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Abstract Amaryllidaceae alkaloids (AAs) are diverse bioactive metabolites with significant pharmaceutical potential, derived from 4´-O-methylnorbelladine (4´OM). The biosynthesis of these compounds involves the condensation of tyramine and 3,4-dihydroxybenzaldehyde by norbelladine synthase (NBS) and/or noroxomaritidine/norcraugsodine reductase (NR), followed by O-methylation. Cytochrome P450 enzymes, particularly the CYP96T family, introduce further structural diversity through C-C couplings, resulting in lycorine, galanthamine, and crinine cores. Despite their importance, the exact biosynthetic pathways remain poorly defined. In this study, we characterized key enzymes from Leucojum aestivum (La), providing crucial insight into AA biosynthesis. Transient expression in Nicotiana benthamiana demonstrated that LaNBS and LaNRII catalyze the conversion of tyramine and 3,4-dihydroxybenzaldehyde to norbelladine, which is subsequently O-methylated by a norbelladine-4´-O-methyltransferase (LaN4´OMT). Co-agroinfiltration of LaNBS, LaNRII, LaN4´OMT, and LaCYP96T1 resulted in the production of various phenol-coupled products, with lycorine as the predominant compound, alongside with haemanthamine, crinine/vittatine, and norgalanthamine. This study identifies LaCYP96T1 and LaCYP96T2 as the first monocot enzymes capable of catalyzing all three regioselective C-C phenol couplings, highlighting the substrate promiscuity of LaNRII. The findings not only elucidate crital steps in AA biosynthesis but also open new avenues for biotechnological application in producing valuable alkaloids, offering potential for novel drug development.
Desgagné‐Penix et al. (Mon,) studied this question.