Corn grain is an important component of feed for non-ruminant animals and
food for humans. It is an excellent source of starch, but is a poor source
of protein nutrition. Protein content is low, usually less than 10% of the
kernel mass, and moreover, the quality of maize protein is not ideal.

Maize protein is deficient in certain amino acids?lysine, tryptophan, and
methionine?that are required by non-ruminant animals (including humans). To
remedy these deficiencies, protein supplements are provided to create a
well-balanced diet, which adds to the cost of feed and food. Genetic
improvements that increase the levels of lysine, tryptophan, and methionine
have been actively sought by researchers for the past fifty years. Recently,
several approaches involving one or more transgenes have been
successful1,2,3.

A recently published approach3 tests the hypothesis that amino acid balance
in maize kernels can be improved by production of a foreign protein to serve
as a sink for amino acids. The foreign protein used in this case is the
porcine milk protein alpha-lactalbumin, which is introduced transgenically.
The transgene contains a maize seed storage protein gene promoter and a
synthetic coding sequence optimized for expression in maize. The coding
sequence is modified by the addition of a maize signal sequence and an
endoplasmic reticulum retention sequence to direct accumulation of the
transgene product only to targeted regions in the cell. This transgene
design was predicted to result in accumulation of alpha-lactalbumin only in
the endoplasmic reticulum of the kernel endosperm, mimicking the pattern of
accumulation of the most abundant natural seed proteins in the kernel, the
zeins. The authors of this study found that transgenic kernels accumulate a
protein that cross-reacts with antiserum raised to alpha-lactalbumin and is
consistent with the size predicted for the transgene product.

The key experiment in this work is a comparison between transgenic and
non-transgenic endosperm tissue produced on the same ear. In two events
evaluated, transgenic and nontransgenic endosperm differ in their amino acid
balance, and the difference generally reflects the amino acid balance of
alpha-lactalbumin. Lysine content is 29 ? 47% higher in transgenic endosperm
than in the non-transgenic sibling kernels. In most cases, total protein
content is not changed. Kernel characteristics such as density, mass, and
seed storage protein content are not altered. Thus it appears that this
modification results in a specific alteration to the endosperm amino acid
balance that does not detectably disrupt kernel development.

The change in amino acid content, together with the accumulation of
alpha-lactalbumin in the kernels, suggests that alpha-lactalbumin may be
acting as an amino acid sink. Closer inspection reveals, however, that the
level of alpha-lactalbumin in the kernels is not sufficient to explain the
change in amino acid composition. Thus it seems that the introduction of
alpha-lactalbumin to the kernels creates a sink for amino acids but the
nature of the sink remains unclear.

The choice of a milk protein is interesting because it could result in
nutritional benefits in addition to improved amino acid balance. Several
studies have shown that derivatives of alpha-lactalbumin have benefits such
as antimicrobial and anti-tumor activities. It is hypothesized that some of
the health benefits enjoyed by nursing infants are conferred by proteins in
milk such as alpha-lactalbumin. The nutritional benefits of
alpha-lactalbumin have led several researchers to conclude that human
alpha-lactalbumin would be a beneficial component of infant formulas. A
logical extension of this idea is to include milk proteins from specific
farm animals as supplements to their diets, especially when young animals
are weaned. By illustrating the feasibility of producing porcine
alpha-lactalbumin in corn, the Bicar study enabled the researchers to test
the hypothesis that corn containing porcine alpha-lactalbumin is beneficial
in swine diets.

One drawback to using a milk protein is its potential for allergenicity in
humans. Porcine alpha-lactalbumin has not been evaluated for its allergenic
potential. Most milk allergies involve a reaction to bovine beta-lactoglobin
protein; however some individuals react to other milk proteins, and some
individuals with bovine milk allergies also react to goat milk proteins. A
thorough evaluation of the allergenicity of porcine alpha-lactalbumin is
therefore a prerequisite for application of this technology. Regardless of
the outcome of this evaluation, this work illustrates the important point
that it is possible to alter amino acid balance in a predictable way by
adding a foreign protein to seeds using genetic engineering.

The challenge of increasing grain amino acids can be thought of in terms of
increasing the strengths of either the source or the sink of the amino acids
of interest. As shown in the Bicar et al. report, adding a foreign protein
to grain may increase the strength of the amino acid sink. Some transgenic
approaches that successfully alter lysine content involve manipulation of
lysine metabolism enzymes, essentially increasing the strength of the source
of lysine. As described by Kirihara et al.4, an effective method to improve
amino acid balance could involve alterations that increase both the source
and the sink strengths for the amino acids of interest. Thus, the pathways
for amino acid metabolism would be altered to increase the level of amino
acids available for incorporation into proteins, and a sink for these amino
acids would be created by manipulation of the seed protein content and/or
composition. This approach has been successfully put into practice1. In this
case, the sink for amino acids is created by reducing the levels of the
native, low lysine proteins of corn, which presumably results in an increase
in high lysine native proteins. The work of Bicar et al. suggests that a
foreign protein can create an effective sink for amino acids and would
therefore be compatible with this approach as well. A carefully selected
protein could not only create an amino acid sink, but could also confer
nutritional or other types of benefit on the seed.

References
1. Huang S, Kruger DE, Frizzi A, D'Ordine RL, Florida CA, Adams WR, Brown
WE, and Luethy MH. (2005) High-lysine corn produced by the combination of
enhanced lysine biosynthesis and reduced zein accumulation. Plant
Biotechnology Journal 3, 555-569

2. Houmard NM, Mainville JL, Bonin CP, Huang S, Luethy MH, and Malvar TM.
(2007) High-lysine corn generated by endosperm-specific suppression of
lysine catabolism using RNAi. Plant Biotechnology Journal 5, 605-614

3. Bicar E, Woodman-Clikeman W, Sangtong V, Peterson JM, Yang S, Lee N, and
Scott MP. (2008) Transgenic maize endosperm containing a milk protein has
improved amino acid balance. Transgenic Research 17, 59-71

4. Kirihara JA, Hibberd KA, and Anthony J. (2005) Method for altering the
nutritional content of plant seed. USA Patent 6,960,709

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