Limitations in the biosynthesis of fucoxanthin as targets for genetic engineering in Phaeodactylum tricornutum

The carotenoid fucoxanthin found in the Phaeophyceae and Bacillariophyceae (diatoms) is an antioxidative agent used as a nutraceutical for humans. In algae, terpenoids (including carotenoids) are synthesized via the 1-deoxy-d-xylulose 5-phosphate synthase pathway. The gene dxs encoding a 1-deoxy-d-xylulose 5-phosphate synthase was identified in the genome of Phaeodactylum tricornutum and its complementary DNA (cDNA) cloned. Its function was demonstrated by an enhanced beta-carotene synthesis due to expression in Escherichia coli transformants with an engineered carotenoid pathway. Dxs as the gateway enzyme of the terpenoid pathway and phytoene synthase controlling the initial step into the specific carotenoid pathway are transcriptionally up-regulated when dark-adapted P. tricornutum cultures were transferred to light. Because of their limiting properties, expression plasmids of dxs and psy cDNA for integration into the genome were constructed. After transformation, several transgenic lines of P. tricornutum with additional copies of integrated dxs or psy were obtained exhibiting higher carotenoid synthesis. The best dxs transformants reached 24.2 mg g(-1) dry weight of the dominating carotenoid fucoxanthin, the best psy transformants 18.4 mg g(-1) dry weight, which is a 2.4-fold and a 1.8-fold higher fucoxanthin content, respectively, than in the wild type. Our approach is the first example for a genetic manipulation of a chloroplast-specific pathway in P. tricornutum. With one psy transformant, we made a comparison of transcript with in situ phytoene synthesis. The results demonstrated that the transcript and product levels were correlated, but not in a linear manner. This is indicative of a further limitation in those engineered strains, most likely by substrate supply implying a combined over-expression of their gene as the next engineering step for a further increase of fucoxanthin production in P. tricornutum.