Metabolic engineering and adaptive evolution ofEscherichia coliKO11 for ethanol production through the Entner-Doudoroff and the pentose phosphate pathways

Abstract

BACKGROUND Ethanologenic Escherichia coli KO11 was modified to channel carbon flux from glucose through the Entner–Doudoroff (ED-P) and pentose phosphate (PP-P) pathways by using a phosphoglucose isomerase (pgi) knockout in the glycolytic pathway. This strain grows very slowly under non-aerated conditions with minimal media supplemented with 4% glucose. To improve the capacity to grow, KO11 Δpgi was evolved for 60 days; the resultant strain was named KO11 E35, which directs the carbon flux through the PP-P and ED-P to lactate and acetate production. RESULTS The activities of glucose-6-phosphate dehydrogenase and the ED-P enzymes increased 17-fold and 2-fold, respectively, in KO11 E35 in comparison with KO11. A homoethanologenic derivative was constructed from KO11 E35 by deleting the pta, ack and ldh genes, yielding the KO11 PPAL strain. This strain channels most of the carbon flux from pyruvate to ethanol and increased expression of heterologous pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis allowed us to obtain specific ethanol production rates similar to those found in KO11, but with half the cell mass, i.e. larger ethanol/glucose and ethanol/biomass yields. CONCLUSIONS These results suggest that it is possible to obtain the same carbon flux using the PP-P and ED-P as when using the Embden–Meyerhof–Parnas pathway for glucose catabolism to ethanol. © 2016 Society of Chemical Industry