Recombineering-Mediated Genome Editing in Burkholderiales Strains

Red/ET recombineering is primarily mediated by the E. coli recombinase pair Redα/Redβ from λ phage or RecE/RecT from Rac prophage, which is applied in E. coli and also closely related Gram-negative bacteria for efficient genome editing. However, some distant bacterial species like Burkholderiales strains require host-specific Redα/Redβ recombinase pair for highly efficient genome editing. A pair of recombinases Redαβ7029 from the Burkholderiales strain DSM 7029, recently identified as Schlegelella brevitalea, were identified for efficient genetic manipulation in the native strain and several other Burkholderiales strains. In this chapter, we describe a detailed protocol for genome engineering in Burkholderiales strains via the Redγ-Redαβ7029 recombineering and Cre/loxP site-specific recombination.