Stable expression of a bifunctional diterpene synthase in the chloroplast of Chlamydomonas reinhardtii
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Stable expression of a bifunctional diterpene synthase in the chloroplast of Chlamydomonas reinhardtii. / Zedler, Julie Annemarie Zita; Gangl, Doris; Hamberger, Björn Robert; Purton, Saul; Robinson, Colin.
In: Journal of Applied Phycology, Vol. 27, No. 6, 2015, p. 2271-2277.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Stable expression of a bifunctional diterpene synthase in the chloroplast of Chlamydomonas reinhardtii
AU - Zedler, Julie Annemarie Zita
AU - Gangl, Doris
AU - Hamberger, Björn Robert
AU - Purton, Saul
AU - Robinson, Colin
PY - 2015
Y1 - 2015
N2 - 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.
AB - 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.
KW - Chlamydomonas
KW - Chlorophyta
KW - Chloroplast transformation
KW - Diterpene synthase
KW - Endogenous marker
KW - Glass bead
KW - Recombinant protein
U2 - 10.1007/s10811-014-0504-2
DO - 10.1007/s10811-014-0504-2
M3 - Journal article
VL - 27
SP - 2271
EP - 2277
JO - Journal of Applied Phycology
JF - Journal of Applied Phycology
SN - 0921-8971
IS - 6
ER -
ID: 162899552