RECC: A Red/ET–CRISPR/Cas9-based system enabling genome mining of marine Pseudoalteromonas for novel natural products

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 R ed/ E T– C RISPR/ C as9 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. • RECC integrates CRISPR/Cas9 with Red/ET for marker-free and electroporation-free gene editing in Pseudoalteromonas. • RECC enables scarless genome editing with an approximately 40 % correct rate, reducing cycle time by 2–3 times. • Using RECC a cryptic NRPS-PKS BGC in P. flavipulchra is activated, revealing two new cyclolipopeptides.