Engineering O-methyltransferase and SAM-ATP cofactor cycle for high-efficiency melatonin biosynthesis in Escherichia coli.

Low O-methyltransferase activity and cofactor insufficiency limit microbial melatonin synthesis. Here, these were addressed through integrated computational enzyme design, pathway optimization, and cofactor network remodeling. Guided by energy‑ and evolution‑based consensus design combined with polar microenvironment reshaping of substrate-binding pocket, a high-stability and high‑activity O-methyltransferase mutant M2 (T32V-S52F-I316S-F321T) was obtained with an 8.1‑fold higher specific activity than the wild‑type. Subsequently, carbon flux distribution between tryptophan and tetrahydrobiopterin synthesis was partially rebalanced through gene expression optimization. Finally, cofactor limitations were substantially alleviated by strengthening SAM synthesis and SAH hydrolysis, introducing an efficient adenosine-to-ATP regeneration pathway, and reducing endogenous consumption of energy and SAM. The final strain produced 26.43-fold higher melatonin in shake flasks than the initial strain, and synthesized 1.48 g/L melatonin in a 5-L bioreactor, representing the highest reported titer for de novo synthesis. This study establishes a coordinated engineering framework that overcomes key barriers in melatonin biosynthesis, offering a transferable paradigm for microbial production of other methylation-dependent natural products.