Journal of Applied Phycology

, Volume 27, Issue 6, pp 2271–2277 | Cite as

Stable expression of a bifunctional diterpene synthase in the chloroplast of Chlamydomonas reinhardtii

  • Julie A. Z. Zedler
  • Doris Gangl
  • Björn Hamberger
  • Saul Purton
  • Colin Robinson


Chlamydomonas reinhardtii has been shown to hold significant promise as a production platform for recombinant proteins, but transformation of the nuclear genome is still a non-trivial process due to random gene insertion and frequent silencing. Insertion of transgenes into the chloroplasts is an alternative strategy, and we report here the stable expression of a large (91 kDa) protein in the chloroplast using a recently developed low-cost transformation protocol. Moreover, selection of transformants is based on restoration of prototrophy using an endogenous gene (psbH) as the marker, thereby allowing the generation of transgenic lines without the use of antibiotic-resistance genes. Here, we have expressed a bifunctional diterpene synthase in C. reinhardtii chloroplasts. Homoplasmic transformants were obtained with the expressed enzyme accounting for 3.7 % of total soluble protein. The enzyme was purified to homogeneity and expression was shown to have a small but reproducible effect on growth rate at the end of log phase growth. These results demonstrate that large recombinant enzymes can be synthesised in the algal chloroplast, and serve to underline its potential as a platform for the biosynthesis of novel metabolites.


Chlamydomonas Chlorophyta Chloroplast transformation Recombinant protein Diterpene synthase Glass bead Endogenous marker 



The authors thank Kevin Howland (Biomolecular Science Facility, School of Biosciences, University of Kent) for his help with peptide mass fingerprint analysis and Umaima Al-Hoqani for the codon optimisation of tps4. The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/ under REA grant agreement no. 317184.

Supplementary material

10811_2014_504_MOESM1_ESM.pdf (837 kb)
ESM 1 (PDF 836 kb)
10811_2014_504_MOESM2_ESM.pdf (291 kb)
ESM 2 (PDF 290 kb)
10811_2014_504_MOESM3_ESM.pdf (405 kb)
ESM 3 (PDF 405 kb)


  1. Campos-Quevedo N, Rosales-Mendoza S, Paz-Maldonado LMT, Martinez-Salgado L, Guevara-Arauza JC, Soria-Guerra RE (2013) Production of milk-derived bioactive peptides as precursor chimeric proteins in chloroplasts of Chlamydomonas reinhardtii. Plant Cell Tiss Organ Cult 113:217–225CrossRefGoogle Scholar
  2. Demurtas OC, Massa S, Ferrante P, Venuti A, Franconi R, Giuliano G (2013) A Chlamydomonas-derived human papillomavirus 16 E7 vaccine induces specific tumor protection. PloS One 8. doi:10.1371/journal.pone.0061473Google Scholar
  3. Economou C, Wannathong T, Szaub J, Purton S (2014) A simple, low-cost method for chloroplast transformation of the green alga Chlamydomonas reinhardtii. Methods Mol Biol 1132:401–411CrossRefPubMedGoogle Scholar
  4. Gorman DS, Levine RP (1965) Cytochrome f and plastocyanin: their sequence in the photosynthetic electron. Proc Natl Acad Sci U S A 54:1665–1669PubMedCentralCrossRefPubMedGoogle Scholar
  5. Gregory JA, Li FW, Tomosada LM, Cox CJ, Topol AB, Vinetz JM, Mayfield S (2012) Algae-produced Pfs25 elicits antibodies that inhibit malaria transmission. PloS One 7. doi:10.1371/journal.pone.0037179Google Scholar
  6. Gregory JA, Topol AB, Doerner DZ, Mayfield S (2013) Alga-produced cholera toxin-Pfs25 fusion proteins as oral vaccines. Appl Environ Microbiol 79:3917–3925PubMedCentralCrossRefPubMedGoogle Scholar
  7. Jin H, Chen L, Wang J, Zhang W (2014) Engineering biofuel tolerance in non-native producing microorganisms. Biotechnol Adv 32:541–548CrossRefPubMedGoogle Scholar
  8. Jones CS, Luong T, Hannon M, Tran M, Gregory JA, Shen ZX, Briggs SP, Mayfield SP (2013) Heterologous expression of the C-terminal antigenic domain of the malaria vaccine candidate Pfs48/45 in the green algae Chlamydomonas reinhardtii. Appl Microbiol Biotechnol 97:1987–1995CrossRefPubMedGoogle Scholar
  9. Keeling C, Weisshaar S, Lin R, Bohlmann J (2008) Functional plasticity of paralogous diterpene synthases involved in conifer defense. Proc Natl Acad Sci U S A 105:1085–1090PubMedCentralCrossRefPubMedGoogle Scholar
  10. Kindle KL, Richards KL, Stern DB (1991) Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 88:1721–1725PubMedCentralCrossRefPubMedGoogle Scholar
  11. Kropat J, Hong-Hermesdorf A, Casero D, Ent P, Castruita M, Pellegrini M, Merchant SS, Malasarn D (2011) A revised mineral nutrient supplement increases biomass and growth rate in Chlamydomonas reinhardtii. Plant J 66:770–780PubMedCentralCrossRefPubMedGoogle Scholar
  12. O’Connor HE, Ruffle SV, Cain AJ, Deak Z, Vass I, Nugent JHA, Purton S (1998) The 9-kDa phosphoprotein of photosystem II. generation and characterisation of Chlamydomonas mutants lacking PSII-H and a site-directed mutant lacking the phosphorylation site. BBA-Bioenerg 1364:63–72CrossRefGoogle Scholar
  13. Pourmir A, Noor-Mohammadi S, Johannes TW (2013) Production of xylitol by recombinant microalgae. J Biotechnol 165:178–183CrossRefPubMedGoogle Scholar
  14. Purton S, Szaub JB, Wannathong T, Young R, Economou CK (2013) Genetic engineering of algal chloroplasts: progress and prospects. Russ J Plant Physiol 60:491–499CrossRefGoogle Scholar
  15. 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:e43349PubMedCentralCrossRefPubMedGoogle Scholar
  16. Rosales-Mendoza S, Paz-Maldonado LMT, Soria-Guerra RE (2012) Chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins: current status and perspectives. Plant Cell Rep 31:479–494CrossRefPubMedGoogle Scholar
  17. Soria-Guerra RE, Ramirez-Alonso JI, Ibanez-Salazar A, Govea-Alonso DO, Paz-Maldonado LMT, Banuelos-Hernandez B, Korban SS, Rosales-Mendoza S (2014) Expression of an HBcAg-based antigen carrying angiotensin II in Chlamydomonas reinhardtii as a candidate hypertension vaccine. Plant Cell Tiss Organ Cult 116:133–139CrossRefGoogle Scholar
  18. Specht EA, Mayfield SP (2014) Algae-based oral recombinant vaccines. Front Microbiol 5Google Scholar
  19. Sueoka N (1960) Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardi. Proc Natl Acad Sci U S A 46:83–91PubMedCentralCrossRefPubMedGoogle Scholar
  20. Tran M, Henry RE, Siefker D, Van C, Newkirk G, Kim J, Bui J, Mayfield SP (2013a) Production of anti-cancer immunotoxins in algae: ribosome inactivating proteins as fusion partners. Biotechnol Bioeng 110:2826–2835CrossRefPubMedGoogle Scholar
  21. Tran M, Van C, Barrera DJ, Pettersson PL, Peinado CD, Bui J, Mayfield SP (2013b) Production of unique immunotoxin cancer therapeutics in algal chloroplasts. Proc Natl Acad Sci U S A 110:E15–E22PubMedCentralCrossRefPubMedGoogle Scholar
  22. Wang CG, Hu ZL, Lei AP, Jin BH (2010) Biosynthesis of poly-3-hydroxybutyrate (PHB) in the transgenic green alga Chlamydomonas reinhardtii. J Phycol 46:396–402CrossRefGoogle Scholar
  23. Zerbe P, Chiang A, Yuen M, Hamberger B, Hamberger B, Draper JA, Britton R, Bohlmann J (2012) Bifunctional cis-abienol synthase from Abies balsamea discovered by transcriptome sequencing and its implications for diterpenoid fragrance production. J Biol Chem 287:12121–12131PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Julie A. Z. Zedler
    • 1
  • Doris Gangl
    • 1
  • Björn Hamberger
    • 2
  • Saul Purton
    • 3
  • Colin Robinson
    • 1
  1. 1.School of BiosciencesUniversity of KentCanterburyUK
  2. 2.Copenhagen Plant Science Centre, Department of Plant and Environmental SciencesUniversity of CopenhagenCopenhagenDenmark
  3. 3.Algal Biotechnology Group, Institute of Structural and Molecular BiologyUniversity College LondonLondonUK

Personalised recommendations