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

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.

Cloning and expression of an anti-cancerous cytokine: human IL-29 gene in Chlamydomonas reinhardtii

“…Chlamydomonas reinhardtii has proven to be an effective platform for recombinant protein/peptide production (Reyes-Barrera et al 2021 ; Li et al 2021 ; Jiang et al 2021 ). In this study, the IL29 protein was first expressed in an algal expression system.…”

Enhancement of β-carotene content in Chlamydomonas reinhardtii by expressing bacterium-driven lycopene β-cyclase

“…To produce target carotenoids in E. coli , plasmids pET-DsLcyb1 and pET-CrtY were transferred into the chemical competent cell of lycopene-producing E. coli using heat-shock method [ 25 ]. To produce target carotenoids in C. reinhardtii , the plasmid pDb-CrtY was transferred into algal cells using glass-bead method with few modifications [ 26 ]. The algal transformation process was performed as described, with additional information that NotI was used to linearize plasmids.…”

Comparing the Ability of Secretory Signal Peptides for Heterologous Expression of Anti-Lipopolysaccharide Factor 3 in Chlamydomonas reinhardtii

“…To evaluate the presence of the protein ALF Pm 3 in T-JaA and T-JcA, the total proteins in cells and the secretory protein in culture supernatant were extracted and subjected to immunoblot analysis. Algal cells were pelleted by centrifugation and total intracellular proteins were extracted based on the reference literature [ 9 ]. The extracellular protein from culture medium was extracted as follows: the culture medium was collected by centrifugation and then freeze dried.…”

“…As one cluster of the known AMPs, anti-lipopolysaccharide factors (ALF) firstly found in Limulus polyphemus displayed high antibacterial activity [ 7 ]. In recent years, it was reported that the anti-lipopolysaccharide factor 3 (ALF Pm 3) from Penaeus monodon with a lipopolysaccharide binding domain (LBD) had strong antibacterial activity against common aquaculture pathogenic bacteria, such as Vibrio harveyi , Vibrio parahaemolyticus , and Staphylococcus aureus , at a very low concentration (0.77 µM), showing its potential for application in the aquaculture industry [ 8 , 9 , 10 ]. ALF Pm 3 is commonly produced via genetic engineering due to its low yield from natural sources.…”