【作者】 王强；

【导师】 邵蔚蓝；

【作者基本信息】 江苏大学 ， 环境科学与工程， 2020， 博士

【摘要】 石油、煤炭等化石能源的生成需要经历上万年的时光,而近代工业的飞速发展使化石能源被迅速消耗、日渐枯竭,成为相对意义上的不可再生能源。而且,化石能源的燃烧会导致温室气体的排放,全球变暖进而导致气候变化、海平面上升、生物多样性丧失和城市污染等一系列环境问题。因此,为了缓解能源供求矛盾、改善人类生存环境,必须大力尝试可再生能源的开发与研究。地球上存在着种类繁多数量巨大的生物质,并且植物每天通过光合作用还会产生新的生物质。而以木质纤维素为原料生产燃料乙醇是最早提出的可再生能源发展策略,也是投入最大、研究最多的科学技术。纤维素乙醇常温发酵的工艺所面临的主要问题包括:用于乙醇发酵的工程菌缺少代谢木糖和降解纤维素的能力、生物质化学水解物产生生长抑制物以及低浓度乙醇蒸馏回收的高能耗等。而利用高温菌发酵,纤维素乙醇有望实现生物质降解、乙醇发酵、乙醇蒸馏过程的同步化,从而最大限度地降低纤维素乙醇的生产成本。但是,高温菌自然菌株降解生物质和发酵乙醇的能力都达不到工业生产的要求,目前还没有菌株同时具备较强的降解生物质和发酵乙醇的能力。因此,纤维素乙醇高温发酵的科学研究极具挑战性,可谓任重而道远。当前最新的研究和发展的目标是创建在更高的温度下有效降解木质纤维素并有效发酵乙醇的菌株。但是目前的国际研究认为,最适生长温度在80℃以上的超高温细菌和古菌中缺少从乙酰辅酶A生成乙醇的三酶途径。新阿波罗栖热袍菌(Thermotoga neapolitana)的生长温度高达90℃,是迄今发现的不多的超高温细菌之一,并且拥有完整的木质纤维素水解酶系统。为了利用微生物同时具有的超高温特性和强力降解木质纤维素的能力,本课题以超高温细菌T.neapolitana为研究对象,研究乙醇超高温发酵的三酶途径。本研究组在以往对其他菌株的代谢途径及调控机理的研究中发现,基因组测序和基因标注并不能完全体现醛/醇脱氢酶的催化活性和生理功能,更不能揭示它们在基因表达和生化反应水平上对乙醇代谢的调控机理。因此,对乙醇代谢途径关键酶及其表达调控进行研究,是针对超高温菌开展分子生物学机理和分子模块设计等合成生物学研究的前提。本论文的研究内容和研究结果主要包括以下五个方面:(1)运用基因工程技术研究T.neapolitana来源的醇脱氢酶在大肠杆菌BL21(DE3)中的高效表达。新阿波罗栖热袍菌中4个醇脱氢酶的基因得到成功克隆,对基因进行测序,证实其未发生突变。所有表达质粒均转化入大肠杆菌,并通过热激诱导的方式,使得目的基因在重组细胞中高效活性表达。(2)重组乙醇脱氢酶的表达纯化和酶学性质的测定。在生化条件下测定和分析4种乙醇脱氢酶的酶学性质,结果表明:Tne-CTN0580、Tne-CTN1655和Tne-CTN1756不仅表现出乙醛还原成乙醇(乙醇脱氢酶正反应)的活性,而且还具有乙醇氧化成乙醛(乙醇脱氢酶逆反应)的活性;而Tne-CTN0257只有乙醇氧化成乙醛的活性;同时发现,Tne-CTN0580还具有催化乙酰辅酶A生成乙醛(乙醛脱氢酶正反应)的作用和乙醛反向生成乙酰辅酶A(乙醛脱氢酶逆反应)的活性。(3)T.neapolitana细胞内微环境的测定和重组醇脱氢酶生理功能的解析。在大体积高温厌氧的流动培养液中,用透析袋对细胞进行小区域限制性培养,获得接近化学静态条件下生长的细胞,以测定胞内生理条件。在模拟的生理条件下,对4种酶的生理功能进行鉴定表明:Tne-CTN0580和Tne-CTN1756表现出乙醛还原成乙醇的功能;Tne-CTN0257有乙醇氧化成乙醛的功能;Tne-CTN0580具有催化乙酰辅酶A生成乙醛的功能;4种酶都不具有催化乙醛生成乙酰辅酶A的功能。(4)双活性醛/醇脱氢酶的生物信息学分析。重组Tne-CTN0580的开放阅读框序列长度为1164bp,可以编码387个氨基酸。重组Tne-CTN0580含有3个半胱氨酸,预测可以形成一个二硫键。蛋白分子量是42.61k Da,等电点PI为5.47。通过分析氨基酸的疏水性,表明该酶是一个相对稳定的亲水性蛋白。NCBI在线blast分析发现,除了超嗜热神袍菌属来源的醇脱氢酶之外,Tne-CTN0580与Thermococcus kodakarensis、Pyrococcus furiosus、Pseudothermotoga thermarum、Kosmotoga spp.以及Pseudothermotoga lettingae来源的醇脱氢酶序列相似性最高,达到90%以上。将Tne-CTN0580与非超高温菌中的醛/醇双活性酶Adh E、Adh B进行同源序列比对,发现,Adh E是一个分子量较大的蛋白,含有醛脱氢酶和醇脱氢酶两个结构域,而Tne-CTN0580和Adh B的双功能酶只含单个醇脱氢酶结构域,并且Tne-CTN0580和Adh B分别属于含铁和含锌醇脱氢酶的两个不同的家族。结果表明,这些双功能酶采用不同的催化机制来进行三酶途径的乙醇发酵。(5)双活性醛/醇脱氢酶体外模拟产乙醇的研究。为了进一步验证Tne-CTN0580具有双活性醛/醇脱氢酶的功能,分别测定了Tne-CTN0580与其他三种醇脱氢酶耦合作用下,催化乙酰辅酶A生成乙醇的情况。研究结果证实Tne-CTN0580可以催化乙酰辅酶A生成乙醇,是一种双功能酶。同时发现Tne-CTN0257和Tne-CTN1756对双活性醛/醇脱氢酶生成乙醇有明显的协同作用。本研究论文的主要创新点在于:首次鉴定了T.neapolitana基因组中标注为醇脱氢酶基因所编码的4种蛋白的酶学性质和生理功能,鉴定了它们在模拟胞内环境下的醛/醇底物特异性以及在乙醇生成或消耗中的作用;探明了T.neapolitana中存在催化乙酰辅酶A产生乙醛的醛脱氢酶,证明超高温细菌中存在乙醇发酵的三酶途径。研究结果为开发新型的超高温纤维素乙醇发酵基因工程菌株奠定了理论基础,同时也为实现木质纤维素类固体废弃物高效制备乙醇提供了科学依据。更多还原

【Abstract】 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.更多还原