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

Anti-lipopolysaccharide factor 3 (ALF Pm 3) possesses a wide antimicrobial spectrum and high antibacterial and viral activities for broad application prospects in the aquaculture industry. However, the application of ALF Pm 3 is limited by its low production in nature, as well as its low activity when expressed in Escherichia coli and yeast. Although it has been proven that its secretory expression can be used to produce antimicrobial peptides with strong antimicrobial activity, there is no study on the high-efficiency secretory expression of ALF Pm 3 in Chlamydomonas reinhardtii . In this study, signal peptides ARS1 and CAH1 were fused with ALF Pm 3 and inserted into the pESVH vector to construct pH-aALF and pH-cALF plasmids, respectively, that were transformed to C. reinhardtii JUV using the glass bead method. Subsequently, through antibiotic screening, DNA-PCR, and RT-PCR, transformants expressing ALF Pm 3 were confirmed and named T-JaA and T-JcA, respectively. The peptide ALF Pm 3 could be detected in algal cells and culture medium by immunoblot, meaning that ALF Pm 3 was successfully expressed in C. reinhardtii and secreted into the extracellular environment. Moreover, ALF Pm 3 extracts from the culture media of T-JaA and T-JcA showed significant inhibitory effects on the growth of V. harveyi , V. alginolyticus , V. anguillarum , and V. parahaemolyticus within 24 h. Interestingly, the inhibitory rate of c-ALF Pm 3 from T-JcA against four Vibrio was 2.77 to 6.23 times greater than that of a-ALF Pm 3 from T-JaA, indicating that the CAH1 signal peptide was more helpful in enhancing the secreted expression of the ALF Pm 3 peptide. Our results provided a new strategy for the secretory production of ALF Pm 3 with high antibacterial activity in C. reinhardtii , which could improve the application potentiality of ALF Pm 3 in the aquaculture industry.