Engineering Escherichia coli Nissle 1917 for the highly efficient biosynthesis of food-grade ergothioneine.

Ergothioneine (EGT) is a natural sulfur-containing amino acid possessing potent antioxidant activity. It demonstrates a range of beneficial biological functions, including anti-inflammatory, neuroprotective, and cardioprotective effects. Microbial fermentation presents a promising alternative to traditional extraction from edible fungi for EGT production, offering greater scalability, though its feasibility hinges critically on production titer and cost. In this study, to achieve food-grade EGT synthesis, the probiotic Escherichia coli Nissle 1917 was employed as the host, and the heterologous enzymes methyltransferase EgtD and C-S lyase EgtE from Mycobacterium, as well as the bifunctional enzyme Egt1 from Neurospora crassa, were introduced. As a result, shake flask fermentations with the designed strain E1 achieved an EGT titer of 109.2 mg/L. Subsequently, overexpression of the EgtE mutant EgtEY106F along with the incorporation of the protein fusion tag NT11 at the N-terminal via a dual-plasmid system increased the yield to 159.9 mg/L. Furtherly, the cellular metabolic homeostasis regulator RpoS was introduced and placed under the control of an arabinose-inducible promoter, thus achieving an EGT titer of 429.2 mg/L for the engineered strain E14 within 24 h of shake flask fermentation. Finally, fed-batch fermentation conducted in a 5-L bioreactor resulted in an EGT titer of 12.3 g/L at 120 h, representing the highest level reported to date. This study developed a scalable and cost-effective platform for food-grade EGT production, paving the way for its industrial application.