CONSTRUCTION OF A FOOD-GRADE MULTIPLE-COPY INTEGRATION SYSTEM FOR LACTOCOCCUS-LACTIS

A food-grade vector system was developed that allows stable integratio n of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori(+)) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacter ium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cl oning site, and a lactococcal DNA fragment of a well-characterized chr omosomal region. The system includes two L. lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori(+) vectors. These helper strains allow the construction and isolation of the replicating form of the integration plasmids from a homologous background. Single-crossover integration of the plasmids in L. lactis MG1363 resulted in amplifications to a level of approximate ly 20 copies/chromosome after selection of the transformants on medium containing sucrose as the only fermentable sugar. The amplifications were stable under selective growth conditions. In glucose-containing m edium a limited loss of integrated plasmid copies was detected at a ra te of (7.5-15)x 10(-2) copies per generation. One strain, MG124, was i solated that had retained 11 integrated copies after a period of 120 g enerations of non-selective growth. These results show that the single -cross-over integration system described here represents a simple proc edure for the engineering of stable food-grade strains carrying multip le copies of a gene of interest.