Optimization of Electroporation Conditions for Introducing Heterologous DNA into <i>Rhodobacter sphaeroides</i>

<i>Rhodobacter sphaeroides</i> is a strain capable of both photoautotrophic and chemoautotrophic growth, with various metabolic pathways that make it highly suitable for converting carbon dioxide into high value-added products. However, its low transformation efficiency has posed challenges for genetic and metabolic engineering of this strain. In this study, we aimed to increase the transformation efficiency of <i>R. sphaeroides</i> by deleting the <i>rshI</i> gene coding for an endogenous DNA restriction enzyme that inhibits. We evaluated the effects of growth conditions for making electrocompetent cells and optimized electroporation parameters to be a cuvette width of 0.1 cm, an electric field strength of 30 kV/cm, a resistance of 200 Ω, and a plasmid DNA amount of 0.5 μg, followed by a 24-h recovery period. As a result, we observed over 7,000 transformants per μg of DNA under the optimized electroporation conditions using the <i>R. sphaeroides</i> <i>ΔrshI</i> strain, which is approximately 10 times higher than that of wild-type <i>R. sphaeroides</i> under standard bacterial electroporation conditions. These findings are expected to enhance the application of <i>R. sphaeroides</i> in various industrial fields in the future.