Development of a novel endolysin, PanLys.1, for the specific inhibition of Peptostreptococcus anaerobius

Abstract

Objective: The objective of this study was to develop a novel endolysin (PanLys.1) for the specific killing of the ruminal hyper-ammonia-producing bacterium <i>Peptostreptococcus anaerobius</i> (<i>P. anaerobius</i>).Methods: Whole genome sequences of <i>P. anaerobius</i> strains and related bacteriophages were collected from the National Center for Biotechnology Information database, and the candidate gene for PanLys.1 was isolated based on amino acid sequences and conserved domain database (CDD) analysis. The gene was overexpressed using a pET system in Escherichia coli BL21 (DE3). The lytic activity of PanLys.1 was evaluated under various conditions (dosage, pH, temperature, NaCl, and metal ions) to determine the optimal lytic activity conditions. Finally, the killing activity of PanLys.1 against <i>P. anaerobius</i> was confirmed using an in vitro rumen fermentation system.Results: CDD analysis showed that PanLys.1 has a modular design with a catalytic domain, amidase-2, at the N-terminal, and a cell wall binding domain, from the CW-7 superfamily, at the C-terminal. The lytic activity of PanLys.1 against <i>P. anaerobius</i> was the highest at pH 8.0 (p<0.05) and was maintained at 37°C to 45°C, and 0 to 250 mM NaCl. The activity of PanLys.1 significantly decreased (p<0.05) after Mn²⁺ or Zn²⁺ treatment. The relative abundance of <i>P. anaerobius</i> did not decrease after administration PanLys.1 under <i>in vitro</i> rumen conditions.Conclusion: The application of PanLys.1 to modulate <i>P. anaerobius</i> in the rumen might not be feasible because its lytic activity was not observed in <i>in vitro</i> rumen system.