Single-Stranded DNA-Binding Protein and Exogenous RecBCD Inhibitors Enhance Phage-Derived Homologous Recombination in Pseudomonas

Jia Yin; Wentao Zheng; Yunsheng Gao; Chanjuan Jiang; Hongbo Shi; Xiaotong Diao; Shanshan Li; Hanna Chen; Hailong Wang; Ruijuan Li; Aiying Li; Liqiu Xia; Yulong Yin; A Francis Stewart; Youming Zhang; Jun Fu

doi:10.1016/j.isci.2019.03.007

Single-Stranded DNA-Binding Protein and Exogenous RecBCD Inhibitors Enhance Phage-Derived Homologous Recombination in Pseudomonas

iScience. 2019 Apr 26:14:1-14. doi: 10.1016/j.isci.2019.03.007. Epub 2019 Mar 12.

Authors

Jia Yin¹, Wentao Zheng², Yunsheng Gao², Chanjuan Jiang², Hongbo Shi², Xiaotong Diao², Shanshan Li², Hanna Chen³, Hailong Wang², Ruijuan Li², Aiying Li², Liqiu Xia³, Yulong Yin⁴, A Francis Stewart⁵, Youming Zhang⁶, Jun Fu⁷

Affiliations

¹ Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, Suzhou Institute of Shandong University, 266235 Qingdao, China; Hunan International Joint Laboratory of Animal Intestinal Ecology and Health, Laboratory of Animal Nutrition and Human Health, College of Life Sciences, Hunan Normal University, 410081 Changsha, China.
² Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, Suzhou Institute of Shandong University, 266235 Qingdao, China.
³ Hunan International Joint Laboratory of Animal Intestinal Ecology and Health, Laboratory of Animal Nutrition and Human Health, College of Life Sciences, Hunan Normal University, 410081 Changsha, China.
⁴ Hunan International Joint Laboratory of Animal Intestinal Ecology and Health, Laboratory of Animal Nutrition and Human Health, College of Life Sciences, Hunan Normal University, 410081 Changsha, China; Chinese Academy of Science, Institute of Subtropical Agriculture, Research Center for Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Science and Key Laboratory for Agroecological Processes in Subtropical Region, Scientific Observation and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, 410125 Changsha, China.
⁵ Biotechnology Research Center, Center for Molecular and Cellular Bioengineering, Dresden University of Technology, BioInnovationsZentrum, Tatzberg 47-51, 01307 Dresden, Germany. Electronic address: francis.stewart@tu-dresden.de.
⁶ Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, Suzhou Institute of Shandong University, 266235 Qingdao, China. Electronic address: zhangyouming@sdu.edu.cn.
⁷ Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, Suzhou Institute of Shandong University, 266235 Qingdao, China. Electronic address: fujun@sdu.edu.cn.

Abstract

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 (RecTE_Psy) 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 Redγ or Pluγ 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.

Keywords: Bioengineering; Biological Sciences; Biotechnology; Cell Biology; Molecular Biology.