Gene Heterologous Coexpression Of [FeFe]-Hydrogenase From Deep-sea Caloranaerobacter Azorensis H53214 And Hydrogen Production Analysis

At present,the rapid development of the economy h as led to the increase of carbon dioxide emissions,the soaring global temperature,and the harsh marine environment.Increased carbon emissions,melting glaciers,thermal expansion of seawater and acidification of the ocean pose a threat to human survival.It is imperative to reduce carbon emissions and the use of fossil fuels and find new non-polluting energy sources.Hydrogen energy is a clean,high quality energy ratio,high output value energy.Bio-hydrogen production has the advantages of wide source of raw materials,low energy consumption and low cost.In this study,three mature genes of a novel[FeFe]-hydrogenase(hydE,hydF,hydG)and its structural gene hyd A2 were co-expressed into K.oxytoca HP1 containing[NiFe]-hydrogenase,a new recombinant strain HP 1-Hyd was constructed.IPTG was used to induce the expression of four proteins.Under anaerobic conditions,mature proteins Hyd E,Hyd F and Hyd G could catalyze the activation of[FeFe]-hydrogenase Hyd A2 protein.The total amount of hydrogen produced by 1 mL fermentation with glucose increased by 26%under the co-catalysis of two hydrogenases.Compared with wild-type strain(118.3 mL/L/h),HPR increased to 145.8 mL/L/h and enzyme activity increased by 1.71 U while wild-type strain was 0.79 U.Studies on the physicochemical properties of hydrogenase showed that the activity of hydrogenase reached the highest level at 60? and pH 7.5,which was also consistent with the environment of K.oxytoca HP1 which was isolated from hot springs and deep-sea thermophile H53214 which provided[FeFe]-hydrogenase.K.oxytoca HP1 mainly produced hydrogen through formic acid pyrolysis during dark fermentation.The results showed that the hydrogen production efficiency of the recombinant strain was only 68.53 mL/L/h compared with that of K.oxytoca HP1,which was 86.35 mL/L/h.The metabolic pathway analysis showed that the content of 2,3-butanediol,ethanol and lactic acid decreased by 0.0201 mol/mol Glu,0.0414 mol/mol Glu and 0.0226 mol/mol Glu in the hydrogen-producing fermentation broth of the recombinant strain compared with the wild strain.Total NADH of metabolic branch decreased.It shows that the hydrogen production capacity of NADH was enhanced.