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Background  Biologically, hydrogen (H 2 ) can be produced through dark fermentation and photofermentation. Dark fermentation is fast in rate and simple in reactor design, but H 2  production yield is unsatisfactorily low as <4 mol H 2 /mol glucose. To address this challenge, simultaneous production of H 2  and ethanol has been suggested. Co-production of ethanol and H 2  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.    Results  Here, 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 H 2  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 H 2  mol −1  glucose, which are close to the theoretically maximal yields, 1.67 mol mol −1  each for ethanol and H 2 . 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.    Conclusions  By 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 H 2 , 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.    Electronic supplementary material  The online version of this article (doi:10.1186/s13068-017-0768-2) contains supplementary material, which is available to authorized users.

biotechnology for biofuels

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)