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食品与发酵工业  2019, Vol. 45 Issue (13): 45-51    DOI: 10.13995/j.cnki.11-1802/ts.020335
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
CRISPR/Cas9介导的低产尿素黄酒酵母工程菌的构建
谢文娟1,2,3, 吴殿辉1,2,3, 李晓敏1,2,3, 蔡国林1,2,3, 谢广发3,4, 陆健1,2,3*
1工业生物技术教育部重点实验室江南大学,江苏 无锡,214122
2粮食发酵工艺与技术国家工程实验室江南大学,江苏 无锡,214122
3江南大学 生物工程学院,江苏 无锡,214122
4浙江树人大学 生物与环境工程学院,浙江 杭州,310015
Metabolic engineering of Chinese rice wine Saccharomyces cerevisiae with reduced urea production by CRISPR/Cas9 system
XIE Wenjuan1,2,3, WU Dianhui1,2,3, LI Xiaomin1,2,3, CAI Guolin1,2,3, XIE Guangfa3,4, LU Jian1,2,3*
1The Key Laboratory of Industrial BiotechnologyJiangnan University,Wuxi 214122, China
2National Engineering Laboratory for Cereal Fermentation TechnologyJiangnan University,Wuxi 214122, China
3School of Biotechnology,Jiangnan University,Wuxi 214122,China
4School of Biological and Enviromental Engineering,Zhejiang Shuren University,Hangzhou 310015,China
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摘要 通过代谢工程改造构建低产尿素的酿酒酵母工程菌,从根源上减少黄酒发酵液中尿素的含量及氨基甲酸乙酯(ethyl carbamate, EC)的形成。该研究利用融合PCR构建DUR3过表达组件“HOL-PGK1p-DUR3-PGK1t-HOR”,通过CRISPR/Cas9介导的基因组编辑技术转化酿酒酵母S. cerevisiae NaDUR1,2-Δcar1,在敲除CAR1和过表达DUR1,2基因的基础上过表达DUR3基因,获得工程菌S. cerevisiae NaDUR1,2/DUR3-Δcar1。实验室黄酒发酵实验结果表明,与亲本菌株S. cerevisiae Na相比,工程菌S. cerevisiae NaDUR1,2/DUR3-Δcar1所酿黄酒发酵液中尿素含量降低了92.1%,EC含量降低了58.6%;与出发菌株S. cerevisiae NaDUR1,2-Δcar1相比,工程菌S. cerevisiae NaDUR1,2/DUR3-Δcar1所酿黄酒发酵液中尿素含量降低了43.4%, EC含量降低了16.2%。过表达DUR3的工程菌S. cerevisiae NaDUR1,2/DUR3-Δcar1具有“尿素吸收”的能力,减少EC的形成。借助CRISPR/Cas9系统,构建的酵母工程菌无外源抗性基因的引入,具有工业化应用的潜在可能性。
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谢文娟
吴殿辉
李晓敏
蔡国林
谢广发
陆健
关键词:  黄酒  酿酒酵母  尿素  氨基甲酸乙酯  CRISPR/Cas9    
Abstract: The low urea-producing Saccharomyces cerevisiae was constructed through metabolic engineering to reduce the content of urea and ethyl carbamate (EC) formation in Chinese rice wine. This study constructed the DUR3 gene expression cassette HOL-PGK1p-DUR3-PGK1t-HOR using fusion PCR and electro-transformed into S. cerevisiae NaDUR1,2-Δcar1 by CRISPR/Cas9 system to obtain the modified strain S. cerevisiae NaDUR1,2/DUR3-Δcar1 with overexpressed DUR3 gene. It showed that the content of urea in Chinese rice wine samples fermented by S. cerevisiae NaDUR1,2/DUR3-Δcar1 reduced by 92.1% and the concentration of EC decreased by 58.6% compared to those of S. cerevisiae Na. Moreover, the content of urea in Chinese rice wine samples fermented by S. cerevisiae NaDUR1,2/DUR3-Δcar1 reduced by 43.4% and the concentration of EC decreased by 16.2% compared to those of S. cerevisiae NaDUR1,2-Δcar1. These results indicated that overexpressed DUR3 could transfer urea from fermentation liquor into yeast cells, which was beneficial to reduce the urea content and EC formation in Chinese rice wine. Furthermore, using CRISPR/Cas9 system introduced no foreign resistant genes to the yeast. In conclusion, S. cerevisiae NaDUR1,2/DUR3-Δcar1 can potentially be applied in industrial production to eliminate the contents of urea and EC in Chinese rice wine.
Key words:  Chinese rice wine    Saccharomyces cerevisiae    urea    ethyl carbamate (EC)    CRISPR/Cas9
收稿日期:  2019-02-26                出版日期:  2019-07-15      发布日期:  2019-07-28      期的出版日期:  2019-07-15
基金资助: 江苏省自然科学基金(BK20170178);国家自然科学基金(31701588和31701730);江苏高校优势学科建设工程资助项目;高等学校学科创新引智计划(111计划)资助项目(111-2-06);江苏省现代工业发酵协同创新中心资助项目
作者简介:  硕士研究生(陆健教授为通讯作者,E-mail: jlu@jiangnan.edu.cn)
引用本文:    
谢文娟,吴殿辉,李晓敏,等. CRISPR/Cas9介导的低产尿素黄酒酵母工程菌的构建[J]. 食品与发酵工业, 2019, 45(13): 45-51.
XIE Wenjuan,WU Dianhui,LI Xiaomin,et al. Metabolic engineering of Chinese rice wine Saccharomyces cerevisiae with reduced urea production by CRISPR/Cas9 system[J]. Food and Fermentation Industries, 2019, 45(13): 45-51.
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http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.020335  或          http://sf1970.cnif.cn/CN/Y2019/V45/I13/45
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