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

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  • Julie Annemarie Zita Zedler
  • Doris Gangl
  • Björn Robert 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.

Original languageEnglish
JournalJournal of Applied Phycology
Volume27
Issue number6
Pages (from-to)2271-2277
Number of pages7
ISSN0921-8971
DOIs
Publication statusPublished - 2015

    Research areas

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

ID: 162899552