Desaturases and methods of using them for synthesis of...

Details Desaturases and methods of using them for synthesis of... Desaturases and methods of using them for synthesis of...

2001-08-17

2004-11-30

Rao, Manjunath N. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S004000, C435S006120, C435S069100, C435S183000, C435S252340, C435S320100, C435S325000, C536S023200, C536S023700

Reexamination Certificate

active

06825017

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to desaturase enzymes that can be used to produce polyunsaturated fatty acids with important dietary applications.
BACKGROUND
Fatty acids are fundamental components of living systems. They make up the major component of cytoplasmic membranes, common to plants, animals and protists alike.
Fatty acids of 20 carbons, with more than one unsaturated carbon—carbon bond along the hydrocarbon chain, are known to be of particular importance. Arachidonate (20:4) (Heinz,
Lipid Metabolism in Plants
, pp. 33-89, 1993; Yamazaki et al.
Biochim. Biophys. Acta
1123:126, 1992; Ulsamer et al.,
J. Cell Biol.
43:105-114, 1969; and Albert et al. Lipids 14:498-500, 1979) and eicosapentaenoate (20:5) (Heinz,
Lipid Metabolism in Plants
, pp. 33-89, 1993; Yamazaki et al.,
Biochim. Biophys
. Acta 1123:18-26, 1992; Ulsamer et al.,
J. Cell Biol.
43:105-114, 1969; Albert et al.
Lipids
14:498-500, 1979; and Cook et al.,
J. Lipid Res.
32:1265-1273, 1991), commonly referred to as EPA, are significant components of mammalian cell membranes and are also precursors of signal molecules including prostaglandins. Certain specialized mammalian tissues such as brain (Naughton,
J. Biochem.
13:21-32, 1981), testes (Wilder and Coniglio,
Proc. Soc. Exp. Biol. Med.
177:399-405, 1984), and retina (Aveldano de Caldironi et al.,
Prog. Lipid Res.
20:49-57, 1981) are especially rich in unsaturated fatty acids.
Arachidonate and eicosapentaenoate serve both as precursors for synthesis of 22-carbon polyunsaturated fatty acids and, with dihomoy-&ggr;-linoleate (20:3) (Yamazaki et al.,
Biochim. Biophys. Acta
1123:18-26, 1992; Ulsamer et al.,
J. Cell Biol.
43:105-114, 1969; and Albert et al., Lipids 14:498-500, 1979), as precursors to the synthesis of eicosanoid metabolic regulators (Hwang,
Fatty Acids in Foods and Their Heath Implications,
545-557, 1992). Key enzymes in the synthesis of 20 carbon fatty acids are desaturases, which introduce cis double bonds by removing two hydrogen atoms at specific locations along the aliphatic hydrocarbon chains. Desaturase enzymes are specific to the position, number, and stereochemistry of the double bonds already present in the target fatty acid (Heinz,
Lipid Metabolism in Plants,
33-89, 1993).
To synthesize 20 carbon polyunsaturated fatty acids, mammals must acquire the essential fatty acids 18:2 (Brenner,
The Role of Fats in Human Nutrition
, pp. 45-79, 1989) and 18:3 (Nelson,
Fatty Acids in Foods and Their Health Implications
, pp. 437-471, 1992; Brenner.
The Role of Fats in Human Nutrition
, pp. 45-79,1989; and Hulanicka et al.
J. Biol Chem.
239:2778-2787,1964) from their diet (Nelson,
Fatty Acids in Foods and Their Health Implications,
437-471,1992). These dietary polyunsaturated fatty acids are metabolized in the endoplasmic reticulum by an alternating series of position-specific desaturases and malonyl-CoA-dependent chain-elongation steps (FIG.
1
A), which results in the characteristic methylene-interrupted double bond pattern. In the liver, which is the primary organ of human lipid metabolism, the first step in biosynthesis of 20-carbon fatty acids is desaturation of the essential fatty acids at the &Dgr;
5
position. The desaturation products are elongated to 20:3 and 20:4 (Cook et al.,
J. Lipid Res.
32:1265-1273, 1991). In turn, these 20-carbon products are desaturated by a &Dgr;
8
-desaturase to produce arachidonate and eicosapentaenoate. The &Dgr;
6
-desaturation step is rate-limiting in this metabolic pathway (Bernet and Sprecher,
Biochim. Biophys. Acta
398:354-363, 1975; and Yamazaki et al.,
Biochim. Biophys. Acta
1123:18-26, 1992) and, not surprisingly, is subject to regulation by dietary and hormonal changes (Brenner,
The Role of Fats in Human Nutrition
, pp. 45-79, 1989).
In contrast to the liver, an alternate pathway for biosynthesis of 20-carbon polyunsaturated fatty acids has been demonstrated in a few organisms and tissues (FIG.
1
B). Instead of desaturation, the first step in the alternate pathway is elongation of the essential 18-carbon fatty acids to 20 carbon chain lengths, producing 20:2 (Ulsamer et al.,
J. Cell Bio.
43:105-114, 1969; and Albert et al.
Lipids
14:498-500, 1979) and 20:3., Subsequent denaturation occurs via a &Dgr;
8
-desaturase (FIG.
1
). The products of this elongation-desaturation, 20:3 and 20:4, are the same as the more usual desaturation-elongation pathway. The &Dgr;
8
pathway is present in the soil amoebae Acanthamoeba sp. (Ulsamer et al,
J. Cell Biol.
43:105-114, 1969), and in euglenoid species, where it is the dominant pathway for formation of 20 carbon polyunsaturated fatty acids (Hulanicka et al.,
Journal of Biological Chemistry
239:2778-2787, 1964).
This &Dgr;
8
-desaturation pathway occurs in mammals, both in rat testis (Albert and Coniglio,
Biochim. Biophys. Acta
489:390-396, 1977) and in human testis (Albert et al.,
Lipids
14:498-500, 1979). While &Dgr;
8
activity has been observed in breast cancer cell lines (Grammatikos et al., Br.
J. Cancer
70:219-227, 1994; and Bardon et al.,
Cancer Lett.
99:51-58, 1996) and in glioma (Cook et al.,
J. Lipid Res.
32:1265-1273, 1991), no &Dgr;
8
activity is detectable in a corresponding non-cancerous breast cell line (Grammatikos et al.,
Br. J. Cancer
70:219-227, 1994) or in the brain (Dhopeshwarkar and Subramanian,
J. Neurochem.
36:1175-1179, 1976). The significance of &Dgr;
8
-desaturation to normal or cancer cell metabolism is unclear, since analysis of desaturase activities in mammalian systems is frequently complicated by the presence of competing &Dgr;
6
reactions and chain-shortening retroconversion of fatty acid substrates in tissue (Sprecher and Lee,
Biochim. Biophys. Acta
388:113-125, 1975; Geiger et al.,
Biochim. Biophys. Acta
1170:137-142, 1993).
Polyunsaturated 20-carbon fatty acids are, for the reasons outlined above, important in the human diet, and there has been considerable recent interest in incorporating such fatty acids into infant food, baby formula, dietary supplements, and nutriceutical formulations.
It would therefore be desirable to produce new transgenic plants and animals with enhanced ability to produce polyunsaturated 20-carbon fatty acids.
SUMMARY OF THE DISCLOSURE
The invention provides novel &Dgr;
5
-(
FIG. 6A
) and &Dgr;
8
(
FIG. 7A
) desaturase enzymes that may be cloned and expressed in the cells of various organisms, including plants, to produce 20-carbon polyunsaturated fatty acids. Expression of such fatty acids enhances the nutritional qualities of such organisms. For instance, oil-seed plants may be engineered to incorporate the &Dgr;
5
- and &Dgr;
8
-desaturases of the invention. Such oil-seed plants would produce seed-oil rich in polyunsaturated 20:3, 20:4, 20:5, 22:4, and 22:5 fatty acids. Such fatty acids could be incorporated usefully into infant formula, foods of all kinds, dietary supplements, nutriceutical, and pharmaceutical formulations.
The invention also provides proteins differing from the proteins of FIG.
6
A and
FIG. 7A
by one or more conservative amino acid substitutions. Also provided are proteins that exhibit “substantial similarity” (defined in the “Definitions” section) with the proteins of FIG.
6
A and FIG.
7
A.
The invention provides isolated novel nucleic acids that encode the above-mentioned proteins, recombinant nucleic acids that include such nucleic acids and cells, and plants and organisms containing such recombinant nucleic acids.
The novel &Dgr;
5
- and &Dgr;
8
desaturase enzymes can be used individually, or in conjunction with one another, for instance in a metabolic pathway, to produce polyunsaturated fatty acids, such as 20:3, 20:4, 20:5, 22:4, and 22:5 fatty acids.
The scope of the invention also includes portions of nucleic acids encoding the novel &Dgr;
5
- and &Dgr;
8
-desaturase enzymes, portions of nucleic acids that encode polypeptides substantially similar to these novel enzymes, and portions of nucleic acids that encode polypeptides that differ from the proteins of FIG.
6
A and
FIG. 7A
by one or

Affiliated with

Also associated with

Rating

Desaturases and methods of using them for synthesis of... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Desaturases and methods of using them for synthesis of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Desaturases and methods of using them for synthesis of... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3358405