Journal of Marine Bioscience and Biotechnology (한국해양바이오학회지)

The Korean Society for Marine Biotechnology (한국해양바이오학회)
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Monitoring and Environmental Risk Assessment of Genetically Modified Microalgae

유전자변형 미세조류의 생태 유출 모니터링 및 위해성평가 연구

  • Cho, Kichul (Department of Genetic Resources Research, National Marine Biodiversity Institute of Korea) ;
  • Jeon, Hancheol (Department of Genetic Resources Research, National Marine Biodiversity Institute of Korea) ;
  • Hwang, Hyun-Ju (Department of Genetic Resources Research, National Marine Biodiversity Institute of Korea) ;
  • Hong, Ji Won (Department of Taxonomy and Systematics, National Marine Biodiversity Institute of Korea) ;
  • Lee, Dae-Sung (Department of Genetic Resources Research, National Marine Biodiversity Institute of Korea) ;
  • Han, Jong Won (Department of Genetic Resources Research, National Marine Biodiversity Institute of Korea)
  • 조기철 (국립해양생물자원관 유전자원연구실) ;
  • 전한철 (국립해양생물자원관 유전자원연구실) ;
  • 황현주 (국립해양생물자원관 유전자원연구실) ;
  • 홍지원 (국립해양생물자원관 분류연구실) ;
  • 이대성 (국립해양생물자원관 유전자원연구실) ;
  • 한종원 (국립해양생물자원관 유전자원연구실)
  • Received : 2019.11.20
  • Accepted : 2019.11.28
  • Published : 2019.12.31
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Abstract

Over the past few decades, microalgae-based biotechnology conjugated with innovative CRISPR/Cas9-mediated genetic engineering has been attracted much attention for the cost-effective and eco-friendly value-added compounds production. However, the discharge of reproducible living modified organism (LMO) into environmental condition potentially causes serious problem in aquatic environment, and thus it is essential to assess potential environmental risk for human health. Accordingly, in this study, we monitored discharged genetically modified microalgae (GMM) near the research complex which is located in Daejeon, South Korea. After testing samples obtained from 6 points of near streams, several green-colored microalgal colonies were detected under hygromicin-containing agar plate. By identification of selection marker genes, the GMM was not detected from all the samples. For the lab-scale environmental risk assessment of GMM, acute toxicity test using rotifer Brachionus calcyflorus was performed by feeding GMM. After feeding, there was no significant difference in mortality between WT and transformant Chlamydomonas reinhardtii. According to further analysis of horizontal transfer of green fluorescence protein (GFP)-coding gene after 24 h of incubation in synthetic freshwater, we concluded that the GFP-expressed gene not transferred into predator. However, further risk assessments and construction of standard methods including prolonged toxicity test are required for the accurate ecological risk assessment.

Keywords

References

  1. Pulz, O. and Gross, W. 2004. Valuable products from biotechnology of microalgae. Appl. Microbiol. Biotechnol. 65(6), 635-648. https://doi.org/10.1007/s00253-004-1647-x
  2. Spolaore, P., Joannis-Cassan, C., Duran, E. and Isambert, A. 2006. Commercial applications of microalgae. J. Biosci. Bioeng. 101(2), 87-96. https://doi.org/10.1263/jbb.101.87
  3. Abdel-Raouf, N., Al-Homaidan, A. A. and Ibraheem, I. B. M. 2012. Microalgae and wastewater treatment. Saudi J. Biol. Sci. 19(3), 257-275. https://doi.org/10.1016/j.sjbs.2012.04.005
  4. Hemaiswarya, S., Raja, R., Kumar, R. R., Ganesan, V. and Anbazhagan, C. 2011. Microalgae: a sustainable feed source for aquaculture. World J. Microb. Biotechnol. 27(8), 1737-1746. https://doi.org/10.1007/s11274-010-0632-z
  5. Metting, B. 1990. Microalgae applications in agriculture. Dev. Ind. Microbiol. 31, 265-270.
  6. Wijffels, R. H., Kruse, O. and Hellingwerf, K. J. 2013. Potential of industrial biotechnology with cyanobacteria and eukaaryotic microalgae. Curr. Opin. Biotech. 24(3), 405-413. https://doi.org/10.1016/j.copbio.2013.04.004
  7. Baek, K., Kim, D. H., Jeong, J., Sim, S. J., Melis, A., Kim, J. S., Jin, E. and Bae, S. 2016. DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Sci. Rep. 6, 30620. https://doi.org/10.1038/srep30620
  8. Baek, K., Yu, J., Jeong, J., Sim, S. J., Bae, S. and Jin, E. 2018. Photoautotrophic production of macular pigment in a Chlamydomonas reinhardtii strain generated by using DNA-free CRISPR-Cas9 RNP-mediated mutagenesis. Biotechnol. Bioeng. 115(3), 719-728. https://doi.org/10.1002/bit.26499
  9. Shin, S. E., Lim, J. M., Koh, H. G., Kim, E. K., Kang, N. K., Jeon, S., Kwon, S., Shin, W. S., Lee, B., Hwangbo, K. and Kim, J. 2016. CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii. Sci. Rep. 6, 27810. https://doi.org/10.1038/srep27810
  10. Tran, Q. G., Cho, K., Park, S. B., Kim, U., Lee, Y. J. and Kim, H. S. 2019a. Impairment of starch biosynthesis results in elevated oxidative stress and autophagy activity in Chlamydomonas reinhardtii. Sci. Rep. 9(1), 1-9. https://doi.org/10.1038/s41598-018-37186-2
  11. Tran, Q. G., Cho, K., Kim, U., Yun, J. H., Cho, D. H., Heo, J., Park, S. B., Kim, J. W., Lee, Y. J., Ramanan, R. and Kim, H. S. 2019b. Enhancement of ${\beta}$-carotene production by regulating the autophagy-carotenoid biosynthesis seesaw in Chlamydomonas reinhardtii. Bioresour. Technol. 292, 121937. https://doi.org/10.1016/j.biortech.2019.121937
  12. Hamilton, M. L., Haslam, R. P., Napier, J. A. and Sayanova, O. 2014. Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids. Metab. Eng. 22, 3-9. https://doi.org/10.1016/j.ymben.2013.12.003
  13. Leon, R., Couso, I. and Fernandez, E. 2007. Metabolic engineering of ketocarotenoids biosynthesis in the unicelullar microalga Chlamydomonas reinhardtii. J. Biotechnol. 130(2), 143-152. https://doi.org/10.1016/j.jbiotec.2007.03.005
  14. Beacham, T. A., Sweet, J. B. and Allen, M. J. 2017. Large scale cultivation of genetically modified microalgae: A new era for environmental risk assessment. Algal Res. 25, 90-100. https://doi.org/10.1016/j.algal.2017.04.028
  15. Glass, D. J. 2015. Government regulation of the uses of genetically modified algae and other microorganisms in biofuel and bio-based chemical production. In Algal Biorefineries. Springer, Cham, pp 23-60
  16. Kumar, S. 2015. GM algae for biofuel production: biosafety and risk assessment. Collect. Biosaf. Rev. 9, 52-75.
  17. Szyjka, S. J., Mandal, S., Schoepp, N. G., Tyler, B. M., Yohn, C. B., Poon, Y. S., Villareal, S., Burkart, M. D., Shurin, J. B. and Mayfield, S. P. 2017. Evaluation of phenotype stability and ecological risk of a genetically engineered alga in open pond production. Algal Res. 24, 378-386. https://doi.org/10.1016/j.algal.2017.04.006
  18. Hwang, H. J., Kim, Y. T., Kang, N. S. and Han, J. W. 2018. A Simple Method for Removal of the Chlamydomonas reinhardtii Cell Wall Using a Commercially Available Subtilisin (Alcalase). J. Mol. Microbiol. Biotechnol. 28(4), 169-178. https://doi.org/10.1159/000495183
  19. Ladygin, V. G. and Boutanaev, A. M. 2002. Transformation of Chlamydomonas reinhardtii CW-15 with the hygromycin phosphotransferase gene as a selectable marker. Russian. J. Genet. 38(9), 1009-1014. https://doi.org/10.1023/A:1020279429009
  20. Wittkopp, T. M. 2018. Nuclear Transformation of Chlamydomonas reinhardtii by Electroporation. Bio-protocol. 8(9).
  21. Dunahay, T. G. 1993. Transformation of Chlamydomonas reinhardtii with silicon carbide whiskers. Biotechniques, 15(3), 452-5.
  22. EL-Sheekh, M. M., Almutairi, A. W. and Touliabah, H. E. 2019. Construction of a novel vector for the nuclear transformation of the unicellular green alga Chlamydomonas reinhardtii and its stable expression. J. Taibah University Sci. 13(1), 529-535. https://doi.org/10.1080/16583655.2019.1603574
  23. Kumar, S. V., Misquitta, R. W., Reddy, V. S., Rao, B. J. and Rajam, M. V. 2004. Genetic transformation of the green alga-Chlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant Sci. 166(3), 731-738. https://doi.org/10.1016/j.plantsci.2003.11.012
  24. Talebi, A. F., Tohidfar, M., Tabatabaei, M., Bagheri, A., Mohsenpor, M. and Mohtashami, S. K. 2013. Genetic manipulation, a feasible tool to enhance unique characteristic of Chlorella vulgaris as a feedstock for biodiesel production. Mol. Biol. Rep. 40(7), 4421-4428. https://doi.org/10.1007/s11033-013-2532-4
  25. Chow, K. C. and Tung, W. L. 1999. Electrotransformation of Chlorella vulgaris. Plant Cell. Rep. 18(9), 778-780. https://doi.org/10.1007/s002990050660
  26. Liu, L., Wang, Y., Zhang, Y., Chen, X., Zhang, P. and Ma, S. 2013. Development of a new method for genetic transformation of the green alga Chlorella ellipsoidea. Mol. Biotechnol. 54(2), 211-219. https://doi.org/10.1007/s12033-012-9554-3
  27. Steinbrenner, J. and Sandmann, G. 2006. Transformation of the green alga Haematococcus pluvialis with a phytoene desaturase for accelerated astaxanthin biosynthesis. Appl. Environ. Microbiol. 72(12), 7477-7484. https://doi.org/10.1128/AEM.01461-06
  28. Apt, K. E., Grossman, A. R. and Kroth-Pancic, P. G. 1996. Stable nuclear transformation of the diatom Phaeodactylum tricornutum. Mol. Gen. Genet. 252(5), 572-579.
  29. Dunahay, T. G., Jarvis, E. E. and Roessler, P. G. 1995. Genetic transformation of the diatoms Cyclotella cryptica and Navicula saprophila. J. Phycol. 31(6), 1004-1012. https://doi.org/10.1111/j.0022-3646.1995.01004.x
  30. Poulsen, N., Chesley, P. M. and Kroger, N. 2006. Molecular genetic manipulation of the diatom Thalassiosira pseudonana (bacillariophyceae) 1. J. Phycol. 42(5), 1059-1065. https://doi.org/10.1111/j.1529-8817.2006.00269.x
  31. Te, M. R. and Miller, D. J. 1998. Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): expression of GUS in microalgae using heterologous promoter constructs. Plant J. 13(3), 427-435. https://doi.org/10.1046/j.1365-313X.1998.00040.x
  32. Shin, W. S. 2018. Genetic modulation of light-harvesting complex in chlorella to improve photosynthetic efficiency and biomass productivity, Ph. D. Thesis in Korea Advanced Science and Technology (KAIST), Korea
  33. Jeong, C. B., Lee, Y. H., Park, J. C., Kang, H. M., Hagiwara, A. and Lee, J. S. 2019. Effects of metal-polluted seawater on life parameters and the induction of oxidative stress in the marine rotifer Brachionus koreanus. Comp. Biochem. Physiol. C. 225, 108576.
  34. Romero-Freire, A., Joonas, E., Muna, M., Cossu-Leguille, C., Vignati, D. A. L. and Giamberini, L. 2019. Assessment of the toxic effects of mixtures of three lanthanides (Ce, Gd, Lu) to aquatic biota. Sci. Total Environ. 661, 276-284. https://doi.org/10.1016/j.scitotenv.2019.01.155
  35. Saavedra, J., Stoll, S. and Slaveykova, V. I. 2019. Influence of nanoplastic surface charge on eco-corona formation, aggregation and toxicity to freshwater zooplankton. Environ. Pollut. 252, 715-722. https://doi.org/10.1016/j.envpol.2019.05.135
  36. Bhatti, F., Asad, S., Khan, Q. M., Mobeen, A., Iqbal, M. J. and Asif, M. 2019. Risk assessment of genetically modified sugarcane expressing AVP1 gene. Food Chem. Toxicol. 130, 267-275. https://doi.org/10.1016/j.fct.2019.05.034
  37. Bertoni, G. and Marsan, P. A. 2005. Safety risks for animals fed genetic modified (GM) plants. Vetres. Commun. 29(2), 13-18.
  38. Domingo, J.L. 2007. Toxicity studies of genetically modified plants: a review of the published literature. Crit. Rev. Food. Sci. 47(8), 721-733. https://doi.org/10.1080/10408390601177670
  39. Seralini, G. E., Cellier, D. and de Vendomois, J. S. 2007. New analysis of a rat feeding study with a genetically modified maize reveals signs of hepatorenal toxicity. Arch. Environ. Con. Tox. 52(4), 596-602. https://doi.org/10.1007/s00244-006-0149-5
  40. Gasson, M. J. 2000. Gene transfer from genetically modified food. Curr. Opin. Biotech. 11(5), 505-508. https://doi.org/10.1016/S0958-1669(00)00136-1
  41. Thomson, J. A. 2001. Horizontal transfer of DNA from GM crops to bacteria and to mammalian cells. J. Food Sci. 66(2), 188-193. https://doi.org/10.1111/j.1365-2621.2001.tb11314.x