Abstract:Ergothioneine(Ergothioneine, ERG) has various physiological functions such as antioxidant, anti-inflammatory and radiation protection, widely applied in food, medicine and cosmetic industries. Microbial fermentation of ergothioneine is of great potential, but engineered strains often have problems such as insufficient precursor supply, resulting in low fermentation concentration and production performance. This study aimed to explore the precursor limitation in different stages of ergothioneine synthesis, and to improve the ergothioneine synthesis capacity of Escherichia coli by optimizing precursor supply and removing the limitation. On the basis of proving that histidine was the key factor limiting the synthesis of ergothioneine, the fermentation concentration of ergothioneine at shake flask level reached 175.81 mg/L by optimizing the supply of histidine. On this basis, additions of cysteine, ammonium ferric citrate and vitamin B6 were orthogonally optimized, and the fermentation concentration was increased to 182.61 mg/L. By further optimizing the additions of methyl donors (1.5 g/L methionine and 0.6 g/L betaine) and by adding 0.2 g/dL CaCl2 to improve the permeability of the cell membrane，the final fermentation concentration of ergothioneine at shake flask level was further increased to 243.06 mg/L. Through fed-batch fermentation for 108 h, the fermentation level of ergothioneine reached 2.01 g/L in a 3 L fermenter, and the titer reached 18.61 mg/L/h. This study could lay a foundation for the large-scale industrial production of ergothioneine.
