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Metabolic engineering of Saccharomyces cerevisiae using the CRISPR/Cas9 system to minimize ethyl carbamate accumulation during Chinese rice wine fermentation

  • Applied genetics and molecular biotechnology
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Abstract

Ethyl carbamate (EC) is a potential carcinogen to humans that is mainly produced through the spontaneous reaction between urea and ethanol during Chinese rice wine brewing. We metabolically engineered a strain by over-expressing the DUR3 gene in a previously modified strain using an improved CRISPR/Cas9 system to further decrease the EC level. Homologous recombination of the DUR3 over-expression cassette was performed at the HO locus by individual transformation of the constructed plasmid CRISPR-DUR3-gBlock-HO, generating the engineered strain N85DUR1,2/DUR3-c. Consequently, the DUR3 expression level was significantly enhanced in the modified strain, resulting in increased utilization of urea. The brewing test showed that N85DUR1,2/DUR3-c reduced urea and EC concentrations by 92.0% and 58.5%, respectively, compared with those of the original N85 strain. Moreover, the engineered strain showed good genetic stability in reducing urea content during the repeated brewing experiments. Importantly, the genetic manipulation had a negligible effect on the growth and fermentation characteristics of the yeast strain. Therefore, the constructed strain is potentially suitable for application to reduce urea and EC contents during production of Chinese rice wine.

Key Points

• An efficient CRISPR vector was constructed and applied for DUR3 over-expression.

• Multi-modification of urea cycle had synergistic effect on reducing EC level.

• Fermentation performance of engineered strain was similar with the parental strain.

• No residual heterologous genes were left in the genome after genetic manipulation.

• An efficient CRISPR vector was constructed and applied for DUR3 over-expression.

• Multi-modification of urea cycle had synergistic effect on reducing EC level.

• Fermentation performance of engineered strain was similar with the parental strain.

• No residual heterologous genes were left in the genome after genetic manipulation.

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Funding

This study is supported by the National Natural Science Foundation of China (31701588 and 31701730), the Natural Science Foundation of Jiangsu Province, China (BK20170178), the Fundamental Research Funds for the Central Universities (JUSRP11965), the Program of Introducing Talents of Discipline to Universities (111 Project) (111-2-06), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Authors and Affiliations

  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, People’s Republic of China

    Dianhui Wu, Wenjuan Xie, Xiaomin Li, Guolin Cai & Jian Lu

  2. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, People’s Republic of China

    Dianhui Wu, Wenjuan Xie, Xiaomin Li, Guolin Cai & Jian Lu

  3. Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, People’s Republic of China

    Dianhui Wu, Xiaomin Li, Guolin Cai & Jian Lu

  4. School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, People’s Republic of China

    Dianhui Wu, Wenjuan Xie, Xiaomin Li, Guolin Cai, Jian Lu & Guangfa Xie

  5. School of Biological and Environmental Engineering, Zhejiang Shuren University, Hangzhou, 310015, People’s Republic of China

    Guangfa Xie

Authors
  1. Dianhui Wu
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  2. Wenjuan Xie
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  3. Xiaomin Li
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  4. Guolin Cai
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  5. Jian Lu
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  6. Guangfa Xie
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Contributions

DW and JL conceived and designed research. DW and WX conducted experiments. GC and GX contributed analytical tools. DW and XL analysed data. DW wrote the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Jian Lu.

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Wu, D., Xie, W., Li, X. et al. Metabolic engineering of Saccharomyces cerevisiae using the CRISPR/Cas9 system to minimize ethyl carbamate accumulation during Chinese rice wine fermentation. Appl Microbiol Biotechnol 104, 4435–4444 (2020). https://doi.org/10.1007/s00253-020-10549-4

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  • DOI: https://doi.org/10.1007/s00253-020-10549-4

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