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Abstract 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 byin plantatransient assays to investigate alkaloid diversity and functionally characterize two candidate cytochrome P450s genes fromAtropa belladonnawithout 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 catalyzeN-demethylation and ring-hydroxylation reactions within the early steps in the biosynthesis of diverseN-demethylated modified tropane alkaloids. 

Summary The mint family (Lamiaceae) is well documented as a rich source of terpene natural products. More than 200 diterpene skeletons have been reported from mints, but biosynthetic pathways are known for just a few of these.We crossreferenced chemotaxonomic data with publicly available transcriptomes to select common selfheal (Prunella vulgaris) and its highly unusual vulgarisin diterpenoids as a case study for exploring the origins of diterpene skeletal diversity in Lamiaceae. Four terpene synthases (TPS) from the TPS‐a subfamily, including two localised to the plastid, were cloned and functionally characterised. Previous examples of TPS‐a enzymes from Lamiaceae were cytosolic and reported to act on the 15‐carbon farnesyl diphosphate. Plastidial TPS‐a enzymes using the 20‐carbon geranylgeranyl diphosphate are known from other plant families, having apparently arisen independently in each family.All four new enzymes were found to be active on multiple prenyl‐diphosphate substrates with different chain lengths and stereochemistries. One of the new enzymes catalysed the cyclisation of geranylgeranyl diphosphate into 11‐hydroxy vulgarisane, the likely biosynthetic precursor of the vulgarisins.We uncovered the pathway to a rare diterpene skeleton. Our results support an emerging paradigm of substrate and compartment switching as important aspects of TPS evolution and diversification.