Biotechnological Production of the Recombinant Two-Component Lantibiotic Lichenicidin in the Bacterial Expression System

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Resumo

Lantibiotics are a family of bacterial antimicrobial peptides synthesized by ribosomes that undergo post-translational modification to form lanthionine (Lan) and methyllanthionine (MeLan) residues. Lantibiotics are considered promising agents for combating antibiotic-resistant bacterial infections. This paper presents a biotechnological method for obtaining two components of the lantibiotic lichenicidin from Bacillus licheniformis B-511 – Lchα and Lchβ. A system has been developed that allows co-expression of the lchA1 or lchA2 genes, encoding the precursors of the α- or β-components, respectively, with the lchM1 or lchM2 genes of the modifying enzymes LchM1 and LchM2 in Escherichia coli cells. The developed system of heterologous expression and purification made it possible to obtain, with high yield, post-translationally modified recombinant Lchβ, completely identical to the natural peptide in structure and biological activity.

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Sobre autores

D. Antoshina

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences

Email: ovch@ibch.ru
Rússia, ul. Miklukho-Maklaya 16/10, Moscow, 117997

S. Balandin

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences; Moscow Institute of Physics and Technology (National Research University)

Email: ovch@ibch.ru

Phystech School of Biological and Medical Physics

Rússia, ul. Miklukho-Maklaya 16/10, Moscow, 117997; Institutskiy per. 9, Dolgoprudny, 141700

A. Tagaev

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences

Email: ovch@ibch.ru
Rússia, ul. Miklukho-Maklaya 16/10, Moscow, 117997

A. Potemkina

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences; Moscow Institute of Physics and Technology (National Research University)

Email: ovch@ibch.ru

Phystech School of Biological and Medical Physics

Rússia, ul. Miklukho-Maklaya 16/10, Moscow, 117997; Institutskiy per. 9, Dolgoprudny, 141700

T. Ovchinnikova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences; Moscow Institute of Physics and Technology (National Research University)

Autor responsável pela correspondência
Email: ovch@ibch.ru

Phystech School of Biological and Medical Physics

Rússia, ul. Miklukho-Maklaya 16/10, Moscow, 117997; Institutskiy per. 9, Dolgoprudny, 141700

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2. Fig. 1. (a) Scheme of biosynthesis and modification of lichenicidin components [4]; (b) structural organization of the lichenicidin biosynthesis cluster from B. licheniformis VK21 [12].

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3. Fig. 2. Schematic representation of expression plasmids for the production of recombinant lichenicidin components.

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4. Fig. 3. Chromatograms of purification of the obtained recombinant lichenicidin components using reversed-phase HPLC. (a) – Lchα and other immature forms of LchA1, (b) – Lchβ and other immature forms of LchA2.

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5. Fig. 4. MALDI mass spectrometric analysis of the obtained recombinant components of lichenicidin. (a) – LchA1, (b) – LchA2. (a) – Lchα and other immature forms of LchA1; (b) – Lchβ and other immature forms of LchA2.

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6. Fig. 5. Chromatogram of purification of natural lichenicidin using reversed-phase HPLC and MALDI mass spectrometric analysis of isolated natural components of lichenicidin.

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7. Fig. 6. Synergistic effect of an equimolar mixture of natural Lchα (Nα) and recombinant Lchβ (Rβ) against L. monocytogenes EGD (total concentration of peptide mixture – 1.0 μg; concentration of Nα or Rβ – 1.0 μg; (+) control – tetracycline at a concentration of 1.0 μg; (–) control – 5% acetonitrile, 0.1% trifluoroacetic acid.

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