The lambda phage Red proteins Reda/Redb/Redc and Rac prophage RecE/RecT proteins are powerful tools for precise and efficient genetic manipulation but have been limited to only a few prokaryotes. Here, we report the development and application of a new recombineering system for Burkholderia glumae and Burkholderia plantarii based on three Rac bacteriophage RecET-like operons, RecEThe BDU8 , RecETh TJI49 and RecETh1h2e YI23 , which were obtained from three different Burkholderia species. Recombineering experiments indicated that RecETh TJI49 and RecETh1h2e YI23 showed higher recombination efficiency compared to RecEThe BDU8 in Burkholderia glumae PG1. Furthermore, all of the proteins currently categorized as hypothetical proteins in RecETh1h2e YI23, RecETh TJI49 and RecEThe BDU8 may have a positive effect on recombination in B. glumae PG1 except for the h2 protein in RecETh1h2e YI23 . Additionally, RecET YI23 combined with exonuclease inhibitors Pluc or Redc exhibited equivalent recombination efficiency compared to Redcba in Escherichia coli, providing potential opportunity of recombineering in other Gram-negative bacteria for its loose host specificity. Using recombinase-assisted in situ insertion of promoters, we successfully activated three cryptic non-ribosomal peptide synthetase biosynthetic gene clusters in Burkholderia strains, resulting in the generation of a series of lipopeptides that were further purified and characterized. Compound 7 exhibited significant potential anti-inflammatory activity by inhibiting lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages. This recombineering system may greatly enhance functional genome research and the mining of novel natural products in the other species of the genus Burkholderia after optimization of a protocol.

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