CONSTRUCTION OF RECOMBINANT E. COLI WITH HIGH GLUTATHIONE BIOSYNTHETIC ACTIVITY AND THE BIOSYNTHETIC PROCESS
具有高谷胱甘肽合成活性重组大肠杆菌的构建及合成反应过程

A recombinant strain E. coli II-1, which exhibited high glutathione (GSH) biosynthetic activity and high stability, was constructed by transforming plasmid pGH501 which contains gene gsh I and gsh II into a wild type strain E. coli II. 4 g/L GSH accumulated extracellularly by using toluene-treated cell. In GSH biosynthetic system, GSH production was improved by increasing the concentration of L-glutamate, while inhibited by L-cysteine if it's concentration was beyond 20 mmol/L. In GSH biosynthetic reaction, the apparent little consumption of L-glutamate and glycine was concluded experimentally to be that toluene-treated E. coli II-1 cells still contained high concentration of L-glutamate and glycine. According to the change of energy cofactor in the GSH biosynthetic process, a possible GSH biosynthetic mechanism controlled by E. coli II-1, was proposed: the energy donator of reaction catalyzed by glutathione synthetase (GSH-II) was ADP but not ATP, the reaction was rate-limited step within the whole GSH biosynthetic process, high concentration of ADP might inhibit the activity of GSH-II. Further degradation of GSH was prevented by the addition of 100 mmol/L L-serine and potassium borate mixture. In such case, 23.0 mmol/L (about 7.1 g/L) GSH accumulated at 3 h.