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.
Wei Li,Chong Qiao,Jing Pang,Guolin Zhang,Yinggang Luo
Amaryllidaceae alkaloids are unique benzylphenethylamine derivatives that comprise of more than 600 members with a huge chemical diversity. Most of them showed interesting bioactivities, for instance, galanthamine (GAL) is clinically used for Alzheimer's disease treatment. All Amaryllidaceae alkaloids had been thought to be derived from 4′- O -methylnorbelladine originated from norbelladine catalyzed by norbelladine 4′- O -methyltransferase (N4OMT). Herein we mined the transcriptome datasets of Lycoris radiata , a GAL-producing plant. LrOMT was cloned, overexpressed in Escherichia coli , and purified to homogeneity. Bioinformatics analysis and enzymatic activity assays revealed that LrOMT is an S-adenosylmethionine-dependent Class I OMT. LrOMT exhibited both para - and meta - O -methylation activities toward norbelladine to give 4′- and 3′- O -methylnorbelladine. Twenty-four analogues, including the proposed biosynthetic intermediates, were introduced to investigate the substrate scope of LrOMT and it showed that the aromatic substrates should have two vicinal hydroxyl groups. The LrOMT-catalyzed O -methylation preference is dependent on the properties of the binding group of the substrates. The transcription levels of LrOMT were positively associated with the accumulation of the Amaryllidaceae alkaloids and the biosynthetic intermediates in L. radiata . The present work revealed that LrOMT catalyzes multiple O -methylation reactions and its characterization will be helpful to uncover novel biosynthetic genes for Amaryllidaceae alkaloids biosynthesis.
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