Efficient ergothioneine production through reconstruction of the methyl and sulfur supply systems in Escherichia coli

Ergothioneine (ERG) is a rare amino acid with diverse biological activities, making it valuable for applications in the food, cosmetics, and pharmaceutical industries. Compared with traditional extraction from edible fungi, microbial fermentation offers a more scalable alternative, but its feasibility is largely determined by production titer and cost. In this study, we developed a novel ERG biosynthesis strategy that eliminates the need for expensive methionine and cysteine supplementation during fermentation. By reconstructing a betaine-driven methyl supply system and an inorganic sulfur supply module in Escherichia coli , we engineered a highly efficient ERG-producing strain capable of utilizing betaine as a methyl donor and inorganic sulfur as a sulfur source. As a result, shake flask fermentations achieved an ERG titer of 456 ± 9 mg/l without exogenous methionine and cysteine supplementation. Further optimization strategies, including enhanced intracellular histidine synthesis, alleviation of methionine feedback inhibition, and improved ERG transport efficiency, increased the shake flask titer to 1.2 g/l, a 24-fold improvement over the initial strain. Scale-up in a 5-l fermenter further optimized fermentation conditions, yielding an ERG titer of 7.2 g/l. Thus, the metabolic engineering strategy described in this study provides a scalable and cost-effective platform for the industrial production of ERG and other methyl- and sulfur-containing natural products.