Russian Journal of Plant Physiology

, Volume 60, Issue 4, pp 491–499

Genetic engineering of algal chloroplasts: Progress and prospects

  • S. Purton
  • J. B. Szaub
  • T. Wannathong
  • R. Young
  • C. K. Economou
Reviews

Abstract

The last few years has seen an ever-increasing interest in the exploitation of microalgae as recombinant platforms for the synthesis of novel bioproducts. These could be biofuel molecules, speciality enzymes, nutraceuticals, or therapeutic proteins, such as antibodies, hormones, and vaccines. This exploitation requires the development of new genetic engineering technologies for those fast-growing, robust species suited for intensive commercial cultivation in bioreactor systems. In particular, there is a need for routine methods for the genetic manipulation of the chloroplast genome, for two reasons: firstly, the chloroplast genetic system is well-suited to the targeted insertion into the genome and high-level expression of foreign genes; secondly, the organelle is the site of numerous biosynthetic pathways and therefore represents the obvious “chassis,” on which to bolt new metabolic pathways that divert the carbon fixed by photosynthesis into novel hydrocarbons, pigments, etc. Stable transformation of the algal chloroplast was first demonstrated in 1988, using the model chlorophyte, Chlamydomonas reinhardtii. Since that time, tremendous advances have been made in the development of sophisticated tools for engineering this particular species, and efforts to transfer this technology to other commercially attractive species are starting to bear fruit. In this article, we review the current field of algal chloroplast transgenics and consider the prospects for the future.

Keywords

Chlamydomonas reinhardtii algae chloroplast genetic engineering transformation transplastomics 

Abbreviations

GOI

gene-of-interest

IPTG

isopropyl-β-D-thiolgalactopyranoside

UTR

untranslated region

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Bhattacharya, D., Yoon, H.S., and Hackett, J.D., Photosynthetic eukaryotes unite: endosymbiosis connects the dots, BioEssays, 2004, vol. 26, pp. 50–60.PubMedCrossRefGoogle Scholar
  2. 2.
    Green, B.R., Chloroplast genomes of photosynthetic eukaryotes, Plant J., 2011, vol. 66, pp. 34–44.PubMedCrossRefGoogle Scholar
  3. 3.
    Barkan, A., Expression of plastid genes: organelle-specific elaborations on a prokaryotic scaffold, Plant Physiol., 2011, vol. 155, pp. 1520–1532.PubMedCrossRefGoogle Scholar
  4. 4.
    Wang, H.H., Yin, W.B., and Hu, Z.M., Advances in Chloroplast Engineering, J. Genet. Genom., 2009, vol. 7, pp. 387–398.CrossRefGoogle Scholar
  5. 5.
    Wani, S.H., Haider, N., Kumar, H., and Singh, N.B., Plant plastid engineering, Curr. Genom., 2010, vol. 11, pp. 500–512.CrossRefGoogle Scholar
  6. 6.
    Bowsher, C.G. and Tobin, A.K., Compartmentation of metabolism within mitochondria and plastids, J. Exp. Bot., 2001, vol. 52, pp. 513–527.PubMedCrossRefGoogle Scholar
  7. 7.
    Purton, S., Tools and techniques for chloroplast transformation of Chlamydomonas, Adv. Exp. Med. Biol., 2007, vol. 616, pp. 34–45.PubMedCrossRefGoogle Scholar
  8. 8.
    Radakovits, R., Jinkerson, R.E., Darzins, A., and Posewitz, M.C., Genetic engineering of algae for enhanced biofuel production, Euk. Cell, 2010, vol. 9, pp. 486–501.CrossRefGoogle Scholar
  9. 9.
    Mayfield, S.P., Manuell, A.L., Chen, S., Wu, J., Tran, M., Siefker, D., Muto, M., and Marin-Navarro, J., Chlamydomonas reinhardtii chloroplasts as protein factories, Curr. Opin. Biotechnol., 2007, vol. 18, pp. 126–133.PubMedCrossRefGoogle Scholar
  10. 10.
    Johanningmeier, U. and Fischer, D., Perspective for the use of genetic transformants in order to enhance the synthesis of the desired metabolites: engineering chloroplasts of microalgae for the production of bioactive compounds, Adv. Exp. Med. Biol., 2010, vol. 698, pp. 144–151.PubMedCrossRefGoogle Scholar
  11. 11.
    Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler, J.P., Johnson, A.M., Jones, A.R., Randolph-Anderson, B.L., Robertson, D., Klein, T.M., and Shark, K.B., Chloroplast transformation in Chlamydomonas with high velocity microprojectiles, Science, 1988, vol. 240, pp. 1534–1538.PubMedCrossRefGoogle Scholar
  12. 12.
    Taylor, N.J. and Fauquet, C.M., Microparticle bombardment as a tool in plant science and agricultural biotechnology, DNA Cell Biol., 2002, vol. 21, pp. 963–977.PubMedCrossRefGoogle Scholar
  13. 13.
    Blowers, A.D., Bogorad, L., Shark, K.B., and Sanford, J.C., Studies on Chlamydomonas chloroplast transformation: foreign DNA can be stably maintained in the chromosome, Plant Cell, 1989, vol. 1, pp. 123–132.PubMedGoogle Scholar
  14. 14.
    Goldschmidt-Clermont, M., Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of Chlamydomonas, Nucleic Acids Res., 1991, vol. 19, pp. 4083–4089.PubMedCrossRefGoogle Scholar
  15. 15.
    Bateman, J.M. and Purton, S., Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker, Mol. Gen. Genet., 2000, vol. 263, pp. 404–410.PubMedCrossRefGoogle Scholar
  16. 16.
    Rochaix, J.D., Chlamydomonas reinhardtii as the photosynthetic yeast, Annu. Rev. Genet., 1995, vol. 29, pp. 209–230.PubMedCrossRefGoogle Scholar
  17. 17.
    Stern, D.B., The Chlamydomonas Sourcebook, New York: Academic, 2009, vol. 2.Google Scholar
  18. 18.
    Cardi, T., Lenzi, P., and Maliga, P., Chloroplasts as expression platforms for plant-produced vaccines, Expert Rev. Vaccines, 2010, vol. 9, pp. 893–911.PubMedCrossRefGoogle Scholar
  19. 19.
    Scotti, N., Rigano, M.M., and Cardi, T., Production of foreign proteins using plastid transformation, Biotechnol. Adv., 2012, vol. 30, pp. 387–397.PubMedCrossRefGoogle Scholar
  20. 20.
    Robertson, D.E., Jacobson, S.A., Morgan, F., Berry, D., Church, G.M., and Afeyan, N.B., A new dawn for industrial photosynthesis, Photosynth. Res., 2011, vol. 107, pp. 269–277.PubMedCrossRefGoogle Scholar
  21. 21.
    Kindle, K.L., Richards, K.L., and Stern, D.B., Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii, Proc. Natl. Acad. Sci. USA, 1991, vol. 88, pp. 1721–1725.PubMedCrossRefGoogle Scholar
  22. 22.
    Doetsch, N.A., Favreau, M.R., Kuscuoglu, N., Thompson, M.D., and Hallick, R.B., Chloroplast transformation in Euglena gracilis: splicing of a group III twintron transcribed from a transgenic PsbK operon, Curr. Genet., 2001, vol. 39, pp. 49–60.PubMedCrossRefGoogle Scholar
  23. 23.
    Gutiérrez, C.L., Gimpel, J., Escobar, C., Marshall, S.H., and Henríquez, V., Chloroplast genetic tool for the green microalgae Haematococcus pluvialis (Chlorophyceae, Volvocales), J. Phycol., 2012, vol. 48, pp. 976–983.CrossRefGoogle Scholar
  24. 24.
    Lapidot, M., Raveh, D., Sivan, A., Arad, S.M., and Shapira, M., Stable chloroplast transformation of the unicellular red alga Porphyridium species, Plant Physiol., 2002, vol. 129, pp. 7–12.PubMedCrossRefGoogle Scholar
  25. 25.
    Remacle, C., Cline, S., Boutaffala, L., Gabilly, S., Larosa, V., Barbieri, M.R., Coosemans, N., and Hamel, P.P., The ARG9 gene encodes the plastid-resident N-acetyl ornithine aminotransferase in the green alga Chlamydomonas reinhardtii, Euk. Cell, 2009, vol. 8, pp. 1460–1463.CrossRefGoogle Scholar
  26. 26.
    Franklin, S., Ngo, B., Efuet, E., and Mayfield, S.P., Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast, Plant J., 2002, vol. 30, pp. 733–744.PubMedCrossRefGoogle Scholar
  27. 27.
    Sakamoto, W., Kindle, K.L., and Stern, D.B., In vivo analysis of Chlamydomonas chloroplast PetD gene expression using stable transformation of beta-glucuronidase translational fusions, Proc. Natl. Acad. Sci. USA, 1993, vol. 90, pp. 497–501.PubMedCrossRefGoogle Scholar
  28. 28.
    Matsuo, T., Onai, K., Okamoto, K., Minagawa, J., and Ishiura, M., Real-time monitoring of chloroplast gene expression by a luciferase reporter: evidence for nuclear regulation of chloroplast circadian period, Mol. Cell. Biol., 2006, vol. 26, pp. 863–870.PubMedCrossRefGoogle Scholar
  29. 29.
    Mayfield, S.P. and Schultz, J., Development of a luciferase reporter gene, LuxCt, for Chlamydomonas reinhardtii chloroplast, Plant J., 2004, vol. 37, pp. 449–458.PubMedCrossRefGoogle Scholar
  30. 30.
    Michelet, L., Lefebvre-Legendre, L., Burr, S.E., Rochaix, J.-D., and Goldschmidt-Clermont, M., Enhanced chloroplast transgene expression in a nuclear mutant of Chlamydomonas, Plant Biotechnol. J., 2011, vol. 9, pp. 565–574.PubMedCrossRefGoogle Scholar
  31. 31.
    Rasala, B.A., Muto, M., Sullivan, J., and Mayfield, S.P., Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5’ untranslated region optimization, Plant Biotechnol. J., 2011, vol. 9, pp. 674–683.PubMedCrossRefGoogle Scholar
  32. 32.
    Fletcher, S.P., Muto, M., and Mayfield, S.P., Optimization of recombinant protein expression in the chloroplasts of green algae, Adv. Exp. Med. Biol., 2007, vol. 616, pp. 90–98.PubMedCrossRefGoogle Scholar
  33. 33.
    Kato, K., Marui, T., Kasai, S., and Shinmyo, A., Artificial control of transgene expression in Chlamydomonas reinhardtii chloroplast using the Lac regulation system from Escherichia coli, J. BioSci. Bioeng., 2007, vol. 104, pp. 207–213.PubMedCrossRefGoogle Scholar
  34. 34.
    Surzycki, R., Cournac, L., Peltier, G., and Rochaix, J.-D., Potential for hydrogen production with inducible chloroplast gene expression in Chlamydomonas, Proc. Natl. Acad. Sci. USA, 2007, vol. 104, pp. 17 548–17 553.CrossRefGoogle Scholar
  35. 35.
    Wu, S., Xu, L., Huang, R., and Wang, Q., Improved biohydrogen production with an expression of codonoptimized hemH and lba genes in the chloroplast of Chlamydomonas reinhardtii, Bioresour. Technol., 2011, vol. 102, pp. 2610–2616.PubMedCrossRefGoogle Scholar
  36. 36.
    Ellis, T., Adie, T., and Baldwin, G.S., DNA assembly for synthetic biology: from parts to pathways and beyond, Integr. Biol. (Camb), 2011, vol. 3, pp. 109–118.CrossRefGoogle Scholar
  37. 37.
    Dreesen, I.A.J., Charpin-El, HamriG., and Fussenegger, M., Heat-stable oral alga-based vaccine protects mice from Staphylococcus aureus infection, J. Biotechnol., 2010, vol. 145, pp. 273–280.PubMedCrossRefGoogle Scholar
  38. 38.
    Yoon, S.-M., Kim, S.Y., Li, K.F., Yoon, B.H., Choe, S., and Kuo, M.M.-C., Transgenic microalgae expressing Escherichia coli AppA phytase as feed additive to reduce phytate excretion in the manure of young broiler chicks, Appl. Microbiol. Biotechnol., 2011, vol. 91, pp. 553–563.PubMedCrossRefGoogle Scholar
  39. 39.
    Gregory, J.A., Li, F., Tomosada, L.M., Cox, C.J., Topol, A.B., Vinetz, J.M., and Mayfield, S., Algaeproduced Pfs25 elicits antibodies that inhibit malaria transmission, PLoS ONE, 2012, vol. 7: e37179.Google Scholar
  40. 40.
    Jones, C.S., Luong, T., Hannon, M., Tran, M., Gregory, J.A., Shen, Z., Briggs, S.P., and Mayfield, S.P., Heterologous expression of the C-terminal antigenic domain of the malaria vaccine candidate Pfs48/45 in the green algae Chlamydomonas reinhardtii, Appl. Microbiol. Biotechnol., 2012, May 18 [Epub ahead of print].Google Scholar
  41. 41.
    Specht, E., Miyake-Stoner, S., and Mayfield, S., Micro-algae come of age as a platform for recombinant protein production, Biotechnol. Lett., 2010, vol. 32, pp. 1373–1383.PubMedCrossRefGoogle Scholar
  42. 42.
    Tran, M., Zhou, B., Pettersson, P.L., Gonzalez, M.J., and Mayfield, S.P., Synthesis and assembly of a full-length human monoclonal antibody in algal chloroplasts, Biotechnol. Bioeng., 2009, vol. 104, pp. 663–673.PubMedGoogle Scholar
  43. 43.
    Fukusaki, E.-I., Nishikawa, T., Kato, K., Shinmyo, A., Hemmi, H., Nishino, T., and Kobayaski, A., Introduction of the archaebacterial geranylgeranyl pyrophosphate synthase gene into Chlamydomonas reinhardtii chloroplast, J. BioSci. Bioeng., 2003, vol. 95, pp. 283–287.PubMedGoogle Scholar
  44. 44.
    Tan, C.-P., Zhao, F.-Q., Su, Z.-L., Liang, C.-W., and Qin, S., Expression of β-carotene hydroxylase gene (crtR-B) from the green alga Haematococcus pluvialis in chloroplasts of Chlamydomonas reinhardtii, J. Appl. Phycol., 2007, vol. 19, pp. 347–355.CrossRefGoogle Scholar
  45. 45.
    Su, Z.-L., Qian, K.-X., Tan, C.-P., Meng, C.-X., and Qin, S., Recombination and heterologous expression of allophycocyanin gene in the chloroplast of Chlamydomonas reinhardtii, Acta Biochim. Biophys. Sinica (Shanghai), 2005, vol. 37, pp. 709–712.CrossRefGoogle Scholar
  46. 46.
    Szaub, J.B., Genetic engineering of green microalgae for the production of biofuel and high value products, Ph.D. Dissertation, London: Univ. College London, 2012.Google Scholar
  47. 47.
    Materna, A.C., Sturm, S., Kroth, P.G., and Lavaud, J., First induced plastid genome mutations in an alga with secondary plastids: PsbA mutations in the diatom Phaeodactylum tricornutum (Bacillariophyceae) reveal consequences on the regulation of photosynthesis, J. Phycol., 2009, vol. 45, pp. 838–846.CrossRefGoogle Scholar
  48. 48.
    Fischer, N., Stampacchia, O., Redding, K., and Rochaix, J.D., Selectable marker recycling in the chloroplast, Mol. Gen. Genet., 1996, vol. 251, pp. 373–380.PubMedCrossRefGoogle Scholar
  49. 49.
    Adachi, T., Takase, H., and Tomizawa, K., Introduction of a 50 kbp DNA fragment into the plastid genome, BoiSci. Biotechnol. Biochem., 2007, vol. 71, pp. 2266–2273.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2013

Authors and Affiliations

  • S. Purton
    • 1
  • J. B. Szaub
    • 1
  • T. Wannathong
    • 1
  • R. Young
    • 1
  • C. K. Economou
    • 1
  1. 1.Algal Biotechnology Group, Institute of Structural and Molecular BiologyUniversity College LondonLondonUK

Personalised recommendations