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.
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pUT 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。.
Keywords: Escherichia coli; Tn5 transposon; genome; high throughput screening; pathway engineering; pyruvate.