Applied and Environmental Microbiology, April 2000, p. 1253-1258, Vol. 66, No. 4
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.
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
*
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.
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