Processing and secretion of non-cognate bacteriocins by EnkT, an ABC transporter from a multiple-bacteriocin producer, Enterococcus faecium NKR-5-3

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EnkT is an ATP-binding cassette (ABC) transporter produced by Enterococcus faecium NKR-5-3, which is responsible for the secretion of multiple bacteriocins; enterocins NKR-5-3A, C, D, and Z (Ent53A, C, D, and Z). EnkT has been shown to possess a tolerant recognition mechanism that enables it to secrete the mature Ent53C from a chimeric precursor peptide containing the leader peptide moieties that are derived from different heterologous bacteriocins. In this study, to further characterize EnkT, we aimed to investigate the capacity of EnkT to recognize, process, and secrete non-cognate bacteriocins, which belong to different subclasses of class II. For this, the non-cognate bacteriocin precursor peptides, including enterocin A, pediocin PA-1, lactococcin Q, lactococcin A, and lacticin Q were co-expressed with EnkT, and thereafter, the production of the mature forms of these non-cognate bacteriocins was assessed. Our results revealed that EnkT could potentially recognize, process, and secrete the non-cognate bacteriocins with an exception of the leaderless bacteriocin, lacticin Q. Moreover, the processing and secretion efficiencies of these heterologous non-cognate bacteriocins by EnkT were further enhanced when the leader peptide moiety was replaced with the Ent53C leader peptide (derived from a native NKR-5-3 bacteriocin). The findings of this study describe the wide substrate tolerance of this ABC transporter, EnkT, that can be exploited in the future in establishing effective bacteriocin production systems adaptive to complex fermentation conditions common in many food systems.

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Bacterial strains, plasmids, and culture conditions

The strains used in this study are listed in Table 1. The heterologous expression host strain, Lactococcus lactis NZ9000 was cultured in M17 medium (Merck, Darmstadt, Germany) supplemented with 0.5 % glucose (Nacalai Tesque, Kyoto, Japan) (GM17) and propagated at 30°C. The indicator strains, E. faecalis JCM 5803T and L. lactis subsp. lactis IL1403 were cultured in MRS broth (Oxoid, Basingstoke, UK) at 37°C, and L. lactis subsp. lactis ATCC 19435T and Lactobacillus sakei subsp. sakei JCM 1157T

Production of non-cognate bacteriocins by EnkT

EnkT is an ABC transporter protein, which is responsible for the processing and secretion of multiple bacteriocins belonging to different subclasses. This allowed us to evaluate the capacity of EnkT to process and secrete non-cognate bacteriocins. Sequence analysis of the non-cognate precursor peptides of EntA, PedA, LcnA, and LaqQ did not reveal the presence of significantly conserved motifs in their leader peptide moieties relative to their Ent53 bacteriocin counterparts except the presence

Discussion

In this present study, we demonstrated the wide substrate tolerance of EnkT for processing different non-cognate bacteriocin precursor peptides to yield their respective mature and active forms. The heterologous non-cognate bacteriocin precursor peptides despite significantly varying in both, the length and the sequence, were still successfully processed and secreted by EnkT (13). Although, previous studies have already described about some ABC transporters that can process heterologous

Acknowledgments

This study was supported in part by the Grants-in-Aid for Scientific Research provided by the Japan Society for the Promotion of Science (JSPS KAKENHI, Grant Number JP17H03797) and the “Innovation inspired by Nature” Research Support Program, Sekisui Chemical Co., Ltd.

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    • Multiple bacteriocin production in lactic acid bacteria

      2022, Journal of Bioscience and Bioengineering
      Citation Excerpt :

      The capacity of EnkT to process the heterogenous module 1 bacteriocins of strain NKR-5-3 contradicts the common understanding that each bacteriocin requires a dedicated ABC-transporter for its processing and secretion. The unique capacity of EnkT in processing heterologous bacteriocin precursor peptides as its substrates was extensively investigated by Sushida et al. (51,58). It was demonstrated that the bioactivity of EnkT requires a specific structural confirmation of the leader peptide rather than specific residues in recognizing and processing of its substrate (bacteriocin precursor peptide).

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