Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters fat – fatty oil – ester-type wax – or...
Reexamination Certificate
1999-07-22
2002-04-30
Nelson, Amy J. (Department: 1639)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters fat, fatty oil, ester-type wax, or...
Reexamination Certificate
active
06380462
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to genetic modification of plants, plant cells and seeds, particularly altering fatty acid composition.
BACKGROUND
Soybean (
Glycine max
) is one of the highest value crops currently grown in the United States (≈$16 billion in 1996). Ranking close to corn (25%) and wheat (22%), soybean accounted for 19% of the United States crop acres planted in 1994. Often referred to as a “miracle crop”, soybean offers tremendous value through the oil, protein and whole soybean products. Agronomic traits, food quality traits related to oils and protein quality are all important for the soybean industry.
More soybeans are grown in the United States than anywhere else in the world (2.4 billion bushels in 1996, 50% of world production). A bushel of soybean (60 pounds) is comprised of 48 pounds of protein meal and 11 pounds of oil. While protein meal is the major component in soybean, oil, lecithin, tocopherols, isoflavones, etc. are all co-products and add value to the bean. Soybean oil is the major edible oil used in the world (40% of the 59.4 million metric tons in 1993). It also accounts for 70% of the 14 billion pounds of edible vegetable oil in the United States. The primary food applications where the oil is used extensively are for baking and frying (40-45%), salad and cooking oil (40-45%), margarine and shortening (15-20%) and a wide spectrum of processed foods. Development of other vegetable oils for specialty uses has recently affected the acreage and production of soybean. The low cost and ready availability of soybean oil provide an excellent opportunity to upgrade this commodity item for specialty uses.
Food fats and oils are chemically composed of triesters of glycerol containing straight chain, normal aliphatic fatty acids, also referred to herein as triacylglycerols or triglycerides (TAG). The properties of food fats and oils are a reflection of the fatty acids contained in the TAG and their distribution on the glycerol backbone. When the melting point of the TAG is below room temperature, the TAG is referred to as an “oil”. Triglycerides that melt above room temperature are referred to as “fat”. Gradients between fluidity and solidity exist. Partially solidified, non-pourable triglycerides are often referred to as “plastic fats”.
Fatty acids are organic acids having a hydrocarbon chain ranging in length from about 4 to 24 carbons. Fatty acids differ from each other in chain length, and in the presence, number and position of double bonds. In cells, fatty acids typically exist in covalently bound forms, the carboxyl portion being referred to as a fatty acyl group. The chain length and degree of saturation of these molecules is often depicted by the formula CX:Y, where “X” indicates number of carbons and “Y” indicates number of double bonds.
Typically, oil derived from commercial soybean varieties is composed of approximately 11% palmitic (C16:0), 4% stearic acid (C18:0), 21% oleic acid (C18:1), 56% linoleic acid (C18:2), and 10% linolenic acid (C18:3). The fatty acid composition of soybean oil, as well as all oils, largely determines its physical and chemical properties, and thus its uses.
Fatty acid biosynthesis has been the subject of research efforts in a number of organisms. For reviews of fatty acid biosynthesis in plants, see Ohlrogge et al., (1995)
Plant Cell,
7:957-970, Ohlrogge et al., (1997)
Annu Rev Plant Physiol Plant Mol Biol,
48:109-136 and Sommerville et al. (1991)
Science,
252:80-87.
As mentioned previously, the fatty acid composition of an oil determines its physical and chemical properties, and thus its uses. Plants, especially plant species which synthesize large amounts of oils in plant seeds, for example soybean, are an important source of oils both for edible and industrial uses. Various combinations of fatty acids in the different positions in the triglyceride will alter the properties of the triglyceride. For example, if the fatty acyl groups are mostly saturated fatty acids, then the triglyceride will be solid at room temperature. In general, however, vegetable oils tend to be mixtures of different triglycerides. The triglyceride oil properties are therefore a result of the combination of triglycerides which make up the oil, which are in turn influenced by their respective fatty acid compositions.
Plant breeders have successfully modified the yield and fatty acid composition of various plant seed oils by introducing desired traits through plant crosses and selection of progeny carrying the desired trait forward. Application of this technique thus is limited to traits which are found within the same plant species. Alternatively, exposure to mutagenic agents can also introduce traits which may produce changes in the composition of a plant seed oil. However, it is important to note that Fatty Acid Synthesis (FAS) occurs in most tissues of the plant including leaf (chloroplasts) and seed tissue (proplastids). Thus, although a mutagenesis approach can sometimes result in a desired modification of the composition of a plant seed oil, it is difficult to effect a change which will not alter FAS in other tissues of the plant.
A wide range of novel vegetable oils compositions and/or improved means to obtain or manipulate fatty acid compositions, from biosynthetic or natural plant sources, are needed. Plant breeding, even with mutagenesis, cannot sufficiently meet this need and provide for the introduction of novel oil.
For example, cocoa-butter has certain desirable qualities (mouthfeel, sharp melting point, etc.) which are a function of its triglyceride composition. Cocoa-butter contains approximately 24.4% palmitate (16:0), 34.5% stearate (18:0), 39.1% oleate (18:1) and 2% linoleate (18:2). Thus, in cocoa butter, palmitate-oleate-stearate (POS) comprises almost 46% of triglyceride composition, with stearate-oleate-stearate (SOS) and palmitate-oleate-palmitate (POP) comprising the major portion of the balance at 33% and 16%, respectively, of the triglyceride composition. Other novel oils compositions of interest might include trierucin (three erucic) or a triglyceride with medium chain fatty acids in each position of the triglyceride molecule.
Plant seed oils contain fatty acids acylated at the sn-1, sn-2, and sn-3 positions of a glycerol backbone, referred to as a triacylglycerol (TAG). The structure of the TAG, as far as positional specificity of fatty acids, is determined by the specificity of enzymes involved in acylating the fatty acyl CoA substrates to the glycerol backbone. For example, there is a tendency for such enzymes from many temperate and tropical crop species to allow either a saturated or an unsaturated fatty acid at the sn-1 or the sn-3 position, but only an unsaturated fatty acid at the sn-2 in the seed TAGs. In some species such as cocoa, TAG compositions suggest that this tendency is carried further in that there is an apparent preference for acylation of the sn-3 position with a saturated fatty acid, if the sn-1 position is esterified to a saturated fatty acid. Thus, there is a higher percentage of structured TAG of the form Sat-Un-Sat (where Sat=saturated fatty acid and Un=unsaturated fatty acid).
Of particular interest are triglyceride molecules in which stearate is esterified at the sn-1 and sn-3 positions of a triglyceride molecule with unsaturates in the sn-2 position particularly oleate. Vegetable oils rich in such SOS (Stearate-Oleate-Stearate) molecules share certain desirable qualities with cocoa butter yet have a degree of additional hardness when blended with other structured lipids. SOS-containing vegetable oils are currently extracted from relatively expensive oilseeds from certain trees grown in tropical areas such as Sal, Shea, and Illipe trees from India, Africa, and Indonesia respectively. Cheaper and more conveniently grown sources for SOS-type vegetable oils are desirable.
In addition, vegetable oils rich in stearate fatty acid content tend to be solid at room temperature. Such vegetable fats can be used directly in shortenings, margarine and other food “
Arnold & Porter
Calgene LLC
Nelson Amy J.
Stierwalt Brian K.
LandOfFree
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