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.