Expression of Anti-Lipopolysaccharide Factor Isoform 3 in Chlamydomonas reinhardtii Showing High Antimicrobial Activity

Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene ( ), encoding an antimicrobial peptide from with a super activity was expressed in , which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized gene was fused with the reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into CC-849. In addition to 8 μg/mL zeocin resistance selection, the transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the -derived ALFPm3 (cALFPm3) was successfully expressed in transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic strain, which can produce the active ALFPm3 driven from , providing a potential strategy to use as the cell factory to produce antimicrobial peptides.