Although several microorganisms can produce pyruvate by fermentation of glucose and other renewable resources, Escherichia coli has many advantages including rapid growth, simple nutritional requirements and the ease of genetic manipulation. A series of steady-state (chemostat) experiments were conducted to evaluate the ability of metabolically engineered E. coli strains (pflB aceEF poxB pps ldhA) to produce pyruvate under several different nutrient-limited conditions. The greatest pyruvate formation rate was found under conditions of acetate-limited growth.  Glucose consumption rate and therefore pyruvate productivity were further increased by introducing ATP synthase knockout, arcA knockout and NADH oxidase.  High pyruvate concentration and productivity were achieved by well-controlled fed-batch fermentations (at a low specific growth rate).