CRISPR/Cas9介导的低产尿素黄酒酵母工程菌的构建  

Metabolic engineering of Chinese rice wine Saccharomyces cerevisiae with reduced urea production by CRISPR/Cas9 system

作　　者：谢文娟 吴殿辉 李晓敏[1,2,3] 蔡国林[1,2,3] 谢广发 陆健[1,2,3] XIE Wenjuan;WU Dianhui;LI Xiaomin;CAI Guolin;XIE Guangfa;LU Jian(The Key Laboratory of Industrial Biotechnology(Jiangnan University),Wuxi 214122,China;National Engineering Laboratory for Cereal Fermentation Technology(Jiangnan University),Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China;School of Biological and Enviromental Engineering,Zhejiang Shuren University,Hangzhou 310015,China)

机构地区：[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

出　　处：《食品与发酵工业》2019年第13期45-51,共7页Food and Fermentation Industries

基　　金：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

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 Na DUR1,2-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 Na DUR1,2/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 Na DUR1,2/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 Na DUR1,2-Δcar1相比,工程菌S.cerevisiae Na DUR1,2/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 Na DUR1,2/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。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 Na DUR1,2-Δcar1 by CRISPR/Cas9 system to obtain the modified strain S.cerevisiae Na DUR1,2/DUR3-Δcar1 with overexpressed DUR3 gene.It showed that the content of urea in Chinese rice wine samples fermented by S.cerevisiae Na DUR1,2/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 Na DUR1,2/DUR3-Δcar1 reduced by 43.4%and the concentration of EC decreased by 16.2%compared to those of S.cerevisiae Na DUR1,2-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 Na DUR1,2/DUR3-Δcar1 can potentially be applied in industrial production to eliminate the contents of urea and EC in Chinese rice wine.

关 键 词：黄酒 酿酒酵母 尿素 氨基甲酸乙酯 CRISPR/Cas9 

分 类 号：TS262.4[轻工技术与工程—发酵工程]