Abstract
Microalgae based systems have the potential to combine the advantages of plants with features of microorganisms, thus becoming an alternative for gene farming. Advantages such as a short time from gene to protein, inexpensive cultivation, fast growth and improved biosafety aspects make microalgae interesting candidates for novel molecular farming systems. As a model organism, the unicellular green alga Chlamydomonas reinhardtii is one of the best studied organisms in this field and provides established methods for transformation, markers and reporters. Its ability for expression of proteins with biopharmaceutical or biotechnological relevance, such as antibodies, enzymes or antigenic peptides, has been demonstrated in a number of cases. Although no commercialized product has been reported so far, the application of algal systems for certain fields, such as edible vaccines, are increasingly gaining interest. Aside from biopharmaceuticals, an additional field for the use of algae for pharmaceutical products could involve novel metabolites, improved by metabolic engineering.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Abe J, Hiwatashi Y, Ito M, Hasebe M, Sekimoto H (2008) Expression of exogenous genes under the control of endogenous HSP70 and CAB promoters in the Closterium peracerosum-strigosum-littorale complex. Plant Cell Physiol 49:625–632
Apt KE, Kroth-Pancic PG, Grossman AR (1996) Stable nuclear transformation of the diatom Phaeodactylum tricornutum. Mol Gen Genet 252:572–579
Bateman JM, Purton S (2000) Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker. Mol Gen Genet 263:404–410
Berthold P, Schmitt R, Mages W (2002) An engineered Streptomyces hygroscopicus aph 7´´ gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. Protist 153:401–412
Blankenship JE, Kindle KL (1992) Expression of chimeric genes by the light-regulated cabII-1 promoter in Chlamydomonas reinhardtii: a cabII-1/nit1 gene functions as a dominant selectable marker in a nit1- nit2- strain. Mol Cell Biol 12:5268–5279
Borowitzka MA (1999) Commercial production of microalgae: ponds, tanks, tubes and fermenters. J Biotechnol 70:313–321
Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, Jones AR, Randolph-Anderson BL, Robertson D, Klein TM, Shark KB, Sanford JC (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240:1534–1538
Brown LE, Sprecher SL, Keller LR (1991) Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation. Mol Cell Biol 11:2328–2332
Cai XH, Brown C, Adhiya J, Traina SJ, Sayre R (1999) Growth and heavy metal binding properties of transgenic Chlamydomonas expressing a foreign metallothionein gene. Int J Phytoremediation 1:53–65
Cerutti H, Johnson AM, Gillham NW, Boynton JE (1997) A eubacterial gene conferring spectinomycin resistance on Chlamydomonas reinhardtii: integration into the nuclear genome and gene expression. Genetics 145:97–110
Chen Y, Li WB, Bai QH, Sun YR (1998) Study on transient expression of GUS gene in Chlorella ellipsoidea (Chlorophyta), by using biolistic particle delivery system. Chin J Oceanol Limnol 47:9–16
Chen HL, Li SS, Huang R, Tsai HJ (2008) Conditional production of a functional fish growth hormone in the transgenic line of Nannochloropsis oculata (Eustigmatophyceae). J Phycol 44:768–776
Chow KC, Tung WL (1999) Electrotransformation of Chlorella vulgaris. Plant Cell Rep 18:778–780
Davies JP, Weeks DP, Grossman AR (1992) Expression of the arylsulfatase gene from the beta 2-tubulin promoter in Chlamydomonas reinhardtii. Nucleic Acids Res 20:2959–2965
Dawson HN, Burlingame R, Cannons AC (1997) Stable transformation of Chlorella: rescue of nitrate reductase-deficient mutants with the nitrate reductase gene. Curr Microbiol 35:356–362
Debuchy R, Purton S, Rochaix JD (1989) The argininosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus. EMBO J 8:2803–2809
Dreesen IA, Charpin-El Hamri G, Fussenegger M (2010) Heat-stable oral alga-based vaccine protects mice from Staphylococcus aureus infection. J Biotechnol 145:273–280
Dunahay TG (1993) Transformation of Chlamydomonas reinhardtii with silicon carbide whiskers. Biotechniques 15:452–455
Dunahay TG, Eric E, Jarvis EE, Roessler PG (1995) Genetic transformation of the diatons Cyclotella cryptica and Navicula saprophila. J Phycol 31:1004–1012
Eichler-Stahlberg A, Weisheit W, Ruecker O, Heitzer M (2009) Strategies to facilitate transgene expression in Chlamydomonas reinhardtii. Planta 229:873–883
El-Sheekh M-M (1999) Stable transformation of the intact cells of Chlorella kessleri with high velocity microprojectiles. Biol Plant Prague 42:209–216
Eriksen NT (2008) The technology of microalgal culturing. Biotechnol Lett 30:1525–1536
Falciatore A, Casotti R, Leblanc C, Abrescia C, Bowler C (1999) Transformation of nonselectable reporter genes in marine diatoms. Mar Biotechnol (NY) 1:239–251
Feng S, Xue L, Liu H, Lu P (2009) Improvement of efficiency of genetic transformation for Dunaliella salina by glass beads method. Mol Biol Rep 36:1433–1439
Fernandez-Sevilla JM, Acien Fernandez FG, Molina Grima E (2010) Biotechnological production of lutein and its applications. Appl Microbiol Biotechnol 86:27–40
Ferris PJ (1995) Localization of the nic-7, ac-29 and thi-10 genes within the mating-type locus of Chlamydomonas reinhardtii. Genetics 141:543–549
Field CB, Behrenfeld MJ, Randerson JT, Falkowski P (1998) Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281:237–240
Fischer N, Rochaix JD (2001) The flanking regions of PsaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii. Mol Genet Genomics 265:888–894
Fischer H, Robl I, Sumper M, Kröger N (1999) Targeting and covalent modification of cell wall and membrane proteins heterologously expressed in the diatom Cylindrotheca fusiformis (Bacillariophyceae). J Phycol 35:113–120
Franklin SE, Mayfield SP (2004) Prospects for molecular farming in the green alga Chlamydomonas reinhardtii. Curr Opin Plant Biol 7:159–165
Franklin SE, Mayfield SP (2005) Recent developments in the production of human therapeutic proteins in eukaryotic algae. Expert Opin Biol Ther 5:225–235
Franklin S, Ngo B, Efuet E, Mayfield SP (2002) Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast. Plant J 30:733–744
Fuhrmann M (2002) Expanding the molecular toolkit for Chlamydomonas reinhardtii – from history to new frontiers. Protist 153:357–364
Fuhrmann M, Oertel W, Hegemann P (1999) A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J 19:353–361
Fuhrmann M, Stahlberg A, Govorunova E, Rank S, Hegemann P (2001) The abundant retinal protein of the Chlamydomonas eye is not the photoreceptor for phototaxis and photophobic responses. J Cell Sci 114:3857–3863
Fuhrmann M, Hausherr A, Ferbitz L, Schödl T, Heitzer M, Hegemann P (2004) Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene. Plant Mol Biol 55:869–881
Goldschmidt-Clermont M (1991) Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of Chlamydomonas. Nucleic Acids Res 19:4083–4089
Griesbeck C, Kobl I, Heitzer M (2006) Chlamydomonas reinhardtii: a protein expression system for pharmaceutical and biotechnological proteins. Mol Biotechnol 34:213–223
Grossman AR (2007) In the grip of algal genomics. Adv Exp Med Biol 616:54–76
Hall LM, Taylor KB, Jones DD (1993) Expression of a foreign gene in Chlamydomonas reinhardtii. Gene 124:75–81
Hawkins RL, Nakamura M (1999) Expression of human growth hormone by the eukaryotic alga, Chlorella. Curr Microbiol 38:335–341
He DM, Qian KX, Shen GF, Zhang ZF, Li YN, Su ZL, Shao HB (2007) Recombination and expression of classical swine fever virus (CSFV) structural protein E2 gene in Chlamydomonas reinhardtii chroloplasts. Colloids Surf B Biointerfaces 55:26–30
Heitzer M, Zschoernig B (2007) Construction of modular tandem expression vectors for the green alga Chlamydomonas reinhardtii using the Cre/lox-system. Biotechniques 43:324–332
Heitzer M, Eckert A, Fuhrmann M, Griesbeck C (2007) Influence of codon bias on the expression of foreign genes in microalgae. Adv Exp Med Biol 616:46–53
Hirakawa Y, Kofuji R, K-i I (2008) Transient transformation of a chlorarachinophyta alga, Lotharella amoebiformis (Chlorarachiophyceae), with uidA and egfp reporter genes. J Phycol 44:814–820
Hosseini Tafreshi A, Shariati M (2009) Dunaliella biotechnology: methods and applications. J Appl Microbiol 107:14–35
Huang J, Liu J, Li Y, Chen F (2008) Isolation and characterization of the phytoene desaturase gene as a potential selective marker for genetic engineering of the astaxanthin-producing green alga Chlorella zofingiensis (Chlorophyta). J Phycol 44:684–690
Kakinuma M, Ikeda M, Coury D, Tominaga H, Kobayashi T, Amano H (2009) Isolation and characterization of the rbcS genes from a sterile mutant of Ulva pertusa (Ulvales, Chlorophyta) and transient gene expression using the rbcS gene promoter. Fish Sci 75:1015–1028
Kathiresan S, Sarada R (2009) Towards genetic improvement of commerically important microalga Haematococcus pluvialis for biotech applications. J Appl Phycol 21:553–558
Kathiresan S, Chandrashekar A, Ravishankar A, Sarada R (2009) Agrobacterium-mediated transformation in the green alga Haematococcus pluvialis (Chlorophyceae volvocales). J Phycol 45:642–649
Kindle KL (1990) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 87:1228–1232
Kindle KL (1998) Nuclear transformation: technology and applications. In: Rochaix JD, Goldschmidt-Clermont M, Merchant S (eds) The molecular biology of chloroplasts and mitochondria in Chlamydomonas. Kluwer Academic, Dordrecht pp 42–61
Kindle KL, Schnell RA, Fernandez E, Lefebvre PA (1989) Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J Cell Biol 109:2589–2601
Koblenz B, Lechtreck K-F (2005) The NIT1 promoter allows inducible and reversible silencing of centrin in Chlamydomonas reinhardtii. Eukaryot Cell 4:1959–1962
Kovar JL, Zhang J, Funke RP, Weeks DP (2002) Molecular analysis of the acetolactate synthase gene of Chlamydomonas reinhardtii and development of a genetically engineered gene as a dominant selectable marker for genetic transformation. Plant J 29:109–117
Kumar SV, Misquitta RW, Reddy VS, Rao BJ, Rajam MV (2004) Genetic transformation of the green alga-Chlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant Sci 166:731–738
Leon-Banares R, Gonzalez-Ballester D, Galvan A, Fernandez E (2004) Transgenic microalgae as green cell-factories. Trends Biotechnol 22:45–52
Lerche K, Hallmann A (2009) Stable nuclear transformation of Gonium pectorale. BMC Biotechnol 9:64–81
Loppes R, Radoux M, Ohresser MC, Matagne RF (1999) Transcriptional regulation of the Nia1 gene encoding nitrate reductase in Chlamydomonas reinhardtii: effects of various environmental factors on the expression of a reporter gene under the control of the Nia1 promoter. Plant Mol Biol 41:701–711
Lumbreras V, Stevens DR, Purton S (1998) Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. Plant J 14:441–447
Manuell A, Beligni MV, Elder JH et al (2007) Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast. Plant Biotechnol J 5:402–412
Matsuo T, Onai K, Okamoto K, Minagawa J, Ishiura M (2006) Real-time monitoring of chloroplast gene expression by a luciferase reporter: evidence for nuclear regulation of chloroplast circadian period. Mol Cell Biol 26:863–870
Mayfield SP, Franklin SE (2005) Expression of human antibodies in eukaryotic micro-algae. Vaccine 23:1828–1832
Mayfield SP, Kindle KL (1990) Stable nuclear transformation of Chlamydomonas reinhardtii by using a C. reinhardtii gene as the selectable marker. Proc Natl Acad Sci USA 87:2087–2091
Mayfield SP, Schultz J (2004) Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast. Plant J 37:449–458
Mayfield SP, Franklin SE, Lerner RA (2003) Expression and assembly of a fully active antibody in algae. Proc Natl Acad Sci USA 100:438–442
Minko I, Holloway SP, Nikaido S, Carter M, Odom OW, Johnson CH, Herrin DL (1999) Renilla luciferase as a vital reporter for chloroplast gene expression in Chlamydomonas. Mol Gen Genet 262:421–425
Molnar A, Schwach F, Studholme DJ, Thuenemann EC, Baulcombe DC (2007) miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii. Nature 447:1126–1129
Molnar A, Bassett A, Thuenemann E, Schwach F, Karkare S, Ossowski S, Weigel D, Baulcombe D (2009) Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. Plant J 58:165–174
Nelson JAE, Savereide PB, Lefebvre PA (1994) The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation. Mol Cell Biol 14:4011–4019
Ohnuma M, Yokoyama T, Inouye T, Sekine Y, Tanaka K (2008) Polyethylene glycol (PEG)-mediated transient gene expression in a red alga, Cyanidioschyzon merolae 10D. Plant Cell Physiol 49:117–120
Plaza M, Herrero M, Cifuentes A, Ibanez E (2009) Innovative natural functional ingredients from microalgae. J Agric Food Chem 57:7159–7170
Potvin G, Zhang Z (2010) Strategies for high-level recombinant protein expression in transgenic microalgae: a review. Biotechnol Adv 28:910–918
Pulz O (2001) Photobioreactors: production systems for phototrophic microorganisms. Appl Microbiol Biotechnol 57:287–293
Quinn JM, Kropat J, Merchant S (2003) Copper response element and Crr1-dependent Ni(2+)-responsive promoter for induced, reversible gene expression in Chlamydomonas reinhardtii. Eukaryot Cell 2:995–1002
Randolph-Anderson BL, Sato R, Johnson AM, Harris EH, Hauser CR, Oeda K, Ishige F, Nishio S, Gillham NW, Boynton JE (1998) Isolation and characterization of a mutant protoporphyrinogen oxidase gene from Chlamydomonas reinhardtii conferring resistance to porphyric herbicides. Plant Mol Biol 38:839–859
Rasala BA, Muto M, Lee PA, Jager M, Cardoso RM, 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 8:719–733
Rechter S, Konig T, Auerochs S, Thulke S, Walter H, Dornenburg H, Walter C, Marschall M (2006) Antiviral activity of Arthrospira-derived spirulan-like substances. Antiviral Res 72:197–206
Remacle C, Cline S, Boutaffala L, Gabilly S, Larosa V, Barbieri MR, Coosemans N, Hamel PP (2009) The ARG9 gene encodes the plastid-resident N-acetyl ornithine aminotransferase in the green alga Chlamydomonas reinhardtii. Eukaryot Cell 8:1460–1463
Ruecker O, Zillner K, Groebner-Ferreira R, Heitzer M (2008) Gaussia-luciferase as a sensitive reporter gene for monitoring promoter activity in the nucleus of the green alga Chlamydomonas reinhardtii. Mol Genet Genomics 280:153–162
Sayre R, Wagner R, Siripornadulsil S, Farias C (2003). Transgenic algae for delivering antigens to an animal. US Patent 7,410,637
Schiedlmeier B, Schmitt R, Muller W, Kirk MM, Gruber H, Mages W, Kirk DL (1994) Nuclear transformation of Volvox carteri. Proc Natl Acad Sci USA 91:5080–5084
Schroda M (2005) RNA silencing in Chlamydomonas: mechanisms and tools. Curr Genet 49:69–84
Schroda M, Blocker D, Beck CF (2000) The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. Plant J 21:121–131
Shimogawara K, Fujiwara S, Grossman A, Usuda H (1998) High-efficiency transformation of Chlamydomonas reinhardtii by electroporation. Genetics 148:1821–1828
Siripornadulsil S, Traina S, Verma DP, Sayre RT (2002) Molecular mechanisms of proline-mediated tolerance to toxic heavy metals in transgenic microalgae. Plant Cell 14:2837–2847
Sizova I, Fuhrmann M, Hegemann P (2001) A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 277:221–229
Sodeinde OA, Kindle KL (1993) Homologous recombination in the nuclear genome of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 90:9199–9203
Specht E, Miyake-Stoner S, Mayfield S (2010) Micro-algae come of age as a platform for recombinant protein production. Biotechnol Lett 32:1373–1383
Steinbrenner J, Sandmann G (2006) Transformation of the green alga Haematococcus pluvialis with a phytoene desaturase for accelerated astaxanthin biosynthesis. Appl Environ Microbiol 72:7477–7484
Stevens DR, Rochaix JD, Purton S (1996) The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas. Mol Gen Genet 251:23–30
Stevens DR, Atteia A, Franzen LG, Purton S (2001) Cycloheximide resistance conferred by novel mutations in ribosomal protein L41 of Chlamydomonas reinhardtii. Mol Gen Genet 264:790–795
Sun M, Qian K, Su N, Chang H, Liu J, Chen G (2003) Foot-and-mouth disease virus VP1 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast. Biotechnol Lett 25:1087–1092
Sun Y, Gao X, Li Q, Zhang Q, Xu Z (2006) Functional complementation of a nitrate reductase defective mutant of a green alga Dunaliella viridis by introducing the nitrate reductase gene. Gene 377:140–149
Surzycki R, Greenham K, Kitayama K, Dibal F, Wagner R, Rochaix JD, Ajam T, Surzycki S (2009) Factors effecting expression of vaccines in microalgae. Biologicals 37:133–138
Tan C, Qin S, Zhang Q, Jiang P, Zhao F (2005) Establishment of a micro-particle bombardment transformation system for Dunaliella salina. J Microbiol 43:361–365
Tang DK, Qiao SY, Wu M (1995) Insertion mutagenesis of Chlamydomonas reinhardtii by electroporation and heterologous DNA. Biochem Mol Biol 36:1025–1035
Ten Lohuis MR, Miller DJ (1998) Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): expression of GUS in microalgae using heterologous promoter constructs. Plant J 13:427–435
Tran M, Zhou B, Pettersson PL et al (2009) Synthesis and assembly of a full-length human monoclonal antibody in algal chloroplasts. Biotechnol Bioeng 104:663–673
Ugwu CU, Aoyagi H, Uchiyama H (2008) Photobioreactors for mass cultivation of algae. Bioresour Technol 99:4021–4028
Walker TL, Becker DK, Dale JL, Collet C (2005a) Towards the development of a nuclear transformation system for Dunaliella tertiolecta. J Appl Phycol 17:363–368
Walker TL, Purton S, Becker DK, Collet C (2005b) Microalgae as bioreactors. Plant Cell Rep 24:629–641
Wang T, Xue L, Hou W, Yang B, Chai Y, Ji X, Wang Y (2007) Increased expression of transgene in stably transformed cells of Dunaliella salina by matrix attachment regions. Appl Microbiol Biotechnol 76:651–657
Wang X, Brandsma M, Tremblay R, Maxwell D, Jevnikar AM, Huner N, Ma S (2008) A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65). BMC Biotechnol 8:87–99
Yang Z, Li Y et al (2006) Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast. Chin Sci Bull 51:1703–1709
Yasuhara-Bell J, Lu Y (2010) Marine compounds and their antiviral activities. Antiviral Res 86:231–240
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 Appl Phycol 36:379–386
Zaslavskaia LA, Lippmeier JC, Shih C, Grossman AR, Apt KE (2001) Trophic conversion of an obligate photoautotrophic organism through metabolic engineering. Science 292:2073–2075
Zhang Y-K, Shen G-F, Ru B-G (2006) Survival of human metallothionein-2 transplastomic Chlamydomonas reinhardtii ultraviolet B exposure. Acta Biochim Biophys Sin 38:187–193
Acknowledgement
This work was supported by the MCI Doctoral Grant Program. We would like to thank Ian Wallace for critical reviewing the manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media B.V.
About this chapter
Cite this chapter
Griesbeck, C., Kirchmayr, A. (2012). Algae: An Alternative to the Higher Plant System in Gene Farming. In: Wang, A., Ma, S. (eds) Molecular Farming in Plants: Recent Advances and Future Prospects. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-2217-0_6
Download citation
DOI: https://doi.org/10.1007/978-94-007-2217-0_6
Published:
Publisher Name: Springer, Dordrecht
Print ISBN: 978-94-007-2216-3
Online ISBN: 978-94-007-2217-0
eBook Packages: Chemistry and Materials ScienceChemistry and Material Science (R0)