Enhanced Production of Ergothioneine in <i>Yarrowia lipolytica</i> through Combined Metabolic and Enzyme Engineering

Ergothioneine (EGT) is a sulfur-containing histidine derivative with antioxidant, antiaging, and antidepressant properties. It is widely used in the food, medicine, and cosmetics industries. However, both the chemical synthesis and biological extraction of EGT are constrained by high cost and low yield. In this study, EGT synthetases EGT1 and EGT2 from Trichoderma reesei were expressed in Yarrowia lipolytica using various expression vectors. Several key sites in TrEgt1 were identified by alanine scanning mutagenesis, mutated to hydrophobic amino acids, and the EGT titer of the Y786A-A492 V-TrEGT1 mutant was 2.41 times higher than that of the wild-type strain. To improve the supply of precursor amino acids, the associated network was divided into four modules, which have been systematically enhanced. Combining the above modifications resulted in an engineered strain that produced 516.3 mg/L EGT in multiwell plates. Fermentation was optimized in a 5 L bioreactor, and EGT accumulation reached 9.3 g/L after 168 h, with a production intensity of 55.35 mg/L/h, the highest reported to date. These strategies provided references for the construction of EGT-producing microorganisms.