Novel Recombineering Systems For Pseudomonas

Pseudomonas are nuclear-free bacteria,which are straight or slightly curved Gram-negative bacteria,with polar flagella movement.Pseudomonas are capable of organic nutrients,strictly aerobic respiratory metabolism,and are distributed in the soil water and various objects planting,which also hasve a strong ability to break down organic matter,with a variety of organic compounds as an energy source.Wherein the genome sequence results of show secondary metabolites are abundant,and the synthesis mechanism of secondary metabolites is complex,involving PKS,NRPS and NRPS-PKS and other gene cluster.Wherein Pseudomonas putida,Pseudomonas aeruginosa,Pseudomonas fluorescens and Pseudomonas syringae grow rapidly,with abundant secondary metabolites,but there are not effective and simple genetic manipulation methods yet.This subject based Red/ET homologous recombination technology,has built an efficient recombination system in Pseudomonas,and used new recombination system for genetically modified in Pseudomonas,so that they can be effective expression of secondary metabolites.Build a series of recombination system with the Red/ET homologous recombination.Hence we explored the properties of lambda Red-like operon(Orf38,Orf37 and Orf36)from Pseudomonas aeruginosa phage vB_PaeP_Tr60_Ab31,and Rac bacteriophages RecET-like operon(RecTEpsy)from Pseudomonas syringae pv.syringae B728a.BLAST results suggest that Orf38 is a single strand annealing protein equivalent to Red?,Orf37 is a 5'-3' exonuclease equivalent to Reda.Compare and select the best recombination system,and make genome modification in different Pseudomonas using the best recombination system.The common Pseudomonas are Pseudomonas putida,Pseudomonas aeruginosa,Pseudomonas fluorescens and Pseudomonas syringae.Firstly,compare and identify the efficiency of several recombination systems in different Pseudomonas,determine the best recombination system in different bacteria.By comparison and identification,select the best recombination system of different bacteria,and make genome modification in Pseudomonas successfully.Recombineering experiments indicate that hybrid systems with Redy/Pluy,based on lambda Red-like operon and RecET-like operon,works in Pseudomonas species,and SSB could increase efficacy significantly.The new gene recombination systems by establishment and optimization in Pseudomonas can make genetic manipulation quickly and easily in Pseudomonas,which consist of dsDNA exonuclease,ssDNA annealed enzymes and exonuclease inhibition proteins.The reaction substrate dsDNA can be obtained by one-step PCR.In the case of genetic manipulation,the desired recombination is reconstituted in one step,which could promote the functional genomics of Pseudomonas and other related strains.The SSB protein of Pseudomonas aeruginosa phage can effectively improve the recombination efficiency of Pseudomonas.Recombineering experiments indicate that hybrid system with Redy/Plu? works well in some Pseudomonas species,and SSB could increase efficiency significantly.Results indicate that the efficiency enhancement from SSB is related to host proteins.Biochemical studies of different recombination systems have revealed that the SSB binds to and removes secondary structure from ssDNA in presynapsis stage,which suggest that SSB could denature secondary structure of ssDNA so as to increase the efficiency in the recombineering.However,our results revealed that the recombineering efficiency was not increased when the secondary structure is high.Therefore,further studies are necessary for illustrating the SSB biological role in recombineering.Allelic exchange is an efficient technique,but still time-consuming for bacterial genome engineering.Lack of effective tools for genetic manipulation of Pseudomonas and limits the investigation of their functional genomics.Hence we explored the properties of lambda Red-like operon(Orf38,Orf37 and Orf36)from Pseudomonas aeruginosa phage Ab31,and Rac bacteriophages RecET-like operon(RecTEPsy)from Pseudomonas syringae pv.syringae B728a.BLAST results suggest that Orf38 is a single strand annealing protein equivalent to Redp,Orf37 is a 5'-3' exonuclease equivalent to Reda.Recombineering experiments indicate that hybrid systems with Redy/Pluy,based on lambda Red-like operon and RecET-like operon,works in Pseudomonas species,and SSB could increase efficacy significantly.This novel recombineering systems were demonstrated by engineering Pseudomonas.Our studies unbolted the potentiality of recombineering for functional genomics in the Pseudomonas and related species.