Stable expression of a bifunctional diterpene synthase in the chloroplast of Chlamydomonas reinhardtii
- 683 Downloads
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.
KeywordsChlamydomonas 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.
- 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
- 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
- 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
- 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
- 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
- Specht EA, Mayfield SP (2014) Algae-based oral recombinant vaccines. Front Microbiol 5Google 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 287:12121–12131PubMedCentralCrossRefPubMedGoogle Scholar