Abstract

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 <4 mol H2/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 (phosphoglucose isomerase) 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 Δpgi can recover its anaerobic growth capability on glucose. Further, with additional deletions of ΔhycA, ΔhyaAB, ΔhybBC, ΔldhA, and ΔfrdAB, the recombinant Δpgi 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 Δpgi 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 ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB, 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.

Highlights

  • Hydrogen ­(H2) can be produced through dark fermentation and photofermentation

  • Our results demonstrate that Δpgi mutants can grow under anaerobic conditions and co-produce ­H2 and Strains, plasmids, and materials The Escherichia coli BW25113 mutant strain (SH5) from our previous study [19] was used as a base strain in this work

  • Growth of E. coli mutant lacking pgi under aerobic and anaerobic conditions To completely block the carbon flux through the EMP pathway and divert it through the PP pathway, the pgi gene was deleted from the E. coli BW25113 mutant strain, which has several deletions in such enzymes as uptake hydrogenases, the negative regulator of formate-hydrogen lyase, lactate dehydrogenase, and fumarate reductase [19]

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Summary

Introduction

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

Methods
Results
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