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Use of Synthetic Genes for Cloning, Production and Functional Expression of the Bacteriocins Enterocin A and Bacteriocin E 50-52 by Pichia pastoris and Kluyveromyces lactis

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Abstract

The use of synthetic genes may constitute a successful approach for the heterologous production and functional expression of bacterial antimicrobial peptides (bacteriocins) by recombinant yeasts. In this work, synthetic genes with adapted codon usage designed from the mature amino acid sequence of the bacteriocin enterocin A (EntA), produced by Enterococcus faecium T136, and the mature bacteriocin E 50-52 (BacE50-52), produced by E. faecium NRRL B-32746, were synthesized. The synthetic entA and bacE50-52 were cloned into the protein expression vectors pPICZαA and pKLAC2 for transformation of derived vectors into Pichia pastoris X-33 and Kluyveromyces lactis GG799, respectively. The recombinant vectors were linearized and transformed into competent cells selecting for P. pastoris X-33EAS (entA), P. pastoris X-33BE50-52S (bacE50-52), K. lactis GG799EAS (entA), and K. lactis GG799BE50-52S (bacE50-52). P. pastoris X-33EAS and K. lactis GG799EAS, but not P. pastoris X-33BE50-52S and K. lactis GG799BE50-52S, showed antimicrobial activity in their supernatants. However, purification of the supernatants of the producer yeasts permitted recovery of the bacteriocins EntA and BacE50-52. Both purified bacteriocins were active against Gram-positive bacteria such as Listeria monocytogenes but not against Gram-negative bacteria, including Campylobacter jejuni.

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Acknowledgments

This work was partially supported by projects AGL2012-34829 from the Ministerio de Economía y Competitividad (MINECO) and AGL2009-08348 from the Ministerio de Ciencia e Innovación (MICINN), by project GR35-10A from the Banco de Santander Central Hispano-Universidad Complutense de Madrid (BSCH-UCM), and by project S2009/AGR-1489 from the Comunidad de Madrid (CAM). J.J. Jiménez and S. Arbulu are recipient of fellowships (FPI) from the MICINN. J. Borrero held a research contract from the CAM, and L. Gútiez holds a fellowship (FPU) from the Ministerio de Educación y Ciencia (MEC), Spain.

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  1. Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid (UCM), Avenida Puerta de Hierro, s/n, 28040, Madrid, Spain

    Juan J. Jiménez, Juan Borrero, Loreto Gútiez, Sara Arbulu, Carmen Herranz, Luis M. Cintas & Pablo E. Hernández

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  1. Juan J. Jiménez
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Correspondence to Pablo E. Hernández.

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Jiménez, J.J., Borrero, J., Gútiez, L. et al. Use of Synthetic Genes for Cloning, Production and Functional Expression of the Bacteriocins Enterocin A and Bacteriocin E 50-52 by Pichia pastoris and Kluyveromyces lactis . Mol Biotechnol 56, 571–583 (2014). https://doi.org/10.1007/s12033-014-9731-7

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