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Isolation of quizalofop-resistant mutants of Nannochloropsis oculata (Eustigmatophyceae) with high eicosapentaenoic acid following N-methyl-N-nitrosourea-induced random mutagenesis

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Abstract

Nannochloropsis oculata was subjected to N-methyl-N-nitrosourea-induced mutagenesis under the selection pressure of quizalofop, a known inhibitor of acetyl-CoA carboxylase (ACCase) activity with the objective of generating genetically tractable mutants with altered fatty acid metabolism. Two mutants, QUIZ1 and QUIZ2, with stable resistance to quizalofop were isolated and partially characterized. The growth properties and morphology of the mutants appeared identical with the parent strain. However thermo-tolerance was observed in the mutants. Enhanced resistance to quizalofop suggested the presence of herbicide resistant isoforms of ACCase. In vitro assays for ACCase activity showed that ACCase in the wild strains was much more sensitive to quizalofop than the mutant strains. Gas chromatographic analysis of fatty acids revealed that the mutant strains were rich in polyunsaturated fatty acids (n− 3PUFAs), as well as total fatty acid contents; this was accompanied by a concomitant increase in triacylglycerol (TAG) followed by linoleic acid (18:2), arachidonic acid (20:4 n− 6) and EPA (20:5 n− 3). These results suggest that an increased substrate pool (malonyl-CoA) (due to increased specific activity of ACCase) in the mutant strains in vivo and in vitro may have led to the increased TAG accumulation. Random mutagenesis was shown to be a good tool to manipulate PUFAs and EPA in Nannochloropsis. The strains developed will be useful in understanding fatty acid metabolism using genetic and biochemical approaches and also for their direct use in mariculture.

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References

  • Bajpai P, Bajpai P.K. (1993). Eicosapentaenoic acid (EPA) produc-tion from microorganisms: A review. J. Biotech. 30: 161–183.

    Google Scholar 

  • Bos C.J. (1987). Induction and isolation of mutants in fungi at low mutagen doses. Curr. Genet. 12: 471–474.

    Google Scholar 

  • Bradford M.M. (1976). Arapid and sensitive method for the quantifi-cation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.

    Google Scholar 

  • Burton J.D., Gronwald J.W., Somers D.A., Connelly J.A., Gengenbach B.G., Wyse D.L. (1987). Inhibition of plant acetyl-CoA carboxylase by the herbicides sethoxydim and haloxyfop. Biochem. Biophys. Res. Commun. 148: 1039–1044.

    Google Scholar 

  • Burton J.D., Gronwald J.W., Somers D.A., Gengenbach B.G., Wyse D.L. (1989). Inhibition of corn acetyl-CoA carboxylase by cyclo-hexanedione and aryloxyphenoxypropionate herbicides. Pestic. Biochem. Physiol. 34: 76–85.

    Google Scholar 

  • Devine M.D., Shimabukuro R.H. (1994). Resisatnce to acetyl coen-zyme A carboxylase inhibiting herbicides. In Powel, SB, Hol-tum, JAM (eds), Herbicide Resistance in Plants: Biology and Biochemistry, Lewis Publishers, Tokyo, pp. 141–169.

    Google Scholar 

  • Egli M.A., Gengenbach B.G., Gronwald J.W., Somers D.A., Wyse D.L. (1993). Characterization of maize acetyl-CoA carboxylase. Plant. Physiol. 101: 499–506.

    Google Scholar 

  • Eisenstadt E. (1987). Analysis of mutagenesis. In Neidhardt FC (eds), Escherichia coliand Salmonella typhimurium.Cellular and Molecular Biology. American Society for Microbiology, Washington, DC, pp. 1016–1033.

    Google Scholar 

  • Gengenbach B.G., Somers D.A., Wyse D.L., Gronwald J.W., Egli M.A., Lutz S.M. (2000). Methods and an acetyl-CoA carboxylase gene for conferring herbicide tolerance and an alteration in oil content of plants. United States Patent No. 6069298: 49–183.

  • Ghirardi M.L., Flynn T., Forestier M., Iyer A., Melis A., Danielson P., Seibert M. (1999).Generation of C. reinhardtiimutants that photo produce H2 from H2O inthe presence of O2.InGarab G (eds), Photosynthesis: Mechanism and Effects.Kluwer Aca-demic Publishers, The Netherlands,pp.1959–1962.

    Google Scholar 

  • Guillard R.R.L., Ryther J.H. (1962). Studies of marine planktonic diatoms. I. Cyclotella nana(Hustedt) and Detinula conferaceae(Cleve). Gran. Can. J. Microbiol. 18: 229–239.

    Google Scholar 

  • Guillard R.R.L. (1973). Division rates. In Sournia A (eds), Hand-book of Phycological Methods. Cambridge University Press, Cambridge, pp. 289–312.

    Google Scholar 

  • Hagemann R. (1982). Induction of plastome mutation by nitroso-urea-compounds. In Edelman M., Hallick R.B., Chua N.H. (eds), Methods in Chloroplast Molecular Biology. Elsevier, Amster-dam, pp. 119–127.

    Google Scholar 

  • Hartnett M.E., Newcomb J.R., Hodson R.C. (1987). Mutations in Chlamydomonas reinhardtiiconferring resistance to the herbi-cide sulfometuron methyl. Plant. Physiol 85: 898–901.

    Google Scholar 

  • Harwood J.L., Ridley S.M., Walker K.A. (1989). Herbicides inhibit-ing lipid synthesis. In Dodge AD (eds), Herbicides and Plant Metabolism. Cambridge University Press, NY, pp. 73–96.

    Google Scholar 

  • Henderson R.J., Hodgson P., Harwood J.L. (1990). Differential ef-fects of the substituted pyridazinone herbicide Sandoz 9785 on lipid composition and biosynthesis in non photosynthetic marine microalgae. 1. Lipid composition and synthesis. J. Exp. Bot. 41: 729–736.

    Google Scholar 

  • Hodgson P.A., Henderson R.J., Sargent J.R., Leftley J.W. (1991). Patterns of variation in the lipid class and fatty acid composition of Nannochloropsis oculata(Eustigmatophyceae) during batch culture. J. Appl. Phycol. 3: 169–181.

    Google Scholar 

  • Horrocks L.A., Yeo Y.K. (1990). Health benefits of docosahexaenoic acid (DHA). Pharm. Res. 40: 211–225.

    Google Scholar 

  • Huang K.P., Stumpf P.K. (1971). Fatty acid synthesis by a soluble fatty acid synthetase from Solanum tuberosum. Arch. Biochem. Biophys. 143: 412–427.

    Google Scholar 

  • James C.M., A l-Hinty S., Salman A.E. (1989). Growth and omega-3 fatty acid composition of marine microalgae under different regimes. Aquaculture 77: 337–351.

    Google Scholar 

  • Kates M. (1972). Isolation, analysis and identification of lipids. In Work T.S., Work E. (eds), Techniques in Lipidology. Elsevier, Amsterdam, pp. 268–618.

    Google Scholar 

  • Ke J., Wen T.N., Nikolau B.J., Wurtele S.E. (2000). Coordinate reg-ulation of the nuclear and plastidic genes coding for the subunits of the heteromeric acetyl-CoA carboxylase. Plant. Physiol 122: 1057–1071.

    Google Scholar 

  • Klug W.S., Cummings M.R. (1983). In Charles E. (eds), Concepts in Genetics, Merrill Publishing Company, Columbus, OH.

    Google Scholar 

  • Konishi T., Sasaki Y. (1994). Compartmentalization of two forms of acetyl-CoA carboxylase in plants and the origin of their tolerance towards herbicides. Proc Natl. Acad. Sci. USA 91: 3598–3601.

    Google Scholar 

  • Kunst L., Browse J., Somerville C. (1989). A mutant of Arabidop-sisdeficient in desaturation of palmitic acid in leaf lipids. Plant Physiol 90: 943–947.

    Google Scholar 

  • Lopez Alonso D., Segura del Castillo C.I., Garcia Sanchez J.L., Sanchez Perez J.A., Garcia Camacho F. (1994). Quantitative ge-netics of fatty acid variation in Isochrysis galbana(Prymnesio-phyceae) and Phaeodactylum tricornutum(Bacillariophyceae). J. Phycol. 30: 553–558.

    Google Scholar 

  • Lopez Alonso D., Segura del Castillo C.I., Grima E.M., Cohen Z. (1996). First insight into improvement of eicosapentaenoic acid content in Phaeodactylium tricornutum(Bacillariophyceae) by induced mutagenesis. J. Phycol. 32: 339–345.

    Google Scholar 

  • Maruyama I., Nakamura T., Matsubayashi T., Ando Y., Naeda T. (1986). Identification of the alga known as 'marine Chlorella'as a member of the Eustigmatophyceae. Jpn. J. Phycol. 34: 319–325.

    Google Scholar 

  • Meireles L.A., Guedes A.C., Malcata F.X. (2003). Increase of the yields of eicosapentaenoic and docosahexaenoic acids by the mi-croalga Pavlova lutherifollowing random mutagenesis. Biotech-nol. Bioeng. 81: 50–55.

    Google Scholar 

  • Miquel M., James D., Dooner H., Browse J. (1993). Arabidopsisre-quires polyunsaturated lipids for low temperature survival. Proc. Natl. Acad. Sci. U.S.A. 90: 6208–6212.

    Google Scholar 

  • Moppes D.V., Barak Z., Chipman D.M., Gollop N., Arad S.M. (1989). An herbicide (sulfometuron methyl) resistant mutant in Porphyridium(Rhodophyta). J. Phycol. 25: 108–112.

    Google Scholar 

  • Nishida I., Murata N. (1996). Chilling sensitivity in plants and cyanobacteria: The crucial contribution of membrane lipids. Ann. Rev. Plant. Physiol. Plant. Mol. Biol. 47: 541–568.

    Google Scholar 

  • Post-Beittenmiller D., Roughan G., Ohlrogge J.B. (1992). Regula-tion of plant fatty acid biosynthesis: Analysis of acyl-CoA and acyl-ACP substrate pools in spinach and pea chloroplasts. Plant. Physiol. 100: 923–930.

    Google Scholar 

  • Rendina A.R., Felts J.M., Beaudoin J.D., Craig-Kennard A.C., Look L.L., Paraskos S.L., Hagenah J.A. (1988). Kinetic characteriza-tion, stereoselectivity, and species selectivity of the inhibition of plant acetyl-CoA carboxylase by aryloxyphenoxypropionic acid grass herbicides. Arch. Biochem. Biophys 265: 219–225.

    Google Scholar 

  • Renaud S.M., Parry D.L., Luong-Van T., Kuo C., Padovan A., Sammy N.L.(1991).Effect of light intensity on the proximate biochemical and fatty acid composition of Isochrysissp. and Nannochloropsis oculatafor use in tropical aquaculture. J. Appl. Phycol. 3: 43–53.

    Google Scholar 

  • Roessler P.G. (1988). Effects of silicon deficiency on lipid composi-tion and metabolism in the diatom cyclotella cryptica.J.Phycol. 24: 394–400.

    Google Scholar 

  • Roessler P.G. (1990). Purification and characterization of acetyl-CoA carboxylase from the diatom Cyclotella cryptica. Plant. Physiol 92: 73–78.

    Google Scholar 

  • Sauer A., Heise K.P. (1984). Regulation of acetyl-CoA carboxy-lase and acetyl-coenzymeA synthetase in spinach chloroplasts. Z. Naturforsch. 39: 268–275.

    Google Scholar 

  • Schneider J.C., Livne A., Sukenik A., Roessler P.G. (1995). A mu-tant of Nannochloropsisdeficient in eicosapentaenoic acid pro-duction. Phytochemistry 40: 807–814.

    Google Scholar 

  • Seto A., Wang H.L., Hesseltine C.W. (1984). Culture conditions af-fect eicosapentaenoic acid content of Chlorella minutissima.J. Am. Oil Soc. 61: 892–894.

    Google Scholar 

  • Shahidi F., Wanasundara U.N. (1998). Omega 3-fatty acid concen-trates: Nutritional aspects and production technologies. Trends Food Sci. Technol 9: 230–240.

    Google Scholar 

  • Simopoulos A.P. (1991). Omega-3 fatty acids in health and disease and in growth and development. Am. J. Clin. Nutr. 54: 438–463.

    Google Scholar 

  • Suen Y., Hubbard J.S., Holzer G., Tornabene T.G. (1987). Total lipid production of the green alga Nannochloropsissp. Q I I under different nitrogen regimes. J. Phycol. 23: 289–296.

    Google Scholar 

  • Sukenik A. (1991). Ecophysiological considerations in the optimiza-tion of eicosapentaenoic acid production by Nannochloropsissp. (Eustigmatophyceae). Bioresources Biotechnol. 36: 263–269.

    Google Scholar 

  • Sukenik A., Carmeli Y. (1990). Lipid synthesis and fatty acid com-position in Nannochloropsissp. (Eustigmatophyceae) grown in light dark cycle. J. Phycol. 26: 463–469.

    Google Scholar 

  • Sukenik A., Carmeli Y., Berner T. (1989). Regulation of fatty acid composition by growth irradiance level in the eustigmatophyte Nannochloropsissp. J. Phycol 25: 686–692.

    Google Scholar 

  • Teshima S., Yamasaki S., Kanazawa A., Koshio S., Mukai H. (1991). Fatty acid composition of Malaysian marine Chlorella. Nippon Suisan Gakkaishi 57: 1985.

    Google Scholar 

  • Thompson P.A., Guo M.X., Harrison P.J., Whyte J.N.C. (1992). Ef-fects of variations in temperature. II. On the fatty acid compo-sition of eight species of marine phytoplankton. J. Phycol. 28: 488–497.

    Google Scholar 

  • Turnham E., Northcote D.H. (1983). Changes in the activity of acetyl-CoA carboxylase during rape seed formation. Biochem. J. 212: 223–229.

    Google Scholar 

  • Wallis J.G., Browse J. (2002). Mutants of Arabidopsisreveal many roles for membrane lipids. Prog. Lipid. Res. 41: 254–278.

    Google Scholar 

  • Zittelli G.C., Lavista F., Bastianini A., Rodolfi L, Vincenzini M, Tredici MR (1999). Production of eicosapentanoic acid by Nan-nochloropsissp. cultures in outdoor tubular photo bioreactors. J. Biotech. 70: 299–312.

    Google Scholar 

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Authors
  1. Ratnesh Chaturvedi
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  2. Srinivasa Rao Uppalapati
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  3. Mohammad Amin Alamsjah
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  4. Yuji Fujita
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Chaturvedi, R., Uppalapati, S.R., Alamsjah, M.A. et al. Isolation of quizalofop-resistant mutants of Nannochloropsis oculata (Eustigmatophyceae) with high eicosapentaenoic acid following N-methyl-N-nitrosourea-induced random mutagenesis. Journal of Applied Phycology 16, 135–144 (2004). https://doi.org/10.1023/B:JAPH.0000044826.70360.8e

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  • DOI: https://doi.org/10.1023/B:JAPH.0000044826.70360.8e

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