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Improved bioethanol production using CRISPR/Cas9 to disrupt the ADH2 gene in Saccharomyces cerevisiae

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Abstract

Bioethanol, as a form of renewable and clean energy, has become increasingly important to the energy supply. One major obstacle in ethanol production is developing a high-capacity system. Existing approaches for regulating the ethanol production pathway are relatively insufficient, with nonspecific genetic manipulation. Here, we used CRISPR/Cas9 technology to disrupt the alcohol dehydrogenase (ADH) 2 gene via complete deletion of the gene and introduction of a frameshift mutation in the ADH2 locus. Sequencing demonstrated the accurate knockout of the target gene with 91.4% and near 100% targeting efficiency. We also utilized genome resequencing to validate the mutations in the ADH2 mutants targeted by various single-guide RNAs. This extensive analysis indicated the mutations in the CRISPR/Cas9-engineered strains were homozygous. We applied the engineered Saccharomyces cerevisiae strains for bioethanol production. Results showed that the ethanol yield improved by up to 74.7% compared with the yield obtained using the native strain. This work illustrates the applicability of this highly efficient and specific genome engineering approach to promote the improvement of bioethanol production in S. cerevisiae via metabolic engineering. Importantly, this study is the first report of the disruption of a target gene, ADH2, in S. cerevisiae using CRISPR/Cas9 technology to improve bioethanol yield.

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Abbreviations

CRISPR:

Clustered regularly interspaced short palindromic repeat

PAM:

Protospacer adjacent motif

sgRNA:

Single-guide RNA

DSB:

Double-strand break

OD600:

Optical density at 600 nm

PCR:

Polymerase chain reaction

ADH:

Alcohol dehydrogenase

GC:

Gas chromatography

WT:

Wild type

TALEN:

Transcription activator-like effector nuclease

DSB:

Double strand break

HR:

Homologous recombination

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Acknowledgements

We thank Liangyi Zhou for help on verification of ADH2 mutants, Heqi Shao for advice on production of enzymatic extract. We thank Nature Research Editing Service (Order ID: 343TY1W5) for editing the English text of a draft of this manuscript.

Funding

This work was supported by the Natural Science Foundation of Fujian Province (Grant No. 2017J01622), the National Sugar Crop Reserach System (Grant No. CARS-170501), and the Education Department Project of Fujian Province (Grant No. JAT160114).

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Author notes

  1. Ting Xue and Kui Liu have contributed equally to this work.

Authors and Affiliations

  1. The Public Service Platform for Industrialization Development Technology of Marine Biological Medicine and Product of State Oceanic Administration, Fujian Normal University, Fuzhou, 350117, China

    Ting Xue, Kui Liu, Xue Yuan, Jingping Fang, Luqiang Huang, Youqiang Chen & Wenjin He

  2. Center of Engineering Technology Research for Microalgae Germplasm Improvement of Fujian, Southern Institute of Oceanography, Fujian Normal University, Fuzhou, 350117, China

    Ting Xue, Kui Liu, Xue Yuan, Jingping Fang, Luqiang Huang, Youqiang Chen & Wenjin He

  3. Key Laboratory of Developmental and Neural Biology, College of Life Sciences, Fujian Normal University, Fuzhou, 350117, China

    Ting Xue, Duo Chen, Youqiang Chen & Wenjin He

  4. FAFU and UIUC-SIB Joint Center for Genomics and Biotechnology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China

    Hansong Yan

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  1. Ting Xue
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Contributions

Ting Xue, Kui Liu and Youqiang Chen designed the project; Kui Liu carried out the experiments; Kui Liu, Duo Chen, Xue Yuan and Jingping Fang analyzed the GC and genome resequencing data; Kui Liu wrote the manuscript; Ting Xue, Jingping Fang, Luqiang Huang, Youqiang Chen, Wenjin He edited the manuscript; All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Youqiang Chen or Wenjin He.

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Xue, T., Liu, K., Chen, D. et al. Improved bioethanol production using CRISPR/Cas9 to disrupt the ADH2 gene in Saccharomyces cerevisiae. World J Microbiol Biotechnol 34, 154 (2018). https://doi.org/10.1007/s11274-018-2518-4

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