Engineering Chlamydomonas Reinhardtii for Efficient Biodegradation of Cyanate

Development of novel technologies for the treatment of industrial and hazardous wastes is increasing rapidly. Particular attention is being focused upon the use of biological treatment systems. Aerobic and anaerobic microbial treatment processes have been successfully employed in the destruction and/or removal of organic compounds, inorganics and metals (Mudder and Botz, 2001). Cyanide is a known toxic chemical produced through anthropogenic activities and industries that use ore leaching, electroplating, steal production, plastics, and synthetic fibers (Hamel 2011; Patil and Paknikar 2000; Sancho and Bellon 2005). Cyanate and its derivatives have been widely used for manufacturing of a broad classes of herbicides and in the synthesis of polymers as well (Koshiishi et al. 1997; Kraus and Kraus 2001). Biologically, cyanate is produced through the breakdown of a number of metabolites such as carbamoylphosphate and urea (Dirnhuber and Schutz 1948; Guilloton and Karst 1987). Moreover, spontaneous photo-oxidation of cyanide as well as the oxidative treatment of cyanide-containing industrial wastes (Mekuto et al. 2016; Nowakowska et al. 2006) are major causes for the release of the toxic cyanate into the environment (Malhotra et al. 2005; Rader et al. 1995). Cyanate and cyanide compounds are detoxified mainly by chemical treatments involving oxidation or chlorination reactions (Akcil and Mudder 2003). However, these chemical treatments methods are disadvantageous due to the high costs and/or production of hazardous byproducts (Srivastava and Muni 2010). Bioremediation systems involving the usage of plants or microorganisms are more eco-friendly and affordable alternatives (Akcil and Mudder 2003). ‎However, microbial biodegradation systems are probably inefficient because of the accumulation of toxic microbial metabolites and/or overloading with excess pollutants (Ebbs 2004). phytoremediation using vascular plants and algal systems might become preferable alternatives for detoxification of cyanate and cyanide contaminants (Bushey et al. 2006; Taebi et al. 2008; Yu et al. 2006).

Cyanase enzyme (EC 4.2.1.104) is able to degrade cyanate into carbon dioxide and ammonium in a bicarbonate-dependent reaction (Anderson 1980; Johnson and Anderson 1987). The enzyme was first identified and fully characterized in Escherichia coli (Anderson and Little 1986; Johnson and Anderson 1987; Sung et al. 1987; Taussig 1960; Walsh et al. 2000). It is found in some Gram-positive bacteria and fungi (Butryn et al. 2015; Kamennaya and Post 2010). Some bacteria was shown to grow on cyanate as sole nitrogen and carbon sources due to the endogenous cyanase activity (Kunz and Nagappan 1989; Luque-Almagro et al. 2008; Taussig 1960). The enzyme was also discovered in cyanobacteria (Blank and Hinman 2016; Garcia-Fernandez and Diez 2004; Harano et al. 1997; Kamennaya et al. 2008; Miller and Espie 1994; Voigt et al. 2014) and plants (Aichi et al. 1998; Qian et al. 2011). In living organisms, cyanase plays a vital role in the detoxification of cyanate and cyanide compounds (Ebbs 2004). Plant cyanase may be involved and plays an integral role in variable physiological and biochemical pathways.

Seed germination and early seedling growth were shown to be inhibited by application of KCNO to Arabidopsis thaliana cyanase knock-out mutants. However, transgenic Arabidopsis thaliana plants overexpressing Arabidopsis thaliana (AtCYN) or Oryza sativa (OsCYN) cyanase genes showed enhanced resistance to cyanate (Qian et al. 2011). In a recent study, involves the overexpression of the cyanobacterial enzyme cyanase in A. thaliana. it was shown that transgenic A. thaliana plants are more tolerant and produce higher biomass compared to wild type plants at high concentrations of KCNO (Kebeish and Al-Zoubi, 2017). Chlamydomonas reinhardtii has attracted more attention as a model for studying biological systems because this organism is the most biologically characterized (Harris, 1989; Maul et al., 2002, Merchant et al., 2007, Popescu and Lee, 2007). Research into recombinant protein production such as expression of enzymes, proteins, human growth factors, antibodies, and vaccine in Chlamydomonas reinhardtii has attracted increasing attention (Ishikura et al., 1999, Mayfield et al., 2003, Purton, 2007, Rasala et al., 2010). So far, no research studies has been implemented to test the efficacy of introducing cyanobacterial cyanase into microalgae for cyanate remediation purposes. In the current study, the cyanobacterial cyanase gene (CYN, gi16329170) was therefore genetically cloned and transferred into C. reinhardtii cells. Transgenic C. reinhardtii lines showed enhanced tolerance to cyanate (up to 30 mM) compared to wild types. The study was extended to assess the biochemical response of the transgenic microalgae under cyanate stress in vivo. Results of the present study can improve our understanding of the ecological risks of CNs and provide effective solutions for CNO- remediation.