Journal of Marine Bioscience and Biotechnology (한국해양바이오학회지)

The Korean Society for Marine Biotechnology (한국해양바이오학회)
권호 표지
DOI QR코드

DOI QR Code

Efficient Expression System of High Value Proteins, hGM-CSF and hEGF, using Agrobacterium-mediated Chlorella Transformation

아그로박테리움 매개 클로렐라 형질전환을 이용하여 유용 단백질인 hGM-CSF와 hEGF의 효율적인 발현 시스템

  • 정유정 ((주)바이오이즈 중앙연구소) ;
  • 박미정 ((주)바이오이즈 중앙연구소) ;
  • 이우정 ((주)바이오이즈 중앙연구소) ;
  • 김성천 ((주)바이오이즈 중앙연구소)
  • Received : 2024.03.02
  • Accepted : 2024.03.09
  • Published : 2024.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

Chlorella has various biotechnological applications, including in the biomedical and pharmaceutical industries, because of its advantages, including rich nutrients, fast growth rate, easy cultivation, and high biomass. We used the Agrobacterium-mediated transformation method to express human GM-CSF and EGF proteins, which are widely used in regenerative medicine, cosmetics, and pharmaceutical materials in Chlorella. The codon-optimized hGM-CSF and hEGF genes were cloned into plant binary vectors and transformed into Chlorella vulgaris using the Agrobacterium-mediated coculture transformation method. After transformation, genomic DNA PCR was performed for each C. vulgaris line that was stably subcultured on an antibiotic-resistant solid medium to confirm the insertion of hGM-CSF and hEGF into the chromosome. Furthermore, PT-PCR and protein expression of hGM-CSF and hEGF in each transformed C. vulgaris were significantly increased compared to the untransformed Chlorella. This study suggests that high-value proteins, including hGM-CSF and hEGF, which are foreign genes of C. vulgaris, can be stably expressed through the Agrobacterium-mediated Chlorella transformation system.

Keywords

References

  1. Khan, M. I., Shin, J. H. and Kim. J. D. 2018. The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products. Microb. Cell Fact. 17(1), 36.
  2. Mehariya, S., Goswami, R. K., Karthikeysan, O. P. and Verma, P. 2021. Microalgae for high-value products: A way towards green nutraceutical and pharmaceutical compounds. Chemosphere. 280, 130553.
  3. Ummalyma, S. B., Sirohi, R., Udayan, A., Yadav, P., Raj, A., Sim, S. J. and Pandey, A. 2022. Sustainable microalgal biomass production in food industry wastewater for low-cost biorefinery products: a review. Phytochem. Rev. 13, 1-23.
  4. Khavari, F., Saidijam, M., Taheri, M. and Nouri, F. 2021. Microalgae: therapeutic potentials and applications. Mol. Biol. Rep. 48(5), 4757-4765. https://doi.org/10.1007/s11033-021-06422-w
  5. Kiran, B. R. and Venkata Mohan, S. 2021. Microalgal Cell Biofactory-Therapeutic, Nutraceutical and Functional Food Applications. Plants (Basel). 10(5), 836.
  6. Xia, D., Qiu, W., Wang, X. and Liu, J. 2021. Recent Advancements and Future Perspectives of Microalgae-Derived Pharmaceuticals. Mar. Drugs. 19(12), 70.
  7. Jareonsin, S. and Pumas, C. 2021. Advantages of Heterotrophic Microalgae as a Host for Phytochemicals Production. Front Bioeng. Biotechnol. 9, 628597.
  8. Siddiqui, A., Wei, Z., Boehm, M. and Ahmad, N. 2020. Engineering microalgae through chloroplast transformation to produce high-value industrial products. Biotechnol. Appl. Biochem. 67(1), 30-40. https://doi.org/10.1002/bab.1823
  9. Yan, N., Fan, C., Chen, Y. and Hu, Z. 2016. The Potential for Microalgae as Bioreactors to Produce Pharmaceuticals. Int. J. Mol. Sci. 17(6), 962.
  10. Sreenikethanam, A., Raj, S., J. R. B., Gugulothu, P. and Bajhaiya, A. K. 2022. Genetic Engineering of Microalgae for Secondary Metabolite Production: Recent Developments, Challenges, and Future Prospects. Front. Bioeng. Biotechnol. 10, 836056.
  11. Yang, B., Liu, J., Liu, B., Sun, P., Ma, X., Jiang, Y., Wei, D. and Chen, F. 2015. Development of a stable genetic system for Chlorella vulgaris: a promising green alga for CO2 biomitigation. Algal Res. 12, 134-141. https://doi.org/10.1016/j.algal.2015.08.012
  12. Ortiz-Matamoros, M. F., Villanueva M, A. and Islas-Flores, T. 2018. Genetic transformation of cell-walled plant and algae cells: delivering DNA through the cell wall. Brief. Funct. Genomics. 17(1), 26-33. https://doi.org/10.1093/bfgp/elx014
  13. Ruiz-Ruiz, F., Torres-Acosta, M. A., Garcia-Echauri, S. A., Aguilar-Yanez, J. M., Rito-Palomares, M., and Ruiz-Ruiz, F. 2018. Genetic Manipulation of Microalgae for the Production of Bioproducts. Front. Biosci. 10, 254-275. https://doi.org/10.2741/e821
  14. Cha, T. S., Yee, W. and 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. https://doi.org/10.1007/s11274-011-0991-0
  15. Siripornadulsil, S., Dabrowski, K. and Sayre, R. 2007. Microalgal vaccines. Adv. Exp. Med. Biol. 616, 122-128. https://doi.org/10.1007/978-0-387-75532-8_11
  16. Hawkins, R. L. and Nakamura, M. 1999. Expression of human growth hormone by the eukaryotic alga, Chlorella. Curr. Microbiol. 38(6), 335-341. https://doi.org/10.1007/PL00006813
  17. Kim, D. H., Kim, Y. T., Cho, J. J., Bae, J. H., Hur, S. B., Hwang, I. and Choi, T. J. 2002. Stable integration and functional expression of flounder growth hormone gene in transformed microalga, Chlorella ellipsoidea. Mar. Biotechnol (NY). 4(1), 63-73. https://doi.org/10.1007/s1012601-0070-x
  18. Shin, J. H., Choi, J., Jeon, J., Kumar, M., Lee, J., Jeong, W. J. and Kim, S. R. 2020. The establishment of new protein expression system using N starvation inducible promoters in Chlorella. Sci. Rep. 10(1), 12713.
  19. Terpe, K. 2006. Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 72(2), 211-222. https://doi.org/10.1007/s00253-006-0465-8
  20. Fu, H., Liang, Y, Zhong, X., Pan, Z., Huang, L., Zhang, H., Xu, Y., Zhou, W. and Liu, Z. 2020. Codon optimization with deep learning to enhance protein expression. Sci. Rep. 10(1), 17617.
  21. Schutz, A., Bernhard, F., Berrow, N., Buyel, J. F., Ferreira-da-Silva, F., Haustraete, J., van den Heuvel, J, Hoffmann J. E., de Marco A., Peleg Y., Suppmann, S., Unger, T., Vanhoucke, M., Witt, S. and Remans, K. 2023. A concise guide to choosing suitable gene expression systems for recombinant protein production. STAR Protoc. 4(4), 102572.
  22. Arai, K. I., Lee, F., Miyajima, A., Miyatake, S., Arai, N. and Yokota, T. 1990. Cytokines: coordinators of immune and inflammatory responses. Annu. Rev. Biochem. 59, 783-836. https://doi.org/10.1146/annurev.bi.59.070190.004031
  23. Anderlini, P., Przepiorka, D., Champlin R. and Korbling M. 1996. Biologic and clinical effects of granulocyte colony-stimulating factor in normal individuals. Blood. 88, 2819-2825. https://doi.org/10.1182/blood.V88.8.2819.bloodjournal8882819
  24. Becher, B., Tugues, S. and Greter, M. 2016. GM-CSF: From Growth Factor to Central Mediator of Tissue Inflammation. Immunity. 45(5), 963-973. https://doi.org/10.1016/j.immuni.2016.10.026
  25. Hamilton, J.A. 2020. GM-CSF in inflammation. J. Exp, Med. 217(1), e20190945.
  26. Pastore, S., Mascia, F., Mariani, V. and Girolomoni, G. 2008. The epidermal growth factor receptor system in skin repair and inflammation. J. Invest. Dermatol. 128(6), 1365-1374. https://doi.org/10.1038/sj.jid.5701184
  27. Berlanga-Acosta, J., Gavilondo-Cowley. J., Lopez-Saura, P., Gonzalez-Lopez, T., Castro-Santana, M. D., Lopez-Mola, E., Guillen-Nieto, G. and Herrera-Martinez, L. 2009. Epidermal growth factor in clinical practice - a review of its biological actions, clinical indications and safety implications. Int. Wound J. 6(5), 331-346. https://doi.org/10.1111/j.1742-481X.2009.00622.x
  28. Shin, S. H., Koh, Y. G., Lee, W. G., Seok, J. and Park, K. Y. 2023. The use of epidermal growth factor in dermatological practice. Int. Wound J. 20(6), 2414-2423. https://doi.org/10.1111/iwj.14075
  29. Fukuzawa, H., Fujiwara, S., Yamamoto, Y., Dionisio-Sese, M. L. and Miyachi, S. 1990. cDNA cloning, sequence, and expression of carbonic anhydrase in Chlamydomonas reinhardtii: regulation by environmental CO2 concentration. Proc. Natl. Acad. Sci USA. 87(11), 4383-4387. https://doi.org/10.1073/pnas.87.11.4383
  30. Froger, A. and Hall, J. E. 2007. Transformation of plasmid DNA into E. coli using the heat shock method. J. Vis. Exp. 6, 253.
  31. Holsters, M., de Waele, D., Depicker, A., Messens, E., van Montagu, M. and Schell, J. 1978. Transfection and transformation of Agrobacterium tumefaciens. Mol. Gen. Genet. 163, 181-187. https://doi.org/10.1007/BF00267408
  32. Hofgen, R. and Willmitzer, L. 1988. Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res. 16(20), 9877.
  33. Weeks, D. P., Beerman, N. and Griffith, O. M. 1986. A small-scale five-hour procedure for isolating multiple samples of CsCl-purified DNA: application to isolations from mammalian, insect, higher plant, algal, yeast, and bacterial sources. Anal. Biochem. 152(2), 376-385. https://doi.org/10.1016/0003-2697(86)90423-9
  34. Vennapusa, A. R., Somayanda1, C. J. and Jagadish, S. V. K. 2020. A universal method for highquality RNA extraction from plant tissues rich in starch, proteins and fiber. Scientific Rep. 10, 16887.
  35. Dasan, Y. K., Lam, M. K., Yusup, S., Lim, J. W., Show, P. L., Tan, I. S. and Lee, K. T. 2020. Cultivation of Chlorella Vulgaris Using Sequential-Flow Bubble Column Photobioreactor: A Stress-Inducing Strategy for Lipid Accumulation and Carbon Dioxide Fixation. J. CO2 Util. 41, 101226.
  36. Diaz C. J., Douglas, K. J., Kang, K., Kolarik, A. L., Malinovski, R., Torres-Tiji, Y., Molino, J. V., Badary, A. and Mayfield, S. P. 2023. Developing algae as a sustainable food source. Front. Nutr. 9, 1029841.
  37. 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, 100-179.
  38. Harmoko, R., Fanata, W. I., Yoo, J. Y., Ko, K. S., Rim, Y. G., Uddin, M. N., Siswoyo, T. A., Lee, S. S., Kim, D. Y., Lee, S. Y. and Lee, K. O. 2013. RNA-dependent RNA polymerase 6 is required for efficient hpRNA-induced gene silencing in plants. Mol. Cells. 35(3), 202-209. https://doi.org/10.1007/s10059-013-2203-2