AP-3复合体在粗糙脉孢菌纤维素酶分泌途径中的功能研究与酶系优化

发布时间：2018-03-25 16:28

  本文选题：粗糙脉孢菌　切入点：分泌途径　出处：《吉林大学》2016年博士论文

【摘要】：植物的木质纤维素是世界上分布最广的可再生资源,目前对于利用降解木质纤维素得到的产物生产生物燃料成为研究的热点。但是由于新兴生物燃料的高成本,使得其与传统石化燃料在应用推广上相比不具有竞争力。在生产生物燃料的过程中,降解木质纤维素所需的纤维素酶的费用在生产成本中所占比重很大。提高纤维素酶产量,构建纤维素酶高产菌株是解决这一问题的主要技术手段。目前工业生产上已经成功构建许多的高产纤维素酶生产菌株,如纤维素酶生产的明星菌株Trichoderma reesei RUT-C30。通过比较基因组学研究发现与出发菌株T.reesei strain QM6a相比RUT-C30菌株中多处发生突变,但是该菌株在遗传操作方面存在一定的困难,所以目前对其高产机制尚不明确。而Neurospora crassa有着与T.reesei有着非常近的亲缘关系,还具有非常简便的遗传操作手段,近年来已逐渐成为研究产酶机理的理想菌株。所以,为研究RUT-C30菌株的高产机制,以N.crassa为研究对象,针对RUT-C30菌株中突变基因,筛选粗糙脉孢菌中的单基因敲除突变体,挖掘这些基因的功能,为改造高产菌株、研究产酶机理提供新的研究方向。木质纤维素的水解需要三种酶的协同作用来完成,而高效的内切纤维素酶与β-葡萄糖苷酶可促使纤维素水解效率得到显著的提高,降低降解木质纤维素的费用,进而促进生物燃料的推广应用。因此在本研究中,分别克隆表达了一个内切纤维素酶AgCel5A和一个β-葡萄糖苷酶NcBGL2,并对其性质进行了分析。以期获得高效的纤维素酶,用来提高纤维素的水解效率,进而达到降低成纤维素酶成本的目的。传统的纤维素酶是由内切纤维素酶、外切纤维素酶和β-葡萄糖苷酶三种组份组成的。但是有没有存在另一种组份,可以使现有的传统纤维素酶系的水解活性得到进一步的提升呢?AA9家族的溶解性多糖单加氧酶就具有这种功能。溶解性多糖单加氧酶(Lytic polysaccharide monooxygenases)简称LPMOs,即原来的GH61蛋白,可以通过与CDH(Cellobiose dehydrogenases)或者一些低分子量的还原剂(如抗坏血酸)发生氧化还原反应,裂解纤维素链上的糖苷键,破坏其结构,从ii而使纤维素更容易被纤维素酶降解,提高传统纤维素酶的水解效率。嗜热毁丝霉(myceliophthorathermophila)是一株嗜热真菌,具有很强的纤维素降解能力,目前已经从该菌株中克隆得到了许多的纤维素酶基因。在本研究中,克隆了两个在m.thermophila表达量最高的lpmo基因,并对其性质进行了分析,以期达到提高原有纤维素酶系水解活性,促进纤维素水解,降低纤维素酶成本,从而促进生物燃料的可持续发展的目的。本研究的主要研究结果如下:(1)利用n.crassa基因敲除突变体库对t.reeseirut-c30中的突变基因进行表型验证,成功筛选得到12株胞外蛋白分泌升高25%以上的突变菌株,4株分泌下降25%以上的菌株;明确编码ap-3复合体μ亚基的ncap3m(ncu03998)和编码β亚基的ncap3b(ncu06569)的缺失可显著提高胞外蛋白的分泌;ncap3m和ncap3b的缺失还会导致n.crassa菌株的分泌应激引起的转录抑制(repressedexpressionofsecretedsequences,ress)现象减弱;最后证明ap-3复合体与纤维素酶分泌途径相关,ap-3复合体功能缺失后会导致纤维素酶的分泌提高。(2)成功克隆得到了一个高效的内切纤维素酶agcel5a和一个β-葡萄糖苷酶ncbgl2,二者在pichapastori中异源表达后,发酵液中均为单一条带;agcel5a最适反应条件为ph5.0,55°c,具有良好的热稳定性和ph稳定性,同时还具有极强的耐盐性,而且co2+可有效促进酶活提高;ncbgl2的最适反应条件为ph5.6,60°c,10%的乙醇可提高80%酶活,还具有非常强的水解大豆异黄酮糖苷活性。(3)水解纤维素的过程中,将mtlpmo9a与mtlpmo9b添加到原有纤维素酶系中时,均可以使最终酶用量减少40%;在n.crassa菌株中表达这两个lpmo蛋白可直接实现纤维素酶系优化,但需保证lpmo蛋白的分泌量。综上所述,通过本文的研究,获得了一个高效的内切纤维素酶agcel5a和一个β-葡萄糖苷酶ncbgl2,而性质优良的内切纤维素酶和β-葡萄糖苷酶在生物炼制特别是生物燃料的可持续发展上具有重要意义;还获得了两个可以有效的提高传统纤维素酶的水解活性lpmo蛋白,降低纤维素酶的用量,节约纤维素酶成本,同样在促进生物燃料的推广应用上具有着重大意义;最后证明了可以利用模式真菌n.crassa,开展对t.reeseirut-c30的突变基因进行功能研究,以明确t.reeseirut-c30高产机理。并确定ap-3复合体与纤维素酶分泌途径相关。该研究结果为纤维素酶分泌方面的研究提供一个新的方向,也为今后高分泌菌株改良提出一个新策略。
[Abstract]:Plant cellulose is the most widely distributed renewable resource in the world, the product of biofuel production using lignocellulose degradation has become a hot research topic. But due to the high cost of new biofuels, with the traditional fossil fuel in the application does not have competitiveness. In the process of production of biofuels, cellulase lignocellulose degradation required for the cost in production accounts for a large proportion. To improve the yield of cellulase, Cellulase Producing Strain construction is the main technical means to solve this problem. The industrial production has been successfully constructed High Cellulase Producing Strains of many, such as Cellulase Producing Strain Trichoderma reesei RUT-C30. star through comparative genomics research found strain QM6a compared with the starting strain T.reesei strain RUT-C30 in multiple inrush Change, but this strain has certain difficulty in the genetic operation, so the yield mechanism is not clear. Neurospora crassa has a very close relationship with T.reesei, also has the genetic manipulation is very simple, in recent years has gradually become the ideal strains for enzymatic production mechanism. So, for study on the mechanism of high yield strain RUT-C30, with N.crassa as the research object, according to the RUT-C30 gene mutation strains, screening of single gene of Neurospora crassa in the knockout mutant, mining the function of these genes, for the transformation of high yield strain, the research of enzyme production mechanism provide new research directions. Synergistic hydrolysis of lignocellulose requires three the enzyme to complete, and endo cellulase and beta glucosidase can promote efficient cellulose hydrolysis efficiency was improved, reducing the cost of lignocellulose degradation, and promote biological Application of fuel. In this research, respectively, cloning and expression of a endoglucanase AgCel5A and a beta glucosidase NcBGL2, and its properties were analyzed. In order to obtain efficient cellulase to improve the hydrolysis efficiency of cellulose, and then reach down into low cost. The traditional cellulase cellulase is by endo cellulase, cellobiohydrolase and p-glucosidase three components. But is there another component, hydrolytic activity can make the existing traditional cellulase system has been further improved? The solubility of AA9 family polysaccharide monooxygenase has the function. Soluble polysaccharide monooxygenase (Lytic polysaccharide monooxygenases) LPMOs, originally called GH61 protein, with CDH (Cellobiose dehydrogenases) reducing agent or some low molecular weight ( Such as ascorbic acid) redox reaction, the glycosidic bond cleavage of cellulose chains, destroy the structure, from the II and the cellulose is easily hydrolyzed degradation, improve the hydrolysis efficiency of cellulase. The traditional Myceliophthora thermophila (myceliophthorathermophila) is a thermophilic fungus, with cellulose degradation ability is very strong, from now this strain was cloned many cellulase genes. In this study, cloned two lpmo gene expression was the highest in m.thermophila, and its properties were analyzed, in order to improve the cellulase hydrolysis activity, promote the hydrolysis of cellulose, reduce the cost of cellulase, so as to promote the sustainable development of biofuels to. The main results of this study are as follows: (1) knockout mutants phenotype verification of mutations in t.reeseirut-c30 genes by N.crassa gene into. The power obtained 12 strains of extracellular protein secretion increased more than 25% of the mutant strains, 4 strains of secretion decreased more than 25%; clear encoding subunits of the AP-3 complex with ncap3m (ncu03998) and encoding the beta subunit of ncap3b (ncu06569) deficiency can significantly increase the secretion of extracellular proteins; ncap3m and ncap3b will be missing the secretion of stress leads to transcriptional suppression caused by N.crassa strains (repressedexpressionofsecretedsequences, RESS) the phenomenon of decline; the last proof of AP-3 complex and AP-3 complex cellulase secretion pathway, loss of function will lead to increased secretion of cellulose enzyme. (2) successfully cloned an efficient endoglucanase agcel5a and a beta glucosidase the enzyme ncbgl2, two heterologous expression in pichapastori after fermentation broth showed a single band of agcel5a; the optimum reaction conditions for ph5.0,55 ~ C, has good thermal stability and pH At the same time also has strong stability, salt tolerance, and co2+ can effectively promote the activity of ncbgl2; the optimum reaction conditions for ph5.6,60 ~ C, 10% ethanol can improve 80% enzyme activity, also has the very strong hydrolysis of soybean isoflavone glucoside activity. (3) the process of cellulose hydrolysis, add mtlpmo9a with mtlpmo9b to the original in the cellulase system, can make the final enzyme dosage reduced 40%; the two lpmo protein can directly realize the cellulase system optimization of expression in the N.crassa strains, but the need to ensure the secretion of lpmo protein. To sum up, through this research, to get an efficient endoglucanase agcel5a and a glucosidase ncbgl2, and excellent properties of Endo cellulase and glucosidase in biorefinery especially has great significance for the sustainable development of biofuels; also received two can effectively improve. The cellulase hydrolysis activity of lpmo protein, reduce cellulase dosage, saving the cost of cellulase, also promote the popularization and application of bio fuels has great significance; and finally proves that we can use the pattern of fungal N.crassa, based on t.reeseirut-c30 to carry out mutation for functional studies to identify t.reeseirut-c30. And to determine the mechanism of high yield AP-3 complex and cellulase secretion methods. The results of this study provide a new direction for the research of cellulase secretion, but also for the future high strain improvement put forward a new strategy.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q93
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本文编号：1663969