大肠杆菌DNA聚合酶III突变体DnaEI171L抑制噬菌体复制的分子机制解析及抗噬菌体底盘细胞构建

噬菌体侵染对发酵工业影响严重，目前关于细菌抗噬菌体侵染的机制解析依然有限。项目前期基于高通量筛选获得抗噬菌体大肠杆菌DNA聚合酶III突变体DnaEI171L，该突变蛋白使噬菌体侵染宿主细胞的滴度下降2800倍，且对宿主细胞本身生长基本无影响，目前其分子机制尚不清楚。项目拟在前期初步明确DnaEI171L与噬菌体基因组复制相关后，组合RT-PCR和Southern Bolt等技术验证DnaEI171L影响噬菌体复制效率。通过转录组学、Pull-down assay和ITC assay等技术确定与DnaEI171L结合的靶点，最终阐述DnaEI171L抑制噬菌体复制的分子机制。针对该突变株抗噬菌体强度仍有一定局限性，拟采用人工辅助蛋白质进化系统对DnaE突变体进行改造，以获得更强的抗噬菌体突变蛋白。并基于合成生物学技术构建高抗、广谱的抗噬菌体大肠杆菌底盘细胞。

Phage pollution does serious harm to fermentation industry, however, the molecular mechanism behind bacterial resistance to phage infection remains limited. In previous work, a DNA polymerase III mutant DnaEI171L resistant to phage Φ21 infection was isolated based on high-throughput screening technology. The titer of phage has reduced by 2800 times when phage was infected to the DnaEI171L mutant compared to wild-type strain BL21, and DnaEI171L mutant had no effect on the growth of the strain BL21.The molecular mechanism of how DnaEI171L regulate the resistance of E. coli to phage infection is not clear. In this project, to confirm that the mechanism of DnaEI171L resistant to phage infection is related to phage genome replication as our previous results, RT-PCR and Southern bolt will be used to confirm that DnaEI171L affect phage genome replication efficiency. Transcriptional assay, pull-down assay and ITC assay will be used to identify the binding site and the molecular mechanism of DnaEI171L regulates in Escherichia coli related to phage resistance. In view of the limitation of the dnaE mutant BL21/DnaEI171L resistant to phage infection, we propose to use the artificial evolution system to carry out the evolution of DnaE mutant to obtain stronger phage resistant mutant protein. Finally, based on the synthetic biology technology, a high resistant and broad-spectrum anti-phage E. coli chassis cell will be constructed.