Cloning and Analysis of Holo-C-Phycocyanin Alpha Subunit Synthesis Genes from Nostoc spp. and Thermosynechococcus sp. TUBT-T01
Abstract
C-phycocyanin is a blue-colored protein used as a fluorescent reagent for immunological analysis and natural pigment in food and cosmetics. Furthermore, many studies reported the pharmaceutical potential of C-phycocyanin due to its antioxidant properties. This study was conducted to isolate and clone the necessary genes for the synthesis of holo-C-phycocyanin alpha subunit including hox1 and pcyA from Nostoc spp. as well as cpcE and cpcF from Thermosynechococcus sp. TUBT-T01. The genes were successfully identified, cloned, and sequenced. Physiochemical properties and secondary structure of proteins were analyzed and predicted. The nucleotide sequences of each gene were aligned with those of other cyanobacterial strains available in the GenBank. The results indicated that the nucleotide sequences of hox1 from Nostoc sp. TUBT04 and pcyA from Nostoc sp. TUBT01 had 97% and 96% identity to those from Nostoc sp. NIES-4103 respectively. While the nucleotide sequences of cpcE and cpcF from Thermosynechococcus sp. TUBT-T01 had 90-92% identity to those from Thermosynechococcus sp. CL-1, Thermosynechococcus elongatus PKUAC-SCTE542, Thermosynechococcus vulcanus NIES-2134, and Thermosynechococcus elongatus BP-1. Results obtained from this study will be very beneficial for the production of holo-C-phycocyanin from Thermosynechococcus sp. TUBT-T01 using Escherichia coli expression system in the near future. Keywords : hox1 ; pcyA ; cpcE ; cpcFReferences
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Dedvisitsakul, P., Huadraksasat, S., Chittapun, S., Charoenrat, T., & Piyapittayanun, C. (2017). Cloning of a C-phycocyanin alpha subunit from Thermosynechococcus sp. TUBT-T01 and prediction of its properties. Walailak Journal of Science and Technology, 15, 857-867.
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Tooley, A. J., Cai, Y. A., & Glazer, A. N. (2001). Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-α subunit in a heterologous host. Proceedings of the National Academy of Sciences of the United States of America, 98(19), 10560–10565.
Wu, F., Zang, X., Zhang, X., Zhang, R., Huang, X., Hou, L., Jiang, M., Liu, C., Pang, C. (2016). Molecular cloning of cpcU and heterodimeric bilin lyase activity analysis of CpcU and CpcS for attachment of phycocyanobilin to Cys-82 on the β-Subunit of phycocyanin in Arthrospira platensis FACHB314. Molecules, 21(3), 357.
Yi, J., Xu, D., Zang, X., Yuan, D., Zhao, B., Tang, L., Tan, Y., Zhang, X. (2014). Lyase activities of heterologous CpcS and CpcT for phycocyanin holo-β-subunit from Arthrospira platensis in Escherichia coli. Journal of Ocean University of China, 13(3), 497–502.
Zhang, R., Feng, X. T., Wu, F., Ding, Y., Zang, X. N., Zhang, X. C., Yuan, D. Y., Zhao, B. R. (2014). Molecular cloning and expression analysis of a new bilin lyase: The cpcT gene encoding a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the β-subunit of phycocyanin in Arthrospira platensis FACHB314. Gene, 544(2), 191–197.
Zhao, K. H., Su, P., Li, J., Tu, J. M., Zhou, M., Bubenzer, C., & Scheer, H. (2006). Chromophore attachment to phycobiliprotein β-subunits: Phycocyanobilin:cysteine-β84 phycobiliprotein lyase activity of CpeS-like protein from Anabaena sp. PCC7120. Journal of Biological Chemistry, 281(13), 857.
Alvey, R., Biswas, A., Schluchter, W., & Bryant, D. (2011). Attachment of noncognate chromophores to CpcA of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002 by heterologous expression in Escherichia coli. Biochemistry, 50(22), 4890-902.
Biswas, A., Vasquez, Y. M., Dragomani, T. M., Kronfel, M. L., Williams, S. R., Alvey, R. M., Bryant, D. A., & Schluchter, W. M. (2010). Biosynthesis of cyanobacterial phycobiliproteins in Escherichia coli: Chromophorylation efficiency and specificity of all bilin lyases from Synechococcus sp. strain PCC 7002. Applied and Environmental Microbiology, 76(9), 2729–2739.
Chittapun, S., & Charoenrat, T. (2015). Isolation and Growth of N2-fixing cyanobacteria from organic agricultural areas in Sanamchaikate, Chachoeng-Sao Province, Thailand. Thammasat International Journal of Science and Technology, 20(2), 27-32.
Dedvisitsakul, P., Huadraksasat, S., Chittapun, S., Charoenrat, T., & Piyapittayanun, C. (2017). Cloning of a C-phycocyanin alpha subunit from Thermosynechococcus sp. TUBT-T01 and prediction of its properties. Walailak Journal of Science and Technology, 15, 857-867.
Doyle, J. J., & Doyle, J. L. (1990). Isolation of plant DNA from fresh tissue. Focus, 12, 13–15.
Garnier, J., Gibrat, J.F., & Robson, B. (1996). GOR method for predicting protein secondary structure from amino acid sequence. Methods in Enzymology, 266, 540-553.
Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D., & Bairoch A. (2005). Protein identification and analysis tools on the ExPASy Server. In J.M. Walker. (Ed.), The Proteomics Protocols Handbook. (pp. 571-607). Humana Press.
Guan, X., Qin, S., Zhao, F., Zhang, X., & Tang, X. (2007). Phycobilisomes linker family in cyanobacterial genomes: Divergence and evolution. International Journal of Biological Sciences, 3(7),
434–445.
Guan, X. Y., Zhang, W. J., Zhang, X. W., Li, Y. X., Wang, J. F., Lin, H. Z., Tang, X. X., & Qin, S. (2009). A potent anti-oxidant property: Fluorescent recombinant α-phycocyanin of Spirulina. Journal of Applied Microbiology, 106(4), 1093–1100.
Huadraksasat, S., Chittapun, S., Charoenrat, T., Dedvisitsakul, P., & Piyapittayanun, C. (2017). Identification and analysis of a C-phycocyanin beta subunit gene from Thermosynechococcus sp. TUBT-T01. Burapha Science Journal, 22(3), 141-156.
Kuddus, M., Singh, P., Thomas, G., & Al-Hazimi, A. (2013). Recent developments in production and biotechnological applications of C-phycocyanin. BioMed Research International, 2013, 742859.
Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Molecular biology and evolution, 35(6), 1547–1549.
Messing, J. (1983). New M13 vectors for cloning. Methods in Enzymology, 101, 20-78.
Rippka, R., Stanier, R. Y., Deruelles, J., Herdman, M., & Waterbury, J. B. (1979). Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria. Microbiology, 111(1), 1–61.
Sambrook, J., Russell, D., & Russell, D. (2001). Molecular cloning: A laboratory manual (3rd ed.). Cold Spring Harbor, New York, N.Y.: Cold Spring Harbor Laboratory Press.
Sidler, W. A. (1994). Phycobilisome and phycobiliprotein structures. In D. A. Bryant (Ed.), The Molecular Biology of Cyanobacteria (pp. 139–216). Dordrecht: Kluwer Academic Publishers.
Suwanmanee, K., Charoenrat, T., & Chittapun, S. (2015). Isolation and cultivation of thermophilic blue green algae from hot springs in Surat Thani province. In Proceeding the 7th National Conference on Algae and Plankton (NCAP) conference. (pp. 1-12), Thailand: Bangkok.
Suwanmanee, K. (2016). Production and determination of thermotolerant properties of C-phycocyanin isolated from hot spring cyanobacterium (Master’s thesis). Thammasat University. Thailand. (in Thai)
Tooley, A. J., Cai, Y. A., & Glazer, A. N. (2001). Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-α subunit in a heterologous host. Proceedings of the National Academy of Sciences of the United States of America, 98(19), 10560–10565.
Wu, F., Zang, X., Zhang, X., Zhang, R., Huang, X., Hou, L., Jiang, M., Liu, C., Pang, C. (2016). Molecular cloning of cpcU and heterodimeric bilin lyase activity analysis of CpcU and CpcS for attachment of phycocyanobilin to Cys-82 on the β-Subunit of phycocyanin in Arthrospira platensis FACHB314. Molecules, 21(3), 357.
Yi, J., Xu, D., Zang, X., Yuan, D., Zhao, B., Tang, L., Tan, Y., Zhang, X. (2014). Lyase activities of heterologous CpcS and CpcT for phycocyanin holo-β-subunit from Arthrospira platensis in Escherichia coli. Journal of Ocean University of China, 13(3), 497–502.
Zhang, R., Feng, X. T., Wu, F., Ding, Y., Zang, X. N., Zhang, X. C., Yuan, D. Y., Zhao, B. R. (2014). Molecular cloning and expression analysis of a new bilin lyase: The cpcT gene encoding a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the β-subunit of phycocyanin in Arthrospira platensis FACHB314. Gene, 544(2), 191–197.
Zhao, K. H., Su, P., Li, J., Tu, J. M., Zhou, M., Bubenzer, C., & Scheer, H. (2006). Chromophore attachment to phycobiliprotein β-subunits: Phycocyanobilin:cysteine-β84 phycobiliprotein lyase activity of CpeS-like protein from Anabaena sp. PCC7120. Journal of Biological Chemistry, 281(13), 857.
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2021-01-04
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