Amaryllidaceae alkaloids (AmAs) exhibit remarkable biochemical properties and pharmacological significance. They include the anti-cancer compounds hemanthamine and lycorine, the anti-viral compound pancratistatin, and galanthamine, which is used to treat Alzheimer’s disease. However, their biosynthetic pathways remain largely unexplored, limiting their potential for microbial production. In this study, we designed and implemented innovative artificial pathways to synthesize 4-O′-Me-norbelladine (4-NB), a valuable branch-point intermediate in the Amaryllidaceae alkaloid family, in Escherichia coli (E. coli). First, retrosynthetic analysis and catalytic mechanism studies guided pathway design, enabling the biosynthesis of 4-NB’s direct precursors, tyramine and 3,4-dihydroxybenzaldehyde (3,4-DHBA), through candidate enzyme screening. Enzyme engineering was further employed to enhance the efficiency of the rate-limiting step-the condensation of tyramine with 3,4-DHBA-catalyzed by norbelladine synthase (NBS). Then, the complete artificial pathway was assembled in E. coli, achieving de novo biosynthesis of 4-NB. Finally, metabolic flux optimization significantly enhanced 4-NB production. This work establishes a microbial platform for 4-NB biosynthesis for the first time, paving the way for the sustainable production of Amaryllidaceae alkaloids.
