Study On The Mechanism Of Ethanol Metabolism Regulation In Clostridium Ljungdahlii Which Can Use Syngas

Due to widely used of oil and coal and the environmental pollution,researchers are looking for more efficient and cleaner energy sources to replace oil and coal.As new renewable resource,ethanol has advantages of high octane and reduced emissions.Syngas is a mixture mainly composed of hydrogen,carbon monoxide and carbon dioxide.Syngas comes from a wide range of sources.Due to its easy availability,syngas has become a potential sustainable energy source.Clostridium ljungdahlii can use syngas as carbon source and energy source to generate acetic acid and ethanol.However,in different fermentation conditions,the difference in ethanol production has made researchers realize that the bacteria's unique energy metabolism regulation mechanism plays a important role in biomass accumulation and ethanol production.Therefore,clarifying the energy metabolism regulation mechanism of the bacteria has become the first problem that we need to solve at present.First,we used the wild type,?adh E1 of knocked adh E1 and?adh E1+2 of knocked adh E1+2 were analyzed in shake flask fermentation experiments.The experimental results showed that the growth and metabolism of them were completely different using fructose,CO+CO₂(80:20,v/v)and CO₂+H₂(40:60,v/v)as carbon source respectively.The wild type can use all three carbon sources,?adh E1 and?adh E1+2 can only grow on the medium of fructose,and they can not grow on gas as carbon source,even unable to produce ethanol.So,adh E played an critical role in growth and metabolism during gas fermentation by C.ljungdahlii.Then using CO+CO₂or CO₂+H₂ as carbon source and energy source respectively,batch fermentation was conducted under the conditions of p H 6.0 and 0.1MPa,and the composition of culture medium as well as the technology of fermentation were adjusted.The results showed that growth of bacteria and the production of acid and alcohol were greatly different under the two conditions of carbon source.With CO+CO₂ as the carbon source and energy source,the biomass(optical density,OD_600nm)reached about 8.0,and the yield of acetic acid reached 12.4 g/L,ethanol32.1 g/L,and 2,3-butadiol 16.6 g/L.When CO₂+H₂ was used as carbon source and energy source,the biomass(OD_600nm)reached about 1.6,the content of acetic acid reached 31.4 g/L,ethanol 2 g/L,and 2,3-butanediol 0.1 g/L.The results showed that CO+CO₂ is a better carbon source and energy source than CO₂+H₂ in the batch fermentation process of C.ljungdahlii.Through RNA-Seq technology,the transcriptomics levels of different genes were compared under two different gas as energy source.The results showed that the transcriptomics level of CODH(CLJU?c09110)was greatly improved under CO+CO₂as carbon source and energy source,so CODH gene played a key role in the metabolic pathway of C.ljungdahlii in gas fermentation.Under two carbon sources,the key enzyme aldehyde ferredoxin oxidoreductase(aor,CLJU?c20120)in ethanol synthesis pathway had high transcriptomics level.But under the conditions of CO+CO₂ as the carbon source and energy source,the enzyme activity of AOR could reach 6.72 U/mg,while when CO₂+H₂ was used as the carbon source and energy source,the enzyme activity was 2.5 U/mg.It further illustrates that when CO+CO₂ are used as carbon sources and energy sources.Therefore,C.ljungdahlii use the AOR pathway to cope with the energy balance under different conditions when growing on syngas.Finally,knockout experiments showed that in the syngas fermentation process of C.ljungdahlii,NADH-dependent aldehyde alcohol dehydrogenase,is the necessary factor in energy metabolism balance.So we mapped the energy metabolism pathway of C.ljungdahlii,under the condition of different carbon sources.According to the results of fermentation,with CO+CO₂ as carbon source and energy source,the generated 1 mol acetic acid,1 mol 2,3-butanedioland 4 mol ethanol could produce 10mol ATP.When CO₂+H₂ was used as carbon sources and energy sources,0.75 ATP was generated by 1mol acetic acid.The analysis of the mechanism of acid and alcohol energy metabolism will provide theoretical support for the industrial application and molecular genetic manipulation of C.ljungdahlii.