Abstract
Marine microorganisms possess vast biosynthetic potential, yet most of their biosynthetic gene clusters (BGCs) remain transcriptionally silent under laboratory conditions. Genetic intractability has been a major barrier to activating these cryptic pathways. Here, we present RECC, an integrated Red/ET-CRISPR/Cas9 system that enables seamless, marker-free genome editing in marine bacteria. RECC couples Red/ET recombineering with CRISPR/Cas9-mediated cleavage, allowing the incorporation of homology arms and protospacers into a single construct through one-step Gibson assembly, thereby substantially simplifying the engineering process. Using Pseudoalteromonas flavipulchra DSM 14401 as a model, we employed RECC to replace the native promoter of a silent nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) hybrid gene cluster with a strong constitutive promoter. This targeted activation led to the production of a series of previously unknown cyclolipopeptides, designated flavipulchrins. Structural characterization and bioinformatic analysis revealed a plausible biosynthetic pathway for these metabolites. Collectively, RECC provides a robust and generalizable genome-editing platform that facilitates the systematic exploration of biosynthetic potential in genetically recalcitrant marine microorganisms.