Applied Microbiology and Biotechnology

, Volume 102, Issue 21, pp 9267–9278 | Cite as

Stable transformation of Spirulina (Arthrospira) platensis: a promising microalga for production of edible vaccines

  • Jaber Dehghani
  • Khosro Adibkia
  • Ali Movafeghi
  • Abolfazl Barzegari
  • Mohammad M. Pourseif
  • Hadi Maleki Kakelar
  • Asal Golchin
  • Yadollah OmidiEmail author
Applied genetics and molecular biotechnology


The planktonic blue-green microalga Spirulina (Arthrospira) platensis possesses important features (e.g., high protein and vital lipids contents as well as essential vitamins) and can be consumed by humans and animals. Accordingly, this microalga gained growing attention as a new platform for producing edible-based pharmaceutical proteins. However, there are limited successful strategies for the transformation of S. platensis, in part because of an efficient expression of strong endonucleases in its cytoplasm. In the current work, as a pilot step for the expression of therapeutic proteins, an Agrobacterium-based system was established to transfer gfp:gus and hygromycin resistance (hygr) genes into the genome of S. platensis. The presence of acetosyringone in the transfection medium significantly reduced the transformation efficiency. The PCR and real-time RT-PCR data confirmed the successful integration and transcription of the genes. Flow cytometry and β-glucuronidase (GUS) activity experiments confirmed the successful production of GFP and the enzyme. Moreover, the western blot analysis showed a ~ 90 kDa band in the transformed cells, indicating the successful production of the GFP:GUS protein. Three months after the transformation, the gene expression stability was validated by histochemical, flow cytometry, and hygromycin B resistance analyses.


Spirulina platensis Arthrospira Algal transformation Agrobacterium tumefaciens Protein expression Edible vaccine 



The authors are grateful for the financial support provided by the Ministry of Health, Care, and Medical Education of Iran and the Research Center for Pharmaceutical Nanotechnology (RCPN) at Tabriz University of Medical Sciences.


This project is part of a postdoc program (grant number: RCPN59560) funded by Tabriz University of Medical Sciences.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Abu-Arish A, Frenkiel-Krispin D, Fricke T, Tzfira T, Citovsky V, Wolf SG, Elbaum M (2004) Three-dimensional reconstruction of Agrobacterium VirE2 protein with single-stranded DNA. J Biol Chem 279(24):25359–25363. CrossRefPubMedGoogle Scholar
  2. Anila N, Chandrashekar A, Ravishankar G, Sarada R (2011) Establishment of Agrobacterium tumefaciens-mediated genetic transformation in Dunaliella bardawil. Eur J Phycol 46(1):36–44. CrossRefGoogle Scholar
  3. Atashpaz S, Khani S, Barzegari A, Barar J, Vahed SZ, Azarbaijani R, Omidi Y (2010) A robust universal method for extraction of genomic DNA from bacterial species. Mikrobiologiia 79(4):562–566PubMedGoogle Scholar
  4. Barzegari A, Saeedi N, Zarredar H, Barar J, Omidi Y (2014) The search for a promising cell factory system for production of edible vaccine. Hum Vaccin Immunother 10(8):2497–2502. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Cha TS, Yee W, Aziz A (2012) Assessment of factors affecting Agrobacterium-mediated genetic transformation of the unicellular green alga, Chlorella vulgaris. World J Microbiol Biotechnol 28(4):1771–1779. CrossRefPubMedGoogle Scholar
  6. Chen YH, Chang GK, Kuo SM, Huang SY, Hu IC, Lo YL, Shih SR (2016) Well-tolerated Spirulina extract inhibits influenza virus replication and reduces virus-induced mortality. Sci Rep 6:24253. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Cheng R, Ma R, Li K, Rong H, Lin X, Wang Z, Yang S, Ma Y (2012) Agrobacterium tumefaciens mediated transformation of marine microalgae Schizochytrium. Microbiol Res 167(3):179–186. CrossRefPubMedGoogle Scholar
  8. de Groot MJ, Bundock P, Hooykaas PJ, Beijersbergen AG (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 16(9):839–842. CrossRefPubMedGoogle Scholar
  9. Dehghani J, Movafeghi A, Barzegari A, Barar J (2017) Efficient and stable transformation of Dunaliella pseudosalina by 3 strains of Agrobacterium tumefaciens. Bioimpacts 7(4):247–254. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Dennis ES, Brettell RI (1990) DNA methylation of maize transposable elements is correlated with activity. Philos Trans R Soc Lond Ser B Biol Sci 326(1235):217–229CrossRefGoogle Scholar
  11. Doron L, Segal N, Shapira M (2016) Transgene expression in microalgae-from tools to applications. Front Plant Sci 7:505. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Dreesen IA, Charpin-El Hamri G, Fussenegger M (2010) Heat-stable oral alga-based vaccine protects mice from Staphylococcus aureus infection. J Biotechnol 145(3):273–280. CrossRefPubMedGoogle Scholar
  13. Fujisawa T, Narikawa R, Okamoto S, Ehira S, Yoshimura H, Suzuki I, Masuda T, Mochimaru M, Takaichi S, Awai K, Sekine M, Horikawa H, Yashiro I, Omata S, Takarada H, Katano Y, Kosugi H, Tanikawa S, Ohmori K, Sato N, Ikeuchi M, Fujita N, Ohmori M (2010) Genomic structure of an economically important cyanobacterium, Arthrospira (Spirulina) platensis NIES-39. DNA Res 17(2):85–103. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol Mol Biol Rev 67(1):16–37CrossRefGoogle Scholar
  15. Godwin I, Todd G, Ford-Lloyd B, Newbury HJ (1991) The effects of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species. Plant Cell Rep 9(12):671–675. CrossRefPubMedGoogle Scholar
  16. Gong Y, Hu H, Gao Y, Xu X, 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. CrossRefPubMedGoogle Scholar
  17. Hempel F, Lau J, Klingl A, Maier UG (2011) Algae as protein factories: expression of a human antibody and the respective antigen in the diatom Phaeodactylum tricornutum. PLoS One 6(12):e28424. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Jeamton W, Dulsawat S, Tanticharoen M, Vonshak A, Cheevadhanarak S (2017) Overcoming intrinsic restriction enzyme barriers enhances transformation efficiency in Arthrospira platensis C1. Plant Cell Physiol 58(4):822–830. CrossRefPubMedGoogle Scholar
  19. Kathiresan S, Chandrashekar A, Ravishankar GA, Sarada R (2009) Agrobacterium-mediated transformation in the green alga Haematococcus pluvialis (Chlorophyceae, Volvocales). J Phycol 45(3):642–649. CrossRefPubMedGoogle Scholar
  20. Kawamura M, Sakakibara M, Watanabe T, Kita K, Hiraoka N, Obayashi A, Takagi M, Yano K (1986) A new restriction endonuclease from Spirulina platensis. Nucleic Acids Res 14(5):1985–1989CrossRefGoogle Scholar
  21. Kawata Y, Yano S, Kojima H, Toyomizu M (2004) Transformation of Spirulina platensis strain C1 (Arthrospira sp. PCC9438) with Tn5 transposase-transposon DNA-cation liposome complex. Mar Biotechnol 6(4):355–363. CrossRefPubMedGoogle Scholar
  22. Khan Z, Bhadouria P, Bisen PS (2005) Nutritional and therapeutic potential of Spirulina. Curr Pharm Biotechnol 6(5):373–379. CrossRefPubMedGoogle Scholar
  23. Kiyokawa K, Yamamoto S, Sato Y, Momota N, Tanaka K, Moriguchi K, Suzuki K (2012) Yeast transformation mediated by Agrobacterium strains harboring an Ri plasmid: comparative study between GALLS of an Ri plasmid and virE of a Ti plasmid. Genes Cells 17(7):597–610. CrossRefPubMedGoogle Scholar
  24. Kulshreshtha A, Zacharia AJ, Jarouliya U, Bhadauriya P, Prasad GB, Bisen PS (2008) Spirulina in health care management. Curr Pharm Biotechnol 9(5):400–405. CrossRefPubMedGoogle Scholar
  25. Kunik T, Tzfira T, Kapulnik Y, Gafni Y, Dingwall C, Citovsky V (2001) Genetic transformation of HeLa cells by Agrobacterium. Proc Natl Acad Sci U S A 98(4):1871–1876. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Lefort F, Calmin G, Crovadore J, Falquet J, Hurni JP, Osteras M, Haldemann F, Farinelli L (2014) Whole-genome shotgun sequence of Arthrospira platensis strain Paraca, a cultivated and edible cyanobacterium. Genome Announc 2(4).
  27. Lohe AR, Hartl DL (1996) Autoregulation of mariner transposase activity by overproduction and dominant-negative complementation. Mol Biol Evol 13(4):549–555. CrossRefPubMedGoogle Scholar
  28. Lycke N (2012) Recent progress in mucosal vaccine development: potential and limitations. Nat Rev Immunol 12(8):592–605. CrossRefPubMedGoogle Scholar
  29. Mahajan G, Kamat M (1995) γ-Linolenic acid production from Spirulina platensis. Appl Microbiol Biotechnol 43(3):466–469. CrossRefGoogle Scholar
  30. McCullen CA, Binns AN (2006) Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer. Annu Rev Cell Dev Biol 22:101–127. CrossRefPubMedGoogle Scholar
  31. Mohseniazar M, Barin M, Zarredar H, Alizadeh S, Shanehbandi D (2011) Potential of microalgae and lactobacilli in biosynthesis of silver nanoparticles. Bioimpacts 1(3):149–152. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Obbard DJ, Gordon KH, Buck AH, Jiggins FM (2009) The evolution of RNAi as a defence against viruses and transposable elements. Philos Trans R Soc Lond Ser B Biol Sci 364(1513):99–115. CrossRefGoogle Scholar
  33. Park SY, Jeong MH, Wang HY, Kim JA, Yu NH, Kim S, Cheong YH, Kang S, Lee YH, Hur JS (2013) Agrobacterium tumefaciens-mediated transformation of the lichen fungus, Umbilicaria muehlenbergii. PLoS One 8(12):e83896. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Pelczar P, Kalck V, Gomez D, Hohn B (2004) Agrobacterium proteins VirD2 and VirE2 mediate precise integration of synthetic T-DNA complexes in mammalian cells. EMBO Rep 5(6):632–637. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Potvin G, Zhang Z (2010) Strategies for high-level recombinant protein expression in transgenic microalgae: a review. Biotechnol Adv 28(6):910–918. CrossRefPubMedGoogle Scholar
  36. Pourseif MM, Moghaddam G, Saeedi N, Barzegari A, Dehghani J, Omidi Y (2018) Current status and future prospective of vaccine development against Echinococcus granulosus. Biologicals 51:1–11. CrossRefGoogle Scholar
  37. Prasad B, Vadakedath N, Jeong HJ, General T, Cho MG, Lein W (2014) Agrobacterium tumefaciens-mediated genetic transformation of haptophytes (Isochrysis species). Appl Microbiol Biotechnol 98(20):8629–8639. CrossRefPubMedGoogle Scholar
  38. Pratheesh PT, Vineetha M, Kurup GM (2014) An efficient protocol for the Agrobacterium-mediated genetic transformation of microalga Chlamydomonas reinhardtii. Mol Biotechnol 56(6):507–515. CrossRefPubMedGoogle Scholar
  39. Pulz O, Gross W (2004) Valuable products from biotechnology of microalgae. Appl Microbiol Biotechnol 65(6):635–648. CrossRefPubMedGoogle Scholar
  40. Rajam MV, Kumar SV (2006) Green alga (Chlamydomonas reinhardtii). Methods Mol Biol 344:421–433. CrossRefPubMedGoogle Scholar
  41. Rasala BA, Lee PA, Shen Z, Briggs SP, Mendez M, Mayfield SP (2012) Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide. PLoS One 7(8):e43349. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Selmi C, Leung PS, Fischer L, German B, Yang CY, Kenny TP, Cysewski GR, Gershwin ME (2011) The effects of Spirulina on anemia and immune function in senior citizens. Cell Mol Immunol 8(3):248–254. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Sheikholeslam SN, Weeks DP (1987) Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol Biol 8(4):291–298. CrossRefPubMedGoogle Scholar
  44. Shiraishi H, Tabuse Y (2013) The AplI restriction-modification system in an edible cyanobacterium, Arthrospira (Spirulina) platensis NIES-39, recognizes the nucleotide sequence 5'-CTGCAG-3′. Biosci Biotechnol Biochem 77(4):782–788. CrossRefPubMedGoogle Scholar
  45. Simon DP, Narayanan A, Mallikarjun Gouda KG, Sarada R (2015) Vir gene inducers in Dunaliella salina; an insight in to the Agrobacterium-mediated genetic transformation of microalgae. Algal Res 11:121–124. CrossRefGoogle Scholar
  46. Specht EA, Mayfield SP (2014) Algae-based oral recombinant vaccines. Front Microbiol 5:60. CrossRefPubMedPubMedCentralGoogle Scholar
  47. Specht E, Miyake-Stoner S, Mayfield S (2010) Microalgae come of age as a platform for recombinant protein production. Biotechnol Lett 32(10):1373–1383. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Spolaore P, Joannis-Cassan C, Duran E, Isambert A (2006) Commercial applications of microalgae. J Biosci Bioeng 101(2):87–96. CrossRefPubMedGoogle Scholar
  49. Toyomizu M, Suzuki K, Kawata Y, Kojima H, Akiba Y (2001) Effective transformation of the cyanobacterium Spirulina platensis using electroporation. J Appl Phycol 13(3):209–214. CrossRefGoogle Scholar
  50. Tragut V, Xiao J, Bylina EJ, Borthakur D (1995) Characterization of DNA restriction-modification systems in Spirulina platensis strain pacifica. J Appl Phycol 7(6):561–564. CrossRefGoogle Scholar
  51. Vonshak A, Boussiba S, Abeliovich A, Richmond A (1983) Production of Spirulina biomass: maintenance of monoalgal culture outdoors. Biotechnol Bioeng 25(2):341–349. CrossRefPubMedGoogle Scholar
  52. Yari Khosroushahi A (2012) Applications of diatoms as potential micro algae in Nanobiotechnology. Bioimpacts 2(2):83–89. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Research Center for Pharmaceutical Nanotechnology, Biomedicine InstituteTabriz University of Medical SciencesTabrizIran
  2. 2.Department of Pharmaceutics, Faculty of PharmacyTabriz University of Medical SciencesTabrizIran
  3. 3.Department of Plant Biology, Faculty of Natural ScienceUniversity of TabrizTabrizIran

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