本文關(guān)鍵詞： 里氏木霉 纖維素酶 Xyr1 異源表達(dá) 菌株分子改造　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：絲狀真菌里氏木霉(Trichoderma reesei)在纖維素底物存在條件下,能夠大量合成分泌包括內(nèi)切酶、外切酶以及β-葡萄糖苷酶在內(nèi)的纖維素酶系,實(shí)現(xiàn)對(duì)底物的高效降解。里氏木霉強(qiáng)大的蛋白合成分泌能力也使其成為異源蛋白表達(dá)的良好宿主,對(duì)一些需要進(jìn)行翻譯后修飾的蛋白具有突出優(yōu)勢(shì)。但里氏木霉中以主要纖維素酶基因cel7a(cbh1)強(qiáng)啟動(dòng)子驅(qū)動(dòng)的蛋白表達(dá)依賴于纖維素誘導(dǎo)條件,而此條件下其自身分泌的大量的纖維素酶不利于異源蛋白的后續(xù)分離純化。另一方面,雖然里氏木霉所產(chǎn)纖維素酶目前已實(shí)現(xiàn)了規(guī)模化生產(chǎn)和應(yīng)用,但是在生物基燃料如乙醇生產(chǎn)中,纖維素酶的使用成本仍然是一個(gè)主要限制因素。因此進(jìn)一步提升工業(yè)菌株的纖維素酶產(chǎn)量對(duì)降低轉(zhuǎn)化成本具有重要意義。本論文主要在以下兩方面對(duì)里氏木霉進(jìn)行了遺傳改造:一是通過(guò)對(duì)主要的纖維素酶編碼基因進(jìn)行敲除以及對(duì)轉(zhuǎn)錄因子X(jué)yr1進(jìn)行過(guò)表達(dá),以降低其作為蛋白表達(dá)宿主菌株的本底分泌蛋白背景,并且使cbh1啟動(dòng)子驅(qū)動(dòng)的蛋白表達(dá)不再依賴于誘導(dǎo)碳源纖維素;二是在里氏木霉高產(chǎn)菌株C10中分別過(guò)表達(dá)了轉(zhuǎn)錄因子X(jué)yr1和β-葡萄糖苷酶Bgl1,進(jìn)一步提升了 C10的纖維素酶產(chǎn)量。主要研究工作總結(jié)如下:一、成功構(gòu)建了主要纖維素酶(Cel5a、Cel7b、Cel6a以及Cel7a)缺失及轉(zhuǎn)錄因子X(jué)yr1過(guò)表達(dá)的兩株里氏木霉菌株△5a7b6aOExyr1和△5a7b6a7aOExyr1,其內(nèi)源分泌蛋白背景低,且纖維素酶合成不依賴于誘導(dǎo)碳源纖維素,是用于以cbh1啟動(dòng)子驅(qū)動(dòng)異源蛋白表達(dá)及純化的理想菌株。我們以里氏木霉QM9414作為出發(fā)菌株,采用基于同源重組原理的基因敲除技術(shù),構(gòu)建了一株內(nèi)切酶Cel5a和Ce17b以及外切酶Cel6a同時(shí)缺失的菌株A5a7b6a,SDS-PAGE分析表明該菌株培養(yǎng)基中分泌蛋白含量明顯下降。我們?cè)谠摼曛羞M(jìn)一步對(duì)纖維素酶基因轉(zhuǎn)錄正調(diào)控因子X(jué)yr1進(jìn)行了過(guò)表達(dá),構(gòu)建了△5a7b6aOExyr1菌株。在非誘導(dǎo)碳源葡萄糖下,該菌株內(nèi)切酶Cel7a(Cbh1)的合成分泌量與野生型誘導(dǎo)碳源纖維素下相當(dāng)。這表明,以低背景的△5a7b6aOExyr1菌株為表達(dá)宿主,可應(yīng)用于分別在纖維素與葡萄糖條件下以cel7a(cbh1)啟動(dòng)子驅(qū)動(dòng)的異源蛋白表達(dá)。我們繼而又在該菌株中對(duì)cel7a基因進(jìn)行了敲除,構(gòu)建了△5a7b6a7aOExyrl菌株,在該菌株中可采用隨機(jī)整合的方式對(duì)異源表達(dá)基因盒進(jìn)行較為方便的操作。雖然該菌株由于cel7a的缺失不能利用纖維素作為碳源,但是其可應(yīng)用于葡萄糖條件下cel7a啟動(dòng)子驅(qū)動(dòng)的異源蛋白表達(dá),并且Ce17a的缺失進(jìn)一步大幅降低了內(nèi)源分泌蛋白的背景,能夠極大便利目標(biāo)蛋白的分離純化。二、以△5a7b6aOExyr1作為宿主菌株,成功地表達(dá)了來(lái)源于特異腐質(zhì)霉(Humicoloinsolens)的內(nèi)切葡聚糖酶NCE5,CMC酶譜電泳顯示該酶的最適pH為6,屬于偏中性內(nèi)切酶。NCE5為來(lái)源于H.insolens 的內(nèi)切葡聚糖酶,理論分子量為23 kDa。我們根據(jù)里氏木霉密碼子使用頻率對(duì)NCE5的編碼序列進(jìn)行優(yōu)化,并構(gòu)建了以cel7a(cbh1)啟動(dòng)子驅(qū)動(dòng)的基因表達(dá)盒,將其定點(diǎn)整合于△5a7b6aOExyr1菌株的cel7a(cbh1)基因位點(diǎn)。CMC酶譜電泳分析表明,該酶成功表達(dá)并分泌到胞外,在pH 6時(shí)CMC酶活最高,屬于偏中性內(nèi)切酶。三、在里氏木霉高產(chǎn)菌株C10中分別過(guò)表達(dá)了轉(zhuǎn)錄正調(diào)控因子X(jué)yr1和β-葡萄糖苷酶Bgl1,分析發(fā)現(xiàn)發(fā)酵液的濾紙酶活分別提升了 25%和30%。里氏木霉C10菌株為實(shí)驗(yàn)室保藏的纖維素酶高產(chǎn)菌株,在葡萄糖條件下可以低水平地解除碳代謝阻遏。我們?cè)贑10中采用tcu-1強(qiáng)啟動(dòng)子對(duì)轉(zhuǎn)錄正調(diào)控因子X(jué)yr1進(jìn)行過(guò)表達(dá),獲得C10-OExyr1菌株。該菌株與C10相比,不僅發(fā)酵液的濾紙酶活提升了約25%,并且纖維素酶分泌時(shí)間提前12h。在C10中以cel7a啟動(dòng)子過(guò)表達(dá)β-葡萄糖昔酶基因bgl1,獲得C10-bgl1菌株,該菌株發(fā)酵液的濾紙酶活比野生型提升約30%。
[Abstract]:The filamentous fungus Trichoderma reesei (Trichoderma reesei) in the presence of conditions of cellulose substrates, including the ability to synthesize enzyme exonuclease, and beta glucosidase, cellulase, efficient degradation of the substrate. Trichoderma reesei strong protein synthesis secretion ability also makes it a good host for heterologous protein expression, on some require post-translational modification of protein has outstanding advantages. But the main Cel7A in Trichoderma reesei cellulase gene (cbh1) promoter driven expression dependent on cellulose induced conditions, purification and subsequent separation under the conditions of its own secretion of cellulase is not conducive to the heterologous protein. On the other hand, although the Richter Trichoderma cellulase has achieved large-scale production and application, but in bio fuels such as ethanol production, the cost is still with Cellulase It is a major limiting factor. Thus further enhance the cellulase production of industrial strains is significant for reducing the cost of conversion. This paper mainly in the following two aspects of Trichoderma reesei were studied by genetic transformation: one is passed on the cellulase gene encoding major knockout and overexpression of transcription factor Xyr1, to reduce the as the expression host strain the secretory protein background, and the expression of cbh1 promoter induced protein is no longer dependent on the carbon source of cellulose; two in Trichoderma reesei producing strain C10 were over expression of transcription factor Xyr1 and beta glucosidase Bgl1, to further enhance the yield of cellulase C10. The main research the work summarized as follows: first, the successful construction of the main cellulase (Cel5a, Cel7b, Cel6a and Cel7a) deletion and transcription factor Xyr1 of two strains of Trichoderma reesei strain expression Delta 5a7b6aOExyr1 and delta 5a7b6a7aOExyr1, the endogenous secretory protein with low background, and does not depend on cellulase synthesis induced by carbon source of cellulose, is used for cbh1 promoter driven expression of heterologous protein and ideal strains purified. We used as the starting strain by Trichoderma reesei QM9414, using the principle of homologous recombination gene knockout technology based on construction one strain A5a7b6a enzyme Cel5a and Ce17b and Cel6a and exonuclease deletions, SDS-PAGE analysis showed that the strains cultured in secretory protein content decreased significantly. We in this strain of cellulose enzyme gene transcription further positive regulatory factor Xyr1 overexpression, construct the delta 5a7b6aOExyr1 strain. Induced by carbon sources of glucose in the presence of the strain Cel7a (Cbh1) enzyme synthesis and secretion induced by wild type cellulose carbon source under quite. This suggests that, with low background for Delta 5a7b6aOExyr1 strains Expression of the host, can be applied respectively in cellulose and glucose conditions with Cel7A (cbh1) expression of heterologous protein promoter. We then in this strain of Cel7A gene was knocked out, the construction of delta 5a7b6a7aOExyrl strains in this strain can be used on the random integration of heterologous expression of gene cassettes convenient operation. Although the strain due to the lack of Cel7A can use cellulose as a carbon source, but it can be applied to glucose conditions Cel7A promoter driven expression of heterologous proteins, and the absence of Ce17a further decrease of endogenous secretory protein can greatly facilitate background, separation and purification of target protein. Two, to 5a7b6aOExyr1 as the host strain was successfully expressed from humicola insolens (Humicoloinsolens) endoglucanase NCE5 spectrum, the optimum electrophoresis showed that the enzyme pH 6 CMC enzyme, which belongs to Neutral endoglucanase enzyme.NCE5 is derived from H.insolens, the theoretical molecular weight of 23 kDa. according to Trichoderma reesei codon use frequency encoding sequences of NCE5 were optimized, and the construction of the Cel7A (cbh1) promoter driven gene expression cassette, the site-specific integration in Delta 5a7b6aOExyr1 strains Cel7A (cbh1) gene locus.CMC isozyme electrophoresis analysis showed that the enzyme was expressed and secreted into the extracellular, at pH 6 CMC the highest enzyme activity, which belongs to the neutral enzyme. In three, C10 in high yield strains of Trichoderma reesei were over expressed positive transcriptional regulation factor Xyr1 and beta glucosidase Bgl1 analysis showed that filter paper enzyme activities were increased by 25% and 30%. of Trichoderma strain C10 for strains with High Cellulase Activity preserved in laboratory, under the condition of low levels of glucose can relieve carbon catabolite repression. We use tcu-1 in the C10 start The positive transcriptional regulatory factor Xyr1 was overexpressed, C10-OExyr1 strains. The strain compared with C10, not only the filter paper enzyme fermentation activity increased by about 25%, and cellulase production time ahead of 12h. in the C10 in the promoter of Cel7A over expression of beta glucosidase gene was bgl1, strain C10-bgl1, filter paper enzyme fermentation the liquid strain live than the wild type increased by approximately 30%.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q933

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 唐雯;嚴(yán)明;;里氏木霉(Trichoderma reesei)分泌組的預(yù)測(cè)及分析[J];微生物學(xué)報(bào);2008年04期

2 陳士華;薛珍;姚金杰;吳興泉;;里氏木霉纖維二糖水解酶Ⅱ的生物信息學(xué)研究[J];河南工業(yè)大學(xué)學(xué)報(bào)(自然科學(xué)版);2008年04期

3 葛春梅;徐娟娟;孫芹英;張潔;蔡敬民;潘仁瑞;;里氏木霉和雞腿菇利用秸稈共發(fā)酵產(chǎn)木質(zhì)降解酶[J];生物工程學(xué)報(bào);2009年12期

4 安莉穎;施思;秦麗娜;陳飛;董志揚(yáng);伍紅;;里氏木霉幾丁質(zhì)酶基因的克隆及其同源轉(zhuǎn)化研究[J];西南民族大學(xué)學(xué)報(bào)(自然科學(xué)版);2013年03期

5 田明;郭宏文;陳連峰;Q，

本文編號(hào)：1461498