Engineering of DAP1 for enhancing the high-temperature fermentation performance of industrial yeast

Abstract

High-temperature fermentation (HTF) can reduce cooling requirements and contamination risks in industrial production, especially for producing bioethanol from lignocellulosic biomass. DAP1 regulates ergosterol biosynthesis and determines thermotolerance, and amino acid position 39 is the key site for its thermostability. Here, we explored and modified the 39th amino acid of Dap1 (valine) via CRISPR/Cas9-assisted precise genome editing technology to enhance the HTF performance of industrial Saccharomyces cerevisiae CEN.PK2-1C. The results showed that converting valine to aspartic acid (V39D) or glutamine (V39Q) increased the growth of the mutants by 12.33% and 12.82%, respectively, at 42 °C. Correspondingly, ethanol yields of the DAP1-V39D and DAP1-V39Q mutants increased by 10.98% and 10.82%, respectively, compared with the wild-type. In addition, overexpressing DAP1-V39Q with the strong promoter pTDH3 in the DAP1-V39Q mutant (DAP1-V39Q-OE) further increased high-temperature growth ability and ethanol production by 3.10% and 0.91%, respectively, compared with the DAP1-V39Q mutant. Finally, from the predicted protein model, we found significant changes in the protein structures of the DAP1-V39D and DAP1-V39Q mutants. Our findings would provide guidance for developing more robust yeast for the industrial production of ethanol at high temperature.

Keywords

Saccharomyces cerevisiae / CRISPR/Cas9 / Saturation mutagenesis / High-temperature fermentation / Bioethanol / DAP1

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Funding

Natural Science Foundation of Hebei Province(C2020204013 and C2025204038)

Research and development project of characteristic industry of dry alkali wheat in Cangzhou City(20241203002D)

the Industry University Research Cooperation Project of Shijiazhuang and University of Hebei province(241490062A)

RIGHTS & PERMISSIONS

Jiangnan University