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

Abstract

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.

FIG. 1

Changes in the OD₆₆₀ (A) and glucose and CO₂ levels (B) in the batch cultures of the wild type (WT) and the pyk mutant (MT) of B. subtilis with an initial glucose concentration of about 8 g/liter. CO₂/N₂ is the molar ratio of CO₂ to N₂ in the effluent gas stream. Since the amount of N₂ does not change, the variation in the CO₂/N₂ ratio represents the changes in the CO₂ concentration.

FIG. 2

Oxaloacetate formation in E. coli and B. subtilis.