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

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 - 2015/12/1

Y1 - 2015/12/1

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

SN - 0921-8971

VL - 27

SP - 2271

EP - 2277

JO - Journal of Applied Phycology

JF - Journal of Applied Phycology

IS - 6

ER -