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

Area of Science:

• Biotechnology
• Microbiology
• Protein Expression

Background:

• Enterocin RM6, an antimicrobial peptide from Enterococcus faecalis, was discovered in 2013.
• Its antibacterial activity, particularly against Listeria monocytogenes, remained largely uninvestigated.
• A need exists for efficient and stable production methods for antimicrobial peptides.

Purpose of the Study:

• To express and characterize enterocin RM6 using the green algae Chlamydomonas reinhardtii.
• To evaluate the stability and antibacterial efficacy of the recombinant enterocin RM6.
• To assess the potential of C. reinhardtii as a host for producing bioactive enterocin RM6.

Main Methods:

• Engineered C. reinhardtii to express N-terminal hemagglutinin and 6×His double-tagged triple tandem repeats of enterocin RM6 (3×Enterocin RM6).
• Cultured transgenic cells for 6 months to assess expression stability and yield (0.45% of total soluble proteins).
• Determined minimum inhibitory concentrations (MICs) against six bacterial strains and evaluated stability across various temperatures, pH levels, and proteolytic enzymes.

Main Results:

• Stable expression of 3×Enterocin RM6 in C. reinhardtii was achieved over 6 months.
• Recombinant enterocin RM6 inhibited Gram-positive and Gram-negative bacteria with MICs of 25–70 μg/mL.
• The peptide showed significant stability, low hemolytic activity, and low cytotoxicity, with effective bacterial cell membrane penetration and morphological disruption.

Conclusions:

• Chlamydomonas reinhardtii is a suitable host for producing bioactive enterocin RM6.
• Recombinant enterocin RM6 exhibits broad-spectrum antibacterial activity and favorable stability characteristics.
• This study validates the potential of algal expression systems for antimicrobial peptide production.