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.

FIG. 1

(A) Effect of pH on Pip activity. (B) Effect of temperature on Pip activity.

FIG. 2

Diagram of the pip expression plasmid pUG39, in which pip is under the control of the lactococcal promoter P32 (black arrow). The pip gene is preceded by the truncated ORF32. ORF32 was translationally fused to a number of codons of the multiple cloning site (ORF32′::aa) to create a gene construct that allows translational coupling of pip as shown in the linear presentation of the expression cassette. RBS32 (black dots), ribosome binding site of ORF32; arrow designated T(prtP), terminator of the lactococcal prtP gene; EmR, erythromycin resistance gene. The origin of replication of pWV01 is indicated by the black box.

FIG. 3

Schematic presentation of the location on the lactococcal chromosome of the chromosomal DNA fragments used for integration (black boxes). The black boxes represent the chromosomal fragments present in the indicated integration vectors. In the DCO vectors pINT29, pINT51, and pINT61 containing two chromosomal fragments the pip gene was cloned in between the adjacent chromosomal fragments. Wavy lines, chromosomal DNA; T, transcriptional terminator; pepX, PepX gene; orf1, open reading frame encoding polypeptide with unknown function; lacR, repressor gene of the lac operon. The interrupted leftward arrow is the L. lactis lacABCDEFG operon.