Advertisement

Journal of Applied Phycology

, Volume 30, Issue 6, pp 3503–3518 | Cite as

Agrobacterium-mediated genetic transformation of Dictyosphaerium pulchellum for the expression of erythropoietin

  • Khawaja Muhammad Imran Bashir
  • Moo-Sang Kim
  • Ulf Stahl
  • Man-Gi ChoEmail author
Article

Abstract

Recombinant proteins are extensively used for a growing number of fields in biology. However, microalgal species have not been widely adopted as cell factories for recombinant protein production. Unique metabolic properties, ease of cultivation, fast growth rates, and continuous progress in genetic engineering of microalgae have raised interest in the use of microalgae species for recombinant protein production. Here, we report an Agrobacterium-mediated genetic transformation system for the heterologous expression of a therapeutic protein, “erythropoietin,” in a nonmodel green microalga, Dictyosphaerium pulchellum. Hygromycin resistance gene (Hyg) was used as a selectable marker. The genetic transformation of D. pulchellum was performed in modified AF6 medium supplemented with 150 μM acetosyringone, co-cultivated for 48 h at 25 ± 2 °C and a light intensity of 18 ± 2 μmol photons m−2 s−1. Co-cultivation of D. pulchellum with Agrobacterium tumefaciens harboring the binary expression vector pCAMBIA1301-Hyg-EPO-Histag yielded hygromycin-resistant colonies on selective medium after 2–3 weeks. Gene integration into the D. pulchellum nuclear genome was confirmed by PCR amplification of T-DNA from the genomic DNA of hygromycin-resistant and wild-type strains. Interestingly, SDS-PAGE and subsequent Western blotting with His-tag monoclonal and anti-erythropoietin monoclonal antibodies revealed an EPO-specific signal slightly below 34 kDa. Furthermore, EPO gene expression and transgene copy numbers were estimated by quantitative real-time PCR. Approximately, 500 μg L−1 of extracellular recombinant erythropoietin protein was purified by His-tag affinity chromatography. The developed genetic transformation system would allow the metabolic engineering and a better alternative to produce recombinant therapeutic proteins from nonmodel freshwater microalgae species.

Keywords

Chlorophyta Heterologous gene expression Hygromycin Microalgae Nuclear genome engineering Protein cell factory Recombinant protein 

Notes

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10811_2018_1483_MOESM1_ESM.pdf (4.5 mb)
ESM 1 (PDF 4649 kb)

References

  1. Ali S, Xianyin Z, Xue Q, Hassan MJ, Qian H (2007) Investigations for improved genetic transformation mediated by Agrobacterium tumefaciens in two rice cultivars. Biotechnology 6:138–147Google Scholar
  2. Anila N, Chandrashekar A, Ravishankar GA, Sarada R (2011) Establishment of Agrobacterium tumefaciens mediated genetic transformation in Dunaliella bardawil. Eur J Phycol 46:36–44Google Scholar
  3. Anwaruzzaman N, Chin BL, Li XP, Lohr M, Martínez DA, Niyogi KK (2004) Genomic analysis of mutants affecting xanthophylls biosynthesis and regulation of photosynthetic light harvesting in Chlamydomonas reinhardtii. Photosynth Res 82:265–276PubMedGoogle Scholar
  4. Barik DP, Mohapatra U, Chand PK (2005) Transgenic grass pea (Lathyrus sativus L.): factors influencing Agrobacterium-mediated transformation and regeneration. Plant Cell Rep 24:523–531PubMedGoogle Scholar
  5. Bashir KMI, Cho M-G (2016) The effect of kanamycin and tetracycline on growth and photosynthetic activity of two chlorophyte algae. Biomed Res Int 2016:5656304Google Scholar
  6. Bashir KMI, Kim M-S, Stahl U, Cho M-G (2016) Microalgae engineering toolbox: selectable and screenable markers. Biotechnol Bioprocess Eng 21:224–235Google Scholar
  7. Bashir KMI, Kim J-S, An JH, Sohn JH, Choi J-S (2017) Natural food additives and preservatives for fish-paste products: a review of the past, present and future states of research. J Food Qual 2017:1–31Google Scholar
  8. Bollig K, Lamshöft M, Schweimer K, Marner FJ, Budzikiewicz H, Waffenschmidt S (2007) Structural analysis of linear hydroxyproline-bound O-glycans of Chlamydomonas reinhardtii-conservation of the inner core in Chlamydomonas and land plants. Carbohydr Res 342:2557–2566PubMedGoogle Scholar
  9. Borovsky D (2003) Trypsin modulating oostatic factor: a potential new larvicide for mosquito control. J Exp Biol 206:3869–3875PubMedGoogle Scholar
  10. Brueggeman AJ, Kuehler D, Weeks DP (2014) Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production. Plant Biotechnol J 12:894–902PubMedGoogle Scholar
  11. Cadoret J-P, Garnier M, Saint-Jean B (2012) Microalgae, functional genomics and biotechnology. Adv Bot Res 64:285–341Google Scholar
  12. Cha TS, Yee W, Aziz A (2012) Assessment of factors affecting Agrobacterium-mediated genetic transformation of the unicellular green alga, Chlorella vulgaris. World J Microbiol Biotechnol 28:771–1779Google Scholar
  13. Cheng R, Ma R, Li K, Rong H, Lin X, Wang Z, Yang S, Ma Y (2012) Agrobacterium tumefaciens mediated transformation of marine microalgae Schizochytrium. Microbiol Res 167:179–186PubMedGoogle Scholar
  14. Cheon BY, Kim HJ, Oh KH, Bahn SC, Ahn JH, Choi JW, Ok SH, Bae JM, Shin JS (2004) Overexpression of human erythropoietin (EPO) affects plant morphologies: retarded vegetative growth in tobacco and male sterility in tobacco and Arabidopsis. Transgenic Res 13:541–549PubMedGoogle Scholar
  15. Chow K-C, Tung WL (1999) Electrotransformation of Chlorella vulgaris. Plant Cell Rep 18:778–780Google Scholar
  16. Chu L, Robinson DK (2001) Industrial choices for protein productions by large-scale cell culture. Curr Opin Biotechnol 12:180–187PubMedGoogle Scholar
  17. Day A, Goldschmidt-Clermont M (2011) The chloroplast transformation toolbox: selectable markers and marker removal. Plant Biotechnol J 9:540–553PubMedGoogle Scholar
  18. Ding XQ, Rao RV, Kuntz SM, Holicky EL, Miller LJ (2000) Impaired resensitization and recycling of the cholecystokinin receptor by co-expression of its second intracellular loop. Mol Pharmacol 58:1424–1433PubMedGoogle Scholar
  19. Doron L, Segal N, Shapira M (2016) Transgene expression in microalgae—from tools to applications. Front Plant Sci 7:505PubMedPubMedCentralGoogle Scholar
  20. Eichler-Stahlberg A, Weisheit W, Ruecker O, Heitzer M (2009) Strategies to facilitate transgene expression in Chlamydomonas reinhardtii. Planta 229:873–883PubMedGoogle Scholar
  21. Feng S, Feng W, Zhao L, Gu H, Li Q, Shi K, Guo S, Zhang N (2014) Preparation of transgenic Dunaliella salina for immunization against white spot syndrome virus in crayfish. Arch Virol 159:519–525PubMedGoogle Scholar
  22. Fullner KJ, Nester EW (1996) Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens. J Bacteriol 178:1498–1504PubMedPubMedCentralGoogle Scholar
  23. Gangl D, Zedler JAZ, Rajakumar PD, Martinez EMR, Riseley A, Włodarczyk A, Purton S, Sakuragi Y, Howe CJ, Jensen PE, Robinson C (2015) Biotechnological exploitation of microalgae. J Exp Bot 66:6975–6990PubMedGoogle Scholar
  24. Geng D, Wang Y, Wang P, Li W, Sun Y (2003) Stable expression of hepatitis B surface antigen in Dunaliella salina (Chlorophyta). J App Phycol 15:451–456Google Scholar
  25. Giddings G, Allison G, Brook D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 18:1151–1155PubMedGoogle Scholar
  26. Głowacka K, Kromdijk J, Leonelli L, Niyogi KK, Clemente TE, Long SP (2016) An evaluation of new and established methods to determine T-DNA copy number and homozygosity in transgenic plants. Plant Cell Environ 39:908–917PubMedPubMedCentralGoogle Scholar
  27. Gong Y, Hu H, Gao Y, Xu X, Gao H (2011) Microalgae as platforms for production of recombinant proteins and valuable compounds: progress and prospects. J Ind Miocrobiol Biotechnol 38:1879–1890Google Scholar
  28. Guo SL, Zhao XQ, Tang Y, Wan C, Alam MA, Ho SH, Bai FW, Chang JS (2013) Establishment of an efficient genetic transformation system in Scenedesmus obliquus. J Biotechnol 163:61–68PubMedGoogle Scholar
  29. Hamilton CM, Frary A, Lewis C, Tanksley S (1996) Stable transfer of intact high molecular weight DNA into plant chromosomes. Proc Natl Acad Sci U S A 93:9975–9979PubMedPubMedCentralGoogle Scholar
  30. Hansen G, Shillito RD, Chilton MD (1997) T-stand integration in maize protoplasts after co-delivery of a T-DNA substrate and virulence genes. Proc Natl Acad Sci U S A 94:11726–11730PubMedPubMedCentralGoogle Scholar
  31. Hiei Y, Ohta S, Komari T, Komashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282PubMedGoogle Scholar
  32. Hoebeeck J, Speleman F, Vandesompele J (2007) Real-time quantitative PCR as an alternative to southern blot or fluorescence in situ hybridization for detection of gene copy number changes. In: Hilario E, Mackay J (eds) Protocols for nucleic acid analysis by nonradioactive probes. Humana Press Inc., Totowa, USA, pp 205–226Google Scholar
  33. Hu Z, Wu Y, Li W, Gao H (2006) Factors affecting Agrobacterium-mediated genetic transformation of Lycium barbarum L. In Vitro Cell Dev Biol Plant 42:461–466Google Scholar
  34. Irfanullah HMD, Moss B (2006) Ecology of Dictyosphaerium pulchellum Wood (Chlorophyta, Chlorococcales) in a shallow, acid, forest lake. Aquat Ecol 40:1–12Google Scholar
  35. Jänne J, Alhonen L, Hyttinen JM, Peura T, Tolvanen M, Korhonen VP (1998) Transgenic bioreactors. Biotechnol Annu Rev 4:55–74PubMedGoogle Scholar
  36. Jarvis EE, Brown LM (1991) Transient expression of Firefly luciferase in protoplasts of the green alga Chlorella ellipsoidea. Curr Genet 19:317–321Google Scholar
  37. Jelkmann W (1992) Erythropoietin: structure, control of production, and function. Physiol Rev 72:449–489PubMedGoogle Scholar
  38. Johnson EA, Rosenberg J, McCarty RE (2007) Expression by Chlamydomonas reinhardtii of a chloroplast ATP synthase with polyhistidine-tagged beta subunits. Biochim Biophys Acta Bioenerg 1767:374–380Google Scholar
  39. Kathiresan S, Chandrashekar A, Ravishankar GA, Sarada R (2009) Agrobacterium-mediated transformation in the green alga Haematococcus pluvialis (Chlorophyceae, Volvocales). J Phycol 45:642–649PubMedGoogle Scholar
  40. Kawar ZS, Haslam SM, Morris HR, Dell A, Cummings RD (2005) Novel poly-GalNAcβ1-4GlcNAc (LacdiNAc) and fucosylated poly-LacdiNAc N-glycans from mammalian cells expressing β1,4-N-acetyl galactosaminyltransferase and α1,3-fucosyltransferase. J Biol Chem 280:12810–12819PubMedGoogle Scholar
  41. Kilian O, Benemann CSE, Niyogi KK, Vick B (2011) High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp. Proc Natl Acad Sci U S A 108:21265–21269PubMedPubMedCentralGoogle Scholar
  42. Kim JT, Sung MB (2001) Occurrence of Dictyosphaerium pulchellum (Chlorophyceae) bloom in a small pond. Korean. J Limnol 34:292–297Google Scholar
  43. Kim YK, Shin HS, Tomiya N, Lee YC, Betenbaugh MJ, Cha HJ (2005) Production and N-glycan analysis of secreted human erythropoietin glycoprotein in stably transfected Drosophila S2 cells. Biotechnol Bioeng 92:452–461PubMedGoogle Scholar
  44. Kim S, Lee Y-C, Cho D-H, Lee H-U, Huh Y-S, Kim G-J, Kim H-S (2014) A simple and non-invasive method for nuclear transformation of intact-walled Chlamydomonas reinhardtii. PLOS One 9:e101018PubMedPubMedCentralGoogle Scholar
  45. Kindle KL (1990) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 87:1228–1232PubMedPubMedCentralGoogle Scholar
  46. Kumar SV, Misquitta RW, Reddy VS, Rao BJ, Rajam MV (2004) Genetic transformation of the green alga Chlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant Sci 166:731–738Google Scholar
  47. Kumar A, Perrine Z, Stroff C, Postier BL, Coury DA, Sayre RT, Allnutt FCT (2016) Molecular tools for bioengineering eukaryotic microalgae. Curr Biotechnol 5:93–108Google Scholar
  48. Lauersen KJ, Berger H, Mussgnug JH, Kruse O (2013a) Efficient recombinant protein production and secretion from nuclear transgenes in Chlamydomonas reinhardtii. J Biotechnol 167:101–110PubMedGoogle Scholar
  49. Lauersen KJ, Vanderveer TL, Berger H, Kaluza I, Mussgnug JH, Walker VK, Kruse O (2013b) Ice recrystallization inhibition mediated by a nuclear-expressed and –secreted recombinant ice-binding protein in the microalga Chlamydomonas reinhardtii. Appl Microbiol Biotechnol 97:9763–9772PubMedGoogle Scholar
  50. Lerche K, Hallmann A (2014) Stable nuclear transformation of Pandorina morum. BMC Biotechnol 14:65PubMedPubMedCentralGoogle Scholar
  51. Lin FK, Suggs S, Lin CH, Browne JK, Smalling R, Egrie JC, Chen KK, Fox GM, Martin F, Stabinsky Z (1985) Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci U S A 82:7580–7584PubMedPubMedCentralGoogle Scholar
  52. Lin J, Ma J, Assad-Garcia N, Kuo J (1996) Hygromycin B as an efficient antibiotic for the selection of transgenic plants. Focus 18:47–49Google Scholar
  53. Mathieu-Rivet E, Kiefer-Meyer M-C, Vanier G, Ovide C, Burel C, Lerouge P, Bardor M (2014) Protein Nglycosylation in eukaryotic microalgae and its impact on the production of nuclear expressed biopharmaceuticals. Front Plant Sci 5:359PubMedPubMedCentralGoogle Scholar
  54. Matsumoto S, Ikura K, Ueda M, Sasaki R (1995) Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells. Plant Mol Biol 27:1163–1172PubMedGoogle Scholar
  55. Mayfield SP, Franklin SE, Lerner RA (2003) Expression and assembly of a fully active antibody in algae. Proc Natl Acad Sci U S A 100:438–442PubMedPubMedCentralGoogle Scholar
  56. Mayfield SP, Manuell AL, Chen S, Wu J, Tran M, Siefker D, Muto M, Marin-Navarro J (2007) Chlamydomonas reinhardtii chloroplasts as protein factories. Curr Opin Biotechnol 18:1–8Google Scholar
  57. Oey M, Ross IL, Hankamer B (2014) Gateway-assisted vector construction to facilitate expression of foreign proteins in the chloroplast of single-celled algae. PLOS One 9:e86841PubMedPubMedCentralGoogle Scholar
  58. Pfaffl MW (2006) Relative quantification. In: Dorak MT (ed) Real-time PCR. Taylor and Francis Group, New York. pp 63–82Google Scholar
  59. Pratheesh PT, Shonima GM, Thomas J, Abraham CI, Muraleedhara KG (2012) Study on efficacy of different Agrobacterium tumefaciens strains in genetic transformation of microalga Chlamydomonas reinhardtii. Adv Appl Sci Res 3:2679–2686Google Scholar
  60. Pratheesh PT, Vineetha M, Kurup GM (2014) An efficient protocol for the Agrobacterium-mediated genetic transformation of microalgae Chlamydomonas reinhardtii. Mol Biotechnol 56:507–515PubMedGoogle Scholar
  61. Rasala BA, Muto M, Lee PA, Jager M, Cardoso RMF, Behnke CA, Kirk P, Hokanson CA, Crea R, Mendez M, Mayfield SP (2010) Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnol J 8:719–733PubMedPubMedCentralGoogle Scholar
  62. Rasala BA, Lee PA, Shen Z, Briggs SP, Mendez M, Mayfield SP (2012) Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide. PloS One 7:e43349PubMedPubMedCentralGoogle Scholar
  63. Rasala BA, Chao S-S, Pier M, Barrera DJ, Mayfield SP (2014) Enhanced genetic tools for engineering multigene traits into green algae. PloS One 9:e94028PubMedPubMedCentralGoogle Scholar
  64. Rathod JP, Prakash G, Pandit R, Lali AM (2013) Agrobacterium-mediated transformation of promising oil-bearing marine algae Parachlorella kessleri. Photosynth Res 118:141–146PubMedGoogle Scholar
  65. Rosenberg JN, Oh VH, Yu G, Guzman BJ, Oyler GA, Betenbaugh MJ (2015) Exploiting the molecular genetics of microalgae: from strain development pipelines to uncharted waters of mass production. In: Kim S-K (ed) Handbook of marine microalgae: biotechnology advances. Elsevier, New York, pp 331–352Google Scholar
  66. Saint-Jore-Dupas C, Faye L, Gomord V (2007) From planta to pharma with glycosylation in the toolbox. Trends Biotechnol 25:317–323PubMedGoogle Scholar
  67. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108PubMedGoogle Scholar
  68. Scott MR, Will R, Ironside J, Nguyen HO, Tremblay P, DeArmond SJ, Prusiner SB (1999) Compelling transgenic evidence for transmission of bovine spongiform encephalopathy prions to humans. Proc Natl Acad Sci U S A 96:15137–15142PubMedPubMedCentralGoogle Scholar
  69. Shamriz S, Ofoghi H (2017) Outlook in the application of Chlamydomonas reinhardtii chloroplast as a platform for recombinant protein production. Biotechnol Genet Eng Rev 32:92–106Google Scholar
  70. Sheikholeslam SN, Weeks DP (1987) Acetosyringone promotes high-efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol Biol 8:291–298PubMedGoogle Scholar
  71. Shrawat AK, Becker D, Lörz H (2007) Agrobacterium tumefaciens mediated genetic transformation of barley (Hordeum vulgare L.). Plant Sci 172:281–290Google Scholar
  72. Smith KJ, Bleyer AJ, Little WC, Sane DC (2003) The cardiovascular effects of erythropoietin. Cardiovasc Res 59:538–548PubMedGoogle Scholar
  73. Specht E, Miyake-Stoner S, Mayfield SP (2010) Micro-algae come of age as a platform for recombinant protein production. Biotechnol Lett 32:1373–1383PubMedPubMedCentralGoogle Scholar
  74. Stachel SE, Messens E, Van Montagu M, Zambryski P (1985) Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318:624–629Google Scholar
  75. Stancheva R, Fuller C, Sheath RG (2016) Soft-bodied stream algae of California. http://dbmuseblade.colorado.edu/DiatomTwo/sbsac_site/species.php?g=Dictyosphaerium&s=pulchellum. Accessed 24 Feb 2017
  76. Steinbrenner J, Sandmann G (2006) Transformation of the green alga Haematococcus pluvialis with a phytoene desaturase for accelerated astaxanthin biosynthesis. Appl Environ Microbiol 72:7477–7484PubMedPubMedCentralGoogle Scholar
  77. Sun M, Qian K, Su N, Chang H, Liu J, Chen GF (2003) Foot-and-mouth disease virus VP1 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast. Biotechnol Lett 25:1087–1092PubMedGoogle Scholar
  78. Tan CP, Qin S, Zhang Q, Jiang P, Zhao FQ (2005) Establishment of a micro-particle bombardment transformation system for Dunaliella salina. J Microbiol 43:361–365PubMedGoogle Scholar
  79. ten Lohuis MR, Miller DJ (1998) Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): expression of GUS in microalgae using heterologous promoter constructs. Plant J 13:427–435Google Scholar
  80. Tzfira T, Citovsky V (2006) Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Curr Opin Biotechnol 17:147–154PubMedGoogle Scholar
  81. Veith N, Ziehr H, MacLeod RA, Reamon-Buettner SM (2016) Mechanisms underlying epigenetic and transcriptional heterogeneity in Chinese hamster ovary (CHO) cell lines. BMC Biotechnol 2016:16Google Scholar
  82. Walker TL, Purton S, Becker DK, Collet C (2005) Microalgae as bioreactors. Plant Cell Rep 24:629–641PubMedGoogle Scholar
  83. Wang W, Jia Y-L, Li Y-C, Jiang C-Q, Guo X, Shang X-F, Zhao C-P, Wang T-Y (2017) Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells. Sci Rep 7:10416PubMedPubMedCentralGoogle Scholar
  84. Watanabe MM, Kawachi M, Hiroki M, Kasai F (2000) NIES collection list of strains, 6th edn. In: Microalgae and protozoa. Microalgae Culture Collections, National Institute for Environmental Studies, Tsukuba, p 159Google Scholar
  85. Weise A, Altmann F, Rodriguez-Franco M, Sjoberg ER, Baumer W, Launhardt H, Kietzmann M, Gorr G (2007) High-level expression of secreted complex glycosylate recombinant human erythropoietin in the Physcomitrella D-fuc-t D-xyl-t mutant. Plant Biotechnol J 5:389–401PubMedGoogle Scholar
  86. Yamano T, Iguchi H, Fukuzawa H (2013) Rapid transformation of Chlamydomonas reinhardtii without cellwall removal. J Biosci Bioeng 115:691–694PubMedGoogle Scholar
  87. Yan N, Fan C, Chen Y, Hu Z (2016) The potential for microalgae as bioreactors to produce pharmaceuticals. Int J Mol Sci 17:962PubMedCentralGoogle Scholar
  88. Zaslavskaia LA, Lippmeier JC, Kroth PG, Grossman AR, Apt KE (2000) Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes. J Phycol 36:379–386Google Scholar
  89. Zorin B, Grundman O, Khozin-Goldberg I, Leu S, Shapira M, Kaye Y, Tourasse N, Vallon O, Boussiba S (2014) Development of a nuclear transformation system for oleaginous green alga Lobosphaera (Parietochloris) incisa and genetic complementation of a mutant strain, deficient in arachidonic acid biosynthesis. PloS One 9:e105223PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018
corrected publication December/2018

Authors and Affiliations

  1. 1.Department of Biotechnology, Division of Energy and BioengineeringDongseo UniversityBusanRepublic of Korea
  2. 2.Seafood Research Center, IACFSilla UniversityBusanRepublic of Korea
  3. 3.Institute of BiotechnologyTechnische Universität BerlinBerlinGermany

Personalised recommendations