低乙醇脱氢酶Ⅱ活性的抗老化啤酒酵母工程菌的构建

Construction of Self-cloning Industrial Brewing Yeast with High-glutathione Production and Low-ADH II Enzyme Activity

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。

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.