Metabolic engineering of ketocarotenoid biosynthesis in leaves and flowers of tobacco species.

Ketocarotenoids and especially astaxanthin are high-valued pigments used as feed additives. Conventionally, they are provided by chemical synthesis. Their biological production is a promising alternative. For the development of a plant production system, Nicotiana glauca, a species with carotenoid-containing yellow pigmented flower petals, was transformed with a cyanobacterial ketolase gene. The resulting plants accumulated 4-ketozeaxantin (adinoxanthin), which is the first ketocarotenoid synthesized in flower petals by genetic modification. Due to the very late flowering in this tobacco species, N. tabacum was used to optimize the yield and ketocarotenoid product pattern by metabolic engineering of the ketolation steps of carotenogenesis. The highly carotenogenic nectary tissue in the flowers represents a model of a flower chromoplast system. By expression of a ketolase gene, it was possible to engineer the biosynthetic pathway towards the formation of 3'-hydroxyechinenone, 3-hydroxyechinenone, 4-ketozeaxanthin, 4-ketozeaxanthin esters, 4-ketolutein and 4-ketolutein esters. Some of these ketocarotenoids were also formed in the leaves of the trangenic plants. In particular, by co-expression of the ketolase gene in combination with a hydroxylase gene under an ubiquitous promoter, the formation of total carotenoids in nectaries increased by more than 2.5-fold. In the nectaries of this type of transformants, more than 50% of the accumulating carotenoids were keto derivatives. In addition, the levels of ketocarotenoid esters were much lower and a higher percentage of the free ketocarotenoids accumulated. These results open new promising perspectives for a successful metabolic engineering of keto-hydroxy carotenoid production in carotenogenic flowers.