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Homogentisic acid

Zeaxanthin

CH3 3 CH3

a-tocopherol

Figure 5.3 Summary of carotenoid (A) and vitamin E (B) biosynthesis pathway in plants. Enzymes carrying out specific reactions (a-h, for carotenoids and i-l, for vitamin E); are named as: a, phytoene synthase (PSY); b and c, phytoene desaturase (PDS); d, Z-carotene desaturase (ZDS); e, the formation of lycopene through a bacterial phytoene desaturase (ctrl) is indicated with a black thick arrow; f, lycopene e-cyclase, g, P-carotene hydroxylase (P-Chy); h, e-hydroxylase (e-Chy); i, hydrogenation enzyme; j, phytyl/prenyl transferase; k, methyl transferase 1 and cyclase; l, tocopherol Y-methyltransferases (y-TMT). Adapted from Ref. 17, 21, 74, 75, 78, 79, 90.

Zeaxanthin

CH3 3 CH3

a-tocopherol

Figure 5.3 Summary of carotenoid (A) and vitamin E (B) biosynthesis pathway in plants. Enzymes carrying out specific reactions (a-h, for carotenoids and i-l, for vitamin E); are named as: a, phytoene synthase (PSY); b and c, phytoene desaturase (PDS); d, Z-carotene desaturase (ZDS); e, the formation of lycopene through a bacterial phytoene desaturase (ctrl) is indicated with a black thick arrow; f, lycopene e-cyclase, g, P-carotene hydroxylase (P-Chy); h, e-hydroxylase (e-Chy); i, hydrogenation enzyme; j, phytyl/prenyl transferase; k, methyl transferase 1 and cyclase; l, tocopherol Y-methyltransferases (y-TMT). Adapted from Ref. 17, 21, 74, 75, 78, 79, 90.

Tomato fruit and its processed products are the principal dietary sources of lycopene, and also useful for their P-carotene content in minor amounts in the red varieties. At the green stage, tomato fruit has a carotenoid content similar to leaves (essentially P-carotene, lutein, and violaxanthin). But during fruit ripening, the genes mediating lycopene synthesis are up regulated whereas those controlling its cyclization are down regulated leading to over accumulation of lycopene in ripe fruit (up to 90% of total carotenoid, followed by P-carotene and only traces of lutein) (Figure 5.3A) (79).

Because the activity of the enzyme phytoene synthase (PSY) increases during tomato fruit ripening and when lycopene deposition occurs, it has been a preferred target to modify carotenoid profiles and levels in the fruit (Figure 5.3A). The constitutive expression of tomato PSY-1 in transgenic tomato plants led to dwarfism (plants with reduced size), due to redirecting GGPP from the gibberellins biosynthetic pathway into the carotenoid pathway, which reduced the amount of those hormones in the plants. Transgenic fruits produced lyco-pene earlier in development, but the final levels in the ripe fruits were lower than those in control samples, due to silencing of the endogenous gene (Table 5.4) (84). On the other hand,

Romer and coworkers (81) achieved a significant increment in the levels of P-carotene (provitamin A) in transgenic tomatoes by manipulating their desaturation activity. They produced transgenic lines overexpressing a PDS from Erwinia uredovora bacterium using a constitutive promoter; an enzyme capable of producing lycopene from phytoene (Figure 5.3A). Although total carotenoid concentration was not affected, ripe tomato fruits from transformed plants showed a threefold increase in P-carotene levels, accounting for up to 45% of total carotenoids. Tomato with improved provitamin A quality possessed about 5 mg/g fresh weight of all-trans-P-carotene (800 retinal equivalents), which satisfy 42% of the RDA, in contrast to normal fruit satisfying only 23% (Tables 5.2, 5.4).

Tomato is one of most productive based on carotenoid production per unit of cultivated area. By metabolic engineering, the large pool of red lycopene could be converted into high value added downstream carotenoids such as xanthophylls, which are an important type of target nutraceuticals, because of their antioxidant properties, their chemical stability and the difficulty in their chemical synthesis (79).

Recently, Dharmapuri et al. (79) reported the hyperexpression in tomato of an Arabidopsis P-lycopene cyclase (P-Lcy) gene with and without pepper P-carotene hydroxylase (P-ChyJ gene under the control of the fruit-specific PDS promoter (Figure 5.3A). They found that the color of the ripe fruit varied from complete red as in wild-type tomato (by natural lycopene accumulation) to red-orange or to complete orange for transgenic tomatoes expressing only recombinant P-lycopene cyclase, or both recombinant P-lycopene cyclase and P-carotene hydroxylase enzymes, suggesting significant changes in carotenoid composition. The transformed fruits showed up to a 12-fold increase in P-carotene content with respect to their untransformed parental line. The transgenic tomato (containing genes P-Lcy + P-ChyJ accumulated P-carotene as much as 63 p,g/g fresh weight as compared to 5 p,g/g fresh weight produced by the untransformed fruit. Notably, modified tomato also stored very good levels of both nutraceutical xanthophylls; P-cryptoxanthin (11 p,g/g fresh weight) and zeaxanthin (13 p,g/g fresh weight), but they are not detectable in control parent fruit or transformed tomato with only P-Lcy gene (Table 5.4). These results demonstrate that P-carotene pool can be converted into xanthophylls by the overexpression of pepper P-Chy and thus adding nutraceutical and commercial value to tomato.

5.4.1.2 Carotenogenesis in Other Food Crops

Another successful genetic manipulation of carotenogenesis has been reported in canola (Brassica napus). The bacterial (Erwinia uredovora) phytoene synthase with a plastid targeting sequence was overexpressed in a seed-specific manner using a napin promoter from rapeseed (Figure 5.3A) (80). Transgenic embryos were visibly orange, as compared with those green from control seeds, and the mature seed exhibited up to 50-fold more carotenoids, mainly a- and P-carotene, both presenting activities of provitamin A. Carotenoid-rich transgenic canola seed reached up to 1.6 mg per gram of fresh weight and produced an oil with 2 mg of carotenoids per gram of oil (Tables 5.2, 5.4).

Worldwide, vitamin A deficiency causes visible eye damage in around 3 million preschool children and up to 500 thousand of those children become partially or totally blind each year because of this deficiency, and approximately two thirds of them die within months of going blind. The estimates of the subclinical prevalence of vitamin A deficiency range between 100 and 200 million across the world. In developing countries several clinical trials have shown that vitamin A capsules can reduce mortality rates among preschool children by 23%, whereas improved vitamin A nutrition could prevent 1.2 million deaths annually among children aged 1-4 years (31). In addition, because vitamin A deficiency is common among vast populations of Asia, Africa, and South America, whose principal source of food is rice, engineering these crops to produce provitamin A-type carotenoids is of great importance (72,74).

Rice is generally consumed in its polished version, as commercial milling removes the oil-rich aleurone layer which becomes rancid during storage mainly in tropical and subtropical regions. Mature rice (Oryza sativa) endosperm is capable of synthesizing and accumulating GGPP but completely lacks carotenoids (Figure 5.3A) (85). In rice endosperm, the additional enzymatic activities needed to produce P-carotene was genetically engineered (82). To achieve this, four plant enzymes are necessary, but alternatively the number may be reduced to three as a bacterial carotene desaturase catalyzes the introduction of four double bonds required to produce lycopene (Figure 5.3A). Therefore three new genes (a plant phytoene synthase gene from daffodil, a bacterial phytoene desaturase gene from Erwinia uredovora and a lycopene P-cyclase also from daffodil plant) were transferred to rice. The first and third genes were driven by the endosperm-specific promoter of the rice glutelin gene, whereas a constitutive CaMV 35S promoter was used for the phytoene desaturase gene. Interestingly, transgenic rice seeds exhibited a beautiful golden yellow color after milling and increased accumulation of P-carotene in edible endosperm; as well as the xanthophylls, zeaxanthin, and lutein were formed to some extent, resulting in a carotenoid qualitative profile somehow analogous to that of green leaves. However, golden rice showed a higher proportion of P-carotene in their endosperm than that of the other two carotenoids, with a maximum amount of 1.6 ^g/g dry weight; however, this quantity only represents 1-2% of carotenoid concentration in transgenic rapeseed (80) (Table 5.4). Nevertheless, it is noteworthy that in a typical Asian diet (about 300 g of rice per day), provitamin A-rich golden rice could provide nearly the full daily vitamin A requirement (85) (Table 5.2). It has been suggested that P-carotene accumulated in golden rice endosperm can be converted to retinol easier than P-carotene in vegetables, where provitamin A is converted to retinol at a rate equivalent to 26 to 1, due to the physico-chemical properties of endosperm matrix (31,85). This significant accomplishment in plant biotechnology of improving the nutritional value of rice with beneficial carotenoids can benefit human nutrition and health (83). A future project aims to join iron-rich rice lines expressing ferritin with golden rice lines because it is known that provitamin A improves the iron bioavailability (Table 5.3) (85).

Nopal cactus (its young cladodes are called nopalitos) is low in calories and since Mesoamerican times has been eaten as a vegetable and fruit source in Mexico. At present, the economic and social importance of nopal is not only because large areas are covered with wild and commercial species in all arid and semiarid Mexican regions, but also because of its remarkable nutritional and nutraceutical qualities. For example, the intake of broiled nopalitos improved glucose control in adult people with noninsulin-dependent diabetes mellitus; it is also known that nopal diminishes human cholesterol levels (35,86). On the other hand, nopal is a good source of dietary fiber, calcium, iron, zinc, and vitamin C, and thereby can also be used in treatments against scurvy. However, although P-caro-tene is accumulated in nopal, its amount is very low; in fact its provitamin A level depends on nopalito development stage and nopal variety (35,86).

In our laboratory, Paredes-López et al. (87) have developed a genetic transformation system for nopal cactus (Opuntia sp.) through Agrobacterium tumefaciens, obtaining regenerated transgenic nopal plants. Thus, it has been proposed to use the nopal plant as a bioreactor for improving the production and storage of provitamin A by introducing additional genes for carotenoid biosynthesis to obtain P-carotene (P. Garcia-Saucedo, O. Paredes-López, personal communication, 2004). Nopal could become an important, cheap, and accessible source of vitamin A, and other nutraceuticals such as lutein or zeaxanthin for a great part of the population in Mexico.

5.4.2 vitamin E

The intense research efforts which have surrounded vitamin E, a lipid-soluble antioxidant, support the hypothesis that preventing free radical-mediated tissue damage, for example to cellular lipids, proteins, or DNA, may play a key role in decreasing or delaying the patho-genesis of a variety of degenerative diseases such as cardiovascular disease, cancer, inflammatory diseases, neurological disorders, cataract, and age-related macular degeneration, and a decline in the immune system function (Table 5.2) (8,21). It has been suggested that vitamin E supplementation of 100 to 400 international units (IU) or around 250 mg of a-tocopherol per day may help reduce the magnitude of occurrence of such health disorders. Vitamin E is represented by a family of structurally related compounds, eight of which are known to occur in nature, being isolated from vegetable oils and other plant materials. The eight naturally occurring compounds are a-, y-, and S-tocopherol (which differ only in the number and position of methyl substituents on the aromatic ring), and a- y-, and S-tocotrienols (8,33). Tocotrienols differ from the corresponding tocopherols in that the iso-prenoid side chain is unsaturated at C3', C7', and C11'. The phenolic hydroxyl group is key for the antioxidant activity of vitamin E, as donation of hydrogen from this group stabilizes free radicals (Figure 5.2). The presence of at least one methyl group on the aromatic ring is also critical. a-tocopherol, with three methyl groups, is the most biologically active of all homologues occurring in nature as a single isomer, followed by ^-tocopherol, y-tocopherol, and S-tocopherol (8,88). Changes in the isoprenoid side chain also influence vitamin E activity; this biological activity is defined in terms of equivalents of a-tocopherol (a-TE). (R, R, R)-a-tocopherol has an activity of 1 a-tocopherol (a-TE) equivalent per milligram of compound. The activities of (R, R, R)-^-, (R, R, R)-y-, and (R, R, R)-S-tocopherols are 0.5, 0.1 and 0.03 per mg of compound, respectively (8,21). Of the tocotrienols, only a-tocotrienol has significant biological activity (0.3 mg a-TE/mg). Lengthening or shortening the side chain results in a progressive loss of vitamin E activity. The previous information is based on all tocopherols which are absorbed to similar extents during digestion, however single (R, R, R)-a-tocopherol is successfully stored and distributed in the entire body, while the other species are not processed with the same efficiency. It has been estimated that one a-TE molecule is capable of protecting 2000 phospholipids (8,88).

The physiological role of vitamin E centers on its ability to react with and quench free radicals in cell membranes and other lipid environments, thereby preventing polyunsaturated fatty acids (PUFAs) from being damaged by lipid oxidation. An imbalance in the production of free radicals and the natural protective system of antioxidants may lead to oxidized products, able to harm tissue; in fact tissue damage due to free radicals has been associated to several human chronic diseases (Table 5.2) (4-8).

Refined and processed foods are usually exposed to light, heat, or metal ions that can cause structural degradation of their constituent lipids by triggering the process of lipid oxidation (42). The rate of lipid oxidation in a food depends on the concentration and type of PUFAs it contains, the amount and effectiveness of the antioxidants present in the food and the heating, processing and storage conditions to which it is subjected to. Tocopherols and tocotrienols are the most important natural antioxidants in fats and oils, acting as primary or chain breaking antioxidants by converting lipid radicals to more stable products (8,42). At normal oxygen pressure, the major lipid radical is the peroxyl radical (ROO°) which can be converted to a hydroperoxide (ROOH) by proton donors such as tocopherols and tocotrienols. The hydrogen is donated from their phenolic groups, stabilizing the radicals and stopping the propagation phase of the oxidative chain reaction.

Supplementation of a-tocopherol has been demonstrated to positively affect sensorial quality of meat as well as saving money (i.e., in the U.S. beef industry meat, color degradation provokes losses up to $1 billion each year). In poultry, the high tocopherol level increased the stability of its meat; whereas in the case of pork and beef, vitamin E protects against rancid flavor, odor, and discoloration improving shelf life of packaged meat (89).

However, traditional plant breeding and food processing technologies have not concerned themselves with maximizing the levels of tocopherols in the human diets and even in diets for domestic animals used for meat, and the supplementation is necessary both for nutritional reasons and for the protection of fat-rich foods against oxidative rancidity (8,42). Unfortunately, synthetic a-tocopherol used as supplement is a complex mixture of stereo-isomers with less biological activity than natural single (R, R, R)-a-tocopherol (16,21,88). Significant changes in the a-tocopherol levels of major edible crops are necessary because there is a growing body of evidence to suggest that the dietary intake of vitamin E is insufficient to protect against the long term health risks associated with oxidative stress (8,16,21,88,89). Normally, the tocopherol composition of cultivated sunflower (Helianthus annuus L.) seed is primarily a-tocopherol, 95-100% of the total tocopherol pool. However, two mutant sunflower lines have been identified with tocopherol compositions of 95% y-tocopherol/5% a-tocopherol, and 50% ^-tocopherol/50% a-tocopherol. Although these presumed tocopherol methylation mutants showed severe alterations in their tocopherol profiles in seeds, their overall levels do not differ significantly from those of wild-type sunflower (8,89). These results suggest that it should be possible to alter the tocopherol profile of different crop species by manipulating the expression of one or both tocopherol methyltransferases (TMT), without having a detrimental effect on the total tocopherol pool size (Figure 5.3B). An exquisite and noteworthy research in the context of increasing the overall level of vitamin E activity available to consumers from plant foods was carried out by Shintani and DellaPenna (90). By overexpressing y-TMT (Figure 5.3B), it was possible to increase a-tocopherol content in the Arabidopsis seed to about 85-95% of the total tocopherol, as compared with levels of 1.1% a-tocopherol and 97% y-tocoph-erol in the untransformed seeds. Transgenic Arabidopsis showed a vitamin E activity about nine times greater than the negative control (Table 5.4). The authors speculate that if y-TMT activity is limiting in commercially important oilseed crops such as soybean, corn, and canola, all of which have low y-tocopherol to a-tocopherol ratios, overexpressing the y-TMT gene in these crops should also elevate a-tocopherol amounts and improve their nutraceutical value (8,16,21,33,34,89).

5.4.3 vitamin c

Vitamin C is used in large scale as an antioxidant in food, animal feed, beverages, pharmaceutical formulations, and cosmetic applications (91). This water-soluble vitamin, defined as l-ascorbic acid (l-AA), structurally is one of the simplest vitamins (Figure 5.1); its oxidation product is termed dehydroascorbate. It is related to the C6 sugars, being the aldono-1,4-lactone of a hexonic acid (l-galactonic or l-gulonic acid) and contains an enediol group on carbons 2 and 3 (92). In animal metabolism, including that of humans, the biological functions of l-AA are centered on its antioxidant properties and on its role to modulate a number of important enzymatic reactions. Thus, generally it acts as an enzyme cofactor, free radical scavenger and donor and acceptor in electron transfer reactions. For example, l-AA is required for collagen synthesis, and consequently in the formation and maintenance of cartilage, bones, gums, skin, teeth, and wound healing. In fact, the Fe-dioxygenases involved in collagen biosynthesis need l-AA for maximal activity, where the function of l-AA is to keep the transition metal ion centers of these enzymes in a reduced form. In the disease scurvy, which is known to be the result of vitamin C deficiency, its symptoms are directly related with the inadequate collagen formation (Table 5.2) (12,92). This micronutri-ent is also crucial for the normal, and enhanced, functioning of immune system, and is required for carnitine synthesis. There is now strong evidence to link high intake dietary vitamin C with reduced risk for several oxidative stress-associated diseases such as cardiovascular diseases, various types of cancers, aging, neurodegenerative diseases, and cataract formation. Cataracts appear to be due to the oxidation of lens protein, and antioxidants such as vitamin C and E, and carotenoids seem to protect against cataracts and macular degeneration of eye in rodents and humans. On the other hand, increased oxidative damage from low vitamin C intake, chronic inflammation, smoking, or radiation, together with elevated levels of uracil in DNA, would be expected to lead to more double strand (chromosome) breaks in individuals who are deficient in both folate and antioxidant (Table 5.2) (4—7,12).

However, there is also large body literature on supplementation studies with vitamin C in humans using biomarkers of oxidative damage to DNA, lipids (its oxidation releases muta-genic aldehydes), and protein. Some studies suggest that blood cell saturation occurs at about 100 mg vitamin C/day and the evidence suggests that this level minimizes DNA damage. Both experimental and epidemiological data support that vitamin C provides protection against stomach cancer, a result that is plausible because of the role of oxidative damage from inflammation by Helicobacter pylori infection, which is the main risk factor for stomach cancer (4—7,12). Unfortunately, the plasma levels of l-AA in large sections of the population around the world are suboptimal for those health benefic effects of this vitamin; in fact about 15% of the population consumes less than half the RDA (60 mg/day) of ascorbate (Table 5.2).

It is thought that l-AA secreted in gastric juices in animals enhances the absorption of iron from plant foods through two mechanisms: by forming Fe(III) complexes and by reducing the less soluble Fe31 to the more soluble and bioavailable Fe21 valence state (Table 5.3) (12,43).

Plants and most animals (i.e., rats, dogs, cats) can synthesize their own vitamin C, but a few mammalian species, including primates, human beings, and guinea pigs have lost this capability, and thus entirely depend upon dietary sources to meet needs for this vital micronutrient. This deficiency has been localized to a lack of the terminal flavo-enzyme l-gulono-1,4-lactone oxidase (l-gulono-y-lactone oxidase, [GuLO]); the gene encoding it was found in the human genome, but was not expressed due to the accumulation of various mutations. In vitamin C-producing animals, GuLO catalyzes the final reaction in the l-AA route corresponding to the oxidation of l-gulono-1,4-lactone, whereas in plants the enzyme l-galactono-1,4-lactone dehydrogenase employs l-galactono-1,4-lactone as a substrate for carrying out the terminal step in the vitamin C production (Figure 5.4) (92-95).

Vitamin C is the single most important specialty chemical manufactured in the world. The current world market of ascorbic acid is 60,000 to 70,000 metric tons each year and generates annual revenues in excess of US$ 500 million (91). But its industrial production is a lengthy procedure involving microbial fermentation and diverse chemical steps (38,91). Until quite recently, little focus has been given to improving the l-AA content of plant foods, either in terms of the amounts present in commercial crop varieties or in minimizing losses prior to consumption. Notably, plants and animal possess different pathway for synthesizing l-AA; the expression in transgenic lettuce plants of an animal cDNA encoding a rat GuLO under the control of CaMV 35S promoter led to accumulation up to seven times more l-AA than untransformed crops (the basal levels of l-AA varied among the three unmodified lettuce cultivars from 0.36-0.58 pmol/g fresh weight) (Table 5.4) (95). In food science and technology, vitamin C as well as bisulfites are used to prevent oxidation in peaches, potato chips, apples, potatoes, peanut butter, beer, fat, and oils (20,42). Therefore, in the future, l-AA-rich transgenic lettuce may diminish the commercial application of bisulfite to avoid browning of its leaf, as well as enhancing the nutritional and nutraceutical value of this food vegetable. A recent report also showed that the l-AA content of Arabidopsis thaliana (untransformed plants have a vitamin C level of about 2 pmol/g fresh weight) was increased two- and threefold by hyperexpression of a d-galacturonic acid reductase gene from strawberry

D-glucose-6-phosphate

D-glucose-1-phosphate i b

UDP-D-glucose

D-mannose-6-phosphate

D-mannose-1-phosphate

UDP-glucuronic acid

D-glucuronic acid-1-phosphate

GDP-D-mannose

GDP-L-galactose

Methyl-D-galacturonic acid

D-glucuronic acid

L-galactose-1-phosphate

D-galacturonic acid

L-gulonic acid i g

Gulono-1,4-lactone

Animals

"A

L-ascorbate

L-galactose i p

L-galactonic acid

L-galactano-1,4-lactone

Plants

Figure 5.4 Proposed pathways for L-ascorbic acid biosynthesis in animals (A) and plants (B and C). Enzymes catalyzing the individuals reactions (a-h, for animals; i-t, for plants) are given next: a, phosphoglucomutase; b, UDP-glucose pyrophosphorylase; c, UDP-glucose dehydrogenase; d, glucuronate-1-phosphate uridylyltransferase; e, glucurono kinase; f, glucuronate reductase; g, aldono-lactonase; h, L-gulono-1,4-lactone oxidase, GuLO; i, glucose-6-phosphate isomerase; j, mannose-6-phosphate isomerase; k, phosphomannomutase; l, GDP-mannose pyrophosphorylase; m, GDP-mannose-3,5-epimerase; n, phosphodiesterase; o, sugar phosphatase; p, L-galactose-1- dehydrogenase; q, L-galactono-1,4-lactone dehydrogenase, r, methylesterase, s, D-galacturonate reductase; t, aldono-lactonase. Adapted from Ref. 92-95.

(Table 5.4) (94). This gene encodes the enzyme d-galacturonate reductase that converts d-galacturonic acid into l-galactonic acid, which is readily transformed to l-galactono-1,4-lactone, the immediate precursor of l-AA (Figure 5.4C).

The previous works demonstrate the possibility that the basal content of minerals and vitamins in important food crops can be augmented by metabolic engineering, and therefore raise the realistic possibility that such increases may substantially benefit vulnerable populations in their daily dietary intakes, without the need for fortification or for a change in dietary habits as a whole.

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