Skip to main content
SpringerLink
Log in

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

  • Published:
Water, Air, & Soil Pollution Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • 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 

  • Akcil, A., & Mudder, T. (2003). Microbial destruction of cyanide wastes in gold mining: process review. Biotechnology Letters, 25, 520–527.

    Article  Google Scholar 

  • Anderson, P. M. (1980). Purification and properties of the inducible enzyme cyanase. Biochemistry, 19, 2882–2888.

    Article  CAS  Google Scholar 

  • Anderson, P. M., & Little, R. M. (1986). Kinetic properties of cyanase. Biochemistry, 25, 1621–1626.

    Article  CAS  Google Scholar 

  • Anderson, P. M., Sung, Y.-C., & Fuchs, J. A. (1990). The cyanase operon and cyanate metabolism. FEMS Microbiology Reviews, 7, 247–252.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Beauchamp, C., & Fridovich, I. (1971). Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Analytical Biochemistry, 44, 276–287.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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. https://doi.org/10.1107/S2053230X15004902.

    Article  CAS  Google Scholar 

  • 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.

    CAS  Google Scholar 

  • Chomczynski, P., & Mackey, K. (1995). Substitution of chloroform by bromochloropropane in the single-step method of RNA isolation. Analytical Biochemistry, 225, 163–164.

    Article  CAS  Google Scholar 

  • Ebbs, S. (2004). Biological degradation of cyanide compounds. Current Opinion in Biotechnology, 15, 231–236.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Guillotonm, M., & Karst, F. (1987). Isolation and characterization of Escherichia coli mutants lacking inducible cyanase. Microbiology, 133, 645–653.

    Article  Google Scholar 

  • Hamel, J. (2011). A review of acute cyanide poisoning with a treatment update. Critical Care Nurse, 82, 31–72.

    Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Chapter  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Johnson, W., & Anderson, P. (1987). Bicarbonate is a recycling substrate for cyanase. The Journal of Biological Chemistry, 262, 9021–9025.

    CAS  Google Scholar 

  • Kamennaya, N. A., & Post, A. F. (2010). Characterization of cyanate metabolism in marine Synechococcus and Prochlorococcus spp. Applied and Environmental Microbiology, 77, 291–301. https://doi.org/10.1128/AEM.01272-10.

    Article  CAS  Google Scholar 

  • Kamennaya, N. A., Chernihovsky, M., & Post, A. F. (2008). The cyanate utilization capacity of marine unicellular cyanobacteria. Limnology and Oceanography, 53, 2485–2495.

    Article  CAS  Google Scholar 

  • Kar, M., & Mishra, D. (1976). Catalase, peroxidase, polyphenol oxidase activities during rice leaf senescence. Journal of Plant Physiology, 57, 315–319.

    Article  CAS  Google Scholar 

  • 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.

    CAS  Google Scholar 

  • 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.

    Article  Google Scholar 

  • 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. https://doi.org/10.1016/j.envexpbot.2014.09.004.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Koshiishi, I., Mamura, Y., & Imanari, T. (1997). Cyanate causes depletion of ascorbate in organisms. Biochimica et Biophysica Acta (BBA)- General Subjects, 1336, 566–574.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Mekuto, L., Ntwampe, S., & Akcil, A. (2016). An integrated biological approach for treatment of cyanidation wastewater. Science of the Total Environment, 571, 711–720.

    Article  CAS  Google Scholar 

  • Metzner, H., Rau, H., & Senger, H. (1965). Untersuchungen zur synchronisierbarkeit einzelner pigment-mangel mutanten von Chlorella. Planta, 65, 186–194.

    Article  CAS  Google Scholar 

  • Mudder, T. I., Botz, M., & Smith, A. (2001). Chemistry and treatment of cyanidation wastes. London: Mining Journal Books.

    Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Racusen, D., & Foote, M. (1965). Protein synthesis in dark grown bean leaves. Canadian Journal of Botany, 43, 817–824.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Robert, R. (1979). Growth measurements. Division rate. Physiological methods. Culture methods and growth measurements (pp. 29–311). Cambridge: Cambridge University. Press.

    Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Siegien, I., & Bogatek, R. (2006). Cyanide action in plants - from toxic to regulatory. Acta Physiologiae Plantarum, 28, 483–497.

    Article  CAS  Google Scholar 

  • 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 

  • Srivastava, A. C., & Muni, R. R. D. (2010). Phytoremediation of cyanide. In Plant adaptation and phytoremediation (pp. 399–426). Springer.

  • 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 

  • 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.

    CAS  Google Scholar 

  • Taussig, A. (1960). The synthesis of the induced enzyme, cyanase, in E. coli. Biochimica et Biophysica Acta, 44, 510–519.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Way, J. L. (1984). Cyanide intoxication and its mechanism of antagonism. Annual Review of Pharmacology and Toxicology, 24, 451–481.

    Article  CAS  Google Scholar 

  • Wishnik, M. W., & Lane, M. D. (1969). Inhibition of ribulose diphosphate carboxylase by cyanide. The Journal of Biological Chemistry, 244, 55–59.

    Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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.

Declarations:

- 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.

Author information

Authors and Affiliations

  1. Plant Biotechnology Laboratory (PBL), Botany and Microbiology Department, Faculty of Science, Zagazig University, El-Gamaa Street 1, Zagazig, Sharkia, 44519, Egypt

    Yassin El-Ayouty, Mostafa Ismaiel, Asmaa Al-Badwy & Rashad Kebeish

  2. Faculty of Science Yanbu, Biology Department, Taibah University, Yanbu, Al-Madinah Almonawarah, Saudi Arabia

    Rashad Kebeish

Authors
  1. Yassin El-Ayouty
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mostafa Ismaiel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Asmaa Al-Badwy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rashad Kebeish
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rashad Kebeish.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

El-Ayouty, Y., Ismaiel, M., Al-Badwy, A. et al. Overexpression of Cyanase in Chlamydomonas reinhardtii: a Promising Approach for Biodegradation of Cyanate in Aquatic Systems. Water Air Soil Pollut 230, 123 (2019). https://doi.org/10.1007/s11270-019-4175-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11270-019-4175-0

Keywords

Search

Navigation