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 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.
Sørensen
K I
KI
Department of Genetics and Microbiology, Chr. Hansen A/S, DK-2970 Hørsholm, Denmark. KimIb.Soerensen@dk.chr-hansen.com
Larsen
R
R
Kibenich
A
A
Junge
M P
MP
Johansen
E
E
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D018274:Electroporation; D019649:Food Industry; D005516:Food Microbiology; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D013294:Lactococcus lactis / Q000235:genetics* / Q000254:growth & development; D008892:Milk / Q000382:microbiology; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011743:Pyrimidines / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D011743:Pyrimidines; D000626:Aminopeptidases; C056860:lysyl aminopeptidase