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Applied and Environmental Microbiology, April 2000, p. 1253-1258, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A Food-Grade Cloning System for Industrial Strains of Lactococcus lactis

Kim I. Sørensen,* Rasmus Larsen, Annette Kibenich, Mette P. Junge, and Eric Johansen

Department of Genetics and Microbiology, Chr. Hansen A/S, DK-2970 Hørsholm, Denmark

Received 18 October 1999/Accepted 6 January 2000

We have previously reported the construction of a food-grade cloning vector for Lactococcus using the ochre suppressor, supB, as the selective marker. This vector, pFG1, causes only a slight growth inhibition in the laboratory strain MG1363 but is unstable in the industrial strains tested. As supB suppresses both amber and ochre stop codons, which are present in 82% of all known lactococcal genes, this undesirable finding may result from the accumulation of elongated mistranslated polypeptides. Here, we report the development of a new food-grade cloning vector, pFG200, which is suitable for overexpressing a variety of genes in industrial strains of Lactococcus lactis. The vector uses an amber suppressor, supD, as selectable marker and consists entirely of Lactococcus DNA, with the exception of a small polylinker region. Using suppressible pyrimidine auxotrophs, selection and maintenance are efficient in any pyrimidine-free medium including milk. Importantly, the presence of this vector in a variety of industrial strains has no significant effect on the growth rate or the rate of acidification in milk, making this an ideal system for food-grade modification of industrially relevant L. lactis strains. The usefulness of this system is demonstrated by overexpressing the pepN gene in a number of industrial backgrounds.

* Corresponding author. Mailing address: Department of Genetics and Microbiology, Chr. Hansen A/S, 10-12 Bøge Allé, DK-2970 Hørsholm, Denmark. Phone: (45) 45 748 354. Fax: (45) 45 748 994. E-mail: KimIb.Soerensen{at}dk.chr-hansen.com.

Applied and Environmental Microbiology, April 2000, p. 1253-1258, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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