Construction and evaluation of food-grade vectors for Lactococcus lactis using aspartate aminotransferase and α-galactosidase as selectable markers

We report development of two food-grade cloning vectors for Lactococcus lactis, which utilize either a lactococcal aspartate aminotransferase gene (aspC), or Bifidobacterium longumα-galactosidase gene (aglL) as selectable markers. The theta-replicon of lactococcal plasmid, pW563, was combined with a...

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Bibliographic Details
Published in:Journal of applied microbiology 2006-07, Vol.101 (1), p.161-171
Main Authors: Sridhar, V.R, Smeianov, V.V, Steele, J.L
Format: Article
Language:English
Subjects:
alpha-galactosidase
alpha-Galactosidase - genetics
aspartate aminotransferase
Aspartate Aminotransferases - genetics
aspartate transaminase
Bifidobacterium
Bifidobacterium - genetics
Biological and medical sciences
Cloning, Molecular
DNA Primers
Food Industry
Food Microbiology
food‐grade cloning
Fundamental and applied biological sciences. Psychology
Gene Expression
genes
Genetic Engineering
Genetic Markers
Genetic Vectors
Lactobacillus helveticus
Lactococcus
Lactococcus lactis
Lactococcus lactis - enzymology
Lactococcus lactis - genetics
Microbiology
milk
mutants
peptidases
Plasmids
Probiotics
α‐galactosidase
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Summary:We report development of two food-grade cloning vectors for Lactococcus lactis, which utilize either a lactococcal aspartate aminotransferase gene (aspC), or Bifidobacterium longumα-galactosidase gene (aglL) as selectable markers. The theta-replicon of lactococcal plasmid, pW563, was combined with aspC and a multiple cloning site. When electroporated into L. lactis JLS400 (AspC⁻), the resulting vector, pSUW611 (3·9 kbp), restores ability of the mutant to grow in milk thus allowing for selection of the transformants. The vector is stable during 100 generations of nonselective growth (0·2% loss per generation). The second vector, pSUW711 (5·1 kbp), was constructed by exchanging aspC with aglL under the control of usp45 promoter. Lactococcus lactis transformed with pSUW711 produced distinctive colonies within 48-72 h on melibiose-containing plates. Expression of two Lactobacillus helveticus peptidases was attempted using these new vehicles. Introduction of pepN on pSUW611 and pSUW711 into L. lactis led to a sixfold, or 27-fold increase in aminopeptidase activity, respectively. However, no changes in endopeptidase activity were recorded upon transformation with pSUW611 carrying pepO2 under control of three different promoters. Attempts were also made to construct high copy variants of pSUW711. The aspC and aglL can be employed as food-grade genetic markers for L. lactis. The vectors, pSUW611 and pSUW711, were successfully used to express Lact. helveticus PepN in L. lactis. Two novel food-grade vectors were developed which provide simple and convenient selection and maintenance in L. lactis.
ISSN:1364-5072
1365-2672
DOI:10.1111/j.1365-2672.2006.02898.x