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CAR1 deletion by CRISPR/Cas9 reduces formation of ethyl carbamate from ethanol fermentation by Saccharomyces cerevisiae

  • Fermentation, Cell Culture and Bioengineering - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Enormous advances in genome editing technology have been achieved in recent decades. Among newly born genome editing technologies, CRISPR/Cas9 is considered revolutionary because it is easy to use and highly precise for editing genes in target organisms. CRISPR/Cas9 technology has also been applied for removing unfavorable target genes. In this study, we used CRISPR/Cas9 technology to reduce ethyl carbamate (EC), a potential carcinogen, which was formed during the ethanol fermentation process by yeast. Because the yeast CAR1 gene encoding arginase is the key gene to form ethyl carbamate, we inactivated the yeast CAR1 gene by the complete deletion of the gene or the introduction of a nonsense mutation in the CAR1 locus using CRISPR/Cas9 technology. The engineered yeast strain showed a 98 % decrease in specific activity of arginase while displaying a comparable ethanol fermentation performance. In addition, the CAR1-inactivated mutants showed reduced formation of EC and urea, as compared to the parental yeast strain. Importantly, CRISPR/Cas9 technology enabled generation of a CAR1-inactivated yeast strains without leaving remnants of heterologous genes from a vector, suggesting that the engineered yeast by CRISPR/Cas9 technology might sidestep GMO regulation.

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Acknowledgments

This research was supported by a grant from Korea Food Research Institute (Project No. E0153106-04) and by High Value-added Food Technology Development Program (2015-314078-3) funded by the Ministry of Agriculture, Food, and Rural Affairs (Republic of Korea).

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Authors and Affiliations

  1. Division of Nutrition and Metabolism Research, Korea Food Research Institute, Seongnam, 13539, Republic of Korea

    Young-Wook Chin, Woo-Kyung Kang & Hyo Jin Kim

  2. Department of Food Science and Engineering, Ewha Womans University, Seoul, 03760, Republic of Korea

    Woo-Kyung Kang

  3. Division of Strategic Food Research, Korea Food Research Institute, Seongnam, 13539, Republic of Korea

    Hae Won Jang

  4. Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, 260 Bevier Hall, Urbana, IL, 61801, USA

    Timothy L. Turner

  5. Department of Food Biotechnology, University of Science and Technology, Daejeon, 34113, Republic of Korea

    Hyo Jin Kim

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  1. Young-Wook Chin
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  2. Woo-Kyung Kang
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Correspondence to Hyo Jin Kim.

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Chin, YW., Kang, WK., Jang, H.W. et al. CAR1 deletion by CRISPR/Cas9 reduces formation of ethyl carbamate from ethanol fermentation by Saccharomyces cerevisiae . J Ind Microbiol Biotechnol 43, 1517–1525 (2016). https://doi.org/10.1007/s10295-016-1831-x

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  • DOI: https://doi.org/10.1007/s10295-016-1831-x

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