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Patent US8143486 - Delta-5 desaturase and uses thereof - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign in<nobr>Advanced Patent Search</nobr>PatentsThe subject invention relates to the identification of genes involved in the desaturation of polyunsaturated fatty acids at carbon 5 (i.e., �Δ5-desaturase�) and at carbon 6 (i.e., �Δ6-desaturase�) and to uses thereof. In particular, Δ5-desaturase may be utilized, for example, in the conversion...http://www.google.com/patents/US8143486?utm_source=gb-gplus-sharePatent US8143486 - Delta-5 desaturase and uses thereofAdvanced Patent SearchPublication numberUS8143486 B2Publication typeGrantApplication numberUS 12/878,788Publication dateMar 27, 2012Filing dateSep 9, 2010Priority dateJan 25, 2001Also published asCA2698579A1, CA2698579C, CA2699611A1, CA2699611C, EP2325300A1, EP2325300B1, EP2333052A1, EP2333052B1, US6635451, US7067285, US7241619, US8143383, US20030157144, US20030167525, US20030190733, US20090265798, US20110003351, US20110003360Publication number12878788, 878788, US 8143486 B2, US 8143486B2, US-B2-8143486, US8143486 B2, US8143486B2InventorsPradip Mukerji, Tapas Das, Yung-Sheng Huang, Jennifer Thurmond, Suzette L. PereiraOriginal AssigneeAbbott LaboratoriesExport CitationBiBTeX, EndNote, RefManPatent Citations (52), Non-Patent Citations (93), Classifications (39) External Links: USPTO, USPTO Assignment, EspacenetDelta-5 desaturase and uses thereofUS 8143486 B2Abstract The subject invention relates to the identification of genes involved in the desaturation of polyunsaturated fatty acids at carbon 5 (i.e., �Δ5-desaturase�) and at carbon 6 (i.e., �Δ6-desaturase�) and to uses thereof. In particular, Δ5-desaturase may be utilized, for example, in the conversion of dihomo-γ-linolenic acid (DGLA) to arachidonic acid (AA) and in the conversion of 20:4n-3 to eicosapentaenoic acid (EPA). Delta-6 desaturase may be used, for example, in the conversion of linoleic (LA) to γ-linolenic acid (GLA). AA or polyunsaturated fatty acids produced therefrom may be added to pharmaceutical compositions, nutritional compositions, animal feeds, as well as other products such as cosmetics.
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).
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates the fatty acid biosynthetic pathway and the roles of Δ5-desaturase and Δ6-desaturase in this pathway.
DETAILED DESCRIPTION OF THE INVENTION The subject invention relates to the nucleotide and translated amino acid sequences of the Δ5-desaturase gene derived from Saprolegnia diclina, the Δ6-desaturase gene derived from Saprolegnia diclina, and the Δ5-desaturase gene derived from Thraustochytrium aureum. Furthermore, the subject invention also includes uses of these genes and of the enzymes encoded by these genes. For example, the genes and corresponding enzymes may be used in the production of polyunsaturated fatty acids such as, for instance, arachidonic acid, eicosapentaenoic acid, and/or adrenic acid which may be added to pharmaceutical compositions, nutritional compositions and to other valuable products.
Example 1 Design of Degenerate Oligonucleotides for the Isolation of Desaturases from Fungi and cDNA Library Construction Analysis of the fatty acid composition of Saprolegnia diclina (S. diclina)(ATCC 56851) revealed the presence of a considerable amount of arachidonic acid (ARA, 20:4 n-6) and eicosapentanoic acid (EPA, 20:5 n-3). Thus, it was thought that this organism contained an active Δ6-desaturase capable of converting linoleic acid (LA, 18:2 n-6) to gamma-linolenic acid (GLA, 18:3 n-6), and an active Δ5-desaturase that would convert dihomo-gamma-linolenic acid (DGLA, 20:3 n-6) to arachidonic acid (ARA, 20:4 n-6) (FIG. 1). In addition, it was thought that S. diclina also contained a Δ17-desaturase capable of desaturating ARA to EPA.
Example 2 Isolation of Δ6-Desaturase Nucleotide Sequences from Saprolegnia diclina (ATCC 56851) Total RNA from Saprolegnia diclina (ATCC 56851) was isolated using the lithium chloride method (Hoge, et al., Exp. Mycology (1982) 6:225-232). Five μg of the total RNA was reverse transcribed, using the SuperScript Preamplification system (LifeTechnologies, Rockville, Md.) and the oligo(dT)12-18 primer supplied with the kit, to generate the first strand cDNA.
a. Clone#20-2 was partially sequenced and the deduced amino acid sequence from 702 by showed 30.2% identity with Δ6-desaturase from Mortierella alpina as the highest scoring match in a TfastA search. b. Clone #30-1 was partially sequenced, and the deduced amino acid sequence of 687 by showed 48.5% amino acid identity with Mortierella alpina's Δ6-desaturase as the highest scoring match in a TfastA search. These two sequences also overlapped each other indicating they belonged to a single putative Δ6-desaturase from S. diclina. This novel Δ6-desaturase sequence was then used to design primers to retrieve the 3′- and the 5′-end of the full-length Δ6-desaturase gene from the cDNA library generated from the mRNA of S. diclina. To isolate the 3′-end, PCR amplification was carried out using plasmid DNA purified from the cDNA library as the template and oligonucleotides RO0923 (SEQ ID NO:7) (5′-CGGTGCAGTGGTGGAAGAACAAGCACAAC-3′) and RO899 (SEQ ID NO:8) (5′-AGCGGATAACAATTTCACACAGGAAACAGC-3′). Oligonucleotide RO923 was designed based on the #20-2 fragment of this putative Δ6-desaturase, and oligonucleotide RO899 corresponded to sequence from the pBluescript II SK(+) vector used for preparation of the cDNA library. Amplification was carried out using 10 pmols of each primer and the Taq PCR Master Mix (Qiagen, Valencia, Calif.). Samples were denatured initially at 94� C. for 3 minutes, followed by 30 cycles of the following: 94� C. for 1 minute, 60� C. for 1 minute, 72� C. for 2 minutes. A final extension cycle at 72� C. for 10 minutes was carried out before the reaction was terminated. The PCR fragments were resolved on a 0.8% agarose gel and gel purified using the Qiagen Gel Extraction Kit. The staggered end on these fragments were �filled-in� using T4 DNA polymerase (LifeTechnologies, Rockville, Md.) as per manufacturer's specifications, and these DNA fragments were cloned into the PCR-Blunt vector (Invitrogen, Carlsbad, Calif.). The recombinant plasmids were transformed into TOP10 supercompetent cells (Invitrogen, Carlsbad, Calif.), and clones were sequenced. Clone sd2-2 contained a 958 by insert which was identified to contain the 3′-end of the putative Δ6-gene based on sequence homology with known Δ6-desaturases and the presence of the �TAA� stop codon and Poly A tail.
To isolate the 5′-end of this Δ6-desaturase from Saprolegnia diclina, the oligonucleotide RO939 (SEQ ID NO:9) (5′-CGTAGTACTGCTCGAGGAGCTTGAGCGCCG-3′) was designed based on the sequence of the #30-1 fragment identified earlier. This oligonucleotide was used in combination with RO898 (SEQ ID NO:10) (5′-CCCAGTCACGACGTTGTAAAACGACGGCCAG-3′) (designed based on the sequence of from the pBluescript SK(+) vector) to PCR amplify the 5′-end of the Δ6-desaturase from the cDNA library. In this case, the Advantage-GC cDNA PCR kit (Clonetech, Palo Alto, Calif.) was used to overcome PCR amplification problems that occur with GC rich regions, predicted to be present at the 5′-end of this β6-desaturase. PCR thermocycling conditions were as follows: The template was initially denatured at 94� C. for 1 minute, followed by 30 cycles of [94� C. for 30 seconds, 68� C. for 3 minutes], and finally an extension cycle at 68� C. for 5 minutes. The PCR products thus obtained were cloned into the PCR-Blunt vector (Invitrogen, Carlsbad, Calif.) following the same protocol as described above. Clone sd21-2 was thus obtained that contained a 360 by insert that contained the putative �ATG� start site of the novel Δ6-desaturase. The deduced amino acid sequence of this fragment, when aligned with known Δ6-desaturases showed 37-45% identity.
The Δ6-desaturase full-length gene insert was 1362 by (SEQ ID NO:13, FIG. 2) in length and, beginning with the first ATG, contained an open reading frame encoding 453 amino acids. (The nucleotide sequence encoding the Δ6-desaturase was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110 under the terms of the Budapest Treaty on Jan. 23, 2001 and was accorded accession number PTA-2929.) The amino acid sequence of the full-length gene (SEQ ID NO:14, FIG. 3) contained regions of homology to Δ6-desaturases from Mortierella alpina, Caenorhabditis elegans and Borago officinalis. It also contained the three conserved �histidine boxes� found in all known membrane-bound desaturases (Okuley, et al. (1994) The Plant Cell 6: 147-158). These were present at amino acid positions 171-176, 208-212, and 391-395. As with other membrane-bound Δ6-desaturases, the third Histidine-box motif (HXXHH) in the S. diclina Δ6-desaturase was found to be QXXHH. This sequence also contained a cytochrome b5 domain at the 5′-end. This cytochrome b5 domain is found in a number of membrane-bound desaturase enzymes, and cytochrome b5 is thought to function as an electron donor in these enzymes. The presence of this domain may be advantageous when expressing the desaturase in heterologous systems for PUFA production. Since the proposed use of this gene is for the reconstruction of the PUFA biosynthetic pathway in plants, the base composition of this gene may be important. (It is known that some recombinant genes show poor expression because of variations in their base composition as compared to that of the host. The overall G+C content of this gene was 59%, which is close to that of the M. alpina desaturases that have been successfully expressed in plants.)
Example 3 Isolation of Δ5-Desaturase Nucleotide Sequences from Saprolegnia diclina (ATCC 56851) Saprolegnia diclina (ATCC 56851) produces both arachidonic acid (ARA, 20:4 n-6) and eicosapentanoic acid (EPA, 20:5 n-3); thus, it was thought to have, perhaps, a Δ5-desaturase which can convert dihomo-gamma-linolenic acid (DGLA, 20:3n-6) to arachidonic acid (ARA, 20:4 n-6). As with the Δ6-desaturase isolation, for the β5-desaturase isolation from S. diclina, various combinations of the degenerate primers were used in PCR reactions, using first strand cDNA as the template. The primer combination, RO753 and RO754, generated a distinct band of 588 by using the following PCR conditions: 2 μl of the first strand cDNA template, 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 μM each deoxyribonucleotide triphosphate, 2 pmole of each primer and 1U cDNA polymerase (Clonetech, Palo Alto, Calif.), in a final reaction volume of 50 μl. Thermocycling was carried out as follows: an initial denaturation at 94� C. for 3 minutes, followed by 35 cycles of: denaturation at 94� C. for 30 seconds, annealing at 60� C. for 30 seconds and extension at 72� C. for 1 minute. This was followed by a final extension at 72� C. for 7 minutes, and the reaction was terminated at 4� C. This fragment thus generated was cloned (clone #18-1), sequenced and, when translated, showed 43% amino acid identity with Mortierella alpina Δ5-desaturase (Genbank accession # AF067654) and 38.7% identity with Dictyostelium discoideum Δ5-desaturase (Genbank accession # AB029311). The second PCR fragment was identified using Primers RO834 and RO838 in the reaction described in Example 2. This fragment, of approximately 1000 by in length, was cloned (Clone #20-8) and the deduced amino acid sequence derived from 775 by showed 42% identity with Δ5-desaturase from Dictyostelium discoideum Δ5-desaturase (Genbank accession # AB029311). These two sequences, #18-1 and #20-8, overlapped each other indicating they belonged to a single putative Δ5-desaturase from S. diclina. These sequences were then used to design primers to retrieve the 3′- and the 5′-end of the novel Δ5-desaturase gene from the cDNA library generated from the mRNA of S. diclina. To isolate the 3′-end of this putative Δ5-desaturase, PCR amplification was carried out using plasmid DNA purified from the cDNA library, as the template and oligonucleotides RO851 (SEQ ID NO:15) (5′-CCATCAAGACGTACCTTGCGATC-3′) and RO899 (SEQ ID NO:8) (5′-AGCGGATAACAATTTCACACAGGAAACAGC-3′). Oligonucleotide RO851 was designed based on the #18-1 fragment of this putative Δ5-desaturase, and oligonucleotide RO899 corresponded to sequence from the pBluescript II SK(+) vector. Amplification was carried out using 200 ng of template plasmid DNA, 10 pmoles of each primer and the Tag PCR Master Mix (Qiagen, Valencia, Calif.). Samples were denatured initially at 94� C. for 3 minutes, followed by 35 cycles of the following: 94� C. for 30 seconds, 60� C. for 30 seconds, 72� C. for 1 minutes. A final extension cycle at 72� C. for 7 minutes was carried out before the reaction was terminated. The PCR fragments were cloned into the PCR-Blunt vector (Invitrogen, Carlsbad, Calif.) as per the protocol described in Example 2. The recombinant plasmids were transformed into TOP10 supercompetent cells (Invitrogen, Carlsbad, Calif.), and clones were sequenced. Clone sd12-11 contained a 648 by insert which contained the 3′-end of the putative Δ5-gene based on sequence homology with known Δ5-desaturases and the presence of the �TAA� stop codon and polyA tail.
The 5′-end of this Δ5-desaturase from Saprolegnia diclina was isolated using primers RO941 and RO898. The oligonucleotide RO941 (SEQ ID NO:16) (5′-GCTGAACGGGTGGTACGAGTCGAACGTG-3′) was designed based on the sequence of the #20-8 fragment identified earlier. This oligonucleotide was used in combination with RO898 (SEQ ID NO:10) (5′-CCCAGTCACGACGTTGTAAAACGACGGCCAG-3′) (designed based on the sequence of from the pBluescript II SK(+) vector) in a PCR amplification reaction using the cDNA library plasmid DNA as the template. Here the Advantage-GC cDNA PCR kit (Clonetech, Palo Alto, Calif.) was used as per the manufacturer's protocol, and the thermocycling conditions were as follows: an initial denaturation was carried out at 94� C. for 1 minute, followed by 30 cycles of [denaturation at 94� C. for 30 seconds, annealing and extension 68� C. for 3 minutes], and a final extension cycle at 68� C. for 5 minutes. These PCR products were purified, cloned into the PCR-Blunt vector (Invitrogen, Carlsbad, Calif.), and sequenced as described above. Clone sd24-1 was identified to contain a 295 by insert that contained the putative �ATG� start site of the novel Δ5-desaturase. Analysis of the deduced amino acid sequence of this fragment showed regions of high homology with known Δ5-desaturases and also the presence of a cytochrome b5 domain.
Conditions for the PCR amplification of the �full length� gene were similar to those described for the amplification of the Δ6-desaturase from genomic DNA (Example 2). The PCR product thus obtained was digested with EcoRI/XhoI and cloned into the yeast expression vector pYX242 (Invitrogen, Carlsbad, Calif.). Clone pRSP3 (genomic DNA-derived) was shown to contain a 1413 by insert and was used for expression studies.
The 1413 by full-length gene (SEQ ID NO:19, FIG. 4) of the putative Δ5-desaturase from S. diclina contained an open reading frame encoding 471 amino acids (SEQ ID NO:20, FIG. 5). (The nucleotide sequence encoding the Δ5-desaturase was deposited with the ATCC, 10810 University Boulevard, Manassas, Va. 20110 under the terms of the Budapest Treaty on Jan. 23, 2001 and was accorded accession number PTA-2928.) This translated protein showed 40.5% overall identity with the Mortierella alpina Δ5-desaturasae (Genbank accession # AF067654) and 39.5% identity with the Dictyostelium discoideum Δ5-desaturase (Genbank accession # AB022097). It also contained the three conserved �histidine boxes� at amino acid positions 186-190, 223-228, 406-410. Like the Δ6-desaturase, this sequence also contained a cytochrome b5 domain at the 5′-end. The overall G+C content of this gene was 61.5%.
Example 4 Expression of S. diclina Desaturase Genes in Baker's Yeast Clone pRSP2, which consisted of the full length Δ6-desaturase cloned into PYX242 (Invitrogen, Carlsbad, Calif.), and clone pRSP3, which consisted of the full-length Delta 5-desaturase gene in pYX242, were transformed into competent Saccharomyces cerevisiae strain 334. Yeast transformation was carried out using the Alkali-Cation Yeast Transformation Kit (BIO 101, Vista, Calif.) according to conditions specified by the manufacturer. Transformants were selected for leucine auxotrophy on media lacking leucine (DOB [-Leu]). To detect the specific desaturase activity of these clones, transformants were grown in the presence of 50 μM specific fatty acid substrates as listed below:
Example 5 Co-Expression of S. diclina Desaturases with Elongases The plasmid pRSP1 (Δ6) and pRSP3 (Δ5) were individually co-transformed with pRAE73-Δ3, a clone that contains the Human Elongase gene (SEQ ID NO:21) in the yeast expression vector pYES2, into yeast as described in Example 4. This elongase gene catalyzes some of the elongation steps in the PUFA pathway. Co-transformants were selected on minimal media lacking leucine and uracil (DOB[-Leu-Ura]).
Example 6 Isolation of Δ5-Desaturase Nucleotide Sequences from Thraustochytrium aureum (ATCC 34303) To isolate putative desaturase genes, total RNA was Isolated as described in Example 2. Approximately 5 μg was reverse transcribed using the SuperScript Preamplification system (LifeTechnologies, Rockville, Md.) as shown in Example 2 to produce first strand cDNA. Using the degenerate primers RO834 (SEQ ID NO:1) and 838 (SEQ ID NO:4) designed with the block maker program in a 50 μl reaction, the following components were combined: 2 μl of the first strand cDNA template, 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 μM each deoxyribonucleotide triphosphate, 2 pmole final concentration of each primer and cDNA polymerase (Clonetech, Palo Alto, Calif.). Thermocycling was carried out as follows: an initial denaturation at 94� C. for 3 minutes, followed by 35 cycles of denaturation at 94� C. for 30 seconds, annealing at 60� C. for 30 seconds and extension at 72� C. for 1 minute. This was followed by a final extension at 72� C. for 7 minutes. Two faint bands of approximately 1000 by were separated on a 1% agarose gel, excised, and purified with the QiaQuick Gel Extraction Kit (Qiagen, Valencia, Calif.). The ends were filled in with T4 DNA polymerase and the blunt-end fragments cloned into PCR Blunt as described in Example 2. Sequencing of the obtained clones identified the partial sequence of 680 by from clone 30-9 whose translation of 226 amino acids had 31.5% identity with Δ6-desaturase from adult zebrafish (Genbank accession number AW281238). A similar degree of amino acid (29.6%-28.7%) homology was found with human Δ6-desaturase (Genbank accession number AF126799), Physcomitrella patens (moss) Δ6-desaturase (Genbank accession number AJ222980), Brassica napus (canola) Δ8-sphingolipid desaturase (Genbank accession number AJ224160), and human Δ5-desaturase (ATCC accession number 203557, Genbank accession number AF199596). Since there was a reasonable degree of amino acid homology to known desaturases, a full-length gene encoding a potential desaturase was sought to determine its activity when expressed in yeast.
Example 7 Expression of T. aureum Desaturase Gene in Baker's Yeast The clone pRTA4 containing the full-length gene was transformed into the yeast host S. cerevisiae 334 and plated on selective media as described in Example 4. The cultures were grown at 24� C. for 48 hours in minimal media lacking leucine with 50 μM of exogenous free fatty acid added as a substrate as shown in Table 5. The only conversion of a substrate was DGLA (20:3n-6) to ARA (20:4n-6). The conversion of 23.7% of the added DGLA indicates that this gene encodes for a Δ5-desaturase.
Example 8 Co-Expression of T. aureum Desaturase Gene with Elongases The plasmid pRTA4 was co-transformed with an additional enzyme in the PUFA pathway, pRAE73-A3 which contains the human elongase gene in the yeast expression vector pYES2 as described in Example 4, and co-transformants were selected on minimal media lacking leucine and uracil.
Soy protein isolate 74% Milk proteins 26% Fat: Honey Graham Crunch�The fat source is a blend of partially hydrogenated cottonseed and soybean, canola, high oleic safflower, oils, and soy lecithin.
Partially hydrogenated cottonseed and soybean oil 76% Canola oil 8% High-oleic safflower oil 8% Corn oil 4% Soy lecithin 4% Carbohydrate: Honey Graham Crunch�The carbohydrate source is a combination of high-fructose corn syrup, brown sugar, maltodextrin, honey, crisp rice, glycerine, soy polysaccharide, and oat bran.
Oxepa contains 22.2 g of fat per 8-fl oz serving (93.7 g/L). The fat source is an oil blend of 31.8% canola oil, 25% medium-chain triglycerides (MCTs), 20% borage oil, 20% fish oil, and 3.2% soy lecithin. The typical fatty acid profile of Oxepa is shown in Table B. Oxepa provides a balanced amount of polyunsaturated, monounsaturated, and saturated fatty acids, as shown in Table VI. Medium-chain triglycerides (MCTs)�25% of the fat blend�aid gastric emptying because they are absorbed by the intestinal tract without emulsification by bile acids.
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