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。
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.
作者
史晓荣
刘俊
彭彦峰
李林
王文科
王钦宏
Xiaorong Shi;Jun Liu;Yanfeng Peng;Lin Li;Wenke Wang;Qinhong Wang(School of Life Sciences, Shanxi Normal University, Linfen 041004, Shanxi, China;Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute oflndustrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China)
出处
《生物工程学报》
CAS
CSCD
北大核心
2017年第12期1913-1922,共10页
Chinese Journal of Biotechnology
基金
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~~
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
Escherichia coli, pyruvate, Tn5 transposon, genome, pathway engineering, high throughput screening