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Proline iminopeptidase produced byPropionibacterium 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 inEscherichia coli andLactococcus lactis , the latter species being an extensively studied, primary cheese starter culture that is less fastidious in its growth condition requirements thanP. shermanii . The levels of expression of thepip gene could be enhanced with a factor 3 to 5 by using a strong constitutive promoter inL. lactis or the inducibletac promoter inE. coli . Stable replication of the rolling-circle replicating (rcr) plasmid, used to expresspip inL. lactis , could only be obtained by providing therepA gene intrans . Upon the integration ofpip , 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 inL. lactis.

applied and environmental microbiology

Cloning, Expression, and Chromosomal Stabilization of the Propionibacterium shermanii Proline Iminopeptidase Gene ( pip ) for Food-Grade Application in Lactococcus lactis