Dataset Information

Heterologous production of antimicrobial peptides in Propionibacterium freudenreichii.

ABSTRACT: 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.

SUBMITTER: Brede DA

PROVIDER: S-EPMC1317351 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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Heterologous production of antimicrobial peptides in Propionibacterium freudenreichii.

Brede Dag Anders DA Faye Therese T Stierli Melanie Patricia MP Dasen Gottfried G Theiler Anita A Nes Ingolf F IF Meile Leo L Holo Helge H

Applied and environmental microbiology 20051201 12

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 ...[more]

PMID: 16332788

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Project description:Cobalamins (vitamin B12) are required by humans for their essential roles as enzyme cofactors in diverse metabolic processes. The four most common cobalamin vitamers are hydroxocobalamin (OHCbl), adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), and cyanocobalamin (CNCbl). Humans are not able to synthesise cobalamins de novo and thus must acquire them from external sources. Therefore, a reliable and robust analytical method to determine the cobalamins in dietary sources is highly required. For such a purpose, stable isotope dilution assays (SIDAs) with LC-MS/MS are most suited due to their superior sensitivity, specificity, and ability to compensate for matrix effects and analyte loss during sample work-up. However, a critical bottleneck for developing a SIDA method for cobalamins is the availability of stable isotope-labelled internal standards. In the present study, we harnessed the potential of Propionibacterium (P.) freudenreichii for the biosynthesis of 15N-labelled cobalamins. First, we developed a chemically defined medium (CDM) containing ammonium sulphate as a single nitrogen source except three essential vitamins that supported long-term stable growth of P. freudenreichii throughout continuous transfers. The CDM was further optimised for cobalamin production under different incubation schemes. With the optimised CDM and incubation scheme, fully 15N-labelled cobalamins were obtained in P. freudenreichii with a final yield of 312 ± 29 μg/L and 635 ± 102 μg/L, respectively, for [15N]-OHCbl and [15N]-AdoCbl. Additionally, an optimised incubation process under anaerobic conditions was successfully employed to produce specifically labelled [15N, 14N2]-cobalamins, with a yield of 96 ± 18 μg/L and 990 ± 210 μg/L, respectively, for [15N, 14N2]-OHCbl and [15N, 14N2]-AdoCbl. The labelled substances were isolated and purified by solid phase extraction and semi-preparative HPLC. Chemical modifications were carried out to produce [15N]-CNCbl and [15N]-MeCbl. Eventually, 15N-labelled compounds were obtained for the four cobalamin vitamers in high chromatographic and isotopic purity with desired 15N-enrichment and labelling patterns, which are perfectly suited for future use in SIDAs or other applications that require isotopologues.

Dataset Information

Heterologous production of antimicrobial peptides in Propionibacterium freudenreichii.

Publications

Heterologous production of antimicrobial peptides in Propionibacterium freudenreichii.

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