Develop A Novel Genome Engineering System For Burkholderia Based On The Endogenous Phage Recombinase And Its Application

The genus Burkholderia is a gram-negative bacterium that exists extensively in the environment.By bioinformatics analysis of the genome of the genus Burkholderia,we retrieved a large number of silent NRPS/PKS gene clusters,which are potential resources for new drug discovery.However,due to the lack of efficient and simple genetic tools in the genus Burkholderia,functional genome research of the genus Burkholderia is slow.It is necessary to establish an efficient and simple genetic engineering system to speed up the Burkholderia research and utilization.The Red/ET homologous recombination technique can be used to modify E.coil chromosome.The RecET homologous recombinase proteins are derived from the Escherichia coli Rac prophage.The Reda?? homologous recombinase proteins are found in the Red operon of the lambda phage.The RecET and Reda?? greatly improve the efficiency of recombination in E.coli and provide powerful genetic engineering techniques,RecE and Reda have 5'-3' exonuclease activity;RecT and Red? are single strand annealing protein;·Redy prevent the RecBCD to degrade exogenous DNA and thereby improve the efficiency of recombination.Because of the host specificity of homologous recombinases,RecET and Reda?y cannot efficiently work in other gram-negative bacteria.If two proteins share more than 30%similarity,they might be functional analogues.We took amino acid sequences of RecE,RecT,Reda and Red? as query,using the NCBI BlastP to mine the homologous recombination proteins of genus BurkholderiaBased on E.coli RecET and Reda? y we found the similar operons in Burkholderia sp.BDU8,Burkholderia sp.TJI49 and Burkholderia sp.Y123.Based on these three operons we constructed several recombinase expression plasmids which were evaluated in different species of Burkruholderia aiming for optimal genetic manipulation tools.After the recombination function of the candidate proteins were determined,we systematically optimized the parameters for electrporation and recombination based on Burkholderia glumae PG1.The new homologous recombination system was introduced into different strains of Burkholderia for genome engineering to activate three silent NRPS/PKS gene clusters.The general utility of the new recombineering system for Burkholderia was validated.Development of new recombineering system using the native phage recombinase is the most effective way to build an efficient genetic engineering system for Burkholderia.The innovation of this technology will greatly facilitate the functional genome research and utilization of Burkholderia.