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Ergothioneine (ERG), a unique thiol compound, is suggested to function as an antioxidant and cytoprotectant. Despite several recent attempts to produce ERG using various organisms, its yield was still very low and the costs remained high. Since the level of ERG produced depends strictly on the availability of three distinct precursor amino acids ( l -cysteine (Cys),  l -histidine, and  l -methionine (Met)), metabolic engineering for enhancement of the flux toward ERG biosynthesis is required. Herein, we took advantage of a high-Cys production system using  Escherichia coli  cells, in which Cys biosynthesis and excretion were activated, and applied it to the fermentative production of ERG from glucose. The Cys overproduction in  E .  coli  cells carrying the  egtBCDE  genes from  Mycobacterium smegmatis  was effective for ERG production. Furthermore, coexpression of the  egtA  gene, which encodes γ-glutamylcysteine synthetase that synthesizes the γ-glutamylcysteine used as a sulfur source of ERG biosynthesis, enhanced ERG production even though  E .  coli  intrinsically has γ-glutamylcysteine synthetase. Additionally, disruption of the  metJ  gene that encodes the transcriptional repressor involved in Met metabolism was effective in further increasing the production of ERG. Finally, we succeeded in the high-level production of 1.31 g/L ERG in a fed-batch culture process using a jar fermenter.

Gram-scale fermentative production of ergothioneine driven by overproduction of cysteine in Escherichia coli