Systems metabolic engineering of glutathione biosynthesis in Saccharomyces cerevisiae: Pathway balancing coupled with enzyme screening for high-titer production

• Novel plasmid-free S. cerevisiae produces 997.46 mg·L⁻¹ glutathione. • Gsh1-Gsh2 fusion resolves γ-glutamylcysteine bottleneck in GSH pathway. • DO-coupled fed-batch boosts GSH titer 2.9 × vs. flasks in 5-L bioreactor. Glutathione (GSH), an essential tripeptide thiol critical for cellular redox regulation, has significant value in the pharmaceutical and nutraceutical industries. To overcome limitations of traditional GSH extraction methods, this study established a microbial cell factory platform in Saccharomyces cerevisiae through integrated metabolic engineering strategies: (1) host strain screening identified NJ-SQYY with superior GSH accumulation (74.14 mg·L⁻¹, 8.27 mg·g -1 dry cell weight [DCW]); (2) CRISPR/Cas9-mediated genomic integration of bacterial gshAB introduced with a bifunctional glutathione synthetase; (3) systematic optimization via promoter tuning and Gsh1-Gsh2 enzyme fusion, and CYS3 overexpression-resolved γ-glutamylcysteine bottlenecks. These interventions synergistically enhanced GSH synthesis to 339.3 mg·L⁻¹ in shake flasks (4.6-fold increase), representing the highest reported titer in chromosomally engineered S. cerevisiae . Scaling to dissolved oxygen-coupled fed-batch fermentation in a 5-L bioreactor produced 997.46 mg·L⁻¹ GSH at 33.85 mg·g⁻¹ DCW. This study demonstrated a holistic metabolic engineering-to-bioprocessing approach for industrial GSH biomanufacturing.