J. Pozueta-Romero, Universidad Pu´blica de Navarra
6.2 Changing the nutritional value of foods
6.2.1 Amino acid content of proteins
An early application of biotechnology for improving the nutritional value of foods has involved changing the amino acid composition of some common
6
The use of molecular genetics to improve food properties
I. Amaya, M. A. Botella and V. Valpuesta, Universidad de Ma´laga
proteins of the human diet. It has been long known that humans cannot live on a protein-free diet. The reason is that we are incapable of synthesising half of the 20 standard amino acids present in proteins. These are known as essential amino acids and must be provided in the diet. Consequently, the nutritive value of a protein-based diet is directly related to the content of these essential amino acids. In general, cereals have proteins with a low content of the essential amino acids lysine, threonine and tryptophan, while legume proteins have a deficiency of cystein, methionine and triptophane. Among the most valuable sources of vegetable protein are the grain legumes. In one of these, soybean, genetic engineering has been applied to increase the content of the essential amino acid lysine in the seed proteins. The rationale was that increasing the synthesis of lysine in the seeds of soybean would increase the synthesis of proteins with high lysine content. Lysine synthesis in this species is finely regulated by a feed-back mechanism, i.e., when the lysine content is high there is an inhibition of two of Fig. 6.1 Biochemical regulatory mechanism proposed to regulate the synthesis of the amino acid Lysine, in higher plants, derived from the amino acid Aspartate. Enzyme activities, aspartate kinase (AK) and dihidrodipicolinate synthase (DHDPS) are indicated.
Broken lines indicate the inhibition of these enzymes by Lysine.
the enzymes involved in the metabolic pathway leading to the synthesis of this amino acid (Fig. 6.1).
The strategy consisted of the integration in the genome of soybean of genes from other species encoding for enzymes without the feed-back mechanism. The transformation of soybean with the gene lysCM4 from Escherichia coli (encoding AK) and the dapA gene from Corynebacterium (encoding DHDPS), both insensitive to the feed-back inhibition by lysine, resulted in a transgenic soybean plant with a duplicated pathway of lysine biosynthesis, one sensitive and the other insensitive to lysine. As a result, the lysine content of the transgenic soybean plants was over 100-fold the value of the untransformed plants. Other plant species like corn, wheat and canola have been subjected to the same genetic manipulation to increase their lysine content with similar results to those obtained in soybean.
6.2.2 Fatty acid composition of triacylglycerols
Lipids are also main components of the human diet. The consumer preference for plant-derived oils is increasing to the detriment of animal fats. Annual plant oil production is increasing worldwide and most of it is used for human consumption as margarines, oils and food ingredients. The triacylglycerols are the most important components of plant seed oils. Interestingly, the physical and chemical properties of an edible oil are related to the chemical structure of the fatty acids esterifying the glycerol (Table 6.1). Properties such as melting point, colour, flavour, mouthfeel, spreadability, stability, and effects on human health are determined by the fatty acid composition of the triacylglycerols. Most efforts in developing changes in the lipid composition of plant oils have been directed to change the proportion among the fatty acids of the triacylglycerols.
Common fatty acids in the commercial seed oils are lauric, myristic, palmitic, stearic, oleic, linoleic and linolenic. As is apparent, their differences occur in the length of the carbon skeleton (C12 to C20) as well as in the presence of double bonds (unsaturations). Long chain fatty acids containing two or more double
Table 6.1 Nomenclature and representative examples of naturally occurring fatty acids Systematic nomenclature m:naZbZ. . .
m: carbon atoms n: double bonds
superscript: positions of the double bonds a, b, . . .: carbon numbered from the carboxyl end
Z: configurationcisof the double bond Examples:
Saturated 18:0 Stearic acid
Unsaturated 18:19Z Oleic acid
Polyunsaturated (PUFA) 20:55Z,8Z,11Z,14Z,17Z
Eicosapentaenoic acid
bonds are named polyunsaturated fatty acids (PUFA). Different studies on the effect of dietary fatty acids consumption on human health have noticed the trend of consumers towards a reduction of saturated acids in the diet and, accordingly, an increase in unsaturated acids. Epidemiological studies have shown that intake of monounsaturated acids was associated with a low incidence of coronary artery disease (Keys et al., 1986), which has been explained by its reduction in the low density lipoproteins (LDL) levels and their oxidation (Mata et al., 1997).
Therefore the unsaturation of fatty acids have been the target for modification by genetic engineering studies. Although the metabolic pathways leading to the synthesis of these compounds are not simple, some genes have been adequately selected to be modified. Oleic acid, the major monounsaturated acid of the diet, reduces cholesterol and LDL in the serum. Transgenic plants overexpressing the desaturase gene, that encodes for the enzyme catalysing the conversion of the saturated precursor stearic acid C18 into oleic, have been obtained (Fig. 6.2).
This has determined that oleic content of soybean has been raised to values up to 80% of the total fatty acids content of the seeds (Kinney, 1996). Although the relationship between PUFA and disease remains contentious, there is a consensus among the health organisations that PUFA should form 8–23% of the total lipid intake in the human diet (Gill and Valivety, 1997). However, the production of high PUFA oil plants is not straightforward (Fig. 6.2), and no report on transgenic plants with high PUFA content is known.
Interestingly, there have been cases where saturation of the fatty acids has been the purpose of the plant genetic modification. Saturation of fatty acids determines properties such as melting temperature and viscosity that may be important to a commercial product. Margarine, for example, needs to be easily
Fig. 6.2 Interconversions of the fatty acids indicated in Table 6.1 as examples, Enzymes:nDS: n-desaturase: EL: elongase.
spreadable within a range of temperatures. In addition, saturation may also be beneficial for oil stability since it is known that unsaturated acids are more readily oxidised, resulting in an increased tendency to rancidity and off odours.
Finally, vegetable oils used for frying require partial saturation by hydrogenation in order to give adequate characteristics of stability and melting temperature to these oils. The chemical hydrogenation has been proven to induce also a change in the configuration of the double bonds of the fatty acids, from the naturally occurring cis to trans. The presence of the trans unsaturated fatty acids has been correlated to a risk of coronary heart disease. Therefore, plants with a high content of natural oil and with a high level of saturation have been engineered. High stearate content of the oil in a Brassica plant has been achieved by two methods. One method transformed this plant species with the antisense construct of the gene encoding the stearoyl-ACP desaturase, the enzyme that catalyses the transformation of stearic acid into oleic acid (Fig. 6.2, 9DS) (Knutzonet al. 1992). The silencing of the endogenous gene produced the accumulation of stearic acid up to 40% of the total fatty acids content. The second method transformed theBrassicaplant with a gene encoding the stearoyl -ACP thiosterase specific for the synthesis of stearic acid (Fig. 6.2). Using this approach, the transgenic plants yielded up to 68% of this fatty acid.
6.2.3 Vitamins and diet enrichment compounds
Vitamins are essential compounds for humans and other vertebrates and they must be obtained from the diet. In addition, some vitamins are used as functional additives in food products. Ascorbic acid (vitamin C) is used to prevent oxidation in apples, peaches, apricots, potatoes, peanut butter, potato chips, beer, fat and oils. The carotenoids (vitamin A precursors) are used as colorants in margarine, cheese, ice cream, pasta, juices and beverages (Giese, 1995). It is generally believed that people in Western countries have adequate vitamin intake. There are, however, susceptible groups within the general population who may have inadequate vitamin intake. Such groups include dieters, people on medication, pregnant women, alcoholics, adolescents, and people with diabetes and other chronic ailments. In developing countries very little information exists concerning their nutritional status.
The edible part of rice grains, the endosperm, lacks an essential nutrient as vitamin A. A diet mostly based on rice consumption may eventually cause vitamin A deficiency. It is estimated that improved vitamin A nutrition could prevent worldwide 1–2 million deaths annually among children. A very promising achievement has been the introduction of genes into rice that enabled the biosynthesis in the endosperm of-carotene, the precursor of vitamin A (Ye et al., 2000). -carotene is synthesised from the precursor geranylgeranyl bisphosphate which is converted to the colourless phytoene by the enzyme phytoene synthase (Fig. 6.3). The phytoene undergoes four desaturations to form lycopene, which is red and gives colour to ripened tomato fruits. Further cyclisation of lycopene results in the formation of -carotene. Immature rice
endosperm is capable of synthesising geranylgeranyl biphosphate which can be used to produce phytoene by expression of a phytoene synthase gene. The introduction of three genes in rice via Agrobacterium allowed the expression of the entire -carotene pathway into the endosperm. These genes were a phytoene synthase and a lycopene -ciclase from daffodil, and a bacterial phytoene desaturase from Erwinia uredovora (Figure 6.3). The grain of the transgenic rice had a yellow-golden colour and by itself contained sufficient -carotene for human vitamin A requirements. In rapeseed, transformation with a phytoene synthase gene also increased the level of vitamin A precursor (Kishore and Shewmaker, 1999). In tomato the transformation with a bacterial phytoene desaturase increased up to twofold the-carotene content in fruits (Ro¨meret al., 2000).
Another lipid-soluble vitamin whose function is linked to an antioxidant role is vitamin E (-tocopherol). Daily intake of this vitamin in excess of a recommended minimum is associated with decreased incidence of several diseases. Plant oils are the main source of dietary vitamin E and they generally have a high content of the vitamin E precursor-tocopherol. Overexpression of
Fig. 6.3 Biosynthetic pathway of vitamin A from geranylgeranyl pyrophosphate (GGPP). Vertical arrows indicate steps catalysed by phytoene desaturase (PDS), carotene
desaturase (ZDS), andErwinia uredovoraphytoene desaturase (CRTI).
-tocopherol methyl transferase greatly increased the seed level of-tocopherol in the model plantArabidopsis thaliana(Shintani and DellaPenna, 1998). This process seems ready to be eventually applied to some commercial crops in the future.
Flavonols are another group of secondary metabolites whose inclusion in the human diet may give protection against cardiovascular diseases. The biosynthetic pathway leading to the synthesis of these compounds has been known for a long time. However, recent information regarding the pathway has allowed the design of specific strategies to increase the content of selected bioactive compounds. Thus, the transformation of tomato with a gene from Petunia encoding a chalcone isomerase has produced tomato fruits with a 78- fold increase in the content of flavonols in the peel (Muiret al.2001). What is more important, 65% of the flanonols were retained in the paste obtained after processing the transgenic fruits.
A high risk of iron deficiency has been reported when vegetables are the major components in the diet. Although some plants are rich in this element, its availability is limited by the fact that the same plants contain oxalic acid and phytate-like substances that may complex this element. Some studies have shown that oral administration of ferritin, a protein used by plants and animals to store iron, can provide the iron needed to treat anaemia in rats. With this information, rice has been transformed with a soybean gene encoding ferritin, under the control of a seed-specific promoter (Goto et al. 1999). Transgenic rice plants accumulated ferritin in the endosperm tissue and up to three-fold levels of iron in comparison to normal seeds. Interestingly, plants overexpressing ferritin have been reported to be tolerant to oxidative damage and pathogens. This seems to be an additional agronomic trait for these transgenic plants (Dea´ket al.1999).