LAUSR

Amaryllidaceae alkaloids (AAs) are plant‐derived natural compounds with diverse bioactivities. To biosynthesize AAs, 4′‐O‐methylnorbelladine (4′‐MB) is a key precursor that is first formed by C–N coupling between tyramine and 3,4‐dihydroxybenzaldehyde catalyzed by a NADPH‐dependent reductase. However, 3,4‐dihydroxybenzaldehyde is oxygen sensitive and not stable due to its catechol moiety when constructing an in vitro enzymatic system for 4′‐MB synthesis. To address this issue, we design an artificial biosynthetic route to synthesize 4′‐MB. First, we choose isovanillin instead of 3,4‐dihydroxybenzaldehyde as an alternative substrate. Then, a short‐chain dehydrogenase/reductase (SDR) from Zephyranthes treatiae is utilized to couple tyramine and isovanillin. After demonstrating the synthesis of 4′‐MB, we optimize the whole biocatalytic system with enhanced performance. The final enzymatic system can be reused for multiple cycles with the help of a crystalline inclusion protein (CipB)‐mediated enzyme immobilization. Additionally, we scale up the reaction system from 50 µL to 10 mL, generating approximately 800 µM of 4′‐MB. We envision that our work will provide an efficient and sustainable approach to produce 4′‐MB, which could support the large‐scale production of Amaryllidaceae plant‐originated natural products.