Engineering Methyltransferase\nand Sulfoxide Synthase\nfor High-Yield Production of Ergothioneine

Ergothioneine (ERG) is an unusual sulfur-containing amino\nacid\nwith antioxidant activity that can be synthesized by certain bacteria\nand fungi. Microbial fermentation is a promising method for ERG production.\nIn this study, the bifunctional enzyme methyltransferase–sulfoxide\nsynthase NcEgt1 from Neurospora crassa was truncated to obtain sulfoxide synthase ^TNcEgt1, which\nshowed a higher expression level in Escherichia coli BL21­(DE3). Then, the genes <i>egtD</i> encoding methyltransferase\nEgtD and <i>egtE</i> encoding C–S lyase EgtE from Mycobacterium smegmatis were cloned with ^<i>T</i><i>ncEgt</i>1 into E. coli BL21­(DE3) to produce 70 mg/L ERG. To improve ERG production, ^TNcEgt1 and EgtD were modified, and the resulting mutants were\nscreened with an established high-throughput method which could directly\nanalyze the ERG content in culture broths. After several rounds of\nmutation and screening, the optimal mutant MD4 was obtained and produced\n290 mg/L ERG. Furthermore, a fed-batch culture was conducted in a\n5 L bioreactor. After optimizing the fermentation process, the ERG\nyield reached 5.4 g/L after 94 h of cultivation supplemented with\namino acids and glycerol, which is the highest ERG yield reported\nto date. The results showed that ERG production was significantly\nimproved by modifying the key enzymes, and the engineered strains\nconstructed in this study have potential industrial application prospects.