Switch on a more efficient pyruvate synthesis pathway based on transcriptome analysis and metabolic evolution

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 ΔldhA ΔpflB Δpta-ackA ΔpoxB Δppc ΔfrdBC). 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 g l−1 with a volumetric productivity of 2.3 g l−1 h−1. This was the first time to produce pyruvate via ED pathway, and prove that this was a more effective way. •A novel effective strategy to produce pyruvate was built based on ED pathway.•Transcriptome analysis revealed the metabolic advantages of gluconate.•The EMP and PP pathways were blocked in the new pyruvate producing strain.•High pyruvate concentration and productivity was obtained in inorganic salt medium.