, Volume 248, Issue 2, pp 465–476 | Cite as

High-level production of single chain monellin mutants with enhanced sweetness and stability in tobacco chloroplasts

  • Daniela Castiglia
  • Serena Leone
  • Rachele Tamburino
  • Lorenza Sannino
  • Jole Fonderico
  • Chiara Melchiorre
  • Andrea Carpentieri
  • Stefania Grillo
  • Delia Picone
  • Nunzia ScottiEmail author
Original Article


Main conclusion

Plastid-based MNEI protein mutants retain the structure, stability and sweetness of their bacterial counterparts, confirming the attractiveness of the plastid transformation technology for high-yield production of recombinant proteins.

The prevalence of obesity and diabetes has dramatically increased the industrial demand for the development and use of alternatives to sugar and traditional sweeteners. Sweet proteins, such as MNEI, a single chain derivative of monellin, are the most promising candidates for industrial applications. In this work, we describe the use of tobacco chloroplasts as a stable plant expression platform to produce three MNEI protein mutants with improved taste profile and stability. All plant-based proteins were correctly expressed in tobacco chloroplasts, purified and subjected to in-depth chemical and sensory analyses. Recombinant MNEI mutants showed a protein yield ranging from 5% to more than 50% of total soluble proteins, which, to date, represents the highest accumulation level of MNEI mutants in plants. Comparative analyses demonstrated the high similarity, in terms of structure, stability and function, of the proteins produced in plant chloroplasts and bacteria. The high yield and the extreme sweetness perceived for the plant-derived proteins prove that plastid transformation technology is a safe, stable and cost-effective production platform for low-calorie sweeteners, with an estimated production of up to 25–30 mg of pure protein/plant.


Green factory Plastid transformation Nicotiana tabacum Sweet proteins Protein structure Low-calorie sweeteners 



This work was partially funded by Fondazione con il Sud, Project 2011-PDR-19, and POR Campania FESR207-2013, Project “Bio Industrial Processes (BIP)”. Technical assistance of Mr. G. Guarino, Ms. S. Arcari and Mr. R. Nocerino (CNR-IBBR, Portici, Italy) with artworks and plant growth is gratefully acknowledged. The authors declare that they have no conflict of interests.

Supplementary material

425_2018_2920_MOESM1_ESM.docx (4.9 mb)
Supplementary material 1 (DOCX 5020 kb)


  1. Apel W, Schulze WX, Bock R (2010) Identification of protein stability determinants in chloroplasts. Plant J 63:636–650CrossRefPubMedPubMedCentralGoogle Scholar
  2. Boyhan D, Daniell H (2011) Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C-peptide. Plant Biotechnol J 9:585–598CrossRefPubMedGoogle Scholar
  3. Cai C, Li L, Lu N, Zheng W, Yang L, Liu B (2016) Expression of a high sweetness and heat-resistant mutant of sweet-tasting protein, monellin, in Pichia pastoris with a constitutive GAPDH promoter and modified N-terminus. Biotechnol Lett 38:1941–1946CrossRefPubMedGoogle Scholar
  4. Castiglia D, Sannino L, Marcolongo L, Ionata E, Tamburino R, De Stradis A, Cobucci-Ponzano B, Moracci M, La Cara F, Scotti N (2016) High-level expression of thermostable cellulolytic enzymes in tobacco transplastomic plants and their use in hydrolysis of an industrially pretreated Arundo donax L. biomass. Biotechnol Biofuels 9:154CrossRefPubMedPubMedCentralGoogle Scholar
  5. Chen Z, Heng C, Li Z, Liang X, Xinchen S (2007) Expression and secretion of a single-chain sweet protein monellin in Bacillus subtilis by sacB promoter and signal peptide. Appl Microbiol Biotechnol 73:1377–1381CrossRefPubMedGoogle Scholar
  6. Chen Z, Li Z, Yu N, Yan L (2011) Expression and secretion of a single-chain sweet protein, monellin, in Saccharomyces cerevisiae by an α-factor signal peptide. Biotechnol Lett 33:721–725CrossRefPubMedGoogle Scholar
  7. Cui M, Jiang P, Maillet E, Max M, Margolskee RF, Osman R (2006) The heterodimeric sweet taste receptor has multiple potential ligand binding sites. Curr Pharm Des 12:4591–4600CrossRefPubMedGoogle Scholar
  8. De Lorenzo C, Cozzolino R, Carpentieri A, Pucci P, Laccetti P, D’Alessio G (2005) Biological properties of a human compact anti-ErbB2 antibody. Carcinogenesis 26:1890–1895CrossRefPubMedGoogle Scholar
  9. Demain AL, Vaishnav P (2009) Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv 27:297–306CrossRefPubMedGoogle Scholar
  10. Di Monaco R, Miele NA, Picone D, Masi P, Cavella S (2013) Taste detection and recognition thresholds of the modified monellin sweetener: MNEI. J Sens Stud 28:25–33CrossRefGoogle Scholar
  11. Di Monaco R, Miele NA, Volpe S, Picone D, Cavella S (2014) Temporal sweetness profile of MNEI and comparison with commercial sweeteners. J Sens Stud 29:385–394CrossRefGoogle Scholar
  12. Esposito V, Gallucci R, Picone D, Saviano G, Tancredi T, Temussi PA (2006) The importance of electrostatic potential in the interaction of sweet proteins with the sweet taste receptor. J Mol Biol 360:448–456CrossRefPubMedGoogle Scholar
  13. Giglione C, Vallon O, Meinnel T (2003) Control of protein life-span by N-terminal methionine excision. EMBO J 22:13–23CrossRefPubMedPubMedCentralGoogle Scholar
  14. Giglione C, Boularot A, Meinnel T (2004) Protein N-terminal methionine excision. Cell Mol Life Sci 61:1455–1474CrossRefPubMedGoogle Scholar
  15. Higginbotham JD, Snodin DJ, Eaton KK, Daniel JW (1983) Safety evaluation of thaumatin (Talin protein). Food Chem Toxicol Int J Publ Br Ind Biol Res Assoc 21:815–823CrossRefGoogle Scholar
  16. Hirai T, Kurokawa N, Duhita N, Hiwasa-Tanase K, Kato K, Kato K, Ezura H (2011) The HSP terminator of Arabidopsis thaliana induces a high level of miraculin accumulation in transgenic tomatoes. J Agric Food Chem 59:9942–9949CrossRefPubMedGoogle Scholar
  17. Hiwasa-Tanase K, Hirai T, Kato K, Duhita N, Ezura H (2012) From miracle fruit to transgenic tomato: mass production of the taste-modifying protein miraculin in transgenic plants. Plant Cell Rep 31:513–525CrossRefPubMedGoogle Scholar
  18. Hobbs JR, Munger SD, Conn GL (2007) Monellin (MNEI) at 1.15 Å resolution. Acta Crystallogr, Sect F Struct Biol Cryst Commun 63:162–167CrossRefGoogle Scholar
  19. Inglett GE, May JF (1969) Serendipity berries–source of a new intense sweetener. J Food Sci 34:408–411CrossRefGoogle Scholar
  20. Kant R (2005) Sweet proteins-Potential replacement for artificial low calorie sweeteners. Nutr J 4:5CrossRefPubMedPubMedCentralGoogle Scholar
  21. Kaul T, Reddy CS, Pandey S (2018) Transgenics with monellin. In: Mérillon JM, Ramawat KG (eds) Sweeteners. Pharmacology, biotechnology and applications. Springer, Cham, pp 211–222Google Scholar
  22. Kim IH, Lim KJ (1996) Large-scale purification of recombinant monellin from yeast. J Ferment Bioeng 82:180–182CrossRefGoogle Scholar
  23. Kim SH, Kang CH, Kim R, Cho JM, Lee YB, Lee TK (1989) Redesigning a sweet protein: increased stability and renaturability. Protein Eng Des Sel 2:571–575CrossRefGoogle Scholar
  24. Kuroda H, Maliga P (2001a) Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs. Nucleic Acids Res 29:970–975CrossRefPubMedPubMedCentralGoogle Scholar
  25. Kuroda H, Maliga P (2001b) Sequences downstream of the translation initiation codon are important determinants of translation efficiency in chloroplasts. Plant Physiol 125:430–436CrossRefPubMedPubMedCentralGoogle Scholar
  26. Lamphear BJ, Barker DK, Brooks CA, Delaney DE, Lane JR, Beifuss K, Love R, Thompson K, Mayor J, Clough R, Harkey R, Poage M, Drees C, Horn ME, Streatfield SJ, Nikolov Z, Woodard SL, Hood EE, Jilka JM, Howard JA (2005) Expression of the sweet protein brazzein in maize for production of a new commercial sweetener. Plant Biotechnol J 3:103–114CrossRefPubMedGoogle Scholar
  27. Lee S-B, Kim Y, Lee J, Oh K-J, Byun M-O, Jeong M-J, Bae S-C (2012) Stable expression of the sweet protein monellin variant MNEI in tobacco chloroplasts. Plant Biotechnol Rep 6:285–295CrossRefGoogle Scholar
  28. Lentz EM, Segretin ME, Morgenfeld MM, Wirth SA, Dus Santos MJ, Mozgovoj MV, Wigdorovitz A, Bravo-Almonacid FF (2010) High expression level of a foot and mouth disease virus epitope in tobacco transplastomic plants. Planta 231:387–395CrossRefPubMedGoogle Scholar
  29. Lenzi P, Scotti N, Alagna F, Tornesello ML, Pompa A, Vitale A, De Stradis A, Monti L, Grillo S, Buonaguro FM, Maliga P, Cardi T (2008) Translational fusion of chloroplast-expressed human papillomavirus type 16 L1 capsid protein enhances antigen accumulation in transplastomic tobacco. Transgenic Res 17:1091–1102CrossRefPubMedGoogle Scholar
  30. Leone S, Sannino F, Tutino ML, Parrilli E, Picone D (2015) Acetate: friend or foe? Efficient production of a sweet protein in Escherichia coli BL21 using acetate as a carbon source. Microb Cell Fact 14:106CrossRefPubMedPubMedCentralGoogle Scholar
  31. Leone S, Pica A, Merlino A, Sannino F, Temussi PA, Picone D (2016) Sweeter and stronger: enhancing sweetness and stability of the single chain monellin MNEI through molecular design. Sci Rep 6:34045CrossRefPubMedPubMedCentralGoogle Scholar
  32. Leone S, Picone D (2016) Molecular dynamics driven design of pH-stabilized mutants of MNEI, a sweet protein. PLoS One 11:e0158372CrossRefPubMedPubMedCentralGoogle Scholar
  33. Liu Q, Li L, Yang L, Liu T, Cai C, Liu B (2016) Modification of the sweetness and stability of sweet-tasting protein monellin by gene mutation and protein engineering. Biomed Res Int. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Lomonossoff GP, D’Aoust MA (2016) Plant-produced biopharmaceuticals: a case of technical developments driving clinical deployment. Science 16:1237–1240CrossRefGoogle Scholar
  35. Masuda T, Kitabatake N (2006) Developments in biotechnological production of sweet proteins. J Biosci Bioeng 102:375–389CrossRefPubMedGoogle Scholar
  36. Masuda T, Ohta K, Ojiro N, Murata K, Mikami B, Tani F, Temussi PA, Kitabatake N (2016) A hypersweet protein: removal of the specific negative charge at Asp21 enhances thaumatin sweetness. Sci Rep 6:20255CrossRefPubMedPubMedCentralGoogle Scholar
  37. Merlin M, Gecchele E, Capaldi S, Pezzotti M, Avesani L (2014) Comparative evaluation of recombinant protein production in different biofactories: the green perspective. Biomed Res Int. PubMedPubMedCentralCrossRefGoogle Scholar
  38. Miele NA, Cabisidan EK, Blaiotta G, Leone S, Masi P, Monaco RD, Cavella S (2017a) Rheological and sensory performance of a protein-based sweetener (MNEI), sucrose, and aspartame in yogurt. J Dairy Sci 100:9539–9550CrossRefPubMedGoogle Scholar
  39. Miele NA, Di Monaco R, Dell’Amura F, Rega MF, Picone D, Cavella S (2017b) A preliminary study on the application of natural sweet proteins in agar-based gels. J Texture Stud 48:103–113CrossRefPubMedGoogle Scholar
  40. Morini G, Bassoli A, Temussi PA (2005) From small sweeteners to sweet proteins: anatomy of the binding sites of the human T1R2_T1R3 receptor. J Med Chem 48:5520–5529CrossRefPubMedGoogle Scholar
  41. Morris JA, Cagan RH (1972) Purification of monellin, the sweet principle of Dioscoreophyllum cumminsii. Biochim Biophys Acta 261:114–122CrossRefPubMedGoogle Scholar
  42. Moustafa K, Makhzoum A, Trémouillaux-Guiller J (2016) Molecular farming on rescue of pharma industry for next generations. Crit Rev Biotechnol 36:840–850CrossRefPubMedGoogle Scholar
  43. Murzin AG (1993) Sweet-tasting protein Monellin is related to the cystatin family of thiol proteinase inhibitors. J Mol Biol 230:689–694CrossRefPubMedGoogle Scholar
  44. Oey M, Lohse M, Kreikemeyer B, Bock R (2009) Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic. Plant J 57:436–445CrossRefPubMedGoogle Scholar
  45. Ogata C, Hatada M, Tomlinson G, Shin W-C, Kim S-H (1987) Crystal structure of the intensely sweet protein monellin. Nature 328:739–742CrossRefPubMedGoogle Scholar
  46. Peñarrubia L, Kim R, Giovannoni J, Kim S-H, Fischer RL (1992) Production of the sweet protein monellin in transgenic plants. Nat Biotechnol 10:561–564CrossRefGoogle Scholar
  47. Petersen K, Bock R (2011) High-level expression of a suite of thermostable cell wall-degrading enzymes from the chloroplast genome. Plant Mol Biol 76:311–321CrossRefPubMedGoogle Scholar
  48. Pham NB, Schäfer H, Wink M (2012) Production and secretion of recombinant thaumatin in tobacco hairy root cultures. Biotechnol J 7:537–545CrossRefPubMedGoogle Scholar
  49. Pica A, Leone S, Di Girolamo R, Donnarumma F, Emendato A, Rega MF, Merlino A, Picone D (2018) pH driven fibrillar aggregation of the super-sweet protein Y65R-MNEI: a step-by-step structural analysis. Biochim Biophys Acta 1862:808–815CrossRefPubMedGoogle Scholar
  50. Picone D, Temussi PA (2012) Dissimilar sweet proteins from plants: oddities or normal components? Plant Sci 195:135–142CrossRefPubMedGoogle Scholar
  51. Reddy CS, Vijayalakshmi M, Kaul T, Islam T, Reddy MK (2015) Improving flavour and quality of tomatoes by expression of synthetic gene encoding sweet protein monellin. Mol Biotechnol 57:448–453CrossRefPubMedGoogle Scholar
  52. Rega MF, Di Monaco R, Leone S, Donnarumma F, Spadaccini R, Cavella S, Picone D (2015) Design of sweet protein based sweeteners: hints from structure-function relationships. Food Chem 173:1179–1186CrossRefPubMedGoogle Scholar
  53. Rega MF, Siciliano A, Gesuele R, Lofrano G, Carpentieri A, Picone D, Guida M (2017) Ecotoxicological survey of MNEI and Y65R-MNEI proteins as new potential high-intensity sweeteners. Environ Sci Pollut Res 24:9734–9740CrossRefGoogle Scholar
  54. Roh KH, Shin K-S, Lee Y-H, Seo S-C, Park H-G, Daniell H, Lee S-B (2006) Accumulation of sweet protein monellin is regulated by the psbA 5′UTR in tobacco chloroplasts. J Plant Biol 49:34–43CrossRefGoogle Scholar
  55. Sanchez-Garcia L, Martín L, Mangues R, Ferrer-Miralles N, Vázquez E, Villaverde A (2016) Recombinant pharmaceuticals from microbial cells: a 2015 update. Microb Cell Fact 15:33CrossRefPubMedPubMedCentralGoogle Scholar
  56. Scotti N, Alagna F, Ferraiolo E, Formisano G, Sannino L, Buonaguro L, De Stradis A, Vitale A, Monti L, Grillo S, Buonaguro FM, Cardi T (2009) High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts. Planta 229:1109–1122CrossRefPubMedGoogle Scholar
  57. Scotti N, Cardi T (2012) Plastid transformation as an expression tool for plant-derived biopharmaceuticals. In: Dunwell JM, Wetten AC (eds) Transgenic plants. Methods and protocols, 2nd edn. Humana Press, New York, pp 451–466CrossRefGoogle Scholar
  58. Spadaccini R, Crescenzi O, Tancredi T, De Casamassimi N, Saviano G, Scognamiglio R, DonatoA Di, Temussi PA (2001) Solution structure of a sweet protein: NMR study of MNEI, a single chain monellin. J Mol Biol 305:505–514CrossRefPubMedGoogle Scholar
  59. Spadaccini R, Leone S, Rega MF, Richter C, Picone D (2016) Influence of pH on the structure and stability of the sweet protein MNEI. FEBS Lett 590:3681–3689CrossRefPubMedGoogle Scholar
  60. Sun H-J, Cui M, Ma B, Ezura H (2006) Functional expression of the taste-modifying protein, miraculin, in transgenic lettuce. FEBS Lett 580:620–626CrossRefPubMedGoogle Scholar
  61. Sun H-J, Kataoka H, Yano M, Ezura H (2007) Genetically stable expression of functional miraculin, a new type of alternative sweetener, in transgenic tomato plants. Plant Biotechnol J 5:768–777CrossRefPubMedGoogle Scholar
  62. 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 310:27–30CrossRefPubMedGoogle Scholar
  63. Tancredi T, Pastore A, Salvadori S, Esposito V, Temussi PA (2004) Interaction of sweet proteins with their receptor. Eur J Biochem 271:2231–2240CrossRefPubMedGoogle Scholar
  64. Temussi PA (2002) Why are sweet proteins sweet? Interaction of brazzein, monellin and thaumatin with the T1R2-T1R3 receptor. FEBS Lett 526:1–4CrossRefPubMedGoogle Scholar
  65. Temussi PA (2011a) New insights into the characteristics of sweet and bitter taste receptors. In: Jeon KW (ed) International Review of Cell and Molecular Biology, vol 291. Academic Press, San Diego, pp 191–226Google Scholar
  66. Temussi PA (2011b) Determinants of sweetness in proteins: a topological approach. J Mol Recognit 24:1033–1042CrossRefPubMedGoogle Scholar
  67. Tschofen M, Knopp D, Hood E, Stöger E (2016) Plant molecular farming: much more than medicines. Annu Rev Anal Chem 9:271–294CrossRefGoogle Scholar
  68. van der Wel H, Loeve K (1972) Isolation and characterization of thaumatin I and II, the sweet-tasting proteins from Thaumatococcus daniellii Benth. Eur J Biochem 31:221–225CrossRefPubMedGoogle Scholar
  69. Waheed MT, Ismail H, Gottschamel J, Mirza B, Lössl AG (2015) Plastids: the green frontiers for vaccine production. Front Plant Sci 6:1005CrossRefPubMedPubMedCentralGoogle Scholar
  70. Zheng W, Yang L, Cai C, Ni J, Liu B (2018) Expression, purification and characterization of a novel double-site mutant of the single-chain sweet-tasting protein monellin (MNEI) with both improved sweetness and stability. Protein Expr Purif 143:52–56CrossRefPubMedGoogle Scholar
  71. Zhou Y, Lu Z, Wang X, Selvaraj JN, Zhang G (2018) Genetic engineering modification and fermentation optimization for extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 102:1545–1556CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Daniela Castiglia
    • 1
  • Serena Leone
    • 2
  • Rachele Tamburino
    • 1
  • Lorenza Sannino
    • 1
  • Jole Fonderico
    • 2
  • Chiara Melchiorre
    • 2
  • Andrea Carpentieri
    • 2
  • Stefania Grillo
    • 1
  • Delia Picone
    • 2
  • Nunzia Scotti
    • 1
    Email author
  1. 1.CNR-IBBR, National Research Council of Italy, Institute of Biosciences and BioResourcesPorticiItaly
  2. 2.Department of Chemical SciencesUniversity of Naples Federico IINaplesItaly

Personalised recommendations