Construction of an E. coli cell factory for ergothioneine through SAM-cycle enhancement and pathway reconstruction

Ergothioneine (EGT) is a rare sulfur-containing derivative of methionine with potent antioxidant, anti-inflammatory, and neuroprotective properties. Its unique bioactivities make it a promising ingredient for applications in functional foods, nutraceuticals, and cosmetics. Microbial fermentation offers a sustainable alternative to extraction from natural sources, yet challenges such as precursor limitations, cofactor imbalances, and pathway complexity continue to restrict industrial-scale production. In this study, we engineered Escherichia coli (E. coli) as a microbial chassis for efficient de novo synthesis of EGT. By co-expressing key enzymes derived from bacteria and fungi, enhancing cysteine biosynthesis, and improving methionine utilization, we addressed key bottlenecks in precursor supply. Furthermore, the introduction of a methylation cycle significantly improved the regeneration of S-adenosylmethionine (SAM), relieving cofactor limitations. These combined metabolic engineering strategies led to a substantial increase in EGT production. The final engineered strain achieved a titer of 141.3 mg/L in shake flasks, representing a sixfold improvement over the base strain. In a 5-liter fed-batch fermentation, the titer reached 1.95 g/L without precursor supplementation and further increased to 2.52 g/L upon low-dose amino acid feeding. This work establishes a cost-effective and scalable biosynthetic platform for EGT production in E. coli, offering a viable route for its application in food and health-related industries.