Enhanced glutathione production in Saccharomyces cerevisiae via transporter activation and catabolic pathway engineering

Industrial interest in glutathione, a tripeptide with key roles in cellular redox homeostasis, has increased, particularly in the food and pharmaceutical sectors. This study aimed to engineer Saccharomyces cerevisiae to enhance glutathione production through a dual strategy: overexpression of glutathione exporters (GXA1 and GEX2) and targeted knockout of genes involved in glutathione degradation. Overexpression of either GXA1 or GEX2 resulted in simultaneous increases in both intracellular and extracellular glutathione levels. To further enhance the intracellular glutathione content of the GXA1-overexpressing strain (G1/2-GXA1), seven candidate genes associated with glutathione catabolism were individually knocked out and the subsequent effects evaluated. Among the observed outcomes, the knockout of DUG2, which encodes a γ-glutamyl transpeptidase-like protein involved in glutathione degradation, and URE2, which encodes a glutathione S-transferase-like protein that also functions as a negative regulator of nitrogen catabolite repression, significantly improved glutathione accumulation under batch fermentation. Specifically, the glutathione contents in the G1/2-GXA1 ΔDUG2 and G1/2-GXA1 ΔURE2 strains increased by 27 % and 46 %, respectively, compared to the parental G1/2-GXA1 strain. Under high-cell-density fed-batch fermentation with glucose-limited feeding, the G1/2-GXA1 ΔDUG2 strain achieved a maximum dry cell weight of 97 g/L and a glutathione titer of 2.8 g/L. These values were 11 % and 14 % higher, respectively, than those of the G1/2-GXA1 ΔURE2 strain. Notably, the 2.8 g/L glutathione titer achieved by the G1/2-GXA1 ΔDUG2 strain represents the highest concentration reported to date in S. cerevisiae without the supplementation of pure precursor amino acids.