Antibacterial Efficacy of Chlamydomonas reinhardtii-Expressed Enterocin RM6 Against Gram-Positive and Gram-Negative Bacteria.

In 2013, enterocin RM6, an antimicrobial peptide, was isolated from Enterococcus faecalis in raw milk. This antimicrobial peptide exhibits certain antibacterial activity against Listeria monocytogenes. However, since its discovery, its antibacterial activity remains uninvestigated. In this study, we utilized the green algae Chlamydomonas reinhardtii to express N-terminal hemagglutinin and 6 × His double-tagged triple tandem repeats of enterocin RM6 (3 × Enterocin RM6). The expression of 3 × Enterocin RM6 remained stable after continuously passaging transgenic cells for 6 months; this yielded 0.45% of total soluble proteins. C. reinhardtii-expressed enterocin RM6 inhibited the growth of six bacterial strains, encompassing both gram-positive and gram-negative strains. The minimum inhibitory concentrations were 25-70 μg/mL. Recombinant 3 × Enterocin RM6 exhibited significant stability across a broad spectrum of temperatures and pH levels, exhibited tolerance to proteolytic enzymes, displayed minimal hemolytic activity against rabbit erythrocytes, and demonstrated low cytotoxicity toward Vero, BHK21, HEK293, and MDBK cell lines. In addition, C. reinhardtii-expressed 3 × Enterocin RM6 exhibited effective cell membrane penetration and caused morphological disruption in the targeted bacterial cells. Our study results indicate that C. reinhardtii is a viable host for producing bioactive enterocin RM6.