An efficient platform for markerless large-scale genomic deletions in Lactococcus lactis

The strain fitness and stress resistance of the starters, which are important to industrial applications, can be enhanced by large-scale genomic deletions. However, the low efficiency of large-scale genomic deletions has limited the efforts to improve the Lactococcus lactis fermentation performance through this method. In this study, we developed an efficient and time-saving platform for markerless large-scale genomic deletions in L. lactis by integrating temperature-sensitive plasmids, the SacB/pG15A counterselection system, and the Cre/loxP site-specific recombination system. First, the p15A origin, upstream and downstream homologies flanking the galK gene from MG1363 were integrated into the temperature-sensitive pG⁺Host9 plasmid, enabling the resulting plasmid pG15A-UD to replicate in Escherichia coli. The SacB gene from Bacillus subtilis, which encodes levansucrase, was then expressed from the plasmid pG15A-UD to establish the SacB/pG15A counterselection system. Furthermore, the SacB/pG15A counterselection system was demonstrated to be functional in L. lactis MG1363 and N8, achieving 40 %–60 % efficiency for markerless gene deletion and insertion. Finally, by combining the temperature-sensitive plasmid, SacB/pG15A and Cre/loxP system, we successfully deleted the 14,646 bp nisin biosynthetic gene cluster and a ∼58.5 kb nonessential genomic region in L. lactis N8. Our results provide an effective, rapid platform for large-scale genomic deletion in L. lactis to enhance the strain fitness and stress resistance and investigate their mechanisms.