Regulation Of NADH Supply By Deletion Of NuoE Subunit Of Complex ? And Its Effect On Hydrogen Production In Enterobacter Aerogenes

Hydrogen(H₂)is not only a commonly used chemical raw material,but also a clean and renewable energy carrier with high energy density.It is widely used and has attracted great attention.Biological hydrogen production can be divided into fermentation and photosynthetic.Among them,dark fermentation is hydrogen production by microorganisms using carbohydrates or organic wastewater rich in sugars as substrates.Dark fermentation has many advantages including mild reaction conditions,low cost and no requirement of complex equipment and it is highly valued by people.In this study,Enterobacter aerogenes with outstanding potential for hydrogen production,was used as the starting strain to knock out the nuoE subunit of Complex?with which competes for hydrogen production to regulate NADN supply and explore the hydrogen production capacity of the strain.What's more,pncB,encoding the key enzyme transphosphoribokinase(NAPRTase)in NAD(H)synthesis system,was also overexpressed to further redistribute carbon flux.This study will lay a theoretical foundation for further improving the hydrogen production capacity of E.aerogenes and provide excellent engineering strains for hydrogen production.The main research contents and results are as following:1.nuoE gene was knocked out in IAM1183 and IAM1183-L(?ldh A)respectively by?-Red homologous recombination technology.The constructed mutants were designated as IAM1183-E(?nuoE)and IAM1183-EL(?nuoE/?ldh A).After 20 h anaerobic flask fermentation,hydrogen yield of IAM1183-E,IAM1183-L and IAM1183-EL has increased by 23.3,81.7 and 97.9%compared with the wild type strain.What's more,hydrogen yield of IAM1183-E and IAM1183-EL was 23 and 9%higher than that of IAM1183 and IAM1183-L respectively.In addition,NADH/NAD⁺was also improved with nuo E deletion and hydrogen yield form NADH pathway was further increased.While significant change in hydrogen yield from formate pathway was not observed.2.The constructed plasmid pET-28a-pncB was electroporated into IAM1183 and its mutants respectively and IAM1183/P,IAM1183-E/P,IAM1183-L/P and IAM1183-EL/P were obtained.Hydrogen production of the four strains was increased by 39.0,6.5,5.9and 5.1%respectively with pnc B overexpression.IAM1183-EL/P has the highest hydrogen production and its titer was 2.35 mol/mol glucose,which is 2.08 times that of the wild-type strain.The ratio of NADH/NAD⁺in the cell were also decreased significantly with pncB overexpression.The hydrogen production of formate pathway in IAM1183-L/P was significantly increased,and the hydrogen production of the NADH pathway in other three strains were also increased.Much more NADH was utilized by NADH dehydrogenase with nuo E deletion thus facilitating hydrogen biosynthesis of NADH pathway.The interaction of ldh A impairment and overexpression of pnc B blocking the lactate synthesis pathway.3.The strain IAM1183-EL/P with outstanding hydrogen production potential was selected to scaled up fermentation in a 5 L fermentor to test its capacity for industrial application.With 45 g glucose as the substrate,the hydrogen production was completed within 16 h,and the yield was 6.18 L,which was 1.58 times that of the wild-type strain.After 44 h fermentation,the production of hydrogen,acetic acid,ethanol and 2,3butanediol by IAM1183-EL/P were significantly improved compared with IAM1183,but production of acetoin was greatly reduced.And lactate production was not observed.