CIFR (Clone–Integrate–Flip-out–Repeat): A toolset for iterative genome and pathway engineering of Gram-negative bacteria

Advanced genome engineering enables precise and customizable modifications of bacterial species, and toolsets that exhibit broad-host compatibility are particularly valued owing to their portability. Tn5 transposon vectors have been widely used to establish random integrations of desired DNA sequences into bacterial genomes. However, the iteration of the procedure remains challenging because of the limited availability and reusability of selection markers. We addressed this challenge with CIFR, a mini-Tn5 integration system tailored for iterative genome engineering. The pCIFR vectors incorporate attP and attB sites flanking an antibiotic resistance marker used to select for the insertion. Subsequent removal of antibiotic determinants is facilitated by the Bxb1 integrase paired to a user-friendly counter-selection marker, both encoded in auxiliary plasmids. CIFR delivers engineered strains harboring stable DNA insertions and free of any antibiotic resistance cassette, allowing for the reusability of the tool. The system was validated in Pseudomonas putida, Escherichia coli, and Cupriavidus necator, underscoring its portability across diverse industrially relevant hosts. The CIFR toolbox was calibrated through combinatorial integrations of chromoprotein genes in P. putida, generating strains displaying a diverse color palette. We also introduced a carotenoid biosynthesis pathway in P. putida in a two-step engineering process, showcasing the potential of the tool for pathway balancing. The broad utility of the CIFR toolbox expands the toolkit for metabolic engineering, allowing for the construction of complex phenotypes while opening new possibilities in bacterial genetic manipulations.