Study On The Relationship Between Complex?and Hydrogen Metabolism Of Enterobacter Aerogenes

Background:Enterobacter aerogenes is a facultative anaerobe and is one of the most widely studied bacterial strains because of its ability to use a variety of substrates,to produce hydrogen at a high rate,and its high growth rate during dark fermentation.However,the rate of hydrogen production has not been optimized.In this present study,three strategies to improve hydrogen production in E.aerogenes,namely the disruption of nuoCDE,overexpression of the small RNA RyhB and of NadE to regulate global anaerobic metabolism,and the redistribution of metabolic flux.The goal was to clarify the effect of nuoCDE,RyhB,and NadE on hydrogen production and how the perturbation of NADH influences the yield of hydrogen gas from E.aerogenes.Results:NADH dehydrogenase activity was impaired by knocking out nuoCD or nuoCDE in E.aerogenes IAM1183 using the CRISPR-Cas9 system to explore the consequent effect on hydrogen production.The hydrogen yield from IAM1183-CD??nuoC/?nuoD?and IAM1183-CDE??nuoC/?nuoD/?nuo E?increased by 24.5%and 45.6%in batch culture?100mL serum bottles?.The hydrogen produced via the NADH pathway increased significantly in IAM1183-CDE,suggesting that nuoE plays an important role in regulating NADH concentration in E.aerogenes.Batch cultivating experiments showed that by the overexpression of NadE?N?,the hydrogen yield of IAM1183/N,IAM1183-CD/N,and IAM1183-CDE/N increased1.06-,1.35-,and 1.55-fold,respectively,compared with IAM1183.Particularly worth mentioning is the strain IAM118-CDE/N reached 2.28 mol in H₂ yield,per mole of glucose consumed.IAN1183/R,IAM1183-CD/R,and IAM1183-CDE/R showed increasing H₂ yields in batch culture.Metabolic flux analysis indicated that increased expression of RyhB led to a significant shift in metabolic patterns.We further investigated IAM1183-CDE/N,which had the best hydrogen-producing traits,as a potential candidate for industry applications using a 5-L fermenter;hydrogen production was up to 1.95 times greater than that measured for IAM1183.Conclusions:Knockout of nuoCD or nuoCDE and the overexpression of nadE in E.aerogenes resulted in a redistribution of metabolic flux and improved hydrogen yield.Overexpression of RyhB had an significant change on the hydrogen production via NADH pathway.A combination of strategies would be a novel approach for developing a more economical and efficient bioprocess for hydrogen production in E.aerogenes.Finally,the latest CRISPR-Cas9 technology was successful for editing genes in E.aerogenes to develop our engineered strain for hydrogen production.