Overexpression of Cyanase in Chlamydomonas reinhardtii: a Promising Approach for Biodegradation of Cyanate in Aquatic Systems

  • Yassin El-Ayouty
  • Mostafa Ismaiel
  • Asmaa Al-Badwy
  • Rashad KebeishEmail author


Cyanate and its derivatives are considered as highly dangerous materials that threaten human health and environment. Cyanate arises from both natural resources and anthropogenic activities including various chemical industries, herbicide production, and mining wastewater. Despite its toxicity, cyanate is considered as an important nitrogen (N) source in marine ecosystems. Cyanase (CYN) catalyzes the decomposition of cyanate into CO2 and NH3 in a bicarbonate-dependent reaction. In marine cyanobacteria, endogenous cyanases participate in detoxification of low concentrations of cyanate. However, this cyanate biodegradation system is seemingly inconvenient especially at contaminated sites due to high cyanate concentrations. In the current study, we have transferred the activity of the cyanobacterial enzyme cyanase into the micro-alga, Chlamydomonas reinhardtii, via Agrobacterium tumefaciens–mediated transformation method. The recombinant cyanase enzyme was shown to be active in transgenic C. reinhardtii lines. When variable concentrations of cyanate (up to 30 mM) is applied to growth medium, transgenic lines showed higher rate of NH3 release, reduced loss of pigmentation symptoms, decreased levels of induced antioxidant enzymes, and low percentage of growth retardation compared to wild-type controls. Results of this study provide an effective eco-friendly phytoremediation system for cyanate detoxification using micro-algae compared to previously reported plant systems.


Cyanate Phytoremediation Transgenic Chlamydomonas reinhardtii Synthetic biology 



This work is in part under the supervision of the Applied Scientific Research Center, Herbal and Medicinal Plants research group, Taibah University. We are thankful to Prof. Dr. Mohammed Ismaeil (Botany Department, Faculty of Science, Mansoura University, Egypt) for providing us with C. reinhardtii culture.


- All authors participate equally in this publication.

- We do not have any conflict of interest with other research institutions or any potential financial support that could be perceived to influence the outcomes of the research.

- No conflicts, informed consent, human or animal rights applicable.

- All authors agreed to the authorship and submission of the manuscript for peer review in its current form.


  1. Aichi, M., Nishida, I., & Omata, T. (1998). Molecular cloning and characterization of a cDNA encoding cyanase from Arabidopsis thaliana. Plant & Cell Physiology, 39, S135–S135.Google Scholar
  2. Akcil, A., & Mudder, T. (2003). Microbial destruction of cyanide wastes in gold mining: process review. Biotechnology Letters, 25, 520–527.CrossRefGoogle Scholar
  3. Anderson, P. M. (1980). Purification and properties of the inducible enzyme cyanase. Biochemistry, 19, 2882–2888.CrossRefGoogle Scholar
  4. Anderson, P. M., & Little, R. M. (1986). Kinetic properties of cyanase. Biochemistry, 25, 1621–1626.CrossRefGoogle Scholar
  5. Anderson, P. M., Sung, Y.-C., & Fuchs, J. A. (1990). The cyanase operon and cyanate metabolism. FEMS Microbiology Reviews, 7, 247–252.CrossRefGoogle Scholar
  6. Askari, H., Edqvist, J., Hajheidari, M., Kafi, M., & Salekdeh, G. H. (2006). Effects of salinity levels on proteome of Suaeda aegyptiaca leaves. Proteomics, 6, 2542–2554.CrossRefGoogle Scholar
  7. Beauchamp, C., & Fridovich, I. (1971). Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Analytical Biochemistry, 44, 276–287.CrossRefGoogle Scholar
  8. Bowler, C., Montagu, M. V., & Inze, D. (1992). Superoxide dismutase and stress tolernace. Annual Review of Plant Physiology and Plant Molecular Biology, 43, 83–116.CrossRefGoogle Scholar
  9. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1–2), 248–254.CrossRefGoogle Scholar
  10. Butryn, A., Stoehr, G., Linke-Winnebeck, C., & Hopfner, K.-P. (2015). Serendipitous crystallization and structure determination of cyanase (CynS) from Serratia proteamaculans. Acta. Crystallographica. Section F: Structural Biology Communications, 71, 471–476. Scholar
  11. Cerutti, H., Johnson, A. M., Gillham, W. N., & Boynton, J. E. (1997). A eubacterial gene conferring spectinomycin resistance on Chlamydomonas reinhardtii: Integration into the nuclear genome and gene expression. Genetics, 145, 97–110.Google Scholar
  12. Chomczynski, P., & Mackey, K. (1995). Substitution of chloroform by bromochloropropane in the single-step method of RNA isolation. Analytical Biochemistry, 225, 163–164.CrossRefGoogle Scholar
  13. Ebbs, S. (2004). Biological degradation of cyanide compounds. Current Opinion in Biotechnology, 15, 231–236.CrossRefGoogle Scholar
  14. Espie, G. S., Jalali, F., Tong, T., Zacal, N. J., & So, A. K.-C. (2007). Involvement of the cynABDS operon and the CO2-concentrating mechanism in the light-dependent transport and metabolism of cyanate by cyanobacteria. Journal of Bacteriology, 189, 1013–1024.CrossRefGoogle Scholar
  15. Gorman, D. S., & Levine, R. P. (1965). Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinharditi. Proceedings of the National Academy of Sciences of the United States of America, 54(6), 1665–1669.CrossRefGoogle Scholar
  16. Guillotonm, M., & Karst, F. (1987). Isolation and characterization of Escherichia coli mutants lacking inducible cyanase. Microbiology, 133, 645–653.CrossRefGoogle Scholar
  17. Hamel, J. (2011). A review of acute cyanide poisoning with a treatment update. Critical Care Nurse, 82, 31–72.Google Scholar
  18. Harano, Y., Suzuki, I., Maeda, S.-I., Kaneko, T., Tabata, S., & Omata, T. (1997). Identification and nitrogen regulation of the cyanase gene from the cyanobacteria Synechocystis sp. strain PCC 6803 and Synechococcus sp. strain PCC 7942. Journal of Bacteriology, 179, 5744–5750.CrossRefGoogle Scholar
  19. Heitzer, M., Eckert, A., Fuhrmann, M., & Griesbeck, C. (2007). Influence of codon bias on the expression of foreign genes in microalgae. In Transgenic microalgae as green cell factories (pp. 46–53). New York: Springer.CrossRefGoogle Scholar
  20. Jeffrey, S. W., & Humphrey, G. F. (1975). New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochemie und Physiologie der Pflanzen, 167, 191–194.CrossRefGoogle Scholar
  21. Johnson, W., & Anderson, P. (1987). Bicarbonate is a recycling substrate for cyanase. The Journal of Biological Chemistry, 262, 9021–9025.Google Scholar
  22. Kamennaya, N. A., & Post, A. F. (2010). Characterization of cyanate metabolism in marine Synechococcus and Prochlorococcus spp. Applied and Environmental Microbiology, 77, 291–301. Scholar
  23. Kamennaya, N. A., Chernihovsky, M., & Post, A. F. (2008). The cyanate utilization capacity of marine unicellular cyanobacteria. Limnology and Oceanography, 53, 2485–2495.CrossRefGoogle Scholar
  24. Kar, M., & Mishra, D. (1976). Catalase, peroxidase, polyphenol oxidase activities during rice leaf senescence. Journal of Plant Physiology, 57, 315–319.CrossRefGoogle Scholar
  25. Karuppanapandian, T., Moon, J. H., Kim, C., Manoharan, K., & Kim, W. (2011). Reactive oxygen species in plants: their generation, signal transduction, and scavenging mechanisms. Australian Journal of Crop Science, 5(6), 709–725.Google Scholar
  26. Kebeish, R., & Al-Zoubi, O. (2017). Expression of the cyanobacterial enzyme cyanase increases cyanate metabolism and cyanate tolerance in Arabidopsis. Environmental Science and Pollution Research International, 12(24), 11825–11835.CrossRefGoogle Scholar
  27. Kebeish, R., Aboelmy, M., El-Naggar, A., El-Ayouty, Y., & Peterhansel, C. (2015). Simultaneous overexpression of cyanidase and formate dehydrogenase in Arabidopsis thaliana chloroplasts enhanced cyanide metabolism and cyanide tolerance. Environmental and Experimental Botany, 110, 19–26. Scholar
  28. Koncz, C., & Schell, J. (1986). The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of agrobacterium binary vector. Molecular and General Genetics MGG, 204, 383–396.CrossRefGoogle Scholar
  29. Koshiishi, I., Mamura, Y., & Imanari, T. (1997). Cyanate causes depletion of ascorbate in organisms. Biochimica et Biophysica Acta (BBA)- General Subjects, 1336, 566–574.CrossRefGoogle Scholar
  30. Kumar, S. V., Misquitta, R. W., Reddy, V. S., Rao, B. J., & Rajam, M. V. (2004). Genetic transformation of the green alga-Chlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant Science, 166, 731–738.CrossRefGoogle Scholar
  31. Leavesley, H. B., Li, L., Prabhakaran, K., Borowitz, J. L., & Isom, G. E. (2008). Interaction of cyanide and nitric oxide with cytochrome c oxidase: implications for acute cyanide toxicity. Toxicological Sciences, 101, 101–111.CrossRefGoogle Scholar
  32. Liu, Q., Zhang, G., Ding, J., Zou, H., Shi, H., & Huang, C. (2018). Evaluation of the removal of potassium cyanide and its toxicity in green algae (Chlorella vulgaris). Bulletin of Environmental Contamination and Toxicology, 100(2), 228–233.CrossRefGoogle Scholar
  33. Luque-Almagro, V. M., Huertas, M.-J., Sáez, L. P., et al. (2008). Characterization of the Pseudomonas pseudoalcaligenes CECT5344 cyanase, an enzyme that is not essential for cyanide. Assimilation. Applied and Environmental Microbiology, 74, 6280–6288.CrossRefGoogle Scholar
  34. Malhotra, S., Pandit, M., Kapoor, J., & Tyagi, D. (2005). Photo-oxidation of cyanide in aqueous solution by the UV/H2O2 process. Journal of Chemical Technology and Biotechnology, 80, 13–19.CrossRefGoogle Scholar
  35. Mekuto, L., Ntwampe, S., & Akcil, A. (2016). An integrated biological approach for treatment of cyanidation wastewater. Science of the Total Environment, 571, 711–720.CrossRefGoogle Scholar
  36. Metzner, H., Rau, H., & Senger, H. (1965). Untersuchungen zur synchronisierbarkeit einzelner pigment-mangel mutanten von Chlorella. Planta, 65, 186–194.CrossRefGoogle Scholar
  37. Mudder, T. I., Botz, M., & Smith, A. (2001). Chemistry and treatment of cyanidation wastes. London: Mining Journal Books.Google Scholar
  38. Niessen, M., Thiruveedhi, K., Rosenkranz, R., Kebeish, R., Hirsch, H.-J., Kreuzaler, F., et al. (2007). Mitochondrial glycolate oxidation contributes to photorespiration in higher plants. Journal of Experimental Botany, 58, 2709–2715.CrossRefGoogle Scholar
  39. Oracz, K., El-Maarouf-Bouteau, H., Kranner, I., Bogatek, R., Corbineau, F., & Bailly, C. (2009). The mechanisms involved in seed dormancy alleviation by hydrogen cyanide unravel the role of reactive oxygen species as key factors of cellular signaling during germination. Plant Physiology, 150, 494–505.CrossRefGoogle Scholar
  40. Qian, D., Jiang, L., Lu, L., Wei, C., & Li, Y. (2011). Biochemical and structural properties of cyanases from Arabidopsis thaliana and Oryza sativa. PLoS One, 6(3), e18300.CrossRefGoogle Scholar
  41. Racusen, D., & Foote, M. (1965). Protein synthesis in dark grown bean leaves. Canadian Journal of Botany, 43, 817–824.CrossRefGoogle Scholar
  42. Rasala, B. A., Muto, M., Lee, P. A., Jager, M., Cardoso, R. M. F., Behnke, C. A., et al. (2010). Production of therapeutic proteins in algae, analysis of expression of seven human proteins 215 in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnology Journal, 8, 719–733.CrossRefGoogle Scholar
  43. Rasco-Gaunt, S., Riley, A., Lazzeri, P., & Barcelo, P. (1999). A facile method for screening for phosphinothricin (PPT)-resistant transgenic wheats. Molecular Breeding, 5, 255–262.CrossRefGoogle Scholar
  44. Reichel, C., Mathur, J., Eckes, P., Langenkemper, K., Koncz, C., Schell, J., et al. (1996). Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono-and dicotyledonous plant cells. Proceedings of the National Academy of Sciences of the United States of America, 93, 5888–5893.CrossRefGoogle Scholar
  45. Robert, R. (1979). Growth measurements. Division rate. Physiological methods. Culture methods and growth measurements (pp. 29–311). Cambridge: Cambridge University. Press.Google Scholar
  46. Rosales-Mendoza, S., Paz-Maldonado, L. M. T., & Soria-Guerra, R. E. (2012). Chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins: current status and perspectives. Plant Cell Reports, 31, 479–494.CrossRefGoogle Scholar
  47. Samuilov, V. D., Kiselevsky, D. B., Sinitsyn, S. V., Shestak, A. A., Lagunova, E. M., & Nesov, A. V. (2006). H2O2 intensifies CN-induced apoptosis in pea leaves. Biochemistry-Moscow, 71, 384–394.CrossRefGoogle Scholar
  48. Siegien, I., & Bogatek, R. (2006). Cyanide action in plants - from toxic to regulatory. Acta Physiologiae Plantarum, 28, 483–497.CrossRefGoogle Scholar
  49. Solomonson, L. P. (1981). Cyanide as a metabolic inhibitor. In B. Vennesland, E. E. Conn, C. J. Knowles, J. Westley, & F. Wissing (Eds.), Cyanide in biology (pp. 11–28). London: Academic Press.Google Scholar
  50. Srivastava, A. C., & Muni, R. R. D. (2010). Phytoremediation of cyanide. In Plant adaptation and phytoremediation (pp. 399–426). Springer.Google Scholar
  51. Taebi, A., Jeirani, K., Mirlohi, A., & Zadeh Bafghi, A. R. (2008). Phytoremediation of cyanide-polluted soils by non-woody plants. JWSS-Isfahan University of Technology, 11, 515–523.Google Scholar
  52. Tang, D., Qiao, S.-Y., & Wu, M. (1995). Insertion mutagenesis of Chlamydomonas reinhardtii by electroporation and heterologous DNA. Biochemistry and Molecular Biology International, 36, 1025–1035.Google Scholar
  53. Taussig, A. (1960). The synthesis of the induced enzyme, cyanase, in E. coli. Biochimica et Biophysica Acta, 44, 510–519.CrossRefGoogle Scholar
  54. Voigt, K., Sharma, C. M., Mitschke, J., Lambrecht, S. J., Voss, B., Hess, W. R., et al. (2014). Comparative transcriptomics of two environmentally relevant cyanobacteria reveals unexpected transcriptome diversity. The ISME Journal, 8, 2056–2068.CrossRefGoogle Scholar
  55. Walsh, M. A., Otwinowski, Z., Perrakis, A., Anderson, P. M., & Joachimiak, A. (2000). Structure of cyanase reveals that a novel dimeric and decameric arrangement of subunits is required for formation of the enzyme active site. Structure, 8, 505–514.CrossRefGoogle Scholar
  56. Way, J. L. (1984). Cyanide intoxication and its mechanism of antagonism. Annual Review of Pharmacology and Toxicology, 24, 451–481.CrossRefGoogle Scholar
  57. Wishnik, M. W., & Lane, M. D. (1969). Inhibition of ribulose diphosphate carboxylase by cyanide. The Journal of Biological Chemistry, 244, 55–59.Google Scholar
  58. Xu, P., Zou, J., Meng, Q., Zou, J., Jiang, W., & Liu, D. (2008). Effects of Cd2+ on seedling growth of garlic (Allium sativum L.) and selected physiological and biochemical characters. Bioresource Technology, 99(14), 6372–6378.CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Plant Biotechnology Laboratory (PBL), Botany and Microbiology Department, Faculty of ScienceZagazig UniversityZagazigEgypt
  2. 2.Faculty of Science Yanbu, Biology DepartmentTaibah UniversityYanbuSaudi Arabia

Personalised recommendations