888 NATURE BIOTECHNOLOGY VOL 18 AUGUST 2000 http://biotech.nature.com RESEARCH ARTICLES Carotenoids constitute a group of natural pigments that are ubiqui- tous throughout nature. Over 600 different carotenoid species are known to exist in bacteria, plants, fungi, and animals. Their colors range from yellow to red with variations of brown and purple. The typical colors of many flowers, fruits, and vegetables are determined by carotenoids, the accumulation of which in these organs is an important agronomic property. Carotenoids also provide colors to certain animals such as insects, fish, and birds. As precursors of vitamin A, carotenoids are fundamental components in our diet and they play additional important roles in human health 1 . Industrial uses of carotenoids include pharmaceuticals, food supplements, animal feed additives, and colorants in cosmetics. Carotenoids are synthesized in all photosynthetic organisms and in some bacteria and fungi. Because animals are unable to synthesize them de novo, they must obtain them by dietary means. Hence, manipulation of carotenoid content and composition in plants can improve their agronomic and nutritional value and provide a new source for valuable materials for industry. Nearly all of the enzymes required for carotenoid biosynthesis in plants have been cloned and characterized in recent years 2 . This achievement has provided the molecular tools needed for genetic manipulations of carotenoids in vivo. In this work, we describe the genetic engineering of tobacco plants to produce astaxanthin. Astaxanthin (3,3′-dihydroxy- β,β- carotene-4,4′-dione) is a ketocarotenoid that occurs in the natural diet of many aquatic animals. It provides a characteristic pink color to salmon, trout, and shrimp. In salmon and trout, astaxanthin was shown to provide protection of the eggs from damage by UV radia- tion and to improve survival and growth rate of juveniles 3,4 . Reduced astaxanthin in Baltic salmon was correlated with higher mortality from the M74 syndrome 5 . Astaxanthin was reported to boost immune functions in humans 6 , to reduce oral carcinogenesis in rat 7 , and to inhibit the growth of mammary tumors in mice 8 . These properties of astaxanthin are probably related to its function as a powerful antioxidant 9–11 . In nature, astaxanthin is synthesized by marine bacteria and microalgae and then passed on to fish through the food chain. Because fish grown in aquaculture are separated from their natural food chain, astaxanthin must be added to their artificial feed to give the typical pink color to their flesh. Currently, astaxanthin is com- mercially produced by chemical synthesis and sold as a feed formula. It is one of the most expensive components of salmon farming, accounting for about 15% of total production costs. Since the astax- anthin molecule has two identical chiral centers, the synthetic astax- anthin exists in three configurational (stereo) isomers: (3S,3′S) (3S,3′R), and (3R,3′R). The natural astaxanthin synthesized by marine organisms is in the 3S,3′S configuration. The significance of this phenomenon is as yet unknown. In previous work, we have cloned from the unicellular green alga Haematococcus pluvialis a cDNA, CrtO, that encodes β-carotene ketolase ( β-C-4 oxygenase). This enzyme catalyzes the two-step conversion of β-carotene to canthaxanthin by adding keto groups in positions C4 and C4′ (ref. 12). Transgenic expression of CrtO in β-carotene producing Escherichia coli cells that also con- tained crtZ from Erwinia, which encodes β-carotene hydroxylase, brought about synthesis of astaxanthin 13 . When the cyanobacterium Synechococcus PCC7942, which normally accumulates β-carotene and zeaxanthin, was transformed with CrtO, it accumulated signifi- cant amounts of astaxanthin as well as other ketocarotenoid interme- diates 13 . These results confirmed that β-carotene ketolase (CRTO) can function in conjunction with the intrinsic β-carotene hydroxy- lase, which adds hydroxyls at C3 and C3′, to produce astaxanthin. A schematic depiction of the pathway is given in Figure 1. Here we report the successful manipulation of the carotenoid biosynthesis pathway in a higher plant to produce natural astaxan- thin. This was achieved by expressing the β-carotene ketolase gene from the alga H. pluvialis in the chromoplast-containing tissue of the nectary that normally synthesizes β-carotene and xanthophylls. Consequently, novel ketocarotenoids accumulated in the cells and changed the color of the nectary from yellow to red. Metabolic engineering of astaxanthin production in tobacco flowers Varda Mann 1 , Mark Harker 1,2 , Iris Pecker 1 , and Joseph Hirschberg 1 * 1 Department of Genetics, The Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem, 91904 Israel. 2 Current address: Unilever Research, Colworth House Laboratory, Colworth House, Sharnbrook, Bedford, MK44 1LQ, UK. * Corresponding author (hirschu@vms.huji.ac.il). Received: 14 February 2000; accepted 2 May 2000 Using metabolic engineering, we have modified the carotenoid biosynthesis pathway in tobacco (Nicotiana tabacum) to produce astaxanthin, a red pigment of considerable economic value. To alter the carotenoid pathway in chromoplasts of higher plants, the cDNA of the gene CrtO from the alga Haematococcus pluvialis, encoding β-carotene ketolase, was transferred to tobacco under the regulation of the tomato Pds (phytoene desaturase) promoter. The transit peptide of PDS from tomato was used to target the CRTO polypeptide to the plastids. Chromoplasts in the nectary tissue of transgenic plants accumulated (3S,3′S) astaxanthin and other ketocarotenoids, changing the color of the nectary from yellow to red. This accomplishment demonstrates that plants can be used as a source of novel carotenoid pigments such as astaxanthin. The procedures described in this work can serve as a platform technology for future genetic manipulations of pigmentation of fruits and flowers of horticultural and floricultural importance. Keywords: Chromoplasts, plant biotechnology, isoprenoids, nectary, transit peptide, protein import, cartenoid biosynthesis © 2000 Nature America Inc. • http://biotech.nature.com © 2000 Nature America Inc. • http://biotech.nature.com