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
1997-09-09
2002-07-30
McElwain, Elizabeth F. (Department: 1638)
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...
C800S298000, C435S419000, C435S468000
Reexamination Certificate
active
06426447
ABSTRACT:
FIELD OF TECHNOLOGY
This invention relates to the application of genetic engineering techniques to plants. More specifically, the invention relates to a strategy for effecting changes in the composition of plant seed oils through the use of foreign DNA sequences which are derived from sources outside of the target plant gene pool.
BACKGROUND
Fatty acids are organic acids having a hydrocarbon chain of from about 4 to 24 carbons. Many different kinds of fatty acids are known which 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. As the carbon chain of fatty acyl molecules always contains an even number of carbons, the formula “C
2X
” may also be used to represent carbon chain length.
Fatty acyl groups are major components of many lipids, and their long, non-polar hydrocarbon chain is responsible for the water-insoluble nature of these lipid molecules. The type of covalent linkage of the fatty acyl group to other factors can vary. For example, in biosynthetic reactions they may be covalently bound via a thioester linkage to an acyl carrier protein (ACP) or to CoenzymeA (CoA), depending on the particular enzymatic reaction. In waxes, fatty acyl groups are linked to fatty alcohols via an ester linkage, and triacylglycerols have three fatty acyl groups linked to a glycerol molecule via an ester linkage.
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, are an important source of oils both for edible and industrial uses.
The fatty acid composition of major oilseeds, ordered here by palmitate content, is shown in Table I. With the exception of laurate (C12:0) sources of coconut endosperm and palm kernel, the common edible oils all basically consist of 16:0, 18:0, 18:1 (oleate), 18:2 (linoleate), and 18:3 (linolenate).
TABLE I
12:0
14:0
16:0
18:0
18:1
18:2
18:3
20:1
22:1
rape
3
0.8
9.9
13.5
9.8
6.8
53.6
(HEAR)
rape
4.9
1.4
56.4
24.2
10.5
(LEAR)
sun-
0.1
5.8
5.2
16
71.5
0.2
flower
peanut
6.7
4.3
71.4
11.1
6.5
saf-
7.6
2
10.8
79.6
flower
coconut
40.2
15.5
7.6
2.4
5.2
1.2
oil palm
50.9
18.4
8.7
1.9
14.6
1.2
kernel
15.3
3.8
20.7
55.8
9.4
soybean
cotton
1
23.4
2.5
17.9
54.2
oil palm
0.1
1.2
46.8
3.8
37.6
meso-
carp
Plant breeders have successfully modified the yield and fatty acid composition of various plant seed oils through programs of introducing desired traits by 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 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 for a variety of intended uses. Plant breeding, even with mutagenesis, cannot meet this need and provide for the introduction of any oil traits which are outside of the target plant's gene pool.
Various oils compositions are now in demand. For example, edible oil sources containing the minimum possible amounts of saturates, palmitate (C16:0) and stearate (C18:0) saturated fatty acids, are desired for dietary reasons and alternatives to current sources of highly saturated oil products, such as tropical oils, are also needed. Generating a spread of C4, C6 and C8 short chain 3-keto fatty acids could become a key improvement in polyhydroxybutyrate (PHB)-based biodegradable plastics made in bacteria and plants. Medium-chain fatty acids have special importance in the detergent and lubricant industries or in the formulation of edible oils with reduced caloric value or other health benefits. See for example, U.S. Pat. No. 4,863,753 and Barch, A. C. & Babayan, V. K.,
Am. J. Clin. Nat
. (1982) 36:950-962. Longer chain fatty acids may have certain other utilities, i.e., C16 and C18 have particular uses in margarine and other solid oil-based products and very long chain fatty acids also have specialized uses, i.e., C22 is used to make peanut butter smoother. As such, a ready source of a variety of fatty acid lengths, including storage lipids which have incorporated differing chain length fatty acids in desired ratios, are desired for a variety of industrial and food use fields. Improved yield of current oilseed crops and the development of novel plant fatty acid compositions and oils products are also needed. Examples of novel plant fatty acid and oils products include fatty alcohols, epoxy fatty acids (e.g., biodegradable paint thinner), long chain liquid wax (e.g., jojoba oil substitute), hydroxylated fatty acids (motor lubricants) or cyclopropanated fatty acids (motor lubricants).
With the advent of genetic engineering, the ability to produce a transgenic plant containing any desired DNA sequence of interest is a reality. And with the development of basic plant biotechnology methodologies, many suggestions have been proposed for fatty acid modification. A good number of these strategies however, rely upon the insertion of genes isolated from organisms outside of the target plant species oftentimes traits from very divergent type plants to alter plant oils. It was not known whether such traits were limited to certain plant types. As one example, certain oil compositions appear to be limited to certain climates. Highly saturated oils, especially those high stearate (C18:0), are strongly correlated with tropical plant sources, e.g., oil palm, coconut. Temperate zone oilseeds are very typically highly unsaturated, e.g., corn, soybean, canola. Thus, the insertion of genes to achieve high stearate in a temperate crop would not meet the usual climatic condition for such trait.
Additionally, it was not known whether the introduced enzymes could effectively compete with the natural enzymes for substrate or whether it would be necessary to reduce the level of the endogenous enzymes to observe a modified fatty acid oil phenotype. Also, it was not known whether antisense technology could be used to influence the fatty acid pathway. In addition, it was not known, in the event that the composition of fatty acids were modified, whether the incorporation of such fatty acids into triglycerides would occur, whether transgenic seed with an altered oils composition would germinate, and to what extent if any, whether seed yield and/or oil yield from such seeds would be affected.
Moreover, in order to genetically engineer plants one must have in place the means to transfer genetic material to the plant in a stable and heritable manner. Additionally, one must have nucleic acid sequences capable of producing the desired phenotypic result, regulatory regions capable of directing the correct application of such sequences, and the like. Moreover, it should be appreciated that to produce a desired modified oils phenotype requires that the FAS pathway of the plant is modified to the extent that the ratios of reactants are modulated or changed.
Higher plants appear to synthesize fatty acids via a common metabolic pathway in plant plastid organelles (i.e., chloroplasts, proplastids, or other related organelles) as part of the FAS complex. (By fatty acid is meant free fatty acids and acyl-fatty acid groups.) Outside of plastid organelles, fatty acids are
Knauf Vic C.
Thompson Gregory A.
McElwain Elizabeth F.
Monsanto Technology LLC
Senniger Powers Leavitt & Roedel
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