Quantitative determination of metabolic fluxes during coutilization of twocarbon sources: Comparative analyses with corynebacterium glutamicum during growth on acetate and/or glucose

Growth of Corynebacterium glutamicum on mixtures of the carbon sources gluc ose and acetate is shown to be distinct from growth on either substrate alo ne. The organism showed nondiauxic growth on media containing acetate-gluco se mixtures and simultaneously metabolized these substrates. Compared to th ose for growth on acetate or glucose alone, the consumption rates of the in dividual substrates were reduced during acetate-glucose cometabolism, resul ting in similar total carbon consumption rates for the three conditions. By C-13-labeling experiments with subsequent nuclear magnetic resonance analy ses in combination with metabolite balancing, the in vivo activities for pa thways or single enzymes in the central metabolism of C. glatamicum were qu antified for growth on acetate, on glucose, and on both carbon sources. The activity of the citric acid cycle was high on acetate, intermediate on ace tate plus glucose, and low on glucose, corresponding to in vivo activities of citrate synthase of 413, 219, and 111 nmol (mg of protein)(-1) min(-1), respectively. The citric acid cycle was replenished by carboxylation of pho sphoenolpyruvate (PEP) and/or pyruvate (30 nmol [mg of protein](-1) min(-1) ) during growth on glucose. Although levels of PEP carboxylase and pyruvate carboxylase during growth on acetate were similar to those for growth on g lucose, anaplerosis occurred solely by the glyoxylate cycle (99 nmol [mg of protein](-1) min(-1)). Surprisingly, the anaplerotic function was fulfille d completely by the glyoxylate cycle (50 nmol [mg of protein](-1) min(-1)) on glucose plus acetate also. Consistent with the predictions deduced from the metabolic flux analyses, a glyoxylate cycle-deficient mutant of C. glut amicum, constructed by targeted deletion of the isocitrate lyase and malate synthase genes, exhibited impaired growth on acetate-glucose mixtures.