title: Expression and membrane-targeting of an active plant cytochrome P450 in the chloroplast of the green alga Chlamydomonas reinhardtii.
abstract: The unicellular green alga Chlamydomonas reinhardtii has potential as a cell factory for the production of recombinant proteins and other compounds, but mainstream adoption has been hindered by a scarcity of genetic tools and a need to identify products that can be generated in a cost-effective manner. A promising strategy is to use algal chloroplasts as a site for synthesis of high value bioactive compounds such as diterpenoids since these are derived from metabolic building blocks that occur naturally within the organelle. However, synthesis of these complex plant metabolites requires the introduction of membrane-associated enzymes including cytochrome P450 enzymes (P450s). Here, we show that a gene (CYP79A1) encoding a model P450 can be introduced into the C. reinhardtii chloroplast genome using a simple transformation system. The gene is stably expressed and the P450 is efficiently targeted into chloroplast membranes by means of its endogenous N-terminal anchor domain, where it is active and accounts for 0.4% of total cell protein. These results provide proof of concept for the introduction of diterpenoid synthesis pathways into the chloroplast of C. reinhardtii.
authors.affiliation: Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom.
authors.forename: Doris
authors.initials: D
authors.lastname: Gangl
authors.affiliation: Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom.
authors.forename: Julie A Z
authors.initials: JA
authors.lastname: Zedler
authors.affiliation: Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Artur
authors.initials: A
authors.lastname: Włodarczyk
authors.affiliation: Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Poul Erik
authors.initials: PE
authors.lastname: Jensen
authors.affiliation: Institute of Structural & Molecular Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
authors.forename: Saul
authors.initials: S
authors.lastname: Purton
authors.affiliation: Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom. Electronic address: c.robinson-504@kent.ac.uk.
authors.forename: Colin
authors.initials: C
authors.lastname: Robinson
mesh_terms: D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000737:chemistry / Q000235:genetics*; D002736:Chloroplasts / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects / Q000378:metabolism*; D004224:Diterpenes / Q000378:metabolism; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism*; D010940:Plant Proteins / Q000032:analysis
chemical_list: D004224:Diterpenes; D045322:Photosynthetic Reaction Center Complex Proteins; D010940:Plant Proteins; D003577:Cytochrome P-450 Enzyme System; C089817:cytochrome P450TYR
keywords: Chlamydomonas; Chloroplast transformation; Cytochrome P450; Diterpenoids; Thylakoid membrane