Developing High-Efficiency Electroporation Protocols for Hard-To-Transform Halomonas spp

Halomonas species have recently emerged as attractive candidates for next-generation industrial biotechnology, due to their ability to thrive under high-salt conditions, which allows for fermentation under open, unsterile conditions. However, their genetic manipulation has long been hindered by difficulties in genetic transformation. In this study, we report the development of a highly efficient electroporation protocol for Halomonas elongata DSM 2581. By optimising competent cell preparation and electroporation parameters, and using plasmid DNA purified from dam/dcm methylation-deficient Escherichia coli, we achieved a maximum transformation efficiency of 2.8 × 10⁸ ± 0.2 × 10⁸ CFU/μg DNA-the highest efficiency reported for any Halomonas species to date. Notably, we observed that growing cells in low-salt medium and harvesting them at late-stationary phase considerably improved electroporation efficiency. Moreover, we further demonstrated that the use of non-methylated plasmids helped evade the defence systems of H. elongata DSM 2581 that target foreign DNA. Importantly, the protocol proved transferable to other industrially relevant strains, achieving efficiencies of 5.3 × 10⁷ ± 0.1 × 10⁷ and 5.4 × 10⁶ ± 1.2 × 10⁶ CFU/μg DNA in Halomonas boliviensis LC1 and Halomonas campaniensis LS21, respectively. Altogether, this work establishes a robust, high-efficiency electroporation method for Halomonas spp., facilitating future genetic manipulation and strain engineering work, as well as encouraging further research into underexplored Halomonas species.