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Based on measurements and theoretical analyses, we identified deletion of pyruvate kinase (PYK) activity as a possible route for elimination of acid formation in Bacillus subtilis cultures grown on glucose minimal media. Evidence consistent with the attenuation of PYK flux has come from metabolic flux calculations, metabolic pool and enzymatic activity measurements, and a series of nuclear magnetic resonance experiments, all suggesting a nearly complete inhibition of PYK activity for glucose-citrate fed cultures in which the amount of acid formation was nearly zero. In this paper, we report the construction and characterization of a pyk mutant of B. subtilis. Our results demonstrate an almost complete elimination of acid production in cultures of the pyk mutant in glucose minimal medium. The substantial reduction in acid production is accompanied by increased CO 2 production and a reduced rate of growth. Metabolic analysis indicated a dramatic increase in intracellular pools of phosphoenolpyruvate (PEP) and glucose-6-P in the pyk mutant. The high concentrations of PEP and glucose-6-P could explain the decreased growth rate of the mutant. The substantial accumulation of PEP does not occur in Escherichia coli pyk mutants. The very high concentration of PEP which accumulates in the B. subtilis pyk mutant could be exploited for production of various aromatics.Acid production is among the important factors that limit process stability and cell concentration and thus cell-based biotechnological processes (e.g., see references 14, 23, and 25). Numerous approaches have been used in an attempt to reduce acid formation. One mechanism involves maintaining low levels of glucose in fed batch reactors (24,27). While this can lead to increased cell mass and product formation, it is a capitalintensive method. Manipulation of the growth media might also be used to reduce acid formation relative to the amount of glucose consumed (13,24).Majewski and Domach (17) used a constrained network analysis of the main metabolic pathways in conjunction with reported measurements of enzymatic activity levels to explain acid production by bacterial cells. This analysis suggested that Escherichia coli and Bacillus subtilis have excess glycolytic capacity relative to the Krebs cycle. This idea is consistent with the notion that given all the anabolic and catabolic tasks that metabolic networks must perform, stoichiometric conflicts and other conflicts arise, leading to imperfect coordination of all tasks. It is thus an overflow or "spilling" of excess carbon that leads to acid production.In experiments using B. subtilis to test the overflow hypothesis, it was found that a small amount of citrate added to glucose minimal medium (0.1 mol of citrate/1 mol of glucose) caused the rate of glucose (or total carbon) use per cell to decline several-fold, while the maximal growth rate was not diminished (12). Further, acid production was undetectable in the glucose-citrate cultures. Subsequent work showed that productivity of recombinant protein (units o...

In this paper, we report on the analysis of acid formation in an E. coli pyk mutant. The results demonstrate that acid formation is insignificant for both the wild-type and the mutant at low glucose concentrations. However, at relatively high glucose concentrations, acid formation remains very low for the mutant but is significant for the wild-type. This substantial reduction in acids is accompanied by an increase in CO(2) production. Moreover, unlike the B. subtilis pyk mutant, the E. coli pyk mutant did not show a substantial increase in the PEP pool.

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When batch and continuous Bacillus subtilis cultures are provided with a small amount of citrate, acid production ceases, carbon yield increases by more than 2-fold, and the productivity of recombinant protein increases. It has been hypothesized that pyruvate kinase activity is attenuated, which in turn lowers glucose flux and minimizes the acid overflow prompted by low Krebs cycle capacity. To complement existing enzyme activity, linear programming, and metabolite pool studies, (13)C NMR studies were performed. Atom mapping and isotopomer mapping matrix methods were used to select the best glucose label. "Best" was defined such that the NMR spectra of glutamate associated with metabolizing labeled glucose via the different candidate metabolic trafficking scenarios would differ considerably in fine structure (e.g., relative singlet intensities). When experiments were performed with 1-(13)C 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 successfully basing a metabolic engineering strategy on reducing pyruvate kinase activity to better match glycolytic and Krebs cycle capacities.