RAISE技术改组转录因子RpoD调控大肠杆菌的低pH值耐受性  

Tailoring of Transcription Factor RpoD by RAISE to Regulate Low pH Value Tolerance of Escherichia Coli

作　　者：高茜[1] 朱丽英[2] 周伟[1] 梁世中[3] 江凌[1,4] 

机构地区：[1]南京工业大学生物与制药工程学院,江苏南京210009 [2]南京工业大学化学与分子工程学院,江苏南京210009 [3]华南理工大学生物科学与工程学院,广东广州510641 [4]南京工业大学食品与轻工学院,江苏南京210009

出　　处：《化学与生物工程》2016年第3期14-18,52,共6页Chemistry & Bioengineering

基　　金：国家自然科学基金青年基金资助项目(21506101);国家973计划资助项目(2012CB725204);江苏省自然科学基金资助项目(BK20131046;BK20130917);江苏省高校自然科学基金面上项目(14KJB530003);教育部高等学校博士学科点专项科研基金资助项目(20123221120011);广东省优秀博士专项基金资助项目(sybjjxm201217)

摘　　要：在生物炼制生产过程中,微生物由于自身对酸的抗逆性差,其代谢生产过程会受到严重的影响。为解决该问题,本实验采用PCR方法从E.coli DH5α中扩增分离得到分子量为2 075bp的RpoD完整序列(包含天然启动子区域和终止子区域),并利用RAISE方法,通过突变全局调控因子RpoD来提高大肠杆菌的耐酸能力。通过对104随机突变文库的多轮筛选,获得最佳突变菌株MutantⅦ。在pH值为3.0、3.5、4.0、5.0、6.0和7.0的条件下,对MutantⅦ和对照菌株的生长情况进行比较分析,结果表明,在酸性条件下MutantⅦ较对照菌株具有明显的生长优势,尤其在pH值为3.0时,相比对照菌株0.015h-1的生长速率,MutantⅦ的生长速率达到0.022h-1,具有较高应用价值。In biorefinery processes,microbial metabolism was seriously suffered by the acid resistance of microbes themselves.To solve this problem,2 075 bp DNA fragment encoding RpoD,including promoter regions and termination regions,was cloned fromE.coli DH5αby PCR method.And RAISE method was adopted to engineer the components of global regulator RpoD of E.coli to improve its acid tolerance.The best strain MutantⅦ was identified from 104 random mutagenesis libraries based on the growth performance.Results showed that,under the pH value of 3.0,3.5,4.0,5.0,6.0and 7.0conditions,MutantⅦ grew much better than the control strain.It was found that MutantⅦ exhibited a significantly improved growth rate through multiwheel of acidshock screening,which was 0.022h-1 as compared to that of 0.015h-1 with the control strain at pH value of3.0.

关 键 词：大肠杆菌 耐酸性 RPOD RAISE方法 

分 类 号：Q754[生物学—分子生物学]