Jiang Kai, Li Qi, Gu Guo-Xian
The Key Laboratory of Industrial Biotechnology, Ministry of Education, Southern Yangtze University, Wuxi 214122, China.
Sheng Wu Gong Cheng Xue Bao. 2007 Nov;23(6):1071-6. doi: 10.1016/s1872-2075(07)60065-x.
Based on homologous recombination, recombinant plasmid pRKG was constructed by replacing the internal fragment of 18S rDNA of pRJ-5 with a copy of gamma-glutamylcysteine synthetase gene (GSH1) from the industrial brewing yeast strain G03 and a copy of G418 resistance gene (Kan) used as the dominant selection marker respectively. The fragment 18s rDNA::( Kan-GSH1) obtained through the PCR reaction was integrated to the chromosomal DNA of G03 strain, and recombinants were screened by G418 resistance. It was shown that the GSH content of beer fermented with the recombinant strain SG1 was 16.6% higher than that of G03, and no significant difference in routine fermentation parameters was found. To test the genetic stability, strains SG1 was inoculated into flasks and transfered continuously 5 times. The intracellular glutathione content of strain kept constant basically. It is an instructive attempt of genetically modifing industrial brewing yeast, as GSH1 was obtained from the host itself.
基于同源重组，通过用来自工业酿造酵母菌株G03的γ-谷氨酰半胱氨酸合成酶基因（GSH1）拷贝和用作显性选择标记的G418抗性基因（Kan）拷贝分别替换pRJ-5的18S rDNA内部片段，构建了重组质粒pRKG。通过PCR反应获得的片段18s rDNA::(Kan-GSH1)整合到G03菌株的染色体DNA中，并通过G418抗性筛选重组体。结果表明，用重组菌株SG1发酵的啤酒中GSH含量比G03高16.6%，常规发酵参数未发现显著差异。为了测试遗传稳定性，将菌株SG1接种到烧瓶中并连续传代5次。菌株的细胞内谷胱甘肽含量基本保持恒定。由于GSH1是从宿主自身获得的，这是对工业酿造酵母进行基因改造的一次有指导意义的尝试。
