Metabolomics-guided discovery of cytochrome P450s involved in pseudotropine-dependent biosynthesis of modified tropane alkaloids

Cytochrome P450s drive the structural diversity of plant alkaloids, many of which have biotechnological uses. Here the authors use reverse genetics and metabolomics to identify two Atropa belladonna cytochrome P450s that synthesize pseudotropine-derived alkaloids. Plant alkaloids constitute an important class of bioactive chemicals with applications in medicine and agriculture. However, the knowledge gap of the diversity and biosynthesis of phytoalkaloids prevents systematic advances in biotechnology for engineered production of these high-value compounds. In particular, the identification of cytochrome P450s driving the structural diversity of phytoalkaloids has remained challenging. Here, we use a combination of reverse genetics with discovery metabolomics and multivariate statistical analysis followed by in planta transient assays to investigate alkaloid diversity and functionally characterize two candidate cytochrome P450s genes from Atropa belladonna without a priori knowledge of their functions or information regarding the identities of key pathway intermediates. This approach uncovered a largely unexplored root localized alkaloid sub-network that relies on pseudotropine as precursor. The two cytochrome P450s catalyze N-demethylation and ring-hydroxylation reactions within the early steps in the biosynthesis of diverse N-demethylated modified tropane alkaloids.

Automated summary

By using reverse genetics and metabolomics, the authors identified two cytochrome P450 genes in Atropa belladonna that are involved in the synthesis of alkaloids derived from pseudotropine. This study reveals an unexplored alkaloid sub-network in the roots, which relies on pseudotropine as a precursor. The two cytochrome P450s catalyze N-demethylation and ring-hydroxylation reactions in the early steps of the biosynthesis of diverse N-demethylated modified tropane alkaloids.