Delta-15 desaturase genes suitable for increasing levels of omega-3 fatty acids   

Abstract: The present invention relates to fungal Δ-15 fatty acid desaturases that are able to catalyze the conversion of linoleic acid (18:2, LA) to alpha-linolenic acid (18:3, ALA). Nucleic acid sequences encoding the desaturases, nucleic acid sequences which hybridize thereto, DNA constructs comprising the desaturase genes, and recombinant host plants and microorganisms expressing increased levels of the desaturases are described. Methods of increasing production of specific omega-3 and omega-6 fatty acids by over-expression of the Δ-15 fatty acid desaturases are also described herein.

This application is a divisional of U.S. application Ser. No. 12/420,345 filed Apr. 8, 2009, now Allowed, which is a Continuation of U.S. application Ser. No. 10/985,109, filed Nov. 10, 2004, now Abandoned, which claims the benefit of U.S. Provisional Application No. 60/519,191, filed Dec. 11, 2003, now Expired, the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention is in the field of biotechnology. More specifically, this invention pertains to the identification of nucleic acid fragments encoding Δ-15 fatty acid desaturase enzymes useful for disrupting or enhancing the production of polyunsaturated fatty acids in plants and organisms, including those microorganisms known as as oleaginous yeast.

BACKGROUND OF THE INVENTION

It has long been recognized that certain polyunsaturated fatty acids, or PUFAs, are important biological components of healthy cells. For example, such PUFAs are recognized as: “Essential” fatty acids that can not be synthesized de novo in mammals and instead must be obtained either in the diet or derived by further desaturation and elongation of linoleic acid (LA) or -α-linolenic acid (ALA); Constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or triglycerides; Necessary for proper development, particularly in the developing infant brain, and for tissue formation and repair; and, Precursors to several biologically active eicosanoids of importance in mammals, including prostacyclins, eicosanoids, leukotrienes and prostaglandins.

In the 1970\'s, observations of Greenland Eskimos linked a low incidence of heart disease and a high intake of long-chain omega-3 PUFAs (Dyerberg, J. et al., Amer. J. Clin Nutr. 28:958-966 (1975); Dyerberg, J. et al., Lancet 2(8081):117-119 (Jul. 15, 1978)). More recent studies have confirmed the cardiovascular protective effects of omega-3 PUFAs (Shimokawa, H., World Rev Nutr Diet, 88:100-108 (2001); von Schacky, C., and Dyerberg, J., World Rev Nutr Diet, 88:90-99 (2001)). Further, it has been discovered that several disorders respond to treatment with omega-3 fatty acids, such as the rate of restenosis after angioplasty, symptoms of inflammation and rheumatoid arthritis, asthma, psoriasis and eczema. Gamma-linolenic acid (GLA, an omega-6 PUFA) has been shown to reduce increases in blood pressure associated with stress and to improve performance on arithmetic tests. GLA and dihomo-gamma-linolenic acid (DGLA, another omega-6 PUFA) have been shown to inhibit platelet aggregation, cause vasodilation, lower cholesterol levels and inhibit proliferation of vessel wall smooth muscle and fibrous tissue (Brenner et al., Adv. Exp. Med. Biol. 83: 85-101 (1976)). Administration of GLA or DGLA, alone or in combination with eicosapentaenoic acid (EPA, an omega-3 PUFA), has been shown to reduce or prevent gastrointestinal bleeding and other side effects caused by non-steroidal anti-inflammatory drugs (U.S. Pat. No. 4,666,701). Further, GLA and DGLA have been shown to prevent or treat endometriosis and premenstrual syndrome (U.S. Pat. No. 4,758,592) and to treat myalgic encephalomyelitis and chronic fatigue after viral infections (U.S. Pat. No. 5,116,871). Other evidence indicates that PUFAs may be involved in the regulation of calcium metabolism, suggesting that they may be useful in the treatment or prevention of osteoporosis and kidney or urinary tract stones. Finally, PUFAs can be used in the treatment of cancer and diabetes (U.S. Pat. No. 4,826,877; Horrobin et al., Am. J. Clin. Nutr. 57 (Suppl.): 732S-737S (1993)).

PUFAs are generally divided into two major classes (consisting of the omega-6 and the omega-3 fatty acids) that are derived by desaturation and elongation of the essential fatty acids, LA and ALA, respectively. Despite a variety of commercial sources of PUFAs from natural sources [e.g., seeds of evening primrose, borage and black currants; filamentous fungi (Mortierella), Porphyridium (red alga), fish oils and marine plankton (Cyclotella, Nitzschia, Crypthecodinium)], there are several disadvantages associated with these methods of production. First, natural sources such as fish and plants tend to have highly heterogeneous oil compositions. The oils obtained from these sources therefore can require extensive purification to separate or enrich one or more of the desired PUFAs. Natural sources are also subject to uncontrollable fluctuations in availability (e.g., due to weather, disease, or over-fishing in the case of fish stocks); and, crops that produce PUFAs often are not competitive economically with hybrid crops developed for food production. Large-scale fermentation of some organisms that naturally produce PUFAs (e.g., Porphyridium, Mortierella) can also be expensive and/or difficult to cultivate on a commercial scale.

As a result of the limitations described above, extensive work has been conducted toward: 1.) the development of recombinant sources of PUFAs that are easy to produce commercially; and 2.) modification of fatty acid biosynthetic pathways, to enable production of desired PUFAs. For example, advances in the isolation, cloning and manipulation of fatty acid desaturase and elongase genes from various organisms have been made over the last several years. Knowledge of these gene sequences offers the prospect of producing a desired fatty acid and/or fatty acid composition in novel host organisms that do not naturally produce PUFAs. The literature reports a number of examples in Saccharomyces cerevisiae, such as: Domergue, F., et al. (Eur. J. Biochem. 269:4105-4113 (2002)), wherein two desaturases from the marine diatom Phaeodactylum tricornutum were cloned into S. cerevisiae, leading to the production of EPA; Beaudoin F., et al. (Proc. Natl. Acad. Sci. U.S.A. 97(12):6421-6 (2000)), wherein the omega-3 and omega-6 PUFA biosynthetic pathways were reconstituted in S. cerevisiae, using genes from Caenorhabditis elegans; Dyer, J. M., et al. (Appl. Eniv. Microbiol., 59:224-230 (2002)), wherein plant fatty acid desaturases (FAD2 and FAD3) were expressed in S. cerevisiae, leading to the production of ALA; and, U.S. Pat. No. 6,136,574 (Knutzon et al., Abbott Laboratories), wherein one desaturase from Brassica napus and two desaturases from the fungus Mortierella alpina were cloned into S. cerevisiae, leading to the production of LA, GLA, ALA and STA.

There remains a need, however, for an appropriate plant and/or microbial system in which these types of genes can be expressed to provide for economical production of commercial quantities of one or more PUFAs. Additionally, a need exists for oils enriched in specific PUFAs, notably EPA and DHA.

One class of microorganisms that has not been previously examined as a production platform for PUFAs are the oleaginous yeast. These organisms can accumulate oil up to 80% of their dry cell weight. The technology for growing oleaginous yeast with high oil content is well developed (for example, see EP 0 005 277B1; Ratledge, C., Prog. Ind. Microbiol. 16:119-206 (1982)), and may offer a cost advantage compared to commercial micro-algae fermentation for production of ω-3- or ω-6 PUFAs. Whole yeast cells may also represent a convenient way of encapsulating omega-3- or omega-6 PUFA-enriched oils for use in functional foods and animal feed supplements.

Despite the advantages noted above, most oleaginous yeast are naturally deficient in omega-6 PUFAs, since naturally produced PUFAs in these organisms are usually limited to 18:2 fatty acids. Thus, the problem to be solved is to develop an oleaginous yeast that accumulates oils enriched in omega-3 and/or omega-6 fatty acids. Toward this end, it is not only necessary to introduce the required desaturases and elongases that allow for the synthesis and accumulation of omega-3 and/or omega-6 fatty acids in oleaginous yeast, but also to increase the availability of the 18:3 substrate (i.e., ALA for ω-3 production). Generally, the availability of this substrate is controlled by the activity of Δ-15 desaturases that catalyze the conversion of LA to ALA.

There were a variety of known Δ-15 desaturases disclosed in the public literature, including those from photosynthetic organisms (e.g., plants) and Caenorhabditis elegans at the time that the instant invention was made. These desaturases are not known to be effective for altering fatty acid composition in oleaginous yeast and are not preferred for use in oleaginous yeast. Furthermore, heterologous expression of these desaturases in the non-oleaginous yeast Saccharomyces cerevisiae has resulted in production of less than 5% ALA (Reed, D. et al. Plant Physiol. 122:715-720 (2000); Meesapyodsuk, D. et al. Biochem. 39:11948-11954 (2000); WO 2003/099216). Thus, there is need for the identification and isolation of genes encoding Δ-15 desaturases that are able to support production of high levels of 18:3 (ALA) and higher ratios of omega-3 to omega-6 fatty acids in oleaginous microorganisms (e.g., oleaginous yeast) for use in the production of PUFAs.

The instant invention concerns, inter alia, isolation of the gene encoding a Δ-15 desaturase from the fungus Fusarium moniliforme and demonstrating surprisingly efficient conversion of 18:2 (LA) to 18:3 (ALA) upon expression in an oleaginous yeast. Orthologs of this Δ-15 desaturase were identified in Magnaporthe grisea, Fusarium graminearium, Aspergillus nidulans and Neurospora crassa. Upon further experimental analysis of the Fusarium moniliforme and Magnaporthe grisea desaturases\' activity, however, it was surprisingly shown that both Δ-15 desaturases also have Δ-12 desaturase activity (and thus the enzymes are characterized herein as having bifunctional Δ-12/Δ-15 desaturase activity).

In addition to the interest in oleaginous yeast as a production platform for PUFAs, there has also been interest in plants as an alternative production platform for PUFAs.

WO 02/26946, published Apr. 4, 2002, describes isolated nucleic acid fragments encoding FAD4, FAD5, FAD5-2 and FAD6 fatty acid desaturase family members which are expressed in LCPUFA-producing organisms, e.g., Thraustochytrium, Pythium irregulare, Schizichytrium and Crypthecodinium. It is indicated that constructs containing the desaturase genes can be used in any expression system including plants, animals, and microorganisms for the production of cells capable of producing LCPUFAs.

WO 02/26946, published Apr. 4, 2002, describes FAD4, FAD5, FAD5-2, and FAD6 fatty acid desaturase members and uses thereof to produce long chain polyunsaturated fatty acids.

WO 98/55625, published Dec. 19, 1998, describes the production of polyunsaturated fatty acids by expression of polyketide-like synthesis genes in plants.

WO 98/46764, published Oct. 22, 1998, describes compositions and methods for preparing long chain fatty acids in plants, plant parts and plant cells which utilize nucleic acid sequences and constructs encoding fatty acid desaturases, including Δ-5 desaturases, Δ-6 desaturases and Δ-12 desaturases.

U.S. Pat. No. 6,075,183, issued to Knutzon et al. on Jun. 13, 2000, describes methods and compositions for synthesis of long chain polyunsaturated fatty acids in plants.

U.S. Pat. No. 6,459,018, issued to Knutzon on Oct. 1, 2002, describes a method for producing stearidonic acid in plant seed utilizing a construct comprising a DNA sequence encoding a Δ-six desaturase.

Spychalla et al., Proc. Natl. Acad. Sci. USA, Vol. 94, 1142-1147 (February 1997), describes the isolation and characterization of a cDNA from C. elegans that, when expressed in Arabidopsis, encodes a fatty acid desaturase which can catalyze the introduction of an omega-3 double bond into a range of 18- and 20-carbon fatty acids.

WO 2004/071467 published on Aug. 26, 2004 describes the production of very long chain polyunsaturated fatty acids in plants.

SUMMARY

In one embodiment, the invention concerns a recombinant construct for altering the total fatty acid profile of mature seeds of an oilseed plant to produce an oil having an omega 3 to omega 6 ratio greater than 0.4, said construct comprising an isolated nucleic acid fragment selected from the group consisting of: (a) an isolated nucleic acid fragment encoding all or part of the amino acid sequence as set forth in SEQ ID NO:2; (b) an isolated nucleic acid fragment that hybridizes with (a) when washed with: 0.1×SSC, 0.1% SDS, 65° C.; (c) an isolated nucleic acid fragment encoding an amino acid sequence having at least 46.2% sequence identity with the amino acid sequences set forth in SEQ ID NOs:2, 6, 10, 14, 18 based on the Clustal V method of alignment; or (d) an isolated nucleic acid fragment that is completely complementary to (a), (b), or (c)

wherein said isolated nucleic acid fragment is operably linked to at least one regulatory sequence.

In a second embodiment, this invention concerns oilseed plants, plant cells, plant tissues or plant parts comprising in their genomes the recombinant construct of the invention.

In a third embodiment, this inventions also concerns seeds obtained from such plants, oil obtained from these seeds and by-products obtained from the processing of this oil.

In a fourth embodiment, this invention concerns use of the oil of the invention in food, animal feed or an industrial application and use of the by-products of the invention in food or animal feed.

In a fifth embodiment, this invention concerns a method for increasing the ratio of omega-3 fatty acids to omega-6 fatty acids in an oilseed plant comprising: a) transforming an oilseed plant cell of with the recombinant construct of the invention; b) regenerating an oilseed plant from the transformed plant cell of step (a); c) selecting those transformed plants having an increased ratio of omega-3 fatty acids to omega-6 fatty acid compared to the ratio of omega-3 fatty acids to omega-6 fatty acid in an untransformed plant.

In a sixth embodiment, this invention concerns oilseed plants made by this method, seeds obtained from such plants, oil obtained from these seeds, use of this oil in food or animal feed, by-products obtained from the processing of this oil and use of these by-products in food or animal feed.

In a seventh embodiment, this invention concerns a method for producing alpha-linolenic acid in seed of an oilseed plant wherein the alpha-linolenic acid content of the oil in the seed is at least 25% of the total fatty acid content of the seed oil, said method comprising: a) transforming an oilseed plant cell of with the recombinant construct of the invention; b) regenerating an oilseed plant from the transformed plant cell of step (a); c) selecting those transformed plants having at least 25% alpha-linolenic acid of the total fatty acid content of the seed oil.

In an eighth embodiment, this invention concerns oilseed plants made by this method, seeds obtained from such plants, oil obtained from these seeds, use of this oil in food or animal feed, by-products obtained from the processing of this oil and use of these by-products in food or animal feed.

Alternatively, the invention provides an isolated nucleic acid fragment encoding a fungal Δ15 desaturase enzyme, selected from the group consisting of: (a) an isolated nucleic acid fragment encoding the amino acid sequence as set forth in SEQ ID NO:2; (b) an isolated nucleic acid fragment that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; or, an isolated nucleic acid fragment that is complementary to (a) or (b).

Alternatively the invention provides an isolated nucleic acid fragment comprising a first nucleotide sequence encoding a Δ15 desaturase enzyme of at least 402 amino acids that has at least 86% identity based on the Clustal method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:2; or a second nucleotide sequence comprising the complement of the first nucleotide sequence.

Additionally the invention provides polypeptides encoded by the nucleic acids described herein as well as geneic chimera and transformed host comprising the same. Preferred host cells for use in the invention include, but are not limited to plants, algae, bacteria, yeast and fungi

In another embodiment the invention provides a method for the production of α-linolenic acid comprising: a) providing a host cell comprising: (i) an isolated nucleic acid fragment encoding a protein having Δ15 desaturase activity that has at least 46.2% identity based on the Clustal method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:2; and (ii) a source of linoleic acid;

b) growing the host cell of step (a) under conditions wherein the nucleic acid fragment encoding a protein having Δ15 desaturase activity is expressed and the linoleic acid is converted to α-linolenic acid; and

c) optionally recovering the α-linolenic acid of step (b).

Similarly the invention provides a method for the production of α-linolenic acid comprising: a) providing a host cell comprising: (i) an isolated nucleic acid fragment encoding a protein having Δ15 desaturase activity that has at least 46.2% identity based on the Clustal method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:2; and (ii) a source of oleic acid; b) growing the host cell of step (a) under conditions wherein the nucleic acid fragment encoding a protein having Δ15 desaturase activity is expressed and the oleic acid is converted to α-linolenic acid; and c) optionally recovering the α-linolenic acid of step (b).

Alternatively the invention provides a method for the production of ω-3 fatty acids in a host cell comprising: a) providing a host cell comprising: (i) an isolated nucleic acid fragment encoding a protein having Δ15 desaturase activity that has at least 46.2% identity based on the Clustal method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:2; and (ii) genes encoding a functional ω-31 ω-6 fatty acid biosynthetic pathway; b) providing a source of desaturase substrate consisting of oleic acid; c) growing the host cell of step (a) with the desaturase substrate of step (b) under conditions wherein ω-3 fatty acids are produced; and d) optionally recovering the ω-3 fatty acids of step (c).

In an alternate embodiment the invention provides a method of increasing the ratio of ω-3 fatty acids to ω-6 fatty acids in a host cell producing ω-3 fatty acids and ω-6 fatty acids comprising: a) providing a host cell producing ω-3 fatty acids and ω-6 fatty acids; b) introducing into the host cell of (a) an isolated nucleic acid fragment encoding a protein having at least 46.2% identity based on the Clustal method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:2, wherein the polypeptide binds both oleic acid and linolenic acid as an enzyme substrate, wherein the ratio of ω-3 fatty acids to ω-6 fatty acids are increased.

Additionally the invention provides microbial oils produced by the methods of the invention.

In yet another embodiment, the invention concerns a recombinant construct for altering the total fatty acid profile of mature seeds of an oilseed plant to produce an oil having an omega 3 to omega 6 ratio greater than 2, wherein said oil has an eicosapentaenoic acid content greater than 2%, said construct comprising an isolated nucleic acid fragment selected from the group consisting of: (a) an isolated nucleic acid fragment encoding all or part of the amino acid sequence as set forth in SEQ ID NO:2; (b) an isolated nucleic acid fragment that hybridizes with (a) when washed with 0.1×SSC, 0.1% SDS, 65° C.; (c) an isolated nucleic acid fragment encoding an amino acid sequence having at least 46.2% sequence identity with the amino acid sequences set forth in SEQ ID NOs:2, 6, 10, 14, 18 based on the Clustal V method of alignment; or (d) an isolated nucleic acid fragment that is completely complementary to (a), (b), or (c) wherein said isolated nucleic acid fragment is operably linked to at least one regulatory sequence.

In a further embodiment, this invention concerns oilseed plants, plant cells, plant tissues, or plant parts comprising in their genomes the recombinant construct of the invention. The invention also concerns the seeds obtained from such plants, oil obtained from these seeds, use of this oil in food or animal feed, by-products obtained from the processing of this oil and use of these by-products in food or animal feed.

Additionally the invention provides microbial oils produced by the methods of the invention.

In another embodiment, the present invention concerns a method for producing eicosapentaenoic acid in seed of an oilseed plant to produce an oil having an omega 3 to omega 6 ratio greater than 2, wherein said oil has an eicosapentaenoic acid content greater than 2% of the total fatty acid content of the seed oil, said method comprising: a) transforming an oilseed plant cell of with the recombinant construct of the present invention; b) regenerating an oilseed plant from the transformed plant cell of step (a); c) selecting those transformed plants having at least 2% eicosapentaenoic acid of the total fatty acid content of the seed oil.

In a further embodiment, this invention concerns oilseed plants, plant cells, plant tissues, or plant parts comprising in their genomes the recombinant construct of the invention. The invention also concerns the seeds obtained from such plants, oil obtained from these seeds, use of this oil in food or animal feed, by-products obtained from the processing of this oil and use of these by-products in food or animal feed.

Additionally the invention provides microbial oils produced by the methods of the invention.

The following plasmids have been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, and bears the following designation, accession number and date of deposit.

Plasmid Accession Number Date of Deposit pKR274 ATCC PTA-4988 Jan. 30, 2003 pKKE2 ATCC PTA-4987 Jan. 30, 2003 pKR578 ATCC PTA-XXXX Nov. 4, 2004 pKR585 ATCC PTA-XXXX Nov. 4, 2004

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic illustration of the biochemical mechanism for lipid accumulation in oleaginous yeast.

FIG. 2 illustrates the omega-3 and omega-6 fatty acid biosynthetic pathways.

FIG. 3 illustrates the construction of the plasmid vector pY5 for gene expression in Yarrowia lipolytica.