Carotenoids

Carotenoids comprise a group of natural pigments that are ubiquitous throughout nature. Over 600 different carotenoids with diverse chemical structures have been identified in bacteria, fungi, algae, and plants (Shew-maker et al., 1999; Mann et al., 2000). Their colors range from yellow to red, with variations of brown and purple; in addition, carotenoids as colorants take advantage of their good pH stability and their insensitivity to reducing agents such as ascorbic acid (Mann et al., 2000). Not to be outdone, humanity through the ages has learned to exploit the pleasing visual properties of carotenoid pigments by supplementing feedstocks and incorporating caro-tenoid pigments into cosmetics and foods (Delgado-Vargas et al., 2000; Jez and Noel, 2000). As precursors of vitamin A, they are fundamental components in our diet and play additional important roles in human health (Delgado-Vargas et al., 2000; Van den Berg et al., 2000; Ye et al., 2000). Because animals are unable to synthesize them de novo, they must obtain them by dietary means.

Other outstanding industrial uses of carotenoids include pharmaceuticals and nutraceuticals. In fact, they are important nutraceutical compounds and natural lipophilic antioxidants, whose sale as food and feed supplements is estimated to be approximately U.S.$500 million, and the market is expanding (Albrecht et al., 2000). All this is due in part to the discovery that these natural products can play a role in the prevention of cancer and chronic disease

(mainly because of their antioxidant properties) and, more recently, that they exhibit significant tumor suppression activity as a result of specific interactions with cancer cells (Sandmann et al., 1999; Albrecht et al., 2000; Van den Berg et al., 2000).

The commercial demand for carotenoids is mainly met by chemical synthesis and, to a minor extent, by extraction from natural sources or microbial fermentation (Sandmann et al., 1999; Shewmaker et al., 1999; Sandmann, 2001). Moreover, although a wide range of natural carotenoid derivatives is known to date, most of these are biosynthetic intermediates that accumulate only in trace amounts, making it very difficult to extract sufficient material for purification (Albrecht et al., 2000). Some important dietary carotenoids are not abundant in the human diet. Zeaxantin, for instance, is a rare caro-tenoid, which together with lutein is the essential component of the macular pigment in the eye (Delgado-Vargas et al., 2000; Van den Berg et al., 2000). Low levels of intake increase the risk of age-related macular degeneration. Marigold extracts from Tagetes erecta or the dried flowers themselves are well known as supplements for chicken feed to color the eggs and the chicken skin (Delgado-Vargas et al., 2000). Interestingly, marigold flowers contain high concentrations of lutein as the major pigment (Delgado-Vargas and Paredes-López, 1997).

During ingestion of carotenoids, the efficiency of their absorption depends largely on the type of food, its processing, and the amount of dietary fat or oil. Whether the presence of a carotenoid in the food matrix might facilitate its bioavailability is still not known (Van den Berg et al., 2000; Sandmann, 2001). As a result of this, the number of carotenoids available for assessing their biological function and pharmaceutical and nutraceutical potential by in vivo and in vitro assay systems is very limited.

Carotenoids are a large family of C40 isoprenoid pigments. Their colorant and biological action, such as antioxidant activity, are related to the number and location of conjugated double bonds within their structure, cyclization of the ends of the molecules, and their modification by oxygen-containing R groups such as hydroxyl, keto, and epoxi groups (Albrecht et al., 2000; Mann et al., 2000; Schmidt-Dannert et al., 2000).

The first committed step in carotenoid biosynthesis is the condensation of two geranyl-geranyl diphosphate (GGDP) molecules to form the C40 backbone, the colorless phytoene (Figure 2.2). Phytoene desaturases from bacteria can introduce four double bonds, yielding red carotenoid lycopene, whereas plants utilize two desaturase enzymes to complete this conversion. Phytoene desaturase (PDS) catalyzes the first two desaturations (phytoene to phyto-fluene to Z-carotene), whereas the conversion of Z-carotene to lycopene via neurosporene is performed by Z-carotene desaturase (ZDS). The cyclization of lycopene, by lycopene cyclase, forms either a- or p-carotene, and subsequent hydroxylation reactions produce the xanthophylls, lutein, and zeax-anthin (Sandmann, 2001).

Different approaches have been followed to modify the carotenoid content in plants to enhance their nutritional value: (1) modification of carotenoid products in tomato, (2) increasing the amounts of preexisting carotenoids in rapeseed (Brassica napus), and (3) engineering a carotenogenic pathway in tissue that is completely devoid of carotenoids, such as rice endosperm (Table 1.3) (Shewmaker et al., 1999; Romer et al., 2000; Schmidt-Dannert et al., 2000; Ye et al., 2000).

During lycopene deposition in tomato fruit ripening, the activity of phy-toene synthase is the major controlling factor of the route; therefore, this enzyme should be an ideal target for the genetic manipulation of the caro-tenoid composition of tomato fruit (Fray et al., 1995). The constitutive highlevel expression of tomato phytoene synthase-1 in transgenic tomato has resulted in carotenoid-rich seed coats, cotyledons, and hypocotyls, but also in reduced levels of carotenoids in ripe tomato fruit due to gene silencing with the endogenous gene and dwarfism due to redirection of GGDP into

TABLE 1.3

Selected Essential Micronutrients for Human Diet, Their Daily Allowances, Manipulation by Plant Biotechnology, and Potential Applications

Nutrient

Maximum Adult RDAa

Vitamin A 1 mg REb

Iron

Zinc

Calcium

Phosphorus

15 mg

15 mg 1200 mg 1000 mg

Engineered Plant

Tomato, rapeseed,

Result

Vitamin

Increased levels of

Potential Application

Provitamin A deficiency

ß-carotene nutraceutical

Vitamin E 10 mg TEc Arabidopsis

Elevated content of a-

tocopherol andreduced ^-tocopherol content

Minerals Tobacco, rice Improved Fe content

Vitamin E deficiency nutraceutical

Tobacco, rapeseed

Reduced phytic acid levels

Ref.

Shewmaker et al., 1999; Romer et al., 2000; Ye et al., 2000

Shintani and DellaPenna, 1998

Anemia nutraceutical

Improvement in mineral bioavailability

Pen et al., 1993; Tramper, 2000

a Recommended dietary allowances per day; values represent the highest RDA either for male or female adults, except for pregnant or lactating women. b Vitamin A activity is expressed in retinol equivalent (RE). One RE is equal to 1 mg of all-frans-

retinol, 6 mg of all-frans-ß-carotene, or 12 mg of other provitamin A carotenoids. c One TE (a-tocopherol equivalent) is equal to 1 mg (R,R,R)-a-tocopherol. Source: Adapted and modified from Shintani and DellaPenna, 1998; DellaPenna, 1999; Guzmän-Maldonado and Paredes-Lopez, 1999.

the gibberellin pathway (Fray et al., 1995). The resultant plants were reduced in size. This work illustrates how problems arise when a balanced metabolism is disturbed (Sandmann, 2001). On the other hand, elevation of the provitamin A (p-carotene) content in transgenic tomato plants was achieved by manipulation of the desaturation activity. Romer et al. (2000) overex-pressed a single carotenoid gene encoding PDS, which converts phytoene into lycopene, from Erwinia uredovora, under the control of a constitutive promoter and with the protein being targeted to the plastid by pea ribulose biphosphate carboxylase small subunit transit sequence. These researchers found that the expression of that gene in transformed tomatoes did not elevate total carotenoid levels. However, the p-carotene content increased about threefold, representing up to 45% of the total carotenoid level. The transgenic tomato fruit contained approximately 5 mg all-trans-p-carotene or 800 retinal equivalents (Table 1.3). Thus, 42% of the RDA is contained in a single provitamin A tomato fruit, as compared to 23% of the control fruit. The advantage of p-carotene instead of retinol (vitamin A) in the diet is that it is nontoxic and can be stored by the body. The alteration in carotenoid content of these transgenic plants did not affect growth and development and their phenotype was stable and reproducible over at least four generations.

Also, the genetic manipulation of canola seeds to increase the carotenoid content to high levels was a tremendous success (Shewmaker et al., 1999). Overexpression of a bacterial phytoene synthase gene extended with a plas-tid-targeting sequence under a seed-specific promoter increased the caro-tenoid content of mature canola seed by up to 50-fold. In the transformant, the embryos were bright orange, as compared to the green embryos in control canola. In the transgenic seeds, concentrations of carotenoids (mainly a- and p-carotene) of more than 1 mg/g fresh weight accumulated, yielding oil with 2 mg carotenoids per g oil (Table 1.3).

Other unexpected results were obtained upon transformation of tobacco with an algal p-carotene ketolase gene. Mann et al. (2000) expanded upon the metabolic framework to redirect metabolic flux of the tobacco carotenoid biosynthetic pathway to produce the marine compound astaxanthin (Figure 1.2). Introducing p-carotene ketolase from unicellular algae into tobacco, astaxanthin could be synthesized using the endogenous pool of p-carotene in tobacco flowers. Tissue-specific synthesis was accomplished by linking a gene promoter for flower petal expression to a fused gene encompassing a transit peptide sequence for plastid localization and the algal ketolase coding sequence. Expression was high in flowers as visualized by the red nectar pigmentation caused by astaxanthin and other ketocarotenoids (Mann et al., 2000). Total carotenoid levels were increased to 140% compared with the wild type. It is important to note that the astaxanthin produced in the transgenic plants had the same chirality as the natural astaxanthin found in marine organisms. In contrast, the synthetic astaxanthin that is currently used as fish feed is a mixture of stereoisomers, of which 75% have an unnatural chiral structure (Hirschberg, 1999). These results demonstrate the

3,4- didehydrolycopene

2 GGPP

Plant PDS Phytofluene

^ Plant PDS

Z-Carotene

Z-ZDS

Lycopene M~

Bacterial PDS

3,4,3',4'-Tetradehydrolycopene Desaturase

^ 3,4,3',4'-Tetradehydrolycopene Desaturase Desaturase-Cyclase

Torulene

Lycopene cyclase

Lutein

ß-Carotene (Provitamin A)

Hydrolase

Hydrolase-Ketolase

Zeaxanthin

Astaxanthin

FIGURE 1.2

Biosynthetic pathway of a- and p-carotene, their oxo derivaties, and schematic view of some the transformations of novel carotenoids obtained by using molecular breeding and an in vitro evolution approach. Production of p- carotene is universal in plants, fungi, and bacteria. Other carotenoids of biotechnological, nutraceutical, and pharmaceutical interest are lutein, zeaxan-thin, and astaxanthin, including the novel and engineered carotenoids (3,4-didehydrolycopene, 3,4,3',4'-tetradehydrolycopene, and torulene), which present improved antioxidant properties. Abbreviations: GGDP, geranyl-geranyl diphosphate; PDS, phytoene desaturase; Ç-ZDS, Ç-car-otene desaturase. (Adapted and modified from Jez and Noel, 2000; Sandmann, 2001.)

prospect of genetically engineering carotenoid biosynthesis toward the production of naturally and commercially valuable compounds in plants.

Rice, a major staple food, is usually milled to remove the oil-rich aleurone layer that turns rancid upon storage. The endosperm, the remaining edible part of rice grains, lacks several essential nutrients, such as provitamin A. In fact, rice in its milled form contains neither p-carotene nor any of its immediate precursors. Thus, predominant rice consumption promotes vitamin A deficiency, a serious public health problem in at least 26 countries, including highly populated areas of Asia, Africa, and Latin America (Kishore and Shewmaker, 1999; Ye et al., 2000; Potrykus, 2001).

Immature rice endosperm synthesizes the carotenoid precusor GGDP. To convert GGDP to p-carotene, Ye et al. (2000) programmed the endosperm to carry out the necessary additional enzymatic reactions leading to the formation of cyclic carotenoids (p-carotene) in the rice endosperm. Transformation was carried out with a plasmid containing a plant phytoene synthase gene and a bacterial phytoene desaturase gene, which together should mediate the synthesis of lycopene from GGDP (Figure 1.2). Both reading frames were extended with transit sequences for targeting the endosperm plastids. One was under control of the endosperm-specific glutelin and the other under one constitutive promoter.

Surprisingly, such transgenic plants did not accumulate lycopene as predicted. Instead, these plants produced essentially the same end products (p-carotene, lutein, and zeaxanthin). The authors speculate that the enzymes necessary to convert lycopene into p-carotene, lutein, and zeax-anthin are constitutively expressed in normal rice endosperm or are induced when lycopene is produced. Co-transformation with another construct that carried the third gene of interest, lycopene p-cyclase, increased the p-carotene content of the rice endosperm to a maximum level of 1.6 mg/g dry weight (Ye et al., 2000). The resulting yellow-colored endosperm, containing provitamin A (p-carotene) and other carotenoids of nutritional importance, could provide additional health benefits. This type of transformed rice accumulating large levels of p-carotene is known as golden rice, and as little as 300 g of the cooked golden rice, a typical Asian diet, should provide almost the entire daily vitamin A requirement (Table 1.3). Golden rice exemplifies the best that agricultural biotechnology has to offer a world whose population is predicted to reach 7 billion by 2013 (Potrykus, 2001).

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