Dataset Information

Cloning, expression, and chromosomal stabilization of the Propionibacterium shermanii proline iminopeptidase gene (pip) for food-grade application in Lactococcus lactis.

ABSTRACT: Proline iminopeptidase produced by Propionibacterium shermanii plays an essential role in the flavor development of Swiss-type cheeses. The enzyme (Pip) was purified and characterized, and the gene (pip) was cloned and expressed in Escherichia coli and Lactococcus lactis, the latter species being an extensively studied, primary cheese starter culture that is less fastidious in its growth condition requirements than P. shermanii. The levels of expression of the pip gene could be enhanced with a factor 3 to 5 by using a strong constitutive promoter in L. lactis or the inducible tac promoter in E. coli. Stable replication of the rolling-circle replicating (rcr) plasmid, used to express pip in L. lactis, could only be obtained by providing the repA gene in trans. Upon the integration of pip, clear gene dosage effects were observed and stable multicopy integrants could be maintained upon growth under the selective pressure of sucrose. The multicopy integrants demonstrated a high degree of stability in the presence of glucose. This study examines the possibilities to overexpress genes that play an important role in food fermentation processes and shows a variety of options to obtain stable food-grade expression of such genes in L. lactis.

SUBMITTER: Leenhouts K

PROVIDER: S-EPMC90916 | biostudies-literature | 1998 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Cloning, expression, and chromosomal stabilization of the Propionibacterium shermanii proline iminopeptidase gene (pip) for food-grade application in Lactococcus lactis.

Leenhouts K K Bolhuis A A Boot J J Deutz I I Toonen M M Venema G G Kok J J Ledeboer A A

Applied and environmental microbiology 19981201 12

Proline iminopeptidase produced by Propionibacterium shermanii plays an essential role in the flavor development of Swiss-type cheeses. The enzyme (Pip) was purified and characterized, and the gene (pip) was cloned and expressed in Escherichia coli and Lactococcus lactis, the latter species being an extensively studied, primary cheese starter culture that is less fastidious in its growth condition requirements than P. shermanii. The levels of expression of the pip gene could be enhanced with a f ...[more]

PMID: 9835556

Similar Datasets

Project description:A phage-resistant mutant with a defect in a membrane component required for phage infections in Lactococcus lactis subsp. lactis C2 was transformed with a chromosomal library of the wild-type, phage-sensitive strain. Of the 4,200 transformants screened for phage sensitivity, three were positively identified as phage sensitive. A cause-and-effect relationship between the cloned chromosomal fragments and the phage-sensitive phenotype was established on the basis of the following two criteria: (i) the frequency of loss of the cloned fragments in the absence of antibiotic selection pressure correlated with the frequency of loss of phage sensitivity; and (ii) phage sensitivity was transferred to 100% of recipient, phage-resistant cells transformed with the cloned fragment. The cloned chromosomal DNA from the three independent isolates was physically mapped with restriction endonucleases. The sizes of the cloned fragments were 9.6, 11.8, and 9.5 kb. Each fragment contained an identical stretch of DNA common to all three, which was 9.4 kb. The gene that conferred phage sensitivity was localized by subcloning to a 4.5-kb region. Further subcloning indicated that a single EcoRI site within the 4.5-kb region must lie within the gene or its promoter. The required 4.5-kb region was sequenced and found to code for one partial and two complete open reading frames. The gene required for complementation was functionally mapped by Tn5 mutagenesis and localized to one of the two complete open reading frames, which was designated pip (an acronym for phage infection protein). pip is 2,703 bases in length. Potential promoters start 206 and 212 bases upstream of the open reading frame. A ribosome binding site and a seven-base spacer precede the GTG (Val) translation initiation codon. The amino acid sequence deduced from the gene has 901 residues and an M(r) of 99,426. Hydropathy analysis revealed four to six potential membrane-spanning regions, one near the amino terminus and the others at the extreme carboxyl terminus. The amino terminus has characteristics of a signal sequence. The putative protein would have a 650-residue, central polar domain.

Dataset Information

Cloning, expression, and chromosomal stabilization of the Propionibacterium shermanii proline iminopeptidase gene (pip) for food-grade application in Lactococcus lactis.

Publications

Cloning, expression, and chromosomal stabilization of the Propionibacterium shermanii proline iminopeptidase gene (pip) for food-grade application in Lactococcus lactis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets