Cloning and expression of synthetic genes encoding angiotensin-I converting enzyme (ACE)-inhibitory bioactive peptides in Bifidobacterium pseudocatenulatum

Authors

  • Luca Losurdo,

    1. Dipartimento di Bioscienze, Biotecnologie e Scienze Farmacologiche, Università degli Studi di Bari ‘Aldo Moro’, Bari, Italy
    2. Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos (IPLA-CSIC), Villaviciosa, Asturias, Spain
    3. Istituto di Biomembrane e Bioenergetica (IBBE), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy
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  • Laura Quintieri,

    1. Istituto di Scienze delle Produzioni Alimentari (ISPA), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy
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  • Leonardo Caputo,

    1. Istituto di Scienze delle Produzioni Alimentari (ISPA), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy
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  • Raffaele Gallerani,

    1. Dipartimento di Bioscienze, Biotecnologie e Scienze Farmacologiche, Università degli Studi di Bari ‘Aldo Moro’, Bari, Italy
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  • Baltasar Mayo,

    1. Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos (IPLA-CSIC), Villaviciosa, Asturias, Spain
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  • Francesca De Leo

    Corresponding author
    1. Istituto di Biomembrane e Bioenergetica (IBBE), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy
    • Dipartimento di Bioscienze, Biotecnologie e Scienze Farmacologiche, Università degli Studi di Bari ‘Aldo Moro’, Bari, Italy
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Correspondence: Francesca De Leo, Istituto di Biomembrane e Bioenergetica-CNR c/o Dipartimento di Bioscienze, Biotecnologie e Scienze Farmacologiche Campus “Ernesto Quagliariello” Via Amendola 165/A 70126 Bari, Italy. Tel.:+39 080 5443311; fax: +39 080 5443317; e-mail: f.deleo@ibbe.cnr.it

Abstract

A wide range of biopeptides potentially able to lower blood pressure through inhibition of the angiotensin-I converting enzyme (ACE) is produced in fermented foods by proteolytic starter cultures. This work applies a procedure based on recombinant DNA technologies for the synthesis and expression of three ACE-inhibitory peptides using a probiotic cell factory. ACE-inhibitory genes and their pro-active precursors were designed, synthesized by PCR, and cloned in Escherichia coli; after which, they were cloned into the pAM1 E. coli-bifidobacteria shuttle vector. After E. coli transformation, constructs carrying the six recombinant clones were electrotransferred into the Bifidobacterium pseudocatenulatum M115 probiotic strain. Interestingly, five of the six constructs proved to be stable. Their expression was confirmed by reverse transcription PCR. Furthermore, transformed strains displayed ACE-inhibitory activity linearly correlated to increasing amounts of cell-free cellular lysates. In particular, 50 μg of lysates from constructs pAM1-Pro-BP3 and pAM1-BP2 showed a 50% higher ACE-inhibitory activity than that of the controls. As a comparison, addition of 50 ng of Pro-BP1 and Pro-BP3 synthetic peptides to 50 μg of cell-free extracts of B. pseudocatenulatum M115 wild-type strain showed an average of 67% of ACE inhibition; this allowed estimating the amount of the peptides produced by the transformants. Engineering of bifidobacteria for the production of biopeptides is envisioned as a promising cell factory model system.

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