Purification And Biochemical Properties Of Key Enzymes MTHFR And AOR For Syngas Fermentation In Clostridium Ljungdahlii

With the development of industrialization,the demand for energy and industrial chemicals is increasing.Syngas is a kind of cheap gas mainly composed of CO,CO2 and H2.It is generally produced by the gasification of oil and natural gas.It can also be gained from industrial production or the gasification of biomass and urban waste.They are usually directly discharged into the atmosphere or burned.Recently,the technology of using anaerobic autotrophic acetogens to ferment syngas or waste gas rich in CO to produce biofuels and chemicals has attracted people's attention.This kind of anaerobes can grow with CO2 or CO as the sole carbon source and synthesize acetyl CoA by fixing CO2 and co through wood ljungdahl pathway(WLP),then produce acetate,ethanol,2,3-butanediol and other compounds.Compared with chemical conversion of syngas,microbial fermentation has many advantages,for example,bacteria have strong tolerance to pollutants in the gas source;bacteria have no specific requirements for CO/H2 content ratio,and the metabolic pathway of bacteria can be designed through gene operation to diversify the fermentation products.Although the fermentation is limited by the transfer of gas-liquid substances and requires anaerobic conditions,its advantages have brought a broad application prospect for bacterial fermentation of syngas and become a highly competitive technology.Using acetogens to ferment syngas can not only reduce pollution,but also transform wastes into valuable resources.In this thesis,the physiological and biochemical characteristics of methylenetetrahydro folate reductase(MTHFR)and aldehyde:ferredoxin oxidoreductase(AOR),which are the key enzymes in the metabolic pathway of Clostridium ljungdahli syngas fermentation,were studied,and the energy metabolism of C.ljungdahli growth on syngas was also discussed.The details are as follows:MTHFR is a key enzyme in syngas fermentation metabolic pathway in C.ljungdahli.A large number of C.ljungdahli cells were successfully cultured with CO as the sole carbon source.In order to further determine the MTHFR coding gene,the protein was purified from the wild-type bacterial cells under strict anaerobic conditions.Firstly,the activity of the artificial electron acceptor benzyl viologen(BV)reduction with methyltetrahydrofolate was measured for the cell extracts,and then the MTHFR protein was successfully obtained through four-step-purification:ammonium sulfate precipitation,Phenyl Sepharose,Q-Sepharose and DEAE Sepharose.The results showed that the MTHFR of C.ljungdahli was composed of two subunits:MetF andMetV.By this method,the enzyme was purified by 44 folds and the average specific activity was 30.7 U/mg.In order to study the electronic donors of MTHFR under the physiological conditions,NAD(P)H,dihydrolipoamide reductase(DLDH),thioredoxin reduction system and ferredoxin(Fd)were tested in the thesis.It was confirmed that Fdred2-was the electron donor of MTHFR under the physiological conditions,and its specific enzyme activity reached 7.4 U/mg.It was found that the brownish yellow MTHFR bound two[4Fe4S]clusters,which was consistent with the bioinformatics analysis.A large number of MTHFR proteins were also obtained by heterologous expression of MTHFR,which provided a possibility for protein crystalization and structure resolution.In addition,based on the number of subunits and phylogenetic analysis,the MTHFR from acetogens can be divided into three types,which provides a new insight for the classification of anaerobic microorganisms.Moreover,an recombinant E.coli was successfully constructed for highly-efficient expression and overproduction of anaerobic 2[4Fe4S]type Fd,its yield is much higher than that of the same type of Fd in other wild bacteria and heterologous expression strains,which provides a new help for the study of acid and alcohol metabolism of anaerobic acetogen.AOR is the key enzyme of acid-alcohol transformation in C.ljungdahli.Under physiological conditions,it uses Fdred2-to reduce acetate to acetaldehyde,and then aldehyde dehydrogenase AdhE is used to generate ethanol.In C.ljungdahli,AOR has two isoenzymes AOR1 and AOR2.Their genes have been knocked out,respectively,and the consequences suggest that the two enzymes may perform different functions according to the literature.However,there is no evidence for the prediction at the protein level.In this thesis,the AOR2 of C.ljungdahli was purified from wild-type bacteria under strict anaerobic conditions.After four steps of ammonium sulfate precipitation,Phenyl Sepharose,Q-Sepharose and DEAE Sepharose,a red brown protein was finally obtained.By this method,the enzyme was purified by 77 folds,and the average specific activity was 46 U/mg(Fd reduction with aldehyde).It was proved that AOR2 was composed of a single subunit of 67 kDa and its molecular weight was determined by gel filtration,which showed that AOR2 is a dimer protein.The result of UV-visible absorption spectra and iron content determination demonstrated that AOR2 binds a[4Fe4S]cluster,which is consistent with bioinformatics analysis.The thesis confirms that the physiological electron acceptor of AOR2 in C.ljungdahlii is Fd and the physiological electron donor is acetaldehyde.In addition,AOR1 was also partially purified and characterized,and its activity was also determined,which are helpful forfurther purification and characterization of the enzyme.This research provides the necessary theoretical basis for the syngas fermentation of the clostridia,and for the metabolic engineering of the strain.In thesis,the specific activity of oxidoreductases in crude extracts of C.ljungdahlii grown on CO as the sole carbon source was determined,and the coding gene composition and their transcription level were analyzed,from which the electron donors under physiological conditions of most oxidoreductases were predicted.Based on the enzyme essays and the predictions,the energy metabolism model of C.ljungdahlii grown on syngas(H2 and CO2,CO)was discussed,and new ideas on the complex energy metabolism mechanism were put forward.In conclusion,C.ljungdahlii grown on CO as the sole carbon source and energy source were successfully obtained by anaerobic culture and syngas fermentation.The MTHFR protein was successfully purified and characterized.It was found that the MTHFR consists of only two subunits and catalyzes a new type of physiological reaction.The electron donor under physiological conditions was Fdred 2-.And AOR,the key protein of acid-alcohol transformation,was also purified by C.ljungdahlii.It was found that the electron acceptor of AOR under physiological conditions was Fdox,and its two isoenzymes may have different physiological functions.Through the research of key enzymes,the energy flow direction of the clostridia was analyzed,the energy metabolism mechanism was discussed.A new energy metabolism model of syngas fermentation was proposed,which provided a theoretical basis for the industrialization of alcohol production by syngas fermentation.