[Construction of recombinant E. coli with high glutathione biosynthetic activity and the biosynthetic process].
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.
Li
H
H
School of Biotechnology, Wuxi University of Light Industry, Wuxi 214036, China.
Li
Y
Y
Lin
J
J
Chen
J
J
D000244:Adenosine Diphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D005721:Glutamate-Cysteine Ligase / Q000235:genetics*; D018698:Glutamic Acid / Q000378:metabolism; D005978:Glutathione / Q000096:biosynthesis* / Q000235:genetics; D005981:Glutathione Synthase / Q000235:genetics*; D005998:Glycine / Q000378:metabolism; D010957:Plasmids; D014050:Toluene / Q000494:pharmacology; D014169:Transformation, Bacterial
D014050:Toluene; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; D005721:Glutamate-Cysteine Ligase; D005981:Glutathione Synthase; D005978:Glutathione; D005998:Glycine