Development of the Efficient Electroporation Protocol for Leuconostoc mesenteroides

Leuconostoc mesenteroides is a key microorganism in food biotechnology, valued for its production of flavor-forming metabolites and exopolysaccharides, and its inclusion in starter cultures and biocatalytic systems. However, the application of advanced genetic tools to L. mesenteroides remains hindered by multiple barriers, including inefficient DNA transfer, elevated endogenous nuclease activity, and restriction-modification systems sensitive to plasmid methylation patterns. As a result, even widely accepted electroporation methodologies often yield inconsistent or irreproducible transformation results, limiting the strain's amenability to metabolic engineering and synthetic biology applications. In this study, a reproducible electroporation protocol for the L. mesenteroides strain H32-02 Ksu is developed and experimentally validated. The protocol concept relies on the sequential optimization of key process steps: targeted weakening of the cell wall followed by osmotic protection, the development of a gentle electrical stimulus that ensures membrane permeability without critical damage, and the creation of recovery conditions that minimize loss of viability and degradation of incoming DNA. Matching plasmid methylation to the recipient's restriction profile proved critical: choosing a source for plasmid DNA production with a compatible methylation pattern dramatically increased the likelihood of successful transformation. In our case, the selection of an E. coli strain with a more suitable methylation profile increased the yield of transformants by 3.5 times. It was also shown that reducing the pulse voltage increase transformant number by 3 times. The combined optimization resulted in an approximately 40-fold increase in transformation efficiency compared to the baseline level and, for the first time, provided consistently reproducible access to transformants of this strain. The highest transformation efficiency was achieved: 8 × 10² CFU µg^-1 DNA. The presented approach highlights the strain-specificity of barriers in Leuconostoc and forms a technological basis for constructing strains with desired properties, expressing heterologous enzymes, and subsequently scaling up bioprocesses in food and related industries. The methodological principles embodied in the protocol are potentially transferable to other lactic acid bacteria with similar limitations.