本文選題：粗糙脈孢菌　切入點(diǎn)：分泌途徑　出處：《吉林大學(xué)》2016年博士論文

【摘要】：植物的木質(zhì)纖維素是世界上分布最廣的可再生資源,目前對(duì)于利用降解木質(zhì)纖維素得到的產(chǎn)物生產(chǎn)生物燃料成為研究的熱點(diǎn)。但是由于新興生物燃料的高成本,使得其與傳統(tǒng)石化燃料在應(yīng)用推廣上相比不具有競(jìng)爭(zhēng)力。在生產(chǎn)生物燃料的過程中,降解木質(zhì)纖維素所需的纖維素酶的費(fèi)用在生產(chǎn)成本中所占比重很大。提高纖維素酶產(chǎn)量,構(gòu)建纖維素酶高產(chǎn)菌株是解決這一問題的主要技術(shù)手段。目前工業(yè)生產(chǎn)上已經(jīng)成功構(gòu)建許多的高產(chǎn)纖維素酶生產(chǎn)菌株,如纖維素酶生產(chǎn)的明星菌株Trichoderma reesei RUT-C30。通過比較基因組學(xué)研究發(fā)現(xiàn)與出發(fā)菌株T.reesei strain QM6a相比RUT-C30菌株中多處發(fā)生突變,但是該菌株在遺傳操作方面存在一定的困難,所以目前對(duì)其高產(chǎn)機(jī)制尚不明確。而Neurospora crassa有著與T.reesei有著非常近的親緣關(guān)系,還具有非常簡(jiǎn)便的遺傳操作手段,近年來(lái)已逐漸成為研究產(chǎn)酶機(jī)理的理想菌株。所以,為研究RUT-C30菌株的高產(chǎn)機(jī)制,以N.crassa為研究對(duì)象,針對(duì)RUT-C30菌株中突變基因,篩選粗糙脈孢菌中的單基因敲除突變體,挖掘這些基因的功能,為改造高產(chǎn)菌株、研究產(chǎn)酶機(jī)理提供新的研究方向。木質(zhì)纖維素的水解需要三種酶的協(xié)同作用來(lái)完成,而高效的內(nèi)切纖維素酶與β-葡萄糖苷酶可促使纖維素水解效率得到顯著的提高,降低降解木質(zhì)纖維素的費(fèi)用,進(jìn)而促進(jìn)生物燃料的推廣應(yīng)用。因此在本研究中,分別克隆表達(dá)了一個(gè)內(nèi)切纖維素酶AgCel5A和一個(gè)β-葡萄糖苷酶NcBGL2,并對(duì)其性質(zhì)進(jìn)行了分析。以期獲得高效的纖維素酶,用來(lái)提高纖維素的水解效率,進(jìn)而達(dá)到降低成纖維素酶成本的目的。傳統(tǒng)的纖維素酶是由內(nèi)切纖維素酶、外切纖維素酶和β-葡萄糖苷酶三種組份組成的。但是有沒有存在另一種組份,可以使現(xiàn)有的傳統(tǒng)纖維素酶系的水解活性得到進(jìn)一步的提升呢?AA9家族的溶解性多糖單加氧酶就具有這種功能。溶解性多糖單加氧酶(Lytic polysaccharide monooxygenases)簡(jiǎn)稱LPMOs,即原來(lái)的GH61蛋白,可以通過與CDH(Cellobiose dehydrogenases)或者一些低分子量的還原劑(如抗壞血酸)發(fā)生氧化還原反應(yīng),裂解纖維素鏈上的糖苷鍵,破壞其結(jié)構(gòu),從ii而使纖維素更容易被纖維素酶降解,提高傳統(tǒng)纖維素酶的水解效率。嗜熱毀絲霉(myceliophthorathermophila)是一株嗜熱真菌,具有很強(qiáng)的纖維素降解能力,目前已經(jīng)從該菌株中克隆得到了許多的纖維素酶基因。在本研究中,克隆了兩個(gè)在m.thermophila表達(dá)量最高的lpmo基因,并對(duì)其性質(zhì)進(jìn)行了分析,以期達(dá)到提高原有纖維素酶系水解活性,促進(jìn)纖維素水解,降低纖維素酶成本,從而促進(jìn)生物燃料的可持續(xù)發(fā)展的目的。本研究的主要研究結(jié)果如下:(1)利用n.crassa基因敲除突變體庫(kù)對(duì)t.reeseirut-c30中的突變基因進(jìn)行表型驗(yàn)證,成功篩選得到12株胞外蛋白分泌升高25%以上的突變菌株,4株分泌下降25%以上的菌株;明確編碼ap-3復(fù)合體μ亞基的ncap3m(ncu03998)和編碼β亞基的ncap3b(ncu06569)的缺失可顯著提高胞外蛋白的分泌;ncap3m和ncap3b的缺失還會(huì)導(dǎo)致n.crassa菌株的分泌應(yīng)激引起的轉(zhuǎn)錄抑制(repressedexpressionofsecretedsequences,ress)現(xiàn)象減弱;最后證明ap-3復(fù)合體與纖維素酶分泌途徑相關(guān),ap-3復(fù)合體功能缺失后會(huì)導(dǎo)致纖維素酶的分泌提高。(2)成功克隆得到了一個(gè)高效的內(nèi)切纖維素酶agcel5a和一個(gè)β-葡萄糖苷酶ncbgl2,二者在pichapastori中異源表達(dá)后,發(fā)酵液中均為單一條帶;agcel5a最適反應(yīng)條件為ph5.0,55°c,具有良好的熱穩(wěn)定性和ph穩(wěn)定性,同時(shí)還具有極強(qiáng)的耐鹽性,而且co2+可有效促進(jìn)酶活提高;ncbgl2的最適反應(yīng)條件為ph5.6,60°c,10%的乙醇可提高80%酶活,還具有非常強(qiáng)的水解大豆異黃酮糖苷活性。(3)水解纖維素的過程中,將mtlpmo9a與mtlpmo9b添加到原有纖維素酶系中時(shí),均可以使最終酶用量減少40%;在n.crassa菌株中表達(dá)這兩個(gè)lpmo蛋白可直接實(shí)現(xiàn)纖維素酶系優(yōu)化,但需保證lpmo蛋白的分泌量。綜上所述,通過本文的研究,獲得了一個(gè)高效的內(nèi)切纖維素酶agcel5a和一個(gè)β-葡萄糖苷酶ncbgl2,而性質(zhì)優(yōu)良的內(nèi)切纖維素酶和β-葡萄糖苷酶在生物煉制特別是生物燃料的可持續(xù)發(fā)展上具有重要意義;還獲得了兩個(gè)可以有效的提高傳統(tǒng)纖維素酶的水解活性lpmo蛋白,降低纖維素酶的用量,節(jié)約纖維素酶成本,同樣在促進(jìn)生物燃料的推廣應(yīng)用上具有著重大意義;最后證明了可以利用模式真菌n.crassa,開展對(duì)t.reeseirut-c30的突變基因進(jìn)行功能研究,以明確t.reeseirut-c30高產(chǎn)機(jī)理。并確定ap-3復(fù)合體與纖維素酶分泌途徑相關(guān)。該研究結(jié)果為纖維素酶分泌方面的研究提供一個(gè)新的方向,也為今后高分泌菌株改良提出一個(gè)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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q93
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本文編號(hào)：1663969