Engineering Amino Acid Metabolism to Improve the Nutritional Quality of Plants for Nonruminants and Ruminants

The aspartate family amino acids, lysine, methionine, and threonine, and the aromatic amino acid tryptophan are the most important essential amino acids required in human foods and livestock feeds. They are the most limiting essential amino acids in the major crop plants that serve as human foods and animal feeds, particularly cereals and legumes that are supplied as grain and/or as forage (Galili et al., 2002). Cereals are deficient mainly in lysine and tryptophan, while legumes are mainly deficient in methionine (Syed Rasheeduddin and Mcdonald, 1974). Thus, many of the commonly used diet formulations based on these crops contain limiting amounts of these essential amino acids.

Livestock that are consumed as human foods are nonruminant animals, such as poultry or pigs, and ruminants, such as cattle or sheep, which differ in feed requirements for optimal incorporation of essential amino acids. The nonruminants or monogastric animals, like humans, cannot synthesize essential amino acids and thus depend entirely on the external supply of essential amino acids. Ruminant animals also cannot synthesize these essential amino acids; however, the microbial flora inhabiting their rumen can metabolize nonessential into essential amino acids and incorporate them into microbial proteins that later become nutritionally available. Nevertheless, these microbial proteins, although of better nutritional quality than plant proteins, do not provide sufficient essential amino acids for optimal growth and milk production (Leng, 1990). Moreover, although the rumen microflora can produce essential amino acids, it can also oppositely metabolize essential amino acids into nonessential ones. Hence, in contrast to nonruminant animals that can utilize either free or protein-incorporated essential amino acids, ruminant feeds should contain the essential amino acids in proteins that are highly stable in the rumen to minimize their degradation by the rumen microflora.

Improving Lysine Levels in Crops: A Comprehensive Approach
Although free lysine content could be significantly improved in legume and cereal grain crops by expression of a bacterial feedback–insensitive DHPS (Avraham and Amir, 2005), such transgenic plants may not be optimal foods and feeds. These plants accumulate relatively high levels of intermediate products of lysine catabolism, such as α-amino adipic acid, which may act as neurotransmitters in animals and can be toxic at high levels (Bonaventure et al., 1985; Karlsen et al., 1982; Reichenbach and Wohlrab, 1985; Welinder et al., 1982). In addition, these plants overaccumulate free lysine rather than lysine-rich proteins and are therefore not suitable for feeding of ruminant animals (National Research Council, 2001). To address this issue, Jung and Falco (2000) used a composite approach to generate lysine-overproducing transgenic maize grains. This included combined expression of two transgenes. One encoded a bacterial feedback-insensitive DHPS under an embryo-specific promoter since lysine overproduction is achieved only in maize embryos (see above). The second encoded a lysine-rich protein (either hordothionine HT12 or the barley high-lysine protein BHL8, containing 28% and 24% lysine, respectively) under an endosperm-specific promoter since the endosperm consists a major part of the maize grain. Two types of maize plants were transformed with these genes, wild-type maize and a maize mutant lacking lysine catabolism due to a knockout of the maize LKR/SDH gene. The HT12 and BHL8 proteins accumulated between 3% and 6% of total grain proteins, and when introduced together with the bacterial DHPS resulted in a marked elevation of total lysine to over 0.7% of seed dry weight (Jung and Falco, 2000), for example, as compared to around 0.2% in wild-type maize. Combination of these genes into a homozygous LKR/SDH knockout background increased grain lysine level further and alleviated the problem of high-level accumulation of lysine catabolic products (Jung and Falco, 2000).

The additive effect of free lysine overproduction in the maize embryo and its incorporation into lysine-rich proteins in the endosperm on total grain lysine content suggests that free lysine is effectively transported between the two tissues. Should the dramatic elevation of lysine levels, obtained by this composite approach, not interfere with yield and other grain quality factors, the commercial application of such high-lysine transgenic maize plants for feeding human and nonruminant livestock looks very promising. Maize is also a suitable crop for ruminant feeding because maize seed proteins are on average highly resistant to rumen proteolysis (National Research Council, 2001). Moreover, the endogenous maize seed proteins may protect transgenic high-lysine proteins from rumen degradation.

Improving Methionine Levels in Plant Seeds: A Source–Sink Interaction
Most attempts to improve the methionine contents of seeds have focused on overexpression of methionine-rich seed storage proteins, such us Brazil nut 2S albumin, sunflower 2S albumin (SSA), and maize methionine-rich zeins (for review see Avraham and Amir, 2005). The SSA was also found highly resistance to rumen proteolysis (Mcnabb et al., 1994), suggesting that transgenic plants overexpressing it may be beneficial not only for nonruminants but also for ruminant feeding. Indeed, feeding experiments with transgenic lupin grains, which expressed the SSA gene, enhanced both rat growth (Molvig et al., 1997) and sheep live weight gain and wool production (White et al., 2000).

Although transgenic methionine-rich proteins can accumulate to high levels in plant seeds, in most cases the total methionine is still less than necessary for optimal feeding (Avraham and Amir, 2005; Demidov et al., 2003; Galili and Hofgen, 2002). This is largely because production of transgenic methionine-rich protein is associated with a compensatory decrease in the levels of endogenous sulfur-rich proteins. This phenomenon implies the presence of limiting levels of free methionine, whose synthesis in the seeds may be regulated by limited availability of its precursor metabolites cysteine, O-phosphohomoserine, or N-methyltetrahydrofolate. Combined seed-specific overexpression of a bacterial feedback-insensitiveAK (apparently to increase the level ofO-phosphohomoserine) as well as Brazil nut 2S albumin in transgenic narbon beans resulted in an additive increase of seed methionine, compared to the parental plants expressing each of these transgenes alone (Demidov et al., 2003). This suggests that methionine accumulation in seeds depends on the pool size of O-phosphohomoserine. In addition, when the SSA was expressed in seeds of transgenic lupin and rice plants (Hagan et al., 2003; Molvig et al., 1997; Tabe and Droux, 2002), although seed methionine levels were increased, there was no increase in seed cysteine and the total seed sulfur content, implying that the vegetative cysteine pool and the extent of sulfur transport from the canopy to the seeds represent two additional ratelimiting factors. Thus, possible additional target genes for genetic engineering of plants with high seed methionine would be genes controlling the assimilation, metabolism, and transport of sulfur. Indeed, constitutive overexpression of serine acetyl transferase, an important regulatory enzyme in cysteine biosynthesis (Fig. 3.2), enhanced seed methionine content in transgenic maize (Tarczynski et al., 2001).

Limited levels of sulfur-containing metabolites in seeds retard the synthesis of endogenous sulfur-rich proteins by negatively regulating the expression of their genes (Tabe and Droux, 2002; Tabe et al., 2002). One way to overcome this negative regulation is by replacing regulatory elements of endogenous genes encoding sulfur-rich proteins with analogous elements derived from endogenous genes whose expression is not responsive to sulfur availability. In a recent study, the promoter and 50 untranslated regions of a maize gene encoding a methionine-rich d-zein were substituted with analogous sequences derived from another gene encoding a γ-zein gene and transformed back into transgenic maize plants (Lai and Messing, 2002). Expression of this chimeric transgene caused an ~30% increase in total seed methionine.

Improving the Nutritional Quality of Hay for Ruminant Feeding
Improving the nutritional quality of hay for ruminant feeding requires the expression of proteins, which are both nutritionally balanced and resistant to rumen proteolysis in vegetative tissues. When genes encoding vacuolar methionine-rich seed storage proteins, which stably accumulate in seeds, were constitutively expressed in various transgenic plants, their encoded proteins failed to accumulate in the protease-rich vegetative vacuoles because of extensive degradation (see Avraham and Amir, 2005 for review). This was partially overcome by preventing the trafficking of these proteins from the endoplasmic reticulum (ER) to the vegetative vacuole, by engineering of an ER retention signal (KDEL or HDEL) into their C-terminus (see Avraham and Amir, 2005 for review).

Vegetative storage proteins (VSPs) may be preferred alternatives to seed storage proteins because they are nutritionally balanced and also stably accumulate in vacuoles of vegetative cells (Staswick, 1994). Galili and associates tested the potential of constitutive expression of genes encoding the α- and β-subunits of soybean VSPs to improve the nutritional quality of vegetative tissues of heterologous plants. The soybean VSPα-subunit accumulated to high levels (up to 3% of total leaf soluble proteins) and its levels remained stable also in mature leaves of transgenic tobacco plants (Guenoune et al., 1999). However, this subunit was totally unstable to rumen proteolysis (Guenoune et al., 2002b). The soybean VSPb was however more resistance to rumen proteolysis (Guenoune et al., 2002b), but accumulated only in young leaves and its levels declined with leaf age (Guenoune et al., 2003). Coexpression of both subunits in the same transgenic plant resulted in stable accumulation of both proteins in older leaves and also improved their stability to rumen degradation (Guenoune et al., 2002b).

Accumulation of transgenic proteins in vegetative tissues may be further improved by their targeting to more than one organelle. Directing the soybean VSPα to plastids resulted in a similar level to that of the vacuole-targeted counterpart (Guenoune et al., 2002a). Targeting of the soybean VSPα to these two organelles in a single transgenic plant resulted in its significantly high accumulation to up to 7.5% of the total soluble proteins (Guenoune et al., 2002a).