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Applied and Environmental Microbiology, December 2005, p. 8077-8084, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8077-8084.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Heterologous Production of Antimicrobial Peptides in Propionibacterium freudenreichii

Dag Anders Brede,1* Therese Faye,1 Melanie Patricia Stierli,3 Gottfried Dasen,3 Anita Theiler,3 Ingolf F. Nes,1 Leo Meile,3 and Helge Holo1,2

Laboratory of Microbial Gene Technology, Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences (UMB), P.O. Box 5003, N-1432 Ås, Norway,1 TINE BA, Oslo, Norway,2 Institute of Food Science and Nutrition, Laboratory of Food Biotechnology, ETH Zuerich, Schmelzbergstrasse 7, CH-8092 Zuerich, Switzerland3

Received 13 April 2005/ Accepted 17 August 2005

Heterologous bacteriocin production in Propionibacterium freudenreichii is described. We developed an efficient system for DNA shuttling between Escherichia coli and P. freudenreichii using vector pAMT1. It is based on the P. freudenreichii rolling-circle replicating plasmid pLME108 and carries the cml(A)/cmx(A) chloramphenicol resistance marker. Introduction of the propionicin T1 structural gene (pctA) into pAMT1 under the control of the constitutive promoter (P4) yielded bacteriocin in amounts equal to those of the wild-type producer Propionibacterium thoenii 419. The P. freudenreichii clone showed propionicin T1 activity in coculture, killing 90% of sensitive bacteria within 48 h. The pamA gene from P. thoenii 419 encoding the protease-activated antimicrobial peptide (PAMP) was cloned and expressed in P. freudenreichii, resulting in secretion of the pro-PAMP protein. Like in the wild type, PAMP activation was dependent on externally added protease. Secretion of the antimicrobial peptide was obtained from a clone in which the pamA signal peptide and PAMP were fused in frame. The promoter region of pamA was identified by fusion of putative promoter fragments to the coding sequence of the pctA gene. The P4 and Ppamp promoters directed constitutive gene expression, and activity of both promoters was enhanced by elements upstream of the promoter core region.

* Corresponding author. Mailing address: Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences (UMB), P.O. Box 5003, N-1432 Ås, Norway. Phone: 47 64 96 58 97. Fax: 47 64 94 14 65. E-mail: dag.anders.brede{at}umb.no.

Applied and Environmental Microbiology, December 2005, p. 8077-8084, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8077-8084.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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