Ergothioneine (ERG) is a histidine-derived thiol compound suggested to function as an antioxidant and cytoprotectant in humans. Therefore, experimental trials have been conducted applying ERG from mushrooms in dietary supplements and as a cosmetic additive. However, this method of producing ERG is expensive; therefore, alternative methods for ERG supply are required. Five Mycobacterium smegmatis genes, egtABCDE, have been confirmed to be responsible for ERG biosynthesis. This enabled us to develop practical fermentative ERG production by microorganisms. In this study, we carried out heterologous and high-level production of ERG in Escherichia coli using the egt genes from M. smegmatis. By high production of each of the Egt enzymes and elimination of bottlenecks in the substrate supply, we succeeded in constructing a production system that yielded 24 mg/L (104 μM) secreted ERG.

We previously constructed a heterologous production system for ergothioneine (ERG) in Escherichia coli using five ERG biosynthesis genes (egtABCDE) from Mycobacterium smegmatis.However, significant amounts of hercynine (HER), an intermediate of ERG, as ERG was accumulated, suggesting that the reaction of EgtB catalyzing the attachment of γ-glutamylcysteine to HER to yield hercynyl-γ-glutamylcysteine sulfoxide was a bottleneck. In this study, we searched for other EgtBs and found many egtB orthologs in diverse microorganisms. Among these, Methylobacterium strains possessed EgtBs that catalyze direct conversion of HER into hercynylcysteine sulfoxide with L-Cys as a sulfur donor, in a manner similar to those of acidobacterial CthEgtB and fungal Egt1. In vitro study with recombinant EgtBs from Methylobacterium brachiatum and M. pseudosasicola clearly showed that both enzymes accepted L-Cys but not γ-glutamylcysteine. We reconstituted the ERG production system in E. coli with egtB from M. pseudosasicola; ERG productivity reached 657 mg L -1 .