Co-production of hydrogen and ethanol from glucose in Escherichia coli by activation of pentose-phosphate pathway through deletion of phosphoglucose isomerase (pgi) and overexpression of glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconate dehydrogenase (gnd)

摘要

BackgroundBiologically, hydrogen (H₂) can be produced through dark fermentation and photofermentation. Dark fermentation is fast in rate and simple in reactor design, but H₂ production yield is unsatisfactorily low as 2/mol glucose. To address this challenge, simultaneous production of H₂ and ethanol has been suggested. Co-production of ethanol and H₂ requires enhanced formation of NAD(P)H during catabolism of glucose, which can be accomplished by diversion of glycolytic flux from the Embden–Meyerhof–Parnas (EMP) pathway to the pentose-phosphate (PP) pathway in Escherichia coli . However, the disruption of pgi ( p hospho g lucose i somerase) for complete diversion of carbon flux to the PP pathway made E. coli unable to grow on glucose under anaerobic condition. ResultsHere, we demonstrate that, when glucose-6-phosphate dehydrogenase (Zwf) and 6-phosphogluconate dehydrogenase (Gnd), two major enzymes of the PP pathway, are homologously overexpressed, E. coli Δ pgi can recover its anaerobic growth capability on glucose. Further, with additional deletions of Δ hycA , Δ hyaAB , Δ hybBC , Δ ldhA , and Δ frdAB , the recombinant Δ pgi mutant could produce 1.69?mol?H₂ and 1.50?mol ethanol from 1?mol glucose. However, acetate was produced at 0.18?mol?mol?1 glucose, indicating that some carbon is metabolized through the Entner–Doudoroff (ED) pathway. To further improve the flux via the PP pathway, heterologous zwf and gnd from Leuconostoc mesenteroides and Gluconobacter oxydans , respectively, which are less inhibited by NADPH, were overexpressed. The new recombinant produced more ethanol at 1.62?mol?mol?1 glucose along with 1.74?mol?H₂?mol?1 glucose, which are close to the theoretically maximal yields, 1.67?mol?mol?1 each for ethanol and H₂. However, the attempt to delete the ED pathway in the Δ pgi mutant to operate the PP pathway as the sole glycolytic route, was unsuccessful. ConclusionsBy deletion of pgi and overexpression of heterologous zwf and gnd in E. coli Δ hycA Δ hyaAB Δ hybBC Δ ldhA Δ frdAB , two important biofuels, ethanol and H₂, could be successfully co-produced at high yields close to their theoretical maximums. The strains developed in this study should be applicable for the production of other biofuels and biochemicals, which requires supply of excessive reducing power under anaerobic conditions.