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.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...

Engineering of Bacillus subtilis for Enhanced Total Synthesis of Folic Acid

“…3 and 4) indicates that attenuating PYK flux to a near-zero level may also be a fruitful starting point for enhancing folic acid production by B. subtilis. Moreover, controlling pyk activity at a low value via IPTG induction may overcome the low growth rate limitation of a pyk knockout (8). Overall, we found that when provided with a low level of IPTG (0.02 mM), an inducible pyk mutant, BSTZ0402, exhibits a threefold higher folic acid yield compared to the parent strain B. subtilis 168.…”

“…In contrast, a pyk knockout of E. coli grows nearly as fast as the wild type (29). The difference in the growth rates of B. subtilis and E. coli can be attributed to different anaplerotic routes to oxaloacetate formation are used by the two species as was explained in a prior publication (8).…”

“…Additionally, the strain BSZT0425 indicates that overexpressing aroH in tandem with low induction of pyk further increases the folic acid yield. The yield enhancement conceivably results from the elevated PEP pool that has been found in B. subtilis mutants with impaired pyruvate kinase activity (8).…”

“…Using the data in Table 3, BSZT0437 exhibits about fourfold greater bioreactor volume productivity (g folic acid/liter h) compared to B. subtilis 168. B. subtilis strains with attenuated pyruvate kinase activity also produce less acetate (8). Thus, apart from increased folic acid yield, another attribute is that BSZT0437 may prove easier to culture to a high cell concentration using fed-batch techniques.…”

“…One means to accomplish this strategy is to create a B. subtilis mutant deficient in the enzyme pyruvate kinase (PYK) by interrupting the pyk gene. A prior publication reported that such a mutant exhibited significantly increased PEP and glucose-6-phosphate pools (8). Moreover, acetate production was significantly reduced, which could ultimately enable high density cell culture and thus improved volumetric yield.…”