Comparing the Ability of Secretory Signal Peptides for Heterologous Expression of Anti-Lipopolysaccharide Factor 3 in <i>Chlamydomonas reinhardtii</i>
Anti-lipopolysaccharide factor 3 (ALF<i>Pm</i>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<i>Pm</i>3 is limited by its low production in natu...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-06-01
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| Series: | Marine Drugs |
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| Online Access: | https://www.mdpi.com/1660-3397/21/6/346 |
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| author | Huilin Zhuang Yaohui Ou Ruoyu Chen Danqiong Huang Chaogang Wang |
| author_facet | Huilin Zhuang Yaohui Ou Ruoyu Chen Danqiong Huang Chaogang Wang |
| author_sort | Huilin Zhuang |
| collection | DOAJ |
| description | Anti-lipopolysaccharide factor 3 (ALF<i>Pm</i>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<i>Pm</i>3 is limited by its low production in nature, as well as its low activity when expressed in <i>Escherichia coli</i> 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<i>Pm</i>3 in <i>Chlamydomonas reinhardtii</i>. In this study, signal peptides ARS1 and CAH1 were fused with ALF<i>Pm</i>3 and inserted into the pESVH vector to construct pH-aALF and pH-cALF plasmids, respectively, that were transformed to <i>C. reinhardtii</i> JUV using the glass bead method. Subsequently, through antibiotic screening, DNA-PCR, and RT-PCR, transformants expressing ALF<i>Pm</i>3 were confirmed and named T-JaA and T-JcA, respectively. The peptide ALF<i>Pm</i>3 could be detected in algal cells and culture medium by immunoblot, meaning that ALF<i>Pm</i>3 was successfully expressed in <i>C. reinhardtii</i> and secreted into the extracellular environment. Moreover, ALF<i>Pm</i>3 extracts from the culture media of T-JaA and T-JcA showed significant inhibitory effects on the growth of <i>V. harveyi</i>, <i>V. alginolyticus</i>, <i>V. anguillarum</i>, and <i>V. parahaemolyticus</i> within 24 h. Interestingly, the inhibitory rate of c-ALF<i>Pm</i>3 from T-JcA against four Vibrio was 2.77 to 6.23 times greater than that of a-ALF<i>Pm</i>3 from T-JaA, indicating that the CAH1 signal peptide was more helpful in enhancing the secreted expression of the ALF<i>Pm</i>3 peptide. Our results provided a new strategy for the secretory production of ALF<i>Pm</i>3 with high antibacterial activity in <i>C. reinhardtii</i>, which could improve the application potentiality of ALF<i>Pm</i>3 in the aquaculture industry. |
| first_indexed | 2024-03-11T02:13:49Z |
| format | Article |
| id | doaj.art-c8f79b5b7cb140e68f12c3cfe63b3f1c |
| institution | Directory Open Access Journal |
| issn | 1660-3397 |
| language | English |
| last_indexed | 2024-03-11T02:13:49Z |
| publishDate | 2023-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Marine Drugs |
| spelling | doaj.art-c8f79b5b7cb140e68f12c3cfe63b3f1c2023-11-18T11:22:37ZengMDPI AGMarine Drugs1660-33972023-06-0121634610.3390/md21060346Comparing the Ability of Secretory Signal Peptides for Heterologous Expression of Anti-Lipopolysaccharide Factor 3 in <i>Chlamydomonas reinhardtii</i>Huilin Zhuang0Yaohui Ou1Ruoyu Chen2Danqiong Huang3Chaogang Wang4Guangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, ChinaGuangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, ChinaGuangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, ChinaGuangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, ChinaGuangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, ChinaAnti-lipopolysaccharide factor 3 (ALF<i>Pm</i>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<i>Pm</i>3 is limited by its low production in nature, as well as its low activity when expressed in <i>Escherichia coli</i> 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<i>Pm</i>3 in <i>Chlamydomonas reinhardtii</i>. In this study, signal peptides ARS1 and CAH1 were fused with ALF<i>Pm</i>3 and inserted into the pESVH vector to construct pH-aALF and pH-cALF plasmids, respectively, that were transformed to <i>C. reinhardtii</i> JUV using the glass bead method. Subsequently, through antibiotic screening, DNA-PCR, and RT-PCR, transformants expressing ALF<i>Pm</i>3 were confirmed and named T-JaA and T-JcA, respectively. The peptide ALF<i>Pm</i>3 could be detected in algal cells and culture medium by immunoblot, meaning that ALF<i>Pm</i>3 was successfully expressed in <i>C. reinhardtii</i> and secreted into the extracellular environment. Moreover, ALF<i>Pm</i>3 extracts from the culture media of T-JaA and T-JcA showed significant inhibitory effects on the growth of <i>V. harveyi</i>, <i>V. alginolyticus</i>, <i>V. anguillarum</i>, and <i>V. parahaemolyticus</i> within 24 h. Interestingly, the inhibitory rate of c-ALF<i>Pm</i>3 from T-JcA against four Vibrio was 2.77 to 6.23 times greater than that of a-ALF<i>Pm</i>3 from T-JaA, indicating that the CAH1 signal peptide was more helpful in enhancing the secreted expression of the ALF<i>Pm</i>3 peptide. Our results provided a new strategy for the secretory production of ALF<i>Pm</i>3 with high antibacterial activity in <i>C. reinhardtii</i>, which could improve the application potentiality of ALF<i>Pm</i>3 in the aquaculture industry.https://www.mdpi.com/1660-3397/21/6/346antimicrobial peptide<i>Chlamydomonas reinhardtii</i>anti-lipopolysaccharide factor 3secretory expressionarylsulfatase 1 signal peptidecarbonic anhydrase 1 signal peptide |
| spellingShingle | Huilin Zhuang Yaohui Ou Ruoyu Chen Danqiong Huang Chaogang Wang Comparing the Ability of Secretory Signal Peptides for Heterologous Expression of Anti-Lipopolysaccharide Factor 3 in <i>Chlamydomonas reinhardtii</i> Marine Drugs antimicrobial peptide <i>Chlamydomonas reinhardtii</i> anti-lipopolysaccharide factor 3 secretory expression arylsulfatase 1 signal peptide carbonic anhydrase 1 signal peptide |
| title | Comparing the Ability of Secretory Signal Peptides for Heterologous Expression of Anti-Lipopolysaccharide Factor 3 in <i>Chlamydomonas reinhardtii</i> |
| title_full | Comparing the Ability of Secretory Signal Peptides for Heterologous Expression of Anti-Lipopolysaccharide Factor 3 in <i>Chlamydomonas reinhardtii</i> |
| title_fullStr | Comparing the Ability of Secretory Signal Peptides for Heterologous Expression of Anti-Lipopolysaccharide Factor 3 in <i>Chlamydomonas reinhardtii</i> |
| title_full_unstemmed | Comparing the Ability of Secretory Signal Peptides for Heterologous Expression of Anti-Lipopolysaccharide Factor 3 in <i>Chlamydomonas reinhardtii</i> |
| title_short | Comparing the Ability of Secretory Signal Peptides for Heterologous Expression of Anti-Lipopolysaccharide Factor 3 in <i>Chlamydomonas reinhardtii</i> |
| title_sort | comparing the ability of secretory signal peptides for heterologous expression of anti lipopolysaccharide factor 3 in i chlamydomonas reinhardtii i |
| topic | antimicrobial peptide <i>Chlamydomonas reinhardtii</i> anti-lipopolysaccharide factor 3 secretory expression arylsulfatase 1 signal peptide carbonic anhydrase 1 signal peptide |
| url | https://www.mdpi.com/1660-3397/21/6/346 |
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