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

Abstract

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.

Nicotiana glauca engineered for the production of ketocarotenoids in flowers and leaves by expressing the cyanobacterial crtO ketolase gene

“…One of the goals of engineering carotenoid biosynthesis is the introduction of ketocarotenoid formation. Following initial attempts to establish ketocarotenoid formation in carotenogenic flowers of N. glauca by transformation with the crtW ketolase gene (Gerjets et al 2007), the present study describes the utilization of the crtO encoded ketolase, instead of crtW in order to improve ketocarotenoid yield.…”

“…Also in the petals of the transgenic line CrtO-N9, the same ketocarotenoids as found in leaves were present e.g., 4-ketolutein (peak 6), echinenone (peak 8), 3 0 -hydroxyechinenone (peak 10) and 4-ketozeaxanthin (peak 7) (Fig. 2d) carotenogenic (Gerjets et al 2007). Determination of carotenoids in different flower tissues indicate that the highest concentration is in the nectaries (Table 2) but they resemble only a minor part of the flower.…”

“…For carotenoid analysis, the extracts were partitioned against 10% (v/v) diethyl ether in petrol and subjected to HPLC analysis. The system used was a Hypersil HyPurity Elite C18, column (5 l particle size) run with actonitrile/2-propanol/methanol/water 83:10:5:2 (v/v/v/v) at 32°C (Gerjets et al 2007). Standards for the identification of carotenoids by co-chromatography and comparison of their spectra were obtained from E. coli transformed with appropriate carotenogenic genes for keto carotenoid production and their molecular weight confirmed by mass spectroscopy (Sandmann 2002).…”

“…In general, transformation with a ß-carotene ketolase gene may result in the formation of different 3-hydroxy-4-keto ß-carotene derivatives since a 3-hydroxylase already exists in plants. To date, ketocarotenoid synthesis was successful by transformation with ketolase genes in tobacco nectary tissue and leaves (Mann et al 2000;Ralley et al 2004;Gerjets et al 2007), Arabidopsis thaliana seeds (Stalberg et al 2003) and potato tuber (Gerjets and Sandmann 2006;Morris et al 2006). Alternatively, a ß-carotene accumulating tomato variety was cotransformed simultaneously with a ß-carotene ketolase and a hydroxlyase gene (Ralley et al 2004).…”

“…For both Lotus japonicum (Suzuki et al 2007) and Nicotiana glauca (Gerjets et al 2007) a crtW type ketolase was used for transformation. Although, L. japonicum exhibited the formation of astaxanthin (3,3 0 -dihydroxy-4,4 0 -keto-ß-carotene) and other keto intermediates, 4-ketozeaxanthin (3,3 0 -dihydroxy-4-keto-ß-carotene) was the only ketolated product in N. glauca.…”