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Antimicrobial Agents and Chemotherapy, July 2005, p. 3004-3008, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.3004-3008.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Production of Enterocin P, an Antilisterial Pediocin-Like Bacteriocin from Enterococcus faecium P13, in Pichia pastoris

Jorge Gutiérrez, Raquel Criado, María Martín, Carmen Herranz, Luis M. Cintas, and Pablo E. Hernández*

Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain

Received 24 November 2004/ Returned for modification 14 January 2004/ Accepted 25 February 2005


   ABSTRACT
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The gene encoding mature enterocin P (EntP), an antimicrobial peptide from Enterococcus faecium P13, was cloned into the pPICZ{alpha}A expression vector to generate plasmid pJC31. This plasmid was integrated into the genome of P. pastoris X-33, and EntP was heterologously secreted from the recombinant P. pastoris X-33t1 derivative at a higher production and antagonistic activity than from E. faecium P13.


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Compounds with antimicrobial activity are produced by a wide range of mammals, birds, insects, plants, and microorganisms, while ribosomally synthesized antimicrobial peptides produced by bacteria are generally referred to as bacteriocins (14, 29). Bacteriocin production is common in lactic acid bacteria (LAB), and since bacteriocins produced by LAB may inhibit gram-positive spoilage bacteria and food-borne pathogens (9, 31), they are attracting considerable interest for their potential use as natural and nontoxic food preservatives (10, 30). Enterocin P (EntP) is a class IIa bacteriocin produced by Enterococcus faecium P13 (7). The EntP structural gene (entP) encodes a 71-amino-acid prepeptide consisting of a 44-amino-acid mature bacteriocin and a 27-amino-acid signal peptide. The mature EntP is a bacteriocin with a broad antimicrobial spectrum that inhibits food-borne pathogens such as Listeria monocytogenes, Clostridium perfringens, Clostridium botulinum, and Staphylococcus aureus (7), while the entP gene seems to be widely distributed in E. faecium strains of different origins (8, 12, 21). EntP has also been shown to dissipate the membrane potential of energized cells and to form specific potassium ion-conducting pores in the cytoplasmic membranes of target cells (19, 20). The broad antimicrobial spectrum of EntP suggests its potential application as a natural antimicrobial additive in the food industry. However, the use of enterococci as producers of bacteriocins should be approached with caution, since many Enterococcus isolates code for potential virulence factors and carry antibiotic resistance genes (13, 15, 16). For biotechnological, hygienic, and safety reasons, the cloning, production, and functional expression of bacteriocins produced by enterococci in heterologous hosts should be evaluated. The yeast Pichia pastoris has proven valuable for the heterologous production of peptides and proteins due to its ability to produce foreign proteins at high levels (6, 17). Thus, we report in this communication the cloning, production, and functional expression of EntP by P. pastoris.

Bacterial strains, plasmids, growth conditions, and basic genetic techniques. E. faecium P13 (7) and E. faecium T136 (4) were grown in MRS broth (Oxoid). The pCR2.1-TOPO vector, Escherichia coli TOP-10 and E. coli MAX Efficiency DH5{alpha} competent cells, the pPICZ{alpha}A expression vector, and the Pichia pastoris X-33 host were from Invitrogen. The E. coli cells were propagated in Luria-Bertani (LB) broth (Sigma). The P. pastoris X-33t1 clone was grown in buffered methanol minimal (BMM) medium (1.34% yeast nitrogen base, 4 x 10–5% biotin, 100 mM potassium phosphate [pH 6], 0.5% methanol) and BMMY (BMM with 1% yeast extract and 2% peptone) to induce production of EntP. Total genomic DNA was isolated using the Wizard DNA purification kit (Promega). All DNA-modifying enzymes were from New England Biolabs, and Platinum Taq DNA polymerase was from Invitrogen. The PCR-generated fragments were extracted from agarose gels using the Real Clean Matrix kit (C. E. Durviz, Madrid, Spain) and purified by the QIAquick PCR purification kit (QIAGEN). Competent P. pastoris X-33 cells were obtained as recommended (Invitrogen).

Construction of the recombinant plasmid pJC31. Primers JC1 (5'-AATTATACTCGAGAAAAGAGCTACGCGTTCATATGGTAATGGTG-3') and JC2 (5'-ATTAGTTTCTAGAATATTAATGTCCCATACCTGCCAAACCAG-3') were used for PCR amplification from total genomic DNA of E. faecium P13 of a 170-bp XhoI-XbaI fragment (insert JC) carrying the {alpha}-factor kex2 signal cleavage fused to the mature EntP (entP) nucleotide sequence. Cloning of fragment JC in plasmid pCR2.1-TOPO and transformation of E. coli TOP-10 cells permitted selection of a clone containing plasmid pJC14. Plasmid pJC14 was digested with XhoI and XbaI, and the resulting 154-bp cleaved fragment was ligated into plasmid pPICZ{alpha}A, to give plasmid pJC31. Competent E. coli DH5{alpha} cells were transformed with pJC31 and the resulting transformants confirmed by PCR amplification and sequencing. Purified pJC31 was linearized with SacI and used to transform competent P. pastoris X-33 cells, in which the presence of the integrated pJC31 genes was confirmed by a bacteriocinogenicity test, PCR, and DNA sequencing of the inserts.

Heterologous production and functional expression of EntP by P. pastoris. Heterologous expression systems for production and secretion of cloned bacteriocins have already been developed in bacteria, mostly in LAB hosts using dedicated ABC transport signals (1, 2, 23, 24, 28) or the Sec pathway (2, 25-27). However, yeasts have not yet been fully exploited as alternative heterologous hosts for production of bacteriocins. Although several yeast species are being used as heterologous production systems, Saccharomyces cerevisiae has not always proven to be ideal as a foreign gene expression host, including the heterologous production of bacteriocins (34, 35). In this work we have produced EntP, a pediocin-like bacteriocin, by using the methylotrophic yeast P. pastoris. Plasmid pPICZ{alpha}A was selected as the expression vector because it contains the S. cerevisae alpha mating factor ({alpha}-MF prepro) leader sequence (11) to target fused proteins to the secretory pathway, a methanol-inducible promoter, and the AOX1 region, which allows integration of the vector into the P. pastoris genome. The gene encoding mature EntP was cloned into pPICZ{alpha}A in frame to the {alpha}-factor secretion signal peptide without the Glu-Ala spacers adjacent to the Kex2 protease cleavage site (3). When the recombinant vector pJC31 was linearized and transformed into P. pastoris X-33 competent cells, the P. pastoris X-33t1 derivative showed a potent direct antimicrobial activity (Fig. 1).



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  		FIG. 1. Direct antimicrobial activities of P. pastoris X-33 (spot 1) and P. pastoris X-33t1 (spot 2) by the stab-on-agar test using (A) E. faecium T136 or (B) E. faecium P13 as the indicator strain.

 
The heterologous production of EntP by P. pastoris X-33t1 was further evaluated and quantified by an agar well diffusion test and a microtiter plate assay (MPA) performed as previously described (8), and by specific anti-EntP antibodies in a noncompetitive indirect enzyme-linked immunosorbent assay (18). Results from Fig. 2 indicate that supernatants of P. pastoris X-33t1 grown in the minimal medium BMM produced smaller halos of inhibition (Fig. 2A) than supernatants of the same culture grown in the complex medium BMMY (Fig. 2B). Moreover, the halos generated by the supernatant of the culture grown in BMMY were larger, and its activity lasted longer during the fermentation period, than those of the culture grown in BMM. Table 1 also indicates that maximum production of EntP during the growth of P. pastoris X-33t1 in BMM (22.8 µg/ml) represents a threefold increase over production by E. faecium P13 (7.5 µg/ml), but its antimicrobial activity (163 bacteriocin units [BU]/ml) was only 26% of that of the EntP produced by E. faecium P13 (625 BU/ml). However, maximum production of EntP by P. pastoris X-33t1 grown in BMMY (28.2 µg/ml) was 3.7-fold higher, and its antimicrobial activity (10,240 BU/ml) 16-fold higher, than production and activity of EntP by E. faecium P13. The specific antimicrobial activity of EntP from the supernatant of P. pastoris X-33t1 grown in BMMY (363 BU/µg) was also higher than that of EntP in the supernatant of E. faecium P13 (83 BU/µg). Salts and other components of the BMM medium may be responsible for the low antimicrobial activity of EntP in the presence of reasonable amounts of the bacteriocin (Table 1). Salt inhibition has previously been reported to reduce the inhibitory activity of antimicrobial peptides, including nisin (37). However, neutral proteases (34) might also be responsible for the observed reduced antigen epitope recognition, leading to a decrease in the EntP production and activity of the supernatants of P. pastoris X-33t1 grown in BMMY (Table 1).



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  		FIG. 2. Agar well diffusion test for detection of enterocin P activity against E. faecium T136. Wells contain supernatants of P. pastoris X-33t1 cultures, grown in the minimal medium BMM (A) or the complex medium BMMY (B), after 0 (d), 4 (e), 6 (f), 8 (g), 10 (h), 12 (i), or 24 (j) hours of incubation. Supernatants of P. pastoris X-33 cultures propagated in BMM or BMMY after 0 (a), 6 (b), or 12 (c) hours of growth were used as negative controls. Supernatants of E. faecium P13 containing 8 (l), 2 (m), and 0.5 (n) µg/ml of EntP were used as positive controls, and the supernatant of E. faecium T136 (k) was used as a negative control.

 

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  		TABLE 1. Production and antimicrobial activities of enterocin P from supernatants of P. pastoris X-33t1 grown in different mediaa

 
Purification of EntP, Western blotting, and mass spectrometry analysis. Purification of EntP from the supernatants of E. faecium P13 and P. pastoris X-33t1 generated two chromatographic peaks with antagonistic activity (Table 2). The existence of multiple chromatographic peaks after purification of EntP has been reported previously (18, 21) and is ascribed to the coexistence after reversed-phase fast protein liquid chromatography (RP-FPLC) of oxidized and nonoxidized forms of the bacteriocin. However, the yield of EntP from the P. pastoris X-33t1 culture was lower than that from E. faecium P13. Reduced adsorption/desorption of EntP to the cation-exchange, hydrophobic-interaction, and RP-FPLC supports are rate-limiting steps for purification of larger quantities of this bacteriocin from P. pastoris. Thus, the overproduction of EntP from recombinant yeast strains would not be useful without the development and optimization of more-efficient purification procedures for this bacteriocin. Further characterization of EntP in fractions B from E. faecium P13 and P. pastoris X33t1 was performed by protein electrophoresis, Western blotting, and an overlay assay, as previously described (18). The results shown in Fig. 3 suggest that EntP produced by P. pastoris X-33t1 has a strong tendency to form aggregates with antimicrobial activity. After matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry analysis in a Voyager-DE STR Instrument (PerSeptive Biosystems), both purified EntP from E. faecium P13 and that from P. pastoris X-33t1 gave a major fragment of identical molecular mass (results not shown), suggesting that the pJC31 vector directs the processing and secretion of EntP adequately in P. pastoris X-33t1.


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  		TABLE 2. Purification of enterocin P produced by E. faecium P13 and P. pastoris X-33t1

 


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  		FIG. 3. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of different bacteriocins after (A) silver staining, (B) overlay with the indicator strain E. faecium T136, and (C) Western blotting with specific anti-EntP antibodies. M, molecular weight markers, with sizes (in kDa) given in the left margin. Lanes 1, 1 µg of pure enterocin Q; lanes 2, 1 µg of pure pediocin PA-1; lanes 3, 1 µg of pure enterocin P from E. faecium P13; lanes 4, 1 µg of pure enterocin P from P. pastoris X-33t1.

 
From the results of this work, it is clear that the heterologous production and functional expression of EntP in P. pastoris have become successful and readily quantifiable events. The heterologous production of proteins and peptides in yeasts permits posttranslational events such as proteolytic maturation, glycosylation, and disulfide bond formation. Although the amino acid sequence of EntP contains a potential glycosylation site (Asn-Asn-Ser), the fact that the purified EntP proteins from E. faecium P13 and P. pastoris X-33t1 show identical molecular masses suggests that the EntP glycosylation site was not recognized. Pediocin PA-1 (32) and plantaricin 423 (35) have been produced by recombinant S. cerevisiae hosts, but no inhibitory activity was detected in the supernatants of the recombinant yeasts without concentration; it is speculated that the bacteriocins remained cell wall associated. Moreover, EntP is the first fully active bacteriocin produced by P. pastoris. The large production and high antimicrobial activity of the EntP in the supernatants of P. pastoris X-33t1 may facilitate future applications of this bacteriocin in the food industry. Many pharmaceuticals and enzymes derived from methylotrophic yeasts either have entered the market or are expected to do so in the near future (17, 22). Further strategies for optimal production of EntP by P. pastoris may include the use of protease-deficient host strains and unbuffered media to inactivate neutral proteases (34), production in fermentors (5), an increase in the cellular mass by using carbon sources that support growth and do not repress methanol induction (33), and the use of appropriate flask designs (36).


   ACKNOWLEDGMENTS
 
This work was partially supported by grant 07G/0026/2000 from the Comunidad de Madrid and grants AGL2000-0706 and AGL2003-01508 from the Ministerio de Educación y Cultura, Spain. J.G. is the recipient of a fellowship from the Ministerio de Ciencia y Tecnología (MCYT), and R.C. and M.M. hold fellowships from the Ministerio de Educación, Cultura y Deporte (MECD), Spain.


   FOOTNOTES
 
* Corresponding author. Mailing address: Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain. Phone: 34-913943752. Fax: 34-913943743. E-mail: ehernan{at}vet.ucm.es. Back


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Antimicrobial Agents and Chemotherapy, July 2005, p. 3004-3008, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.3004-3008.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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