Pyruvate is a three-carbon (triose) ketoacid central to metabolism that could potentially be produced by fermentation. Previous studies observed pyruvate accumulation in strains having mutations in the acetate pathway. The present study applies metabolic engineering strategies to E. coli strains that generate limited acetate under normal growth conditions. Of the various acetate synthesis mutants, strains with a mutation in pyruvate dehydrogenase (PDH) complex (aceE, aceF, lpd) produced more pyruvate than pta, ack or rpoS mutants. Higher growth and pyruvate production rates were observed in complex media compared to minimal media. pH and temperature also affected pyruvate generation, with a pH of 7.0 and a temperature of 32 C optimum for pyruvate generation in strains with a PDH complex mutation. The highest rate of pyruvate generation occurred during the growth phase. A deletion in the ppc gene encoding for phosphoenolpyruvate carboxylase was observed to direct more carbon flux to pyruvate. Acetate generation by PDH mutant strains was attributed to pyruvate oxidase activity. A final concentration of 38 g/L was achieved using a fed-batch strategy.