Journal Article

Elucidating the enzyme network driving Amaryllidaceae alkaloids biosynthesis in Leucojum aestivum

Koirala, Manoj, dos Santos, Karen Cristine Gonçalves, Lamichhane, Basanta, Gélinas, Sarah‐Eve, Merindol, Natacha, Desgagné‐Penix, Isabel, Germain, Hugo

2025

Overview

Summary 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 describe key enzymes from Leucojum aestivum (La), providing crucial insight into AA biosynthesis. Transient expression in Nicotiana benthamiana demonstrated that LaNBS and LaNRII catalyse the conversion of tyramine and 3,4‐dihydroxybenzaldehyde to norbelladine, which is subsequently O‐methylated by a norbelladine‐4′‐O‐methyltransferase (LaN4′OMT) in planta. 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 haemanthamine, crinine/vittatine and norgalanthamine. This study identifies LaCYP96T1 and LaCYP96T2 as the first monocot enzymes capable of catalysing all three regioselective C‐C phenol couplings and also highlights the substrate promiscuity of LaNRII. The findings not only elucidate critical steps in AA biosynthesis but also open new avenues for biotechnological application in producing valuable alkaloids, offering potential for novel drug development. Schematic representation of Amaryllidaceae alkaloid biosynthesis in Leucojum aestivum. Norbelladine synthase (LaNBS) and noroxomaritidine/norcraugsodine reductase (LaNRII) catalyze the condensation of tyramine and 3,4‐dihydroxybenzaldehyde to form norbelladine, which is subsequently O‐methylated by norbelladine‐4′‐O‐methyltransferase (LaN4′OMT). Cytochrome P450 enzymes LaCYP96T1 and LaCYP96T2 mediate C–C phenol couplings, leading to the formation of lycorine, haemanthamine, crinine/vittatine, and norgalanthamine. Transient expression in Nicotiana benthamiana confirms enzymatic activities and alkaloid production, providing new insights into Amaryllidaceae alkaloid biosynthesis and biotechnological applications.

• Publisher: John Wiley & Sons, Inc,John Wiley and Sons Inc
• Subject: Alkaloids / Amaryllidaceae / Amaryllidaceae - metabolism / Amaryllidaceae Alkaloids - metabolism / Amino acids / Bioactive compounds / Biosynthesis / biosynthetic pathway / Biosynthetic Pathways / Biotechnology / Candidates / Condensates / Couplings / cytochrome P-450 / Cytochrome P-450 Enzyme System - genetics / Cytochrome P-450 Enzyme System - metabolism / Cytochrome P450 / Cytochromes P450 / Drug development / Enzymes / family / Leucojum aestivum / Ligands / Liliaceae - enzymology / Liliaceae - genetics / Liliaceae - metabolism / lycorine / metabolic engineering / Metabolism / Metabolites / Methylation / Methyltransferase / Methyltransferases - metabolism / Nicotiana - genetics / Nicotiana - metabolism / Nicotiana benthamiana / oxidoreductases / pharmaceuticals / phenol / phenol coupling / Phenols / Plant Proteins - genetics / Plant Proteins - metabolism / Proteins / Reductases / regioselectivity / specialized metabolism / Tyramine