JP2002538826A - Polyunsaturated fatty acid (PUFA) elongase from Kaenorabuditis elegans - Google Patents

Abstract

(57) Abstract: An isolated polypeptide comprising a functional long chain polyunsaturated fatty acid (PUFA) elongase.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a polyunsaturated fatty acid (PUFA) elongase. More specifically, the present invention relates to C. elegans encoding PUFA elongase. Elegance (C.eleg
ans).

[0002] Unsaturated fatty acids are essential components required for normal cell function and are deeply involved in a number of roles ranging from membrane fluidity to acting as signal molecules (Gill, I.
Valivety, R. (1997). Trends Biotechnol. 15, 401-409; Broun, P. et al., (
1999) Ann. Rev. Nutr. 19, 197-216). In particular, polyunsaturated fatty acids (PUFA)
A class of fatty acids, known as steroids, has attracted great interest as pharmaceutical and nutritional compounds (Broun, supra; Horrobin, DF (1990) Reviews in Contemp Pharmacoth.
erpy 1, 1-45).

[0003] The synthesis of PUFAs, a fatty acid that is 18 carbons or longer and contains two or more double bonds, is believed to be catalyzed by specific fatty acid elongases in various organisms. This elongase is 18 carbon PUFA
To give a 20 carbon fatty acid. Illustrative of this reaction, .gamma.-linolenic acid (GLA; 18: 3Δ ^6,9,12) of dihomo -γ- linolenic acid (DHGLA; 20: 3Δ ^8,11,14) is extended to, here 3 Valun unsaturated 1
Eight carbon fatty acids are extended by the addition of two carbons to yield triunsaturated 20 carbon fatty acids. Chain length exceeds 18 carbons and great interest is being held for the production of long chain PUFA, for example arachidonic acid and eicosapentaenoic acid (eicosapenta e no
For ic acid), the identification of this enzyme is of both academic and commercial interest.

[0004] At present, there is no example that the gene encoding PUFA elongase has been identified.
But encodes an enzyme that is thought to be deeply involved in other aspects of lipogenesis.
Many genes have been identified. For example, the Arabidopsis gene (FAE)
1) is a very long-chain monounsaturated fatty acid (erucic acid; 20: 1Δ¹¹Etc.)
Have been shown to be required for synthesis (James, D.W. et al. (1995) Plant Cell
7, 309-319). However, this enzyme is a divalent and trivalent unsaturated 18 carbon oil.
Fatty acids such as linoleic acid, 18: 2Δ^9,12Or α-linoleic acid, 18: 3Δ ^{9, 12,15} Do not recognize each as a substrate, and therefore are profound for the synthesis of long chain PUFAs.
Clearly unrelated (Miller and Kunst (1997) Plant Jou
rnal 12, 121-131). This is not surprising in itself, but it does
In the physical world, Physcomicotrella patens (Girke et al., (1998)
), Plant J. 15: 39-48) and only a small number of lower plant species such as mosses
Is capable of synthesizing long chain PUFAs, and thus Arabidopsis
It is not expected to contain enzymes (Napier et al., (1997), Biochem J. 3).
28: 717-720; Napier et al., (1999) Trends in Plant Sci 4, 2-5).

[0005] A schematic diagram showing the general route of PUFA production is shown in FIG. A biochemical feature of the mammalian elongation system (particularly from liver microsomes) is that mammalian elongase consists of four subunits composed of condensing enzymes, β-keto reductase, dehydrase, and enoyl reductase. (Reviewed in Cinti, DL, et al. (1992) Prog. Lipid Res. 31, 1-15). The Arabidopsis thaliana FAE1 gene product encodes a 56 kDa polypeptide that shows negligible homology to condensing enzymes such as chalcone synthase and stilbene synthase (James, DW, supra). FAE1
Is normally only expressed in seed tissues, but ectopic expression in non-seed tissues (or in yeast as xenogeneic) indicated that FAE1 can direct the synthesis of erucic acid (Millar , AA, Kunst, L. (1997) Plant J. 12
, 121-131).

[0006] The activity of three fatty acid elongases has been characterized from baker's yeast (S. cerevisiae). Again, this microorganism does not synthesize PUFA, and
Does not contain the gene encoding elongase. One gene, ELO1, was identified based on a screen to isolate mutants defective in elongation of 14 carbon chain (ie, medium chain) saturated fatty acids (Toke and Martin (1996) J. Bio.
l Chem 271, 18413-18422). Complementation of the elo1 mutant restored viability, indicating that the ELO1 gene product encodes a polypeptide that makes a specific extension of 14: 0 fatty acids to 16: 0 fatty acids. .

[0007] Also, two related genes have been detected in the genome of baker's yeast, and their function has been determined by disruption. These two genes were later transformed into ELO2 and EL
Although named O3, it was shown to be deeply involved in the extension of very long chain saturated fatty acids found in sphingolipid molecules (Oh et al., (1997), J. Biol Chem 27
2, 17376-17384). In particular, ELO2 was required for extension of fatty acids up to 24 carbons, and ELO3 was required for extension of 24 carbon fatty acids to 26 carbons. However, neither gene was essential for viability. Examination of these three fatty acid elongases reveals a conservative “histidine box” motif toward the center of the polypeptide sequence (Shanklin et al., (1994) Biochemistry, 33, 1).
2787-12794) (His-XX-His-His, where X is an amino acid). Importantly, no homology could be detected between the yeast elongase (ELO1, 2, 3) and the plant's very long-chain monounsaturated fatty acid elongase (FAE1) (Oh et al., Supra).

[0008] To identify the gene encoding PUFA elongase, PUFA elongase
It is necessary to study systems whose synthesis has been well documented. A good example of this is the model animal strain C. Elegance (C. elegans), a small, non-parasitic helminth (Tanaka et al., (1996), Lipids 31, 1173-1178). C. Elegance, like most other animals, also in contrast to higher plants, arachidonic acid as a precursor known as eicosanoids ^{(AA; 20; 4Δ 5,8,11,14)} PUF such
A is synthesized and it is utilized as a precursor for compounds such as prostaglandins and leukotrienes. (Horrobin, (1990), Reviews in Contemp Ph
armacotherpy; 1: 1-45). C. The presence of AA and other long chain polyunsaturated fatty acids in Elegance is well documented (Tanaka et al., (1996), Lipids 31,
1173-1178). The complete sequence of the nematode genome is now publicly available (The C. elegans consortium, 1998, Science 282, 2012-2018: database http://www.sanger.ac.uk/Projects/ C_elegans / blast_server.shtml).

It is an object of the present invention to provide an isolated PUFA elongase.

[0010] The present inventors have proposed the above-described C.I. By using the Elegans genomic sequence with the appropriate search strings, eight related putative open reading frames (ORFs) encoding PUFA elongase were identified. Several different search criteria were used to identify a large number (ORFs) that appeared to encode a polypeptide with fatty acid elongase activity. These ORFs were then functionally characterized by heterologous expression in yeast, allowing the identification of PUFA elongase.

[0011] Accordingly, a first aspect of the present invention provides a functional long chain polyunsaturated fatty acid (PUFA).
C) providing an isolated polypeptide comprising elongase, ie, a polypeptide having the function of extending the chain length of an 18 carbon PUFA to 20 carbon lengths. This polypeptide can be used to increase the level of PUFA in an animal, thereby providing a convenient source of PUFA.

[0012] The polypeptide may be derived from a eukaryote.

[0013] The polypeptide may comprise at least a portion of the amino acids set forth in SEQ ID NO: 15, or a variant thereof.

For the purposes of this application, the term “variant” in reference to a sequence refers to the insertion or deletion of one or more amino acid residues, or one or more amino acid residues Substitution of one polar amino acid residue with another polar amino acid residue or another nonpolar amino acid residue with another nonpolar amino acid residue Means a protein or polypeptide derived from the sequence through substitution with a group. In each case, the variant must have elongase function as defined herein.

[0015] A second aspect of the present invention provides a polypeptide having at least 60% homology to a polypeptide according to the first aspect of the present invention. The polypeptide may have at least 80%, or even 90%, or higher homology to the polypeptide according to the first aspect of the invention.

A polypeptide according to any of the aspects of the invention may include a sequence motif that is responsible for maintaining the endoplasmic reticulum (ER). This allows the polypeptide to be specifically localized or targeted to the ER of the cell.

The polypeptide may also allow the extension of palmitoleic acid (PA; 16: 1Δ ⁹ ) to vacceric acid (VA; 18: 1Δ ¹¹ ). Thus, the polypeptides can also extend Δ ⁹ -1 unsaturated 16 carbon fatty acids.

[0018] Preferably, the polypeptide is from an animal, more preferably the animal is an invertebrate, such as a helminth. If the animal is a helminth, it is preferably C. elegans. Elegance. Alternatively, the animal is a vertebrate, preferably a mammal, such as a human, rat, or mouse.

A third aspect of the present invention provides an isolated DNA sequence, preferably a cDNA sequence encoding a polypeptide according to the first or second aspect of the present invention. The DNA
The sequences may be used to engineer transgenic organisms.

Preferably, the DNA sequence comprises the sequence shown in SEQ ID NO: 7 or a variant thereof, for example by substitution, deletion and / or addition of bases.

[0021] A fourth aspect of the present invention provides an engineered organism, such as a transgenic animal engineered to express a polypeptide according to the first or second aspects of the present invention. The engineered organism may be engineered to express the polypeptide at an enhanced level, thereby reducing the number of organisms that must be used, thus reducing the cost of supplying the polypeptide. Provided at

Preferably, the engineered organism is a mammal, such as a rat, mouse, or monkey.

[0023] A fifth aspect of the present invention provides an engineered organism comprising a synthetic pathway for the production of a polypeptide according to the first or second aspects of the present invention. This has the advantage that the production of PUFAs by this route can be better controlled by the organism.

[0024] The route delta ⁵ - may comprise fatty acid desaturase - fatty acid desaturases, and / or delta ^6.

The engineered organism according to the fourth or fifth aspect of the invention may be a lower eukaryote, such as yeast. On the other hand, the transgenic organism may be a fish.

[0026] A sixth aspect of the present invention provides a transgenic plant engineered to express a polypeptide according to the first aspect of the present invention.

[0027] A seventh aspect of the present invention provides a transgenic plant comprising a DNA sequence according to the third aspect of the present invention.

An eighth aspect of the present invention provides a method for producing a PUFA, comprising performing an elongase reaction catalyzed by a polypeptide according to the first or second aspect of the present invention.

[0029] The PUFA is, dihomo -γ- linoleic acid (20: 3Δ ^8,11,14), arachidonic acid (20: 4Δ ^5,8,11,14), eicosapentaenoic acid (20: 5Δ ^{5,8 , 11,14,17}
), Docosatrienoic acid (22: 3Δ ^3,16,19 ), ^{docosatetraenoic} acid (22: 4
Δ ^7,10,13,16), docosapentaenoic acid (22: 5Δ ^{7,10,13,16,19),} or docosahexaenoic acid (22: 6Δ ^{4,7,10,13,16,19)} may be .

[0030] The PUFA may be a 24-carbon fatty acid having at least four double bonds.

A ninth aspect of the present invention is directed to a P-produced by a method according to the eighth aspect of the present invention.
Provide UFA.

[0032] PUFAs may be used in foodstuffs, supplements, or pharmaceutical compositions.

[0033] A tenth aspect of the present invention provides a food product comprising a PUFA according to the fifth aspect of the present invention. The food can be given to animals.

An eleventh aspect of the present invention provides a supplement comprising a PUFA according to the fifth aspect of the present invention. The supplement can be provided to the animal to increase its PUFA level.

A twelfth aspect of the present invention provides a pharmaceutical composition comprising a polypeptide according to the first or second aspect of the present invention or a PUFA according to the ninth aspect of the present invention.

[0036] Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable diluent, carrier, excipient, or bulking agent. This allows the composition to be supplied in a form that is most suitable for the pharmaceutical application in question. For example, a topical application will preferably be a cream or lotion, but different shapes will be more suitable if the composition is to be ingested.

A thirteenth aspect of the present invention relates to a DNA sequence according to the third aspect of the invention, a foodstuff according to the tenth aspect of the invention, a supplement according to the eleventh aspect of the invention, Or a PUFA according to the ninth aspect of the invention
A method of treating an animal or a plant, such as a mammal, comprising supplying the animal or the plant with

[0038] Preferably, the animal is a human. The present invention will now be further described with reference to SEQ ID NOs: 1 to 16 and FIGS. 2 to 11 by way of example only: SEQ ID NOs: 1 to 8 encode PUFA elongases, A to H, respectively. SEQ ID NOs: 9-16 show the deduced amino acid sequences of the putative ORFs of SEQ ID NOs: 1-8, respectively; FIGS. 2-9 show each PUFA elongase A 9 shows hydrophobicity plots for each of ~ H. FIG. 1 shows an amino acid sequence alignment comparing ORC F. Elegance ORF F56H11.4 (Z68749) with related sequences. FIG. 11 shows a chromatogram of fatty acid methyl esters from the transformed yeast.

(Introduction to Overall Strategy) The Elegance database lists TBLAST for sequences showing low levels of homology to the yeast ELO genes (ELO2 and ELO3).
Searched using the N program. A similar search revealed a short (20-50 amino acid) stretch of the ELO gene that was conserved between the three ELO polypeptide sequences.
(stretches). Seeds identified by this method. Elegance sequences were then used as search probes themselves, by the relevant C. elegans that were not initially identified in a search using yeast sequences. Elegance was used to identify genes. This was necessary because the level of homology between the yeast ELO gene and the helminth gene was always low (see BLAST score below). To enable a more sensitive search for helminth sequences, a novel approach has been applied to circumvent the major drawbacks of searching using the BLAST program,
That is, the search string (ie, the input search motif) must be longer than 15 characters for the algorithm to work. Thus, if one wanted to search for short motifs (like the histidine box), the BLAST program would not have been able to do this. C. A complete list of all predicted ORFs present in the Elegance genome
It exists as a database called Wormpep, which
r WWW site (http://www.sanger.ac.uk/Pr
objects / C_elegans / webbase_front_end. sh
tml) for free. The latest version of Wormpep was downloaded to the hard disk of a Pentium (Pentium) PC and re-formatted as a Microsoft Word 6 document, resulting in a 3,500 page document. This was then searched using the "Search &Replace" function of Word 6, which could also introduce "wildcard" characters into the search chief. Thus, for example, one could search for a short text string HPGG identifying the predicted helminth ORF present in the 3,500 page document of Wormpep containing this motif, and instead, instead of HPGX (X Can be searched using wildcard characters. Obviously, such a (manual) search for a 3,500 page document is very time consuming and severe, and also requires a visual inspection of each and every identified ORF. I need it. For example, a search using a motif such as HXXHH identifies over 300 different ORFs. However, by using some short search strings (as outlined below) and by linking them with other methods to identify putative elongase enzymes, some ORFs were Candidates have been identified.

(Database Search Using FAE1 Polypeptide Sequence) As a negative control, the FAE1 gene sequence was a DNA sequence encoding PUFA elongase. To demonstrate that it does not provide a useful search sequence in the identification of Elegance sequences, GenBank was used to identify related genes or expressed sequence transcripts (ESTs) using the Arabidopsis thaliana FAE1 polypeptide sequence. Database (http: //www.ncbi.nlm.n)
ih. gov / Web / Search / index. html).
GenBank is the NIH gene sequence database, an annotated collection of all publicly available DNA sequences (Nucleic Acid Research (1998
) 26, 1-7). As of December 1998, about 2,2 in the 3,044,000 sequence
16,000,000 bases have been recorded. Search is BLAST2 (Basi
c Performed using the Local Alignment Search Tool algorithm (Altschul et al., (1990) J. Mol Biol 215, 403, 410).
Although the ORFs and ESTs of some plants were reported to be related, this search did not identify any animal sequences, and the observation that FAE1 was not likely to be a suitable candidate as a PUFA elongase search template. Confirmed.

(Database search using yeast ELO sequence) Using three yeast elongase sequences (ELO 1, 2, 3) as probes, C. C. forming the elegance genome sequence database. Several putative ORFs in the DNA of the cosmid sequence from Elegance were identified. Furthermore, the Wormpep database (ftp: //ftp.san)
ger. ac. uk. An extensive and time-consuming search for the downloaded copy of (/ pub / dataabaes / wormpep) using a manual search string of MS word 6 results in several seeds containing a putative histidine box. Elegance ORF was identified. Wormpep is C. Contains proteins predicted from the Elegance Genome Sequence project, which are located at the Sanger Center in Cambridge, UK.
) And the Genom Sequencing Center in St. Louis, USA
Center). Current Wompep database,
Wormpep 16 contains 16,332 protein sequences (7,120,115 residues). The search strings used are [HXXHH], [HXXXHH]
], [QXXHH], and [YHH]. Comparison of data from two different searches showed a small number (<10) of putative ORFs as candidate elongases. The histidine box motif is shown in bold print in SEQ ID NOs: 9-16.

(Hydrophobic plot analysis) Since the fatty acid elongase reaction is performed on the cytosol surface of the inner membrane system,
Toke and Martin (1996) supra; Oh et al. (1997) supra);
The ORF of Elegance was determined by the hydrophobicity plot of kyte and Doolittle (J. Mol
Biol. (1982), 157, 105-132). This is because several ORs with potential domains spanning the membrane
F and revealed some similarity to the secondary structure of some of the identified ORFs.

(Screening for ER Retention Signal Sequence) Since the fatty acid elongase is expected to be a membrane protein of the endoplasmic reticulum (ER), the present inventors have proposed the peptide signal responsible for “ER-retention”. It is assumed that it is expected to have In the case of ER membrane proteins, this signal often takes the form of the C-terminal motif [KK-X _2.3 -Stop] or similar variants thereof (Jackson et al., (1990), EMBO J. 9, 3153? 3162).
C. Further sequence analysis of the putative elongase of Elegance revealed four ORFs
(F41H10.7, F41H10.8, F56H11.4, Y53F4B.c
) Has a C-terminal motif that exactly matches this search pattern, and two additional ORFs (F11E6.5, C40H1.4) have related sequences. These sequence motifs are underlined in SEQ ID NOs: 9-13, 15, and 16.

(Chromosome Mapping) The present inventors have previously described C. et al., Which are deeply involved in PUFA synthesis. Because we have observed that the elegance gene may be present in tandem (eg, the Δ5 and Δ6 desaturases required for AA and GLA synthesis, respectively, have <1 on chromosome IV).
kb away (Michaelson et al., (1998), FEBS Letts 439, 215-218)),
Estimated sea. The position of the ORF of the elongase of Elegance was determined by the Web Access C.A. elegans server (http: //www.s
angel. ac. uk / Projects / C_elegans / webac
e_front_end. (shtml). This indicates that two pairs of putative elongases are This indicates that they were in close proximity to each other on chromosome IV of elegance.

F41H10.7 and F41H10.8 were identified about 10 Kb apart on chromosome IV, and F56H11.3 and F56H11.4 were identified about 2 Kb on chromosome IV.

(Estimated C. elegans fatty acid elongase) The location of the putative ORF in the Elegance genome is shown below, ie, the chromosome number, and the location on the map in centimorgan, along with the GenBank database accession number.

The names used were Sanger Center's C.I. The same as that used in the Elegance Database, ie ORFC40H1.4 is cosmid C40
Predicted to encode sequence 4 on H1.

[0048]

[Table 1]

Comparison of the C. elegans putative elongase ORF with the yeast gene: Each of the three ELO polypeptides of yeast was compared to the translated ORF sequence of all putative helminth elongases; They were then ordered by similarity (as measured by BLAST score) (Altschul et al., (1990), supra).

The result is shown below in the order of O, sorted from most similar to least similar.
Shown using RF sequence, BLAST score is shown in parentheses.

Yeast ELO1 (14-16 carbon fatty acid elongase) G (262)> E (241)> D (225)> C (219)> A (216)> F
(215)> H (197)> B (172) Yeast ELO2 (24 carbon sphingolipid elongase) E (231)> C (226)> G (189)> A (181)> F (166)> D
(150)> H (141)> B (140) Yeast ELO3 (24-26 sphingolipid elongase) D (171)> G (163)> F (154)> A (152)> E (150)> C
(131)> B (132)> H (128)

From the BLAST score values, it is clear that the sequences are related, but the level of homology is low. As a comparison, two related helminth proteins, Δ5
The BLAST score for homology between and and Δ6 desaturase is 500 or more.

Analysis of Possible Sources of Sphingolipids We have previously noted the similarity between fatty acid Δ6 desaturase and plant sphingolipid desaturase, and the two separate The enzyme could have been derived from one ancestral gene. Furthermore, sphingolipid desaturases are thought to have preceded fatty acid desaturases, and may in fact have been primitive ancestors. Therefore, it is plausible that the next step in the arachidonic acid biosynthetic pathway also evolved from the metabolic pathway of sphingolipids. Therefore C. It is very significant that some of the putative elongases of the Elegance ORF have similarities to sphingolipid enzymes. For these reasons, these ORFs are considered to be very distinct candidates for PUFA elongase. Previously, Sea. Elegance Δ5 and Δ6 fatty acid desaturases were thought to have evolved from one ancestral gene (Michaelson et al., (1998), FEBS Letts 439).
, 215-218). C. Elegance pair of putative elongase ORFs (F
And G) are genetically mapped in close proximity to the Δ5 / Δ6 fatty acid desaturase gene, and it is also significant that both gene pairs are located at the top of chromosome IV.

[0054]

[Table 2]

Cloning of Desaturase and Elongase Genes in Yeast Expression Vector The putative elongase sequences, F56H11.4 and F41H10.8,
It was cloned into the pYES2 vector (Invitrogen) by PCR. C. A cDNA library from a mixed period of elegance was used as a template for PCR. F56H11.4 is a primer:

56h114. Forward 5'-GCG GGTACC ATGGCTCAGCATC
CGCTC-3 ′ and; 56h114. Reverse 5'-GCG GGATCC TTAGTTGTTCTTC
Amplified using TTCTT-3 '. F41H10.8 is a primer: 41h108. Forward 5'-GCG GGTAC ATGCCACAGGGAG
AAGTC-3 'and; 41h108. Reverse 5'-GCG GGATCC TTATTCAATTTTTT
Amplified using CTTTT-3 ′.

The amplified sequence was then restricted using Kpnl and BamHI (forward and reverse primers are each underlined), purified using a Qiagen PCR purification kit, and further purified using Kpnl / BamHI. PYes2 cut at
Ligated into vector.

[0058] Mortierella alpina (Mortierella alpina) of Δ ⁵ -.. ORF encoding fatty acid desaturase (Michaelson, LV, et al., (1998) J Biol Chem.
273, 19055-19059) are primers:

Mad5. Forward 5'-GC GAATTC ACCATGGGGTACGGACC
AAGGA-3 ′ and; Mad5. Reverse 5'-GCG GAGCTC CTACTCTTCCTTGGG
ACG-3 '

The plasmid was amplified using Escherichia coli, restricted using EcoRI and SacI, gel purified as described, and ligated into an EcoRI / SacI cut pEST-TRP vector (Stratagene) to ligate pESC / Δ ⁵ was caused.

The ORF encoding borage Δ6-fatty acid desaturase (Sayanova,
O., et al., (1997) Proc. Natl. Acad. Sci USA 94, 4211-4216)
3 using the BamHI and XhoI restricted from yielded pESC / delta ⁶ is connected to the pESC-TRP vector is BamHI / XhoI cleavage.

[0062] The double construct also a BamHI / XhoI borage delta ⁶ insert was ligated into pESC / delta ⁵ constructs previously ^{described, pESC / (Δ 5, Δ} 6) generated by causing.

(Characterization of Functionality in Yeast) The elongase and desaturase constructs were prepared from baker's yeast (Saccharomy) by a method based on lithium acetate (Elble, R. (1972) Biotechniques 13, 18-20).
ces cerevisiae) W303-1A, and the expression of the transgene is
er et al. (Napier, JA et al. (1998) Biochem J 330, 611-614; Michaelson L.
V., supra; Michaelson, LV, ((1998) FEBS Letts 439, 215-218)), induced by adding galactose to 2% (w / v). Yeast transformants containing the construct derived from pYES2 were synthesized minimal media (SD,
This composition is defined in Sherman, F (1991) Methods in Enzymology 194, 3-21), grown on synthetic minimal medium without uracil, and the pESC-derived construct is grown on SD minimal medium except tryptophan. Was. Co-transformed yeast (
(including both pYES2 and pESC derivatives) were grown on SD minimal medium without uracil and tryptophan. Prior to induction, cultures were grown in the presence of 2% raffinose and supplemented with 0.5 mM of the appropriate fatty acid substrate in the presence of 1% tergitol- (NP40) (Sigma). All cultures were then grown for an additional 48 hours unless indicated.

(Fatty Acid Analysis) In order to identify the elongation reaction of dihomo-γ-linolenic acid (DHGLA; 20: 3Δ ^8,11,14 ) that undertakes synthesis from GLA, the latter fatty acid (exogenous ) Given as substrate.

Lipids were obtained from transformed and control yeast by homogenization in MeOH-CHCl ₃ by Blight and Dyer (Dickenson and Lester (1
999) Extracted using a modification of Biochim Biophys Acta 1426, 347-357). The resulting CHCl ₃ phase was evaporated to dryness under nitrogen gas and the sample was transmethylated with 1M HCl in methanol at 80 ° C. for 1 hour. Fatty acid methyl esters (FAMES) were extracted into hexane and purified using a small column packed with florisil. The analysis of FAMES was 25M x
The measurements were performed using a Hewlett Packard 5880A series gas chromatograph equipped with a 0.32 mm RSL-500BP fixed capillary column and a flame ion detector. Fatty acids are FAME separated by the same GC
It was identified by comparing the retention time with a standard (Sigma). Quantification was performed using the area integral of the peak height, expressed as the sum of all integrals (Bligh, E
G. and Dyer, WJ (1959) Can. J. Biochem. Physiol. 37, 911-917). Total fatty acids extracted from yeast cultures were analyzed by gas chromatography (GC) for methyl ester derivatives. Lipids were extracted and transmethylated, and fatty acid methyl esters (FAME) were analyzed as indicated by Sayanova et al.

FIG. 11 uses control (empty) plasmid pYES2 (FIG. 11A) or p
FIG. 11 shows a chromatogram of fatty acid methyl esters from yeast transformed using the ORF in YES2, F56H11.4 (FIG. 11B). GLA
The exogenous substrate in the form was applied to the culture. Two new peaks (B)
These peaks (annotated as 20: 3 and 18: 1 *) were identified (relative to known standards) as DHGLA and vaccenic acid, respectively. Detection was by flame ionization.

One cDNA ORF tested in this manner showed a high level of elongase activity on the GLA substrate, converting 44% to DHGLA. The identity of the extension product was determined by comparison to a known standard (the standard used was a known standard for any of DHGLA, AA, EPA, or VA from Sigma Chemicals) by Kratos ( Kratos) GCMS analysis using MS80 RFA (Napier, JA, supra; Michaelson, LV, supra; Michaelson)
, LV, supra) as DHGLA. The deduced amino acid sequence for a functional elongase clone was determined by C.I. Elegance gene F5
Identified as encoded by 6H11.4, and comparison with the yeast ELO gene showed low homology limited to a few short amino acid motifs (see FIG. 10). Mouse gene Ci has been implicated in brown adipose tissue recruitment in the liver
Some similarity to g30 (Tvrdik, P., (1977) J. Biol. Chem. 272, 31738-31746) indicates a potential human homology encoded by the gene located on chromosome 4q25 BAC 207d4. Observed as well as The most closely related sea. Elegance ORF, F41H10.8 (U61954) and F56H
11.3 (Z68749) has also been shown as part of a related human gene present on chromosome IV (present on BAC clone B207d4; AC00405).
0). GenBank accession numbers are given for all sequences.

C. Fatty acids synthesized by elegance require many different elongation reactions (Tanaka, T., (1996) Lipids 31, 1173-1178). Therefore, F56H1
The substrate specificity of the 1.4 PUFA elongase was measured using certain exogenously provided fatty acids. This revealed that GLA was the major substrate and some other fatty acids were extended with low efficiency (see Table I). Most of these substrates are polyunsaturated fatty acids, but surprisingly, palmitoleoic acid (pa
rmitoleoic acid) (PA; 16: 1Δ ⁹ ) was also extended by F56H11.4 to yield vaccenic acid (VA; 18: 1Δ ¹¹ ). Although the biosynthetic pathway of VA is not known, this data indicates that it may be synthesized by elongation of Δ ⁹ -1 unsaturated 16 carbon fatty acids.

C. Elegance PUFA elongase ORF56H11.4 contains chromosome IV
At the top (at 4.32 cM), a related sequence (F56) located 1,824 bp downstream
H11.3; 51% similarity). Another sea. The elegance gene (F41H10.8) was also observed, which is present on chromosome IV and has slightly higher levels of PUFA elongase than F56H11.3 (F56H11.3).
53%) (see FIG. 10). However, ORF F41H10
. When the PCR product encoding 8 was expressed in yeast in a manner equivalent to that used for F56H11.4, the former could contribute to the elongation of any fatty acid, despite the widespread supply of substrate. None (see Table II).

To reconstruct the PUFA biosynthetic pathway in a heterologous system, the PUFA elongase F56H11.4 was isolated and characterized by a Δ ⁵ previously isolated by the present inventors.
-Or Δ ⁶ -fatty acid desaturase (Napier, JA, supra; Michaelson, LV
, Supra) in yeast. Δ ⁶ -fatty acid desaturase and F56
Expression of the H11.4 has been carried out in the presence of two different substrates (LA or ALA), whereas delta ⁵ - the fatty acid desaturases and the elongase was expressed in the presence of GLA only. This demonstrated that desaturase and elongase could be combined in yeast to produce significant amounts of the final "product" (see Table III). In the case of elongase and Δ ⁶ -fatty acid desaturase,
The reaction demonstrated a high efficiency of 4.5% DHGLA production from the LA substrate.
This was due to the 25% desaturation of the LA substrate to GLA,
It was then extended to DHGLA with a similar level of efficiency (18%). This is lower than the% conversion observed for GLA when given exogenously (Table I).
), Indicating that in vivo production of the substrate for extension may be rate-limiting.

If ALA was used as the substrate, 27% of this would first be Δ ⁶ −
Desaturated has been octadecatetraenoic acid (OTA; 18: 4Δ ^8,9,12,15) produced a, only the 8% is subsequently extended eicosatetraenoic acid (20: 4Δ ^{8, 11,14,17} ). Thus, the final conversion efficiency of ALA to the 20 carbon tetraene PUFA was only about 2.2%. Since DHGLA is an n-6 fatty acid, whereas eicostetraenoic acid from OTA is of the n-3 type, this suggests that elongases may have both types of essential fatty acids, albeit with different efficiencies. Has proved acceptable. Also, since the ⁶ -desaturase did not show any activity on the 20: 2 or 20: 3 substrates, the 20-carbon PUFA synthesized in the yeast expression system was generated by the ⁶ -desaturation of the 18-carbon substrate, It was also proved that it had been extended thereafter (see Table III).

[0072] delta ⁵ - combination of desaturases and elongases also, these two enzymes have proven to be able to operate in tandem, the efficiency of the overall conversion is low (from GLA 3.3% of AA), which previously observed a delta ⁵ - desaturase enzyme itself low activity (Michaelson, LV, supra; was due Michaelson, LV, above). Thus, although almost 45% of the GLA substrate was extended to DHGLA, only 7.5% of this was subsequently desaturated to AA (see Table III).

Finally, AA or eicosapentaenoic acid (EPA; 20: 5Δ) in yeast ^5,8,11,14,17 ) Produced from a diene or triene 18 carbon substrate by three enzymes
Element (two desaturases and F56H11.4 PUFA elongase)
Was achieved by co-expression. As shown in Table IV, 18 carbon diene fat
When acid LA was given to yeast, small but significant amounts of AA were produced.

(Analysis by GC-Mass Spectroscopy (MS)) Peak identification and confirmation use Kratos MS80RFA.
The analysis was performed by GC-MS using a known standard (Sigma). This
Of the 20-carbon PUFA was confirmed by GCMS and the conversion efficiency from LA was 0
. It was found to be 65%. When ALA was used as the substrate, (Δ ⁶ 12.5 of eicosatetraene n-3 fatty acid (unsaturated and elongated)
% Is Δ^Five-Unsaturated, overall 0.3% of ALA substrate is converted to EPA
(EPA identification was confirmed by GCMS).

(Expression of C. elegans elongase in plants) In order to express Elegance elongase in plants, the following protocol is an example of a process that can be used to make transgenic plants. C. The Elegance ORF sequence can be subcloned into a plant expression vector pJD330 containing a viral 35S promoter and a Nos terminator. The resulting cassette or promoter / coding sequence / terminator is then subcloned into the plant binary transformation vector pBin 19 and the resulting plasmid is introduced into Agrobacterium tumefaciens. This Agrobacterium line can be used to transform Arabidopsis by vacuum infiltration of the flower, and seeds are plated on a selective medium containing kanamycin. pBin 19 confers resistance to this antibiotic,
Only transformed plant material will grow. Thus, resistant lines are identified and self-pollinated to yield homozygous material. The leaves are made of sea. Elegance elongase can be analyzed for expression.

[0076] Fatty acid methyl esters were analyzed as previously described.

[0077]

[Table 3]

[0078]

[Table 4]

[0079]

[Table 5]

[0080]

[Table 6]

[0081]

[Table 7]

[0082]

[Table 8]

[0083]

[Table 9]

[0084]

[Table 10]

[0085]

[Table 11]

[0086]

[Table 12]

[0087]

[Table 13]

[0088]

[Table 14]

[0089]

[Table 15]

[0090]

[Table 16]

[0091]

[Table 17]

[0092]

[Table 18]

[0093]

[Table 19]

[0094]

[Table 20]

[0095]

[Table 21]

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a schematic diagram illustrating a generalized route for PUFA production.

FIG. 2 shows a hydrophobicity plot for each of PUFA elongase A.

FIG. 3 shows a hydrophobicity plot for each of PUFA elongase B.

FIG. 4 shows a hydrophobicity plot for each of PUFA elongase C.

FIG. 5 shows a hydrophobicity plot for each of PUFA Elongase D.

FIG. 6 shows a hydrophobicity plot for each of PUFA Elongase E.

FIG. 7 shows a hydrophobicity plot for each of PUFA elongase F.

FIG. 8 shows a hydrophobicity plot for each of PUFA elongase G.

FIG. 9 shows a hydrophobicity plot for each of PUFA Elongase H.

FIG. 1 shows an amino acid sequence alignment comparing ORC F. Elegance ORF F56H11.4 (Z68749) with related sequences.

FIG. 11 shows a chromatogram of fatty acid methyl esters from transformed yeast.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 38/00 C12N 1/19 4C084 A61P 43/00 105 9/00 4C206 C12N 1/19 C12P 7/64 9 / 00 C12N 15/00 ZNAA C12P 7/64 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU , MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K) E, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), E, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI , GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA , UG, US, UZ, VN, YU, ZA, ZWF term (reference) 4B018 MD10 MD11 MD12 MD13 ME04 4B024 AA01 AA05 BA07 CA03 DA02 DA03 DA12 HA17 4B050 CC01 CC03 CC08 DD11 KK06 LL01 LL02 LL05 4B064 AD12 BH07 BH07 CE08 CE10 DA01 DA10 4B065 AA72X AA90X AA90Y AA91X AB01 BB08 BD29 CA10 CA27 CA41 CA4 4 4C084 AA02 AA06 AA07 DC01 MA52 NA14 ZB212 ZC612 4C206 AA01 AA02 DA04 DA05 MA04 MA72 ZB21 ZC61

Claims (40)

[Claims] 1. A functional long chain polyunsaturated fatty acid (PUFA) as defined herein
An isolated polypeptide comprising an elongase. 2. The polypeptide according to claim 1, wherein the polypeptide is derived from a eukaryote. 3. at least a part of the amino acid sequence shown in SEQ ID NO: 15,
3. The polypeptide according to claim 1 or 2 having a mutant thereof. 4. A polypeptide having at least 60% homology to the polypeptide according to claim 3, and having a PUFA elongase function. 5. The polypeptide according to claim 4, which has at least 80% homology. 6. The polypeptide according to claim 5, which has at least 90% homology. 7. The polypeptide according to any of the preceding claims, characterized in that it comprises a sequence motif responsible for the retention of the endoplasmic reticulum (ER). 8. Palmitoleic acid (PA; 16: 1Δ ⁹ ) is converted to vaccenic acid (PA
VA; 18: 1 Δ ¹¹ ). 9. The polypeptide according to claim 1, wherein the polypeptide is derived from an animal. 10. The polypeptide according to claim 9, wherein said animal is an invertebrate. 11. The method according to claim 10, wherein the invertebrate is a helminth.
The polypeptide of any one of the preceding claims. 12. The helminth according to claim 12, wherein The polypeptide according to claim 11, which is C. elegans. 13. The polypeptide according to claim 9, wherein said animal is a vertebrate. 14. The method according to claim 13, wherein the vertebrate is a mammal.
The polypeptide of any one of the preceding claims. 15. The polypeptide according to claim 14, wherein said mammal is a human, rat, or mouse. 16. A DNA sequence encoding a polypeptide according to any of the preceding claims. 17. The DNA sequence according to claim 16, comprising the sequence shown in SEQ ID NO: 7, or a variant obtained by substitution, deletion, and / or addition of bases in the sequence. 18. An engineered organism engineered to express the polypeptide according to any one of claims 1 to 15. 19. The engineered organism according to claim 18, wherein said animal is a mammal. 20. The engineered organism according to claim 19, wherein the mammal is a rat, a mouse, or a monkey. 21. An engineered organism comprising a synthetic route for the polypeptide of any one of claims 1 to 15. 22. The path delta ⁵ - engineered organism of claim 21, characterized in that it comprises a fatty acid desaturase. 23. The path delta ⁶ - engineered organism of claim 21 or 22 further characterized in that comprises a fatty acid desaturase. 24. The animal of claim 2, wherein said animal is a lower eukaryote.
24. The engineered organism according to any one of 1 to 23. 25. The engineered organism of claim 24, wherein said lower eukaryote is yeast. 26. The manipulated organism according to claim 18, wherein the animal is a fish. 27. A transgenic plant engineered to express the polypeptide according to any one of claims 1 to 15. 28. A transgenic plant comprising the DNA sequence according to claim 16 or 17. 29. A method for producing a PUFA, comprising performing an elongase reaction catalyzed by the polypeptide according to any one of claims 1 to 15. 30. The PUFA is dihomo -γ- linolenic acid ^{(20: 3Δ 8,11, 14)} , arachidonic acid (20: 4Δ ^5,8,11,14), eicosapentaenoic acid (20: 5
Δ ^{5,8,11,14,17),} docosatriene acid (22: 3Δ ^3,16,19), docosatetraenoic acid (22: 4Δ ^7,10,13,16), docosapentaenoic acid (22: 5Δ ^{7,10,13,16,1 9),} or docosahexaenoic acid (22: 6Δ ^{4,7,10,13,16,19)} the method of claim 29, wherein the a. 31. The method of claim 29, wherein said PUFA is a 24-carbon fatty acid having at least four double bonds. 32. PUFA produced by the method according to any one of claims 29 to 31. 33. A foodstuff comprising the PUFA of claim 32. 34. A supplement comprising the PUFA of claim 32. 35. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 15. 36. A pharmaceutical composition comprising the PUFA according to claim 32. 37. The pharmaceutical composition according to claim 35, comprising a pharmaceutically acceptable diluent, carrier, excipient, or bulking agent. 38. A method for increasing PUFA levels in an animal or plant, comprising:
A polypeptide according to any of claims 1 to 15, a DNA sequence according to claim 16 or 17, a food product according to claim 33, a food supplement according to claim 34, claim 3
A method for providing the animal or plant with the pharmaceutical composition according to any one of claims 5 to 37, or the PUFA according to claim 32. 39. The method of claim 38, wherein said animal is a mammal. 40. The method of claim 39, wherein said mammal is a human.