Harnessing membrane vesicles to activate silent bacterial metabolite production

Most bacterial biosynthetic gene clusters (BGCs) are considered "silent" because they are poorly expressed or entirely inactive under standard laboratory conditions. This results in a significant gap between natural products that have been isolated and the hidden biosynthetic potential of bacteria. Several strategies have been developed to fill this gap by the activation of these silent BGCs. Among them, genetics-free approaches that employ environmental substances to mimic native bacterial growth conditions have proven effective. These methods enhance the chemical diversity of bacterial culture extracts, which can be integrated with diverse screening systems to discover unique natural products. In this study, we employed bacterial membrane vesicles (MVs) to induce silent metabolite production, since MVs are naturally present in bacterial habitats, likely function in interbacterial communication, and can mimic bacterial original growth habitats to trigger natural product biosynthesis. Indeed, Burkholderia multivorans MVs induced the production of a wide variety of natural products in diverse bacterial strains. Herein, we provide guidelines for isolating and characterizing MVs, preparing bacterial culture extracts using MVs, and performing LC-MS-guided screening to identify MV-induced metabolites. This approach not only uncovers the hidden biosynthetic potential of bacteria but also offers valuable insights into the ecological roles of natural products in their native environments.