초록
<P>Pyruvate kinase is one of the three rate-limiting glycolytic enzymes that catalyze the last step of glycolysis, conversion of phosphoenolpyruvate (PEP) into pyruvate, which is associated with ATP generation. Two isozymes of pyruvate kinase, PykF and PykA, are identified in <I>Escherichia coli</I>. PykF is considered important, whereas PykA has a less-defined role. Prior studies inactivated the <I>pykA</I> gene to increase the level of its substrate, PEP, and thereby increased the yield of end products derived from PEP. We were surprised when we found a <I>pykA</I>::Tn<I>5</I> mutant in a screen for increased yield of an end product derived from pyruvate (<I>n</I>-butanol), suggesting that the role of PykA needs to be reexamined. We show that the <I>pykA</I> mutant exhibited elevated intracellular ATP levels, biomass concentrations, glucose consumption, and <I>n</I>-butanol production. We also discovered that the <I>pykA</I> mutant expresses higher levels of a presumed pyruvate transporter, YhjX, permitting the mutant to recapture and metabolize excreted pyruvate. Furthermore, we demonstrated that the nucleotide diphosphate kinase activity of PykA leads to negative regulation of the intracellular ATP levels. Taking the data together, we propose that inactivation of <I>pykA</I> can be considered a general strategy to enhance the production of pyruvate-derived metabolites under anaerobic conditions.</P><P><B>IMPORTANCE</B> This study showed that knocking out <I>pykA</I> significantly increased the intracellular ATP level and thus significantly increased the levels of glucose consumption, biomass formation, and pyruvate-derived product formation under anaerobic conditions. <I>pykA</I> was considered to be encoding a dispensable pyruvate kinase; here we show that <I>pykA</I> negatively regulates the anaerobic glycolysis rate through regulating the energy distribution. Thus, knocking out <I>pykA</I> can be used as a general strategy to increase the level of pyruvate-derived fermentative products.</P>