Glutathione production by using a cyanobacterial ATP regeneration system

1. PCR克隆大肠杆菌MV1184株gsh- 1和gsh-II基因及含有gsh-II基因质粒的大肠杆菌细胞：分别用gsh- 1和gsh-II末端区设计的引物和MV1184株的模板DNA进行PCR克隆。将扩增的基因插入pUC18载体，将含有gsh- 1和gsh- 2的质粒导入MV1184菌株。与不含质粒的MV1184相比，含质粒的MV1184的gsh - 1活性（21.5倍）和GSH-II活性（475倍）显著提高。2. 用甲苯处理克隆的大肠杆菌细胞：用含质粒的MV1184菌株在25‰的温度下用甲苯（0- 10%）处理1小时。在累积谷胱甘肽水平中，甲苯的最佳浓度为4%左右。处理后的细胞在- 20c .3下稳定至少1.5年。克隆大肠杆菌细胞与蓝藻细胞（紫紫紫）混合生产谷胱甘肽：gsh- 1克隆细胞与gsh-II克隆细胞的最佳比例（以湿重为基础）为30:1 （gsh- 1活性：gsh-II活性= 1:3）。在最佳条件下，谷胱甘肽水平为7mumol/wet g，持续6小时。4. 大肠杆菌gsh- 1和II基因转化蓝藻细胞：为了开发高效的蓝细菌谷胱甘肽生产，初步将大肠杆菌gsh- 1和II基因转化到蓝藻细胞中。为了增加细胞内底物（谷氨酸、半胱氨酸、甘氨酸）的供应，还研究了涉及这些氨基酸生物合成的酶。

1. PCR Clonings of gsh-I and gsh-II from E.coli MV1184 strain and E.coli cells with plasmid containing those genes : PCR clonings were carried out with the primer set designed from the terminal regions of gsh-I and gsh-II,respectively, and a template DNA from MV1184 strain. The amplified genes were inserted to pUC18 vector and the plasmids containing gsh-I and II were introduced into MV1184 strain. The MV1184 with the plasmid showed significantly high GSH-I (21.5-fold) and GSH-II (475-fold) activities compared to the strain without plasmid. 2. Treatment of the cloned E.coli cells with toluene : The MV1184 strains with plasmids were treated with toluene (0-10 %) at 25ﾟC for 1 hr. The optimum concentration of toluene in the accumulated glutathione level was around 4 %. The treated cells were stable at least for 1.5 year at -20ﾟC.3. Glutathione production by the mixture of the cloned E.coli cells and cyanobacterial cells (Phormidium lapideum) : The optimum ratio (based on wet weight) of gsh-I cloned cells and gsh-II cloned cells was 30 : 1 (GSH-I activity : GSH-II activity =1 : 3). Under the optimum condition, the glutathione level was 7mumol/wet g for 6 hr. 4. Transformation of E.coli gsh-I and II genes into cyanobacterial cells : To develop an efficient cyanobacterial glutathione production, the E.coli gsh-I and II genes were preliminary transformed into cyanobacterial cells.To increase intracellular supply of the substrates (glutamate, cysteine, glycine), the enzymes involving biosynthesis of those amino acids were also studied.