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
1999-03-29
2002-12-17
McElwain, Elizabeth (Department: 1649)
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, C536S023200, C435S069100, C435S468000, C435S419000
Reexamination Certificate
active
06495738
ABSTRACT:
FIELD OF INVENTION
This invention relates to the preparation and use of nucleic acid fragments or genes which encode fungal palmitoyl-CoA &Dgr;-9 desaturase enzymes to create transgenic plants having altered oil profiles.
BACKGROUND OF THE INVENTION
Plant-produced oils can be found in a wide variety of products including lubricants and foods. Interestingly, different plant species synthesize various oil types. For example, coconut and palm plants produce oils that are abundant in fatty acids having medium chain lengths (10-12 carbon atoms). These oils are used in manufacturing soaps, detergents and surfactants, and represent a U.S. market size greater than $350 million per year. Other plants, such as rape, produce oils abundant in long chain fatty acids (22 carbon atoms) and are used as lubricants and anti-slip agents. Additional applications of plant oils include their use in plasticizers, coatings, paints, varnishes and cosmetics (Volker et al., (1992) Science 257:72-74; Ohlrogge, (1994) Plant Physiol. 104:821-826). However, the predominant use of plant oils is in the production of food and food products.
Over the years, vegetable-derived oils have gradually replaced animal-derived oils and fats as the major source of dietary fat intake. However, saturated fat intake in most industrialized nations has remained at about 15% to 20% of total caloric consumption. In efforts to promote healthier lifestyles, the United States Department of Agriculture(USDA) has recently recommended that saturated fats make up less than 10% of daily caloric intake. To facilitate consumer awareness, current labeling guidelines issued by the USDA now require total saturated fatty acid levels be less than 1.0 g per 14 g serving to receive the “low-sat” label and less than 0.5 g per 14 g serving to receive the “no-sat” label. This means that the saturated fatty acid content of plant oils needs to be less than 7% and 1.75% to receive the “low sat” and “no sat” label, respectively. Since issuance of these guidelines, there has been a surge in consumer demand for “low-sat” oils. To date, this has been met principally with canola oil, and to a much lesser degree with sunflower and safflower oils.
The characteristics of oils, whether of plant or animal origin, are determined predominately by the number of carbon and hydrogen atoms, as well as the number and position of double bonds comprising the fatty acid chain. Most oils derived from plants are composed of varying amounts of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic (18:3) fatty acids. Conventionally, palmitic and stearic acids are designated as “saturated” since the fatty acid chains have 16 and 18 carbon atoms, respectively, and no double bonds. They therefore contain the maximal number of hydrogen atoms possible. However, oleic, linoleic, and linolenic are 18-carbon fatty acid chains having one, two, and three double bonds, respectively, therein. Oleic acid is typically considered a mono-unsaturated fatty acid, whereas linoleic and linolenic are considered to be poly-unsaturated fatty acids.
Saturated fatty acids are linear molecules and tend to form self-stacked structures thereby resulting in high melting temperatures; a characteristic that is quite desirable when producing foods like chocolate. Animal fats, which are also solid at room temperature, are another readily available source of saturated fatty acids. However, use of said oil is often discouraged due to the high levels of cholesterol associated therewith. In comparison, unsaturated fatty acid chains are nonlinear due to bending induced by double bond insertion. The bending of the molecule impedes the ability of the fatty acid chains to stack thus causing them to remain fluid at lower temperatures. Vegetable oils, for example, are high in unsaturated fatty acids, and therefore are typically liquid at room temperature. Furthermore, saturated fatty acid can be modified to become unsaturated fatty acids by removal of hydrogen atoms and insertion of double bonds between two carbon atoms on the fatty acid chain. Desaturation can be achieved either enzymatically or chemically and decreases melting points due to the inability of the fatty acid molecules to self-stack.
The total saturated fatty acid level of corn oil, averaging about 13.9%, does not meet the current labeling guidelines discussed above. On average, corn oil is comprised of 11.5% palmitic acid, 2.2% stearic acid, 26.6% oleic acid, 58.7% linoleic acid, and 0.8% linolenic acid. Corn oil also contains 0.2% arachidic acid, a twenty-carbon saturated fatty acid (Dunlap et. al., (1995) J. Amer. Oil Chem. Soc. 72:981-987). The composition of corn oil instills it with properties that are most desirable in edible oils. These include properties such as heat stability, flavor, and long shelf life. However, consumer demand for “low sat” oils has resulted in a significant decrease in corn oil utilization and thus decreased market share. Therefore, a corn oil with modified levels of saturated fatty acids is highly desirable and would have practical use in that it would meet the consumer demand for healthier oils while having most or all of the properties that made corn oil a popular and preferred oil in the past.
Corn is typically not considered to be an oil crop as compared to soybean, canola, sunflower and the like. In fact, the oil produced and extracted from corn is considered to be a byproduct of the wet milling process used in starch extraction. Because of this, there has been little interest in modifying the saturate levels of corn oil until those efforts disclosed herein.
As disclosed herein, saturate levels of fatty acids comprising plant oils can be altered by expressing a fungal palmitate-CoA &Dgr;-9 desaturase within a plant cell. These proteins most likely enzymatically desaturate palmitate-CoA molecules by removing two hydrogen atoms and adding a double bond between the 9
th
and 10
th
carbon atoms from the CoA portion of the molecule, thus producing palmitoleic-CoA (16:1
&Dgr;9
) . The palmitoleic-CoA is ultimately incorporated into seed oil thus lowering the total saturate levels of said oil.
SUMMARY OF THE INVENTION
In the present invention, a gene encoding a fungal palmitate-CoA &Dgr;-9 desaturase has been isolated and cloned from
Aspergillus nidulans.
The saturate level of oils found in plant cells can be altered by expressing said palmitate-CoA &Dgr;-9 desaturase from
Aspergillus nidulans.
One aspect of the disclosed invention is a gene encoding said palmitate-CoA &Dgr;-9 desaturase, said gene being isolated and purified from
Aspergillus nidulans.
An additional aspect of the present invention relates to producing a gene wherein the codon bias of a gene from a non-plant source has been modified to look similar to genes from a plant source.
Another aspect of the invention relates to altering oil saturate levels within a plant cell by expressing said genes encoding palmitate-CoA &Dgr;-9 desaturase from
Aspergillus nidulans.
Genes disclosed herein can be used to alter saturate levels by placing said genes in the sense orientation. Plants cells being transformed with genes encoding palmitate-CoA &Dgr;-9 desaturase from
Aspergillus nidulans
in the sense orientation results in the oils of said plants having increased 16:1 levels and decreased total saturate levels over non-transformed plants.
An additional aspect of the present invention is the production of chimeric genes using the genes disclosed herein encoding for palmitoyl CoA-&Dgr;-9 desaturase in combination with promoter regulatory elements and the use of said chimeric genes within a plant cell.
Yet an additional aspect of the present invention is the transformation of plant species disclosed herein with said chimeric genes.
Other aspects, embodiments, advantages, and features of the present invention will become apparent from the following specification.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods and compositions for obtaining transgenic plants wherein plant oils produced thereby have altered sa
Folkerts Otto
Merlo Donald J.
Dow AgroSciences LLC
Ludwig Kenneth B.
McElwain Elizabeth
Stuart Donald R.
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