LB medium supplemented with 50 µg/ml chloramphenicol to OD600=0.5

Measurement of metabolite concentrations - A sample was added to 100 mM MOPS buffer (pH 7.2) containing 0.1 mM NADH, 20 mM MgCl2, 5 mM ATP, and 2 mM DTT (I), and maintained at 37 ºC. Then, 1.8 U/ml ALD, 31 U/ml G3PDH, and 90 U/ml TIM (II), 3 U/ml PfkA (III), 18 U/ml Pgi (IV) ... , and 10 U/ml Glk (V) were added sequentially, and A340 was recorded at each step. Concentrations of fructose-1,6-diphosphate (FDP), F6P, G6P, and glucose were calculated from the difference in A340 between I and II, II and III, III and IV, and IV and V, respectively. The FDP concentration of each sample was calibrated by the measured value of a known concentration standard solution of FDP treated using the same procedure employed for sample preparation.