Effects of nutritional conditions on plasmid stability and production of tryptophan synthase by a recombinant Escherichia coli.

Abstract

Effects of nutritional conditions and insertion direction of the tryptophan synthase (TSase) gene into a plasmid vector on the plasmid stability and the production of TSase in high cell concentration cultures were examined using recombinant Escherichia coli (E. coli K12 IFO 3301) harboring pBR322trpAB(1) (the TSase gene was inserted at the EcoRI site of pBR322 in the clockwise direction) and pBR322trpAB(2) (counterclockwise direction). As to the effects of the insertion direction, the cells harboring pBR322trpAB(2) were slightly lower in the growth rate and the plasmid stability than those harboring pBR322trpAB(1). However, the former was higher in the productivity of TSase and the final cell concentration attained than the latter. On the other hand, the addition of organic nutrients, especially yeast extract, to TK-25 medium was very effective to improve the plasmid stability. Among the components of yeast extract, L-glutamic acid was found to be effective to improve both the plasmid stability and the production of TSase. When 1 g/l of L-glutamic acid was added to TK-25 medium, a mineral synthetic medium developed for a high concentration culture, 115g (dry basis)/l of recombinant cells were obtained in 14 hr and the expression of TSase was maintained at 240-300 U/mg-protein during the cultivation.