C-13 NMR evidence for pyruvate kinase flux attenuation underlying suppressed acid formation in Bacillus subtilis

When batch and continuous Bacillus subtilis cultures are provided with a sm all amount of citrate, add production ceases, carbon yield increases by mor e than 2-fold, and the productivity of recombinant protein increases. It ha s been hypothesized that pyruvate kinase activity is attenuated, which in t urn lowers glucose flux and minimizes the acid overflow prompted by low Kre bs cycle capacity. To complement existing enzyme activity, linear programmi ng, and metabolite pool studies, C-13 NMR studies were performed. Atom mapp ing and isotopomer mapping matrix methods were used to select the best gluc ose label. "Best" was defined such that the NMR spectra of glutamate associ ated with metabolizing labeled glucose via the different candidate metaboli c trafficking scenarios would differ considerably in fine structure (e.g., relative singlet intensities). When experiments were performed with 1-C-13 glucose, the observed NMR spectra corresponded well to the one predicted to arise when the metabolic trafficking occurs according to a pyruvate kinase attenuation scenario. This evidence further fortifies the prospects for su ccessfully basing a metabolic engineering strategy on reducing pyruvate kin ase activity to better match glycolytic and Krebs cycle capacities.