Characterization of growth and acid formation in a Bacillus subtilis pyruvate kinase mutant

Based on measurements and theoretical analyses, we identified deletion of p yruvate kinase (PYK) activity as a possible route for elimination of acid f ormation in Bacillus subtilis cultures grown on glucose minimal media. Evid ence consistent with the attenuation of PYK flux has come from metabolic fl ux calculations, metabolic pool and enzymatic activity measurements, and a series of nuclear magnetic resonance experiments, all suggesting a nearly c omplete inhibition of PYK activity for glucose-citrate fed cultures in whic h the amount of acid formation was nearly zero. In this paper, we report th e construction and characterization of a pyk mutant of B. subtilis. Our res ults demonstrate an almost complete elimination of acid production in cultu res of the pgk mutant in glucose minimal medium. The substantial reduction in acid production is accompanied by increased CO2 production and a reduced rate of growth. Metabolic analysis indicated a dramatic increase in intrac ellular pools of phosphoenolpyruvate (PEP) and glucose-6-P in the pyk mutan t. The high concentrations of PEP and glucose-6-P could explain the decreas ed growth rate of the mutant. The substantial accumulation of PEP does not occur in Escherichia coli pyk mutants. The very high concentration of PEP w hich accumulates in the B. subtilis pyk mutant could be exploited for produ ction of various aromatics.