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途径工程及Tn5转座子介导突变提高大肠杆菌丙酮酸生产
引用本文:史晓荣,刘俊,彭彦峰,李林,王文科,王钦宏.途径工程及Tn5转座子介导突变提高大肠杆菌丙酮酸生产[J].生物工程学报,2017,33(12):1913-1922.
作者姓名:史晓荣  刘俊  彭彦峰  李林  王文科  王钦宏
作者单位:1 山西师范大学 生命科学学院,山西 临汾 041004;2 中国科学院天津工业生物技术研究所 中国科学院系统微生物工程重点实验室,天津 300308,1 山西师范大学 生命科学学院,山西 临汾 041004;2 中国科学院天津工业生物技术研究所 中国科学院系统微生物工程重点实验室,天津 300308,2 中国科学院天津工业生物技术研究所 中国科学院系统微生物工程重点实验室,天津 300308,2 中国科学院天津工业生物技术研究所 中国科学院系统微生物工程重点实验室,天津 300308,1 山西师范大学 生命科学学院,山西 临汾 041004,2 中国科学院天津工业生物技术研究所 中国科学院系统微生物工程重点实验室,天津 300308
基金项目:天津市科技计划项目 (No. 14ZCZDSY00066),中国科学院科技服务网络计划STS (No. KFJ-SW-STS-165) 资助。
摘    要:为了开发丙酮酸高产菌株,以大肠杆菌MG1655为出发菌株,通过基因敲除阻断副产物途径构建了产丙酮酸大肠杆菌工程菌KLPP。进一步利用p UT Mini-Tn5载体进行转座子随机突变,构建了含有7 197个单克隆的突变体文库。使用基于丙酮酸的二硝基苯肼显色法,建立了96孔板-酶标仪快速筛选方法,经过两轮的筛选,成功筛选到了6个突变体菌株,比KLPP丙酮酸产量提高了38%、31%、19%、28%、44%和14%。利用全基因组重测序确定了其转座子插入的位置,进而确定了可能影响丙酮酸产量的基因位点,为后续菌株改造工作奠定了基础。

关 键 词:大肠杆菌,丙酮酸,Tn5转座子,基因组,途径工程,高通量筛选
收稿时间:2017/3/7 0:00:00

Improvement of pyruvate production by Escherichia coli via pathway engineering and Tn5 transposon mediated mutagenesis
Xiaorong Shi,Jun Liu,Yanfeng Peng,Lin Li,Wenke Wang and Qinhong Wang.Improvement of pyruvate production by Escherichia coli via pathway engineering and Tn5 transposon mediated mutagenesis[J].Chinese Journal of Biotechnology,2017,33(12):1913-1922.
Authors:Xiaorong Shi  Jun Liu  Yanfeng Peng  Lin Li  Wenke Wang and Qinhong Wang
Institution:1 School of Life Sciences, Shanxi Normal University, Linfen 041004, Shanxi, China; 2 Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China,1 School of Life Sciences, Shanxi Normal University, Linfen 041004, Shanxi, China; 2 Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China,2 Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China,2 Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China,1 School of Life Sciences, Shanxi Normal University, Linfen 041004, Shanxi, China and 2 Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
Abstract:To develop a high-yield pyruvate strain, we first engineered a pyruvate-producing Escherichia coli KLPP from wild-type E. coli MG1655 by blocking the pathways for byproduct formation via gene knockout. Then, we built a library of mutant containing 7 197 monoclones by using the pUT Mini-Tn5 transposon vector for random mutagenesis with E. coli KLPP. We developed a high-throughput method for pyruvate detection based on dinitrophenylhydrazine reaction using 96-well microplate reader. After two-round screening we successfully obtained six mutants with increased pyruvate titer using this method, the titer of pyruvate was increased by 38%, 31%, 19%, 28%, 44% and 14%, respectively. The position of transposon insertion was determined by whole genome re-sequencing, and the gene locus possibly influencing pyruvate production was analyzed, which laid the foundation for subsequent strain improvement by metabolic engineering.
Keywords:Escherichia coli  pyruvate  Tn5 transposon  genome  pathway engineering  high throughput screening
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