Optimization of Electroporation Conditions for Introducing Heterologous DNA into Rhodobacter sphaeroides.

Abstract

Rhodobacter sphaeroides 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 R. sphaeroides by deleting the rshI 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 R. sphaeroides ΔrshI strain, which is approximately 10 times higher than that of wild-type R. sphaeroides under standard bacterial electroporation conditions. These findings are expected to enhance the application of R. sphaeroides in various industrial fields in the future.