Due to the decrease of intracellular NADH availability, gluconate metabolism is more conducive to pyruvate production than glucose. Transcriptome analysis revealed that the Entner-Doudoroff (ED) pathway was activated by gluconate in Escherichia coli YP211 (MG1655 Delta IdhA Delta pflB Delta pta-ackA Delta poxB Delta ppc Delta frdBC). To construct a new pyruvate producing strain with glucose metabolism via ED pathway, the genes ppsA, ptsG, pgi and gnd were deleted sequentially to reduce the demand for PEP and block the Embden-Meyerhor-Parnas pathway and Pentose-Phosphate pathway. After nearly 1000 generations of growth-based selection, the evolved strain YP404 was isolated and the ED pathway was proved to be activated as the primary glycolytic pathway. Comparing with YP211, the pyruvate concentration and yield increased by 59% and 10.1%, respectively. In fed-batch fermentation, the pyruvate concentration reached 83.5 gl(-1) with a volumetric productivity of 2.3 gl(-1) h(-1). This was the first time to produce pyruvate via ED pathway, and prove that this was a more effective way. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.