Metabolic Engineering and Genome-Wide Adaptive Evolution for Efficient Reduction of Glycerol in Industrial Saccharomyces cerevisiae

Abstract

The production of glycerol as a major by-product during yeast-based bioethanol fermentation arises directly from the need to re-oxidize excess NADH, which reduces conversion efficiency. In this study, an optimized Cas9-based genome editing method was performed to develop a mixotrophic CO₂-fixing industrial Saccharomyces cerevisiae by heterologous expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO form Pseudomonas sp.) and phosphoribulokinase (PRK form Spinach). Additionally, the gene encoding alcohol dehydrogenase (ADH2) responsible for converting ethanol to acetaldehyde was deleted, while the great wall-family protein kinase Rim15 gene was overexpressed to facilitate the reduction in glycerol content. The resulting CO₂-fixing yeast M-2 led to a 21.5% reduction of the by-product glycerol in corn mash fermentation cultures at 39 ℃. Moreover, we established a novel gene mutators mediated genome-wide mutations system that accumulates distinct mutations in the industrial S. cerevisiae strains under the stress conditions to improve the robustness in the S. cerevisiae strains efficiently.

Keywords

Saccharomyces cerevisiae / Gene editing / Glycerol yield / Genome-wide mutation / Adaptive evolution / Robustness

Acknowledgments

We would like to thank Editage (www.editage.com) for English language editing.

Author Contributions

N.X. and X.G. designed the study, analyzed the data, and wrote the paper. N.X., Y.Y., H.C., Y.Z., B.L., N.P. and Y.W. conducted the experiments. All the authors approved the manuscript.

Ethics Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

Funding

This work was financially supported by the Hubei Provincial Natural Science Foundation Yichang Innovation and Development Joint Fund Project (2024AFD125).

Declaration of Competing Interest

The authors declare no competing financial interest.

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