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蓝藻回补途径扰动及其代谢响应
Alternative TitleAnaplerosis pathway perturbation and metabolic response in cyanobacteria
颜飞
Subtype博士
Thesis Advisor丛威
2014-05
Degree Grantor中国科学院研究生院
Degree Discipline生物化工
Keyword蓝藻   磷酸烯醇式丙酮酸羧化酶   基因敲除   铜离子诱导表达   细胞碳流分布
Abstract随着全球能源与环境问题的日益突出,围绕光能自养原核生物蓝藻的研究成为热点。由于蓝藻具有相对清晰的遗传背景和丰富稳定的基因操作方法,可以对其进行系统的遗传改造和代谢修饰,使得细胞具有高水平的目标代谢产物产率。目前蓝藻代谢途径的分析和改造多是围绕目标产物合成途径进行,这些途径多数为特异化的分支代谢途径,而针对细胞中心代谢途径的研究相对较少。但由于中心代谢途径在细胞物质和能量代谢的核心作用,有必要对其代谢角色和特征进行深入分析,从而引导相关代谢途径修饰并进一步强化目标产物的合成。本研究选择了磷酸烯醇式丙酮酸羧化酶(PEPC)所催化的回补反应途径作为研究对象,通过基因敲除和铜离子诱导表达两种方式对回补途径进行扰动,对途径扰动下细胞代谢响应特征进行了分析,并以此对途径修饰的实际应用意义进行了评价。研究工作主要包括以下几个方面: 对高碳底物浓度下鱼腥藻7120细胞吸收和同化氮源的特性进行了研究。结果表明该条件对细胞吸收和同化氮源NO3-速率没有显著影响,与高光合固碳速率协同强化的氮同化速率由细胞分化的异形胞固氮同化来实现。 对鱼腥藻7120磷酸烯醇式丙酮酸羧化酶基因(ppc)进行基因敲除并考查该扰动所产生的代谢变化,主要是PEPC活性变化和细胞碳流分布变化。结果表明该方案不能得到完全分离的敲除突变株,部分突变株PEPC活性显著下调,与野生型对照相比活性降低可达50%。突变株PEPC活性的显著下调致使细胞胞内总糖含量显著增高,与野生型对照相比含量增高可达100%。由此推断PEPC活性的改变产生了该途径上下游碳流分布的改变,活性下降将增加上游碳流而减少下游碳流。胞内PEPC途径上下游小分子代谢物的含量变化支持了这一推论。 对鱼腥藻7120和集胞藻6803 ppc基因进行了铜离子诱导启动子插入构建并考查了不同铜离子浓度下该扰动产生的代谢变化,主要包括PEPC活性调控范围和细胞碳流分布变化。结果表明使用该方案鱼腥藻7120不能得到完全分离的启动子片段插入突变株,但集胞藻6803可以得到完全分离的突变株。胞内不同铜离子浓度下集胞藻6803突变株的PEPC活性可以被有效调节在一个区间,下限值活性由上游转录背景所致,上游壮观霉素(Sp)抗性标记基因(Spr)的转录是其中一个因素;上限值活性可能由其它调控机制所控制和影响。突变株PEPC活性变化与胞内总糖含量变化呈负相关关系,与胞内总蛋白含量变化呈正相关关系。突变株PEPC活性变化引起的细胞两大组分的含量变化这一结果再一次支持了PEPC活性的改变可以产生该途径上下游碳流分布改变的推断。
Other AbstractDue to the effects of global warming and the decreasing availability of fossil energy, research on cyanobacteria which is a photoautotroph prokaryote has been highlighted. As the detailed genetic background and abundance of genetic tools for cyanobacteria, it is possible to modify metabolic pathways and attain stable mutant in order to improve the transformation rate of target metablolite product. For the moment most work focuses on pathways that are directly relevant to synthesis of target product, while seldom work has been done on central metabolic pathways. Due to the core role of central metabolic pathways for mass and energy metabolism, it is necessary to explore their functions and roles for enhancing the target metabolite production. In this study, anaplerosis reaction which is catalyzed by PEPC(phosphoenolpyruvate carboxylase) was chosen. In vivo PEPC activity was perturbed by gene-knockout and copper-induced expression methods in order to analyse characteristics of cell metabolic response. The results can be applied to estimate future application of PEPC node modification. The study includes three parts as below: The effect of high inorganic carbon substrate concentration on NO3- assimilation rate was investigated in Nostoc sp. PCC 7120. The result showed that there was little effect of high carbon substrate concentration on NO3- assimilation rate. In accordance with the high carbon fixation rate, nitrogen assimilation rate of the cell was enhanced by increasing heterocyst differentiation frequency. The gene encoding PEPC(ppc) was tried to be knocked-out in Nostoc sp. PCC 7120 in order to investigate the cell metabolic response, mainly the change of PEPC activity and carbon flux distribution. The result showed that a completely segregated mutant could not be obtained by this method. PEPC activity of some mutants was about 50% of that of wild type control. As the PEPC activity was reduced significantly in the mutant, total carbohydrate content of the cell in late exponential phase increased significantly, more than 100% of that of wild type control. It is reasonable to infer that as the change of PEPC activity, carbon flux distribution surrounding this node altered. When PEPC activity was reduced, more carbon flux flowed into upstream pathways, while fewer carbon flux flowed into downstream pathways. The results of some small molecular metabolite content supported this conclusion. The copper-induced expression method was used in both Nostoc sp. PCC 7120 and Synechocystis sp. PCC 6803, in order to investigate PEPC acitivity regulation capacity and the change of carbon flux distribution. The result showed that a completely segregated and stable mutant was obtained for Synechocystis sp. PCC 6803 but not for Nostoc sp. PCC 7120. Cultured in different copper titers, the mutant presented narrow enzyme activity variance in the late exponential phase. The minimum activity was contributed by a background transcription, which included the transcription of upstream anti- spectinomycin gene. The maximum activity was limited by an unknown metabolic regulation mechanism. When PEPC activity was downregulated, the content of intracellular total carbonhydrate increased significantly and the content of intracellular total protein decreased. This result supported the conclusion that the change of PEPC activity caused carbon flux distribution altering surrounding PEPC node.
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/15515
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
颜飞. 蓝藻回补途径扰动及其代谢响应[D]. 中国科学院研究生院,2014.
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