Markerless large DNA integration in Lactococcus lactis through the coupling of homologous single-crossover and...

Lactococcus lactis is widely used in food fermentation and has great potential as a microbial cell factory for producing high-value compounds. Currently, heterologous gene expression in this host mainly relies on plasmid-based systems, which suffer from segregational instability without antibiotic selection. To address this problem, several genome integration tools have been developed, yet most are limited to single gene insertions or leave selection markers. In this study, we developed an efficient and markerless platform for integration single genes and large DNA fragments combining a temperature-sensitive plasmid for homologous single-crossover and the Cre/loxP system for plasmid backbone excision. First, using CRISPR/Cas9 assisted ssDNA recombineering, we introduced a loxP site into the ribB gene of L. lactis NZ9000/RecT, creating the chassis strain L. lactis loxP. We then constructed a donor plasmid, pG15AribB-Up-Cat-loxP, which carries a homology arm of ribB, the chloramphenicol resistance gene cat, and a second loxP site. This donor plasmid was integrated into the chromosome next to the existing loxP site through a single-crossover event. Subsequent expression of Cre recombinase then removed the plasmid backbone, leaving only the target gene cat at the insertion site. After 99 generations without chloramphenicol selection, the integrant L. lactis IMT maintained nearly 100% genetic stability. Using this method, we next successfully markerless integrated the ∼4.8 kb crtEBI cluster, and the seven gene tagatose-6-phosphate pathway (∼7.0 kb). This work provides an efficient platform for markerless and stable large DNA integration in L. lactis for constructing microbial cell factory to produce high-value compounds.