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Abstract

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.