Metabolic engineering in the chloroplast of Chlamydomonas reinhardtii
Thanyanun Ninlayarn and Saul Purton
Structural & Molecular Biology, UCL, Gower St, London,UK, WC1E 6BT
Carotenoids are C40 hydrocarbon pigments synthesised in the plastid organelle of plant and algae. These pigments, especially the oxygenated ketocarotenoid derivatives, are of considerable commercial interest because of their strong antioxidant properties and their use in the aquaculture, food and pharmaceutical industries. We are investigating whether C. reinhardtii can be engineered to produce a novel and/or high levels of ketocarotenoids. Our approach is to target foreign genes involved in carotenogenesis into the chloroplast genome by using a chloroplast expression vector (pASapI). Following DNA introduction into the chloroplast by microparticle bombardment, the gene cassettes are inserted via homologous recombination into the chloroplast genome at a neutral locus downstream of the PSII gene, psbH. The coding sequences for several algal beta-cartotene ketolases (BKT) were cloned into the pASapI, and their catalytic activities in E.coli were verified by complementation and HPLC analysis since the chloroplast promoter element is active also in E. coli, allowing us to confirm the functionality of the gene cassettes prior to chloroplast transformations. C. reinhardtii transformants have been obtained with a codon-optimised version of the Muriella zofingiensis BKT. Molecular analysis of the transformants lines has confirmed that the gene cassette has integrated successfully into the chloroplast genome. In addition, phytoene synthase (PSY/crtB) genes derived from cyanobacteria, archaea and Chlamydomonas itself have been cloned into pASapI and transformant lines are being produced using a nuclear mutant (FN68) that is defective in phytoene synthase.
e-mail address of presenting author: