Single-Stranded DNA-Binding Protein and Exogenous RecBCD Inhibitors Enhance Phage-Derived Homologous Recombination in Pseudomonas
Single-Stranded DNA-Binding Protein and Exogenous RecBCD Inhibitors Enhance Phage-Derived Homologous Recombination in Pseudomonas
复制标题
单链 DNA 结合蛋白和外源 RecBCD 抑制剂增强假单胞菌中噬菌体衍生的同源重组
DOI：
10.1016/j.isci.2019.03.007
复制
发表时间：
2019-04-26
期刊：
影响因子：
5.8
通讯作者：
Fu, Jun
中科院分区：
文献类型：
--
作者：
Yin, Jia;Zheng, Wentao;Fu, Jun
现有的用于假单胞菌遗传操作的工具的有限效率限制了该属的基础研究和利用。我们探索了来自铜绿假单胞菌噬菌体Ab 31的λ Red样操纵子（BAS）和来自铜绿假单胞菌致病变种的Rac噬菌体RecET样操纵子（Rec-TEPsy）的性质。菜B728 a。与RecTE（Psy）相比，BAS操纵子在更高的温度下起作用，表明可能是假单胞菌的通用系统。由于在BAS操纵子中缺乏RecBCD抑制剂，我们添加了Red γ或Plu γ，并发现在铜绿假单胞菌和荧光假单胞菌中重组效率增加，但在恶臭假单胞菌和P.单链DNA结合蛋白的过表达在几种情况下增强了重组工程，包括大肠杆菌中的RecET重组。杆菌这些系统的效用通过工程化铜绿假单胞菌基因组以产生减毒的鼠李糖脂生产者来证明。我们的工作增强了假单胞菌功能基因组学的潜力。
The limited efficiency of the available tools for genetic manipulation of Pseudomonas limits fundamental research and utilization of this genus. We explored the properties of a lambda Red-like operon (BAS) from Pseudomonas aeruginosa phage Ab31 and a Rac bacteriophage RecET-like operon (Rec-TEPsy) from Pseudomonas syringae pv. syringae B728a. Compared with RecTE(Psy), the BAS operon was functional at a higher temperature indicating potential to be a generic system for Pseudomonas. Owing to the lack of RecBCD inhibitor in the BAS operon, we added Red gamma or Plu gamma and found increased recombineering efficiencies in P. aeruginosa and Pseudomonas fluorescens but not in Pseudomonas putida and P. syringae. Overexpression of single-stranded DNA-binding protein enhanced recombineering in several contexts including RecET recombination in E. coli. The utility of these systems was demonstrated by engineering P. aeruginosa genomes to create an attenuated rhamnolipid producer. Our work enhances the potential for functional genomics in Pseudomonas.