Journal
FOOD RESEARCH INTERNATIONAL
Volume 121, Issue -, Pages 888-899Publisher
ELSEVIER
DOI: 10.1016/j.foodres.2019.01.015
Keywords
Synthetic genes; Native bacteriocins; Hybrid- and bacteriocin-derived chimeras; Heterologous bacteriocin production; Pichia pastoris; MALDI-TOF MS analysis; MRM-ESI-LC-MS/MS (QTRAP) analysis
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Funding
- Ministerio de Economia y Competitividad (MINECO) [AGL2012-34829]
- Comunidad de Madrid (CAM) [S2013/ABI-2747]
- Universidad Complutense of Madrid (UCM), Spain [FEI 16/54]
- MINECO
- Ministerio de Ciencia e Innovacion (MICINN)
- Ministerio de Education y Ciencia (MEC), Spain
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In this work, synthetic genes designed from (a), the native amino acid sequence of the class IIa bacteriocins enterocin HF (EntHF) and enterocin CRL35 (EntCRL35), (b) from hybrid bacteriocins derived from fusion of enterocin A (EntA) to itself and to EntHF and EntCRL35 through a tri-glycine peptide linker, and (c) from bacteriocin-derived chimeras devised from fusion of the N-terminal region of EntA and enterocin P (EntP) to the C-terminal end of EntHF and EntCRL35, were cloned in plasmid pPICZ alpha A for expression by P. pastoris X-33. Synthetic genes encoding EntHF and EntCRL35 were also cloned in plasmid pP-alpha hSUMO3 for expression of the hSUMO3-fused bacteriocins by P. pastoris. Only recombinant P. pastoris expressing the bacteriocin-derived chimeras displayed a direct antimicrobial activity whereas P. pastoris X-33, producer of EntP::EntHF, showed the highest antimicrobial activity in their supernatants and in the multi-step chromatographic purified fractions. The MRM-ESI-LC-MS/MS (QTRAP) analysis of purified fractions from P. pastoris producers of hybrid- and bacteriocin-derived chimeras, permitted detection in the samples of peptides with the expected molecular mass of the bacteriocins produced. The antimicrobial activity of the EntP::EntHF chimera compared to that of the synthetic EntP::EntHF peptide, suggest that the biologically-produced bacteriocin-derived chimera shows a higher specific antimicrobial activity than its synthetic counterpart against different Listeria strains, including L. monocytogenes. More important, the N-terminal region of EntA and EntP seems to drive the production, processing and secretion of hybrid- and bacteriocin-derived chimeras, by P. pastoris X-33.
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