# Production of a Bacteriocin Like Protein PEG 446 from Clostridium tyrobutyricum NRRL B-67062

## TL;DR

This paper reports the discovery of an antibacterial protein, PEG 446, produced by a strain of Clostridium tyrobutyricum that also generates butyric acid.

## Contribution

The study identifies and characterizes a novel bacteriocin-like protein, PEG 446, from a butyric acid-producing bacterium.

## Key findings

- PEG 446 exhibits antibacterial activity against several Gram-positive bacteria.
- Molecular modeling reveals structural features of PEG 446, including an alpha-helix and beta-sheets.
- The peg446 gene was successfully cloned and expressed in E. coli and B. subtilis.

## Abstract

Clostridium tyrobutyricum strain NRRL B-67062 was previously isolated from an ethanol production facility and shown to produce high yields of butyric acid. In addition, the cell-free supernatant of the fermentation broth from NRRL B-67062 contained antibacterial activity against certain Gram-positive bacteria. To determine the source of this antibacterial activity, we report the genome and genome mining of this strain. The complete genome of NRRL B-67062 showed one circular chromosome of 3,242,608 nucleotides, 3114 predicted coding sequences, 79 RNA genes, and a G+C content of 31.0%. Analyses of the genome data for genes potentially associated with antimicrobial features were sought after by using BAGEL-4 and anti-SMASH databases. Among the leads, a polypeptide of 66 amino acids (PEG 446) contains the DUF4177 domain, which is an uncharacterized highly conserved domain (pfam13783). The cloning and expression of the peg446 gene in Escherichia coli and Bacillus subtilis confirmed the antibacterial property against Lactococcus lactis LM 0230, Limosilactobacillus fermentum 0315-25, and Listeria innocua NRRL B-33088 by gel overlay and well diffusion assays. Molecular modeling suggested that PEG 446 contains one alpha-helix and three anti-parallel short beta-sheets. These results will aid further functional studies and facilitate simultaneously fermentative production of both butyric acid and a putative bacteriocin from agricultural waste and lignocellulosic biomass materials.

The online version contains supplementary material available at 10.1007/s12602-023-10211-1.