低乙醇脱氢酶ⅱ活性的抗老化啤酒酵母工程菌的构建-生物谷.doc

低乙醇脱氢酶Ⅱ活性的抗老化啤酒酵母工程菌的构建 蔡 勇1 母 茜1 王肇悦2 张博润2 晏本菊1* (1. 四川农业大学生命科学与理学院 雅安 625014) (2. 中国科学院微生物研究所 北京 100101) 摘 要: 采用自克隆技术, 破坏啤酒酵母工业菌株YSF31的ADH2基因, 在ADH2基因位点插入来源于YSF31的编码γ-谷氨酰半胱氨酸合成酶的GSH1基因和铜抗性筛选标记CUP1基因。通过铜抗性筛选转化子, 经PCR和乙醇脱氢酶Ⅱ (ADHⅡ) 活性测定验证, 获得了1株啤酒酵母工程菌。10(P麦芽汁发酵实验显示, 自克隆菌株的乙醇脱氢酶Ⅱ活性是受体菌的65%, 谷胱甘肽含量比受体菌YSF31的高34%。其他发酵指标并没有发生明显改变。由于DNA操作过程中没有外源基因介入, 因此啤酒酵母工程菌为生物安全的自克隆菌株, 具有重要的应用价值。 关键词: 啤酒酵母工业菌株, 谷胱甘肽, 乙醇脱氢酶Ⅱ, 自克隆 Construction of Self-cloning Industrial Brewing Yeast with High-glutathione Production and Low-ADH II Enzyme Activity CAI Yong1 MU Qian1 WANG Zhao-Yue2 ZHANG Bo-Run2 YAN Ben-Ju1* (1. College of Life Sciences, Sichuan Agricultural University, Ya’an 625014) (2. Institute of Microbiology, Chinese Academy of Science, Beijing 100101) Abstract: Self-cloning strains of industrial brewing yeast were constructed by integrating Saccharomyces cerevisiae genes, γ-glutamylcysteine synthetase gene (GSH1) and copper resistant gene (CUP1) into the locus of alcohol dehydrogenase II (ADH II) gene (ADH2). The self-cloning strains were selected for their resistance to CuSO4 and identified by PCR amplification. The results of ADH II and glutathione (GSH) assay from fermentation with the self-cloning strains in 500ml conical flask showed that ADH II activity decreased to 65% and GSH content was 1.3 fold compared with that of host yeasts. The self-cloning strains do not contain any heterologous DNA; they may be more acceptable to the public. Keywords: Industrial brewing yeast, Glutathione, Alcohol dehydrogenase II, Self-cloning乙醛(CH3CHO)是啤酒中主要的风味物质之 一[1]。一般啤酒中的含量为5 mg/L~12 mg/L。国外优质啤酒的乙醛含量多在3 mg/L以下。低浓度的乙醛使啤酒具有芳香味, 高浓度则产生像青草或者苹果腐烂的味道[2]。乙醛含量已成为国内啤酒风味改良的瓶颈。降低啤酒中乙醛的含量, 主要有两种手段：一是通过对发酵工艺的改进降低乙醛含量; 二是通过代谢工程, 修饰工业菌株的代谢途径达到降低乙醛含量的目的。关于前者有大量的文献报道, 但由于啤酒生产对发酵条件要求严格, 使此类工艺改进很难在生产中应用。国内外对啤酒酵母(Saccha- romyces cerevisiae)开展的代谢工程有大量报道, 但针对乙醛含量开展的代谢工程研究报道很少。随着对啤酒酵母中乙醛代谢途径的揭示, 以及相关基因的克隆, 利用基因工程的手段对乙醛代谢过程进行修饰已成为可能。 谷胱甘肽是三肽小分子, 具有抗氧化的功能, 对细胞有保护作用。增加谷胱甘肽的含量可以提高啤酒的抗老化能力[3]。(-谷氨酰