# Tandem solubility enhancing tags enable the heterologous expression and in vitro maturation of a class IIa bacteriocin, clesteriocin a, identified in Candidatus Clostridium mucoides CM038

## TL;DR

Researchers discovered a new class IIa bacteriocin from a meat-related bacterium and successfully produced it in a lab, showing it can fight harmful bacteria like Listeria.

## Contribution

The study reports the first class IIa bacteriocin from the Clostridium estertheticum complex and demonstrates its heterologous expression and maturation.

## Key findings

- Class IIa bacteriocin clesteriocin A was identified and heterologously expressed in Escherichia coli.
- Tandem solubility tags enabled soluble expression of CleA and CleB150 proteins.
- Mature clesteriocin A showed antimicrobial activity against Listeria monocytogenes via the mptC gene.

## Abstract

The Clostridium estertheticum complex (CEC) consists of closely related bacterial species that are mostly isolated from meat processing environment. Genome mining studies have recently established CEC as a source of class I bacteriocins. However, up until now, class II bacteriocins have not been reported from CEC. In the present study, we determined the presence of class II bacteriocin biosynthetic clusters in 33 CEC genomes through genome mining followed by bacteriocin production through heterologous expression in Escherichia coli. Six biosynthetic gene clusters belonging to class IIa (n = 1), IIb (n = 2), IId (n = 2) and an undefined class containing three precursor peptides were identified in six different CEC strains. Using molecular biology, we developed dedicated expression vectors for the class IIa bacteriocin, clesteriocin A. Its precursor peptide, CleA, and protease, CleB150, were initially expressed in insoluble form in E. coli. Through a systematic analysis of suitable solubility enhancing protein tags, both CleA and CleB150 were expressed in significant amounts of soluble fractions using tandem tags derived from Small Ubiquitin-like Modifier, superfolder Green fluorescent protein, Maltose binding protein and N-utilization substance. Mature clesteriocin A was obtained following in vitro maturation reactions between the tagged CleA and CleB150. The novel bacteriocin displayed antimicrobial activity against Listeria monocytogenes, which was mediated by the mptC gene. By combining genome mining and molecular biology, we have shown CEC is a source of novel class II bacteriocins that have potential for application as food biopreservatives.