title: Co-production of hydrogen and ethanol from glucose inEscherichia coliby activation of pentose-phosphate pathway through deletion of phosphoglucose isomerase (pgi) and overexpression of glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconate dehydrogenase (gnd).
abstract: BACKGROUND
  Biologically, hydrogen (H2) can be produced through dark fermentation and photofermentation. Dark fermentation is fast in rate and simple in reactor design, but H2production yield is unsatisfactorily low as <4 mol H2/mol glucose. To address this challenge, simultaneous production of H2and ethanol has been suggested. Co-production of ethanol and H2requires 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 inEscherichia coli. However, the disruption ofpgi(phosphoglucoseisomerase) for complete diversion of carbon flux to the PP pathway madeE. coliunable 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. coliΔpgican recover its anaerobic growth capability on glucose. Further, with additional deletions of ΔhycA, ΔhyaAB, ΔhybBC, ΔldhA, and ΔfrdAB, the recombinant Δpgimutant could produce 1.69 mol H2and 1.50 mol ethanol from 1 mol glucose. However, acetate was produced at 0.18 mol mol-1glucose, indicating that some carbon is metabolized through the Entner-Doudoroff (ED) pathway. To further improve the flux via the PP pathway, heterologouszwfandgndfromLeuconostoc mesenteroidesandGluconobacter oxydans, respectively, which are less inhibited by NADPH, were overexpressed. The new recombinant produced more ethanol at 1.62 mol mol-1glucose along with 1.74 mol H2mol-1glucose, which are close to the theoretically maximal yields, 1.67 mol mol-1each for ethanol and H2. However, the attempt to delete the ED pathway in the Δpgimutant to operate the PP pathway as the sole glycolytic route, was unsuccessful.
  
  
  CONCLUSIONS
  By deletion ofpgiand overexpression of heterologouszwfandgndinE. coliΔhycAΔhyaABΔhybBCΔldhAΔfrdAB, 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Pusan National University, 2, Busandaehak-ro 63 beon-gil, Geumjeong-gu, Busan, 46241 Republic of Korea.
authors.forename: Balaji
authors.initials: B
authors.lastname: Sundara Sekar
authors.affiliation: Department of Chemical and Biomolecular Engineering, Pusan National University, 2, Busandaehak-ro 63 beon-gil, Geumjeong-gu, Busan, 46241 Republic of Korea.
authors.forename: Eunhee
authors.initials: E
authors.lastname: Seol
authors.affiliation: Department of Chemical and Biomolecular Engineering, Pusan National University, 2, Busandaehak-ro 63 beon-gil, Geumjeong-gu, Busan, 46241 Republic of Korea.
authors.forename: Sunghoon
authors.initials: S
authors.lastname: Park
keywords: Biohydrogen; Co-production of hydrogen and ethanol; Escherichia coli; Pentose-phosphate pathway; Phosphoglucose isomerase deletion