**5. Health and nutrition**

Soybean is considered a dual use crop since it is a valuable source of seed protein and seed oil. Due to consumer health awareness and an increased demand for vegetable oil, much at‐ tention has been drawn to seed oil quality and content. To address these needs, efforts have been made with soybean to increase oxidative stability of soybean oil, enhance fatty acid content of oil, and increase total oil content within the seed. Significant progress has been made by breeders to improve overall yield of soybean, however minimal advancements have been made in the development of high-yield germ lines that have a major shift in car‐ bon flux for increased total protein or oil content in the seed. This may be attributed to the inverse correlation between absolute oil and protein contents within soybean seeds. Biotech‐ nology offers new tools for the development of soybeans that have improved oil quality for use in food, feed, and industrial applications. These nutritional enhancements have been achieved by directed modification of fatty acid biosynthesis to alter amounts of fatty acids that naturally occur in soybean, or to produce novel fatty acids. Two fatty acid profiles that have been targeted through genetic strategies include soybeans with low linolenic acid oil content and high oleic acid oil content.

#### **5.1. Fatty acid content**

Linolenic acid (LA) accounts for 10-13% of the total fatty acid content of soybean oil. This fatty acid reduces oxidative stability of oil which results in rancidity and decreased shelflife. A family of three desaturase genes (*Gm*FAD3) contribute to LA biosynthesis in soybean. Flores et al., employed a targeted gene silencing approach to suppress the *Gm*FAD3 gene family using a single RNAi construct [44]. The down regulation of this gene family resulted in low linolenic soybeans with LA contents below 2%. Oleic acid (OA) is a pre-cursor of LA and is considered a healthy source of fat. Conventional soybean oil contains ~18% OA. While high oleic soybean oil has obvious nutritional value, conventional breeding of high oleic soybean lines have not materialized, in part due to the decrease of yield and environ‐ mental instability associated with bred traits. The observed yield drag may be attributed to an alteration in the fatty acid profiles within vegetative tissues of soybean. Progress toward decreasing this yield drag has been made by down regulating Δ12 desaturases (FAD2-1A and -1B) which converts OA to LA [45, 46]. Seeds resulting from this genetic approach have an increased OA content to ~80% without alterations of vegetative tissue fatty acid contents. In combination with high levels of OA, elevated steric acid levels in soybean oil is also desir‐ able to meet needs of confectionary applications, and development of such soybeans may be possible in the future using similar approaches.

Nutritional enhancements such as increased ω-3 fatty acid levels are also desired for hu‐ man food and animal feed consumption. Two fatty acids, γ-linolenic acid (GLA) and stear‐ idonic acid (SDA) are of particular interest since they exhibit pharmacological properties and nutritional value, respectively. Sato et al., were successful at increasing these impor‐ tant fatty acids in soybeans by overexpressing a borage Δ<sup>6</sup> desaturase which converts LA and α-linolenic (ALA) to GLA and SDA, respectively [47]. Field studies with transgenic soybean harboring the borage Δ<sup>6</sup> desaturase produced GLA to ~27% and SDA to ~3% in seed oil [48]. To increase SDA levels in soy, Eckert et al., pyramided the borage Δ<sup>6</sup> desatur‐ ase with an *Arabidopsis* Δ15desaturase which converts LA to ALA [49]. It was reasoned that increased pools of ALA would lead to increased levels of SDA via the Δ<sup>6</sup> desaturase. This strategy resulted in soybean lines with 21.6% SDA when grown under greenhouse condi‐ tions. SDA in turn is a precursor for the long chain polyunsaturated fatty acids eicosapen‐ taenoic acid (EPA) and docosahexaenoic acid (DHA). Diets rich in these fatty acids are associated with cardiovascular fitness. While humans and animals possess the enzymatic machinery to convert SDA to EPA and DHA, diets are often supplemented with EPA and DHA derived from fish oil since vascular plants lack the genes for synthesis of these fatty acids. Attempts have been made to assemble these pathways in soybean by using biosyn‐ thetic genes from fungi, algae, and protists along with seed specific co-expression of these genes [50]. In such experiments EPA levels approached 20% and DHA levels represented up to 3% of total seed fatty acid content in soybean. The production of such important fat‐ ty acids has significant market potential, particularly with respect to human, poultry, pet, and aquaculture feed applications.

#### **5.2. Tocopherols**

**5. Health and nutrition**

Relationships

458

content and high oleic acid oil content.

possible in the future using similar approaches.

tant fatty acids in soybeans by overexpressing a borage Δ<sup>6</sup>

**5.1. Fatty acid content**

Soybean is considered a dual use crop since it is a valuable source of seed protein and seed oil. Due to consumer health awareness and an increased demand for vegetable oil, much at‐ tention has been drawn to seed oil quality and content. To address these needs, efforts have been made with soybean to increase oxidative stability of soybean oil, enhance fatty acid content of oil, and increase total oil content within the seed. Significant progress has been made by breeders to improve overall yield of soybean, however minimal advancements have been made in the development of high-yield germ lines that have a major shift in car‐ bon flux for increased total protein or oil content in the seed. This may be attributed to the inverse correlation between absolute oil and protein contents within soybean seeds. Biotech‐ nology offers new tools for the development of soybeans that have improved oil quality for use in food, feed, and industrial applications. These nutritional enhancements have been achieved by directed modification of fatty acid biosynthesis to alter amounts of fatty acids that naturally occur in soybean, or to produce novel fatty acids. Two fatty acid profiles that have been targeted through genetic strategies include soybeans with low linolenic acid oil

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

Linolenic acid (LA) accounts for 10-13% of the total fatty acid content of soybean oil. This fatty acid reduces oxidative stability of oil which results in rancidity and decreased shelflife. A family of three desaturase genes (*Gm*FAD3) contribute to LA biosynthesis in soybean. Flores et al., employed a targeted gene silencing approach to suppress the *Gm*FAD3 gene family using a single RNAi construct [44]. The down regulation of this gene family resulted in low linolenic soybeans with LA contents below 2%. Oleic acid (OA) is a pre-cursor of LA and is considered a healthy source of fat. Conventional soybean oil contains ~18% OA. While high oleic soybean oil has obvious nutritional value, conventional breeding of high oleic soybean lines have not materialized, in part due to the decrease of yield and environ‐ mental instability associated with bred traits. The observed yield drag may be attributed to an alteration in the fatty acid profiles within vegetative tissues of soybean. Progress toward decreasing this yield drag has been made by down regulating Δ12 desaturases (FAD2-1A and -1B) which converts OA to LA [45, 46]. Seeds resulting from this genetic approach have an increased OA content to ~80% without alterations of vegetative tissue fatty acid contents. In combination with high levels of OA, elevated steric acid levels in soybean oil is also desir‐ able to meet needs of confectionary applications, and development of such soybeans may be

Nutritional enhancements such as increased ω-3 fatty acid levels are also desired for hu‐ man food and animal feed consumption. Two fatty acids, γ-linolenic acid (GLA) and stear‐ idonic acid (SDA) are of particular interest since they exhibit pharmacological properties and nutritional value, respectively. Sato et al., were successful at increasing these impor‐

and α-linolenic (ALA) to GLA and SDA, respectively [47]. Field studies with transgenic

desaturase which converts LA

Tocopherols are lipid-soluble antioxidants that are extracted during the commercial process‐ ing of soybean seeds and add to the stability of the oil. In soybean there are four forms of tocopherols (α, β, γ and δ,) classified by the number of methyl groups present on the mole‐ cule. These molecules are collectively referred to as vitamin E, with δ - and γ -tocopherols being the most predominant forms in seeds. Biotechnological enhancements of tocopherols in soybean have mainly focused on increasing the amounts of α-tocopherol since this form has the highest nutritional value. Attempts to increase total levels of tocopherols have only been marginally successful. Examples of studies to increase total levels have focused on upregulating homogentisatephytyltransferase (HPT) activity in seeds. While HPT catalyzes the first step in tocopherol synthesis, overexpressing lines resulted in little increase of tocopher‐ ol in transgenic seeds [51, 52]. A modest increase in tocopherol content was achieved by the expression of corresponding HPT genes from *Arabidopsis* and *Synechocystis* with a strong seed specific promoter [52]. This approach resulted in a 1.5-fold increase of total tocopherol content. This same group also expressed a bacterial chorismatemutase-prephenate dehydro‐ genase (TYRA) gene with several other enzymes under the control of seed specific promot‐ ers and observed a >10- fold increase in total vitamin E type molecules.

To generate soybean lines with increased levels of tocopherols a more direct approach has involved overexpression of homogentisategeranylgeranlytransferase (HGGT), an enzyme involved in the biosynthesis of tocopherols in monocots. Recently transgenic expression of rice HGGT was expressed in soybean with a seed-specific and constitutive promoter [53]. Transgenic soybean expressing the HGGT gene had significantly higher levels of antioxi‐ dant activities and showed enhanced vitamin E levels associated with the presence of all forms of tocopherols, including tocotrienols (with the exception of the β- form) which are not found naturally in soybean.

Enhanced amounts of α-tocopherol in soybean have proven easier to metabolically engineer. Expression of genes for two enzymes responsible for methylation of tocopherol head groups (VTE3 and VTE4) from *Arabidopsis* were co-expressed within the seed and generated plants with α-tocopherol levels greater than 90% of total tocopherol content [54]. The total levels of tocopherol remained the same in these seeds, showing a shift in tocopherol to mainly the αform yielding a 5-fold increase in vitamin E activity. This research can lead to soybean oil with enhanced vitamin E and more nutritional value for consumers.

### **5.3. Dietary amino acids**

Soy is also deficient in several essential dietary amino acids, most notably methionine and cysteine due to their high sulfur contents. Albumins from Brazil nuts, sunflowers, and corn have been expressed in soybean and although they resulted in increased methionine and cysteine levels, they are not adequate enough to avoid supplementation of these amino acids in animal feed and human diets. The physical synthesis of cysteine is carried out by the en‐ zyme O-acetylserinesulfhydrylase (OASS). In an attempt to increase sulfur containing amino acids in soybean, Kim et al., overexpressed cytosolic OASS and found that transgenic seeds contained elevated levels of both protein bound cysteine (58-74%) and free cysteine (22-32%) [55]. Another approach used to increase sulfur amino acid content in soybean is the use of a maize zein gene which gives rise to several species of insoluble proteins containing high lev‐ els of methionine. Dinkins et al., overexpressed maize zein in soybean seeds and observed a 12-20% increase in methionine and 15-35% increase in cysteine without adverse effects on protein composition [56].

The essential amino acids lysine and threonine have also been explored for their potential to create nutritionally enhanced soybean. An increased level of lysine was observed by geneti‐ cally engineering the lysine biosynthetic pathway to circumvent the normal feedback regu‐ lation of the enzymes aspartokinase and dihydrodipicolinic acid synthase in soybean [57]. In this case, a >100-fold increase of free lysine and 5-fold increase of total seed lysine content was observed. More recently, soybeans showing enhanced threonine levels have been engi‐ neered using seed-specific expression of lysine-insensitive variants of aspartate kinases from bacteria [58]. This strategy produced transgenic soybeans with a 100-fold increase in threo‐ nine levels and 3.5-fold increase in total free amino acid content without negative impacts on seed morphology or germination. While enhancement of essential amino acids in soy‐ bean seeds has clear potential for commercial applications, it will be important to demon‐ strate that transgenic soybean with increased nutritional enhancement traits also maintain optimal agronomic characteristics when grown in the field under a variety of conditions.
