| 题名 | 解纤维梭菌遗传体系的建立及其纤维素乙醇代谢工程研究 |
| 作者 | 崔古贞 |
| 学位类别 | 博士 |
| 答辩日期 | 2012-11 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 崔球 研究员 |
| 关键词 | 纤维素乙醇 解纤维梭菌 遗传改造 底盘细胞 代谢工程 纤维小体 Clostron |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | 纤维素乙醇具有环境友好、原料供应丰富的特征，是一种重要的生物能源产品。整合生物加工（CBP，Consolidated bioprocessing）技术实现了从纤维素到乙醇的反应器生产，能够极大简化纤维素乙醇的生产工艺，降低成本。解纤维梭菌（Clostridum cellulolyticum）作为嗜中温的产纤维小体的模式菌株，是最有希望用于CBP纤维素乙醇生产的的优良候选菌株之一。本文以C. cellulolyticum H10为研究对象，在建立其遗传改造体系的同时，从乙醇合成途径改造和纤维素降解效率改进两方面入手对其纤维素乙醇的合成进行详细的代谢工程研究：1、解纤维梭菌H10的遗传操作系统建立；通过筛选包括甘氨酸、苏氨酸、氨苄和异烟肼等不同的细胞壁弱化剂，将解纤维梭菌的转化效率提高到3.1×103 CFU/mg DNA，利用团队自主研制的电脉冲系统，将转化效率进一步提高到1.05×104 CFU/mg DNA。在获得稳定高效的遗传转化方法后，建立了基于厌氧绿色荧光蛋白的解纤维梭菌基因表达荧光报告系统，为研究蛋白的功能、相互作用和定位等问题提供了工具。同时建立了高效可靠的依赖于Clostron技术的基因靶向敲除系统，并基于该方法，构建了解纤维梭菌的无甲基化基因操作底盘细胞以及基于pyrF的双向遗传筛选底盘细胞，从而简化了遗传操作，提高了遗传操作的效率。2、解纤维梭菌H10的乙醇代谢途径改造；我们首先通过对乙酸激酶（ccel2136）、乳酸脱氢酶（ccel2485和ccel0137）的注释编码基因分别进行靶向失活，切断H10的乙酸及乳酸合成途径。发酵试验表明，与野生型菌株相比，工程菌株的代谢物谱发生了变化，即ccel2136失活突变株的乙酸产量降低了18.7%，说明H10中可能存在未知的乙酸激酶或其他乙酸生产途径；ccel2485突变株的乳酸产量降低了85%，乙醇和乙酸的产量分别提高了36%和26%；ccel2485和ccel0137双突变株的代谢物谱与ccel2485突变株相似，说明ccel0137可能没有乳酸脱氢酶的功能。上述结果说明通过改造乙醇代谢途径可以提高解纤维梭菌的乙醇产量。3、解纤维梭菌H10的纤维素降解酶系的功能分析及改造；提高纤维素降解的效率是提高纤维素乙醇产量的重要手段，而解纤维梭菌是通过纤维小体这一多酶复合体实现纤维素的降解，因此我们对解纤维梭菌的纤维小体的重要元件进行了初步的功能研究。解纤维梭菌基因组中与纤维小体相关且参与纤维素降解的基因主要位于cip-cel基因簇上，我们选择了该基因簇上编码脚手架蛋白的基因cipC、关键的纤维素酶编码基因cel48F以及未知功能的基因orfX作为敲除对象，并获得了相应的突变株。表型分析发现，cipC和cel48F的突变株在纤维素培养基中不能生长或生长速度非常缓慢，说明这两个基因对纤维小体的形成以及纤维素的降解具有至关重要的作用，而未发现orfX的突变株有明显的表型变化。此外，通过对野生菌株及上述三个突变株的cip-cel基因簇进行RT-qPCR分析发现，cipC和cel48F的突变严重抑制了整个cip-cel基因簇的转录，orfX基因的突变严重抑制了其后面基因的转录，但对前面基因的转录没有影响。这说明cip-cel基因簇可能是在同一个操纵子调控下进行转录的，且该操纵子位于cipC基因之前。总之，本论文建立的遗传改造的工具以及对解纤维梭菌进行的代谢工程改造和纤维小体元件的初步研究，为纤维素乙醇的研究和开发奠定了基础。 |
| 英文摘要 | Cellulosic ethanol is one of the most promising new bioenergy as an environmentally friendly fuel with rich source (i.e., lignincellulose). Consolidated bioprocessing (CBP) is believed to be one of the preferred strategies to produce biofuels directly from cellulose, which can greatly reduce the costs. Clostridium cellulolyticum, a typical mesophilic, gram-positive anaerobe, is one of the prominent candidates for CBP production of cellulosic ethanol. This work, aiming at higher cellulosic ethanol production, provided a reliable genetic manipulation system of C. cellulolyticum H10, and presented the metabolic engineering and the function study of cellulosome components in this microorganism. We firstly established the genetic manipulation methods of C. cellulolyticum H10. The transformation efficiency of C. cellulolyticum was improved up to 3.1×103 CFU/ug DNA by employing the cell wall weakening agent.With the new developed custom-built pulse generation system, the transformation efficiency was then improved to 1.05×104 CFU/ug DNA. A green fluorescent reporter system of C. cellulolyticum was successfully constructed, which could be used to evaluate the expression of heterologous protein in C. cellulolyticum conveniently by detecting green fluorescence. This reporter system provided a tool for the study of protein function, interaction, positioning, etc. A targeted gene inactivation method based on the clostron technology was established and laid the foundation for the study of gene function and genetic engineering. Based on the genetic tools, we developed a methylation-free cell chassis and a screening cell chassis by disruption of mspI and pyrF, respectively, which simplified the genetic manipulation and improved efficiency of genetic manipulation. Secondly, we improved the ethanol production of C. cellulolyticum H10 via metabolic engineering. We inactivated the acetate kinase (ccel2136) and lactate dehydrogenase (ccel2485 and ccel0137) to eliminate the production of by-products (e.g., actate and lactate). The preliminary fermentation results indicated that the metabolite patterns of C. cellulolyticum were changed due to the gene disruption. Compared with the wild type strain, the acetate production in ccel2136 inactivited mutant decreased only 18.7%, which indicated that there might be hitherto unknown acetate kinase or other pathways in C. cellulolyticum. The ccel2485 inactivated strain showed 85% decrease of lactate production, and 36% and 26% increase of ethanol and acetate production, respectively; the mutant with double distuption of ccel2485 and ccel0137 showed similar fermentation products with the ccel2485 inactivated strain, which indicated that ccel0137 might not be lactate dehydrogenase in C. cellulolyticum. Thus, our results demonstrated that the biofuel (e.g., ethanol) production of C. cellulolyticum could be improved by metabolic engineering. Function analysis and engineering of cellulsome of C.cellulolyticum H10. In order to improve the production of cellulosic ethanol, it is important to enhance the cellulose degradation of C. cellulolycium. Thus, it is nessesary to understand the function of the essential components of cellulosome, the multienzyme complex employed for cellulose degradation in this microorganism. Most of the genes encoding cellulosomal enzymes located in the cip-cel cluster in C. cellulolycium H10. Three genes in this cluster were chosen to disrupt, i.e., gene cipC (scaffoldin encoding gene), cel48F (cellulase encoding gene), and orfX (unknown function). cipC and cel48F inactivated mutants were unable to grown on cellulose or grew very slowly, which indicated that these two genes were essential for the degradation of cellulose, while no significant difference was observed in the orfX inactivated mutant. The transcription analyses based on RT-qPCR showed that the mutations of cipC and cel48F severely inhibited the transcription of the cip-cel gene cluster, and the disruption of gene orfX only blocked the transcription of genes located after orfX. These results argued that the cip-cel cluster might be co-transcribed under the control of the promoter located in front of gene cipC. In summary, a reliable genetic manipulation system was established in this work, and the metabolic engineering and the preliminarily study of the cellulosome would lay the foundation for the further development and utilization of cellulosic ethanol. |
| 语种 | 中文 |
| 学科主题 | 代谢物组学 |
| 公开日期 | 2012-12-12 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.qibebt.ac.cn:8080/handle/337004/1436]  |
| 专题 | 青岛生物能源与过程研究所_代谢物组学团队 |
| 推荐引用方式 GB/T 7714 | 崔古贞. 解纤维梭菌遗传体系的建立及其纤维素乙醇代谢工程研究[D]. 北京. 中国科学院研究生院. 2012. |
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