Advertisement

Heterologous Expression and Characterization of Mimosinase from Leucaena leucocephala

  • Vishal Singh Negi
  • Dulal BorthakurEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1405)

Abstract

Heterologous expression of eukaryotic genes in bacterial system is an important method in synthetic biology to characterize proteins. It is a widely used method, which can be sometimes quite challenging, as a number of factors that act along the path of expression of a transgene to mRNA, and mRNA to protein, can potentially affect the expression of a transgene in a heterologous system. Here, we describe a method for successful cloning and expression of mimosinase-encoding gene from Leucaena leucocephala (leucaena) in E. coli as the heterologous host. Mimosinase is an important enzyme especially in the context of metabolic engineering of plant secondary metabolite as it catalyzes the degradation of mimosine, which is a toxic secondary metabolite found in all Leucaena and Mimosa species. We also describe the methods used for characterization of the recombinant mimosinase.

Key words

Mimosine Mimosinase Leucaena 3-Hydroxy-4-pyridone C-N lyase Aminotransferase Secondary metabolite Heterologous expression 

Notes

Acknowledgement

We thank James L Brewbaker (College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa) and Edward J. Behrman for generously providing us leucaena seeds and synthetic 3H4P, respectively. This work was supported by the National Science Foundation Award No. CBET 08-27057 and partially by a HATCH grant (HAW00551-H). VSN was supported by IFP fellowship from the Ford Foundation for 3 years.

References

  1. 1.
    Wink M (2010) Introduction: biochemistry, physiology and ecological functions of secondary metabolites. In: Wink M (ed) Biochemistry of plant secondary metabolism, vol 40, Annual plant reviews. Wiley-Blackwell, Chichester, pp 1–19CrossRefGoogle Scholar
  2. 2.
    Kroymann J (2011) Natural diversity and adaptation in plant secondary metabolism. Curr Opin Plant Biol 14:246–251CrossRefPubMedGoogle Scholar
  3. 3.
    Garcia GW, Ferguson TU, Neckles FA, Archibald KAE (1996) The nutritive value and forage productivity of Leucaena leucocephala. Anim Feed Sci Technol 60:29–41CrossRefGoogle Scholar
  4. 4.
    Soedarjo M, Borthakur D (1998) Mimosine, a toxin produced by the tree-legume leucaena provides a nodulation competition advantage to mimosine-degrading rhizobium strains. Soil Biol Biochem 30:1605–1613CrossRefGoogle Scholar
  5. 5.
    Pal A, Negi VS, Borthakur D (2012) Efficient in vitro regeneration of Leucaena leucocephala using immature zygotic embryos as explants. Agroforest Syst 84:131–140CrossRefGoogle Scholar
  6. 6.
    Lalande M (1990) A reversible arrest point in the late G1 phase of the mammalian cell cycle. Exp Cell Res 186:332–339CrossRefPubMedGoogle Scholar
  7. 7.
    Soedarjo M, Hemscheidt TK, Borthakur D (1994) Mimosine, a toxin present in leguminous trees (Leucaena spp.), induces a mimosine-de grading enzyme activity in some Rhizobium strains. Appl Environ Microbiol 60:4268–4272PubMedCentralPubMedGoogle Scholar
  8. 8.
    Negi VS, Bingham J-P, Li QX, Borthakur D (2014) A carbon-nitrogen lyase from Leucaena leucocephala catalyzes the first step of mimosine degradation. Plant Physiol 164:922–934PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Facchini PJ, Bohlmann J, Covello PS, De Luca V, Mahadevan R, Page JE, Ro D-K, Sensen CW, Storms R, Martin VJ (2012) Synthetic biosystems for the production of high-value plant metabolites. Trends Biotechnol 30:127–131CrossRefPubMedGoogle Scholar
  10. 10.
    Negi VS, Pal A, Singh R, Borthakur D (2011) Identification of species-specific genes from Leucaena leucocephala using interspecies suppression subtractive hybridisation. Ann Appl Biol 159:387–398CrossRefGoogle Scholar
  11. 11.
    Fink AL (1998) Protein aggregation: folding aggregates, inclusion bodies and amyloid. Fold Des 3:R9–R23CrossRefPubMedGoogle Scholar
  12. 12.
    Pal A, Negi VS, Khanal S, Borthakur D (2012) Immunodetection of curcin in seed meal of Jatropha curcas using polyclonal antibody developed against Curcin-L. Curr Nutr Food Sci 8:213–219CrossRefGoogle Scholar
  13. 13.
    Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–1016CrossRefPubMedGoogle Scholar
  14. 14.
    Behrman EJ (2009) Synthesis of 4-Pyridone-3-sulfate and an improved synthesis of 3-Hydroxy-4-Pyridone. Chem Cent J 3:1PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Smith IK, Fowden L (1966) A study of mimosine toxicity in plants. J Exp Bot 17:750–761CrossRefGoogle Scholar
  16. 16.
    Negi VS, Bingham J-P, Li QX, Borthakur D (2013) midD-encoded ‘rhizomimosinase’from Rhizobium sp. strain TAL1145 is a C–N lyase that catabolizes L-mimosine into 3-hydroxy-4-pyridone, pyruvate and ammonia. Amino Acids 44:1537–1547CrossRefPubMedGoogle Scholar
  17. 17.
    Curran JF, Yarus M (1989) Rates of aminoacyl-tRNA selection at 29 sense codons in vivo. J Mol Biol 209:65–77CrossRefPubMedGoogle Scholar
  18. 18.
    Welch M, Villalobos A, Gustafsson C, Minshull J (2009) You’re one in a googol: optimizing genes for protein expression. J R Soc Interface 6:S467–S476PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Richardson JP (1991) Preventing the synthesis of unused transcripts by Rho factor. Cell 64:1047–1049CrossRefPubMedGoogle Scholar
  20. 20.
    Proshkin S, Rahmouni AR, Mironov A, Nudler E (2010) Cooperation between translating ribosomes and RNA polymerase in transcription elongation. Science 328:504–508PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Molecular Biosciences and BioengineeringUniversity of Hawaii at ManoaHonoluluUSA

Personalised recommendations