High‐Level Production of Ergothioneine in <i>Yarrowia lipolytica</i> via Systematic Metabolic Engineering Strategies

Ergothioneine (ERG), a food-derived antioxidant and cytoprotectant, has broad application prospects in food, nutraceuticals, cosmetics, and biomedical fields. The growing market demand for ERG has driven the development of microbial cell factories as a sustainable production platform. However, the biosynthetic production of ERG faces challenges including poor substrate channeling, limited precursor supply, and potential inhibition by intracellular product accumulation. To construct a promising cell factory for high-level ERG production, firstly, the ERG biosynthetic pathway from Trichoderma reesei was heterologously expressed in Y. lipolytica Po1f (Δku70). Secondly, the application of Tregt1-Tregt2 fusion with a flexible (GGGGS)₂ linker and multi-copy integration led to an ERG production of 618.97 mg/L, an 83% improvement. Then, carbon flux was systematically redirected toward ERG production via modular engineering to enhance the precursor supply. Finally, fermentation conditions were optimized, resulting in an ERG production of 1075.27 mg/L in shake flask after 120 h fermentation. When scaled up to 5 L bioreactor, the strain achieved a final ERG production of 8.03 g/L within 168 h (47.79 mg/L/h), the highest reported to date without the supplementation of exogenous precursors. This study provides an effective strategy for utilizing Y. lipolytica as a microbial cell factory for ERG biosynthesis.