Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Utility Patent
1998-06-16
2001-01-02
Hutzell, Paula K. (Department: 1649)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C536S023600, C435S412000, C435S419000, C435S069100, C530S370000, C530S350000, C800S295000, C800S298000
Utility Patent
active
06169232
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the preparation and use of genes encoding various sink proteins and the use thereof to improve the nutritional quality of animal feeds.
BACKGROUND OF THE INVENTION
Each year, over 2.5 billion bushels of corn seed are processed as animal feeds (hereinafter feed(s)) for cattle, poultry, swine, and the like. This single use accounts for approximately 35% of the total United States corn production. However, corn seed is not an optimal feed source due to the low abundance of essential amino acids such as methionine, tryptophan, lysine and the like. Low abundance of these amino acids in corn seed is thought to be due to the absence of appropriate “sink proteins” which act as reservoirs for said amino acids. Therefore, current use of corn for feeds requires the addition of supplements obtained from other sources, such as soybeans or purified amino acids, to address these deficiencies and thus prevent stunted animal growth and development.
The term “sink protein” is frequently associated with the term “storage protein” albeit there are differences between the two. Storage proteins are thought to fulfill a role as a nitrogen reservoir within cells; a pool in which a variety of different amino acids can be deposited for future use. These proteins can then be proteolyzed at a later time, such as germination, to provide the amino acids necessary for proper cell growth and development. Amino acids found in storage proteins are typically used directly for incorporation into other proteins or used as substrates in the biosynthesis of additional amino acids or other metabolites [Staswick (1990) The Plant Cell, (1990) 2:1-6.]. Many examples of storage proteins are noted in the art including the beta-conglycinins found in soybean seed, and patatin, the major storage protein found in potato tubers.
Sink proteins, often found in large abundance, differ from storage proteins in that they contain a higher than the averaged amount of one or more specific amino acids. They are usually classified by their most abundant amino acid and are thought to serve as stable reservoirs for such. The sulfur-rich sink proteins, those being rich in cysteine and methionine, and their corresponding genes have been studied most thus far. These include a 15 kDa zein-class sink protein for corn [Pedersen et al., (1986) J. Biol. Chem. 261:6279-6284]; a 10 kDa zein-class sink protein from corn [Kirihara et al., (1988) Gene 71:359-370]; two genes from pea seed encoding albumins [Higgins et al., (1986) J. Biol. Chem. 261:11124-11130]; and a gene from Brazil nut encoding a seed 2S albumin [Altenbach et al., (1987) Plant Mol. Biol. 8:239-250].
Evidence presented in the art to date suggests that biosynthesis rates of amino acids in plants do not vary substantially when compared one to another. However, the final averaged amino acid content of a seed by dry weight can vary dramatically depending on the plant species. In some cases, variations observed in final amino acid content are related to the tolerance of the cell to free amino acid levels (those not incorporated into protein). Tolerance levels of said free amino acids are most often maintained and regulated through feedback mechanisms involving enzymes that are sensitive to the size of free amino acid pools. In addition, free amino acids may be degraded. For example, the enzyme lysine-ketoglutarate reductase monitors and degrades free lysine in corn endosperm thereby preventing its accumulation to levels that may disrupt cell metabolism [Arruda and Silva, (1982) Eur. J. Biochem. 209:933-937; Brochetta-Braga et al., (1992) Plant Physiol. 98:1139-1147]. However, if amino acids are incorporated into protein, they are removed from the “free” pool and thus prevented from exceeding tolerance limits. Incorporation into protein also prevents limitations placed on biosynthesis rates through biochemical feedback mechanisms.
The low abundance of certain amino acids accumulated in a cell by dry weight can also be correlated to the low abundance of certain amino acids in specific sink or storage proteins. For example, while seeds may not need high levels of certain amino acids to maintain their physiological viability, these low levels result in the feeds derived thereof to be nutritionally unbalanced. Low levels of tryptophan, cysteine and methionine in corn kernels may be traced directly to the nominal frequency of these amino acid in zeins, the major storage protein in corn kernels. Although there may be other proteins in corn kernels having higher levels of tryptophan, cysteine, and methionine, accumulated levels of these proteins are not high enough to result in a substantial contribution to the total amino acid profile. Increased expression of proteins which can act as sink proteins or introduction of new sink proteins in a seed can improve the total amino acid profile.
Currently, nutritional deficiencies in feeds are augmented by supplementation with soybean meal and/or the purified amino acids of interest. However, this results in overall higher feed cost due to the cost associated with supplements as well as increased handling and processing requirements. Therefore, it would be quite desirous for feeds to be obtained from genetically engineered seeds endogenously expressing sink proteins that would improve the nutritional balance of said feed. It would also be desirous for the sink proteins used as amino acid supplements to improve the nutritional balance of feeds to be produced less expensively through the use of molecular biology and heterologous expression systems. The inventions, as described herein, address these problems and therefore will allow small farm owners/operators to produce nutritionally balanced feeds at reduced cost.
SUMMARY OF THE INVENTION
In the present invention, genes encoding storage proteins have been isolated, cloned and modified to encode sink proteins. Furthermore, genes encoding naturally occurring sink proteins have been isolated and cloned. Said genes can be expressed in cells to produce proteins. These expressed proteins can be added to feeds to improve the nutritional value thereof.
One aspect of the disclosed invention is the development of methods whereby genes encoding storage proteins can be modified to encode sink proteins. More specifically, the modified genes encode Tryptophan sink protein (hereinafter TSP), which has been created from bark storage protein (hereinafter BSP) by genetically performing conservative substitutions of tryptophan for specific phenylalanine residues. The addition of TSP to feeds can improve the tryptophan content therein and the nutritional value thereof. Another aspect of the present invention relates to methods for increasing the specific amino acid content of proteins having naturally occurring proteolytically processed regions. Specifically, the genes encoding proteolytically processed regions are modified to encode peptides wherein conservative substitutions are made. More specifically, the modified genes encode for lysine enriched ribosome inactivating protein (KRIP) and derivatives thereof. Expressing genes encoding for KRIP and derivatives thereof can produce proteins which can improve the nutritional balance of feeds.
Another aspect of the present inventions relates to methods of creating a single gene encoding for two or more individual sink proteins being linked together. More specifically, the genes encode for RIP-5, containing a derivative of KRIP and a sub-domain of potato multicystatin (PMC). Different sink proteins covalently linked together can improve the nutritional value of feeds by increasing the levels of either the same or different amino acids.
Another aspect of the present invention relates to isolating and cloning gene fragments encoding naturally occurring sink proteins. More specifically, the isolated genes encode potato multicystatin and sub-domains thereof (hereinafter PMC) and the 10 kDa rice prolamin protein (hereinafter RP-10). Expressing said genes can increase the n
Hey Timothy D.
Merlo Ann Owens
Walsh Terence A.
Dow AgroSciences LLC
Hutzell Paula K.
Stuart Donald R.
Zaghmout Ousama
LandOfFree
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