Building a Balanced Amino Acid Soybean Meal

Source: Hari B. Krishnan - Plant Genetics Research Unit, USDA/ARS ; John O. Bennett - Division of Plant Sciences, University of
Missouri, Columbia, MO, USA
(dated 10/04/2006)

ABSTRACT
Poultry, swine, beef, and dairy rations account for 94% of the domestic utilization of soybean meal,
totaling over 28 million metric tons annually. Recent developments resulting in methods to enhance
levels of methionine and other essential amino acids of soybeans have the potential to impact the
animal feed industry. Animal feeds derived from the improved soybean meal could enhance feed
efficiency and minimize problems linked to excess nitrogenous waste. Development of soybean
varieties, which express the improved amino acid traits, will be facilitated by the combined use of
genetic engineering and plant breeding.
Introduction
Providing adequate nutrition for a growing human population, while sustaining the very environment
and resources essential for continued food production, is a challenge requiring the best efforts of
agriculture, industry, and science. Soybeans are an integral component of the food chain, supplying
two-thirds of the worlds vegetable protein. Competition for market-share among soybean producing
nations necessitates our continued diligence toward improving the quality of the seed protein. The
onus is on soybean researchers to develop cultivars that provide an adequate and balanced supply of
amino acids necessary for optimal growth of livestock and poultry with the concomitant minimization of
nitrogenous effluents. Such varieties must be acceptable to farmers, producing competitive yields and
displaying good agronomic characteristics. At present, supplementation of feeds with synthetic amino
acids allows for lowering of crude protein while meeting the nutritional needs of the animal. Synthetic
lysine and methionine, the primary limiting amino acids in swine and poultry rations are routinely
utilized to build balanced animal rations. Although the cost of these two synthetic amino acids is
minimal, feed manufacturers and producers spend approximately 100 million dollars annually on the
methionine analog alone (Imsande, 2001). After lysine and methionine requirements are met, other
amino acids such as arginine, isoleucine, tryptophan, and valine can become limiting. However, the
higher cost of some synthetic analogs minimizes their use in swine and poultry rations (Kerley and
Allee, 2003). Soybean varieties expressing sufficient quantities of essential amino acids necessary to
meet the nutritional demands of growing livestock and poultry would greatly enhance the utility and
value of soybean seed meal as a feed ingredient.
Soybean seed composition
Soybeans grown in the United States contain approximately 35% protein and 20% oil, the two
principal ingredients responsible for the economic value of the crop. Remaining seed components
include carbohydrates, phospholipids, isoflavones, saponins, phytic acid, minerals, and vitamins. In
the United States, the protein fraction isolated as soybean meal is primarily utilized in domestic
livestock and poultry rations. The continual demand for increased animal production efficiency and
stringent waste management regulations require improvement in both the quantity and quality of the
soybean protein. The oil component of soybeans is predominantly used for human
consumption. Increasing the oleic acid component of the soybean oil is an area, which is currently
attracting interest. Oil with higher content of this monounsaturated fatty acid has increased heat
stability and minimizes the need for hydrogenation, a process generating undesirable trans fatty
acids. Feeding soybeans with modified fatty acid profiles also is of interest to the animal industry
(Kerley and Allee, 2003). Carbohydrates, the third major constituent of the soybean, include starch,
sucrose, and a variety of other sugars. Two of the sugars raffinose and stachyose have a low
digestibility in monogastric animals resulting in gastric distress and a reduction in metabolizable
energy. Concerted efforts are being made to minimize the content of these sugars in
soybean. Partitioning of the carbon fixed by photosynthesis among these seed components is a

process not completely understood. Shifting the allocation of the fixed carbon from carbohydrate
synthesis has potential for increasing the accumulation of protein or oil.
Soybean seed protein
Glycinin (11S) and b-conglycinin (7S), which belong to a class of proteins extractable from the seed by
salt solutions, constitute 70% of the soybean seed-storage proteins. A family of five genes and two
additional glycinin-related genes encode the glycinin proteins (Nielsen et al., 1989). The proteins are
synthesized, moved to protein storage vacuoles, and then cleaved at a specific location to form acidic
and basic subunits. These subunits are joined by a single disulfide bond, re-assembled, and stored as
hexamers (Nielsen et al., 1989). Glycinins begin to accumulate from two to three weeks after
pollination reaching a maximum rate of deposition during the middle stages of seed development. bconglycinins are trimers encoded by at least 15 different genes. Composed of three subunits
designated a, a, and b, the b-conglycinins are associated with carbohydrates (Thanh and Shibasaki,
1976). Between two and three weeks after pollination, the a and a subunits begin to accumulate in
the protein storage vacuoles followed by the deposition of the b subunit. The 11S glycinins with higher
sulfur amino acid content have a greater nutritional value than the 7S b-conglycinins (Krishnan,
2005). Plant nutrient availability influences the final ratio of these proteins in mature soybean
seed. An abundance of nitrogen or deficiency of sulfur results in the enhanced accumulation of the 7S
b-subunit of b-conglycinin, which is devoid of the sulfur amino acids (Paek et al., 1997).
In addition to the 11S glycinins, other proteins, some of which are rich in the sulfur amino acids, are
found in seed but in lower abundance (Choi et al., 1995). The albumin or water-soluble seed proteins
include Bowman-Birk protease inhibitors, which can account for 4-6% of the total seed protein are rich
in cysteine. Methionine-rich proteins have also been identified in the albumin fraction. Other seed
storage proteins include lectins, lipoxygenases, and ureases. These proteins have been attributed to
play a role in various physiological functions such as germination, plant defense, and growth and
development.
Importance of sulfur nutrition in plant growth and development
To increase the cysteine and methionine content of the soybean protein an optimal supply of sulfate
must first be absorbed and transported from the root to the leaf or developing seed. Second, optimal
performance of key enzymes involved in the chemical reduction of sulfate and subsequent assimilation
into cysteine needs to be attained. Finally, we must effectively enhance synthesis and storage of
stable sulfur rich proteins. There are a myriad of biochemical processes between sulfate uptake from
the soil to final synthesis of the amino acid and then assimilation into the proteins. Cysteine in
essence becomes the sulfur donor for the synthesis of methionine and other compounds such as
glutathione and phytochelatins, which enable the plant to cope with pathogens, insects, and
environmental stresses (Saito, 2004). Additionally, sulfur-containing compounds are components of
cell membranes and cofactors, the latter of which work in conjunction with plant enzymes. The amino
acids cysteine and methionine due to the chemically active thiol group are components of many
enzymes, which carry out chemical reactions in the plant cell (Saito, 2004).
Improvement of sulfur amino acid content of soybeans
Soybean breeding
Twelve ancestral lines constitute more than 80% of the genetic base of North American soybean
cultivars (Gizlice et al., 1994). Although progress has been made toward improving soybean protein
content, this limited genetic base could hamper future success (Wilcox and Shibles,
2001). Additionally, the inverse relationship between seed yield and protein content is another
impediment in the selection and breeding of soybeans expressing both characteristics (Burton,
1987). A 20-year breeding project resulted in the production of a high protein soybean with a yield
potential comparable to commercial varieties showing that the barrier could be overcome (Wilcox and
Cavins, 1995). Cultivars exhibiting protein concentrations of 50% or more are present in germplasm
collections in the United States and Asia. These cultivars could be utilized by breeders to increase the
protein content of commercial varieties. Another technique being employed, chemical mutagenesis,

has resulted in lines which produce seeds of high methionine content (Imsande, 2001).
Genetic engineering of soybean protein
The technology of genetic engineering has provided additional avenues for improving soybean seed
protein. Seed from annual and perennial plants such as corn, sunflower, and Brazil nut contain
proteins, which are rich in the sulfur amino acids. Although expression of some of these proteins in
soybean has been successful, the resulting increase in the sulfur amino acid content of the soybean
has been modest. An early attempt to introduce a heterologous high methionine protein into the
soybean involved the use of the 2S albumin from the Brazil nut (Townsend and Thomas,
1994). Although the 2S albumin was expressed successfully and the methionine content improved,
the introduced protein was found to be an allergen, thus development of this approach was not
rigorously pursued (Nordlee et al., 1996). Subsequent transformation of soybean using a corn 15
kilodalton methionine-rich zein protein resulted in a modest increase of methionine and cysteine
content in transgenic lines (Dinkins et al., 2001). An 11 kilodalton zein when expressed in soybean
resulted in the formation of novel protein storage structures, which were located only in cells close to
the vascular tissue of the seed (Kim and Krishnan, 2004). The reason for restricted accumulation of
the introduced protein to an area near the vascular tissue of the seed has not been determined, but is
possibly related to the availability and transport of methionine.
Other options that are being considered in the attempt to increase protein quality are enhanced
expression of native soybean proteins that contain substantial quantities of sulfur amino
acids. Glycinins, encoded by a multigene family, have normal quantities of cysteine and methionine
and account for approximately 40% of the seed storage protein. A specific section of these proteins
showing natural variation in structure has been targeted for modification using cysteine and
methionine residues to replace the original amino acids. Attempts to express a gene encoding the
modified protein in tobacco (Nicotiana tabacum L.) were not entirely successful, as the encoded protein
was degraded in the storage vacuoles (Nielsen et al., 1995). Introduction of a similarly modified gene
has not been attempted in soybean, but the approach seemingly has merit and should be revisited.
Methionine rich proteins, in which this essential amino acid represents up to 12.9% of the protein
weight, have been isolated from soybean (de Lumen et al., 1999). Currently, little information is
available as to the regulation of genes encoding these proteins or the effects of over-expression on
agronomic traits of the plant. Another class of native proteins, the Bowman-Birk protease inhibitors, is
rich in cysteine. These proteins however interfere with the action of digestive enzymes in monogastric
animals. Obviously, they would not be candidates for over-expression without modification to
eliminate the deleterious effect on animal nutrition.
Another approach being considered is the construction a synthetic protein whose sequence is based on
that of a high sulfur amino acid corn storage protein (Kim et al., 1992). Eighty percent of the amino
acids in this designer protein are essential to monogastric animals. Successfully expressed in rice,
sweet potato, and tobacco this synthetic protein increased the essential amino acid content of each
and thus holds the potential to improve the protein quality of soybeans.
Although sulfur rich proteins from the other plant species have been successfully expressed in
soybean, only modest increases in the methionine and cysteine contents have resulted. Analyses
indicate that accumulation of these introduced proteins comes at the expense of sulfur-rich native
proteins. Availability of cysteine and methionine is thought to be insufficient to meet the demand for
synthesis of both the native and introduced proteins. Research is being conducted to pinpoint the
steps in sulfate transport and assimilation, which could be limiting synthesis of these amino acids
(Leustek et al., 2000). Sulfate is moved from the soil into the plant vascular system and subsequently
into specific tissues and cell organs for assimilation by membrane associated transporter proteins
(Buchner et al., 2004). A possible method of enhancing the availability of sulfate for assimilation into
the organic form, cysteine, is over expression of the transporter proteins. Within plant cells sulfate is
chemically reduced to sulfide by a series of enzyme-catalyzed reactions in preparation for reaction with
the amino acid serine to form cysteine. Cysteine is pivotal in the synthesis of methionine and other
sulfur containing compounds required by the plant (Saito, 2004). The process is a tightly regulated
system of enzyme-catalyzed reactions designed to produce cysteine in the proportions required for the

plants needs (Saito, 2004). A common scenario in biosynthetic pathways is the inhibition of key
enzyme activity by the end-product of the pathway. Alteration of this feedback control is a possible
means to increase cysteine synthesis. There is a precedent for the success of this
approach. Transgenic soybeans, which exhibit altered control mechanisms in the lysine and
tryptophan biosynthetic pathways exhibit significantly enhanced levels of these amino
acids. Increasing the rate of sulfate transport and assimilation may be the key to enhancing the
methionine content of the seed protein and thus its nutritional quality.
Genetic engineering along with increased knowledge of the soybean genome and the identification of
chromomosomal regions responsible for the traits allow for map based breeding to improve the protein
content and quality of the soybean. Recent developments in genetic engineering techniques coupled
with a greater understanding of biochemical pathways involved in amino acid biosynthesis and protein
accumulation will facilitate progress in the development of soybean varieties containing amino acids
that provide optimal nutrition for swine and poultry.
References
Buchner. P., Takahashi, H., and Hawkesford, M. J. 2004. Plant sulphate transporters: Co-ordination of uptake, intracellular,
and long-distance transport. Journal of Experimental Botany, 55:1765-1773.
Burton, J. W. 1987. Quantitative genetics: Results relevant to soybean breeding. p. 211-247. In: J. R. Wilcox (ed.),
Soybeans: Improvement, production, and uses, 2nd ed. Agron. Monogr. 16. ASA, CSSA, and SSSA, Madison, WI
Choi, Y., Ahn, J. H., Choi, Y. D., and Lee, J. S. 1995. Tissue-specific and developmental regulation of a gene encoding a low
molecular weight protein in soybean seeds. Molecular and General Genetics, 246:266-268.
de Lumen, B. O., Galvez, A. F., Revilleza, M. J., and Krenz, D. C. 1999. Molecular strategies to improve the nutritional
quality of legume proteins. Advances in Experimental Medicine and Biology, 464:117-126.
Dinkins, R. D., Reddy, M. S. S., Meurer, C. A., Yan, B., Trick, H., Thibaud-Nissan, F. et al. 2001. Increased sulfur amino
acids in soybean plants overexpressing the maize 15 kDa zein protein. In Vitro Cellular & Developmental Biology, 37:742747.
Gizlice, Z. Carter, T. E. Jr., and Burton, J. W. 1994. Genetic base of North American public soybean cultivars released
between 1947 and 1988. Crop Science, 34:1143-1151.
Imsande, J. 2001. Selection of soybean mutants with increased concentrations of seed methionine and cysteine. Crop
Science, 41:510-515.
Kerley, M. S. and Allee, G. L. 2003. Modifications in soybean seed composition to enhance animal feed use and value:
Moving from a dietary ingredient to a functional dietary component. AgBioForum, 6:14-17.
Kim, J. H., Cetiner, S., and Jaynes, J. M. 1992. Enhancing the nutritional quality of crop plants: Design, construction and
expression of an artificial plant storage protein gene. p. 1-36. In: D. Bhatnagar and T. E. Cleveland (eds.), Molecular
approaches to improving food quality and safety. AVI, New York
Kim, W-S. and Krishnan, H. B. 2004. Expression of an 11 kD methionine-rich delta zein in transgenic soybean results in the
formation of two types of novel protein bodies in transitional cells situated between the vascular tissue and storage
parenchyma cells. Plant Biotechnology Journal, 2:199-210.
Krishnan, H. B. 2005. Engineering soybean for enhanced sulfur amino acid content. Crop Science, 45:454-461.
Leustek, T., Martin, M. N., Bick, J-A., and Davies, J. P. 2000. Pathways and regulations of sulfur metabolism revealed
through molecular and genetic studies. Annual Review of Plant Physiology and Plant Molecular Biology, 51:141-165.
Nielsen, N. C., Dickinson, C. D., Cho, T. J., Thanh, V. H., Scallon, B. J., Fischer, R. L., et al. 1989. Characterization of the
glycinin gene family. Plant Cell, 1:313-328.
Nielsen, N. C., Jung, R., Nam, Y.-W., Beaman, T. W., Oliveira, L. O., and Bassner, R. 1995. Synthesis and assembly of

11S globulins. Journal of Plant Physiology, 145:641-647.

Nordlee, J. A., Taylor, S. L., Townsend, J. A., Thomas, L. A., and Bush, R. K. 1996. Identification of Brazil-nut allergen in
transgenic soybeans. New England Journal of Medicine, 334:688-692.
Paek, N. C., Imsande, J., Shoemaker, R. C., and Shibles, R. 1997. Nutritional control of soybean seed storage protein. Crop
Science, 37:498-503.
Saito, K. 2004. Sulfur assimilatory metabolism. The long and smelling road. Plant Physiology, 136:2443-2450.
Thanh, V. H. and Shibasaki, K. 1976. Major proteins of soybean seeds. A straightforward fractionation and their
characterization. Journal of Agricultural and Food Chemistry, 24:1117-1121.
Townsend, J. A. and Thomas, L. A. 1994. Factors which influence the Agrobacterium-mediated transformation of soybean.
Journal of Cellular Biochemistry Supplement, 18A:78.
Wilcox, J. R. and Cavins, J. F. 1995. Backcrossing high seed protein to a soybean cultivar. Crop Science, 35:1036-1041.
Wilcox, J. R. and Shibles, R. M. 2001. Interrelationships among seed quality attributes in soybean. Crop Science, 41:11-14.

For further information on this research, feel free to contact:

Hari B. Krishnan, Ph.D.
USDA/ARS
Plant Genetics Research Unit
108W Curtis Hall
University of Missouri
Columbia, MO 65211
USA
Tel: +1 573-882-8151
Fax: +1 573-884-7850
E-mail: KrishnanH@missouri.edu