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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)

Sundara Sekar, Balaji, Seol, Eunhee, Park, Sunghoon
Biotechnology for biofuels 2017 v.10 no.1 pp. 85
Escherichia coli, Gluconobacter oxydans, Leuconostoc mesenteroides, NADP (coenzyme), acetates, anaerobic conditions, biofuels, carbon, ethanol, fermentation, glucose, glucose-6-phosphate 1-dehydrogenase, glucose-6-phosphate isomerase, glycolysis, hydrogen, hydrogen production, mutants, phosphogluconate dehydrogenase
BACKGROUND: Biologically, 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. 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 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. CONCLUSIONS: By 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.