Reexamination of the physiological role of PykA in Escherichia coli revealed that it negatively regulates the intracellular ATP levels under anaerobic conditions

Pyruvate kinase is one of the three rate-limiting glycolytic enzymes that catalyzes 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 Escherichia coli . The PykF is considered important, whereas the PykA has less-defined role. Prior studies inactivated the pykA 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 pykA ::Tn5 mutant in a screen for increased yield of an end-product derived from pyruvate ( n -butanol), suggesting the role of PykA needs to be reexamined. We show that the pykA mutant exhibited elevated intracellular ATP levels, increased biomass concentration, glucose consumption, and n -butanol production. We also discovered that the pykA mutant expresses higher levels of a presumed pyruvate transporter, YhjX, permitting the mutant to recapture and metabolize excreted pyruvate. Furthermore, we demonstrated the nucleotide diphosphate kinase activity of PykA leads to negative regulation on the intracellular ATP levels. Taken together, we propose that inactivation of pykA can be considered as a general strategy to enhance the production of pyruvate-derived metabolites under anaerobic conditions. IMPORTANCE This study discovered that knocking out pykA significantly increased the intracellular ATP level and thus significantly increased the glucose consumption, biomass formation, and pyruvate-derived product formation under anaerobic conditions. pykA was considered encoding a dispensable pyruvate kinase, here we show that pykA negatively regulates the anaerobic glycolysis rate through regulating the energy distribution. Thus, knocking out pykA can be used as a general strategy to increase the productivity of pyruvate-derived fermentative products.