Healthy omega-3 enhancement in Echium acanthocarpum transformed hairy roots by overexpression of a 6-desaturase gene from Primula vialli

Volume 1, Issue 1, October 2016     |     PP. 1-40      |     PDF (1260 K)    |     Pub. Date: October 13, 2016
DOI:    535 Downloads     5930 Views  


Rafael Zárate, Canary Islands Cancer Research Institute (ICIC), Ave. La Trinidad 61, Torre A. Arévalo, 7th floor, 38204 La Laguna, Tenerife, Spain.
Elena Cequier-Sánchez, Canary Islands Cancer Research Institute (ICIC), Ave. La Trinidad 61, Torre A. Arévalo, 7th floor, 38204 La Laguna, Tenerife, Spain.
Nabil El Jaber-Vazdekis, Institute of Microbiology CAS, Laboratory of Photosynthesis, Novohradska 237, Opatovicky mlyn. Trebon 379 81, Czech Republic.
Covadonga Rodríguez, Animal Biology, Edaphology and Geology Dept. (Animal Physiology Unit), Faculty of Sciences, Universidad de La Laguna, Ave. Fco. Sánchez, 38206 La Laguna, Tenerife, Spain.
Roberto Dorta-Guerra, Statistics and Computation Dept., Maths Faculty, Universidad de La Laguna, Ave. Fco. Sánchez, 38206 La Laguna, Tenerife, Spain
Ángel G. Ravelo, Canary Islands Cancer Research Institute (ICIC), Ave. La Trinidad 61, Torre A. Arévalo, 7th floor, 38204 La Laguna, Tenerife, Spain.

Omega-3 long change polyunsaturated fatty acids in higher plants are limited with just a few plant genus showing the accumulation of stearidonic acid (SDA) being also the longest and more unsaturated omega-3 fatty acid present. Echium acanthocarpum has been proven to be an efficient and attractive producer of SDA. Improved production of this fatty acid was attained by overexpression of a Δ6-desaturase gene from Primula vialii in transgenic E. acanthocarpum hairy roots. In this transgenic line, a drastic reduction of the substrates LA (linoleic acid) and ALA (α-linolenic acid)(40 and 30%, respectively) was parallel to the dramatic increase in GLA (γ-linolenic acid) and SDA in the total fatty acids extracted. Especially, SDA reached a percentage of 4.7% of total fatty acids, demonstrating the successful manipulation of this biosynthetic pathway in E. acanthocarpum hairy roots by overexpression of this gene. The temperature per se, was also a highly influential factor governing the fatty acid profiles in this novel transgenic hairy root culture. In terms of absolute values, the data were even more evident, due to the significant increase in total lipid extracted from the transgenic hairy root. The amount of SDA and GLA was increased 7.5 and 3 fold, respectively, compared to the control. In this transgenic culture, decreasing the culture temperature influenced directly the increments of polyunsaturated fatty acids, but did not affect lipid classes except when this factor interacted with the overexpression of the P. vialii Δ6-desaturase gene. The activation of the transgene did modify significantly the phospholipids, phosphatidylglycerol and phosphatidylcholine, whose percentages were significantly higher in these cultures compared to the control.

Echium acanthocarpum, fatty acids, hairy roots, omega-3, overexpression, stearidonic acid, polyunsaturated fatty acids

Cite this paper
Rafael Zárate, Elena Cequier-Sánchez, Nabil El Jaber-Vazdekis, Covadonga Rodríguez, Roberto Dorta-Guerra, Ángel G. Ravelo, Healthy omega-3 enhancement in Echium acanthocarpum transformed hairy roots by overexpression of a 6-desaturase gene from Primula vialli , SCIREA Journal of Biology. Volume 1, Issue 1, October 2016 | PP. 1-40.


[ 1 ] Oresic M, Hanninen VA, Vidal-Puig A. Lipidomics: a new window to biomedical frontiers. Trends Biotechnol. 2008; 26(12): 647-52.
[ 2 ] Siddiqui RA, Harvey K, Stillwell W. Anticancer properties of oxidation products of docosahexaenoic acid. Chem Phys Lipids. 2008; 153(1): 47-56.
[ 3 ] Calder PC. Omega-3 Fatty Acids and Inflammatory Processes. Nutrients. 2010; 2(3): 355-74.
[ 4 ] Horrobin DF. Nutritional and medical importance of gamma-linoleic acid. Prog Lipid Res. 1992; 31(2): 163-94.
[ 5 ] van Gool CJ, Thijs C, Henquet CJ, van Houwelingen AC, Dagnelie PC, Schrander J, et al. γ-Linolenic acid supplementation for prophylaxis of atopic dermatitis—a randomized controlled trial in infants at high familial risk. The American Journal of Clinical Nutrition. 2003; 77(4): 943-51.
[ 6 ] Schuchardt JP, Huss M, Stauss-Grabo M, Hahn A. Significance of long-chain polyunsaturated fatty acids (PUFAs) for the development and behaviour of children. Eur J Pediatr. 2010; 169(2): 149-64.
[ 7 ] Gunstone FD. Gamma linolenic acid - Occurence and physical chemical properties Prog Lipid Res. 1992; 31(2): 145-61.
[ 8 ] Tsevegsuren N, Aitzetmuller K. gamma-Linolenic and stearidonic acids in Mongolian Boraginaceae. J Am Oil Chem Soc. 1996; 73(12): 1681-4.
[ 9 ] Guil-Guerrero JL, Lopez-Martinez JC, Gomez-Mercado F, Campra-Madrid P. Gamma-linolenic and stearidonic acids from Moroccan Boraginaceae. Eur J Lipid Sci Technol. 2006; 108(1): 43-7.
[ 10 ] Ozcan T. Analysis of the total oil and fatty acid composition of seeds of some Boraginaceae taxa from Turkey. Plant Syst Evol. 2008; 274(3-4): 143-53.
[ 11 ] Portolesi R, Powell BC, Gibson RA. Competition between 24 : 5n-3 and ALA for D6 desaturase may limit the accumulation of DHA in HepG2 cell membranes. J Lipid Res. 2007; 48(7): 1592-8.
[ 12 ] Voss A, Reinhart M, Sankarappa S, Sprecher H. The metabolism of 7,10,13,16,19-docosapentanoic acid to 4,7,10,13,16,19-docosahexanoic acid in rat-liver independent of a 4-desaturase. J Biol Chem. 1991; 266(30): 19995-20000.
[ 13 ] Sayanova O, Smith MA, Lapinskas P, Stobart AK, Dobson G, Christie WW, et al. Expression of a borage desaturase cDNA containing an N-terminal cytochrome b(5) domain results in the accumulation of high levels of Delta(6)-desaturated fatty acids in transgenic tobacco. Proc Natl Acad Sci U S A. 1997; 94(8): 4211-6.
[ 14 ] Chen Q, Nimal J, Li WL, Liu X, Cao WG. Delta-6 desaturase from borage converts linoleic acid to gamma-linolenic acid in HEK293 cells. Biochem Biophys Res Commun. 2011; 410(3): 484-8.
[ 15 ] Whitney HM, Michaelson LV, Sayanova O, Pickett JA, Napier JA. Functional characterisation of two cytochrome b(5)-fusion desaturases from Anemone leveillei: the unexpected identification of a fatty acid Delta(6)-desaturase. Planta. 2003; 217(6): 983-92.
[ 16 ] Garcia-Maroto F, Garrido-Cardenas JA, Rodriguez-Ruiz J, Vilches-Ferron M, Adam AC, Polaina J, et al. Cloning and molecular characterization of the Delta 6-desaturase from two Echium plant species: Production of GLA by heterologous expression in yeast and tobacco. Lipids. 2002; 37(4): 417-26.
[ 17 ] Zhou XR, Robert S, Singh S, Green A. Heterologous production of GLA and SDA by expression of an Echium plantagineum Delta 6-desaturase gene. Plant Sci. 2006; 170(3): 665-73.
[ 18 ] Kajikawa M, Yamato KT, Kohzu Y, Nojiri M, Sakuradani E, Shimizu S, et al. Isolation and characterization of Delta(6)-desaturase, an ELO-like enzyme and Delta(5)-desaturase from the liverwort Marchantia polymorpha and production of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris. Plant MolBiol. 2004; 54(3): 335-52.
[ 19 ] Sayanova OV, Beaudoin F, Michaelson LV, Shewry PR, Napier JA. Identification of Primula fatty acid Delta(6)-desaturases with n-3 substrate preferences. FEBS Lett. 2003; 542(1-3): 100-4.
[ 20 ] Sayanova O, Haslam R, Venegas-Caleron M, Napier JA. Identification of Primula "front-end" desaturases with distinct n-6 or n-3 substrate preferences. Planta. 2006; 224(6): 1269-77.
[ 21 ] Zhang RJ, Zhu YM, Ren L, Zhou PP, Hu JR, Yu LJ. Identification of a fatty acid a Delta(6)-desaturase gene from the eicosapentaenoic acid-producing fungus Pythium splendens RBB-5. Biotechnology Letters. 2013; 35(3): 431-8.
[ 22 ] Hong H, Datla N, Reed DW, Covello PS, MacKenzie SL, Qiu X. High-level production of gamma-linolenic acid in Brassica juncea using a Delta 6 desaturase from Pythium irregulare. Plant Physiol. 2002; 129(1): 354-62.
[ 23 ] Sakuradani E, Shimizu S. Gene cloning and functional analysis of a second Delta 6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus. Biosci Biotechnol Biochem. 2003; 67(4): 704-11.
[ 24 ] Domergue F, Lerchl J, Zahringer U, Heinz E. Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis. Eur J Biochem. 2002; 269(16): 4105-13.
[ 25 ] Sayanova O, Haslam RP, Caleron MV, Ruiz-Lopez N, Worthy C, Rooks P, et al. Identification and functional characterisation of genes encoding the omega-3 polyunsaturated fatty acid biosynthetic pathway from the coccolithophore Emiliania huxleyi. Phytochemistry. 2011; 72(7): 594-600.
[ 26 ] Sperling P, Lee M, Thomas G, Zahringer U, Stymne S, Heinz E. A bifunctional Delta(6)-fatty acyl acetylenase/desaturase from the moss Ceratodon purpureus - A new member of the cytochrome b(5) superfamily. Eur J Biochem. 2000; 267(12): 3801-11.
[ 27 ] Girke T, Schmidt H, Zahringer U, Reski R, Heinz E. Identification of a novel Delta 6-acyl-group desaturase by targeted gene disruption in Physcomitrella patens. Plant J. 1998; 15(1): 39-48.
[ 28 ] Domergue F, Abbadi A, Zahringer U, Moreau H, Heinz E. In vivo characterization of the first acyl-CoA Delta(6)-desaturase from a member of the plant kingdom, the microalga Ostreococcus tauri. Biochem J. 2005; 389: 483-90.
[ 29 ] Petrie JR, Liu Q, Mackenzie AM, Shrestha P, Mansour MP, Robert SS, et al. Isolation and Characterisation of a High-Efficiency Desaturase and Elongases from Microalgae for Transgenic LC-PUFA Production. Mar Biotechnol. 2010; 12(4): 430-8.
[ 30 ] Xie DZ, Chen F, Lin SY, Wang SQ, You CH, Monroig O, et al. Cloning, Functional Characterization and Nutritional Regulation of Delta 6 Fatty Acyl Desaturase in the Herbivorous Euryhaline Teleost Scatophagus Argus. PLoS One. 2014; 9(3): 10.
[ 31 ] Tanomman S, Ketudat-Cairns M, Jangprai A, Boonanuntanasarn S. Characterization of fatty acid delta-6 desaturase gene in Nile tilapia and heterogenous expression in Saccharomyces cerevisiae. Comp Biochem Physiol B-Biochem Mol Biol. 2013; 166(2): 148-56.
[ 32 ] Ruiz-Lopez N, Haslam RP, Venegas-Caleron M, Larson TR, Graham IA, Napier JA, et al. The synthesis and accumulation of stearidonic acid in transgenic plants: a novel source of 'heart-healthy' omega-3 fatty acids. Plant Biotechnol J. 2009; 7(7): 704-16.
[ 33 ] Abbadi A, Domergue F, Bauer J, Napier JA, Welti R, Zahringer U, et al. Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: Constraints on their accumulation. Plant Cell. 2004; 16(10): 2734-48.
[ 34 ] Napier JA, Usher S, Haslam RP, Ruiz-Lopez N, Sayanova O. Transgenic plants as a sustainable, terrestrial source of fish oils. Eur J Lipid Sci Technol. 2015; 117(9): 1317-24.
[ 35 ] Guil-Guerrero JL. Stearidonic acid (18 : 4n-3): Metabolism, nutritional importance, medical uses and natural sources. Eur J Lipid Sci Technol. 2007; 109(12): 1226-36.
[ 36 ] James MJ, Ursin VM, Cleland LG. Metabolism of stearidonic acid in human subjects: comparison with the metabolism of other n-3 fatty acids. Am J Clin Nutr. 2003; 77(5): 1140-5.
[ 37 ] Burdge GC, Calder PC. Conversion of alpha-linolenic acid to longer-chain polyunsaturated fatty acids in human adults. Reprod Nutr Dev. 2005; 45(5): 581-97.
[ 38 ] Cequier-Sanchez E, Rodriguez C, Dorta-Guerra R, Ravelo AG, Zarate R. Echium acanthocarpum hairy root cultures, a suitable system for polyunsaturated fatty acid studies and production. BMC Biotechnol. 2011; 11: 14.
[ 39 ] Zarate R, Cequier-Sanchez E, Rodriguez C, Dorta-Guerra R, El Jaber-Vazdekis N, Ravelo AG. Improvement of Polyunsaturated Fatty Acid Production in Echium acanthocarpum Transformed Hairy Root Cultures by Application of Different Abiotic Stress Conditions. ISRN biotechnology. 2013; 2013: 169510.
[ 40 ] Gamborg OL, Miller RA, Ojima K. Nutrient requirements od suspension cultures of Soybean root cells Exp Cell Res. 1968; 50(1): 151-&.
[ 41 ] Zarate R, El Jaber-Vazdekis N, Medina B, Ravelo AG. Tailoring tropane alkaloid accumulation in transgenic hairy roots of Atropa baetica by over-expressing the gene encoding hyoscyamine 6 beta-hydroxylase. Biotechnology Letters. 2006; 28(16): 1271-7.
[ 42 ] Mattanovich D, Ruker F, Machado AD, Laimer M, Regner F, Steinkellner H, et al. Efficient transformation of agrobacterium of Agrobacterium spp by electroporation Nucleic Acids Res. 1989; 17(16): 6747-.
[ 43 ] Wise AA, Liu ZY, Binns AN. Three methods for the introduction of foreign DNA into Agrobacterium. Agrobacterium Protocols, Second Edition, Vol 1. 2006; 343: 43-53.
[ 44 ] Folch J, Lees M, Stanley GHS. A simple method for the isolation and purification of total lipids from animal tissues J Biol Chem. 1957; 226(1): 497-509.
[ 45 ] Cequier-Sanchez E, Rodriguez C, Ravelo AG, Zarate R. Dichloromethane as a solvent for lipid extraction and assessment of lipid classes and fatty acids from samples of different natures. J Agric Food Chem. 2008; 56(12): 4297-303.
[ 46 ] Christie WW. The isolation of lipids from tissues. The preparation of derivates of lipids. . In: Christie WW, editor. Lipid Analysis. 17-23. Canada: Pergamon Press Cnada Ltd.; 1982. p. 55-61.
[ 47 ] Garcia-Maroto F, Manas-Fernandez A, Garrido-Cardenas JA, Alonso DL. Substrate specificity of acyl-Delta(6)-desaturases from Continental versus Macaronesian Echium species. Phytochemistry. 2006; 67(6): 540-4.
[ 48 ] Garcia-Maroto F, Manas-Fernandez A, Garrido-Cardenas JA, Alonso DL, Guil-Guerrero JL, Guzman B, et al. Delta(6)-Desaturase sequence evidence for explosive Pliocene radiations within the adaptive radiation of Macaronesian Echium (Boraginaceae). Mol Phylogenet Evol. 2009; 52(3): 563-74.
[ 49 ] Zhang M, Barg R, Yin MG, Gueta-Dahan Y, Leikin-Frenkel A, Salts Y, et al. Modulated fatty acid desaturation via overexpression of two distinct omega-3 desaturases differentially alters tolerance to various abiotic stresses in transgenic tobacco cells and plants. Plant J. 2005; 44(3): 361-71.
[ 50 ] Beremand PD, Nunberg AN, Reddy AS, Thomas TL. Production of gamma-linolenic acid by transgenic plants expressing cyanobacterial or plant Delta(6)-desaturase genes. Williams JP, Khan MU, Lem NW, editors. Dordrecht: Kluwer Academic Publ; 1997. 351-3 p.
[ 51 ] Qiu X, Hong HP, Datla N, MacKenzie SL, Taylor DC, Thomas TL. Expression of borage Delta 6 desaturase in Saccharomyces cerevisiae and oilseed crops. Can J Bot-Rev Can Bot. 2002; 80(1): 42-9.
[ 52 ] Cook D, Grierson D, Jones C, Wallace A, West G, Tucker G. Modification of fatty acid composition in tomato (Lycopersicon esculentum) by expression of a borage Delta(6)-desaturase. Mol Biotechnol. 2002; 21(2): 123-8.
[ 53 ] Sato S, Xing AQ, Ye XG, Schweiger B, Kinney A, Graef G, et al. Production of gamma-linolenic acid and stearidonic acid in seeds of marker-free transgenic soybean. Crop Sci. 2004; 44(2): 646-52.
[ 54 ] de Gyves EM, Sparks CA, Sayanova O, Lazzeri P, Napier JA, Jones HD. Genetic manipulation of gamma-linolenic acid (GLA) synthesis in a commercial variety of evening primrose (Oenothera sp.). Plant Biotechnol J. 2004; 2(4): 351-7.
[ 55 ] Chen R, Tsuda S, Matsui K, Fukuchi-Mizutani M, Ochiai M, Shimizu S, et al. Production of gamma-linolenic acid in Lotus japonicus and Vigna angularis by expression of the Delta 6-fatty-acid desaturase gene isolated from Mortierella alpina. Plant Sci. 2005; 169(3): 599-605.
[ 56 ] Guil-Guerrero JL, Gomez-Mercado F, Garcia-Maroto F, Campra-Madrid P. Occurrence and characterization of oils rich in gamma-linolenic acid - Part I: Echium seeds from Macaronesia. Phytochemistry. 2000; 53(4): 451-6.
[ 57 ] Guil-Guerrero JL, Garcı́a-Maroto F, Campra-Madrid P, Gómez-Mercado F. Occurrence and characterization of oils rich in γ-linolenic acid Part II: fatty acids and squalene from Macaronesian Echium leaves. Phytochemistry. 2000; 54(5): 525-9.
[ 58 ] Griffiths G, Stobart AK, Stymne S. Delta-6-desaturase and delta-12-desaturase activities and phosphatidic-acid formation in microsomal preparation from the developing cotyledons of common Borage ( Borago officinalis). Biochem J. 1988; 252(3): 641-7.
[ 59 ] Somerville C, Browse J. Plants Lipids - Metabolism, Mutants and Membranes Science. 1991; 252(5002): 80-7.
[ 60 ] Sakuradani E, Kobayashi M, Shimizu S. Delta 6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus - Gene cloning and its heterologous expression in a fungus, Aspergillus. Gene. 1999; 238(2): 445-53.
[ 61 ] Yang NS, Christou P. Cell type specifc expression of a CAMV 35S-GUS gene in transgenic soybean plants. Dev Genet. 1990; 11(4): 289-93.
[ 62 ] Sayanova O, Davies GM, Smith MA, Griffiths G, Stobart AK, Shewry PR, et al. Accumulation of Delta(6)-unsaturated fatty acids in transgenic tobacco plants expressing a Delta(6)-desaturase from Borago officinalis. J Exp Bot. 1999; 50(340): 1647-52.
[ 63 ] Hoffmann M, Wagner M, Abbadi A, Fulda M, Feussner I. Metabolic engineering of omega 3-very long chain polyunsaturated fatty acid production by an exclusively acyl-CoA-dependent pathway. J Biol Chem. 2008; 283(33): 22352-62.
[ 64 ] Petrie JR, Shrestha P, Mansour MP, Nichols PD, Liu Q, Singh SP. Metabolic engineering of omega-3 long-chain polyunsaturated fatty acids in plants using an acyl-CoA Δ6-desaturase with ω3-preference from the marine microalga Micromonas pusilla. Metab Eng. 2010; 12(3): 233-40.
[ 65 ] Shi HS, Chen HQ, Gu ZA, Song YD, Zhang H, Chen W, et al. Molecular mechanism of substrate specificity for delta 6 desaturase from Mortierella alpina and Micromonas pusilla. J Lipid Res. 2015; 56(12): 2309-21.
[ 66 ] Ursin VM. Modification of plant lipids for human health: Development of functional land-based omega-3 fatty acids. J Nutr. 2003; 133(12): 4271-4.
[ 67 ] Eckert H, LaVallee B, Schweiger BJ, Kinney AJ, Cahoon EB, Clemente T. Co-expression of the borage Delta(6) desaturase and the Arabidopsis Delta(15) desaturase results in high accumulation of stearidonic acid in the seeds of transgenic soybean. Planta. 2006; 224(5): 1050-7.
[ 68 ] Leonard EC. Camelina oil : Alpha-linolenic source Champaign, IL, ETATS-UNIS: American Oil Chemists' Society; 1998. 6 p.
[ 69 ] Lu CF, Kang JL. Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation. Plant Cell Reports. 2008; 27(2): 273-8.
[ 70 ] Chen G, Qu SJ, Wang Q, Bian F, Peng ZY, Zhang Y, et al. Transgenic expression of delta-6 and delta-15 fatty acid desaturases enhances omega-3 polyunsaturated fatty acid accumulation in Synechocystis sp PCC6803. Biotechnol Biofuels. 2014; 7: 10.
[ 71 ] Petrie JR, Singh SP. Expanding the docosahexaenoic acid food web for sustainable production: engineering lower plant pathways into higher plants. AoB PLANTS. 2011; 2011: plr011.
[ 72 ] Kinney A, Cahoon E, Damude H, Hitz W, Liu ZB, Kolar C. Production of very long chain polyunsaturated fatty acids in oilseed plants. Google Patents; 2004.