High Electroporation Efficiency of Corynebacterium glutamicum with Xenogeneic Plasmid DNA

摘要

The restriction-modification systems of C. glutamicum impede its uptake of foreign DNA and thus limiting its transformation efficiency. Here we investigated the possibility of improving the transformation efficiency of C. glutamicum by characterizing and circumventing their restriction/modification barrier, as well as by optimizing the conditions of electroporation. We found that, among various conditions tested for electroporation, the most important factor is the source of plasmid DNA. When plasmid DNA isolated from the dam--dcm- E. coli mutant JM110 was used for transformation of C. glutamicum that had been grown in BHIS supplemented with glycine 2.5% (w/v), INH (4 mg/ml) and tween80 0.1% (w/v) at 180;C, and harvested at an OD600 of 0.5, the maximum transformation efficiency of 6.57;106 transformants/µg of plasmid DNA was obtained at a field strength of 20 kV/cm, while plasmid DNA isolated from the other E. coli strains such as DH5α led to a extremely low level of transformation efficiency. Our observations strongly indicate that the use of unmethylated plasmid DNA is critical to achieve high transformation efficiency in C. glutamicum.