| With the development of global warming and the huge increase in fossil fuel consumption,hydrogen has attracted a lot of attention as a renewable,zero-pollution and efficient clean energy in today’s society.Among different hydrogen production methods,biological hydrogen production has become the focus of research on hydrogen production in the energy industry due to its lowest production cost,abundant raw material sources,and low environmental pollution.In the development of biological hydrogen production,catalase is a particularly important link.Catalase is a redox enzyme containing metal centers.It exists widely in nature.The characteristic of catalase is to reversibly catalyze the oxidation and reduction of h2(2H++2e-?h2).Studies have investigated that the homologous expression of EcHydA protein of Enterobacter cloacae IIT-BT 08 can improve the hydrogen production in fermentation of Klesiella oxytoca HP1 which is efficient hydrogen production characteristics,rapid growth,and aerobic hydrogen production characteristics,which has a good development prospect in the dark fermentation hydrogen production industry.The overall goal of this study is to further enhance the fermentation hydrogen production of strain k.oxytoca HP1 by transferring the[Fe-Fe]hydrogenase gene EchydA from E.cloacae IIT-BT 08 into K.oxytoca HP1.Through RT-qPCR study,it was found that the expression of EcHydA in the recombinant strain reduced the expression of[Ni-Fe]hydrogenase gene.Further studies showed that the hydrogen-producing activity of the recombinant strain was decreased by the formic acid pathway(the hydrogen-producing pathway of[Ni-Fe]hydrogenase).This indicated that exogenous EcHydA in the recombinant strain enhanced the overall hydrogen production activity of the recombinant strain through NADH pathway rather than formic acid pathway.The amount of products dependent on NADH or NADPH in fermentation products was analyzed at the metabolomic level.Further validate the NADH pathway of EcHydA.This study confirmed that EcHydA is a complete hydrogenase that promotes hydrogen production by k.oxytoca HP1 through the NADH pathway.At the same time,the results of this experiment also indicate that EcHydA,as a kind of hydrogen enzyme,can be expressed and matured in K.oxytoca HP1 with catalytic activity.Through structural comparison,it was found that compared with the traditional[Fe-Fe]hydrogenase,the EcHydA from E.cloacae IIT-BT 08 lacks the[Fe-S]cluster that transmits electrons.In order to study the effect of[Fe-S]cluster on the catalytic activity of[Fe-Fe]hydrogenase,a conservative[Fe-S]cluster was added to the Nterminal of EcHydA protein in this study,so that the structure of EcHydA was similar to that of traditional[Fe-Fe]hydrogenase.The results showed that compared with EcHydA,the[Fe-S]-HydA protein had no significant change in the hydrogenase activity and its influence on the hydrogen-producing activity of recombinant k.oxytoca HP1,indicating that the[Fe-S]cluster had little influence on the catalytic activity of EcHydA,and the heterogenic expression of EcHydA could play a strong role in promoting the hydrogen production of k oxytoca HP1.The results of this study confirmed that EcHydA heterologous expression can promote hydrogen production by K.oxytoca HP1 through NADH pathway,which provides a new idea for improving the hydrogen production efficiency of K.oxytoca HP1 and further industrial application. |
