Abstract
Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene (ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, 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 ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii 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 C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.
Highlights
Nowadays, people are suffering from the multidrug-resistant (MDR) microorganisms caused by the overuse of antibiotics [1,2,3]
Our results demonstrated that C. reinhardtii is suitable for Antimicrobial peptides (AMPs) production, showing its potentiality to be used as a replacement of antibiotics on aquaculture
The ble gene was fused to the N-terminal of optimized ALFPm3 gene by Foot and Mouth Disease
Summary
People are suffering from the multidrug-resistant (MDR) microorganisms caused by the overuse of antibiotics [1,2,3]. Antimicrobial peptides (AMPs) are one of the promising candidates due to their positive effect on killing drug-resistant microorganisms [4,5]. AMPs own a broad-spectrum antimicrobial activity [10,11], anti-cancer cell activity [12,13], and antiviral activity [14]. As one of the known AMPs, anti-lipopolysaccharide factors (ALFs) were firstly identified from the hemocytes of Limulus polyphemus [15]. ALFs have the lipopolysaccharide (LPS) binding domain (LBD), which can bind to LPS to exhibit antimicrobial activity. ALFPm3 has a wider anti-bacteria spectrum than other ALFs [19,20,21,22]. The ALFPm3 could hardly be expressed in E. coli possibly because of its strong antimicrobial activity [20]
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