Construction of a food-grade multiple-copy integration system for Lactococcus lactis |
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Authors: | K Leenhouts A Bolhuis G Venema J Kok |
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Institution: | (1) Biomolecular Sciences and Biotechnology Institute, Department of Genetics, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands Tel.: +31 50 632111 Fax: +31 50 632348 e-mail: kokj@biol.rug.nl, NL |
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Abstract: | 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-cross-over 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) × 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.
Received: 23 September 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997 |
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