Engineered maize as a source of astaxanthin: processing and application as fish feed.

Astaxanthin from a transgenic maize line was evaluated as feed supplement source conferring effective pigmentation of rainbow trout flesh. An extraction procedure using ethanol together with the addition of vegetal oil was established. This resulted in an oily astaxanthin preparation which was not sufficiently concentrated for direct application to the feed. Therefore, a concentration process involving multiple phase partitioning steps was implemented to remove 90 % of the oil. The resulting astaxanthin raw material contained non-esterified astaxanthin with 12 % 4-keto zeaxanthin and 2 % zeaxanthin as additional carotenoids. Isomeric analysis confirmed the exclusive presence of the 3S, 3'S astaxanthin enantiomer. The geometrical isomers were 89 % all-E, 8 % 13-Z and 3 % 9-Z. The incorporation of the oily astaxanthin preparation into trout feed was performed to deliver 7 mg/kg astaxanthin in the final feed formulation for the first 3.5 weeks and 72 mg/kg for the final 3.5 weeks of the feeding trial. The resulting pigmentation of the trout fillets was determined by hue values with a colour meter and further confirmed by astaxanthin quantification. Pigmentation properties of the maize-produced natural astaxanthin incorporated to 3.5 µg/g dw in the trout fillet resembles that of chemically synthesized astaxanthin. By comparing the relative carotenoid compositions in feed, flesh and feces, a preferential uptake of zeaxanthin and 4-keto zeaxanthin over astaxanthin was observed.

Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways.

Vitamin deficiency affects up to 50% of the world's population, disproportionately impacting on developing countries where populations endure monotonous, cereal-rich diets. Transgenic plants offer an effective way to increase the vitamin content of staple crops, but thus far it has only been possible to enhance individual vitamins. We created elite inbred South African transgenic corn plants in which the levels of 3 vitamins were increased specifically in the endosperm through the simultaneous modification of 3 separate metabolic pathways. The transgenic kernels contained 169-fold the normal amount of beta-carotene, 6-fold the normal amount of ascorbate, and double the normal amount of folate. Levels of engineered vitamins remained stable at least through to the T3 homozygous generation. This achievement, which vastly exceeds any realized thus far by conventional breeding alone, opens the way for the development of nutritionally complete cereals to benefit the world's poorest people.

Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and its assignment as a beta-carotene 3-hydroxylase/4-ketolase.

A gene has been cloned from Xanthophyllomyces dendrorhous by complementation of astaxanthin formation in a beta-carotene accumulating mutant. It consists of 3,166 bp and contains 17 introns. For the beta-carotene mutant ATCC 96815, a single point mutation in the splicing sequence of intron 8 was found. The resulting improper splicing of the mRNA results in an inactive protein. The cDNA of this beta-carotene oxygenase encodes a cytochrome P450 monooxygenase belonging to the 3A subfamily. P450-specific domains were identified including a cytochrome P450 and an oxygen binding motif. Electrons are provided by a cytochrome P450 reductase. Functional characterization of the enzyme by genetic modification of X. dendrorhous demonstrated that this P450 monooxygenase is multifunctional catalyzing all steps from beta-carotene to astaxanthin formation by oxygenation of carbon 3 and 4. The reaction sequence is first 4-ketolation of beta-carotene followed by 3-hydroxylation. A hydroxylation mechanism at allylic carbon atoms has been proposed for the generation of 4-keto and 3-hydroxy groups at both beta-ionone ends.