钙离子介导的热激蛋白dnaK操纵子对拜氏梭菌代谢的调控研究

项目名称： 钙离子介导的热激蛋白dnaK操纵子对拜氏梭菌代谢的调控研究

项目编号： No.31200031

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 微生物学、植物学

项目作者： 韩蓓

作者单位： 西安交通大学

项目金额： 23万元

中文摘要： 本实验室研究发现CaCO3促进了拜氏梭菌产溶剂代谢，可增加总溶剂产量达125%，并显著提高了胞内热激蛋白dnaK操纵子的转录和表达,推测拜氏梭菌中,钙离子可通过促进热激蛋白高表达来正调控全局代谢,并提高细胞的抗逆性。本课题拟对热激蛋白dnaK操纵子在响应CaCO3后对细胞代谢的调控途径及作用靶标进行研究，内容包括热激蛋白dnaK体外抗逆活性测定分析；对dnaK操纵子进行不同的上调表达后的产溶剂代谢途径及代谢产物分析；并比较野生菌株及重组菌株对Ca2+的添加与否的不同表现，最终确定Ca2+促进热激蛋白dnaK操纵子高表达的方式，进一步阐明Ca2+通过对热激蛋白dnaK操纵子的调控进而促进细胞产溶剂代谢的机制，并以此为基础构建高产丁醇的工程菌株，为深入研究拜氏梭菌代谢，提高发酵产率，进行工业发酵生产提供数据支持，奠定理论基础。

中文关键词： 拜氏梭菌；产溶剂代谢；钙离子；调控；退化菌株

英文摘要： CaCO3 addtition led to at least 125% higher total solvent production by Clostridum beijerinckii NCIMB8052, and the heat shock protein dnaK was highly transcripted and expressed in the CaCO3 treatment cell. It was hypothesized that the calcium ion could stimulate the expression of dnaK operon, as general positive regulator. In this proposal, the responses mechnism and corresponding target in the dnaK operon to the addition of CaCO3 will be investigated, which inculding the anti-stress activity assay of heat shock protein DnaK in vitro; the up-regulation of dnaK operon, and the transcription, expression, and activity analysis of the key enzymes during the solventogenic metabolism; comparasion of the fermentation parameters between the wild-type strain and recombinant strains with and without addition of CaCO3. The objective of this study is to identify and illustrate the mechnism of high expression of dnaK operon by stimulating of calcium ion. Based on this research of calcium-mediated heat shock protein dnaK operon on the metabolism regulation of C. beijerinckii, and the constructed high-butanol producing recombinant strains, it will provide significant experimental data and more theoretical support to the industrial bio-butanol fermentation.

英文关键词： Clostridum beijerinckii；solventogenic metabolism；Ca2+；regulation；degenerated strain