Welcome to Chinese Agricultural Science Bulletin,

Chinese Agricultural Science Bulletin ›› 2020, Vol. 36 ›› Issue (29): 69-77.doi: 10.11924/j.issn.1000-6850.casb2020-0127

Special Issue: 生物技术

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Effects on 2,3-butanediol Production of Saccharomyces cerevisiae: gpd2 Gene Knockout by CRISPR/Cas9 Technology

Liu Lei1,2(), Li Na1,2, Jiang Xueyong1,2, Sun Jian1,2, Lv Yuze1,2, Ge Jingping1,2()   

  1. 1Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150500
    2 Key Laboratory of Microbiology, College of Heilongjiang Province, School of Life Sciences, Heilongjiang University, Harbin 150080
  • Received:2020-05-25 Revised:2020-07-27 Online:2020-10-15 Published:2020-10-16
  • Contact: Ge Jingping E-mail:liuleiheida@163.com;gejingping@126.com

Abstract:

This study aims at using CRISPR/Cas9 gene editing technology to knock out the glycerol-3-phosphate dehydrogenase gene (gpd2) in Saccharomyces cerevisiae, and investigating its effect on 2,3-butanediol production. Designing donor and guide RNA (gRNA) based on glycerol-3-phosphate dehydrogenase gene (gpd2) of S. cerevisiae W5, and linking the knockout vector could express Cas9 protein to the gRNA fragment, then the recombinant plasmid and donor DNA fragment were transformed into S. cerevisiae W5 cells. The gpd2 gene knocked-out strain was obtained through phenotypic screening and PCR validation, indicating that the gpd2 gene was successfully knocked out. Compared with the original strain, the gpd2 gene knocked-out strain increased ethanol production by 24.65%, and decreased glycerol production by 22.01% and 2,3-butanediol production by 10.60%. The production of 2,3-butanediol did not increase by knocking out the gpd2 gene, probably because that gradually accumulated NADH was preferentially oxidized by a large amount of alcohol dehydrogenase in the cell, and affected the production of ethanol rather than the synthesis of 2,3-butanediol. In this study, a suitable gene knockout system is constructed for S. cerevisiae, which has practical reference for further exploring the relationship between other metabolites of S. cerevisiae synthesis and 2,3-butanediol synthesis.

Key words: Saccharomyces cerevisiae, CRISPR/Cas9, 2,3-butanediol, vector construction, metabolic engineering

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