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

Abstract

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.

Keywords

Glutathione biosynthesis / Saccharomyces cerevisiae / Enzyme fusion / CRISPR/Cas9 / Bioreactor scale-up

Data Availability Statement

Data will be made available on request.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Yingjun Li reports financial support was provided by Key Research and Development Project of Hubei Province. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Zhiqi Hu: Writing - original draft, Visualization, Validation, Software, Resources, Methodology, Investigation, Formal analysis. Mengyuan Su: Visualization, Validation, Resources, Methodology, Data curation. Qibing Liu: Validation, Software, Resources, Formal analysis, Data curation. Ying Li: Visualization, Software, Methodology, Data curation. Yunxiang Liang: Visualization, Resources, Conceptualization. Shuangquan Li: Project administration, Funding acquisition, Conceptualization. Yingjun Li: Writing - review & editing, Supervision, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization.

Acknowledgements

We would like to thank the National Key Laboratory of Agricultural Microbiology Core Facility for technical support. This work was supported by the National Natural Science Foundation of China (32170096); Key Research and Development Project of Hubei Province (2023BBB025).

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