低Mg2+诱导的大肠杆菌裂解系统的构建

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	低Mg2+诱导的大肠杆菌裂解系统的构建
其他题名	无
	姚岚
导师	赵广 ; 刘辉
	2016-05
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	生物工程
关键词	大肠杆菌 Λ噬菌体裂解基因 Mg2++ 聚-3羟基丙酸
摘要	微生物代谢产物可以分为胞外产物和胞内产物两类。与胞外产物相比，胞内产物的分离首先要通过物理或化学手段进行细胞破碎，使代谢产物释放。目前常用的细胞破碎方法存在着能耗高、污染大等弊端，因此我们希望构建一个自诱导裂解系统，使细胞在发酵后期裂解并释放产物。大肠杆菌λ噬菌体裂解基因（SRRZ）编码三个蛋白，其中基因S编码穿孔素（holin），攻击内膜蛋白；基因R编码裂解素（endolysin），使细胞壁水解；基因RZ编码辅助裂解因子。将裂解基因连接到质粒pBAD18上表达，构建了重组菌株Q2537。在Q2537培养过程加入阿拉伯糖诱导后，菌体迅速裂解，证明了裂解基因的有效性。考虑到实际生产过程，我们希望建立一种更为经济、简单的裂解系统。基因mgtA的转录受到二组分系统PhoPQ和其mRNA 5’非翻译区（5’UTR）内核糖开关的共同控制。Mg2+浓度较低的条件会激活受PhoP调节基因mgtA的表达；在Mg2+浓度较高的条件下，基因mgtA的表达受到抑制。mgtA核糖开关可作为细胞内Mg2+浓度传感器，并根据Mg2+浓度形成不同的高级结构，当细胞内Mg2+浓度升高时，核糖开关的特定结构会在转录进行到编码区之前将其提前终止。在本研究中，我们克隆了λ噬菌体裂解基因SRRZ，对裂解基因的裂解效果进行了验证，发现在1小时内，菌株即表现出明显的裂解效果；然后在裂解基因前插入基因mgtA的启动子PmgtA. 重组质粒在不同宿主中均表现出明显的裂解效果，裂解效率在90%以上，但是在聚3-羟基丙酸（P3HP）的发酵过程中发现，质粒丢失现象严重，产物释放率较低。因此，我们将片段PmgtA-SRRZ克隆到P3HP生产菌株Q1638携带的质粒pW01中，使双质粒系统转变为单质粒系统，质粒稳定性提高2.7倍，同时在聚3-羟基丙酸（P3HP）的发酵过程中，P3HP的产量占细胞干重的70 %以上，比原始菌株提高了2.37倍，单质粒裂解系统产物释放率大幅提高；为进一步提高基因的稳定性，同时减少抗生素的使用，降低生产成本，我们利用染色体整合（Chromosome Gene Integration，CGI）手段，将裂解系统的基因片段整合到基因组染色体上进行表达，但是CGI裂解效果较差，将启动子更换为PT7或Plac后，短时间内仍无明显的裂解效果。随着现代生物技术手段的不断发展，微生物法合成生物化工产品得到了迅速地发展。经济方便的产品分离提纯过程，对微生物发酵的发展十分重要。我们建立的肠杆菌自诱导裂解系统，可以通过微生物代谢过程中对Mg2+的消耗，在发酵后期自行裂解并释放产物，操作方便；裂解基因的表达不需要使用昂贵的诱导剂，经济性高；同时，裂解系统的裂解效率高，产物释放率高。Mg2+诱导的大肠杆菌自诱导裂解系统除用于P3HP的产物回收外，还可用于其他胞内产物如蛋白质等的分离，为微生物代谢产物的分离提供了新思路，对微生物发酵产业的发展意义深远。
其他摘要	Microbial metabolites can be divided into two categories, extracellular products and intracellular products. Compared with the extracellular products, the separation of intracellular products is more complicated. First the cells should be broken up by physical or chemical methods to release the metabolites. The common methods of cell disruption has big drawbacks such as high energy consumption, high pollution. So we want to build an auto-inducible lysis system which can make the cells break up during the later period of fermentation. λ bacteriophage lysis genes (SRRZ) has three subunits, gene S encodes the protein of holin and holin accumulates in the cell membrane, gene R encodes the protein of endolysin and endolysin hydrolyzes the cell wall, gene RZ encodes the cofactor. The lysis genes were inserted into the plasmid pBAD18 and the recombinant strains Q2537 was constructed. In the process of the culture of Q2537, arabinose was added to induce the lysis genes expression. The cell concentration dropped quickly by induction. And considering the practical production proces, we hope to establish a more economic and simple lysis system. It is suggested that two independent mechanisms are involved in Mg2+-dependent transcriptional regulation of mgtA. (i) The PhoP/PhoQ two-component system responds to micromolar levels of environmental Mg2+ and activates transcription initiation; or to milli-molar levels of Mg2+ and represses transcription initiation. (ii) Once transcription is initiated, the 50 untranslated region of nascent mgtA transcripts functions as an alternative Mg2+-sensing system. If the Mg2+ concentration increases in the bacterial cytoplasm, the latter system interrupts mgtA transcription before it is extended to the downstream coding region. In this study, λ bacteriophagelysis genes (SRRZ) was cloned, and then the promter of mgtA was inserted before SRRZ. Because the promter PmgtA will activate genes expression in milli-molar levels of Mg2+ and more Mg2+ will be applied to keep the regular growth of cells which will adversely affect the accumulation of metabolites. So the 5' untranslated region (5'-UTR) of mgtA and the promter PmgtA were both inserted to ensure SRRZ express in micromolar levels of environmental Mg2+. To improve the plasmid stability, genetic fragments PmgtA and PmgtA-UTR were cloned into the plasmid PW01 respectively and the double plasmid system was changed into single plasmid system. In order to further increase the stability of the genes and reduce antibiotic use simultaneously, the Mg2+-inducible lysis system was integrated into the host genome using chromosome gene integration（CGI） method. In lysis capacity experiments, the lysis rates of all the recombinant strains except CGI strains were all more than 90%. The plasmid stability was 2.7 times higher in single plasmid system than the double plasmid system. P3HP was accounted for 50% of total dried cell，which was 2.37 times higher than the original strains. But the CGI strains did not show obviouslysis capacity. With the development of modern bio-technology, microbial synthesis of bio-chemicalproducts developed rapidly. Aeconomical convenience method is very important for the developmentof microbial fermentation. The auto-inducible lysis system can make cells break up during the later period of fermentation by microbial metabolism of Mg2+ and it waseasy to operate; the expression of lysis genes did not need expensive inducer and the cracking efficiency and products release rate were high. The auto-inducible Escherichia coli lysis system can be applied on other products separation such as extracellular proteins. It provided new ideas to the isolation of metabolism products which has important significance to the development of animalcule fermentation engineering.
作者部门	材料生物技术研究中心
学科领域	工业催化
公开日期	2019
学位类型	硕士 ; 学位论文
语种	中文
文献类型	学位论文
条目标识符	http://ir.qibebt.ac.cn/handle/337004/9773
专题	生物基材料组群
作者单位	中国科学院青岛生物能源与过程研究所
推荐引用方式 GB/T 7714	姚岚. 低Mg2+诱导的大肠杆菌裂解系统的构建[D]. 北京. 中国科学院研究生院,2016.

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