The Burkholderia genus possesses ecological and metabolic diversities. A large number of silent biosynthetic gene clusters (BGCs) in the Burkholderia genome remain uncharacterized and represent a promising resource for new natural product discovery. However, exploitation of the metabolomic potential of Burkholderia is limited by the absence of efficient genetic manipulation tools. Here, we screened a bacteriophage recombinase system Redγ-BAS, which was functional for genome modification in the plant pathogen Burkholderia gladioli ATCC 10248. By using this recombineering tool, the constitutive promoters were precisely inserted in the genome, leading to activation of two silent nonribosomal peptide synthetase gene clusters (bgdd and hgdd) and production of corresponding new classes of lipopeptides, burriogladiodins A-H (1-8) and haereogladiodins A-B (9-10). Structure elucidation revealed an unnatural amino acid Z- dehydrobutyrine (Dhb) in 1-8 and an E-Dhb in 9-10. Notably, compounds 2-4 and 9 feature an unusual threonine tag that is longer than the predicted collinearity assembly lines. The structural diversity of burriogladiodins was derived from the relaxed substrate specificity of the fifth adenylation domain as well as chain termination conducted by water or threonine. The recombinase-mediating genome editing system is not only applicable in B. gladioli, but also possesses great potential for mining meaningful silent gene clusters from other Burkholderia species.

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