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Marine Biotechnology

, Volume 21, Issue 2, pp 171–185 | Cite as

A Novel Dietary Source of EPA and DHA: Metabolic Engineering of an Important Freshwater Species—Common Carp by fat1-Transgenesis

  • Xiaofan Zhang
  • Shaochen Pang
  • Chengjie Liu
  • Houpeng Wang
  • Ding Ye
  • Zuoyan Zhu
  • Yonghua SunEmail author
Original Article

Abstract

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), are essential for neural development and human health. The n-3 PUFAs are mainly obtained from marine fish by dietary intake. Freshwater fish species usually contain low level of n-3 PUFAs due to the lack of n-3 PUFAs in their food chain. In this study, we report on the substantial production of EPA and DHA in a globally important freshwater fish species, common carp (Cyprinus carpio). This was achieved by introducing an “all-fish” transgene CA:fat1 containing the fish codon-optimized omega-3 desaturase gene (fat1) driven by the common carp β-actin promoter (CA). Through a sperm sample screening method, we successfully generated fat1-positive F1 transgenic population with high efficiency. In F1 population, the muscle contents of ALA (18:3n-3), EPA and DHA were significantly increased when compared with non-transgenic siblings. Thereafter, four independent F2 heterozygous lines were obtained from 4 F1 transgenic males and a detailed comparison of fatty acids profile and growth performance was carried out for these 4 lines. All fat1-transgenic common carps from 4 lines showed an evident decrease in n-6 PUFA contents and a substantial increase in n-3 PUFA contents, among which line 4 stands out, showing a statistically significant increase in all 4 types of n-3 PUFAs including ALA (4.4-fold increase, p < 0.001), EPA (4.8-fold increase, p < 0.01), C22:5n-3 (DPA, 2.4-fold increase, p < 0.05), and DHA (1.9-fold increase, p < 0.05). Therefore, the line 4 was selected as the optimized breeding stock for further study, and the proximate nutrition composition and PUFA synthesis pathway were analyzed. Our study demonstrates that in the transgenic group, the muscular lipid content did not change, while fat accumulations in the internal organs and especially in the liver were significantly decreased as a result of hyperactivation of fatty acid oxidation process. Finally, we conclude that the “all-fish” CA:fat1-transgenic freshwater fish—common carp—can serve as a novel healthy dietary source of n3-PUFAs, especially EPA and DHA.

Keywords

Omega-3 fatty acids Omega-3 desaturase Gene transfer Common carp Transcriptional regulation 

Notes

Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant nos. 31721005, 31222052 & 31671501), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no: XDA08010106), the State Key Laboratory of Freshwater Ecology and Biotechnology (grant no: 2016FBZ03) and the Youth Innovation Promotion Association of CAS. We thank Yongming Li for his help in fish culture.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflicts of interest.

Supplementary material

10126_2018_9868_MOESM1_ESM.docx (339 kb)
ESM 1 (DOCX 338 kb)

References

  1. Agaba M, Tocher DR, Dickson CA, Dick JR, Teale AJ (2004) Zebrafish cDNA encoding multifunctional fatty acid elongase involved in production of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids. Mar Biotechnol 6:251–261CrossRefGoogle Scholar
  2. Calon F, Cole G (2007) Neuroprotective action of omega-3 polyunsaturated fatty acids against neurodegenerative diseases: evidence from animal studies. Prostaglandins Leukot Essent Fat Acids 77:287–293CrossRefGoogle Scholar
  3. Chatzifotis S, Takeuchi T, Seikai T (1996) The effect of dietary carnitine supplementation on growth of red sea bream (Pagrus major) fingerlings at two levels of dietary lysine. Aquaculture 147:235–248CrossRefGoogle Scholar
  4. Cheng Q, Su B, Qin Z, Weng CC, Yin F, Zhou Y, Fobes M, Perera DA, Shang M, Soller F, Shi Z, Davis A, Dunham RA (2014) Interaction of diet and the masou salmon Delta5-desaturase transgene on Delta6-desaturase and stearoyl-CoA desaturase gene expression and N-3 fatty acid level in common carp (Cyprinus carpio). Transgenic Res 23:729–742CrossRefGoogle Scholar
  5. Cheng G, Fu C, Wang H, Adoligbe C, Wei S, Li S, Jiang B, Wang H, Zan L (2015) Production of transgenic beef cattle rich in n-3 PUFAs by somatic cell nuclear transfer. Biotechnol Lett 37:1565–1571CrossRefGoogle Scholar
  6. Chomczynski P, Mackey K (1995) Short technical reports. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide-and proteoglycan-rich sources. BioTechniques 19:942–945Google Scholar
  7. Chow CK (2007) Fatty acids in foods and their health implications. CRC Press, Boca RatonCrossRefGoogle Scholar
  8. Dong Z, Nguyen NH, Zhu W (2015) Genetic evaluation of a selective breeding program for common carp Cyprinus carpio conducted from 2004 to 2014. BMC Genet 16:94CrossRefGoogle Scholar
  9. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226:497–509Google Scholar
  10. Henderson RJ, Tocher DR (1987) The lipid composition and biochemistry of freshwater fish. Prog Lipid Res 26:281–347CrossRefGoogle Scholar
  11. Horwitz W, Chichilo P, Reynolds H (1970) Official methods of analysis of the Association of Official Analytical Chemists. Association of Official Analytical Chemists, Washington, DCGoogle Scholar
  12. Janssen CI, Kiliaan AJ (2014) Long-chain polyunsaturated fatty acids (LCPUFA) from genesis to senescence: the influence of LCPUFA on neural development, aging, and neurodegeneration. Prog Lipid Res 53:1–17CrossRefGoogle Scholar
  13. Jia G, Feng J, Qin Z (2006) Studies on the fatty liver diseases of Sciaenops ocellatus caused by different ether extract levels in diets. Front Biol China 1:9–12CrossRefGoogle Scholar
  14. Jump DB (2008) N-3 polyunsaturated fatty acid regulation of hepatic gene transcription. Curr Opin Lipidol 19:242–247CrossRefGoogle Scholar
  15. Jump DB, Botolin D, Wang Y, Xu J, Christian B, Demeure O (2005) Fatty acid regulation of hepatic gene transcription. J Nutr 135:2503–2506CrossRefGoogle Scholar
  16. Kang JX, Wang J, Wu L, Kang ZB (2004) Transgenic mice: Fat-1 mice convert n-6 to n-3 fatty acids. Nature 427:504CrossRefGoogle Scholar
  17. Kidd PM (2007) Omega-3 DHA and EPA for cognition, behavior, and mood: clinical findings and structural-functional synergies with cell membrane phospholipids. Altern Med Rev 12:207–227Google Scholar
  18. Kris-Etherton PM, Harris WS, Appel LJ, American Heart Association. Nutrition, C (2002) Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation 106:2747–2757CrossRefGoogle Scholar
  19. Lai L, Kang JX, Li R, Wang J, Witt WT, Yong HY, Hao Y, Wax DM, Murphy CN, Rieke A, Samuel M, Linville ML, Korte SW, Evans RW, Starzl TE, Prather RS, Dai Y (2006) Generation of cloned transgenic pigs rich in omega-3 fatty acids. Nat Biotechnol 24:435–436CrossRefGoogle Scholar
  20. Leaver MJ, Bautista JM, Bjornsson BT, Jonsson E, Krey G, Tocher DR, Torstensen BE (2008) Towards fish lipid nutrigenomics: current state and prospects for fin-fish aquaculture. Rev Fish Sci 16:73–94CrossRefGoogle Scholar
  21. Ledford H (2015) Transgenic salmon leaps to the dinner table: long-awaited decision by US government authorizes the first genetically engineered animal to be sold as food. Nature 527:417–419CrossRefGoogle Scholar
  22. Lee JM, Lee H, Kang S, Park WJ (2016) Fatty acid desaturases, polyunsaturated fatty acid regulation, and biotechnological advances. Nutrients 8:23CrossRefGoogle Scholar
  23. Liang XF, Ogata HY, Oku H (2002) Effect of dietary fatty acids on lipoprotein lipase gene expression in the liver and visceral adipose tissue of fed and starved red sea bream Pagrus major. Comp Biochem Physiol A Mol Integr Physiol 132:913–919  https://doi.org/10.1016/S1095-6433(02)00118-6 CrossRefGoogle Scholar
  24. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408CrossRefGoogle Scholar
  25. Maclean CH, Newberry SJ, Mojica WA, Khanna P, Issa AM, Suttorp MJ, Lim YW, Traina SB, Hilton L, Garland R, Morton SC (2006) Effects of omega-3 fatty acids on cancer risk: a systematic review. JAMA 295:403–415CrossRefGoogle Scholar
  26. Mehlem A, Hagberg CE, Muhl L, Eriksson U, Falkevall A (2013) Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease. Nat Protoc 8:1149–1154CrossRefGoogle Scholar
  27. Pan D, Zhang L, Zhou Y, Feng C, Long C, Liu X, Wan R, Zhang J, Lin A, Dong E, Wang S, Xu H, Chen H (2010) Efficient production of omega-3 fatty acid desaturase (sFat-1)-transgenic pigs by somatic cell nuclear transfer. Sci China Life Sci 53:517–523CrossRefGoogle Scholar
  28. Pang SC, Wang HP, Li KY, Zhu ZY, Kang JX, Sun YH (2014) Double transgenesis of humanized fat1 and fat2 genes promotes omega-3 polyunsaturated fatty acids synthesis in a zebrafish model. Mar Biotechnol 16:580–593CrossRefGoogle Scholar
  29. Pang SC, Wang HP, Zhu ZY, Sun YH (2015) Transcriptional activity and DNA methylation dynamics of the Gal4/UAS system in zebrafish. Mar Biotechnol (NY) 17:593–603CrossRefGoogle Scholar
  30. Prchal M, Kause A, Vandeputte M, Gela D, Allamellou J-M, Kumar G, Bestin A, Bugeon J, Zhao J, Kocour M (2018) The genetics of overwintering performance in two-year old common carp and its relation to performance until market size. PLoS One 13:e0191624CrossRefGoogle Scholar
  31. Rasmussen RS (2001) Quality of farmed salmonids with emphasis on proximate composition, yield and sensory characteristics. Aquac Res 32:767–786CrossRefGoogle Scholar
  32. Reddy JK, Rao MS (2006) Lipid metabolism and liver inflammation. II. Fatty liver disease and fatty acid oxidation. Am J Physiol Gastrointest Liver Physiol 290:G852–G858CrossRefGoogle Scholar
  33. Simopoulos AP (2002a) The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomed Pharmacother 56:365–379CrossRefGoogle Scholar
  34. Simopoulos AP (2002b) Omega-3 fatty acids in inflammation and autoimmune diseases. J Am Coll Nutr 21:495–505CrossRefGoogle Scholar
  35. Simopoulos AP (2008) The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases. Exp Biol Med 233:674–688CrossRefGoogle Scholar
  36. Spiller GA (1995) Handbook of lipids in human nutrition. CRC Press, Boca RatonGoogle Scholar
  37. Steffens W (1997) Effects of variation in essential fatty acids in fish feeds on nutritive value of freshwater fish for humans. Aquaculture 151:97–119CrossRefGoogle Scholar
  38. Swanson D, Block R, Mousa SA (2012) Omega-3 fatty acids EPA and DHA: health benefits throughout life. Adv Nutr 3:1–7CrossRefGoogle Scholar
  39. Turchini GM, Torstensen BE, Ng WK (2009) Fish oil replacement in finfish nutrition. Rev Aquac 1:10–57CrossRefGoogle Scholar
  40. Venegas-Caleron M, Sayanova O, Napier JA (2010) An alternative to fish oils: metabolic engineering of oil-seed crops to produce omega-3 long chain polyunsaturated fatty acids. Prog Lipid Res 49:108–119CrossRefGoogle Scholar
  41. Whelan J, Rust C (2006) Innovative dietary sources of n-3 fatty acids. Annu Rev Nutr 26:75–103CrossRefGoogle Scholar
  42. Xu P, Zhang X, Wang X, Li J, Liu G, Kuang Y, Xu J, Zheng X, Ren L, Wang G, Zhang Y, Huo L, Zhao Z, Cao D, Lu C, Li C, Zhou Y, Liu Z, Fan Z, Shan G, Li X, Wu S, Song L, Hou G, Jiang Y, Jeney Z, Yu D, Wang L, Shao C, Song L, Sun J, Ji P, Wang J, Li Q, Xu L, Sun F, Feng J, Wang C, Wang S, Wang B, Li Y, Zhu Y, Xue W, Zhao L, Wang J, Gu Y, Lv W, Wu K, Xiao J, Wu J, Zhang Z, Yu J, Sun X (2014) Genome sequence and genetic diversity of the common carp, Cyprinus carpio. Nat Genet 46:1212–1219CrossRefGoogle Scholar
  43. Yan Y, Jiang W, Spinetti T, Tardivel A, Castillo R, Bourquin C, Guarda G, Tian Z, Tschopp J, Zhou R (2013) Omega-3 fatty acids prevent inflammation and metabolic disorder through inhibition of NLRP3 inflammasome activation. Immunity 38:1154–1163CrossRefGoogle Scholar
  44. Zhang P, Zhang Y, Dou H, Yin J, Chen Y, Pang X, Vajta G, Bolund L, Du Y, Ma RZ (2012) Handmade cloned transgenic piglets expressing the nematode fat-1 gene. Cellular Reprogram 14:258–266CrossRefGoogle Scholar
  45. Zhang P, Liu P, Dou H, Chen L, Chen L, Lin L, Tan P, Vajta G, Gao J, Du Y, Ma RZ (2013) Handmade cloned transgenic sheep rich in omega-3 fatty acids. PLoS One 8:e55941CrossRefGoogle Scholar
  46. Zhang L, Gozlan RE, Li Z, Liu J, Zhang T, Hu W, Zhu Z (2014) Rapid growth increases intrinsic predation risk in genetically modified Cyprinus carpio: implications for environmental risk. J Fish Biol 84:1527–1538CrossRefGoogle Scholar
  47. Zhong S, Wang YP, Pei DS, Luo DJ, Liao LJ, Zhu ZY (2009) A one-year investigation of the relationship between serum GH levels and the growth of F(4) transgenic and non-transgenic common carp Cyprinus carpio. J Fish Biol 75:1092–1100CrossRefGoogle Scholar
  48. Zhong CR, Song YL, Wang YP, Li YM, Liao LJ, Xie SQ, Zhu ZY, Hu W (2012) Growth hormone transgene effects on growth performance are inconsistent among offspring derived from different homozygous transgenic common carp (Cyprinus carpio L.). Aquaculture 356:404–411CrossRefGoogle Scholar
  49. Zhu Z (1993) Growth hormone gene and the transgenic fish. In: You C, Chen Z, Ding Y (eds) Biotechnology in agriculture. Springer, NetherlandsGoogle Scholar
  50. Zhu T, Zhang T, Wang Y, Chen Y, Hu W, Zhu Z (2013) Effects of growth hormone (GH) transgene and nutrition on growth and bone development in common carp. J Exp Zool A Ecol Genet Physiol 319:451–460CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of HydrobiologyChinese Academy of SciencesWuhanChina
  2. 2.College of Advanced Agricultural SciencesUniversity of Chinese Academy of SciencesBeijingChina
  3. 3.Institute of Environment and HealthJianghan UniversityWuhanChina

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