Establishment of recombineering genome editing system in Paraburkholderia megapolitana empowers activation of silent biosynthetic gene clusters

Summary The Burkholderiales are an emerging source of bioactive natural products. Their genomes contain a large number of cryptic biosynthetic gene clusters (BGCs), indicating great potential for novel structures. However, the lack of genetic tools for the most of Burkholderiales strains restricts the mining of these cryptic BGCs. We previously discovered novel phage recombinases Redαβ7029 from Burkholderiales strain DSM 7029 that could help in efficiently editing several Burkholderiales genomes and established the recombineering genome editing system in Burkholderialse species. Herein, we report the application of this phage recombinase system in another species Paraburkholderia megapolitana DSM 23488, resulting in activation of two silent non‐ribosomal peptide synthetase/polyketide synthase BGCs. A novel class of lipopeptide, haereomegapolitanin, was identified through spectroscopic characterization. Haereomegapolitanin A represents an unusual threonine‐tagged lipopeptide which is longer than the predicted NRPS assembly line. This recombineering‐mediated genome editing system shows great potential for genetic manipulation of more Burkholderiales species to activate silent BGCs for bioactive metabolites discovery. We establish the optimal genome editing system for P. megapolitana DSM 23488 by screening different recombineering systems from Burkholderiales and Pseudomonas species, and implement recombineering‐assisted promoter insertion to awaken two silent BGCs. A class of threonine‐tagged lipopeptide, haereomegapolitanin, is identified, and haereomegapolitanin A is found to have biosurfactant activity and promotion of bacterial cell motility. This recombineering‐mediated genome editing system offers more possibility for manipulation of more Burkholderiales species, facilitating secondary metabolite mining, and accelerating the discovery of potentially novel bioactive compounds.