Construction of a food-grade multiple-copy integration system for Lactococcus lactis.
A food-grade vector system was developed that allows stable integration 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 bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal 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 approximately 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 medium a limited loss of integrated plasmid copies was detected at a rate of (7.5-15) x 10(-2) copies per generation. One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations of non-selective growth. These results show that the single-cross-over integration system described here represents a simple procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest.
Leenhouts
K
K
Department of Genetics, University of Groningen, Haren, The Netherlands.
Bolhuis
A
A
Venema
G
G
Kok
J
J
D015139:Blotting, Southern; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D013294:Lactococcus lactis / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism; D013997:Time Factors
D011994:Recombinant Proteins; D013395:Sucrose