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Efficient targeted mutation of genomic essential genes in yeast Saccharomyces cerevisiae

  • Applied genetics and molecular biotechnology
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Abstract

Targeted gene mutation by allelic replacement is important for functional genomic analysis and metabolic engineering. However, it is challenging in mutating the essential genes with the traditional method by using a selection marker, since the first step of essential gene knockout will result in a lethal phenotype. Here, we developed a two-end selection marker (Two-ESM) method for site-directed mutation of essential genes in Saccharomyces cerevisiae with the aid of the CRISPR/Cas9 system. With this method, single and double mutations of the essential gene ERG20 (encoding farnesyl diphosphate synthase) in S. cerevisiae were successfully constructed with high efficiencies of 100%. In addition, the Two-ESM method significantly improved the mutation efficiency and simplified the genetic manipulation procedure compared with traditional methods. The genome integration and mutation efficiencies were further improved by dynamic regulation of mutant gene expression and optimization of the integration modules. This Two-ESM method will facilitate the construction of genomic mutations of essential genes for functional genomic analysis and metabolic flux regulation in yeasts.

Key Points

• A Two-ESM strategy achieves mutations of essential genes with high efficiency of 100%.

• The optimized three-module method improves the integration efficiency by more than three times.

• This method will facilitate the functional genomic analysis and metabolic flux regulation.

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Acknowledgments

We thank Prof. Jens Nielsen (Chalmers University of Technology) for kindly sharing the plasmid pECAS9-gRNA-KanMX-tHFD1 carrying the Cas9 encoding gene and Prof. Jack Pronk (Delft University of Technology) for sharing the pROS10 and the amdSYM encoding gene.

Funding

This study was funded by the National Key Research and Development Program of China (2018YFA0900300), National Natural Science Foundation of China (21877111), LiaoNing Revitalization Talents Program (XLYC1807191), and the DICP&QIBEBT program (DICP & QIBEBT UN201706).

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Authors and Affiliations

  1. Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023, China

    Shan Yang, Xuan Cao, Wei Yu & Yongjin J. Zhou

  2. Dalian Key Laboratory of Energy Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China

    Shan Yang & Yongjin J. Zhou

  3. University of Chinese Academy of Sciences, Beijing, 100049, China

    Shan Yang

  4. Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, 266101, Shandong, China

    Shengying Li

  5. CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China

    Yongjin J. Zhou

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  1. Shan Yang
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  2. Xuan Cao
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  3. Wei Yu
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  4. Shengying Li
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Contributions

SY conducted the experiments and wrote the manuscript. XC assisted the construction of the CRISPR-Cas9 system. WY and SL assisted the designing part of experiments and revised the manuscript. YJZ conceived the study, designed the experiments and revised the manuscript.

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Correspondence to Yongjin J. Zhou.

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Dedicated to the 70th anniversary of Dalian Institute of Chemical Physics, Chinese Academy of Sciences

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Yang, S., Cao, X., Yu, W. et al. Efficient targeted mutation of genomic essential genes in yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 104, 3037–3047 (2020). https://doi.org/10.1007/s00253-020-10405-5

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