Metabolic engineering of Escherichia coli strains for co-production of hydrogen and ethanol from glucose

The co-production of H2 and ethanol from glucose was studied to address the low H2 production yield in dark fermentation. Several mutant strains devoid of ackA-pta, pfkA or pgi were developed using Escherichia coli BW25113 ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB as base strain. Disruption of ackA-pta eliminated acetate production during glucose fermentation but resulted in the secretion of a significant amount of pyruvate (0.73 mol mol−1 glucose) without improving the co-production of H2 and ethanol. When pfkA or pgi was further disrupted to enhance NAD(P)H supply by diverting the carbon flux from Embden-Meyerhof-Parnas (EMP) pathway to the pentose phosphate pathway (PPP), the cell growth of both strains was severely impaired under anaerobic conditions, and only the ΔpfkA mutant could recover its growth after adaptive evolution. The production yields of the ΔpfkA strain (H2, 1.03 mol mol−1 glucose and ethanol, 1.04 mol mol−1 glucose) were higher than those of the pfkA+ strain (H2, 0.69 mol mol−1 glucose and ethanol, 0.95 mol mol−1 glucose), but pyruvate excretion was not reduced. The excessive excretion of pyruvate in the ΔpfkA mutant was attributed to an insufficient NAD(P)H supply caused by the diversion of carbon flux from the EMP pathway to the Entner-Doudoroff pathway (EDP), rather than the PPP as intended. This study suggests that co-production of H2 and ethanol from glucose is possible, but further metabolic pathway engineering is required to fully activate PPP under anaerobic conditions.