Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Stable expression of brazzein protein, a new type of alternative sweetener in transgenic rice

형질전환 벼에서 brazzein 감미단백질의 안정적인 발현

  • Lee, Ye Rim (Department of Horticultural Life Science, Hankyong National University) ;
  • Akter, Shahina (Department of Horticultural Life Science, Hankyong National University) ;
  • Lee, In Hye (Biological Resources Division, National Institute of Biological Resources) ;
  • Jung, Yeo Jin (Department of Horticultural Life Science, Hankyong National University) ;
  • Park, So Young (Department of Horticultural Science, Chungbuk National University) ;
  • Cho, Yong-Gu (Department of Crop Science, Chungbuk National University) ;
  • Kang, Kwon Kyoo (Department of Horticultural Life Science, Hankyong National University) ;
  • Jung, Yu Jin (Department of Horticultural Life Science, Hankyong National University)
  • 이예림 (국립한경대학교 원예생명과학과) ;
  • 샤히나 (국립한경대학교 원예생명과학과) ;
  • 이인혜 (국립생물자원관 생물소재연구단 연구팀) ;
  • 정여진 (국립한경대학교 원예생명과학과) ;
  • 박소영 (충북대학교 응용생명공학부 원예과학전공) ;
  • 조용구 (충북대학교 식물자원학과) ;
  • 강권규 (국립한경대학교 원예생명과학과) ;
  • 정유진 (국립한경대학교 원예생명과학과)
  • Received : 2018.03.26
  • Accepted : 2018.03.26
  • Published : 2018.03.31
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Abstract

Brazzein is the smallest sweet protein and was isolated from the fruit pulp of Pentadiplandra brazzeana Baillon, native to tropical Africa. From ancient times, the indigenous people used this fruit in their diet to add sweetness to their daily food. Brazzein is 500 to 2000 times sweeter than sucrose on a weight basis and 9500 times sweeter on a molar basis. This unique property has led to increasing interest in this protein. However, it is expensive and difficult to produce brazzein other than in its native growing conditions which limits its availability for use as a food additive. In this study, we report high production yields of, brazzein protein in transgenic rice plants. An ORF region encoding brazzein and driven by the $2{\times}CaMV\;35S$ promoter was introduced into rice genome (Oryza sativa Japonica) via Agrobacterium-mediated transformation. After transformation, 17 regenerated plant lines were obtained and these transgene-containing plants were confirmed by PCR analysis. In addition, the selected plant lines were analyzed by Taqman PCR and results showed that 9 T0 lines were found to have a single copy out of 17 transgenic plants. Moreover, high and genetically stable expression of brazzein was confirmed by western blot analysis. These results demonstrate that recombinant brazzein was efficiently expressed in transgenic rice plants, and that we have developed a new rice variety with a natural sweetener.

Brazzein은 열대식물인 P. brazzeana Baillon의 과실에서 분리된 가장 작은 감미단백질로 토착민들의 단맛원료로 사용되어 왔다. Brazzein은 sucrose보다 분자량 기준으로 500 ~ 2000배, 몰 기준으로 9500배 당도가 높아 감미료로써 매우 높은 평가를 받고 있다. 그러나 이 감미단백질은 재배가 어렵고 생산 비용이 높아서 brazzein 단백질의 이용 가능성을 높이기 위한 대체 생산 시스템으로 형질전환 식물체 육성 하고자 하였다. 본 연구에서는 brazzein 관련 유전자를 벼에 도입하기 위하여 식물형질전환용 Ti-plasmid에 $2{\times}CaMV\;35S$ 프로모터에 의해 지배되어 발현하도록 하고, 선발 마커로 bar 유전자가 삽입된 식물발현 벡터를 구축하여 A. tumefaciens EHA105에 형질전환시켜 17개의 재분화 식물체를 육성하였다. 17개 재분화 식물체는 PCR 및 RT-PCR 분석을 통하여 유전자 도입 및 발현을 확인하였으며, TaqMan PCR을 통해 single copy로 도입된 T0 세대 9개체를 선발하였다. 또한 FST 분석을 통하여 도입 유전자가 intergenic으로 삽입된 개체 5개를 선발하였다. 이들 5개체를 이용하여 western blot 분석에 의해 단백질 발현량을 분석한 결과 선발된 모든 개체에서 발현 밴드를 확인하였다. 그 중 brazzein 단백질의 발현량이 높은 개체를 TG11으로 계통화하여 후대 종자를 육성하였다. TG11 계통은 천연 감미료 brazzein을 생산하는 새로운 벼 품종을 개발하기 위한 육종 소재로 활용 가능하다고 시사된다.

Keywords

References

  1. Akter S, Huq MA, Jung YJ, Cho YG, Kang KK (2016) Application of sweet and taste modifying genes for development in plants: current status and prospects. J Plant Biotechnol 43:397-404 https://doi.org/10.5010/JPB.2016.43.4.397
  2. Assadi-Porter FM, Aceti DJ, Cheng H, Markley JL (2000) Efficient production of recombinant brazzein, a small, heat-stable, sweet-tasting protein of plant origin. Arch Biochem Biophys 376:252-258
  3. Assadi-Porter FM, Aceti DJ, Markley JL (2000) Sweetness Determinant Sites of Brazzein, a Small, Heat-Stable, Sweet-Tasting Protein. Arch Biochem Biophys 376:259-265. https://doi.org/10.1006/abbi.2000.1726
  4. Assadi-Porter FM, Patry S, Markley JL (2008) Efficient and rapid protein expression and purification of small high disulfide containing sweet protein brazzein in E. coli. Protein Expr Purif 58:263-268 https://doi.org/10.1016/j.pep.2007.11.009
  5. Berlec A, Jevnikar Z, Majhenic AC, Rogelj I, Strukelj B (2006) Expression of the sweet-tasting plant protein brazzein in Escherichia coli and Lactococcus lactis: a path toward sweet lactic acid bacteria. Appl microbiol biot 73:158-165 https://doi.org/10.1007/s00253-006-0438-y
  6. Berlec A, Strukelj B (2009) Large increase in brazzein expression achieved by changing the plasmid/strain combination of the NICE system in Lactococcus lactis. Lett Appl Microbiol 48:750-755
  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  8. Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 6:219-226 https://doi.org/10.1016/S1360-1385(01)01922-7
  9. Faus I (2000) Recent developments in the characterization and biotechnological production of sweet-tasting proteins. Appl Microbiol Biot 53:145-151 https://doi.org/10.1007/s002530050001
  10. Hood EE, Jilka JM (1999) Plant-based production of xenogenic proteins. Curr Opin Biotech 10:382-386 https://doi.org/10.1016/S0958-1669(99)80069-X
  11. Hood EE, Woodard SL, Horn ME (2002) Monoclonal antibody manufacturing on transgenic plants - myths and realities. Curr Opin Biotech 13:630-635 https://doi.org/10.1016/S0958-1669(02)00351-8
  12. Horn ME, Woodard SL, Howard JA (2004) Plant molecular farming: systems and products. Plant Cell Rep 22:711-720 https://doi.org/10.1007/s00299-004-0767-1
  13. Howard JA, Hood E (2005) Bioindustrial and biopharmaceutical products produced in plants. Adv Agron 85:91-124
  14. Jo HJ, Noh JS, Kong KH (2013) Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis. Protein Expres Purif 90:84-9 https://doi.org/10.1016/j.pep.2013.05.001
  15. Jung YJ, Bae SS, Lee GJ, Seo PJ, Cho Y G, Kang KK (2017) A novel method for high-frequency genome editing in rice, using the CRISPR/Cas9 system. J Plant Biotechnol 44:89-96 https://doi.org/10.5010/JPB.2017.44.1.089
  16. Jung YJ, Nogoy FM, Cho Y G, Kang KK (2015) Development of high tryptophan GM rice and its transcriptome analysis. J Plant Biotechnol 42:186-195 https://doi.org/10.5010/JPB.2015.42.3.186
  17. Jung YJ, Nou IS, Kang KK (2014) Overexpression of Oshsp16.9 gene encoding small heat shock protein enhances tolerance to abiotic stresses in rice. Plant Breed Biotech 2(4):370-379 https://doi.org/10.9787/PBB.2014.2.4.370
  18. Kant R (2005) Sweet proteins, potential replacement for artificial low calorie sweeteners. Nutrition 4:5 https://doi.org/10.1186/1475-2891-4-5
  19. Kurihara Y, Beidler LM (1968) Taste-modifying protein from miracle fruit. Science 161:1241-1243 https://doi.org/10.1126/science.161.3847.1241
  20. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 https://doi.org/10.1038/227680a0
  21. 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 Biotechnology Journal 3:103-114
  22. Lee JJ, Kong JN, Do HD, Jo DH, Kong KH (2010) Design and efficient soluble expression of a sweet protein, brazzein and minor-form mutant. B Kor Chem Soc 31:3830-3833 https://doi.org/10.5012/bkcs.2010.31.12.3830
  23. Liu X, Hu Z, Maeda S, Aiuchi T, Nakaya K, Kurihara Y (1993) Purification, complete amino acid sequence and structure characterization of the heat stable sweet protein, mabinlin II. Eur J Biochem 211:281-287
  24. Masuda T, Kitabatake N (2006) Developments in biotechnological production of sweet proteins. J Biosci Bioeng 102:375-389
  25. Masuda T, Ueno Y, Kitabatake N (2001) Sweetness and enzymatic activity of lysozyme. J Agr Food Chem 49:4937-4941
  26. Ming D, Hellekant G (1994) Brazzein, a new high-potency thermostable sweet protein from Pentadiplandra byamarazzeana B. FEBS Lett 355:106-108 https://doi.org/10.1016/0014-5793(94)01184-2
  27. Moris JA, Cagan RH (1972) Purification of monellin, the sweet principal for Dioscoreophyllum cumminsii. Biochim Biophys Acta 261:114-122
  28. Price JM, Biava CG, Oser BL, Vogin EE, Steinfield J, Ley HL (1970) 'Bladder tumors in rats fed cyclohexylamine or high doses of a mixture of cyclamate and saccharin'. Science 167:1131-1132 https://doi.org/10.1126/science.167.3921.1131
  29. Rachid A, Belloir C, Chevalier J, Desmetz C, Miller ML, et al. (2009) Optimization of the Production of Recombinant Brazzein Secreted by the Yeast Pichia pastoris. Chem senses 34:A80-A80
  30. Shirasuka Y, Nakajima K, Asakura T, Yamashita H, Yamamoto A, Hata S, Nagata S, Abo M, Sorimachi H, Abe K (2004) Neoculin as a new taste-modifying protein occurring in the fruit of Curculigo latifolia. Biosci Biotech Bioch 68:1403-1407 https://doi.org/10.1271/bbb.68.1403
  31. Streatfield SJ (2007) Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnol J 5:2-15 https://doi.org/10.1111/j.1467-7652.2006.00216.x
  32. Streatfield SJ, Howard JA (2003) Plant-based vaccines. Int J Parasitol 33:479-493
  33. Sun HJ, Cui ML, Ma B, Ezura H (2006) Functional expression of the taste-modifying protein, miraculin, in transgenic lettuce. FEBS Lett 580:620-626
  34. Sun HJ, 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 5:768-777 https://doi.org/10.1111/j.1467-7652.2007.00283.x
  35. Thole V, Alves SC, Worland B, Bevan MW, Vain P (2009) A protocol for efficiently retrieving and characterizing flanking sequence tags (FSTs) in Brachypodium distachyon T-DNA insertional mutants. Nature 4:650-661
  36. Van der Wel H (1972) Isolation and characterization of the sweet principal for Dioscoreophyllum cumminsii (Stapf) Diels. FEBS Lett 21:88-90 https://doi.org/10.1016/0014-5793(72)80170-4
  37. Van der Wel H and Loeve K (1972) Isolation and characterization of thaumatin I and II, the sweet-tasting proteins from Thaumatococcus daniellii Benth. Eur J Biochem 31:221-225
  38. Van der Wel H, Larson G, Hladik A, Hladik CM, Hellekant G, Glaser D (1989) Isolation and characterization of pentadin, the sweet principle of Pentadiplandra brazzeana Baillon. Chem Senses 264:6655-6659
  39. Yamashita H, Theeraship A, Nakaya T, Nakamura Y, Kurihara Y (1990) Purification and complete amino acid sequence of a new type of sweet protein with taste-modifying activity, curculin. J Biol Chem 265:15770-15775
  40. Yan S, Song H, Pang D, Zou Q, Li L, Yan Q, Fan N, Zhao X, Yu H, Li Z, Wang H, Gao F, Ouyang H, Lai L (2013) Expression of Plant Sweet Protein Brazzein in the Milk of Transgenic Mice. PloS one 8:e76769
  41. Yoshida K, Shinmyo A (2000) Transgenic expression systems in plants, a natural bioreactor. J Biosci Bioeng 90:353-362 https://doi.org/10.1016/S1389-1723(01)80001-3