The Regulation Mechanism Of Nitrogenase And Cytochrome C During Methane Biosynthesis And Oxidation

Carbon is one of the first elements discovered and used by mankind,and the carbon cycle in ecological communities is driven by microorganisms.Nitrogen is also an important element of organisms,and the abundant nitrogen in the air can only be converted into a compound state before it can be used by organisms.In recent years,the promotion of carbon and nitrogen cycles in ecological communities by microorganisms is of great ecological significance.It has also been found that methane can be produced during the expression of iron-only nitrogenase.At the same time,methane anaerobic oxidizing microorganisms in ecological communities oxidize and utilize methane directly.The whole process involves carbon and nitrogen,which also shows that the biological nitrogen fixation process has an important position.Industrial synthetic ammonia requires high temperature and high pressure,requires particular equipment and consumes huge energy,while biological nitrogen fixation is green and environmentally-friendly and has many obvious advantages.The research fields of biological nitrogen fixation include biological nitrogen fixation mechanism,symbiotic nitrogen fixation mechanism,molecular genetics of nitrogen fixation and nitrogen fixation microorganisms.As early as 1942,the ¹⁵N experiment tracer confirmed that NH₃ was the product of biological nitrogen fixation,which opened the research on the mechanism of biological nitrogen fixation.At present,nitrogenase is divided into molybdenum nitrogenase and alternative nitrogenase（vanadium nitrogenase and iron nitrogenase）,and alternative nitrogenase is only found in organisms with molybdenum nitrogenase.It is speculated that the biosynthesis and assembly of its cofactors require molybdenum nitrogenase nif gene clusters,which means that alternative nitrogenases depend on the molybdenum nitrogenase to exist.In recent years,it has been discovered that compared with molybdenum nitrogenase and vanadium nitrogenase,iron nitrogenase can produce more hydrogen and can directly reduce CO₂ to methane.It also plays an untapped important role in the global carbon and nitrogen cycle and climate change.Nitrogenase is structurally assembled from Fe protein and MFe protein（M=Mo,V,Fe）,and these two parts are susceptible to oxygen.The two-component system is a basic regulatory system that responds to and adapts to changes in the environment or intracellular state,and plays a broad and relatively upstream regulatory role in the process of nitrogen fixation.Therefore,this project started with a two-component system,looking for a more direct regulatory relationship between the two-component system and the regulation of nitrogenase expression,and perfecting the regulatory pathway for the expression of iron nitrogenase.Methane（CH₄）is the second most important greenhouse gas that cannot be ignored.It has been proved that anaerobic oxidation of methane（AOM）as one of the important ways can control effectively the global methane emissions.Anaerobic oxidation of methane can use different electron acceptors(SO₄^2-,NO₂^-/NO₃^-,Fe³⁺,Mn⁴⁺,Cr⁵⁺,Mn O₄^-,etc.),but cytochrome c plays an important role in electron donor,so the heterologous expression of cytochrome c is of great significance for studying the electron transport mechanism of anaerobic oxidation of methane.In this project,we mined the genomic information of the uncultured archaea Candidatus Methanoperedens sp.to obtain a coding sequence with four heme-binding motifs（-CXXCH-）,which was expressed heterologously for providing the foundation of subsequent electrochemical analysis to analyze the electron transfer in the anaerobic oxidation mechanism of methane.This article draws the following conclusions:NtrC is not a direct regulator of PII protein（Gln K2）;The two-component system ntrXY may play a more important role in the regulation of nitrogenase expression;Fix LJ is the key regulator of nitrogenase reaction oxygen signal and it is in a relatively upstream position in the regulation of nitrogenase expression;The heterologous expression of cytochrome c requires an appropriate maturation system,and the expression level in Shewanella oneidensis MR-1 significantly exceeds the expression level in the expression host of E.coli.