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SUMMARY: Plant‐specialized metabolism represents an inexhaustible source of active molecules, some of which have been used in human health for decades. Among these, monoterpene indole alkaloids (MIAs) include a wide range of valuable compounds with anticancer, antihypertensive, or neuroactive properties. This is particularly the case for the pachysiphine derivatives which show interesting antitumor and anti‐Alzheimer activities but accumulate at very low levels in several Tabernaemontana species. Unfortunately, genome data in Tabernaemontanaceae are lacking and knowledge on the biogenesis of pachysiphine‐related MIAs in planta remains scarce, limiting the prospects for the biotechnological supply of many pachysiphine‐derived biopharmaceuticals. Here, we report a raw version of the toad tree (Tabernaemontana elegans) genome sequence. These new genomic resources led to the identification and characterization of a couple of genes encoding cytochrome P450 with pachysiphine synthase activity. Our phylogenomic and docking analyses highlight the different evolutionary processes that have been recruited to epoxidize the pachysiphine precursor tabersonine at a specific position and in a dedicated orientation, thus enriching our understanding of the diversification and speciation of the MIA metabolism in plants. These gene discoveries also allowed us to engineer the synthesis of MIAs in yeast through the combinatorial association of metabolic enzymes resulting in the tailor‐made synthesis of non‐natural MIAs. Overall, this work represents a step forward for the future supply of pachysiphine‐derived drugs by microbial cell factories. [ABSTRACT FROM AUTHOR]

Monoterpene indole alkaloids (MIAs) are valuable metabolites produced in numerous medicinal plants from the Apocynaceae family such as Alstonia scholaris, which synthesizes strictamine, a MIA displaying neuropharmacological properties of a potential importance. To get insights into the MIA metabolism in A. scholaris, we studied here both the spatial and transcriptional regulations of MIA genes by performing a robust transcriptomics analysis of the main plant organs, leaf epidermis but also by sequencing RNA from leaves transiently overexpressing the master transcriptional regulator MYC2. These transcriptomic studies notably demonstrated that the first steps of the MIA pathway are successively distributed in the internal phloem associated parenchyma and epidermis, and that MYC2 exerts a remarkable transcriptional effect by modulating the expression of around 1000 genes. By combining these distinct datasets, we initiated the search for MIA-related genes encoding CYP71, based on the similarity of expression compared to already known MIA genes. Transient expression of these candidates in Nicotiana benthamiana leaves and yeast notably led to the identification of a related isoform of rhazimal synthase (RHS) capable of converting the MIA precursor geissoschizine into akuammicine, strictamine and 16-epi-pleiocarpamine. Investigating its catalytic mechanism revealed that strictamine results from rhazimal deformylation and that a similar mechanism may also explain 16-epi-pleiocarpamine synthesis. This prompted us to rename these enzymes geissoschizine cyclase due to their capacity of cyclizing geissoschizine into three different MIA scaffolds and to form both C-C and C-N bonds. This identification thus illustrates the potential of integrating spatial and transcriptional regulation analysis for MIA gene identification., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)