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Amino Acids

, Volume 46, Issue 9, pp 2177–2188 | Cite as

Expression of threonine-biosynthetic genes in mammalian cells and transgenic mice

  • Yurui Zhang
  • Zhaolai Dai
  • Guoyao Wu
  • Ran Zhang
  • Yunping Dai
  • Ning LiEmail author
Original Article

Abstract

Threonine is a nutritionally essential amino acid (EAA) for the growth and development of humans and other nonruminant animals and must be provided in diets to sustain life. The aim of this study was to synthesize threonine in mammalian cells through transgenic techniques. To achieve this goal, we combined the genes involved in bacterial threonine biosynthesis pathways into a single open reading frame separated by self-cleaving peptides (2A) and then linked it into a transposon system (piggyBac). The plasmids pEF1a-IRES-GFP-E2F-his and pEF1a-IRES-GFP-M2F-his expressed Escherichia coli homoserine kinase and threonine synthase efficiently in mouse cells and enabled cells to synthesize threonine from homoserine. This biosynthetic pathway occurred with a low level of efficiency in transgenic mice. Three transgenic mice were identified by Southern blot from 72 newborn mice, raising the possibility that a high level of expression of these genes in mouse embryos might be lethal. The results indicated that it is feasible to synthesize threonine in animal cells using genetic engineering technology. Further work is required to improve the efficiency of this method for introducing genes into mammals. We propose that the transgenic technology provides a promising means to enhance the synthesis of nutritionally EAAs in farm animals and to eliminate or reduce supplementation of these nutrients in diets for livestock, poultry and fish.

Keywords

Threonine biosynthesis Transgene Homoserine 2A peptide 

Abbreviations

EAA

Nutritionally essential amino acid

HPLC

High-performance liquid chromatography

KHB

Krebs–Henseleit bicarbonate buffer

PCR

Polymerase chain reaction

RT-PCR

Reverse transcription polymerase chain reaction

Notes

Acknowledgments

We thank Dr. Sen Wu for providing us with the pEF1a-PB plasmid and materials for cell culture. This study was supported by the National Transgenic Breeding Project of China (grant 2013ZX08006-004), Chinese Universities Scientific Funds (2012RC024), and the Thousand-People Talent program at China Agricultural University.

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Bach A, Calsamiglia S, Stern MD (2005) Nitrogen metabolism in the rumen. J Dairy Sci 88(Suppl 1):E9–E21. doi: 10.3168/jds.S0022-0302(05)73133-7 PubMedGoogle Scholar
  2. Bawden CS, Sivaprasad AV, Verma PJ, Walker SK, Rogers GE (1995) Expression of bacterial cysteine biosynthesis genes in transgenic mice and sheep: toward a new in vivo amino acid biosynthesis pathway and improved wool growth. Transgenic Res 4:87–104PubMedGoogle Scholar
  3. Bi M, Tong J, Chang F, Wang J, Wei H, Dai Y, Chu M, Zhao Y, Li N (2012) Pituitary-specific overexpression of porcine follicle-stimulating hormone leads to improvement of female fecundity in BAC transgenic mice. PLoS ONE 7:e42335. doi: 10.1371/journal.pone.0042335 PubMedCentralPubMedGoogle Scholar
  4. Bird MI, Nunn PB (1983) Metabolic homoeostasis of l-threonine in the normally-fed rat. Importance of liver threonine dehydrogenase activity. Biochem J 214:687–694PubMedCentralPubMedGoogle Scholar
  5. Chassagnole C, Rais B, Quentin E, Fell DA, Mazat JP (2001) An integrated study of threonine-pathway enzyme kinetics in Escherichia coli. Biochem J 356:415–423PubMedCentralPubMedGoogle Scholar
  6. Ciftci I, Ceylan N (2004) Effects of dietary threonine and crude protein on growth performance, carcase and meat composition of broiler chickens. Br Poult Sci 45:280–289. doi: 10.1080/00071660410001715894 PubMedGoogle Scholar
  7. Dai ZL, Li XL, Xi PB, Zhang J, Wu G, Zhu WY (2013) l-Glutamine regulates amino acid utilization by intestinal bacteria. Amino Acids 45:501–512. doi: 10.1007/s00726-012-1264-4 PubMedGoogle Scholar
  8. Dale RA (1978) Catabolism of threonine in mammals by coupling of l-threonine 3-dehydrogenase with 2-amino-3-oxobutyrate-CoA ligase. Biochim Biophys Acta 544:496–503PubMedGoogle Scholar
  9. Darling PB, Dunn M, Sarwar G, Brookes S, Ball RO, Pencharz PB (1999) Threonine kinetics in preterm infants fed their mothers’ milk or formula with various ratios of whey to casein. Am J Clin Nutr 69:105–114PubMedGoogle Scholar
  10. de Felipe P, Luke GA, Hughes LE, Gani D, Halpin C, Ryan MD (2006) E unum pluribus: multiple proteins from a self-processing polyprotein. Trends Biotechnol 24:68–75. doi: 10.1016/j.tibtech.2005.12.006 PubMedGoogle Scholar
  11. Eikmanns BJ, Eggeling L, Sahm H (1993) Molecular aspects of lysine, threonine, and isoleucine biosynthesis in Corynebacterium glutamicum. Antonie Van Leeuwenhoek 64:145–163PubMedGoogle Scholar
  12. Fenderson CL, Bergen WG (1975) An assessment of essential amino acid requirements of growing steers. J Anim Sci 41:1759–1766PubMedGoogle Scholar
  13. Golovan SP, Hayes MA, Phillips JP, Forsberg CW (2001) Transgenic mice expressing bacterial phytase as a model for phosphorus pollution control. Nat Biotechnol 19:429–433. doi: 10.1038/88091 PubMedGoogle Scholar
  14. Gum JR Jr (1992) Mucin genes and the proteins they encode: structure, diversity, and regulation. Am J Respir Cell Mol Biol 7:557–564. doi: 10.1165/ajrcmb/7.6.557 PubMedGoogle Scholar
  15. Horn NL, Donkin SS, Applegate TJ, Adeola O (2009) Intestinal mucin dynamics: response of broiler chicks and White Pekin ducklings to dietary threonine. Poult Sci 88:1906–1914. doi: 10.3382/ps.2009-00009 PubMedGoogle Scholar
  16. Katinka M, Cossart P, Sibilli L, Saint-Girons I, Chalvignac MA, Le Bras G, Cohen GN, Yaniv M (1980) Nucleotide sequence of the thrA gene of Escherichia coli. Proc Natl Acad Sci USA 77:5730–5733PubMedCentralPubMedGoogle Scholar
  17. Li P, Yin YL, Li DF, Kim SW, Wu G (2007) Amino acids and immune function. Br J Nutr 98:237–252PubMedGoogle Scholar
  18. Rees WD, Hay SM (1993) The expression of Escherichia coli threonine synthase and the production of threonine from homoserine in mouse 3T3 cells. Biochem J 291(Pt 1):315–322PubMedCentralPubMedGoogle Scholar
  19. Rees WD, Hay SM (1995) The biosynthesis of threonine by mammalian cells: expression of a complete bacterial biosynthetic pathway in an animal cell. Biochem J 309(Pt 3):999–1007PubMedCentralPubMedGoogle Scholar
  20. Rees WD, Flint HJ, Fuller MF (1990) A molecular biological approach to reducing dietary amino acid needs. Biotechnology (NY) 8:629–633Google Scholar
  21. Rees WD, Hay SM, Flint HJ (1992) Expression of Escherichia coli homoserine kinase in mouse 3T3 cells. Biochem J 281(Pt 3):865–870PubMedCentralPubMedGoogle Scholar
  22. Rees WD, Grant SD, Hay SM, Saqib KM (1994) Threonine synthesis from homoserine as a selectable marker in mammalian cells. Biochem J 299(Pt 3):637–644PubMedCentralPubMedGoogle Scholar
  23. Remond D, Buffiere C, Godin JP, Mirand PP, Obled C, Papet I, Dardevet D, Williamson G, Breuille D, Faure M (2009) Intestinal inflammation increases gastrointestinal threonine uptake and mucin synthesis in enterally fed minipigs. J Nutr 139:720–726. doi: 10.3945/jn.108.101675 PubMedGoogle Scholar
  24. Ryu JM, Han HJ (2011) l-threonine regulates G1/S phase transition of mouse embryonic stem cells via PI3K/Akt, MAPKs, and mTORC pathways. J Biol Chem 286:23667–23678. doi: 10.1074/jbc.M110.216283 PubMedCentralPubMedGoogle Scholar
  25. Saini KS, Byrne CR, Leish Z, Pruss CA, Rigby NW, Brownlee AG, Nancarrow CD, Ward KA (1996) Introduction and expression of the bacterial glyoxylate cycle genes in transgenic mice. Transgenic Res 5:467–473PubMedGoogle Scholar
  26. Shyh-Chang N, Locasale JW, Lyssiotis CA, Zheng Y, Teo RY, Ratanasirintrawoot S, Zhang J, Onder T, Unternaehrer JJ, Zhu H, Asara JM, Daley GQ, Cantley LC (2013) Influence of threonine metabolism on S-adenosylmethionine and histone methylation. Science 339:222–226. doi: 10.1126/science.1226603 PubMedCentralPubMedGoogle Scholar
  27. Stoll B, Henry J, Reeds PJ, Yu H, Jahoor F, Burrin DG (1998) Catabolism dominates the first-pass intestinal metabolism of dietary essential amino acids in milk protein-fed piglets. J Nutr 128:606–614PubMedGoogle Scholar
  28. Szymczak AL, Workman CJ, Wang Y, Vignali KM, Dilioglou S, Vanin EF, Vignali DA (2004) Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nat Biotechnol 22:589–594. doi: 10.1038/nbt957 PubMedGoogle Scholar
  29. Trichas G, Begbie J, Srinivas S (2008) Use of the viral 2A peptide for bicistronic expression in transgenic mice. BMC Biol 6:40. doi: 10.1186/1741-7007-6-40 PubMedCentralPubMedGoogle Scholar
  30. Wang X, Qiao S, Yin Y, Yue L, Wang Z, Wu G (2007) A deficiency or excess of dietary threonine reduces protein synthesis in jejunum and skeletal muscle of young pigs. J Nutr 137:1442–1446PubMedGoogle Scholar
  31. Wang J, Alexander P, Wu L, Hammer R, Cleaver O, McKnight SL (2009) Dependence of mouse embryonic stem cells on threonine catabolism. Science 325:435–439. doi: 10.1126/science.1173288 PubMedGoogle Scholar
  32. Wang WW, Zeng XF, Mao XB, Wu G, Qiao SY (2010) Optimal dietary true ileal digestible threonine for supporting mucosal barrier in the small intestine of weanling pigs. J Nutr 140:981–986PubMedGoogle Scholar
  33. Ward KA (2000) Transgene-mediated modifications to animal biochemistry. Trends Biotechnol 18:99–102PubMedGoogle Scholar
  34. Wei J, Yang X, Zheng M, Wang M, Dai Y, Chen Z, Li N (2011) The recombinant chimeric antibody chHAb18 against hepatocellular carcinoma can be produced in milk of transgenic mice. Transgenic Res 20:321–330. doi: 10.1007/s11248-010-9408-3 PubMedGoogle Scholar
  35. Wu G (2009) Amino acids: metabolism, functions, and nutrition. Amino Acids 37:1–17. doi: 10.1007/s00726-009-0269-0 PubMedGoogle Scholar
  36. Wu G (2010) Functional amino acids in growth, reproduction, and health. Adv Nutr 1:31–37. doi: 10.3945/an.110.1008 PubMedCentralPubMedGoogle Scholar
  37. Wu G (2013a) Functional amino acids in nutrition and health. Amino Acids 45:407–411. doi: 10.1007/s00726-013-1500-6 PubMedGoogle Scholar
  38. Wu G (2013b) Amino acids: biochemistry and nutrition. CRC Press, Boca RatonGoogle Scholar
  39. Wu G, Knabe DA, Flynn NE (1994) Synthesis of citrulline from glutamine in pig enterocytes. Biochem J 299(Pt 1):115–121PubMedCentralPubMedGoogle Scholar
  40. Wu G, Wu ZL, Dai ZL, Yang Y, Wang WW, Liu C, Wang B, Wang JJ, Yin YL (2013a) Dietary requirements of “nutritionally nonessential amino acids” by animals and humans. Amino Acids 44:1107–1113PubMedGoogle Scholar
  41. Wu G, Bazer FW, Satterfield MC, Li XL, Wang XQ, Johnson GA, Burghardt RC, Dai ZL, Wang JJ, Wu ZL (2013b) Impacts of arginine nutrition on embryonic and fetal development in mammals. Amino Acids 45:241–256PubMedGoogle Scholar
  42. Wu G, Bazer FW, Dai ZL, Li DF, Wang JJ, Wu ZL (2014) Amino acid nutrition in animals: protein synthesis and beyond. Annu Rev Anim Biosci 2:387–417Google Scholar

Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Yurui Zhang
    • 1
  • Zhaolai Dai
    • 2
  • Guoyao Wu
    • 2
    • 3
  • Ran Zhang
    • 1
  • Yunping Dai
    • 1
  • Ning Li
    • 1
    Email author
  1. 1.State Key Laboratory for Agrobiotechnology, College of Biological SciencesChina Agricultural UniversityBeijingChina
  2. 2.State Key Laboratory of Animal NutritionChina Agricultural UniversityBeijingChina
  3. 3.Department of Animal ScienceTexas A and M UniversityCollege stationUSA

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