An automated operon refactoring workflow for heterologous expression of non-ribosomal peptide biosynthetic gene clusters

Microbial genomes encode numerous biosynthetic gene clusters, yet most remain experimentally uncharacterized, particularly large non-ribosomal peptide synthetase (NRPS) clusters that resist conventional cloning. Existing refactoring approaches are labor-intensive, depend on de novo DNA synthesis and are difficult to scale. Here we show that OSAR (One-Step Assembly and Refactoring)—an automated platform integrating algorithmic primer design with yeast homologous-recombination cloning—directly assembles and refactors NRPS clusters without custom DNA synthesis. We apply OSAR to 34 low- to medium-GC bacterial NRPS clusters (7–78 kb) and successfully assemble 31. Expression in Bacillus subtilis yields 29 peptides from 10 clusters. Several products arose from cryptic biosynthetic behaviors such as potential module skipping and premature release. OSAR provides a practical workflow for assembly and refactoring of NRPS clusters and may facilitate the translation of sequence information into natural products. Existing approaches for biosynthetic gene cluster (BGC) discovery are labor-intensive, depend on de novo DNA synthesis and are difficult to scale. Here we show that an automated platform for one-step assembly and refactoring of BGCs facilitates facile novel product discovery.