里氏木霉异源蛋白表达及高产纤维素酶菌株的遗传改造

发布时间：2018-01-25 00:42

本文关键词： 里氏木霉纤维素酶 Xyr1 异源表达菌株分子改造　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：丝状真菌里氏木霉(Trichoderma reesei)在纤维素底物存在条件下,能够大量合成分泌包括内切酶、外切酶以及β-葡萄糖苷酶在内的纤维素酶系,实现对底物的高效降解。里氏木霉强大的蛋白合成分泌能力也使其成为异源蛋白表达的良好宿主,对一些需要进行翻译后修饰的蛋白具有突出优势。但里氏木霉中以主要纤维素酶基因cel7a(cbh1)强启动子驱动的蛋白表达依赖于纤维素诱导条件,而此条件下其自身分泌的大量的纤维素酶不利于异源蛋白的后续分离纯化。另一方面,虽然里氏木霉所产纤维素酶目前已实现了规模化生产和应用,但是在生物基燃料如乙醇生产中,纤维素酶的使用成本仍然是一个主要限制因素。因此进一步提升工业菌株的纤维素酶产量对降低转化成本具有重要意义。本论文主要在以下两方面对里氏木霉进行了遗传改造:一是通过对主要的纤维素酶编码基因进行敲除以及对转录因子Xyr1进行过表达,以降低其作为蛋白表达宿主菌株的本底分泌蛋白背景,并且使cbh1启动子驱动的蛋白表达不再依赖于诱导碳源纤维素;二是在里氏木霉高产菌株C10中分别过表达了转录因子Xyr1和β-葡萄糖苷酶Bgl1,进一步提升了 C10的纤维素酶产量。主要研究工作总结如下:一、成功构建了主要纤维素酶(Cel5a、Cel7b、Cel6a以及Cel7a)缺失及转录因子Xyr1过表达的两株里氏木霉菌株△5a7b6aOExyr1和△5a7b6a7aOExyr1,其内源分泌蛋白背景低,且纤维素酶合成不依赖于诱导碳源纤维素,是用于以cbh1启动子驱动异源蛋白表达及纯化的理想菌株。我们以里氏木霉QM9414作为出发菌株,采用基于同源重组原理的基因敲除技术,构建了一株内切酶Cel5a和Ce17b以及外切酶Cel6a同时缺失的菌株A5a7b6a,SDS-PAGE分析表明该菌株培养基中分泌蛋白含量明显下降。我们在该菌株中进一步对纤维素酶基因转录正调控因子Xyr1进行了过表达,构建了△5a7b6aOExyr1菌株。在非诱导碳源葡萄糖下,该菌株内切酶Cel7a(Cbh1)的合成分泌量与野生型诱导碳源纤维素下相当。这表明,以低背景的△5a7b6aOExyr1菌株为表达宿主,可应用于分别在纤维素与葡萄糖条件下以cel7a(cbh1)启动子驱动的异源蛋白表达。我们继而又在该菌株中对cel7a基因进行了敲除,构建了△5a7b6a7aOExyrl菌株,在该菌株中可采用随机整合的方式对异源表达基因盒进行较为方便的操作。虽然该菌株由于cel7a的缺失不能利用纤维素作为碳源,但是其可应用于葡萄糖条件下cel7a启动子驱动的异源蛋白表达,并且Ce17a的缺失进一步大幅降低了内源分泌蛋白的背景,能够极大便利目标蛋白的分离纯化。二、以△5a7b6aOExyr1作为宿主菌株,成功地表达了来源于特异腐质霉(Humicoloinsolens)的内切葡聚糖酶NCE5,CMC酶谱电泳显示该酶的最适pH为6,属于偏中性内切酶。NCE5为来源于H.insolens 的内切葡聚糖酶,理论分子量为23 kDa。我们根据里氏木霉密码子使用频率对NCE5的编码序列进行优化,并构建了以cel7a(cbh1)启动子驱动的基因表达盒,将其定点整合于△5a7b6aOExyr1菌株的cel7a(cbh1)基因位点。CMC酶谱电泳分析表明,该酶成功表达并分泌到胞外,在pH 6时CMC酶活最高,属于偏中性内切酶。三、在里氏木霉高产菌株C10中分别过表达了转录正调控因子Xyr1和β-葡萄糖苷酶Bgl1,分析发现发酵液的滤纸酶活分别提升了 25%和30%。里氏木霉C10菌株为实验室保藏的纤维素酶高产菌株,在葡萄糖条件下可以低水平地解除碳代谢阻遏。我们在C10中采用tcu-1强启动子对转录正调控因子Xyr1进行过表达,获得C10-OExyr1菌株。该菌株与C10相比,不仅发酵液的滤纸酶活提升了约25%,并且纤维素酶分泌时间提前12h。在C10中以cel7a启动子过表达β-葡萄糖昔酶基因bgl1,获得C10-bgl1菌株,该菌株发酵液的滤纸酶活比野生型提升约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%.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q933

【相似文献】