Ethyl Methane Sulfonate-Induced Dunaliella salina KU11 Mutants Affected for Growth Rate, Cell Accumulation and Biomass

Authors: Vongsathorn Ngampuak, Yutachai Chookaew, Wipawee Dejtisakdi

Abstract:

Dunaliella salina has great potential as a system for generating commercially valuable products, including beta-carotene, pharmaceuticals, and biofuels. Our goal is to improve this potential by enhancing growth rate and other properties of D. salina under optimal growth conditions. We used ethyl methane sulfonate (EMS) to generate random mutants in D. salina KU11, a strain classified in Thailand. In a preliminary experiment, we first treated D. salina cells with 0%, 0.8%, 1.0%, 1.2%, 1.44% and 1.66% EMS to generate a killing curve. After that, we randomly picked 30 candidates from approximately 300 isolated survivor colonies from the 1.44% EMS treatment (which permitted 30% survival) as an initial test of the mutant screen. Among the 30 survivor lines, we found that 2 strains (mutant #17 and #24) had significantly improved growth rates and cell number accumulation at stationary phase approximately up to 1.8 and 1.45 fold, respectively, 2 strains (mutant #6 and #23) had significantly decreased growth rates and cell number accumulation at stationary phase approximately down to 1.4 and 1.35 fold, respectively, while 26 of 30 lines had similar growth rates compared with the wild type control. We also analyzed cell size for each strain and found there was no significant difference comparing all mutants with the wild type. In addition, mutant #24 had shown an increase of biomass accumulation approximately 1.65 fold compared with the wild type strain on day 5 that was entering early stationary phase. From these preliminary results, it could be feasible to identify D. salina mutants with significant improved growth rate, cell accumulation and biomass production compared to the wild type for the further study; this makes it possible to improve this microorganism as a platform for biotechnology application.

Keywords: Dunaliella salina, mutant, ethyl methane sulfonate, growth rate, biomass.

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1125619

References:

[1] K. E. Apt and P. W. Behrens, “Commercial developments in microalgal biotechnology,” J. Phycol., vol. 35, no. 2, pp. 215–226, Apr. 1999.
[2] W. Becker and A. Richmond, “Microalgae for aquaculture: the nutritional value of microalgae for aquaculture.,” pp. 380–391, 2004.
[3] Q. Hu, M. Sommerfeld, E. Jarvis, M. Ghirardi, M. Posewitz, M. Seibert, and A. Darzins, “Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances.,” Plant J., vol. 54, no. 4, pp. 621–39, May 2008.
[4] R. Raja, S. Hemaiswarya, N. A. Kumar, S. Sridhar, and R. Rengasamy, “A Perspective on the Biotechnological Potential of Microalgae,” Crit. Rev. Microbiol., vol. 34, no. 2, pp. 77–88, Jan. 2008.
[5] A. Martins, N. S. Caetano, and T. M. Mata, “Microalgae for biodiesel production and other applications: A review,” vol. 14, pp. 217–232, 2010.
[6] Y. Gong, H. Hu, Y. Gao, X. Xu, and H. Gao, “Microalgae as platforms for production of recombinant proteins and valuable compounds: progress and prospects.,” J. Ind. Microbiol. Biotechnol., vol. 38, no. 12, pp. 1879–90, Dec. 2011.
[7] R. Sathasivam, N. Juntawong, and B. Program, “Modified medium for enhanced growth of Dunaliella strains,” pp. 67–73, 2013.
[8] Z. Wu, P. Duangmanee, P. Zhao, N. Juntawong, and C. Ma, “The Effects of Light, Temperature, and Nutrition on Growth and Pigment Accumulation of Three Dunaliella salina Strains Isolated from Saline Soil.,” Jundishapur J. Microbiol., vol. 9, no. 1, p. e26732, Jan. 2016.
[9] M. Schroda, D. Blöcker, and C. F. Beck, “The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas.,” Plant J., vol. 21, no. 2, pp. 121–31, Jan. 2000.
[10] T. Wang, L. Xue, W. Hou, B. Yang, Y. Chai, X. Ji, and Y. Wang, “Increased expression of transgene in stably transformed cells of Dunaliella salina by matrix attachment regions.,” Appl. Microbiol. Biotechnol., vol. 76, no. 3, pp. 651–7, Sep. 2007.
[11] R. P. Sinha and D.-P. Häder, “UV-induced DNA damage and repair: a review,” Photochem. Photobiol. Sci., vol. 1, no. 4, pp. 225–236, Apr. 2002.
[12] Y. Kim, K. S. Schumaker, and J.-K. Zhu, “EMS mutagenesis of Arabidopsis.,” Methods Mol. Biol., vol. 323, no. 6, pp. 101–3, Jan. 2006.
[13] M. Hlavova, Z. Turoczy, and K. Bisova, “Improving microalgae for biotechnology — From genetics to synthetic biology,” Biotechnol. Adv., vol. 33, no. 6, pp. 1194–1203, 2015.
[14] B. Lee, G.-G. Choi, Y.-E. Choi, M. Sung, M. S. Park, and J.-W. Yang, “Enhancement of lipid productivity by ethyl methane sulfonate-mediated random mutagenesis and proteomic analysis in Chlamydomonas reinhardtii,” Korean J. Chem. Eng., vol. 31, no. 6, pp. 1036–1042, Jun. 2014.
[15] R. Radakovits, R. E. Jinkerson, A. Darzins, and M. C. Posewitz, “Genetic engineering of algae for enhanced biofuel production.,” Eukaryot. Cell, vol. 9, no. 4, pp. 486–501, Apr. 2010.
[16] F. Ahmed, K. Fanning, M. Netzel, and P. M. Schenk, “Induced carotenoid accumulation in Dunaliella salina and Tetraselmis suecica by plant hormones and UV-C radiation.,” Appl. Microbiol. Biotechnol., vol. 99, no. 22, pp. 9407–16, Nov. 2015.
[17] H. Mendoza, A. de la Jara, K. Freijanes, L. Carmona, A. A. Ramos, V. de Sousa Duarte, and J. C. Serafim Varela, “Characterization of Dunaliella salina strains by flow cytometry: a new approach to select carotenoid hyperproducing strains,” Electron. J. Biotechnol., vol. 11, no. 4, Oct. 2008.
[18] B. Sandesh Kamath, R. Vidhyavathi, R. Sarada, and G. A. Ravishankar, “Enhancement of carotenoids by mutation and stress induced carotenogenic genes in Haematococcus pluvialis mutants,” Bioresour. Technol., vol. 99, no. 18, pp. 8667–8673, 2008.
[19] V. H. Work, R. Radakovits, R. E. Jinkerson, J. E. Meuser, L. G. Elliott, D. J. Vinyard, L. M. L. Laurens, G. C. Dismukes, and M. C. Posewitz, “Increased lipid accumulation in the Chlamydomonas reinhardtii sta7-10 starchless isoamylase mutant and increased carbohydrate synthesis in complemented strains.,” Eukaryot. Cell, vol. 9, no. 8, pp. 1251–61, Aug. 2010.
[20] M. Mobini-Dehkordi, I. Nahvi, H. Zarkesh-Esfahani, K. Ghaedi, M. Tavassoli, and R. Akada, “Isolation of a novel mutant strain of Saccharomyces cerevisiae by an ethyl methane sulfonate-induced mutagenesis approach as a high producer of bioethanol,” J. Biosci. Bioeng., vol. 105, no. 4, pp. 403–408, 2008.
[21] M. H. Huesemann, T. S. Hausmann, R. Bartha, M. Aksoy, J. C. Weissman, and J. R. Benemann, “Biomass productivities in wild type and pigment mutant of Cyclotella sp. (Diatom).,” Appl. Biochem. Biotechnol., vol. 157, no. 3, pp. 507–26, Jun. 2009.
[22] A. Hosseini Tafreshi and M. Shariati, “Dunaliella biotechnology: Methods and applications,” J. Appl. Microbiol., vol. 107, no. 1, pp. 14–35, 2009.