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Engineering soybean for enhanced sulfur amino acid content.

Soybean Is an Excellent Protein Source

Humans have cultivated and utilized seed crops as food source for centuries. Currently, more than 70% of protein consumed by humans is derived from legumes and cereals. Legumes, in contrast to cereals, accumulate higher amounts of protein. Among the legumes, soybean contains a high percentage of protein that varies from 35 to 50% depending on the cultivar and growing conditions. The protein content of soybean exceeds that of the most commonly consumed food sources (Fig. 1). Although soybean is an excellent source of protein, its nutritional quality is compromised by a low concentration of the sulfur amino acids methionine and cysteine. Humans and monogastric animals are unable to synthesize essential amino acids, including methionine, and are dependent on their diets to meet sulfur amino acid requirements. On the basis of feeding studies using rats as model animals, researchers have asserted that soybean protein does not provide adequate methionine to meet human dietary needs (Young, 1991). This conclusion was based on the protein efficiency ratio (PER), an evaluation method that compares weight gain of rats fed casein as a protein source to that of animals consuming the test protein. In 1991, a new evaluation method, protein digestibility corrected amino acid score (PDCAAS), was adopted. This method provides a more concise evaluation of protein quality because it takes into account not only the feed amino acid profile, but also the digestibility of the proteins in the species tested. Recently, the validity of PDCAAS method to predict protein quality has been questioned (Sarwar, 1997; Schaafsma, 2000). Experimental evidence suggests that this method overestimates the quality of proteins containing antinutritional factors (Sarwar, 1997). Nevertheless, on the basis of PDCAAS, it has been established that the quality of soybean protein is equivalent to casein and egg white (Fig. 2, Food and Agricultural Organization-World Health Organization, 1991). In addition to well-established health benefits of soybean isoflavones in reducing the risk of cancer and heart disease, soybean protein consumption has been associated with a diminution of chronic ailments such as osteoporosis, atherosclerosis, and renal disease (Messina, 1999).


Soybean is extensively used as high-protein feed ingredient in livestock and poultry production. Young swine and poultry have higher dietary requirement for sulfur amino acids than provided by grain-soybean meal rations. The poultry and swine industries spend an estimated $100 million annually augmenting feeds with synthetic methionine to promote optimal growth and development of animals consuming grain-soybean meal rations (Imsande, 2001). Since soybean is the principal seed meal used in feeds, developing soybean cultivars with high sulfur amino acid content may have a significant and positive impact on the livestock and poultry industry. With the advent of genetic engineering and establishment of reliable soybean transformation methodology (Somerset al., 2003), efforts to improve the protein quality of soybean have met with increasing success (Wang et al., 2003). This article focuses on the progress and challenges that are involved in improving the sulfur amino acid content of soybean. Progress on the improvement of nutritional quality of other legumes has been reported in several recent publications (Wang et al., 2003; Muntz et al., 1998; Hagan et al., 2003; Tabe and Higgins, 1998).

Abundant Soybean Seed Proteins and Their Sulfur Amino Acid Content

The predominant storage proteins of soybean are salt-soluble globulins. Soybean globulins are denoted 2S, 7S, 11S, or 15S representing the sedimentation coefficients of proteins in sucrose density centrifugation (Thanh and Shibasaki, 1976). The 7S and 11S globulins, designated [beta]-conglycinin and glycinin, respectively, account for 70% of the total seed protein (Nielsen, 1996; Krishnan, 2000). Because of their abundance, these two groups of proteins are essentially responsible for the nutritive value of foods and feeds derived from soybean. The glycinins, comprising approximately 40% of the total seed protein, occur as hexamers with an estimated molecular mass of 320 to 375 kDa (Wolf and Briggs, 1958; Bradley et al., 1975). Glycinins are synthesized as precursor proteins, and then posttranslationally cleaved to yield acidic and basic subunits (Beachy et al., 1981; Barton et al., 1981). Genes encoding the major glycinins Gy1, Gy2, Gy3, Gy4, and Gy5 have been cloned and characterized (Staswick et al., 1984; Nielsen et al., 1989; Xue et al., 1992). In addition, "glycinin-related" genes have been identified that presumably encode glycinin subunits of lower abundance (Lei et al., 1983; Nielsen et al., 1989). [beta]-Conglycinin, isolated as a trimer and having a molecular mass of 150 to 175 kDa (Thanh and Shibasaki, 1976, 1978), is composed of [alpha]' (76 kDa), [alpha] (72 kDa), and [beta] (53 kDa) subunits (Coates et al., 1985; Tierney et al., 1987). Comparison of sulfur amino acid contents indicates that glycinin contains substantially more cysteine and methionine than the [beta]-conglycinin (Table 1). Several studies have established that the accumulation of the [beta]-subunit of [beta]-conglycinin is promoted either by excess nitrogen or by sulfur deficiency (Gayler and Sykes, 1985; Paek et al., 1997, 2000; Imsande and Schmidt, 1998; Imsande, 2003). An increase in the accumulation of [beta]-conglycinin lowers the cysteine and methionine content of soybean seed protein and thus its nutritive quality.

Since commercially grown soybean do not accumulate sufficient methionine and cysteine to meet the needs of growing swine and poultry, researchers are attempting to increase the amounts of these amino acids in soybean protein. Approaches taken to improve protein quality can be grouped into five categories: (i) utilization of traditional plant breeding methods; (ii) expression of sulfur rich heterologous seed proteins; (iii) modification of abundant endogenous proteins; (iv) enhancing the accumulation of low abundance endogenous methionine rich proteins; and (v) expression of synthetic proteins with well-balanced amino acid composition.

Traditional Plant Breeding Methods

Although substantial increases in protein levels have been achieved by traditional breeding (Brim and Burton, 1979; Burton et al., 1982; Wilcox and Shibles, 2001), lack of variability in methionine content among soybean cultivars (Kuiken and Lyman, 1949; Krober, 1956) has limited the use of conventional methods to increase the sulfur amino acid content. In general, the amount of sulfur amino acids has remained constant regardless of the amount of seed protein (Burton et al., 1982; Wilcox and Shibles, 2001). Madison and Thompson (1988) identified several methionine-over-producing soybean cell culture lines following treatment with ethionine, a chemical analog of methionine. These lines accumulate 8.7-fold higher methionine than the parental line, but mature soybean plants were not generated from these cultures. Imsande (2001) mutagenized Kenwood 94 soybean seeds with ethyl methanesulfonate (EMS) and screened for ethionine-resistant plants exhibiting dark-green leaves. This procedure resulted in the identification of several methionine-over-producing lines. The concentration of methionine and cysteine in one mutant was determined to be approximately 20% higher than that of the parent line (Imsande, 2001). This study demonstrated the feasibility of increasing the sulfur amino acid content of soybean by chemical mutagenesis. However, it remains to be seen if the reported 20% increase in the sulfur amino acid content is adequate to meet the nutritional requirement of livestock and poultry.

The ratio of the 7S and 11S globulins influences the sulfur amino acid content of soybean seed proteins (Paeket al., 1997; 2000). Because the 7S globulins are deficient in sulfur-amino acids, either down-regulating or eliminating the expression of these proteins could enhance accumulation of glycinins or other sulfur-rich proteins. Several soybean mutants have been reported that are altered in seed protein composition. Both spontaneous and induced mutants with altered [beta]-conglycinin composition have been characterized (Kitamura and Kaizuma, 1981; Ladin et al., 1984; Tsukada et al., 1986). Unfortunately, these recessive mutants are accompanied by developmental abnormalities. A mutant line lacking all three subunits of [beta]-conglycinin has been obtained by subjecting seeds to [gamma]-ray irradiation (Kitagawa et al., 1991). Genetic analysis has indicated that this mutation resulted from an alteration in the single recessive gene Cgdef. Plants carrying the mutation, however, have exhibited developmental abnormalities and have failed to reproduce.

Another mutant devoid of [beta]-conglycinin was identified in a Japanese wild soybean collection (Hajika et al., 1996). A single dominant gene Scg-1 was responsible for the absence of the 7S globulin. This trait has been successfully incorporated into soybean cultivars without obvious deleterious effect on agronomic performance (Teraishi et al., 2001). It should be interesting to observe how the complete absence of [beta]-conglycinin altered the nutritional and functional quality of seed proteins. Such mutants could serve as valuable sources for improving the sulfur content of soybean seeds.

Expression of Heterologous Seed Proteins Rich in Sulfur Amino Acids

Proteins that are high in sulfur-containing amino acids have been isolated from Brazil nut (Bertholletia excelsa H.B.K.), sunflower (Helianthus annuus L.), and corn (Zea mays L.). The Brazil nut 2S albumin (BNA) contains 18% methionine and 8% cysteine (Altenbach et al., 1987); while, the sunflower 2S albumin (SFA8) contains 23% methionine and cysteine (Kortt et al., 1991). Delta zeins, hydrophobic proteins of corn, contain 23 % methionine and 4% cysteine (Kirihara et al., 1988; Chui and Falco, 1995). As these proteins are highly rich in sulfur amino acids, they have been targeted for expression in legume crops such as soybean, narbon bean (Vicia narbonensis L.), and lupins (Lupinus angustifolius L.). This strategy has been successfully employed to transform soybean with sulfur-rich 2S albumin derived from Brazil nut (Townsend and Thomas, 1994). For some transgenic soybean lines, the 2S albumin comprises 10% of the total seed protein, but the overall methionine content has increased by 15 to 40% (Townsend and Thomas, 1994). It has been reported that these transgenic soybean accumulate lower amounts of protease inhibitors, proteins rich in sulfur amino acids (Streit et al., 2001). The BNA has been determined to be a major allergen (Nordlee et al., 1996) and consequently no commercial soybean cultivars expressing the BNA have been developed.

Amino acid analysis of transgenic soybean seeds expressing a 15-kDa delta zein revealed that methionine was 12-20% and cysteine 15-35% more abundant than in wild-type seeds (Dinkins et al., 2001). However, it is not known if the increase in methionine and cysteine content occurred at the expense of endogenous sulfur-rich proteins. Recently, a novel 11 kDa delta zein containing 32 methionine and 7 cysteine residues, accounting for 25% of the total amino acids of this protein, was isolated (Kim and Krishnan, 2003). When this gene was placed under the [beta]-conglycinin [alpha]'-subunit promoter and introduced into soybean, seed-specific expression was observed. Electron microscopic investigation of seed tissue revealed that 11-kDa zeins accumulated in novel endoplasmic reticulum-derived protein bodies (Fig. 3). The distribution of these protein bodies was confined to cells present between seed vascular tissue and storage parenchyma cells. Amino acid analysis of alcohol-soluble protein fractions isolated from a transgenic soybean line showed a 1.5 to 1.7-fold increase in the methionine content when compared to control (Kim and Krishnan, 2004).


Bovine [beta]-casein, a hypoallergenic milk protein, contains a well-balanced amino acid profile. This protein has also been utilized to improve the protein quality in soybean (Maughan et al., 1999). In transgenic lines, the bovine [beta]-casein has accumulated in soybean seeds at approximately 0.1 to 0.4% of the total soluble seed protein. However, it is not known if the expression of the bovine [beta]-casein increases the protein quality. Consumer acceptance of soybean modified with animal-derived proteins is an area that must be carefully evaluated.

Modification of Abundant Endogenous Proteins

A multigene family consisting of at least five genes encodes glycinin (Nielsen et al., 1989). On the basis of amino acid sequences, all five glycinin genes Gy1 to Gy5 belong to either Group 1 or Group 2. The notable structural difference between the two groups is the presence of glutamate/aspartate-rich insertions in the hypervariable region (HVR) of Group-2 glycinin. The HVR region is characterized by extensive natural variation, and consequently has been targeted for insertion of sulfur amino acids (Nielsen et al., 1990). Oligonucleotides encoding methionine residues have been inserted into the HVR of the Gy4 gene, and the modified glycinin gene has been expressed in tobacco (Nicotiana tabacum L.). Even though transcription of the modified gene has been detected, no accumulation of a protein has been observed, presumably due to proteolytic degradation in storage vacuoles (Nielsen et al., 1995). This may be the result of introduced amino acid residues that interfere with the correct folding of the protein, and leading to rapid degradation.

To ascertain sites where introduction of amino acid residues would be tolerated without affecting folding of the polypeptides and assembly into holoprotein, methionine residues were introduced into different regions of the Gy1 gene (Utsumi et al., 1994). Insertion of these sequences near the HRV C-terminal region and close to the C terminus of the [beta]-chain was found not to interfere with trimer formation. Methionine-enriched glycinins, when expressed in transgenic tobacco seeds and potato (Solanum tuberosum L.) tubers, accumulated stably, thus indicating correct folding and assembly (Gidamis et al., 1995; Takaiwa et al., 1995). These studies indicate the possibility of introducing additional methionine residues in certain regions of glycinin without drastically affecting protein stability. Since multigene families encode glycinins and only one member of this gene family was altered, the overall effect on the methionine content would most likely be below the level required for animal feed.

Lysine and methionine are the two principal limiting amino acids in corn-soybean based rations. In addition, threonine, isoleucine, tryptophan, valine, and arginine have been identified as "second-tier" limiting amino acids in animal feed. Attempts have been made to increase the content of some of these "second-tier" amino acids in soybean by genetic engineering (Rapp et al., 2003). To increase the isoleucine content, oligonucleotides coding for 10 to 20 isoleucine residues have been introduced into the coding region of [beta]-conglycinin. The introduction of isoleucine residues does not appears to interfere with the secondary structure of the protein as evidenced by correct folding of the protein following in vitro assays. High-level expression of isoleucine-enriched [beta]-conglycinin in transgenic soybean has been recently reported (Rapp et al., 2003).

Elevating the Levels of Endogenous Sulfur-Rich Proteins

Methionine-rich proteins (MRPs) have been identified in the soybean albumin fraction (Kho and de Lumen, 1988; George and de Lumen, 1991; Revilleza et al., 1996). The 10.8-kDa MRP contains 12.1% methionine, while the 8-kDa MRP is comprised of 8.6% methionine and 11.4% lysine. These proteins accumulate at low levels in soybean seeds. Because of the high content of methionine, it has been suggested that enhancing the accumulation of these MRPs will increase the methionine content of soybean (de Lumen et al., 1999). A cDNA encoding the 8-kDa MRP has been isolated, and the deduced amino acid sequence revealed 60% homology with a cysteine-rich seed storage protein from lupine (Galvez et al., 1997). Unlike the major storage proteins of soybean, little information is known about MRPs in soybean. To manipulate the expression of low abundance MRPs in soybean, it is imperative that we have a better understanding of structure and regulation of MRP genes.

Soybean accumulate two types of protease inhibitors, Kunitz trypsin inhibitor (KTi) and Bowman-Birk inhibitor (BBI) of chymotrypsin and trypsin. In addition, soybean seeds store a BBI-related family of isoinhibitors. These inhibitory proteins, that are rich in cysteine, contribute significantly to the sulfur amino acid content of soybean. Elevating expression of these proteins can lead to an increase in the sulfur amino acid content of soybean. This approach can be beneficial to human nutrition as recent studies have demonstrated that BBI may have anticarcinogenic properties (Kennedy, 1998). However, protease inhibitors have been shown to have a negative effect on the performance of nonruminants such as poultry and swine. Since soybean are primarily used as adjuvants for animal feed, increasing the concentration of these sulfur-rich proteins by enhancing the levels of protease inhibitors is not desirable.

Expression of Synthetic Gene with Well-Balanced Amino Acid Composition

An alternative approach to increasing the nutritional quality of soybean involves expression of synthetic genes encoding proteins with desirable amino acid composition. A synthetic gene (ASP1) that encodes a protein comprised of 80% essential amino acids has been constructed on the basis of the structural model of corn zeins (Kim et al., 1992). ASP1 is an ideal high-quality protein made up of four 20 amino acid helical-repeating monomers. This synthetic protein has been successfully expressed in tobacco, sweet potato [Ipomoea batatas (L.) Lam], and rice (Oryza sativa L.), resulting in an increase in the essential amino acids in each species (Zhang et al., 2002; Potrykus, 2003). Research is underway to express the ASP1 gene in most staple crops, including soybean. It should be interesting to witness if this strategy results in the development of soybean with improved protein quality.


Substantial progress has been achieved with genetic engineering in facilitating the expression of heterologous sulfur-rich proteins in soybean. Introduction of methionine-rich zeins and BNA has resulted in modest increases in the sulfur amino acid content of soybean seeds. However, this increase is not sufficient to adequately meet the nutritional requirements of livestock and poultry. Although there is a net increase in the sulfur amino acid content of transgenic soybean seeds expressing BNA, this increase is accompanied by a decrease in the endogenous sulfur rich proteins (Streit et al., 2001). This suggests that the synthesis or supply of sulfur amino acids in developing soybean seeds is not adequate to meet the demand for cysteine and methionine created by the introduction of sulfur-rich proteins. Recent studies have shown that the assimilation of sulfur can also occur in developing soybean seeds (Sexton and Shibles, 1999, Chronis and Krishnan, 2003a, 2003b). The activity of three enzymes involved in cysteine synthesis, ATP sulfurylase, O-acetylserine (thiol) lyase (OASTL), and serine acetyl transferase (SATase) have been demonstrated in developing soybean seeds. Serine acetyl transferase and OASTL, two key enzymes in sulfur assimilation pathway, are subject to multiple levels of regulation (Leustek et al., 2000; Noji and Saito. 2002). To increase the biosynthesis of cysteine, it is desirable to engineer SATase and OASTL, insensitive to feedback inhibition, and express these two enzymes in developing soybean seeds. A similar approach has been successfully used to generate transgenic soybean plants expressing high levels of lysine and tryptophan (Falco et al., 1995; Rapp et al., 2003). When feedback-insensitive aspartokinase and dihydrodipicolinic acid synthase, enzymes in the synthetic pathway of lysine, are expressed in soybean, a five-fold increase in the amino acid has been observed (Falco et al., 1995). Similarly, expression of a modified anthranilate synthase, an enzyme involved in tryptophan synthesis, in transgenic soybean seed has resulted in the accumulation of 20 to 30 fold higher tryptophan content than that of nontransgenic seed (Rapp et al., 2003). On the basis of these studies, it appears that by judiciously manipulating key enzymes involved in the sulfur biosynthetic pathway, it should be possible to elevate the sulfur amino acid content of soybean.

To successfully engineer high sulfur content in soybean, several factors, including levels of transgene expression, gene silencing, potential allergenicity of introduced proteins, stability of introduced protein, and subcellular localization of the introduced protein, must be carefully addressed. For example, even though a high level of BNP expression has been obtained in soybean using seed-specific promoters, substantial increases in the sulfur amino acid content of soybean seeds have not been observed. Additionally, this high-level expression of BNP is accompanied by suppression of certain endogenous proteins that are rich in sulfur-amino acids. It is noteworthy to point out that BNA has been found to be a major allergen, rendering it unsuitable for human consumption.

The choice of transformation method utilized may influence expression levels of the gene introduced into soybean. Transgenic plants generated by particle bombardment often contain multiple integration events that result in gene silencing (Reddy et al., 2003). In transgenic plants generated by Agrobacterium tumefaciens-mediated transformation, gene insertion occurs randomly resulting in differential expression of the introduced gene. As a consequence, evaluation of several independent transformation events is necessary to obtain the desired phenotype. Recent developments in transformation methodology bode well for achieving increased soybean transformation efficiencies which should facilitate engineering of sulfur-rich soybean.

In conclusion, a two-prong approach involving metabolic engineering of sulfur assimilatory pathway genes and high level expression of genes encoding sulfur-rich storage proteins, appears to be the most promising way of designing soybean with desired amount of sulfur amino acids.
Table 1. Amino acid composition of 7S and 11S storage proteins of
soybean. ([dagger])

                  [beta]-Conglycinin (7S)

Amino acid      [alpha]'   [alpha]   [beta]

Alanine            4.2        4.5      5.3
Arginine           6.7        7.9      7.0
Asparagine         6.5        7.1      8.0
Aspartic acid      4.5        4.8      5.1
Cysteine           0.8        0.9      0.0
Glutamine          8.9        8.3      8.0
Glutamic acid     13.6       13.6      8.9
Glycine            4.7        4.1      4.3
Histidine          3.5        1.4      1.9
Isoleucine         4.7        5.3      6.3
Leucine            7.2        8.3      9.9
Lysine             7.2        6.2      4.8
Methionine         0.3        0.2      0.0
Phenylalanine      4.5        4.6      6.8
Proline            5.7        6.7      5.1
Serine             7.4        7.2      7.5
Threonine          2.0        1.9      2.4
Tryptophan         0.5        0.3      0.0
Tyrosine           2.5        2.6      2.9
Valine             4.5        4.1      5.8

                          Glycinin (11S)

Amino acid       Gy1    Gy2    Gy3   Gy4   Gy5

Alanine          5.7    6.6    5.8   4.3   3.7
Arginine         5.7    6.2    5.6   7.3   6.3
Asparagine       7.8    8.6    7.5   6.1   6.7
Aspartic acid    3.6    3.9    3.4   6.0   4.7
Cysteine         1.7    1.7    1.7   1.1   1.2
Glutamine       10.1   10.9   10.5   9.5   9.5
Glutamic acid    8.6    7.9    8.6   9.9   8.7
Glycine          7.4    7.3    7.3   6.3   7.9
Histidine        1.7    0.9    1.3   2.8   3.2
Isoleucine       5.5    4.9    5.2   3.9   3.7
Leucine          6.9    7.1    6.9   6.9   6.9
Lysine           5.0    3.9    3.9   4.8   3.7
Methionine       1.3    1.5    1.1   0.4   0.6
Phenylalanine    4.2    4.1    5.2   3.0   3.2
Proline          6.1    5.6    6.2   6.9   7.3
Serine           6.7    6.4    7.3   7.3   7.7
Threonine        4.2    3.9    3.9   3.5   3.9
Tryptophan       0.8    0.9    0.9   0.9   0.8
Tyrosine         2.3    2.4    2.4   2.6   3.2
Valine           4.8    5.6    5.4   6.5   7.1

([dagger]) Amino acid composition of the mature proteins was calculated
from the sequences obtained from the protein database. The protein
accession numbers are as follows: Beta-conglycinin alpha prime subunit
(BAC78524); Beta-conglycinin alpha subunit (BAB56161),
Beta-conaglycinin beta subunit (P25974), Gy1 (S10851), Gy2 (S11002),
Gy3 (S11003), Gy4 (S11004), and Gy5 (CAA55977).

Fig. 2. Protein digestibility corrected amino acid score (PDCAAS) of
selected food proteins. Note that soybean protein quality is equivalent
to that of animal proteins and superior to other plant proteins.

                        Casein   1.00
                     Egg white   1.00
 Soybean protein (concentrate)   0.99
Rapeseed protein (concentrate)   0.93
     Soybean protein (isolate)   0.92
                          Beef   0.92
     Pea protein (concentrate)   0.73
                     Pea flour   0.69
                   Peaunt meal   0.52
                   Whole wheat   0.40
   Sunflower protein (isolate)   0.37
                  Wheat gluten   0.24

Note: Table made from bar graph.

Abbreviations: BNA, Brazil nut albumin; HVR, hypervariable region; MRP, methionine-rich protein; PDCAAS; protein digestibility corrected amino acid score; SAT, serine acetyltransferase.


The author wishes to thank Dr. Larry Darrah and John Bennett for critical review of the manuscript.


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Hari B. Krishnan *

Plant Genetics Research Unit, Agricultural Research Service-USDA and Department of Agronomy, University of Missouri, Columbia, MO 65211. Received 4 Nov. 2003. Symposia. * Corresponding author (
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Title Annotation:Symposium
Author:Krishnan, Hari B.
Publication:Crop Science
Geographic Code:1USA
Date:Mar 1, 2005
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