A food-grade cloning system for industrial strains of Lactococcus lactis

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 laborator y strain MG1363 but is unstable in the industrial strains tested. As supB s uppresses both amber and ochre stop codons, which are present in 82% of all known lactococcal genes, this undesirable finding may result from the accu mulation of elongated mistranslated polypeptides. Here,,ve report the devel opment of a new food-grade cloning vector, pFG200, which is suitable for ov erexpressing a variety of genes in industrial strains of Lactococcus lactis . The vector uses an amber suppressor, supD, as selectable marker and consi sts entirely of Lactococcus DNA, with the exception of a small polylinker r egion. Using suppressible pyrimidine auxotrophs, selection and maintenance are efficient in any pyrimidine-free medium including milk. Importantly, th e presence of this vector in a variety of industrial strains has no signifi cant 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 overexp ressing the pepN gene in a number of industrial backgrounds.