Unlocking genetic accessibility in Halomonas bluephagenesis: A high-efficiency transformation chassis enables linear DNA-mediated genome editing

Halomonas bluephagenesis has emerged as a promising chassis for industrial biotechnology distinguished by its halophilic robustness and capacity for open and continuous fermentation. However, its poor transformability has substantially limited rapid, iterative, and automatable strain engineering. Here, we develop a “restriction release” strategy that increases transformation efficiency from approximately 10 2 to 10 7 cfu/μg DNA, rivaling the efficiency of standard laboratory Escherichia coli . A spontaneous IS30 insertion disrupting the type II restriction endonuclease gene notI increased plasmid transformation efficiency by about 2000-fold. Targeted deletion of notI reproduced this phenotype, confirming its role as a major barrier. Further deletion of the DNA phosphorothioation restriction locus dptFGH yielded an additional 10 to 37-fold improvement, generating the highly transformable ΔEPSΔFlgΔNotIΔPTRes chassis. Notably, the enhanced chassis further facilitated two linear-DNA-based editing strategies: direct RecET-mediated pyrF deletion using a linear donor coupled with phenotypic selection, achieving a recombination efficiency of 16.7%, and CRISPR/Cas9-assisted ribDHBA promoter replacement via co-delivery of a gRNA plasmid and a linear donor, achieving an efficiency of 11.1%. Together, these results identify endogenous restriction systems as primary barriers to genetic manipulation in H. bluephagenesis . Stepwise restriction release markedly improves plasmid transformation and enables linear DNA-mediated genome editing, providing an improved genetic platform for the iterative engineering of this industrial halophile. More broadly, this study suggests a practical strategy for overcoming the genetic inaccessibility of other non-model microorganisms and accelerating iterative strain engineering.