Dynamic responses of the intracellular metabolite concentrations of the wild type and pykA mutant Escherichiacoli against pulse addition of glucose or NH3 under those limiting continuous cultures

The dynamics of the intracellular metabolite concentrations were investigated for the wild type and pykA gene knockout mutant Escherichia coli in responses to the glucose pulse addition during glucose-limited continuous culture and in responses to the ammonia pulse addition during ammonia-limited continuous culture. For this, we developed a new automated rapid sampling device, which enables us to take samples rapidly within a second. The intracellular concentrations of G6P, F6P, 2PG, PEP, OAA, 6PG, Ribu5P, E4P and NADPH were higher for pykA mutant as compared with the wild type under both limited continuous cultures, and the concentrations of PYR, ATP and acetate were much lower for pykA mutant than those of the wild type. These phenomena reflected the fact that the accumulation of PEP caused the increased flux from PEP to OAA and that the accumulated PEP inhibited pfk, which caused the accumulation of G6P and F6P, which in turn increased the flux toward pentose phosphate (PP) pathway and increased the PP pathway metabolite concentrations. Oxygen uptake rate (OUR) was a little higher for pykA mutant as compared with that of its wild type, while CO2 production rate (CER) shows the reverse trend. OUR and CER were much less for NH3-limited condition than that of NH3-rich condition. The intracellular concentrations of PEP, ATP and PYR decreased rapidly within several seconds, whereas the concentrations of G6P, F6P FBP, 6PG, ADP, NADH and NADPH increased after glucose pulse addition during glucose-limited condition for both wild type and pykA knockout mutant. Initial decrease in PEP concentration was considered to be due to PTS system. The intracellular concentration of NADPH decreased after NH3 pulse addition under NH3-limited condition for both strains, which is due to the fact that NADPH is utilized through glutamate production under NH3 addition.