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Transgenic Research

, 18:661 | Cite as

Folate biofortification of lettuce by expression of a codon optimized chicken GTP cyclohydrolase I gene

  • Aline C. S. Nunes
  • Danielle C. Kalkmann
  • Francisco J. L. Aragão
Original Paper

Abstract

Folates are essential coenzymes involved in one-carbon metabolism. Folate deficiency is associated with a higher risk of newborns with neural tube defects, spina bifida, and anencephaly, and an increased risk of cardiovascular diseases, cancer, and impaired cognitive function in adults. In plants folates are synthesized in mitochondria from pterin precursors, which are synthesized from guanosine-5′-triphosphate (GTP) in the cytosol (pterin branch), and p-aminobenzoate (PABA), derived from chorismate in plastids (PABA branch). We generated transgenic lettuce lines expressing a synthetic codon-optimized GTP-cyclohydrolase I gene (gchI) based on native Gallus gallus gene. Immunoblotting analyses confirmed the presence of the gchI in transgenic lines. Twenty-nine transgenic lines were generated and 19 exhibited significant increase in the folate content, ranging from 2.1 to 8.5-fold higher when compared to non-transgenic lines. The folate content in enriched lettuce would provide 26% of the Dietary Reference Intakes for an adult, in a regular serving. Although the lettuce lines generated here exhibited high folate enhancement over the control, better folate enrichment could be further achieved by engineering simultaneously both PABA and pterin pathways.

Keywords

Biofortification Folate GTP cyclohydrolase I Lactuca sativa Nutrigenomics Transgenic lettuce 

Notes

Acknowledgments

We gratefully acknowledge the financial support of Embrapa and Nicolau B. da Cunha for assistance with western blot analyses. A. Nunes was supported by a fellowship from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

References

  1. Balen B, Krsnik-Rasol M (2007) N-Glycosylation of recombinant therapeutic glycoproteins in plant systems. Food Technol Biotechnol 45:1–10Google Scholar
  2. Bonfim K, Faria JC, Nogueira EOPL et al (2007) RNAi-mediated resistance to bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris). Mol Plant Microbe Interact 20:717–726. doi: 10.1094/MPMI-20-6-0717 PubMedCrossRefGoogle Scholar
  3. Cha KW, Jacobson KB, Yim JJ (1991) Isolation and characterization of GTP cyclohydrolase I from mouse liver—comparison of normal and the hph-1 mutant. J Biol Chem 266:12294–12300PubMedGoogle Scholar
  4. Cossins EA (2000) The fascinating world of folate and one-carbon metabolism. Can J Bot 78:691–708. doi: 10.1139/cjb-78-6-691 CrossRefGoogle Scholar
  5. Cossins EA, Chen L (1997) Folates and one-carbon metabolism in plant and fungi. Phytochem 45:437–452. doi: 10.1016/S0031-9422(96)00833-3 CrossRefGoogle Scholar
  6. Datla RSS et al (1991) A bifunctional fusion between b-glucuronidase and neomycin phosphotransferase: a broad-spectrum marker enzyme for plants. Gene 101:239–246. doi: 10.1016/0378-1119(91)90417-A PubMedCrossRefGoogle Scholar
  7. De La Garza RD, Quinlivan EP, Klaus SMJ et al (2004) Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis. Proc Natl Acad Sci USA 101:13720–13725. doi: 10.1073/pnas.0404208101 CrossRefGoogle Scholar
  8. De La Garza RID, Gregory III JF, Hanson AD (2007) Folate biofortification of tomato fruit. Proc Natl Acad Sci USA 104:4218–4222. doi: 10.1073/pnas.0700409104 CrossRefGoogle Scholar
  9. DeVries JW, Keagy PM, Hudson CA et al (2001) AACC collaborative study of a method for determining total folate in cereal products—microbiological assay using trienzyme extraction (AACC method 86-47). Cereal Foods World 46:216–219Google Scholar
  10. Dias BBA, Cunha WG, Vianna GR et al (2006) Expression of an oxalate decarboxylase gene from Flammulina sp. in transgenic lettuce (Lactuca sativa) plants and resistance to Sclerotinia sclerotiorum. Plant Pathol 55:187–193. doi: 10.1111/j.1365-3059.2006.01342.x CrossRefGoogle Scholar
  11. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bull 19:11–15Google Scholar
  12. Eskes TKAB (2000) From anemia to spina bifida—the story of folic acid—a tribute to Professor Richard Smithells. Eur J Obstet Gynecol Reprod Biol 90:119–123. doi: 10.1016/S0301-2115(00)00257-8 PubMedCrossRefGoogle Scholar
  13. French AE, Grant R, Weitzman S et al (2003) Folic acid food fortification is associated with a decline in neuroblastoma. Clin Pharmacol Ther 74:288–294. doi: 10.1016/S0009-9236(03)00200-5 PubMedCrossRefGoogle Scholar
  14. Gallucci M, Zanardo A, De Valentin L et al (2004) Homocysteine in Alzheimer disease and vascular dementia. Arch Gerontol Geriatr 38:195–200. doi: 10.1016/j.archger.2004.04.027 CrossRefGoogle Scholar
  15. Goyer A, Navarre DA (2007) Determination of folate concentrations in diverse potato germplasm using a trienzyme extraction and a microbiological assay. J Agric Food Chem 55:3523–3528. doi: 10.1021/jf063647x PubMedCrossRefGoogle Scholar
  16. Gupta R, Brunak S (2002) Prediction of glycosylation across the human proteome and the correlation to protein function. Pac Symp Biocomput 7:310–322Google Scholar
  17. Hall J, Solehdin F (1998) Folic acid for the prevention of congenital anomalies. Eur J Pediatr 157:445–450. doi: 10.1007/s004310050850 PubMedCrossRefGoogle Scholar
  18. Hesslinger C, Kremmer E, Hültner L et al (1998) Phosphorylation of GTP cyclohydrolase I and modulation of its activity in rodent mast cells. J Biol Chem 273:21616–21622. doi: 10.1074/jbc.273.34.21616 PubMedCrossRefGoogle Scholar
  19. Honein MA, Paulozzi LJ, Mathews TJ et al (2001) Impact of folic acid fortification of the US food supply on the occurrence of neural tube defects. JAMA 285:2981–2986. doi: 10.1001/jama.285.23.2981 PubMedCrossRefGoogle Scholar
  20. Hossain T, Rosenberg I, Selhub J et al (2004) Enhancement of folates in plants through metabolic engineering. Proc Natl Acad Sci USA 101:5158–5163. doi: 10.1073/pnas.0401342101 PubMedCrossRefGoogle Scholar
  21. Hyun TH, Tamura T (2005) Trienzyme extraction in combination with microbiologic assay in food folate analysis: an updated review. Soc Exp Biol Med 230:444–454Google Scholar
  22. Konings EJ, Roomans HH, Dorant E et al (2001) Folate intake of the Dutch population according to newly established liquid chromatography data for foods. Am J Clin Nutr 73:765–776PubMedGoogle Scholar
  23. Krishnaswamy K, Madhavan NK (2001) Importance of folate in human nutrition. Br J Nutr 85:S115–S124PubMedGoogle Scholar
  24. Lopez-Camelo JS, Orioli IM, da Graca Dutra M et al (2005) Reduction of birth prevalence rates of neural tube defects after folic acid fortification in Chile. Am J Med Genet 135A:120–125. doi: 10.1002/ajmg.a.30651 CrossRefGoogle Scholar
  25. Lucock M (2000) Folic acid: nutritional biochemistry, molecular biology, and role in disease processes. Mol Genet Metab 71:121–138. doi: 10.1006/mgme.2000.3027 PubMedCrossRefGoogle Scholar
  26. Ma JK-C, Drake PMW, Christou P (2003) The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 4:794–805. doi: 10.1038/nrg1177 PubMedCrossRefGoogle Scholar
  27. Molloy AM, Scott JM (2001) Folates and prevention of disease. Public Health Nutr 4:601–609. doi: 10.1079/PHN2001144 PubMedCrossRefGoogle Scholar
  28. Morris MC, Tangney CC (2007) Is dietary intake of folate too low? Lancet 369:166–167. doi: 10.1016/S0140-6736(07)60083-X PubMedCrossRefGoogle Scholar
  29. MRC Vitamin Study Research Group (1991) Prevention of neural tube defects: results of the medical research council vitamin study. Lancet 338:131–137. doi: 10.1016/0140-6736(91)90133-A CrossRefGoogle Scholar
  30. Pandrangi S, LaBorde LF (2004) Optimization of microbiological assay of folic acid and determination of folate content in spinach. Int J Food Sci Technol 39:525–532. doi: 10.1111/j.1365-2621.2004.00812.x CrossRefGoogle Scholar
  31. Persad VL, Van den Hof MC, Dubé JM et al (2002) Incidence of open neural tube defects in Nova Scotia after folic acid fortification. Can Med Assoc J 167:241–245Google Scholar
  32. Scott J, Rébeillé F, Fletcher J (2000) Folic acid and folates: the feasibility for nutritional enhancement in plant foods. J Sci Food Agric 80:795–804. doi: 10.1002/(SICI)1097-0010(20000515)80:7<795::AID-JSFA599>3.0.CO;2-K CrossRefGoogle Scholar
  33. Sohta Y, Ohta T, Masada M (1997) Purification and some properties of GTP cyclohydrolase I from spinach leaves. Biotechnol Biosci Biotechnol Biochem 61:1081–1085CrossRefGoogle Scholar
  34. Storozhenko S, Ravanel S, Zhang G-F et al (2005) Folate enhancement in staple crops by metabolic engineering. Trends Food Sci Technol 16:271–281. doi: 10.1016/j.tifs.2005.03.007 CrossRefGoogle Scholar
  35. Storozhenko S, De Brouwer V, Volckaert M et al (2007) Folate fortification of rice by metabolic engineering. Nat Biotechnol 25:1277–1279. doi: 10.1038/nbt1351 PubMedCrossRefGoogle Scholar
  36. Streatfield SJ (2007) Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnol J 5:2–15. doi: 10.1111/j.1467-7652.2006.00216.x PubMedCrossRefGoogle Scholar
  37. Wilson SD, Horne DW (1982) Use of glycerol-cryoprotected Lactobacillus casei for microbiological assay of folic acid. Clin Chem 28:1198–1200PubMedGoogle Scholar
  38. Yang Q, Botto LD, Erickson JD et al (2006) Improvement in stroke mortality in Canada and the United States, 1990 to 2002. Circulation 113:1335–1343. doi: 10.1161/CIRCULATIONAHA.105.570846 PubMedCrossRefGoogle Scholar
  39. Yoneyama T, Hatakeyama K (1998) Decameric GTP cyclohydrolase I forms complexes with two pentameric GTP cyclohydrolase I feedback regulatory proteins in the presence of phenylalanine or of a combination of tetrahydrobiopterin and GTP. J Biol Chem 273:20102–20108. doi: 10.1074/jbc.273.32.20102 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Aline C. S. Nunes
    • 1
    • 2
  • Danielle C. Kalkmann
    • 1
  • Francisco J. L. Aragão
    • 1
  1. 1.Embrapa Recursos Genéticos e Biotecnologia, PqEB W5 NorteBrasíliaBrazil
  2. 2.Departamento de Biologia CelularUniversidade de BrasíliaBrasíliaBrazil

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