Journal of Marine Bioscience and Biotechnology (한국해양바이오학회지)

The Korean Society for Marine Biotechnology (한국해양바이오학회)
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Efficient Extracellular Secretion of the Antimicrobial Peptide Magainin 2 in the Chlorella-based System

클로렐라 시스템에서 항균펩타이드 Magainin 2의 효율적인 세포외 분비

  • 정유정 ((주)바이오이즈 중앙연구소) ;
  • 황재윤 ((주)바이오이즈 중앙연구소) ;
  • 김성천 ((주)바이오이즈)
  • Received : 2024.03.19
  • Accepted : 2024.04.09
  • Published : 2024.06.30
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Abstract

Various antimicrobial peptides (AMPs) from microalgae have shown antibacterial, antiviral, antifungal, anticancer, and antioxidant effects, and play crucial roles in medical applications, aquaculture-related disease management, and the food industry. Magainin 2 (MAG2), an AMP, exhibits high antibacterial and antitumor activity, necessitating an efficient recombinant expression system for low-cost, large-scale production. To enhance MAG2 secretion efficiency in Chlorella, we constructed the SS:MAG2:His vector using the known Chlamydomonas reinhardtii CA1 signal sequence (SS) and obtained a stable transformant via an Agrobacterium-mediated transformation method and RT-qPCR. ELISA results revealed that the MAG2 content secreted into the medium by the SS:MAG2:His transformants increased proportionally with mRNA expression. These findings offer a strategy for high MAG2 secretion in the Chlorella vulgaris platform, potentially minimizing downstream processing costs.

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References

  1. Marrez, D. A., Naguib, M. M., Sultan, Y. Y. and Higazy, A. M. 2019. Antimicrobial and anticancer activities of Scenedesmus obliquus metabolites. Heliyon. 5(3), e01404.
  2. Shannon, E. and Abu-Ghannam, N. 2016. Antibacterial derivatives of marine algae: An overview of pharmacological mechanisms and applications. Mar. Drugs. 14(4), 81.
  3. Gong, Y., Hu, H., Gao, Y., Xu, X. and Gao, H. 2011. Microalgae as platforms for production of recombinant proteins and valuable compounds: progress and prospects. J. Ind. Microbiol. Biotechnol. 38(12), 1879-1890.
  4. Lauritano, C., Andersen, J. H., Hansen, E., Albrigtsen, M., Escalera, L., Esposito, F., Helland, K., Hanssen K. O., Romano, G. and Ianora, A. 2016. Bioactivity screening of microalgae for antioxidant, anti-inflammatory, anticancer, anti-diabetes, and antibacterial activities. Front. Mar. Sci. 3, 1-12.
  5. Patra J. K., Patra A. P., Mahapatra N. K., Thatoi H. N., Das S., Sahu R. K. and Swain G. C. 2009. Antimicrobial activity of organic solvent extracts of three marine macroalgae from Chilika Lake, Orissa. India. Malays. J. Microbiol. 5, 128-131.
  6. Kim, S. K. and Wijesekara I. 2010. Development and biological activities of marine-derived bioactive peptides: A review. J. Funct. Foods. 2010, 2:1-9.
  7. Ayswaria, R., Vijayan, J. and Nathan, V. K. 2023. Antimicrobial peptides derived from microalgae for combating antibiotic resistance: Current status and prospects. Cell Biochem. Funct. 41(2), 142-151.
  8. Dingmann, B. J. 2018. Searching for New Antibiotics Right Under our Feet. J. Public Health Issues Pract. 2, 111.
  9. Lagadinou, M., Onisor, M. O., Rigas, A., Musetescu, D. V. Gkentzi, D., Assimakopoulos, S. F., Panos, G. and Marangos, M. 2020. Antimicrobial Properties on Non-Antibiotic Drugs in the Era of Increased Bacterial Resistance. Antibiotics. 9, 107.
  10. Huan, Y., Kong, Q., Mou H. and Yi, H. 2020. Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields. Front. Microbiol. 11, 582779.
  11. Zhang, Q. Y., Yan, Z. B., Meng, Y. M., Hong, X. Y., Shao, G., Ma, J. J., Cheng, X. R., Liu, J., Kang, J. and Fu, C. Y. 2021. Antimicrobial peptides: mechanism of action, activity and clinical potential. Mil. Med. Res. 8(1), 48.
  12. Kang, X., Dong, F., Shi, C., Liu, S., Sun, J., Chen, J., Li, H., Xu, H., Lao, X. and Zheng H. 2019. DRAMP 2.0, an updated data repository of antimicrobial peptides. Sci. Data. 6(1), 148.
  13. Shi, G., Kang, X., Dong, F., Liu, Y., Zhu, N., Hu, Y., Xu, H., Lao, X. and Zheng, H. 2022. DRAMP 3.0: an enhanced comprehensive data repository of antimicrobial peptides. Nucleic Acids Res. 50(D1), D488-D496.
  14. Barrell, P. J., Liew, O. W. and Conner, A. J. 2004. Expressing an antibacterial protein in bacteria for raising antibodies. Prot Expr Purif. 33, 153-159.
  15. Li Y. 2011. Recombinant production of antimicrobial peptides in Escherichia coli: a review. Prot Expr Purif. 80, 260-267.
  16. Imperial, I. C. V. J. and Ibana, J. A. 2016. Addressing the Antibiotic Resistance Problem with Probiotics: Reducing the Risk of Its Double-Edged Sword Effect. Front. Microbiol. 7, 1983.
  17. Zasloff M. 1987. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc. Natl. Acad. Sci. USA. 84(15), 5449-5453.
  18. Imura, Y., Choda, N. and Matsuzaki, K. 2008. Magainin 2 in action: distinct modes of membrane permeabilization in living bacterial and mammalian cells. Biophys. J. 95(12), 5757-5765.
  19. Chen, H. C, Brown, J. H., Morell, J. L. and Huang, C. M. 1988. Synthetic magainin analogues with improved antimicrobial activity. FEBS Lett. 236, 462-466.
  20. Tachi, T., Epand, R. F., Epand, R. M. and Matsuzaki, K. 2002. Position-dependent hydrophobicity of the antimicrobial magainin peptide affects the mode of peptide-lipid interactions and selective toxicity. Biochemistry. 41(34), 10723-10731.
  21. Zasloff, M. 2002. Antimicrobial peptides of multicellularorganisms. Nature. 415, 389-395.
  22. Gottler, L. M. and Ramamoorthy, A. 2009. Structure, membrane orientation, mechanism, and function of pexiganan-a highly potent antimicrobial peptide designed from magainin. Biochim. Biophys. Acta. 1788(8), 1680-1686.
  23. Wright O, Yoshimi T. and Tunnacliffe A. 2012. Recombinant production of cathelicidin-derived antimicrobial peptides in Escherichia coli using an inducible autocleaving enzyme tag. N. Biotechnol. 29, 352-358.
  24. Zorko, M. and Jerala, R. 2010. Production of recombinant antimicrobial peptides in bacteria. Methods Mol. Biol. 618, 61-76.
  25. Stanier, R. Y., Kunisawa, R., Mandel, M. and Cohen-Bazire, G. 1971. Purifcation and properties of unicellular blue-green algae (order Chroococcales). Bac. Rev. 35, 171-205.
  26. Fujiwara, S., Fukuzawa, H., Tachiki, A. and Miyachi, S. 1990. Structure and differential expression of two genes encoding carbonic anhydrase in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA. 87(24), 9779-9783.
  27. Guerrero, E., Saugar, J. M., Matsuzaki, K. and Rivas, L. 2004. Role of positional hydrophobicity in the leishmanicidal activity of magainin 2. Antimicrob. Agents Chemother. 48(8), 2980-2986.
  28. Holsters, M., de Waele, D., Depicker, A. and Messens, E,, van Montagu, M., and Schell, J. 1978. Transfection and transformation of Agrobacterium tumefaciens. Mol. Gen. Genet. 163, 181-187.
  29. Futatsumori-Sugai, M. and Tsumoto, K. 2010. Signal peptide design for improving recombinant protein secretion in the baculovirus expression vector system. Biochem. Biophys. Res. Commun. 391(1), 931-935.
  30. Lauersen, K. J., Berger, H., Mussgnug, J. H. and Kruse, O. 2013. Efficient recombinant protein production and secretion from nuclear transgenes in Chlamydomonas reinhardtii. J. Biotechnol. 167, 101-110.
  31. Amack, S. C. and Antunes, M. S. 2020. CaMV35S promoter-a plant biology and biotechnology workhorse in the era of synthetic biology. Curr. Opin. Plant Biol. 24, 100179.
  32. Montero-Lobato, Z., Vazquez, M., Navarro, F., Fuentes, J.L., Bermejo, E., Garbayo, I., and Vilchez, C. and Cuaresma, M. 2018. Chemically-Induced Production of Anti-Inflammatory Molecules in Microalgae. Mar. Drugs. 16(12), 478.
  33. Bai, L. L., Yin, W. B., Chen, Y. H., Niu, L. L., Sun, Y. R., Zhao, S. M., Yang, F. Q., Wang, R. R., Wu, Q., Zhang, X. Q. and Hu, Z. M. 2013. A new strategy to produce a defensin: stable production of mutated NP-1 in nitrate reductase-deficient Chlorella ellipsoidea. PLoS One. 8(1), e54966.
  34. Kong, F., Yamasaki, T., Kurniasih, S. D., Hou, L., Li, X., Ivanova, N., Okada, S. and Ohama T. (2015) Robust expression of heterologous genes by selection marker fusion system in improved Chlamydomonas strains. J. Biosci. Bioeng. 120, 239-245.
  35. Barahimipour, R., Strenkert, D., Neupert, J., Schroda, M., Merchant, S. S. and Bock, R. (2015) Dissecting the contributions of GC content and codon usage to gene expression in the model alga Chlamydomonas reinhardtii. Plant J. 84, 704-717.
  36. Scranton, M. A., Ostrand, J. T., Georgianna, D. R., Lofgren, S. M., Li, D., Ellis, R. C., Carruthers, D. N., Drager A., David L., Masica D. L. and Mayfield S. P. (2016) Synthetic promoters capable of driving robust nuclear gene expression in the green alga Chlamydomonas reinhardtii. Algal Res. 15, 135-142.
  37. Erdene-Ochir, E., Shin, B. K., Kwon, B., Jung, C. and Pan, C. H. 2019. Identification and characterisation of the novel endogenous promoter HASP1 and its signal peptide from Phaeodactylum tricornutum. Sci. Rep. 9(1), 9941.