title: CAR1 deletion by CRISPR/Cas9 reduces formation of ethyl carbamate from ethanol fermentation by Saccharomyces cerevisiae.
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.
authors: Division of Nutrition and Metabolism Research, Korea Food Research Institute, Seongnam, 13539, Republic of Korea.
authors: Young-Wook
authors: YW
authors: Chin
authors: Division of Nutrition and Metabolism Research, Korea Food Research Institute, Seongnam, 13539, Republic of Korea.|Department of Food Science and Engineering, Ewha Womans University, Seoul, 03760, Republic of Korea.
authors: Woo-Kyung
authors: WK
authors: Kang
authors: Division of Strategic Food Research, Korea Food Research Institute, Seongnam, 13539, Republic of Korea.
authors: Hae Won
authors: HW
authors: Jang
authors: Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, 260 Bevier Hall, Urbana, IL, 61801, USA.
authors: Timothy L
authors: TL
authors: Turner
authors: Division of Nutrition and Metabolism Research, Korea Food Research Institute, Seongnam, 13539, Republic of Korea. erythritol@kfri.re.kr.|Department of Food Biotechnology, University of Science and Technology, Daejeon, 34113, Republic of Korea. erythritol@kfri.re.kr.
authors: Hyo Jin
authors: 0000-0002-5757-8773
authors: HJ
authors: Kim
mesh_terms: D001119:Arginase / Q000235:genetics*; D064113:CRISPR-Cas Systems; D000431:Ethanol / Q000378:metabolism*; D005285:Fermentation; D017353:Gene Deletion; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D014520:Urethane / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D014520:Urethane; D000431:Ethanol; D001119:Arginase; C490028:CAR1 protein, S cerevisiae
keywords: Arginase; CRISPR/Cas9; Engineered Saccharomyces cerevisiae; Ethyl carbamate