Exploiting phenotypic heterogeneity to improve production of glutathione by yeast

Xu, Mingzhi; Vallières, Cindy; Finnis, Chris; Winzer, Klaus; Avery, Simon V.

Abstract

Background: Gene expression noise (variation in gene expression among individual cells of a genetically uniform cell population) can result in heterogenous metabolite production by industrial microorganisms, with cultures containing both low- and high-producing cells. The presence of low-producing individuals may be a factor limiting the potential for high yields. This study tested the hypothesis that low-producing variants in yeast cell populations can be continuously counter-selected, to increase net production of glutathione (GSH) as an exemplar product. Results: A counter-selection system was engineered in Saccharomyces cerevisiae based on the known feedback inhibition of gamma-glutamylcysteine synthetase (GSH1) gene expression, which is rate limiting for GSH synthesis: the GSH1 ORF and the counter-selectable marker GAP1 were expressed under control of the TEF1 and GSH-regulated GSH1 promoters, respectively. An 18% increase in the mean cellular GSH level was achieved in cultures of the engineered strain supplemented with D-histidine to counter-select cells with high GAP1 expression (i.e. low GSH-producing cells). The phenotype was non-heritable and did not arise from a generic response to D-histidine, unlike that with certain other test-constructs prepared with alternative markers. Conclusions: The results corroborate that the system developed here improves GSH production by targeting low-producing cells. This supports the potential for exploiting end-product/promoter interactions to enrich high-producing cells in phenotypically heterogeneous populations, in order to improve metabolite production by yeast.

Citation

Xu, M., Vallières, C., Finnis, C., Winzer, K., & Avery, S. V. (2024). Exploiting phenotypic heterogeneity to improve production of glutathione by yeast. Microbial Cell Factories, 23(1), Article 267. https://doi.org/10.1186/s12934-024-02536-5

• Journal Article Type: Article
• Acceptance Date: Sep 25, 2024
• Online Publication Date: Oct 7, 2024
• Publication Date: Oct 7, 2024
• Deposit Date: Oct 3, 2024
• Publicly Available Date: Oct 7, 2024
• Journal: Microbial Cell Factories
• Electronic ISSN: 1475-2859
• Publisher: Springer Verlag
• Peer Reviewed: Peer Reviewed
• Volume: 23
• Issue: 1
• Article Number: 267
• DOI: https://doi.org/10.1186/s12934-024-02536-5
• Public URL: https://nottingham-repository.worktribe.com/output/40286192
• Publisher URL: https://link.springer.com/article/10.1186/s12934-024-02536-5
• Additional Information: Received: 3 May 2024; Accepted: 25 September 2024; First Online: 7 October 2024; : ; : Not applicable.; : Not applicable.; : The authors declare no competing interests.