SpringerLink
Log in

Stable expression of the sweet protein monellin variant MNEI in tobacco chloroplasts

  • Original Article
  • Published:
Plant Biotechnology Reports Aims and scope Submit manuscript

Abstract

Monellin is a naturally sweet protein that consists of two polypeptide chains and has potential uses as a highly potent non-carbohydrate sweetener. We aimed to make this protein more usable by increasing its stability and expressing it in a high-yielding system. MNEI is a modified version of the protein that consists of the two natural chains of monellin joined via a dipeptide linkage. In the thermostability analysis of MNEI variants, four mutated MNEIs, MNEI-E24L, MNEI-E24F, MNEI-E24W, and MNEI-E24A, had higher melting temperatures than wild-type MNEI and retained their sweet flavor even at temperatures above 70 °C. Our findings indicate that the increased stability of monellin allows it to retain its strong sweetness even under extreme conditions. We successfully overexpressed the thermostable MNEI mutants in tobacco chloroplasts. Here, we report that the MNEI mutants showed enhanced thermostability, and the stable forms of MNEI can be produced through plastid transformation in tobacco.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Chen Z, Cai H, Lu F, Du L (2005) High-level expression of a synthetic gene encoding a sweet protein, monellin, in Escherichia coli. Biotechnol Lett 27:1745–1749

    Article  PubMed  CAS  Google Scholar 

  • Cheng L, Li HP, Qu B, Huang T, Tu JX, Fu TD, Liao YC (2010) Chloroplast transformation of rapeseed (Brassica napus) by particle bombardment of cotyledons. Plant Cell Rep 29:371–381

    Article  PubMed  CAS  Google Scholar 

  • Choi WC, Kim MH, Ro HS, Ryu SR, Oh TK, Lee JK (2005) Zinc in lipase L1 from Geobacillus stearothermophilus L1 and structural implications on thermal stability. FEBS Lett 579:3461–3466

    Article  PubMed  CAS  Google Scholar 

  • Daniell H (2007) Transgene containment by maternal inheritance: effective or elusive? Proc Natl Acad Sci USA 104:6879–6880

    Article  PubMed  CAS  Google Scholar 

  • Daniell H, Datta R, Varma S, Gray S, Lee SB (1998) Containment of herbicide resistance through genetic engineering of the chloroplast genome. Nat Biotechnol 16:345–348

    Article  PubMed  CAS  Google Scholar 

  • Daniell H, Lee SB, Panchal T, Wiebe PO (2001) Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J Mol Biol 311:1001–1009

    Article  PubMed  CAS  Google Scholar 

  • Dansby R (1997) Sweet science: overexpression of monellin in yeast. Nat Biotechnol 15:419–420

    Article  PubMed  CAS  Google Scholar 

  • Dufourmantel N, Pelissier B, Garcon F, Peltier G, Ferullo JM, Tissot G (2004) Generation of fertile transplastomic soybean. Plant Mol Biol 55:479–489

    Article  PubMed  CAS  Google Scholar 

  • Edens L, Heaslinga L, Klok R, Ledeboer MNJ, Yoonen MY, Visser C, Verrips CT (1982) Cloning of cDNA encoding the sweet-tasting plant protein thaumatin and its expression in Escherichia coli. Gene 18:1–12

    Article  PubMed  CAS  Google Scholar 

  • Faus I (2000) Recent developments in the characterization and biotechnological production of sweet-tasting proteins. Appl Microbiol Biotechnol 53:145–151

    Article  PubMed  CAS  Google Scholar 

  • Ferro M, Tardif M, Reguer E, Cahuzac R, Bruley C, Vermat T, Nugues E, Vigouroux M, Vandenbrouck Y, Garin J, Viari A (2008) PepLine: a software pipeline for high-throughput direct map** of tandem mass spectrometry data on genomic sequences. J Proteome Res 7:1873–1883

    Article  PubMed  CAS  Google Scholar 

  • Hobbs JR, Munger SD, Conn GL (2007) Monellin (MNEI) at 1.15 Å resolution. Acta Crystallogr Sect F Struct Biol Cryst Commun 63:162–167

    Article  PubMed  CAS  Google Scholar 

  • Hou BK, Zhou YH, Wan LH, Zhang ZL, Shen GF, Chen ZH, Hu ZM (2003) Chloroplast transformation in oilseed rape. Transgenic Res 12:111–114

    Article  PubMed  CAS  Google Scholar 

  • Hu Z, He M (1983) Studies on mabinlin, a sweet protein from the seeds of Capparis masaikai levl. I. extraction, purification and certain characteristics. Acta Bot Yunnan 5:207–212

    CAS  Google Scholar 

  • Inglett GE, May JF (1969) Serendipity berries—source of a new intense sweetener. J Food Sci 34:408–411

    Article  CAS  Google Scholar 

  • Jha SK, Undgaonkar BJ (2009) Direct evidence for a dry molten globule intermediate during the unfolding of a small protein. Proc Natl Acad Sci USA 106:12289–12294

    Article  PubMed  CAS  Google Scholar 

  • Kanamoto H, Yamashita A, Asao H, Okumura S, Takase H, Hattori M, Yokota A, Tomizawa K (2006) Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids. Transgenic Res 15:205–217

    Article  PubMed  CAS  Google Scholar 

  • Kim SH, Kang CH, Kim R, Cho JM, Lee YB, Lee TK (1989) Redesigning a sweet protein: increased stability and renaturability. Protein Eng 2:571–575

    Article  PubMed  CAS  Google Scholar 

  • Kim HJ, Moon EJ, Moon SC, Jung HJ, Yang YL, Park YH, Heo MY, Cheon MK, Chang IS, Han DS (2007) New method of evaluation relative thermal stabilities of proteins based on their amino acid sequences; TargetStar. Int J Modern Phys C 18:1513–1526

    Article  CAS  Google Scholar 

  • Kimura T, Uzawa T, Ishimori K, Morishima I, Takahashi S, Konno T, Akiyama S, Fujisawa T (2005) Specific collapse followed by slow hydrogen-bond formation of beta-sheet in the folding of single-chain monellin. Proc Natl Acad Sci USA 102:2748–2753

    Article  PubMed  CAS  Google Scholar 

  • Kondo K, Miura Y, Sone H, Kobayashi K, Iijima H (1997) High-level expression of a sweet protein, monellin, in the food yeast Candida utilis. Nat Biotechnol 15:453–457

    Article  PubMed  CAS  Google Scholar 

  • Korkegian A, Black ME, Baker D, Stoddard BL (2005) Computational thermostabilization of an enzyme. Science 308:857–860

    Article  PubMed  CAS  Google Scholar 

  • Krishnan HB, Natarajan SS (2009) A rapid method for depletion of Rubisco from soybean (Glycine max) leaf for proteomic analysis of lower abundance proteins. Phytochemistry 70:1958–1964

    Article  PubMed  CAS  Google Scholar 

  • Kumar S, Dhingra A, Daniell H (2004) Stable transformation of the cotton plastid genome and maternal inheritance of transgenes. Plant Mol Biol 56:203–216

    Article  PubMed  CAS  Google Scholar 

  • Lee SY, Lee JH, Chang HJ, Cho JM, Jung JW, Lee W (1999) Solution structure of a sweet protein single-chain monellin determined by nuclear magnetic resonance and dynamical simulated annealing calculations. Biochemistry 38:2340–2346

    Article  PubMed  CAS  Google Scholar 

  • Lee SB, Kwon HB, Kwon SJ, Park SC, Jeong MJ, Han SE, Byun MO, Daniell H (2003) Accumulation of trehalose within transgenic chloroplasts confers drought tolerance. Mol Breed 11:1–13

    Article  CAS  Google Scholar 

  • Lee SM, Kang K, Chung H, Yoo SH, Xu XM, Lee SB, Cheong JJ, Daniell H, Kim M (2006) Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny. Mol Cells 21:401–410

    PubMed  CAS  Google Scholar 

  • Lee SB, Li B, ** S, Daniell H (2011) Expression and characterization of antimicrobial peptides Retrocyclin-101 and Protegrin-1 in chloroplasts to control viral and bacterial infections. Plant Biotechnol J 9:100–115

    Article  PubMed  CAS  Google Scholar 

  • Lelivelt CL, McCabe MS, Newell CA, Desnoo CB, van Dun KM, Birch-Machin I, Gray JC, Mills KH, Nugent JM (2005) Stable plastid transformation in lettuce (Lactuca sativa L.). Plant Mol Biol 58:763–774

    Article  PubMed  CAS  Google Scholar 

  • Morris JA, Martenson R, Deibler G, Cagan RH (1973) Characterization of monellin, a protein that tastes sweet. J Biol Chem 248:534–539

    PubMed  CAS  Google Scholar 

  • Niccolai N, Spadaccini R, Scarselli M, Bernini A, Crescenzi O, Spiga O, Ciutti A, Di Maro D, Bracci L, Dalvit C, Temussi PA (2001) Probing the surface of a sweet protein: NMR study of MNEI with a paramagnetic probe. Protein Sci 10:1498–1507

    Article  PubMed  CAS  Google Scholar 

  • Nugent GD, Ten Have M, van der Gulik A, Dix PJ, Uijtewaal BA, Mordhorst AP (2005) Plastid transformants of tomato selected using mutations affecting ribosome structure. Plant Cell Rep 24:341–349

    Article  PubMed  CAS  Google Scholar 

  • Ogata C, Hatada M, Tomlinson G, Shin WC, Kim SH (1987) Crystal structure of the intensely sweet protein monellin. Nature 328:739–742

    Article  PubMed  CAS  Google Scholar 

  • Patra AK, Udgaonkar JB (2007) Characterization of the folding and unfolding reactions of single-chain monellin: evidence for multiple intermediates and competing pathways. Biochemistry 46:11727–11743

    Article  PubMed  CAS  Google Scholar 

  • Peñarrubia L, Kim R, Giovannoni J, Kim SH, Fischer RL (1992) Production of the sweet protein monellin in transgenic plants. Nat Biotechnol 10:561–564

    Article  Google Scholar 

  • Roh KH, Shin KS, Lee YH, Seo SC, Park HG, Daniell H, Lee SB (2006) Accumulation of sweet protein monellin is regulated by the psbA 5′UTR in tobacco chloroplasts. J Plant Biol 49:34–43

    Article  CAS  Google Scholar 

  • Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chunwongse J, Obokata J, Yamaguchi-Shinozaki K, Ohto C, Torazawa K, Meng BY, Sugita M, Deno H, Kamogashira T, Yamada K, Kusuda J, Takaiwa F, Kato A, Tohdoh N, Shimada H, Sugiura M (1986) The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J 5:2043–2049

    PubMed  CAS  Google Scholar 

  • Singh ND, Li M, Lee SB, Schnell D, Daniell H (2008) Arabidopsis Tic40 expression in tobacco chloroplasts results in massive proliferation of the inner envelope membrane and upregulation of associated proteins. Plant Cell 20:3405–3417

    Article  PubMed  CAS  Google Scholar 

  • Spadaccini R, Trabucco F, Saviano G, Picone D, Crescenzi O, Tancredi T, Temussi PA (2003) The mechanism of interaction of sweet proteins with the T1R2–T1R3 receptor: evidence from the solution structure of G16A-MNEI. J Mol Biol 328:683–692

    Article  PubMed  CAS  Google Scholar 

  • Svab Z, Maliga P (2007) Exceptional transmission of plastids and mitochondria from the transplastomic pollen parent and its impact on transgene containment. Proc Natl Acad Sci USA 104:7003–7008

    Article  PubMed  CAS  Google Scholar 

  • Tancredi T, Iijima H, Saviano G, Amodeo P, Temussi PA (1992) Structural determination of the active site of a sweet protein. A 1H NMR investigation of pMNEI. FEBS Lett 310:27–30

    Article  PubMed  CAS  Google Scholar 

  • Tancredi T, Pastore A, Salvadori S, Esposito V, Temussi PA (2004) Interaction of sweet proteins with their receptor—a conformational study of peptides corresponding to loops of brazzein, monellin and thaumatin. Eur J Biochem 271:2231–2240

    Article  PubMed  CAS  Google Scholar 

  • Templeton CM, Ostovar pour S, Hobbs JR, Blanch EW, Munger SD, Conn GL (2011) Reduced sweetness of a monellin (MNEI) mutant results from increased protein flexibility and disruption of a distant poly-(l-proline) II helix. Chem Senses 36:425–434

    Article  PubMed  CAS  Google Scholar 

  • Van der Wel H, Larcon G, Hladika A, Hladik CM, Hellekant G, Glaser D (1989) Isolation and characterization of pentadin, the sweet principle of Pentadiplandra brazzeana Baillon. Chem Senses 14:75–79

    Article  Google Scholar 

  • Verma D, Samson NP, Koya V, Daniell H (2008) A protocol for expression of foreign genes in chloroplasts. Nat Protoc 3:739–758

    Article  PubMed  CAS  Google Scholar 

  • Vigues S, Dotson CD, Munger SD (2009) The receptor basis of sweet taste in mammals. Results Probl Cell Differ 47:187–202

    Article  PubMed  CAS  Google Scholar 

  • Wei-Feng X, Szczepankiewicz O, Thulin E, Linse S, Carey J (2009) Role of protein surface charge in monellin sweetness. Biochim Biophys Acta 1794:410–420

    Article  Google Scholar 

  • Yarmolinsky DA, Zuker CS, Ryba NJ (2009) Common sense about taste: from mammals to insects. Cell 139:234–244

    Article  PubMed  CAS  Google Scholar 

  • Zubkot MK, Zubkot EI, van Zuilen K, Meyer P, Day A (2004) Stable transformation of petunia plastids. Transgenic Res 13:523–530

    Article  PubMed  Google Scholar 

Download references

Acknowledgment

This study was carried out with the support of the “Research Program for Agricultural Science & Technology Development (Project No. PJ006776)”, National Academy of Agricultural Science, Rural Development Administration, Republic of Korea.

Author information

Authors and Affiliations

  1. National Academy of Agricultural Science, Rural Development Administration, Suwon, 441-707, South Korea

    Seung-Bum Lee, Jeonghwa Lee, Myoung-Ok Byun, Mi-Jeong Jeong & Shin-Chul Bae

  2. Department of Biological Substance Research, Ensoltek Co., Ltd., Daejeon, 305-510, Korea

    Yangseon Kim & Kwang-Ji Oh

Authors
  1. Seung-Bum Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yangseon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jeonghwa Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kwang-Ji Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Myoung-Ok Byun
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mi-Jeong Jeong
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shin-Chul Bae
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Seung-Bum Lee.

Additional information

S.-B. Lee and Y. Kim contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 236 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lee, SB., Kim, Y., Lee, J. et al. Stable expression of the sweet protein monellin variant MNEI in tobacco chloroplasts. Plant Biotechnol Rep 6, 285–295 (2012). https://doi.org/10.1007/s11816-012-0223-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11816-012-0223-6

Keywords

Search

Navigation