联苯培养条件下红球菌R04转录应答及其中间代谢物原儿茶酸的生物转化

发布时间：2019-01-27 20:03

【摘要】：随着对多氯联苯(PCBs)生物降解过程中代谢动力学和代谢机理的研究日趋成熟,人们对多氯联苯代谢菌的研究逐渐转移到了基因组和转录组水平上,基因组和转录组研究不仅能够从根本上揭示特定生物学过程的分子机理,还能直观监测整个细胞代谢特征,进而获得联苯/PCBs胁迫下,微生物相关细胞生理调节反应和联苯通路可能存在的辅助机制信息。为了研究不同碳源培养条件下,特别是联苯条件下红球菌的细胞转录应答,挖掘与多氯联苯(PCBs)转运、代谢及其调控相关的基因,为进一步全面理解PCBs微生物降解的分子机制奠定基础。本课题以一株多氯联苯降解菌红球菌(Rhodococcussp.R04)为材料,分别提取不同碳源(乙醇、葡萄糖和联苯)培养条件下菌体的总RNA,反转录合成cDNA。采用高通量测序法分别对这三种样品进行转录组测序,分析测序数据得出全基因组表达模式,并对不同条件下的基因表达进行差示分析,进而对联苯代谢网络和红球菌中其他基因的转录调节和代谢应答反应做出相关性分析。并通过Q-RT-PCR分析不同碳源培养条件下的基因表达情况。测序结果表明,与葡萄糖和乙醇相比,联苯培养条件下显著上调(log2 Ratio"g1)基因个数分别为375和332个。与葡萄糖相比,联苯培养条件下相关基因上调表达量与Q-RT-PCR实验结果基本一致。功能分类获得细胞组分、分子功能和生物学过程三大类别160多个细小分支的差示表达基因,部分基因参与联苯代谢转录调控、联苯转运、抗氧化应激反应以及信号传导通路系统等多种生理过程。参与联苯上游代谢途径的众多同工酶基因中,只有bphC2和bphDl在联苯中大量上调表达,其余同工酶在联苯中基本量不变或下调表达。转录组注释及差示分析推测,红球菌R04中苯甲酸的代谢主要是通过儿茶酚邻位途径、间位途径以及原儿茶酸途径三条代谢途径完成。与葡萄糖和乙醇相比,红球菌R04在联苯培养条件下基因表达差异明显,这为我们进一步解析多氯联苯代谢特征和代谢调控提供理论依据。原儿茶酸是多种中药的主要活性成分和多种药物的前体物质,其生物学功效具有抗氧化、抗菌、抗肿瘤、抗炎等作用。目前国内市场对原儿茶酸需求量逐年增长,其生产方法主要是采用化学化工法从植物中抽提,提取工艺复杂,在多步提取过程中都有不同程度的损耗,提取率低,且对生态环境造成一定破坏。本课题从生物转化角度解决原儿茶酸生产工艺中的各种问题,首次运用生物酶催化法生产原儿茶酸。将对羟基苯甲酸-3-羟化酶基因重组表达在大肠杆菌BL21(DE3)细胞中,成功构建了一株高效生产原儿茶酸的工程菌,实现了原儿茶酸的绿色催化。此外,本课题对实验条件进行优化以提高原儿茶酸的产率,底物对羟基苯甲酸在每OD菌液中转化量可达到250～300μg。
[Abstract]:With the study of metabolic kinetics and metabolic mechanism of PCBs (PCBs) biodegradation, the study of PCBs metabolic bacteria gradually transferred to genome and transcriptome level. Genome and transcriptome studies can not only reveal the molecular mechanism of specific biological processes, but also directly monitor the metabolic characteristics of the whole cell, and then obtain biphenyl / PCBs stress. Microorganism related cell physiological regulation response and biphenyl pathway may exist auxiliary mechanism information. In order to study the transcriptional response of erythrococcus cells in different carbon source cultures, especially in biphenyls, the genes related to (PCBs) transport, metabolism and regulation of PCBs were identified. It lays a foundation for further understanding the molecular mechanism of microbial degradation of PCBs. In this study, a polychlorinated biphenyls degrading bacterium erythrococcus (Rhodococcussp.R04) was used as a material to extract total RNA, reverse transcriptional synthesis of cDNA. from different carbon sources (ethanol, glucose and biphenyls). The three samples were sequenced by high throughput sequencing. The whole genome expression pattern was obtained by analyzing the sequencing data, and the gene expression under different conditions was analyzed by differential analysis. The relationship between biphenyl metabolic network and transcription regulation and metabolic response of other genes in erythrococcus was analyzed. Q-RT-PCR was used to analyze the gene expression under different carbon source culture conditions. Compared with glucose and ethanol, the number of up-regulated (log2 Ratio "g1) genes in biphenyl culture was 375 and 332, respectively. Compared with glucose, the up-regulated expression of related genes in biphenyl culture was basically consistent with that of Q-RT-PCR. The differentially expressed genes of more than 160 small branches of cell components, molecular functions and biological processes were obtained by functional classification. Some of the genes were involved in the regulation of biphenyl metabolism and transcription, and the transport of biphenyls. Antioxidant stress response and signal transduction pathway system and other physiological processes. Among the many isozyme genes involved in the upstream metabolic pathway of biphenyls, only bphC2 and bphDl upregulated the expression in biphenyls, while the other isozymes remained unchanged or down-regulated in biphenyls. Transcription notes and differential analysis suggested that the metabolism of benzoic acid in Rhodococcus R04 was mainly accomplished by three metabolic pathways, namely, catechol-adjacent pathway, meso-site pathway and protocatechuic acid pathway. Compared with glucose and ethanol, the gene expression of Rhodococcus rupestris R04 was significantly different from that of glucose and ethanol, which provided a theoretical basis for further analysis of metabolic characteristics and regulation of PCBs. Protocatechuic acid is the main active component of many kinds of traditional Chinese medicine and the precursor of many kinds of drugs. Its biological effects are antioxidant, antibacterial, anti-tumor, anti-inflammatory and so on. At present, the demand for protocatechuic acid in domestic market is increasing year by year. The main production method is to extract from plants by chemical and chemical methods. The extraction process is complex, and the extraction rate is low and the losses are different in the process of multi-step extraction. And caused certain damage to the ecological environment. In this paper, various problems in protocatechuic acid production were solved from the point of view of biotransformation, and protocatechuic acid was first produced by bioenzyme catalysis. The gene of p-hydroxybenzoic acid-3-hydroxylase was expressed in E. coli BL21 (DE3) cells. An engineering strain producing protocatechuic acid was successfully constructed, and the green catalysis of protocatechuic acid was realized. In addition, the experiment conditions were optimized to improve the yield of protocatechuic acid. The conversion of p-hydroxybenzoic acid in the solution of OD could reach 250 渭 g.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X172;Q78

【共引文献】