Significance Microbes have the genetic capacity to produce large numbers of specialized compounds, yet produce only a small fraction of these in the laboratory. Here we introduce a genetic platform that allows the efficient production of natural product molecules from uncharacterized gene collections. We used transformation-associated recombination in yeast to directly clone and express an orphan biosynthetic gene cluster for the production of the lipopeptide antibiotic taromycin A. With this direct cloning approach, a single genomic capture and expression vector was designed directly from next-generation sequencing data, which precisely captures genetic loci of interest and readily facilitates genetic manipulations. This study highlights a “plug-and-play” approach to cryptic biosynthetic pathways for the discovery and development of natural product drug candidates.

Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A