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类型 基础研究 预答辩日期 2018-03-08
开始(开题)日期 2015-12-15 论文结束日期 2018-01-05
地点 四牌楼校区岩土工程馆西侧小白楼生物表界面实验室会议室305 论文选题来源 国家自然科学基金项目     论文字数 7.2 (万字)
题目 里氏木霉高产纤维素酶的机理研究和应用
主题词 里氏木霉,高产纤维素酶,乳糖,色素,机制
摘要 里氏木霉(Trichoderma reesei)因其蛋白分泌量大(100 g/L)、遗传性状稳定、生长繁殖迅速、培养条件简单和生物安全性等特点,已被广泛用于工业生产及各种异源蛋白的表达载体。并且,该菌在特定条件下可以产生大量的具有抗癌、抗病毒、抗菌特性的次级代谢产物黄色色素类物质,具有广泛的商业价值,次级代谢产物黄色色素的产生对初级代谢产物纤维素酶的产生有一定的影响,二者受到相关调节因子的调控。同时,里氏木霉多个菌株的全基因组测序已经完成,这为研究里氏木霉纤维素酶、色素类物质的高产机制以及初级代谢和次级代谢的相关性奠定了基础,也为基因改造获得具有优良特性的高产菌株提供了辅助手段。 本论文主要以里氏木霉RUT-C30为出发菌株通过基因工程改造获得了在纤维素诱导条件下高产纤维素酶的基因工程菌TRB1、在纤维素和乳糖诱导下都能高产纤维素酶的里氏木霉基因工程菌SEU-7以及高产色素的基因工程菌△121121,并针对以上构建的基因工程菌研究了纤维素酶的高产机制、里氏木霉初级代谢和次级代谢之间的相互转化机制。之后,我们采用荧光标记的办法对里氏木霉三种主要的纤维素酶进行标记以研究纤维素酶的真实分布情况以及分泌机制。最后采用转录组测序及基因敲除等手段对纤维素酶的产生及其和色素产生的转化关系进行了深入研究。具体说来主要包含以下内容: 1. 构建了高产BGL的基因工程菌TRB1,并揭示了CEL3D基因在纤维素酶高产中的作用及机理 以里氏木霉RUT-C30为出发菌株,采用农杆菌介导的方法构建了高产β-葡萄糖苷酶(pNPGase/BGL)的基因工程菌TRB1。qRT-PCR以及基因克隆实验显示TRB1中CEL3D基因受到了干扰,是纤维素酶增加的原因之一。 2. 构建了乳糖诱导下高产纤维素酶的基因工程菌SEU-7并研究其高产机制 首次构建了一株可以在乳糖诱导下高产纤维素酶的基因工程菌SEU-7。与出发菌株RUT-C30相比,SEU-7的纤维素酶和半纤维素酶活性无论在纤维素还是在乳糖诱导条件下都大幅度提高。以乳糖作为诱导物,在分批培养条件下,FPase活性可以达到13 IU/mL;分批补料培养时,FPase活性提高至47 IU/mL,此外,在分批培养过程中,SEU-7在纤维素和乳糖诱导条件下均表现出了极高的pNPGase活性,分别为81和144 IU/mL,是目前已知菌株中最高的。之后采用qRT-PCR、qPCR以及基因组重测序分析研究了其在不同碳源下的高产机理。 3. 基因121121在里氏木霉纤维素酶和黄色色素产生中的开关作用 通过构建敲除基因121121的基因工程菌及过表达121121的菌株,揭示了基因121121在纤维素酶产生和黄色色素类物质sorbicillin分泌过程的开关作用,即,敲除121121会降低纤维素酶产量但极大程度增加黄色色素sorbicillin的产量,并对该基因工程菌进行了RNA-seq分析。同时采用液质联用(HPLC-MS)的方法检测了敲除菌株△121121和过表达菌株OE121121的代谢产物。 4. 里氏木霉纤维素酶的产生、分布以及分泌机制研究 采用红色荧光蛋白对三种主要的纤维素酶BGL、CMC和CBH进行了标记,研究了三种纤维素酶产生的先后顺序、在细胞内的分布情况以及分泌情况,结果发现BGL最早出现在菌丝体内,随后产生CMC和CBH,然而,BGL的分泌确晚于CMC和CBH。共聚焦观察结果表明BGL和CBH是定位到细胞膜上的,而CMC没有定位到膜上,胆固醇-PEG-FITC共染、原生质体制备以及超高分辨结果更进一步的证明了BGL定位到了细胞膜上。通过进一步的内质网共染实验证明了三种主要的纤维素酶都会被输送到内质网进行加工修饰,并且三种酶都存在于囊泡中,高尔基体共染实验证明BGL和CMC都要经过高尔基体分泌到胞外,而CBH则不通过高尔基体分泌。
英文题目 MECHANISM STUDY OF CELLULASE HYPER-PRODUCTION OF TRICHODERMA REESEI AND ITS APPLKICATION
英文主题词 Trichoderma reesei, cellulase hyper-production, lactose, pigment; mechanism
英文摘要 Title: Mechanism study of cellulase hyper-production of Trichoderma reesei and its application Doctoral candidate: Li Chengcheng Supervisor: Chen Zhan and Lin Fengming University: Southeast University T. reesei is widely used in industry and as a vector for the expression of heterologous protein due to its large ammount of secreted protein (100 g/L), genetic stability, rapid growth rate, simple cultivation and safety. In addition, T. reesei can secrete a kind of yellow pigment named as sorbicillin with the character of anti-cancer, anti-viral and anti-bacteria. Otherwise, the accomplishment of the genome sequencing of several strains of T. reesei provides a solid foundation for construction of gene engineering strains with good properties and studying the cellulase production mechanism. In this paper, three gene engineering strains TRB1, SEU-7 and △121121 were obtained. TRB1 produced more cellulases than RUT-C30 and released CCR effect, SEU-7 produced the most FPase to this day on lactose. △121121 could produce abundant yellow pigments. Then DsRed was used to label the major cellulases and study the secretion mechanism of the three major cellulases. Finally, we further investigated the cellulase and sorbicillin production mechanism by RNA-seq and qRT-PCR. The major contributions are as follows: 1. A β-glucosidase hyper-production Trichoderma reesei mutant reveals a potential role and mechanism of CEL3D in cellulase production The T. reesei recombinant strain TRB1 was constructed from T. reesei RUT-C30 by the T-DNA-based mutagenesis. qRT-PCR and gene cloning showed that in TRB1 β-glucosidase cel3D was mutated through the random insertion by AMT and was not expressed. 2. Cellulase hyper-production by Trichoderma reesei mutant SEU-7 on lactose T. reesei mutant strain SEU-7 was constructed from T. reesei RUT-C30 with the overexpression of endogenous gene β-glucosidase (BGL1) by insertional mutagenesis via Agrobacterium tumefaciens-mediated transformation (AMT). Compared to RUT-C30, SEU-7 displays substantially enhanced activities of both cellulase and hemicellulase when grown on either lactose or cellulose. The induction efficiency with lactose was found to be higher than cellulose in strain SEU-7. To the best of our knowledge, we achieved the highest FPase activity in SEU-7 in both batch culture (13.0 IU/mL) and fed-batch culture (47.0 IU/mL) on lactose. Moreover, SEU-7 displayed unrivaled pNPGase activity on lactose in both batch-culture (81.0 IU/mL) and fed-batch culture (144.0 IU/mL) as compared to the other reported T. reesei strains in the literature grown in batch or fed-batch experiments on cellulose or lactose. Afteterwards, qRT-PCR, qPCR and Genome sequencing were performed to study the mechanism for cellulase hyper-production on different carbon sources. 3. Deletion of gene 121121 switched the cellulase production to the yellow pigment production pathway in Trichoderma reesei For the first time, 121121 was proved to be a transcriptional regulator for the convertion between cellulase production and the yellow pigment (sorbicillin) production. Deletion of 121121 reduced cellulase production but increased the production of sorbicillin. We performed RNA-seq for more informations of △121121, then HPLC-MS was used to identify the metabolites of △121121 and OE121121. 4. Tracking the production, distribution and secretion of the main cellulases in Trichoderma reesei In our study, we successfully engineered several transformants overexpressed the CMC, CBH and BGL1 gene labeled with DsRed as a tracking gene, designated as RCMC, RCBH and RBGL, through the AMT method. Results showed that BGL1 appeared earlier than the other two cellulases in vivo, however the secretion of BGL1 was slower than the other two cellulases. Microscopy observation showed that BGL1 and CBH can be secreted onto cell memebrane, but not for CBH. Membrane staining with cholesterol-PEG-FITC reinforced the above result. Moreover, we proved that all three cellulases can be entered into ER for processing by co-staining with ER-Green and BGL1 and CMC secreted through Golgi but not for CBH by co-staining with GolgiGreen.
学术讨论
主办单位时间地点报告人报告主题
生医学院生物表界面实验室 2016.10.9 生物表界面实验室3楼 李程程 Tracking the production, distribution and secretion of the main cellulases in Trichoderma reesei
生医学院生物表界面实验室 2017.3.10 生物表界面实验室3楼 李程程 Deletion of gene 121121 switched the cellulase production to the yellow pigment production pathway in Trichoderma reesei
生医学院生物表界面实验室 2015.12.3 生物表界面实验室3楼 李程程 Mechanism study of β-glucosidase 1 (BGL1)and its production improvement in T. reesei
生医学院生物表界面实验室 2016.4.8 生物表界面实验室3楼 李程程 Overexpression of bgl1 gene in T. reesei improve the production of BGL1.
生医学院生物表界面实验室 2016.6.10 生物表界面实验室3楼 李程程 The nanostructure and the application of EPS produced by Lactobacillus plantarum 605
生医学院生物表界面实验室 2017.7.6 生物表界面实验室3楼 李程程 Cellulase hyper-production by Trichoderma reesei mutant SEU-7 on lactose
SZ研究生会 2015.12.3 中大院2楼报告厅 杜奕瑾 人工智能的过去现在与未来
JLH校研究生会学术部 2016.5.6 纪忠楼报告厅 朱进东 恪守学术道德,得心应手撰写论文
     
学术会议
会议名称时间地点本人报告本人报告题目
International Conference on Biotechnology and Agriculture Engineering 2017.3 日本大阪 Novel Self-assembled Exopolysaccharide Nanoparticles for Bioremediation and Green Synthesis of Noble Metal Nanoparticles
. 7th International Forum on Industrial Bioprocesses 2017.5 中国无锡 Cellulase Hyper-production by Trichoderma reesei SEU-7 Using Soluble Carbon Sources
International Bioenergy Exhibition and Asian Bioenergy Conference 2015.10 中国上海 Overexpression of β-glucosidase gives T.reesei improved cellulose productivity and glucose resistance
     
代表作
论文名称
Self-assembled Exopolysaccharide Nanoparticles for Bioremediation and Green Synthesis of Noble Metal
A β-glucosidase hyper-production Trichoderma reesei mutant reveals a potential role of cel3D in cell
Cellulase hyper-production by Trichoderma reesei mutant SEU-7 on lactose
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
周东山 正高 教授 博导 南京大学
刘宏 正高 教授 博导 东南大学
周晓燕 正高 教授 博导 南京林业大学
吴富根 正高 教授 博导 东南大学
杨芳 正高 教授 博导 东南大学
卢晓林 正高 教授 博导 东南大学
      
答辩秘书信息
姓名职称工作单位备注
韩晓锋 副高 东南大学