Construction of glutathione-producing strains of Escherichia coli B by recombinant DNA techniques

The enzymatic production of glutathione (GSH) has been studied in a bioreactor system using toluene-treated cells of Escherichia coli B transformed with recombinant plasmids for gamma-glutamylcysteine synthetase (GSH-I) and glutathione synthetase (GSH-II). As reported previously the genes for both enzymes were separately cloned onto vector plasmid pBR322. The plasmid for GSH-I was designated pGS100-2 and that for GSH-II as pGS200. The effect on GSH production in the bioreactor system, containing an ATP regenerating system, of using cells containing various hybrid plasmids has now been explored. Three kinds of hybrid plasmids, designated pGS300, pGS400, and pGS500, were constructed by subcloning the genes in pGS100-2 and pGS200 onto vector plasmid pBR325. pGS300 contained the E. coli B chromosomal DNA fragment with a gene for GSH-I in the PstI site of pBR325. pGS400 also contained E. coli B chromosomal DNA fragment with a gene for GSH-II in the HindIII site of pBR325. In contrast, pGS500 contained two kinds of DNA fragments with the genes for GSH-I and GSH-II in the PstI and HindIII sites of pBR325, respectively. All the hybrid plasmids thus prepared were stably maintained in E. coli cells when chloramphenicol was included at 10 micrograms/ml in the medium. The activity of the cells containing pGS300 was higher than that of the cells containing pGS400, although the former activity did not come up to that of cells having both pGS300 and pGS400. The highest glutathione-producing activity was found in the case of the cells transformed with pGS500 carrying both genes for GSH-I and GSH-II on the vector plasmid pBR325. About 5 mg/ml of glutathione was produced by E. coli cells with pGS500 from 80 mM L-glutamate, 20 mM L-cysteine, and 20 mM glycine within 3 h at 37 degrees C.