Skip to main content
SpringerLink
Log in

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

  • Published:
Journal of Applied Phycology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • 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 113:217–225

    Article  CAS  Google Scholar 

  • 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.0061473

  • 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 1132:401–411

    Article  CAS  PubMed  Google Scholar 

  • Gorman DS, Levine RP (1965) Cytochrome f and plastocyanin: their sequence in the photosynthetic electron. Proc Natl Acad Sci U S A 54:1665–1669

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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.0037179

  • Gregory JA, Topol AB, Doerner DZ, Mayfield S (2013) Alga-produced cholera toxin-Pfs25 fusion proteins as oral vaccines. Appl Environ Microbiol 79:3917–3925

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Jin H, Chen L, Wang J, Zhang W (2014) Engineering biofuel tolerance in non-native producing microorganisms. Biotechnol Adv 32:541–548

    Article  CAS  PubMed  Google Scholar 

  • 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 97:1987–1995

    Article  CAS  PubMed  Google Scholar 

  • 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 105:1085–1090

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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 88:1721–1725

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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 66:770–780

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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 1364:63–72

    Article  Google Scholar 

  • Pourmir A, Noor-Mohammadi S, Johannes TW (2013) Production of xylitol by recombinant microalgae. J Biotechnol 165:178–183

    Article  CAS  PubMed  Google Scholar 

  • Purton S, Szaub JB, Wannathong T, Young R, Economou CK (2013) Genetic engineering of algal chloroplasts: progress and prospects. Russ J Plant Physiol 60:491–499

    Article  CAS  Google Scholar 

  • 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:e43349

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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 31:479–494

    Article  CAS  PubMed  Google Scholar 

  • 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 116:133–139

    Article  CAS  Google Scholar 

  • Specht EA, Mayfield SP (2014) Algae-based oral recombinant vaccines. Front Microbiol 5

  • Sueoka N (1960) Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardi. Proc Natl Acad Sci U S A 46:83–91

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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 110:2826–2835

    Article  CAS  PubMed  Google Scholar 

  • 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–E22

    Article  PubMed Central  PubMed  Google Scholar 

  • Wang CG, Hu ZL, Lei AP, Jin BH (2010) Biosynthesis of poly-3-hydroxybutyrate (PHB) in the transgenic green alga Chlamydomonas reinhardtii. J Phycol 46:396–402

    Article  CAS  Google Scholar 

  • 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 287:12121–12131

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

  1. School of Biosciences, University of Kent, Ingram Building, Canterbury, CT2 7NJ, UK

    Julie A. Z. Zedler, Doris Gangl & Colin Robinson

  2. Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark

    Björn Hamberger

  3. Algal Biotechnology Group, Institute of Structural and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK

    Saul Purton

Authors
  1. Julie A. Z. Zedler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Doris Gangl
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Björn Hamberger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Saul Purton
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Colin Robinson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Colin Robinson.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 836 kb)

ESM 2

(PDF 290 kb)

ESM 3

(PDF 405 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zedler, J.A.Z., Gangl, D., Hamberger, B. et al. Stable expression of a bifunctional diterpene synthase in the chloroplast of Chlamydomonas reinhardtii . J Appl Phycol 27, 2271–2277 (2015). https://doi.org/10.1007/s10811-014-0504-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10811-014-0504-2

Keywords

Search

Navigation