Biocatalytic Reductive Amination for In Vitro Biosynthesis of the Amaryllidaceae Alkaloid Precursor

Graphical 4′-O-Methylnorbelladine (4′-MB) is a key precursor for the synthesis of Amaryllidaceae plant-derived alkaloids. In this study, an efficient biosynthetic system for the production of 4′-MB was developed through a comprehensive optimization strategy that includes protein engineering, enzyme immobilization, by-product recycling, and cofactor regeneration. This approach paves the way for the large-scale production of pharmaceuticals derived from Amaryllidaceae plants. 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.