Deciphering the galantamine biosynthetic pathway in Lycoris species lays the foundation for plant chassis-based...

Galantamine, a clinically essential Amaryllidaceae alkaloid widely used to treat Alzheimer's disease, faces a constrained and unsustainable plant-derived supply, yet our understanding of its biosynthesis in Lycoris is relatively limited. Here, we provide the first comprehensive elucidation of galantamine biosynthesis in Lycoris longituba, a species with exceptionally high galantamine content. Comparative alkaloid metabolomics between L. longituba and the low-galantamine species L. insularis revealed ∼10-fold higher galantamine accumulation in L. longituba, particularly at the S1 developmental stage (November), when all precursor metabolites were markedly enriched. Using PacBio full-length and Illumina short-read transcriptomes, we identified 83 differentially expressed isoquinoline alkaloid genes, 53 of which were upregulated in L. longituba. Furthermore, integrative correlation of DEGs-metabolite networks analysis combined with Nicotiana benthamiana transient assays functionally identified LlNMT1, an N-methyltransferase, co-expressed with NtCYP96T6, converting 4'-O-methylnorbelladine to narwedine, and LlAKR4, an aldo-keto reductase mediating narwedine conversion to galantamine. In addition, we cloned cytochrome P450 candidates for the oxidative coupling of 4'-O-methylnorbelladine, obtaining 21 and 18 CYP96T-like sequences from L. longituba and L. insularis, respectively. Phylogenetic screening yielded 10 candidates, of which only LlCYP96T_clone20 and LiCYP96T_clone1 produced a galantamine-matching peak in 35S::LlAKR4-p2A-LlNMT1 tobacco, with LlCYP96T_clone20 showing ∼2.89-fold higher response. Moreover, structural modelling of the ten CYP96T-like proteins further suggested that a conserved residue, Ile386 adjacent to the heme cofactor, plays an important role in catalytic efficiency. Collectively, our work not only elucidates the downstream biosynthetic pathway from 4'-O-methylnorbelladine to galantamine in Lycoris, but also lays a robust molecular foundation for heterologous production in engineered plant chassis and the subsequent development of its clinical and industrial applications.