CRISPR/Cas9介导的低产尿素黄酒酵母工程菌的构建
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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-PGK1tHOR",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。
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-PGK1 p-DUR3-PGK1 t-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.
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-PGK1 p-DUR3-PGK1 t-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.
引文
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[22] FABER K N,HAIMA P,HARDER W,et al. Highly-efficient electrotransformation of the yeast Hansenula polymorpha[J]. Current Genetics,1994,25(4):305-310.
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[24]谢广发.黄酒酿造技术[M].北京:中国轻工业出版社,2010.
[25] GB/T 13662—2008,黄酒[S].北京:中国标准出版社,2008.
[26]邢江涛,钟其顶,熊正河,等.高效液相色谱-荧光检测器法测定黄酒中尿素含量[J].酿酒科技,2011(3):104-106.
[2] LIU Y,DONG B,QIN Z,et al. Ethyl carbamate levels in wine and spirits from markets in Hebei province,China[J]. Food Additives&Contaminants:Part B Surveillance,2011,4(1):1-5.
[3] NOBREGA I C C,PEREIRA J A P,PAIVA J E,et al. Ethyl carbamate in cachaca(Brazilian sugarcane spirit):Extended survey confirms simple mitigation approaches in pot still distillation[J]. Food Chemistry,2011,127(3):1 243-1 247.
[4] QIANG Xia,YUAN Huawei,WU Chongde,et al. An improved and validated sample cleanup method for analysis of ethyl carbamate in Chinese liquor[J]. Journal of Food Science,2014,79(9):T1 854-T1 860.
[5] IARC. Alcoholic beverage consumption and ethyl carbamate(urethane)//IARC Monograph 96 on the Evaluation of Carcinogenic Risks to Humans[C]. Lyon,France,2010.
[6] WEBER J V,SHARYPOV V I. Ethyl carbamate in foods and beverages:A review[J]. Environmental Chemistry Letters,2009,7(3):233-247.
[7]陈小萍,林国斌,林升清,等.蒸馏酒和发酵酒中氨基甲酸乙酯的监测与危害控制[J].海峡预防医学杂志,2009,15(6):54-55.
[8] CHIN Y W,KANG W K,JANG H W,et al. CAR1 deletion by CRISPR/Cas9 reduces formation of ethyl carbamate from ethanol fermentation by Saccharomyces cerevisiae[J].Journal of Industrial Microbiology&Biotechnology,2016,43(11):1-9.
[9] GUO Xuewu,LI Yuanzi,GUO Jian,et al. Reduced production of ethyl carbamate for wine fermentation by deleting CAR1 in Saccharomyces cerevisiae[J]. Journal of Industrial Microbiology&Biotechnology,2016,43(5):671-679.
[10] KITAMOTOK,ODA K,GOMI K,et al. Genetic engineering of a sake yeast producing no urea by successive disruption of arginase gene[J]. Applied and Environmental Microbiology,1991,57(1):301-306.
[11] WU Dianhui,LI Xiaomin,LU Jian,et al. Constitutive expression of the DUR1,2 gene in an industrial yeast strain to minimize ethyl carbamate production during Chinese rice wine fermentation[J]. Fems Microbiology Letters,2016,363(1):fnv214.
[12] COULON J,HUSNIK J I,INGLIS D L,et al. Metabolic engineering of Saccharomyces cerevisiae to minimize the production of ethyl carbamate in wine[J]. American Journal of Enology and Viticulture,2006,57(2):113-124.
[13] DAHABIEH M S,HUSNIK J I,VUUREN H J J V. Functional enhancement of sake yeast strains to minimize the production of ethyl carbamate in Sake wine[J]. Journal of Applied Microbiology,2010,109(3):963-973.
[14]申超,吴殿辉,李晓敏,等.尿素吸收型工业黄酒酵母单倍体工程菌的构建[J].食品与发酵工业,2014,40(3):25-29.
[15]吴殿辉.代谢工程改造黄酒酿造用酵母低产氨基甲酸乙酯的研究[D].无锡:江南大学,2016.
[16] TADAS J,BONDE I,MARKUS H,et al. Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae[J]. Metabolic Engineering,2015,28:213-222.
[17]周文龙,唐亮,成凯,等. CRISPR/Cas9介导的高产谷胱甘肽原养型酵母工程菌的构建[J].生物工程学报,2017,33(12):1 999-2 008.
[18] WU Dianhui,LI Xiaomin,SHEN Chao,et al. Isolation of a haploid from an industrial Chinese rice wine yeast for metabolic engineering manipulation[J]. Journal of the Institute of Brewing,2013,119(4):288-293.
[19] WU Dianhui,LI Xiaomin,LU Jian,et al. The overexpression of DUR1,2 and deletion of CAR1 in an industrial Saccharomyces cerevisiae strain and effects on nitrogen catabolite repression in Chinese rice wine production[J].Journal of the Institute of Brewing,2016,122(3):480-485.
[20]王颖,鲍晓明,杨国梁,等.工业酵母菌株转化体系的建立[J].工业微生物,2004,34(1):6-11.
[21] WU S,LETCHWORTH G J. High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol[J]. Biotechniques,2004,36(1):152-154.
[22] FABER K N,HAIMA P,HARDER W,et al. Highly-efficient electrotransformation of the yeast Hansenula polymorpha[J]. Current Genetics,1994,25(4):305-310.
[23] BOER V M,TAI S L,VURALHAN Z,et al. Transcriptional responses of Saccharomyces cerevisiae to preferred and nonpreferred nitrogen sources in glucose-limited chemostat cultures[J]. Fems Yeast Research,2010,7(4):604-620.
[24]谢广发.黄酒酿造技术[M].北京:中国轻工业出版社,2010.
[25] GB/T 13662—2008,黄酒[S].北京:中国标准出版社,2008.
[26]邢江涛,钟其顶,熊正河,等.高效液相色谱-荧光检测器法测定黄酒中尿素含量[J].酿酒科技,2011(3):104-106.