Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-05-30
2004-11-30
Fox, David T. (Department: 1638)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S023200, C435S183000, C435S189000, C800S281000
Reexamination Certificate
active
06825335
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of genetic engineering, and more particularly to transformation of plants with heterologous fatty acid desaturase genes modified for optimum expression in plants.
BACKGROUND OF THE INVENTION
Several publications are referenced in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
Alteration of fatty acid desaturation in plants is of interest to plant biologists and food scientists alike, due to the influence of unsaturated fatty acids on the health benefits and flavors of foods, as well as the role of these molecules in plant biological processes. For a nation interested in healthy diet, the quality of fats and oils depends on their fatty acid composition, with oils high in monounsaturated fatty acids (e.g., canola, olive) gaining popularity as new health benefits are discovered. Considering the flavors of plant foods, many flavor-producing compounds are derived from peroxidation of unsaturated fatty acids. Thus, efforts are being made to produce plants with increased amounts of unsaturated fatty acids, preferably monounsaturated fatty acids.
In animal and fungal cells, monounsaturated fatty acids are aerobically synthesized from saturated fatty acids by a microsomal &Dgr;-9 fatty acid desaturase that is membrane bound and cytochrome b
5
-dependent. A double bond is inserted between the 9- and 10-carbons of palmitoyl (16:0) and stearoyl (18:0) CoA to form palmitoleic (16:1) and oleic (18:1) acids. In the reaction mechanism, electrons are transferred from NADH-dependent cytochrome b
5
reductase, via the heme-containing cytochrome b
5
(Cyt b
5
) molecule, to the &Dgr;-9 fatty acid desaturase. The major form of cytochrome b
5
in animal, fungal and plant cells exists as an independent protein molecule that is anchored to the membrane by a short, carboxyl terminal, hydrophobic stretch of amino acids. The carboxyl terminal anchor orients the heme group of the Cyt b
5
on the membrane surface and allows it to translationally diffuse across the surface of the membrane. This property of lateral mobility allows this form of cytochrome b
5
to participate as an electron donor to a number of different proteins that catalyze a variety metabolic reactions on the membrane surface, including fatty acid desaturases, various sterol biosynthetic enzymes and a variety of cytochrome P450 mediated reactions. While this contributes to the versatility of Cyt b
5
as an electron donor, it also implies that the major form of cytochrome b
5
shuttles between its redox partners by translational diffusion across the surface of the membrane (Strittmatter and Rogers, Proc. Natl. Acad. Sci. USA, 72: 2658-2661, (1975; Lederer, Biochimie 76: 674-692, 1994). Furthermore, this mechanism suggests that an independent, membrane bound cytochrome b
5
molecule can potentially limit the rate of the metabolic reaction, depending on its abundance, its location on the membrane surface, its proximity to the electron acceptor, and the rate at which it can move and orient itself to the acceptor on the membrane surface.
In plants, unsaturated fatty acids are formed and incorporated into complex lipids in two distinct cellular compartments. De novo fatty acid synthesis occurs almost exclusively in the plastids, producing the saturated species 16:0-ACP (acyl carrier protein) and 18:0-ACP. 18:1-ACP is formed from 18:0-ACP in the plastid by a soluble, ferredoxin-dependent &Dgr;-9 desaturase. These fatty acids are then shunted into one of two routes—a plastid-localized “procaryotic” pathway or a cytosolic/ER (endoplasmic reticulum) “eucaryotic” pathway—for further modification and acylation into glycerolipids (Somerville and Browse, Science 252: 80-87, 1991). The acyl ACPs that are shunted into the prokaryotic pathway remain within the plastid and are used for the synthesis of phosphatidic acid and further conversion to chloroplast glycerolipids. The fatty acyl groups of those lipids may be further desaturated by plastid desaturases that also use ferrodoxin as the electron donor.
Acyl-ACPs that are shunted into the eukaryotic pathway are converted to free fatty acids, transported across the chloroplast membrane into the cytoplasm where they are converted to acyl CoA thioesters by acyl CoA synthetase. Those fatty acids are then converted to cytoplasmic/ER phosphatidic acid which can then be converted to membrane glycerophospholipids, or storage lipids, in the form of triacylglycerols and sterol esters that are the major components of plant oils.
Most polyunsaturated 18-carbon plant fatty acids appear to be formed in the cytosol by the ER-bound desaturases (Table 1). Once the 18:1 fatty acid is incorporated into phospholipid, an ER-bound desaturase can catalyze the formation of a &Dgr;-12 double bond in the fatty acyl chain to form &Dgr;-9,12 18:2. Other ER bound desaturase enzymes can act on 18:2 to introduce a &Dgr;-15 double bond to form &Dgr;9,12,15 18:3. These desaturase are thought to be similar to animal and fungal desaturases because they are membrane bound and appear to require a cytochrome b
5
-mediated electron transport chain.
TABLE 1
Desaturase
Primary
b5
Plant
Gene
Type
Activity
chimera
Reference
Arabidopsis
FAD2
&Dgr;12,
18:1->18:2
no
Okuley J. et al.
microsomal
Plant Cell 6: 147-158,
1994
Arabidopsis
FAD3
&Dgr;15,
18:2->18:3
no
Shah S. & Z. Xin,
microsomal
Plant Physiol. 114:
1533-1539, 1997
Nicotiana
NtFA
&Dgr;15,
18:2->18:3
no
Hamada T. et al.
tabacum
D3
microsomal
Plant & Cell.
Physiol. 37: 606-611,
1996, Hamada T. et al.
Transgenic Res. 5:
115-121, 1996
Soybean
FAD
&Dgr;12,
18:1->
no
Heppard E. P. et al.
2-1
microsomal,
18:2
Plant Physiol. 110:
developing
311-319, 1996
seeds
Soybean
FAD
&Dgr;12,
18:1->18:2
no
Heppard, E. P. et al.
2-2
microsomal
1996, supra
developing
seeds and
vegetative
tissues
Borage
&Dgr;-6
18:2
yes, N-
Sayanova et al.
(9, 12)-18:3
terminal
Proc. Natl. Acad.
(6, 9, 12)
Sci. USA 94: 4211-4216,
1997
The conversion of saturated fatty acyl chains to monounsaturated species in plants appears to be confined to the chloroplasts. No &Dgr;-9 desaturase activity has been identified in the cytoplasm or endoplasmic reticulum of plants. The soluble plant chloroplast &Dgr;-9 desaturase is highly specific for 18:0-ACP as a substrate and does not desaturate 16:0-ACP (Somerville and Browse, supra). As a result, only a small amount of 16:1 is present in most higher plants, while the pool of 16:0 is concomitantly larger due to its disfavor as a substrate for the plant desaturase. By comparison, a larger amount of 18:1 is found in higher plant cells, with a correspondingly lesser amount of 18:0. Thus, for the purpose of increasing the concentration of mono-unsaturated lipids in a plant, the 16:0 fatty acid constitutes a significant pool of available substrate that is under-utilized by the endogenous plant desaturase.
In contrast to the plant &Dgr;-9 desaturase, fungal and animal &Dgr;-9 desaturases efficiently convert a wide range of saturated fatty acids with differing hydrocarbon chain lengths to monounsaturated fatty acids. The
Saccharomyces cerevisiae
enyzme, for example, efficiently desaturates even and odd chain fatty acyl CoA substrates from 13 carbons to 19 carbons in length. A broad functional homology exists among various Cyt b
5
-dependent desaturases, as evidenced, for example, by the successful expression of the rat &Dgr;-9 desaturase in yeast (Stukey et al., J. Biol. Chem. 2: 20144-20149, 1990).
The rat and yeast &Dgr;-9 desaturase genes have been expressed in plants: both the rat and the yeast genes have been expressed in tobacco (Grayburn et al., BioTechnology 10: 675-678, 1992 (rat); Polashock et al., Plant Physiol. 100: 894-901, 1992 (yeast), and the yeast gene has also been expressed in tomato (Wang et al., J. Agric. Food Chem. 44: 3399-3402, 1996). The yeast &Dgr;-9 desaturase has been shown to function in tobacco and tomato, leading to increases in the level of monounsaturated fa
Martin Charles E.
Mitchell Andrew
Fox David T.
Kallis Russell
Rutgers The State University of New Jersey
Woodcock & Washburn LLP
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