Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using a micro-organism to make a protein or polypeptide
Reexamination Certificate
2001-01-25
2003-10-21
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using a micro-organism to make a protein or polypeptide
C536S023100, C536S023200, C435S320100, C435S189000, C424S093700, C424S093210
Reexamination Certificate
active
06635451
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The subject invention relates to the identification and isolation of genes that encodes enzymes (i.e.,
Thraustochytrium aureum
&Dgr;5-desaturase,
Saprolegnia diclina
&Dgr;5-desaturase and
Saprolegnia diclina
&Dgr;6-desaturase) involved in the synthesis of polyunsaturated fatty acids and to uses thereof. In particular, &Dgr;5-desaturase 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). Delta-6 desaturase catalyzes the conversion of, for example, &agr;-linolenic acid (ALA) to stearidonic acid (SDA). The converted products may then be utilized as substrates 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 that have many important functions. For example, polyunsaturated fatty acids (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 in addition to &Dgr;5-desaturase and &Dgr;6-desaturase. 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 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 (SDA, 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 acids 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 and &Dgr;6-desaturase enzymes, the respective genes encoding these enzymes, as well as recombinant methods of producing these enzymes. 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 and &Dgr;6-desaturase genes.
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 comprising SEQ ID NO:13 (FIG.
2
), SEQ ID NO:19 (
FIG. 4
) or SEQ ID NO:28 (FIG.
6
).
The isolated nucleotide sequence may be represented by SEQ ID NO:13, SEQ ID NO:19 or SEQ ID NO:28. These sequences may encode a functionally active desaturase which utilizes a polyunsaturated fatty acid as a substrate. The sequences may be derived from, for example, a fungus such as
Saprolegnia diclina
(SEQ ID NO:13 and SEQ ID NO:19) and
Thraustochytrium aureum
(SEQ ID NO:28).
The present invention also includes purified proteins (SEQ ID NO:14 (FIG.
3
), SEQ ID NO:20 (
FIG. 5
) and SEQ ID NO:29 (FIG.
7
)) encoded by the nucleotide sequences referred to above.
Additionally, the present invention includes a purified polypeptide which desaturates polyunsaturated fatty acids at carbon 5 or carbon 6 and has at least about 50% amino acid similarity to the amino acid sequence of the purified proteins referred to directly above (i.e., SEQ ID NO:14, SEQ ID NO:20 or SEQ ID NO:29).
Furthermore, the present invention also encompasses a method of producing a desaturase (i.e., &Dgr;5 or &Dgr;6). This method comprises the steps of: a) isolating the nucleotide sequence comprising SEQ ID NO:19, SEQ ID NO:28, or SEQ ID NO:13, as appropriate; 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 &Dgr;5-desaturase or &Dgr;6-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
, Kluyveromyces 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:13, SEQ ID NO:19 or SEQ ID NO:28 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, EPA, GLA or SDA, depending upon whether the nucleotide sequence encodes a &Dgr;5- or &Dgr;6-desaturase. 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
Das Tapas
Huang Yung-Sheng
Mukerji Pradip
Pereira Suzette L.
Thurmond Jennifer
Abbott Laboratories
Becker Cheryl L.
Ketter James
Sullivan Daniel
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