaine and (−)-voacangine are of the opposite enantiomeric configuration compared to the other major alkaloids found in this natural product class. Therefore, this discovery provides insight into enantioselective enzymatic formal Diels–Alder reactions.

Strychnine is a natural product that, through isolation, structural elucidation and synthetic efforts, shaped the field of organic chemistry. Currently, strychnine is used as a pesticide to control rodents1 because of its potent neurotoxicity2,3. The polycyclic architecture of strychnine has inspired chemists to develop new synthetic transformations and strategies to access this molecular scaffold4, yet it is still unknown how plants create this complex structure. Here we report the biosynthetic pathway of strychnine, along with the related molecules brucine and diaboline. Moreover, we successfully recapitulate strychnine, brucine and diaboline biosynthesis in Nicotiana benthamiana from an upstream intermediate, thus demonstrating that this complex, pharmacologically active class of compounds can now be harnessed through metabolic engineering approaches.

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.

Nepetalactones are iridoid monoterpenes with a broad range of biological activities produced by plants in the Nepeta genus. However, none of the genes for nepetalactone biosynthesis have been discovered. Here we report the transcriptomes of two Nepeta species, each with distinctive profiles of nepetalactone stereoisomers. As a starting point for investigation of nepetalactone biosynthesis in Nepeta, these transcriptomes were used to identify candidate genes for iridoid synthase homologs, an enzyme that has been shown to form the core iridoid skeleton in several iridoid producing plant species. Iridoid synthase homologs identified from the transcriptomes were cloned, heterologously expressed, and then assayed with the 8-oxogeranial substrate. These experiments revealed that catalytically active iridoid synthase enzymes are present in Nepeta, though there are unusual mutations in key active site residues. Nevertheless, these enzymes exhibit similar catalytic activity and product profile compared to previously reported iridoid synthases from other plants. Notably, four nepetalactone stereoisomers with differing stereochemistry at the 4α and 7α positions – which are generated during the iridoid synthase reaction – are observed at different ratios in various Nepeta species. This work strongly suggests that the variable stereochemistry at these 4α and 7α positions of nepetalactone diastereomers is established further downstream in the iridoid pathway in Nepeta. Overall, this work provides a gateway into the biosynthesis of nepetalactones in Nepeta.