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
Reexamination Certificate
1998-04-22
2001-12-18
Nelson, Amy J. (Department: 1638)
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
C435S196000, C435S320100, C435S419000, C435S468000, C536S023200, C536S023600, C800S281000
Reexamination Certificate
active
06331664
ABSTRACT:
FIELD OF INVENTION
This invention relates to the preparation and use of nucleic acid fragments or genes encoding acyl-acyl carrier protein thioesterase enzymes to create transgenic plants having altered oil profiles.
BACKGROUND OF THE INVENTION
Oils produced by plants can be found in a wide variety of products including soaps, 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 the manufacture of soaps, detergents and surfactants and represent a US 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.
The characteristics of oils are determined predominately by the number of carbon atoms 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. Palmitic and stearic acids are 16- and 18-carbon long saturated fatty acids, respectively. Conventionally, they are designated as “saturated” since the fatty acid chains have no double bonds and therefore contain the maximal number of hydrogen atoms possible. Saturated fatty acids are linear molecules and tend to form self-stacked structures thereby resulting in high melting temperatures. For example, animal fats, which are solid at room temperature, are typically high in saturated fatty acids. The other predominant fatty acids found in plant oils, oleic, linoleic, and linolenic, are 18-carbon long fatty acid chains having one, two, and three double bonds therein, respectively. Oleic acid is typically considered a mono-unsaturated fatty acid, whereas linoleic and linolenic are considered to be poly-unsaturated fatty acids. These fatty acid chains are nonlinear due to bending induced by the insertion of the double bond in the cis conformation. Double bond insertion decreases melting point due to the inability of the fatty acid molecules to self-stack. For example, vegetable oils, which are typically liquid at room temperature, are high in unsaturated fatty acids.
Over the years, vegetable oils have gradually replaced animal-derived oils and fats as the major source of dietary fat intake. However, saturated fat in most industrialized nations has remained at 15 to 20% of total caloric intake. The United States Department of Agriculture 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 United States Food and Drug Administration 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 would need be less than 7% and 1.75% to receive the “low sat” and “no sat” label, respectively. Therefore, there has been a surge in increased 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 total saturated fatty acid level of corn oil, approximately 13.9%, does not meet the 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 fatty acid 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 market size. Therefore, a corn oil with low levels of saturated fatty acids is highly desirable, in that it would meet the consumer demand for healthier oils while having most or all of the properties that made corn oil popular in the past and a preferred oil for many uses.
Although corn oil with low levels of saturated fatty acids is desirable, there is also a demand for corn oil having high levels of saturated fatty acids. For example, about half of all consumption of vegetable oils is in the form of margarine and shortening. However, the use of corn oil for these products requires chemical modification of the oil due to its low melting point. Typically, an increased melting point is achieved through catalytic hydrogenation which increases the level of saturated fatty acids. In this process, hydrogen atoms are added at double bonds found in the fatty acid through the use of a catalyst. An additional side reaction that occurs during hydrogenation is the substantial conversion of the naturally occurring cis double bonds to the trans isomer, which are more stable. There have been some controversies regarding health risks associated with intake of oils containing trans double bonds. In a recent study, it was shown that a diet high in trans isomer consumption actually raised serum lipoprotein profiles and cholesterol levels (Mensink and Katan (1990) N. Eng. J. Med. 323:439-445). Therefore, production of oil containing a higher content of saturated fatty acids would reduce the need for hydrogenation in margarine and shortening production thereby reducing the content of trans isomers in the diet. In addition, partial hydrogenation typically increases cost an additional 2 to 3 cents per pound of oil. Therefore, a corn oil with naturally high saturates levels is also highly desirable for production of margarine and shortening since this would fulfill a market need while reducing manufacture cost.
Corn is typically not considered to be an oil crop as compared to soybean, canola, sunflower and the like. In fact, the oil produced by corn is considered to be a byproduct of the wet milling process used to extract starch. Because of this, there has been little interest in modification of saturate levels of corn oil until that disclosed herein.
SUMMARY OF THE INVENTION
In the present invention, acyl-acyl carrier protein thioesterases (acyl-ACP thioesterases) from maize have been isolated and cloned. The saturate level of oils found in plant cells can be altered by modifying the expression and activity levels of acyl-ACP thioesterases within the cell.
One aspect of the disclosed invention is genes and nucleic acid fragments encoding maize acyl-ACP thioesterases. More specifically, the isolated genes and nucleic acid fragments herein encode maize palmitoyl-ACP thioesterase, hereinafter PTE, and maize oleoyl-ACP thioesterase, hereinafter OTE. Maize PTE and OTE hydrolyze acyl-ACP units into free fatty acids and ACP in somewhat a selective although not specific manner.
Another aspect of the invention relates to altering saturate levels within a cell by modifying expression levels of PTE and OTE. The genes and nucleic acid fragments disclosed herein can be used to alter saturate levels by placing said genes and fragments in the antisense orientation. Plants being transformed with PTE in the antisense orientation results in the oils of said plants having lowered 16:0 and lowered total saturate levels. Plants being transformed with OTE in the antisense orientation results in the oils of said plant having increased 16:0 and increased total saturate levels. Results similar to those disclosed herein with antisense can also be produced through the use of ribozymes designed specifically for PTE and OTE.
Another aspect of the invention relates to alte
Armstrong Katherine
Cowen Neil M.
Folkerts Otto
Guo Lining
Rubin-Wilson Beth
Boruki Andrea T.
Dow AgroSciences LLC
Nelson Amy J.
Stuart Donald R.
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