Engineering Escherichia coli for high-level production of ergothioneine

Ergothioneine (ERG) is a natural sulfur-containing compound derived from histidine that is known for its significant antioxidant properties. Microbial biosynthesis of ERG has attracted considerable interest due to its sustainability and scalability. Despite efforts to construct engineered strains, the ERG yield remains low. In this study, Escherichia coli was engineered for high-level ERG production through a series of metabolic engineering strategies. A promising methyltransferase was identified and an evolved variant with 1.4-fold increase in catalytic activity was obtained through random mutagenesis and screening. Subsequently, combinatorial pathway engineering was conducted by combining various sulfoxide synthases and C-S lyases with the evolved methyltransferase, generating a strain with 297 ± 3 mg/L of ERG. To further improve the pathway efficiency, ribosome binding site and promoter engineering were innovatively employed to fine-tune the expression levels of key enzymes in the ERG synthesis pathway. Furthermore, the supply of S-adenosylmethionine, an essential methyl donor, was enhanced by applying cofactor engineering. Finally, a high ERG-producing strain S5 was obtained, whose ERG yield reached 453 ± 9 mg/L. Upon scale-up in a 5-L fermenter, the strain S5 produced 12161 ± 311 mg/L of ERG at 144 h, representing the highest titer achieved to date in microbial fermentation. This work demonstrated that the effectiveness of synergistic application of multiple metabolic engineering strategies in improving product yield, laying a foundation for the cost-effective biosynthesis of ERG.