【作者】 陈璟；

【导师】 须文波；

【作者基本信息】 江南大学 ， 轻工信息技术与工程， 2013， 博士

【摘要】 由于嗜热菌对高温环境有很强的适应性,近几年来嗜热菌在基因工程、蛋白质工程、发酵工程及矿产资源的开发利用上均有很大的应用价值。为了从系统水平上阐明嗜热菌的耐热机制,并指导嗜热菌在工业生产上的进一步应用,本文以产甲烷菌的代谢网络作为研究对象,从研究代谢网络的拓扑结构以及模块化出发,探索常温产甲烷菌Methanosarcina acetivorans (M. acetivorans)和嗜热产甲烷菌Methanopyrus kandleri (M. kandleri)之间的代谢网络的耐热性的差异。论文首先基于日本京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)数据库重构了常温的产甲烷菌M. acetivorans和嗜热的产甲烷菌M. kandleri的代谢网络,并进一步验证了代谢网络具有小世界、无标度网络等特征。根据全局代谢网络的比较,发现途径00010(糖酵解/葡糖异生)、00020(柠檬酸循环)以及00250(丙氨酸、天冬氨酸和谷氨酸代谢)是常温产甲烷菌M. acetivorans与嗜热产甲烷菌M. kandleri的重要途径,它们的度也是整个网络最高的,这说明它们是在进化过程中是较早出现的途径。同时基于全局代谢网络考察了代谢网络中酶的性质分类与连接度之间的关联,发现常温产甲烷菌M. acetivorans与嗜热产甲烷菌M. kandleri的代谢网络中合成酶类的连接度最低,而连接度最高的是氧化还原酶类与转移酶类。为了比较分析网络拓扑结构与耐热性之间的关联,计算了代谢网络的度、度的中心性、介数中心性、聚集系数以及直径等参数值,发现常温产甲烷菌M. acetivorans和嗜热产甲烷菌M. kandleri的代谢网络的聚集系数远高于同样规模及拓扑结构的随机网络的聚集系数,表明这两个菌具有较高的模块化结构。常温产甲烷菌M. acetivorans的代谢网络比嗜热产甲烷菌M. kandleri有更长的平均路径长度,更大的直径,这表明常温产甲烷菌M. acetivorans的总体结构是相对低密度、松散的网络结构。对于大规模的网络而言,高度紧密的子网络能反映网络的全局特征,并且是比较不同生物体代谢网络的重要因素。发现常温产甲烷菌M. acetivorans最紧密的9-核和嗜热产甲烷菌M. kandleri最紧密的7-核分别包含27和19个酶。其中嗜热产甲烷菌M. kandleri网络中最紧密的7-核分成两个小网络,一个子网中酶是两个产甲烷菌所共有的酶,而另一个子网络中的酶是嗜热产甲烷菌M. kandleri7-核所特有的酶,这些特有的酶全部与酪氨酸的合成有关,推测嗜热产甲烷菌M. kandleri的耐热性可能受到胞内酪氨酸的影响。代谢网络与其他复杂网络系统相类似,也具备一定的模块化组织结构,有效识别并提取代谢网络中的功能模块有助于把握其中蕴含的功能信息。因此在代谢网络全局研究的基础上,从系统水平上分析这些特征蕴含的功能意义,采用了Girvan和Newman提出的GN模块识别算法以及模拟退火算法(Simulated Annealing, SA)分析这两个产甲烷菌的功能模块。采用GN算法后,常温产甲烷菌M. acetivorans的代谢网络由39个模块组成,而嗜热产甲烷菌M. kandleri的代谢网络被划分了30个模块,其中常温产甲烷菌M. acetivorans的代谢网络中模块1、15、16以及模块4,较其它模块来说节点更多,连接也更紧密,所以这四个模块是整个代谢网络中的hub模块。而嗜热产甲烷菌M. kandleri的代谢网络的模块1和11是hub模块。模块化后,识别了具有一定功能意义的网络模块,发现常温产甲烷菌M. acetivorans中有4个模块属于碳水化合物,1个模块属于次级代谢的生物合成,而嗜热产甲烷菌M. kandleri中有2个模块属于碳水化合物代谢。用SA算法对代谢网络模块化后,发现常温产甲烷菌M. acetivorans有39个模块,包括25个与其它模块之间没有连接的独立模块。独立模块中,有15个模块是纯功能模块。而嗜热产甲烷菌M. kandleri有30个模块,其中20个是独立模块。独立模块中14个模块是纯功能模块。同时发现hub模块是核苷酸代谢、氨基酸代谢以及碳水化合物代谢。Hub模块间的酶的度也远高于整个网络的平均度,尤其是酶EC2.6.1.1, EC1.2.1.3,这表明度高的酶是更重要的。生物网络比对是生物体的结构、功能和进化分析的重要研究手段。最后采用MI-GRAAL算法对两个产甲烷菌的整体代谢网络以及模块化后的hub模块进行比对,发现嗜热的产甲烷菌M. kandleri的hub模块K-1,K-11所特有的酶都包含在高度紧密的子网络k-核中,因此对不同物种的代谢网络的比较研究可分别从高度紧密子网络、模块化以及网络比对等几方面进行。更多还原

【Abstract】 Because thermophiles have strong adaptability to high temperature, they have greatapplication value in genetic engineering, protein engineering, fermentation engineering andutilizationof mineral resources in recent years. In order to clarify heat-resistant mechanism ofthese thermophiles from system level and guide further applications of thermophiles inindustry, this paper focuses on the research of the metabolic network of methanogens. Westudied the metabolic network’s topology structure and modular, and explored the differenceof metabolic network of heat resistance between mesophilic methanogen Methanosarcinaacetivorans (M. acetivorans) and thermophilic methanogen Methan opyrus kandleri(M. kandleri).The paper firstly reconstructed the metabolic network of mesophilic methanogen M.acetivorans and thermophilic methanogen M. kandleri based on the KEGG database, thenverified the metabolic networks which had characters such as small world and scale-freenetworks.According to global comparison of metabolic networks, the pathways00010(glycolysis/glucose dysplasia),00020(citric acid cycle) and00250(Alanine, aspartate andglutamate metabolism) are important pathways in mesophilic methanogen M. acetivorans andthermophilic methanogen M. kandleri. Their degrees are the highest of the entire networkwhich mean that they are appeared in early stage of the evolution. Meanwhile, the relationbetween the classification of the enzymes of metabolic network and degree was examinedbased on the global metabolic network. The lowest degree is synthetases and the higest degreeis oxidoreductases. And transferases are found in their metabolic networks. In order tocompare and analyze the relation between topology and heat resistance of their metabolicnetworks, the degree, degree centrality, closeness centrality, betweenness centrality, clusteringcoefficient and diameter of their metabolic network were calculated. The clusteringcoefficients of the mesophilic methanogen M. acetivorans and thermophilic methanogenM. kandleri metabolic network are much higher than random network of the same size andtopology. It indicates that the two species have high modular structure. And the metabolicnetwork of mesophilic methanogen M. acetivorans has longer average path length, largerdiameter than thermophilic methanogen M. kandleri. It shows that the overall structure ofmesophilic methanogen M. acetivorans is relative low density and loose network structure.The highly closeness subnetwork can reflect the network’s global characteristics, so it isan important factor in comparing the metabolic network of different species of large-scalenetwork. Our experiments shows that the9-core has27enzymes of the mesophilicmethanogen M. acetivorans and7-core of the thermophilic methanogen M. kandleri has19enzymes. The7-core of the thermophic methanogen M. kandleri is divided into two smallnetworks. One has shared enzymes of the two methanogens, the other has particular enzymesof the7-core of thermophilic methanogen M. kandleri. These unique enzymes are associatedwith the synthesis of tyrosine, so the heat resistance of thermophilic methanogen M. kandlerimay be affected by the intracellular tyrosine. The metabolic networks are similar to other complex network systems. They havemodular structure. The effective recognition and extracting the function module of metabolicnetworks can help us grasping the implied function information. Therefore, based on theglobal research of metabolic network, Girvan and Newman’s module identification algorithm(GN) and simulated annealing algorithm (SA) were adopted for analysis the functionalsignificance of these characteristics at the system level. The metabolic network of themesophilic methanogen M. acetivorans is composed of39modules. And the metabolicnetwork of the M. kandleri is divided into30modules. The hub modules are1,4,15and16ofM. acetivorans because the connections of these modular nodes are much closer thanother modules. The hub modules are module1and11of the thermophilic methanogenM. kandleri. The certain functional meaning network modules are identified using GNalgorithm. There are four modules belonging to carbohydrates, one module belonging to thebiosynthesis of secondary metabolism of M. acetivorans. And two modules belong to thecarbohydrate metabolism of the thermophilic methanogen M. kandleri.Biological network alignment is an important research method to the study of organism’sstructure, function and evolutionary analysis. Finally, global metabolic networks of the twomethanogens and hub module were compared after using MI-GRAAL algorithm. All specificenzymes of hub modules K-1and K-11of thermophilic methanogen M. kandleri are includedin a highly close k-core subnetwork. So the comparative study on the metabolic network ofdifferent species can use respectively highly closest subnetwork, modular, and networkalignment methods, etc.更多还原