Elongase genes and uses thereof

Details Elongase genes and uses thereof Elongase genes and uses thereof

2001-07-11

2004-01-13

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S252330, C435S320100, C435S348000, C435S419000, C435S254110, C435S254210, C435S254220, C435S254300, C435S254400, C435S254500, C435S254600, C435S252310, C435S328000, C435S254230, C536S023200

Reexamination Certificate

active

06677145

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Technical Field
The subject invention relates to the identification of several genes involved in the elongation of long-chain polyunsaturated fatty acids (i.e., “elongases”) and to uses thereof. In particular, the elongase enzyme is utilized in the conversion of one fatty acid to another. For example, elongase catalyzes the conversion of gamma linolenic acid (GLA) to dihomo-&ggr;-linolenic acid (DGLA, 20:3n-6) and the conversion of stearidonic acid (STA, 18:4n-3) to (n-3)-eicosatetraenoic acid (20:4n-3). Elongase also catalyzes the conversion of arachidonic acid (AA, 20:4n-6) to adrenic acid (ADA, 22:4n-6), the conversion of eicosapentaenoic acid (EPA, 20:5n-3) to &ohgr;3-docosapentaenoic acid (22:5n-3), and the conversation of &agr;-linolenic acid (ALA, 18:3n-3) to 20:3n-3. DGLA, for example, may be utilized in the production of other polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA) which may be added to pharmaceutical compositions, nutritional compositions, animal feeds, as well as other products such as cosmetics.
2. Background Information
The elongases which have been identified in the past differ in terms of the substrates upon which they act. Furthermore, they are present in both animals and plants. Those found in mammals have the ability to act on saturated, monounsaturated and polyunsaturated fatty acids. In contrast, those found in plants are. specific for saturated or monounsaturated fatty acids. Thus, in order to generate polyunsaturated fatty acids in plants, there is a need for a PUFA-specific elongase. In both plants and animals, the elongation process is believed to be the result of a four-step mechanism (Lassner et al.,
The Plant Cell
8:281-292 (1996)). CoA is the acyl carrier. Step one involves condensation of malonyl-CoA with a long-chain acyl-CoA to yield carbon dioxide and a &bgr;-ketoacyl-CoA in which the acyl moiety has been elongated by two carbon atoms. Subsequent reactions include reduction to &bgr;-hydroxyacyl-CoA, dehydration to an enoyl-CoA, and a second reduction to yield the elongated acyl-CoA. The initial condensation reaction is not only the substrate-specific step but also the rate-limiting step.
As noted previously, elongases, more specifically, those which utilize PUFAs as substrates, 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, efficiently. A number of enzymes are involved in PUFA biosynthesis including elongases (elo) (see FIG.
1
). For example, linoleic acid (LA, 18:2-&Dgr;9,12 or 18:2n-6) is produced from oleic acid (OA, 18:1-&Dgr;9 or 18:1n-9) by a &Dgr;12 desaturase. GLA (18:3-&Dgr;6,9,12) is produced from linoleic acid by a &Dgr;6-desaturase. AA (20:4-&Dgr;5,8,11,14) is produced from dihomo-&ggr;-linolenic acid (DGLA, 20:3-&Dgr;8,11,14) by a &Dgr;5-desaturase. As noted above, DGLA is produced from GLA by an elongase.
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 or 18:3n-3) cannot be synthesized by mammals, since they lack &Dgr;15 desaturase activity. 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; see also U.S. Pat. No. 5,552,306), followed by elongation to (n-3)-eicosatetraenoic acid (20:4-&Dgr;8,11,14,17 or 20:4n-3) 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, 20:5-85,8,11,14,17) by a &Dgr;5-desaturase. Other eukaryotes, including fungi and plants, have enzymes which desaturate at carbons 12 (see PCT publication WO 94/11516 and U.S. Pat. No. 5,443,974) and 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 &bgr;-linolenic acid. In view of the inability of mammals to produce these essential long chain fatty acids, it is of significant interest to isolate genes involved in PUFA biosynthesis 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. Consequently, there is a definite need for the elongase enzyme, the gene encoding the enzyme, as well as recombinant methods of producing this enzyme. Additionally, a need exists for oils containing levels of PUFA beyond those naturally present as well as those enriched in novel PUFAs. Such oils-can only be made by isolation and expression of the elongase gene.
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 the adrenal glands. As noted above, AA production from DGLA is catalyzed by a &Dgr;5-desaturase, and DGLA production from &ggr;-linolenic acid (GLA) is catalyzed by an elongase. However, until the present invention, no elongase had been identified which was active on substrate fatty acids in the pathways for the production of long chain PUFAs and, in particular, AA, eicosapentaenoic acid (EPA), adrenic acid, docosahexaenoic acid (DHA, 22:6n-3), &ohgr;3-docosapentaenoic acid (22:5n-3) or &ohgr;6-docosapentaenoic acid (22:5n-6).
Two genes appeared to be of interest in the present search for the elongase gene. In particular, the jojoba &bgr;-ketoacyl-coenzyme A synthase (KCS), or jojoba KCS (GenBank Accession #U37088), catalyzes the initial reaction of the fatty acyl-CoA elongation pathway (i.e., the condensation of malonyl-CoA with long-chain acyl-CoA (Lassner et al., The
Plant Cell
8:281-292 (1996)). Jojoba KCS substrate preference is 18:0, 20:0, 20:1, 18:1, 22:1, 22:0 and 16:0
. Saccharomcyes cerevisiae
elongase (ELO2) also catalyzes the conversion of long chain saturated and monounsaturated fatty acids, producing high levels of 22:0, 24:0, and also 18:0, 18:1, 20:0, 20:1, 22:0, 22:1, and 24:1 (Oh et al.,
The Journal of Biological Chemistry
272 (28):17376-17384 (1997); see also U.S. Pat. No. 5,484,724 for a nucleotide sequence which includes the sequence of ELO2; see PCT publication WO 88/07577 for a discussion of the sequence of a glycosylation inhibiting factor which is described in Example V). The search for a long chain PUFA-specific elongase in
Mortierella alpina
began based upon a review of the homologies shared between these two genes and by expression screening for PUFA-elongase activity.
SUMMARY OF THE INVENTION
The present invention relates to an isolated nucleotide sequence corresponding to or complementary to at least about 50% of the nucleotide sequence shown in SEQ ID NO:1 (FIG.
6
). This isolated sequence may be represented by SEQ ID NO:1. The sequence encodes a functionally active elongase which utilizes a polyunsaturated fatty acid or a monounsaturated fatty acid as a substrate. In particular, the sequence may be derived from a fungus of the genus Mortierella and may specifically be isolated from
Mortierella alpina.
The present invention also includes a purified protein encoded by the above nucleotide sequence as well as a purified polypeptide which elongates polyunsaturated fatty acids or monounsaturated fatty acids and has at least about 50% amino acid similarity to the amino acid sequence of the purified protein encoded by the above nucleotide sequence.
Additionally, the present invent

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