Optimization of electrotransformation conditions for Leuconostoc mesenteroides subsp mesenteroides ATCC8293

Abstract

Aims: To establish an efficient genetic transformation protocol for Leuconostoc species, methods for competent-cell preparation and electroporation conditions were optimized. Methods and Results: Leuconostoc mesenteroides subsp. mesenteroides ATCC8293 cells were sequentially treated with penicillin G and lysozyme, and the plasmid pLeuCM was subsequently transformed into the cells. Our results demonstrated that transformation efficiencies were significantly increased (100-fold), and increased electric field strength also contributed to enhance transformation efficiency. Maximum transformation efficiency (1 X 104 or more transformants per mu g DNA) was achieved when cells were grown in De Man, Rogosa, Sharpe (MRS) media containing 0.25 mol l-1 sucrose and 0.8 mu g ml-1 penicillin G, followed by treatment with 600 U ml-1 lysozyme and electroporation at a field strength of 10 kV cm-1. When this protocol was used to transform pLeuCM into Leuc. mesenteroides, Leuconostoc gelidum, Leuconostoc fallax and Leuconostoc argentinun, successful transformations were obtained in all cases. Furthermore, this procedure was applicable to species belonging to other genera, including Lactobacillus plantarum, Pediococcus pentosaceus and Weissella confusa. Conclusions: The results demonstrate that the transformation efficiency for Leuconostoc spp. could be increased via optimization of the entire electroporation procedures. Significance and Impact of the Study: These optimized conditions can be used for the extensive genetic study and the metabolic engineering of not only Leuconostoc spp. but also different species of lactic acid bacteria.