Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1999-11-12
2002-08-06
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S135000, C435S136000, C435S189000, C435S252300, C435S320100, C536S023200, C530S350000
Reexamination Certificate
active
06428990
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The subject invention relates to the identification and isolation of a gene that encodes an enzyme (i.e., human &Dgr;5-desaturase) involved in the synthesis of polyunsaturated fatty acids and to uses thereof. In particular, &Dgr;5-desaturase VQ catalyzes the conversion of, for example, dihomo-&ggr;-linolenic acid (DGLA) to arachidonic acid (AA) and (n-3)-eicosatetraenoic acid (20:4n-3) to eicosapentaenoic acid (20:5n-3). The converted product may then be utilized as a substrate in the production of other polyunsaturated fatty acids (PUFAs). The product or other polyunsaturated fatty acids may be added to pharmaceutical compositions, nutritional composition, animal feeds as well as other products such as cosmetics.
2. Background Information
Desaturases are critical in the production of long-chain polyunsaturated fatty acids which have many important functions. For example, PUFAs are important components of the plasma membrane of a cell, where they are found in the form of phospholipids. They also serve as precursors to mammalian prostacyclins, eicosanoids, leukotrienes and prostaglandins. Additionally, PUFAs are necessary for the proper development of the developing infant brain as well as for tissue formation and repair. In view of the biological significance of PUFAs, attempts are being made to produce them, as well as intermediates leading to their production, in an efficient manner.
A number of enzymes are involved in PUFA biosynthesis including &Dgr;5-desaturase (see FIG.
11
). For example, elongase (elo) catalyzes the conversion of &ggr;-linolenic acid (GLA) to dihomo-&ggr;-linolenic acid (DGLA) and of stearidonic acid (18:4n-3) to (n-3)-eicosatetraenoic acid (20:4n-3). Linoleic acid (LA, 18:2-&Dgr;9,12 or 18:2n-6) is produced from oleic acid (18:1-&Dgr;9) by a &Dgr;12-desaturase. GLA (18:3-&Dgr;6,9,12) is produced from linoleic &Dgr;5 acid by a &Dgr;6-desaturase.
It must be noted that animals cannot desaturate beyond the &Dgr;9 position and therefore cannot convert oleic acid into linoleic acid. Likewise, &agr;-linolenic acid (ALA, 18:3-&Dgr;9,12,15) cannot be synthesized by mammals. However, &agr;-linolenic acid can be converted to stearidonic acid (STA, 18:4-&Dgr;6,9,12,15) by a &Dgr;6-desaturase (see PCT publication WO 96/13591 and
The Faseb Journal,
Abstracts, Part I, Abstract 3093, page A532 (Experimental Biology 98, San Francisco, Calif., Apr. 18-22, 1998) see also U.S. Pat. No. 5,552,306), followed by elongation to (n-3)-eicosatetraenoic acid (20:4-&Dgr;8,11,14,17) in mammals and algae. This polyunsaturated fatty acid (i.e., 20:4-&Dgr;8,11,14,17) can then be converted to eicosapentaenoic acid (EPA, 20:5-&Dgr;5,8,11,14,17) by a &Dgr;5-desaturase, such as that of the present invention. Other eukaryotes, including fungi and plants, have enzymes which desaturate at carbon 12 (see PCT publication WO 94/11516 and U.S. Pat. No. 5,443,974) and carbon 15 (see PCT publication WO 93/11245). The major polyunsaturated fatty acid of animals therefore are either derived from diet and/or from desaturation and elongation of linoleic acid or &agr;-linolenic acid. In view of these difficulties, it is of significant interest to isolate genes involved in PUFA synthesis from species that naturally produce these fatty acids and to express these genes in a microbial, plant, or animal system which can be altered to provide production of commercial quantities of one or more PUFAs. One of the most important long chain PUFAs, noted above, is arachidonic acid (AA). AA is found in filamentous fungi and can also be purified from mammalian tissues including the liver and adrenal glands. As noted above, AA production from dihomo-&ggr;-linolenic acid is catalyzed by a &Dgr;5-desaturase. EPA is another important long-chain PUFA. EPA is found in fungi and also in marine oils. As noted above, EPA is produced from (n-3)-eicosatetraenoic acid and is catalyzed by a &Dgr;5-desaturase.
In view of the above discussion, there is a definite need for the &Dgr;5-desaturase enzyme, the gene encoding this enzyme, as well as recombinant methods of producing this enzyme. Additionally, a need exists for oils containing levels of PUFAs beyond those naturally present as well as those enriched in novel PUFAs. Such oils can only be made by isolation and expression of the &Dgr;5-desaturase gene.
All U.S. patents and publications referred to herein are hereby incorporated in their entirety by reference.
SUMMARY OF THE INVENTION
The present invention includes an isolated nucleotide sequence corresponding to or complementary to at least about 50% of the nucleotide sequence shown in SEQ ID NO:1 (FIG.
12
). The isolated nucleotide sequence may be represented by SEQ ID NO:1. These sequences may encode a functionally active desaturase which utilizes a polyunsaturated fatty acid as a substrate. The sequences may be derived from a mammal such as, for example, a human.
The present invention also includes purified proteins encoded by the nucleotide sequences referred to above. Additionally, the present invention includes a purified polypeptide which desaturates polyunsaturated fatty acids at carbon 5 and has at least about 50% amino acid similarity to the amino acid sequence of the purified proteins referred to directly above.
Furthermore, the present invention also encompasses a method of producing a human &Dgr;5-desaturase. This method comprises the steps of: a) isolating the nucleotide sequence represented by SEQ ID NO:1 (FIG.
12
); b) constructing a vector comprising: i) the isolated nucleotide sequence operably linked to ii) a promoter; and c) introducing the vector into a host cell under time and conditions sufficient for expression of the human &Dgr;5-desaturase. The host cell may be, for example, a eukaryotic cell or a prokaryotic cell. In particular, the prokaryotic cell may be, for example,
E. coli,
cyanobacteria or
B. subtilis.
The eukaryotic cell may be, for example, a mammalian cell, an insect cell, a plant cell or a fungal cell (e.g., a yeast cell such as
Saccharomyces cerevisiae, Saccharomyces carlsbergensis,
Candida spp.,
Lipomyces starkey, Yarrowia lipolytica,
Kluvveromyces spp., Hansenula Spp., Trichoderma spp. or Pichia spp.).
Additionally, the present invention also encompasses a vector comprising: a) a nucleotide sequence as represented by SEQ ID NO:1 (
FIG. 12
) operably linked to b) a promoter. The invention also includes a host cell comprising this vector. The host cell may be, for example, a eukaryotic cell or a prokaryotic cell. Suitable eukaryotic cells and prokaryotic cells are as defined above.
Moreover, the present invention also includes a plant cell, plant or plant tissue comprising the above vector, wherein expression of the nucleotide sequence of the vector results in production of a polyunsaturated fatty acid by the plant cell, plant or plant tissue. The polyunsaturated fatty acid may be, for example, selected from the group consisting of AA and EPA. The invention also includes one or more plant oils or acids expressed by the above plant cell, plant or plant tissue.
Additionally, the present invention also encompasses a transgenic plant comprising the above vector, wherein expression of the nucleotide sequence of the vector results in production of a polyunsaturated fatty acid in seeds of the transgenic plant.
Also, the invention includes a mammalian cell comprising the above vector wherein expression of the nucleotide sequence of the vector results in production of altered levels of AA or EPA when the cell is grown in a culture media comprising a fatty acid selected from the group consisting of an essential fatty acid, LA and ALA.
It should also be noted that the present invention encompasses a transgenic, non-human mammal whose genome comprises a DNA sequence encoding a human &Dgr;5-desaturase operably linked to a promoter. The DNA sequence may be represented by SEQ ID NO:1 (FIG.
12
). Additionally, the present invention includes a fluid (e.g., milk) produced by the transgenic, non-human mammal wherein the fluid comprises a detectabl
Huang Yung-Sheng
Leonard Amanda Eun-Yeong
Mukerji Pradip
Parker-Barnes Jennifer M.
Abbott Laboratories
Becker Cheryl L.
Saidha Tekchand
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