•NAD synthetase overexpression increased NAD(H) pool, reduced NADH/NAD+ ratio.•Low NADH/NAD+ ratio improved glucose uptake, enhanced hydrogen yield.•High level NAD(H) pool promoted hydrogen production.•Double modification strategy exhibited positive synergistic effect.•The mutant strain produced 2.89 mol H2/mol glucose and 5.1 L H2/L.The effects of combining two strategies, recycling NAD and improving the availability of NADH, on hydrogen production in Enterobacter aerogenes were investigated. The NAD synthetase encoded by nadE gene was homologously overexpressed in AB91002-O, which had been obtained previously, to increase the intracellular concentration of the NAD(H/+) pool. This overexpression was duplicated in mutant strains in which the phosphoenolpyruvate carboxylase (PEPC) gene (ppc) and hybO gene were knocked out, yielding AB91102-OP (ΔhybO/Δppc), AB91102-ON (ΔhybO/nadE), and AB91102-OP/N (ΔhybO/Δppc/nadE). Chemostat experiments showed that the total NAD(H) pool size in AB91102-ON increased 2-fold compared with the control strain AB91102-OC, but the NADH/NAD+ ratio decreased by 24%. Metabolic analysis of batch experiments indicated that a larger NAD(H/+) pool and inactivation of PEPC led to a significant shift in metabolic patterns, whereas a smaller NADH/NAD+ ratio improved glucose uptake. Thus, compared with the control strain, the hydrogen yields per glucose of the mutant strains AB91102-OP, AB91102-ON, and AB91102-OP/N were enhanced by 36.2%, 66.0%, and 149%, respectively, and the total volumes of hydrogen production increased by 27%, 165%, and 301%, respectively. The maximum hydrogen production of 5.1 L/L was achieved by AB91102-OP/N, suggesting that the double modification strategy exhibits markedly positive synergistic effects on hydrogen production.

•Genes encoding hydrogenase-3 were successfully knocked out.•Gene knockout inhibits FHL hydrogen production pathway.•Gene knockout synchronously inhibits NADH pathway.•NADH pathway proven definitely involving hydrogenase-3.In this study, the hydrogenase-3 gene cluster (hycDEFGH) was isolated and identified from Enterobacter aerogenes CCTCC AB91102. All gene products were highly homologous to the reported bacterial hydrogenase-3 (Hyd-3) proteins. The genes hycE, hycF, hycG encoding the subunits of hydrogenase-3 were targeted for genetic knockout to inhibit the FHL hydrogen production pathway via the Red recombination system, generating three mutant strains AB91102-E (ΔhycE), AB91102-F (ΔhycF) and AB91102-G (ΔhycG). Deletion of the three genes affected the integrity of hydrogenase-3. The hydrogen production experiments with the mutant strains showed that no hydrogen was detected compared with the wild type (0.886 mol/mol glucose), demonstrating that knocking out any of the three genes could inhibit NADH hydrogen production pathway. Meanwhile, the metabolites of the mutant strains were significantly changed in comparison with the wild type, indicating corresponding changes in metabolic flux by mutation. Additionally, the activity of NADH-mediated hydrogenase was found to be nil in the mutant strains. The chemostat experiments showed that the NADH/NAD+ ratio of the mutant strains increased nearly 1.4-fold compared with the wild type. The NADH-mediated hydrogenase activity and NADH/NAD+ ratio analysis both suggested that NADH pathway required the involvement of the electron transport chain of hydrogenase-3.