Affiliation:
1. Department of Genetics and Microbiology, Chr. Hansen A/S, DK-2970 Hørsholm, Denmark
Abstract
ABSTRACT
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.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
61 articles.
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