| The generation of fossil energy such as oil and coal takes tens of thousands of years,while the rapid development of modern industry had made the fossil energy rapidly consumed and gradually exhausted,making it a non-renewable energy in a relative sense.Moreover,the burning of fossil energy will lead to greenhouse gas emissions,and global warming will lead to climate change,sea level rise,loss of biodiversity,urban pollution and a series of environmental problems.Therefore,in order to alleviate the contradiction between energy supply and demand and improve the living environment of human beings,we must try to develop and research the renewable energy.There are many species of biomass on earth in huge quantities,and plants produce new biomass every day through photosynthesis.The production of fuel ethanol from lignocellulosic materials was the first proposed renewable energy development strategy,and the most invested and researched science and technology.The main problems in the normal temperature fermentation of cellulosic ethanol included: the lack of the ability of the engineering bacteria used in ethanol fermentation to metabolize xylose and degrade cellulose,the presence of growth inhibitors in the biomass chemical hydrolysates,and the high energy consumption in the distillation of low concentration ethanol.By using thermophilic bacteria fermentation,cellulosic ethanol was expected to realize the synchronization of biomass degradation,ethanol fermentation and ethanol distillation,thus minimizing the production cost of cellulosic ethanol.However,the degradation of biomass and the ability of fermentation ethanol by the natural strains of thermophilic bacteria could not meet the requirements of industrial production.At present,no strain had strong ability to degrade biomass and ferment ethanol at the same time.Therefore,the scientific research of thermophilic fermentation of cellulosic ethanol was very challenging and there was a long way to go.The goal of current research and development is to create strains that degrade lignocellulose effectively and ferment ethanol efficiently at higher temperatures.However,current international studies suggest that there is no trienzyme pathway to produce ethanol from acetyl-Co A in hyperthermophilic bacteria and archaea with optimal growth temperature above 80?.The growth temperature of T.neapolitana is as high as 90?.It is one of the few hyperthermophilic bacteria found so far,and it has a complete lignocellulosic hydrolase system.In order to take advantage of both the hyperthermophilic characteristics and the strong ability of microorganisms to degrade lignocellulose,this project took the hyperthermophilic bacteria T.neapolitana as the research object to study the trienzyme pathway of ethanol hyperthermophilic fermentation.From our previous studies on metabolic pathways and regulatory mechanisms of other strains,it was found that genome sequencing and gene labeling could not reflect the catalytic activity and physiological function of aldehyde/alcohol dehydrogenase.Moreover,it could not reveal their role for regulation of ethanol metabolism on the level of gene expression and biochemical reaction.Therefore,the study on the key enzymes and expression regulation of ethanol metabolism pathway is the prerequisite for the research on molecular biological mechanism and molecular module design of hyperthermophilic bacteria.The research contents and results of this paper mainly include the following five aspects:(1)Study of the expression of alcohol dehydrogenase from T.neapolitana in E.coli BL21(DE3)with the help of genetic engineering techniques.Four genes labeled alcohol dehydrogenase from the T.neapolitana were successfully cloned.The target genes in the expression plasmids were sequenced and it was confirmed that no mutation had occurred in any of them.All the expression plasmids were transformed into E.coli and the target genes were efficiently expressed in recombinant cells through heat shock induction.(2)Purification and characterization of recombinant alcohol dehydrogenase.The enzymatic properties of four alcohol dehydrogenases were determined and analyzed under biochemical conditions,the results showed that Tne-CTN0580,Tne-CTN1655 and Tne-CTN1756 not only showed the activity of acetaldehyde reduction to ethanol(alcohol dehydrogenase forward reaction),but also the activity of ethanol oxidation to acetaldehyde(alcohol dehydrogenase reverse reaction).While Tne-CTN0257 only had the activity of ethanol oxidation to acetaldehyde.At the same time found that Tne-CTN0580 also had the activity of catalyzing acetyl-Co A to generate acetaldehyde(acetaldehyde dehydrogenase forward reaction),and acetaldehyde to acetyl-Co A(acetaldehyde dehydrogenase reverse reaction).(3)Determination of intracellular microenvironment of T.neapolitana and analysis of physiological functions of recombinant alcohol dehydrogenase.In a large volume of thermophilic anaerobic flow culture medium,dialysis bag was used to culture cells in a small restricted area,to obtain cells growing under nearly chemical static conditions,so as to determine the intracellular physiological microenvironmental conditions.Under simulated physiological conditions,the physiological functions of four enzymes were identified,and the results showed that Tne-CTN0580 and Tne-CTN1756 had the function of acetaldehyde reduction to ethanol.Tne-CTN0257 had the activity of ethanol oxidation to acetaldehyde.Tne-CTN0580 could catalyze the formation of acetaldehyde by acetyl-Co A.None had the catalytic activity of acetaldehyde to form acetyl-Co A.(4)Bioinformatics analysis of the bifunctional aldehyde/alcohol dehydrogenase.The sequence length of the open reading frame of the recombinant Tne-CTN0580 was1164 bp,and it could encode 387 amino acids.Recombinant Tne-CTN0580 contained 3cysteines which could form a disulfide bond.The molecular weight of the protein was42.61 k Da,and the isoelectric point PI was 5.47.The hydrophobicity analysis of amino acids indicated that the enzyme was a stable hydrophilic protein.NCBI online blast analysis found that,except for the alcohol dehydrogenase from Thermotoga spp.,the Tne-CTN0580 sequence had the highest similarity with the alcohol dehydrogenase sequence from Thermococcus kodakarensis,Pyrococcus furiosus,Pseudothermotoga thermarum,Kosmotoga spp.and Pseudothermotoga lettingae,reaching more than 90%.The amino acid sequences were also compared between Tne-CTN0580 and the other bifunctional aldehyde/alcohol dehydrogenases,Adh E an Adh B.The results revealed that Adh E was a large protein with two domains of alcohol dehydrogenase and aldehyde dehydrogenase,and Tne-CTN0580 and Adh B bifunctional enzyme contained only a single alcohol dehydrogenase domain,and Tne-CTN0580 and Adh B belonged to the iron-and zinc-containing alcohol dehydrogenase families,respectively.These results indicated that these bifunctional enzymes adopted different catalytic mechanisms to conduct ethanol fermentation by the trienzyme pathway.(5)Study on stimulated ethanol production by the bifunctional aldehyde/alcohol dehydrogenase.In order to further confirm bifunctional nature of Tne-CTN0580 as aldehyde/alcohol dehydrogenase,the production of ethanol directly catalyzed from acetyl-Co A in the presence of Tne-CTN0580 alone and three other alcohol dehydrogenases were determined.The results of the study confirmed that Tne-CTN0580 was a bifunctional aldehyde/alcohol dehydrogenase and could directly catalyze acetyl-Co A to produce ethanol.Tne-CTN0257 and Tne-CTN1756 had obvious synergistic effect on the production of ethanol by bifunctional aldehyde/alcohol dehydrogenase.The main innovations of this paper were as follows: the enzymatic properties and physiological functions of the four proteins encoded by the alcohol dehydrogenase gene in the genome of T.neapolitana were clarified for the first time,and their intracellular aldehydes,alcohol substrate specificity and roles in the generation or consumption of ethanol were identified.The existence of aldehyde dehydrogenase catalyzing acetyl-Co A to produce acetaldehyde in T.neapolitana was investigated,and the existence of the trienzyme pathway of ethanol fermentation in hyperthermophilic bacteria was confirmed.The research results lay a theoretical foundation for the development of new hyperthermophilic cellulosic ethanol fermentation genetic engineering strains,and also provide a scientific basis for the realization of efficient production of ethanol from lignocellulosic solid wastes. |
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