Production of a bacteriocin like protein PEG 446 from Clostridium tyrobutyricum strain NRRL B-67062

Authors: Liu, Siqing; Lu, Shao; PATEL, MAULIK - Oak Ridge Institute For Science And Education (ORISE); Qureshi, Nasib; Dunlap, Christopher; Hoecker, Eric; Skory, Christopher - Chris

Submitted to: Probiotics and Antimicrobial Proteins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/29/2023
Publication Date: 1/22/2024
Citation: Liu, S., Lu, S.Y., Patel, M., Qureshi, N., Dunlap, C.A., Hoecker, E.C., Skory, C.D. 2024. Production of a bacteriocin like protein PEG 446 from Clostridium tyrobutyricum strain NRRL B-67062. Probiotics and Antimicrobial Proteins. https://doi.org/10.1007/s12602-023-10211-1.
DOI: https://doi.org/10.1007/s12602-023-10211-1

Interpretive Summary: Butyric acid, which is produced by animals and certain microbial strains, is commonly used in the chemical, food, and pharmaceutical industries. ARS researchers in Peoria, Illinois previously isolated a bacterial strain, called Clostridium tyrobutyricum, that was capable of producing high yields of this important product from agricultural feedstocks. In this study, the researchers sequenced the genome of this bacterial strain in an effort to further improve productivity. While studying the genome, the researchers identified several unique antibacterial proteins. One of these proteins, named PEG446, was shown to inhibit several bacteria, including a Listeria strain that is closely related to a foodborne pathogen. In addition, PEG446 inhibited growth of another bacteria that commonly contaminates biorefining facilities. The discovery of new antimicrobial compounds that can be made by growing microorganisms on agricultural feedstocks further diversifies production of value-added bioproducts and promotes rural economics.

Technical Abstract: Clostridium tyrobutyricum strain NRRL B-67062 was isolated from an ethanol production facility and shown to produce high yields of butyric acid. In addition, we determined that the fermentation broth of cell-free supernatant (CFS) from NRRL B-67062 contained antibacterial activity against certain Gram-positive cells. Draft genome of NRRL B-67062 showed one circular chromosome of 3,242,608 nucleotides, 3,114 predicted coding sequences (CDSs) and 79 RNA genes and an average 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, one protein PEG446 containing a predicted antibacterial polypeptide of 66 amino acids was studied here. PEG446 belongs to 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 L. lactis LM 0230, Limosilactobacillus fermentum 0315-25 and Listeria innocua B-33088 by gel overlay and well diffusion assays. Molecular modeling suggested PEG446 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 sugars.