JP2002508932A - Desaturase - Google Patents

Abstract

  (57) [Summary] The present invention relates to a cDNA sequence encoding a Δ5-fatty acid desaturase comprising the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a DNA encoding a Δ5-fatty acid desaturase (desaturase)
Sequence, encoded Δ5-fatty acid desaturase and applications of Δ5-fatty acid desaturase.

[0002] Polyunsaturated fatty acids are important as dietary supplements because of their particular health-promoting activities and are of biomedical importance with regard to their potential pharmaceutical application in the treatment of certain disease states. is there.

[0003] Polyunsaturated fatty acids are precursors to two major classes of metabolites: prostanoids (including prostaglandins and thromboxanes) and leukotrienes. The Δ5-fatty acid desaturase is capable of converting arachidonic acid (A) of dihomo-γ-linolenic acid (DHL) by introducing a double bond into the Δ5 carbon of each substrate.
A) and the conversion of eicosatetraenoic acid (ETA) to eicosapentaenoic acid (
It catalyzes the conversion to EPA) and exists in its natural state as an endoplasmic reticulum membrane-associated protein.

Arachidonic acid has a chain of 20 carbon atoms having four double bonds and is a precursor for the synthesis of prostaglandins (fatty acids having 20 carbon atoms including one five-membered ring). As such, it is very important in human metabolism. Prostaglandins are modulators of hormonal action, and their potential effects include stimulating inflammation, controlling blood flow to certain organs, regulating ion transport across several membranes, and Includes the regulation of synaptic transmission. Prostaglandins are also potentially useful as contraceptives due to their ability to suppress progesterone secretion. Therefore, the ability to modulate prostaglandin synthesis by modulating the expression levels of polyunsaturated fatty acid precursor synthesis is of great medical and commercial importance.

As the importance of polyunsaturated fatty acids in the food and pharmaceutical industries increases,
The demands over current production levels and the demand for high quality sources are increasing and low cost polyunsaturated fatty acids are needed.

[0006] Current commercial sources of polyunsaturated fatty acids include selected seed plants, fish and mammals, and traditional processing techniques for extracting polyunsaturated fatty acids from these sources. Includes solvent extraction, antifreeze treatment, urea addition formation and distillation. However, these sources have disadvantages such as variations in production levels and quality due to seasonal and climatic changes, lack of available plant and fish sources, and the high cost of low-grade oil refining. There is a point. Due to high costs and insufficient production levels, developments to increase the marketability of polyunsaturated fatty acids are lagging behind.

Many efforts have been made to develop alternative sources of polyunsaturated fatty acids,
Work is underway to characterize the constituent genes and their biosynthetic coding proteins. For example, techniques for biosynthesis of polyunsaturated fatty acids in oilseeds include, for example, γ-linolenate (GLA), dihomo-γ-linolenate (DHGLA),
It has many advantages in mass production of arachidonic acid (AA), eicosapentaenoate (EPA) and docosahexaenoate (DHA). The utility of this technique has been demonstrated by the production of GLA and octadecatetraenoic acid (18: 4) by expression of the tobacco Borage Δ6 desaturase gene (Soyanova et al., 1997, PNAS, 94, 9411-9414). As many biosynthetic genes for polyunsaturated fatty acid synthesis are becoming available, at least GLA, AA, E
Allows for control of PA and DHA production and the type of lipid collected. Benefits obtained from such crops include the inexpensive and continuous supply of the desired polyunsaturated fatty acids for mass production, unsaturated fatty acids characterized by meeting specific nutritional requirements, and fine chemicals ( In the (pharmaceutical) industry, it involves the production of fatty acids at the prescription level and where no unsaturation sites have ever been.

[0008] Further approaches to the production of polyunsaturated fatty acids are the lower organisms (eg, algae, bacteria, fungi) that can synthesize a full range of polyunsaturated fatty acids and grow on an industrial scale. Algae (including algae)). Genetic transformation of these organisms allows the development of overproducing strains by biosynthetic pathway engineering and manipulation of polyunsaturated features.

Fungal Δ5 and Δ6 fatty acid desaturases have been cloned and their sequences are WO98 / 46763, WO98 / 46764 and WO98 / 4676.
5 is disclosed.

[0010] Polyunsaturated fatty acid metabolism is of paramount importance in human metabolism. These acids, via eicosanoids, are fundamental to proper homeostasis and are associated with severe physiological and pathophysiological syndromes.

Surprisingly, we have found that the zygomycete soil fungus Mortierella alpina, which encodes a functional Δ5 fatty acid desaturase,
alpina) was isolated and characterized.

[0012] In addition, surprisingly, we have found that the nematode Cenoorhabdi encodes a functional Δ5-fatty acid desaturase.
tis elegans) was isolated and characterized. Functional Δ5-
This DNA sequence encoding the fatty acid desaturase appears to be more closely related to the human Δ5-fatty acid desaturase than to the gene sequence of any previously isolated Δ5-fatty acid desaturase.

[0013] As well as being potentially useful in humans from the polypeptides encoded by the DNA sequences of the present invention, the DNA sequences of the present invention allow for the cloning of human gene equivalents, thereby Promotes the overproduction of DNA sequences and enables their biochemical use in the treatment of certain human diseases.

[0014] Plant and fungal desaturases are polypeptides that are primarily integral with the membrane and are difficult to purify and identify by conventional methods. Therefore, molecular biology techniques, including the use of mutants and transformed plants, have been applied to study lipid metabolism in detail.

A first feature of the present invention is to provide a Δ5-fatty acid desaturase from an isolated animal and a functional part thereof.

A second feature of the present invention is that the isolated Senorabditis elegans Δ5-
It is to provide a fatty acid desaturase.

A third feature of the present invention is that the sequence shown in SEQ ID NO: 2 encoding a Δ5-fatty acid desaturase capable of functioning due to the degeneracy of the genetic code, an equivalent of the sequence, or an equivalent of a part of the sequence It is an object of the present invention to provide a DNA sequence according to the first or second aspect of the present invention, which comprises at least a part of: Preferably, the DNA sequence is a DNA sequence from Senolabditis elegans.

Preferably, the gene encoding the Δ5-fatty acid desaturase encoded by the cloned gene is 1341 bases long. This protein is 447 amino acids long and has an estimated molecular weight of 57 kDa.

Alternatively, the DNA sequence encodes a functional Δ5-fatty acid desaturase, and encodes a Δ5-fatty acid desaturase capable of functioning due to the degeneracy of the genetic code. At least some of the equivalents of some of the sequences are included. Preferably, the DNA sequence is a DNA sequence from Mortierella alpina.

Preferably, the gene encoding the Δ5-fatty acid desaturase encoded by the cloned gene is 1338 bases long. This protein is 446 amino acids long and has an estimated molecular weight of 57 kDa.

[0021] Preferably, the DNA sequence according to the third aspect of the invention functions in mammals.

Preferably, the DNA sequence is expressed in a mammal.

[0023] Preferably, the DNA sequence is expressed in humans.

Preferably, the DNA sequence is obtained by modifying a naturally occurring functional gene encoding Δ5-fatty acid desaturase.

[0025] Preferably, the modifications include modifications by chemical, physical or biological means that do not remove the catalytic activity of the encoded enzyme.

Preferably, this modification improves the catalytic activity of the encoded enzyme.

[0027] Preferably, the biological modification includes recombinant DNA techniques and forced mutagenesis.

[0028] Preferably, the forced mutation method is DNA shuffling.

A fourth aspect of the present invention is to provide a polypeptide encoded by the DNA sequence according to the third aspect of the present invention.

Preferably, at least a portion of the polypeptide has the sequence set forth in SEQ ID NO: 3 or a functional equivalent of the sequence or a functional equivalent of a portion of the sequence. Alternatively, at least a portion of the polypeptide has the sequence set forth in SEQ ID NO: 4, or a functional equivalent of the sequence, or a functional equivalent of a portion of the sequence.

[0031] Preferably, the polypeptide catalyzes the conversion of dihomo-γ-linolenic acid to arachidonic acid.

Preferably, the polypeptide has been modified without removing the catalytic activity of the encoded polypeptide.

Preferably, the polypeptide is modified at a particular position in the molecular structure of the substrate in such a way as to induce a specific level of saturation with the substrate.

[0034] A fifth aspect of the present invention is the DN of a part of the DNA sequence according to the third aspect of the present invention.
The object is to provide a vector containing the A sequence.

According to a sixth aspect of the present invention, there is provided a method according to the first or second aspect of the present invention, comprising:
-It is an object of the present invention to provide a method for producing a polyunsaturated fatty acid, comprising a step of contacting a fatty acid desaturase or a polypeptide according to the fourth aspect of the present invention.

[0036] A seventh aspect of the present invention is directed to a Δ5-fatty acid desaturase according to the first or second aspect of the present invention, or a conversion catalyzed by a modified polypeptide according to the fourth aspect of the present invention. And to convert dihomo-γ-linolenic acid into arachidonic acid.

An eighth aspect of the present invention is to provide an organism that has been engineered to produce high levels of a polypeptide according to the fourth aspect of the present invention.

A ninth aspect of the present invention is a reaction catalyzed by a Δ5-fatty acid desaturase according to the first or second aspect of the present invention, or a polypeptide according to the fourth aspect of the present invention. The goal is to provide organisms that have been engineered to produce products at high levels.

Preferably, the organism is manipulated by the method according to the sixth or seventh aspect of the invention.

Preferably, the organism is a microorganism.

Preferably, the microorganism is selected from algae, bacteria and fungi.

[0042] Preferably, the fungi include algal fungi. Alternatively, the microorganism is a yeast.

Alternatively, the organism is a plant. Preferably, the plant is selected from oilseed plants.

[0044] Preferably, the oilseed plants are rape, sunflower, cereals including corn, tobacco, legumes including peanuts and soybeans, safflower, oil palm, coconut and other palms, cotton, sesame, mustard, flaxseed, Selected from castor bean, borage, and primrose.

A tenth aspect of the present invention is to provide an organism-derived seed or other reproductive substance according to the ninth aspect of the present invention.

[0046] Preferably, the organism is a mammal.

An eleventh aspect of the present invention provides an isolated multienzyme biosynthetic pathway, wherein the biosynthetic pathway comprises the Δ5-fatty acid desaturase according to the first or second aspect of the present invention. It is.

The twelfth feature of the present invention is that the conversion is Δ5 according to the first or second feature of the present invention.
-To provide compounds produced by the conversion of a substrate, catalyzed by fatty acid desaturases.

A thirteenth aspect of the present invention is to provide an intermediate compound produced by a catalytic reaction with a Δ5-fatty acid desaturase according to the first or second aspect of the present invention.

A fourteenth aspect of the present invention is to provide a food or supplement comprising a polyunsaturated fatty acid produced by the method according to the sixth aspect of the present invention.

A fifteenth aspect of the present invention is to provide a pharmaceutical preparation comprising a polyunsaturated fatty acid produced by the method according to the sixth aspect of the present invention.

A sixteenth aspect of the present invention is to provide a prostaglandin synthesized by a biosynthetic pathway including the catalytic activity of a Δ5-fatty acid desaturase according to the first or second aspect of the present invention. .

A seventeenth aspect of the present invention is to provide a method for adjusting prostaglandin synthesis by adjusting the expression level of a DNA sequence according to the third aspect of the present invention.

An eighteenth aspect of the present invention is to provide a probe comprising all or part of the DNA sequence according to the third aspect of the present invention or an equivalent RNA sequence.

A nineteenth aspect of the present invention is to provide a probe comprising all or a part of the Δ5-fatty acid desaturase polypeptide according to the fourth aspect of the present invention.

A twentieth aspect of the present invention is to provide a method for isolating a Δ5-fatty acid desaturase using the probe according to the nineteenth aspect of the present invention.

The gene of the present invention is capable of transformation into human cells, and is applied to appropriate levels in vivo by gene therapy techniques to convert fatty acids into polyunsaturated fatty acids in a patient. It is possible to continuously supply the enzyme to be converted. this is,
For example, an effective prophylactic treatment for patients with high cholesterol levels or for patients with other medical disorders for which the administration of polyunsaturated fatty acids would be beneficial.

Further, all or part of the DNA sequence of the present invention or all or part of the polypeptide sequence of the present invention could be used as a search probe for research or diagnostic purposes.

The present invention will now be described, by way of example only, with reference to SEQ ID NO: 1 to SEQ ID NO: 4 and FIGS. 1 to 4 set forth below.

SEQ ID NO: 1 is a cDNA sequence encoding a Δ5-fatty acid desaturase from Mortierella alpina, SEQ ID NO: 2 is a cDNA sequence encoding a Δ5-fatty acid desaturase from Senorabuditis elegans, No. 3 is a peptide sequence obtained by translating the gene sequence of SEQ ID NO: 1, and SEQ ID NO: 4 is a peptide sequence obtained by translating the gene sequence of SEQ ID NO: 2.

Cloning and Sequencing of a Δ5-Fatty Acid Desaturase Gene from Mortierella alpina The DNA sequence of the present invention encodes a Δ5-fatty acid desaturase and combines a cDNA library template with a specifically designed primer.
Cloned using the technique. The function of the DNA sequence, namely the conversion of dihomo-γ-linolenic acid (DHL) to arachidonic acid (AA) and the conversion of eicosatetraenoic acid (ETA) to eicosapentaenoic acid (EPA), is determined by the corresponding c in yeast.
Proven by expressing DNA.

The Δ5-fatty acid desaturase gene from Mortierella alpina was
The first of the plant Δ12, Δ15 desaturases previously identified by nklin
And a third histidine base (Shanklin, J. Whittle, EJ & Fox, BG. Bioche
mistry. 33, 12787-12794, 1994), and a polymerase chain reaction using a cDNA derived from Mortierella alpina as a template and a specifically designed denatured oligonucleotide primer (DP) shown below. PCR) technique.

The modified oligonucleotide primers (DP) are as follows: 5′-GC GAATTA (A / T) TIGGICA (T / C) GA (T / C) T
G (T / C) GICA-3 '5'-GC GAATTC ATTTT (G / T) IGG (A / G) AAIA (G / A
) (A / G) TG (A / G) TG-3 ′ (In the sequence, I indicates inosine and the EcoRI site is underlined.)

The PCR amplification was thermocycled using a program that performed 32 cycles of 94 ° C. for 2 minutes, 94 ° C. for 45 seconds, 55 ° C. for 1 minute, 72 ° C. for 1 minute, followed by amplification at 72 ° C. for another 10 minutes. The procedure was carried out exactly as in the conventional method. PCR amplification products were separated on a 1% agarose gel.

[0065] The PCR products amplified from cDNA template of the Mortierella alpina is gel purified, cloned into pGEM-T (Promega ^RTM), was transformed into DH5α is E. coli expression host, the 660bp Product.

Primers (P) were designed against the 660 bp product sequence and fragment amplification was performed by PCR using the cloned 660 bp product fragment as a template. The sequence-specific primer (P) is 660 bp
Based on the product sequence.

ΔBfor is 5′-GATGCGTCTCACTTTTCA-3 ′, and ΔBrev is 5′-GTGGTGCACAGCCCTGGGTAGTT.

The PCR amplification product was purified on a gel and used as a probe
The Alpina cDNA library was screened. The fragment probe hybridized to 25 of the 3.5 × 10 ⁵ phage clones screened, and one clone was shown by restriction enzyme analysis to have the expected size of 1.5 kb. This clone, designated L11, was selected for further analysis.

According to the sequence analysis of L11, 1 encoding a polypeptide of 446 amino acids
388 bp chain length open reading frame was revealed. GCG8
Analysis of protein and genomic databases using a program (Devereux J. et al., Nucleic Acids. Res., 12, 387-395, 1984) showed that L11 was a Δ6 desaturase from PCC6803 from Synechocystis sp. It showed a low level of identity of 20% with the gene (FIG. 1).

In FIG. 1, the sequences listed have the following accession numbers: S54259 Δ12 Spirulina Accession number: X86736 S54809 Δ6 Spirulina Accession number: X87094 S68358 Putative sphingolipid desaturase Accession number: X87143 S35157 Δ6 Synechocystis accession No .: L11421 PBOR6 Δ6 borage Access number: U79010 FU2 Δ5 desaturase Access number: AF054824

Furthermore, despite all three histidine boxes specific to the desaturase enzyme being present in the translated sequence, the third histidine box located at position 1159 bp in the sequence contains mutant QXXHH. Translated sequence also includes a heme-binding domain of cytochrome b ₅ like the N-terminus containing EHPGG motif, this feature has been observed only at the C-terminus of the desaturase other fungi previously.

Southern Blotting of Genomic DNA Sequence specific primers (P) designed for the L11 sequence between histidine boxes 1 and 3 of L11 were used in a PCR reaction to
The p region was amplified. The 660 bp PCR product was gel purified and Southern blots of restriction fragments of genomic DNA of Mortierella alpina and Mucor circinelloides were performed using the 660 bp fragment as a probe. The results suggest that the gene encoding the Δ5-fatty acid desaturase of the present invention is present only in the copy of Mortierella alpina and does not appear to be present in Muco sarcineroides. Furthermore, Mucor sarcineroides has no detectable Δ5-fatty acid desaturase activity.

Expression of Cloned Mortierella alpina Gene Encoding Δ5-Fatty Acid Desaturase To confirm that the L11 sequence encoded the Δ5-fatty acid desaturase enzyme, the cDNA was cloned from the GAL4 polymerase promoter producing plasmid pYES2 / L11. Under control, it was subcloned into the yeast expression vector pYES2, commercially available from Invitrogen ^™ . Expression of L11 was determined by Prome
using the transcription and translation system of ga ^TM pYES2 / L11 in vitro (in v
It was checked by in vitro) transcription-translation. ³⁵ S methionine labeled translation products were generated, run on SDS-PAGE, and visualized by exposure to autoradiographic film. The evaluated molecular weight of the product was 55-60 kD, and the control plasmid pYES2 without insert did not produce any labeled translation products.

The construction pYES2 / L11 was replaced with yeast (Saccharomyces cerevisiae: Saccha).
Romyces cerevisiae) and grown in uracil-deficient YCA medium. Transformants were selected by the presence of the URA3 selection marker carried by pYES2 / L11, and L11 expression was induced by the addition of a final concentration of 1% mM galactose. The medium was grown overnight in the presence of 0.5 mM dihomo-γ-linoleate, detergent (1% terditol NP-40) and 2% raffinose. Aliquots were collected at t = 0, t = 4 hours and t = 16 hours.

The total fatty acids of the yeast were analyzed by methyl ester GC. Methyl groups were transferred from lipids from induced and uninduced control samples with 1M HCL in methanol at 80 ° C. for 1 hour. Fatty acid methyl ester (F
AMES) was extracted in hexane. FAMES GC analysis was 25M x 0.
Hewlett Packard 58804 Series Ga equipped with a 32 mm RSL-500BP coupled capillary column and a flame photoionization detector
s Chromatograph was used.

The methyl ester of total fatty acids isolated from yeast harboring plasmid pYes2 / L11 and grown in the presence of galactose and dihomo-γ-linoleic acid was G
Analysis at C revealed additional peaks (see FIG. 3). This particular peak has the same retention time as authentic arachidonic acid standard (Sigma),
This indicates that the transgenic yeast can desaturate dihomo-γ-linolenic acid at the Δ5 position. Such a peak is expressed in the control sample (p
Yes2) (transformation using Yes2) (Fig. 3). The additional peak identity was due to the GC that this compound was previously identified as arachidonic acid.
Confirmed by MS (Kratos MS80RFA, scanning range 500-40 daltons, operated at ionization voltage of 70 eV).

This indicates that the DNA sequence from Mortierella alpina encodes a functional polypeptide involved in arachidonic acid synthesis in the presence of galactose and dihomo-γ-linolenate.

Cloning and Sequencing of the Δ5-Fatty Acid Desaturase Gene of Senolabditis elegans Previously, we have determined that Δ5- and Δ6 of fungi from plant and animal species different from the previously identified microsomal desaturase. -A fatty acid desaturase was identified. This difference is due to the presence of N-terminal extension which shows homology to the electron donor protein cytochrome b _5.

During the characterization of the fungus (Mortierella alpina) Δ5-fatty acid desaturase and the Cenolabditis elegans Δ6-fatty acid desaturase (present in cosmid W08D2 (accession number Z70271)), we characterized the fatty acid desaturase. Senoravditis Elegance DN that seems to code
A related sequence of cosmid T13F2.1 (accession number Z81122), which also contains the A sequence, was identified.

Sequence analysis (Genefinder program (Wilson, R. et al., 1994, Nature, 36
8, 32-38)) revealed that cosmids W08D2 and T13F2 contain overlapping regions. Further definition, the cosmid T13F2 have been found to contain an open reading frame (ORF) (indicated as T13F2.1), which is the N-terminal cytochrome b ₅ domain (diagnostic His-Pro-Gly-G ly motif As well as three "histidine boxes" specific to all microsomal desaturases. In addition, this putative desaturase contained a mutated third histidine box in which the first histidine in the His-XX-His-His motif was replaced by H to Q. This substitution for glutamine has been shown in plant and animal Δ6-fatty acid desaturases and in fungal Δ5-fatty acid desaturases from Mortierella alpina.

The overlap between cosmids T13F2 and W08D2 is due to the putative desaturase ORF.
(T13F2.1) and the proximity of the Δ6-fatty acid desaturase allow identification of two sequences separated by 990 bases from the predicted stop codon of T13F2.1 to the methionine start triplet of the Δ6-fatty acid desaturase. It was revealed that they were arranged in a single line on cytochrome IV.

Heterofunctional expression of genomic DNA was not feasible, as sequence analysis predicts that the T13F2.1 ORF is dispersed in a large number of introns. Therefore, the polymerase chain reaction (PCR) using the following primers CEFOR and CEREV was used to amplify a cDNA clone corresponding to the putative long exon at the 5 'end of the T13F2.1 ORF: CEFOR: 5 '-ATGGTATCTAGGAGCAAGA-3' CEREV: 5'-TCTGGGATCTCTGGTTCTTG-3 '

After initial denaturation at 94 ° C. for 2 minutes, amplification was performed as follows: 32 cycles of 94 ° C. for 45 seconds, 55 ° C. for 1 minute, 72 ° C. for 1 minute, and finally at 72 ° C. 1
Amplification was performed for 0 minutes.

The DNA fragment of the correct expected size was amplified (visualized on a 1% agarose gel), the gel band was excised, the DNA was purified and pGEM-T (Prom
ega) and the resulting plasmid was cloned into E. coli. coli DH5α cells. For sequencing, the plasmid DNA was converted to Qiagen.
The insert was purified using the QIAprep miniprep kit and the nucleotide sequence of the insert was identified by automated sequencing using an ABI-377 DNA sequencer.

The isolated 233 bp PCR amplified fragment was used to isolate the complete coding region corresponding to ORF T13F2.1 using Prof Yuji Kohara-M
A cDNA library of Cenolabditis elegans in a mixed state composed in λZapII provided by ishima, Japan was screened. The cloned PCR products were screened using standard techniques using probes (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual). Ready to Go DNL-Labelling reaction mix (P
DNA fragments were labeled with α [ ³² P] dCTP using H. mariacia. Five plaques out of 1.4 × 10 ⁵ pfu screened for hybridization to the 233 bp fragment showed a positive signal and a cylindrical sample was drawn from the agar plate and eluted in SM buffer. The resulting phage suspension was screened for the presence of T13F2.1 by PCR amplification using CEFOR and CEREV. One clone (designated L4) was purified by hybridization screening at 65 ° C. with the 233 bp fragment isolated by two additional platings and PCR. Plasmid L4 was released from lambda clone L4 by excision and cDNA on both strands
Inserts were sequenced using a Perkin Elmer ABI-377 DNA sequencer.

The obtained DNA sequence is shown in SEQ ID NO: 2, and the predicted amino acid sequence is shown in SEQ ID NO: 4.

Functional Analysis of L4 in Yeast The complete coding sequence of L4 (447 amino acid code) was amplified by PCR using primers YCEDFor and TCEDRev shown below (HindIII and BamHI flanking sites were also introduced on the side). ): YCEDFor: 5′-GCG AAGCTT AAAATGGTATTACGAG
AGCAAGAGC-3 '(annealing to the starting methionine is shown in bold and the HindIII restriction site is underlined) YCEDrev: 5'-GCG GGATCC
TTAGTCAA-3 ′ (annealing of the stop codon to the complement is shown in bold and the BamHI restriction is underlined).

The amplified PCR product containing the complete coding region of L4 was prepared using HindIII and Bam
The yeast expression vector -pYES2 (Invitrogen) was ligated downstream of the GAL1 promoter using a III restriction site (enzyme from Boehringer Mannheim). The resulting configuration (denoted as pYES2 / L4)
E. FIG. E. coli and the fidelity of the insert generated by PCR in plasmid pYES2 / L4 was confirmed in vitro by transcription / translation using the TNT system (Promega). The resulting translation is labeled with ³⁵ S methionine,
Separated by SDS-PAGE and visualized by autoradiography.

The translation obtained from pYES2 / L4 has a molecular weight of about M _r 57,000, whereas the control vector pYES2 without insert does not produce a translation.

For functional analysis of the L4 coding region, the recombinant plasmid was transformed into Saccharomyces cerevisiae DBY746 by the lithium acetate method (Elble R., 19).
92, Bio Techniques, 13, 18-20). Cells were incubated with linoleic acid (18: 2Δ ^9,12 ) or di-homogenein in the presence of 1% terrigitol containing raffinose as a carbon source.
Cultured overnight in ^medium supplemented with either addition of γ-linolenic acid (C20: 3Δ8,11,14) (described in Napier et al., 1998, Biochem. J., 330, 611-614). These fatty acids are S.P. cerevisiae does not exist, but Δ6
Alternatively, it acts as a specific substrate for Δ5-desaturase. Vector GAL
Expression of the L4 coding region from one promoter was induced by the addition of up to 1% galactose. Culture growth was continued for 16 hours, after which an aliquot was removed for fatty acid analysis by GC. Total fatty acids extracted from yeast cultures were analyzed by gas chromatography of methyl esters (GC). 8 in methanol
The methyl group in the lipid was transferred using 1 M HCL at 0 ° C. for 1 hour, and then the fatty acid methyl ester (FAME) was extracted in hexane. FAMES GC analysis was performed on a Hewlett Packard 58 equipped with a 25M x 0.32 mm RSL-500BP coupled capillary column and a flame photoionization detector.
This was performed using an 80A Series Gas Chromatograph.
Fatty acids were identified by comparing to the retention time of a FAME standard (Sigma). The relative percentage of fatty acids was estimated from the peak area. Arachidonic acid was treated with Kra operating at a scanning range of 500-40 Daltons and an ionization voltage of 70 eV.
Identified by GC-MS using ts MS80RFA. FIG. 4 shows the results of GC analysis of fatty acid methyl esters of the transformed yeast strain. Induced pYE grown in the presence of di-homo-γ-linolenic acid compared to the empty vector control
It is clear that an additional peak was obtained in the trace from S2 / L4. This peak was also not from uninduced medium grown on di-homo-γ-linolenic acid, and pYES2 / L4 grown in the presence of linolenic acid did not accumulate new peaks, and It is important to note that this indicates that this fatty acid is not a substrate for the enzyme encoded by the Cenolabditis elegans cDNA. The retention times of the additional peaks correspond to those of the authentic methyl arachidonic acid standard. Fatty acids generated from di-homo-γ-linolenic acid were converted to GCMS (Gas Chromatography Mass Spect).
romance) and identified as arachidonic acid. Therefore,
This result indicates that the yeast cells transformed with the plasmid pYES2 / L4 had a functional Δ5
-Obtain desaturase activity, at which point arachidonic acid can be synthesized from the substrate di-homo-γ-linolenic acid. Δ5- in transformed yeast
Clearly, desaturase is an effective catalyst.

This indicates that the DNA sequence from Senolabditis elegans encodes a functional polypeptide involved in the synthesis of arachidonic acid in the presence of galactose and di-homo-γ-linolenate. ing.

[Table 1]

[Table 2]

[Table 3]

[Brief description of the drawings]

FIG. 1 is a list of various Δ6-desaturases and genes encoding Δ12-desaturase and Δ5-fatty acid desaturase of Mortierella alpina.

FIG. 2 is a continuation of FIG. 1;

FIG. 3 is a continuation of FIG. 1;

FIG. 2 is a list of genes encoding Δ6-desaturase derived from Senolabditis elegans, fungi Δ5-desaturase derived from Mortierella alpina, and Δ5-fatty acid desaturase.

FIG. 3 is a trace of gas chromatography of fatty acid methyl esters of an induced yeast cell transformant transformed with Mortierella alpina Δ5-fatty acid desaturase gene and a non-induced yeast cell transformant. .

FIG. 4 shows traces of gas chromatography of fatty acid methyl esters of an induced yeast cell transformant and a non-induced yeast cell transformant transformed with a Δ5-fatty acid desaturase gene of Senorabuditis elegans. It is.

[Procedure amendment]

[Submission date] June 21, 2001 (2001.6.21)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction contents]

[Title of the Invention] Desaturase

[Claims]

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a DNA encoding a Δ5-fatty acid desaturase (desaturase)
Sequence, encoded Δ5-fatty acid desaturase and applications of Δ5-fatty acid desaturase.

[0002] Polyunsaturated fatty acids are important as dietary supplements because of their particular health-promoting activities and are of biomedical importance with regard to their potential pharmaceutical application in the treatment of certain disease states. is there.

[0003] Polyunsaturated fatty acids are precursors to two major classes of metabolites: prostanoids (including prostaglandins and thromboxanes) and leukotrienes. Δ5-fatty acid desaturases are capable of converting arachidonic acid (A) of dihomo-γ-linolenic acid (DHL) by introducing a double bond into the Δ5 carbon of each substrate.
A) and the conversion of eicosatetraenoic acid (ETA) to eicosapentaenoic acid (
It catalyzes the conversion to EPA) and exists in its natural state as an endoplasmic reticulum membrane-associated protein.

Arachidonic acid has a chain of 20 carbon atoms having four double bonds and is a precursor for the synthesis of prostaglandins (20-carbon fatty acids containing one 5-membered ring). As such, it is very important in human metabolism. Prostaglandins are modulators of hormonal action, and their potential effects include stimulating inflammation, controlling blood flow to certain organs, regulating ion transport across several membranes, and Includes regulation of synaptic transmission. Prostaglandins are also potentially useful as contraceptives due to their ability to suppress progesterone secretion. Therefore, the ability to modulate prostaglandin synthesis by modulating the expression levels of polyunsaturated fatty acid precursor synthesis is of great medical and commercial importance.

As the importance of polyunsaturated fatty acids in the food and pharmaceutical industries increases,
The demands over current production levels and the demand for high quality sources are increasing and low cost polyunsaturated fatty acids are needed.

[0006] Current commercial sources of polyunsaturated fatty acids include selected seed plants, fish and mammals, and traditional processing techniques for extracting polyunsaturated fatty acids from these sources. Includes solvent extraction, antifreeze treatment, urea addition formation and distillation. However, these sources have disadvantages, such as variations in production levels and quality due to seasonal and climatic changes, lack of available plant and fish sources, and the high cost of low-grade oil refining. There is a point. Due to high costs and insufficient production levels, developments to increase the marketability of polyunsaturated fatty acids are lagging behind.

Many efforts have been made to develop alternative sources of polyunsaturated fatty acids,
Work is underway to characterize the constituent genes and their biosynthetic coding proteins. For example, techniques for biosynthesis of polyunsaturated fatty acids in oilseeds include, for example, γ-linolenate (GLA), dihomo-γ-linolenate (DHGLA),
It has many advantages in mass production of arachidonic acid (AA), eicosapentaenoate (EPA) and docosahexaenoate (DHA). The utility of this technique has been demonstrated by the production of GLA and octadecatetraenoic acid (18: 4) by expression of the tobacco Borage Δ6 desaturase gene (Soyanova et al., 1997, PNAS, 94, 9411-9414). As many biosynthetic genes for polyunsaturated fatty acid synthesis are becoming available, at least GLA, AA, E
Allows for control of PA and DHA production and the type of lipid collected. The benefits obtained from such crops include the inexpensive and sustained supply of the desired polyunsaturated fatty acids for mass production, unsaturated fatty acids characterized by meeting specific nutritional requirements, and fine chemicals ( In the (pharmaceutical) industry, it involves the production of fatty acids at the prescription level and where no unsaturation sites have ever been.

[0008] Further approaches to the production of polyunsaturated fatty acids are the lower organisms (eg, algae, bacteria, fungi, etc.) that can synthesize the full range of polyunsaturated fatty acids and grow on an industrial scale. Algae (including algae)). Genetic transformation of these organisms allows the development of overproducing strains by biosynthetic pathway engineering and manipulation of polyunsaturated features.

Fungal Δ5 and Δ6 fatty acid desaturases have been cloned and their sequences are WO98 / 46763, WO98 / 46764 and WO98 / 4676.
5 is disclosed.

[0010] Polyunsaturated fatty acid metabolism is of paramount importance in human metabolism. These acids, via eicosanoids, are fundamental to proper homeostasis and are associated with severe physiological and pathophysiological syndromes.

Surprisingly, we have found that the zygomycete soil fungus Mortierella alpina, which encodes a functional Δ5 fatty acid desaturase,
alpina) was isolated and characterized.

[0012] In addition, surprisingly, we have found that the nematode Cenoorhabdit encodes a functional Δ5-fatty acid desaturase.
DNA isolates were isolated and characterized. This DNA sequence encoding a functional Δ5-fatty acid desaturase is believed to be more closely related to human Δ5-fatty acid desaturase than any previously isolated gene sequence for Δ5-fatty acid desaturase.

[0013] As well as being potentially useful in humans from the polypeptides encoded by the DNA sequences of the present invention, the DNA sequences of the present invention allow the cloning of equivalents of the human gene, whereby human Promotes the overproduction of DNA sequences and enables their biochemical use in the treatment of certain human diseases.

[0014] Plant and fungal desaturases are polypeptides that are primarily integral with the membrane and are difficult to purify and identify by conventional methods. Therefore, molecular biology techniques, including the use of mutants and transformed plants, have been applied to study lipid metabolism in detail.

A first feature of the present invention is to provide a Δ5-fatty acid desaturase from an isolated animal and a functional part thereof.

A second aspect of the present invention is to provide an isolated Δ5-fatty acid desaturase of Cenolabditis elegans.

A third feature of the present invention is that the sequence shown in SEQ ID NO: 2, which encodes a Δ5-fatty acid desaturase capable of functioning due to the degeneracy of the genetic code, an equivalent of the sequence, or an equivalent of a part of the sequence It is an object of the present invention to provide a DNA sequence according to the first or second aspect of the present invention, which comprises at least a part of: Preferably, the DNA sequence is a DNA sequence from Senolabditis elegans.

Preferably, the gene encoding the Δ5-fatty acid desaturase encoded by the cloned gene is 1341 bases long. This protein is 447 amino acids long and has an estimated molecular weight of 57 kDa.

Alternatively, the DNA sequence encodes a functional Δ5-fatty acid desaturase, and encodes a Δ5-fatty acid desaturase capable of functioning due to the degeneracy of the genetic code. At least some of the equivalents of some of the sequences are included. Preferably, the DNA sequence is a DNA sequence from Mortierella alpina.

Preferably, the gene encoding the Δ5-fatty acid desaturase encoded by the cloned gene is 1338 bases long. This protein is 446 amino acids long and has an estimated molecular weight of 57 kDa.

[0021] Preferably, the DNA sequence according to the third aspect of the invention functions in mammals.

Preferably, the DNA sequence is expressed in a mammal.

[0023] Preferably, the DNA sequence is expressed in humans.

Preferably, the DNA sequence is obtained by modifying a naturally occurring functional gene encoding Δ5-fatty acid desaturase.

[0025] Preferably, the modifications include modifications by chemical, physical or biological means that do not remove the catalytic activity of the encoded enzyme.

Preferably, this modification improves the catalytic activity of the encoded enzyme.

[0027] Preferably, the biological modification includes recombinant DNA techniques and forced mutagenesis.

[0028] Preferably, the forced mutation method is DNA recombination.

A fourth aspect of the present invention is to provide a polypeptide encoded by the DNA sequence according to the third aspect of the present invention.

Preferably, at least a portion of the polypeptide has the sequence set forth in SEQ ID NO: 3 or a functional equivalent of the sequence or a functional equivalent of a portion of the sequence. Alternatively, at least a portion of the polypeptide has the sequence set forth in SEQ ID NO: 4, or a functional equivalent of the sequence, or a functional equivalent of a portion of the sequence.

[0031] Preferably, the polypeptide catalyzes the conversion of dihomo-γ-linolenic acid to arachidonic acid.

Preferably, the polypeptide has been modified without removing the catalytic activity of the encoded polypeptide.

Preferably, the polypeptide is modified at a particular position in the molecular structure of the substrate in such a way as to induce a specific level of saturation with the substrate.

[0034] A fifth aspect of the present invention is the DN of a part of the DNA sequence according to the third aspect of the present invention.
The object is to provide a vector containing the A sequence.

According to a sixth aspect of the present invention, there is provided a method according to the first or second aspect of the present invention, comprising:
-It is an object of the present invention to provide a method for producing a polyunsaturated fatty acid, comprising a step of contacting with a fatty acid desaturase or a polypeptide according to the fourth aspect of the present invention.

[0036] A seventh aspect of the present invention is directed to a Δ5-fatty acid desaturase according to the first or second aspect of the present invention, or a conversion catalyzed by a modified polypeptide according to the fourth aspect of the present invention. And to convert dihomo-γ-linolenic acid into arachidonic acid.

An eighth aspect of the present invention is to provide an organism that has been engineered to produce high levels of a polypeptide according to the fourth aspect of the present invention.

A ninth aspect of the present invention is a reaction catalyzed by a Δ5-fatty acid desaturase according to the first or second aspect of the present invention, or a polypeptide according to the fourth aspect of the present invention. The goal is to provide organisms that have been engineered to produce products at high levels.

Preferably, the organism is manipulated by the method according to the sixth or seventh aspect of the invention.

Preferably, the organism is a microorganism.

[0041] Preferably, the microorganism is selected from algae, bacteria and fungi.

[0042] Preferably, the fungi include algal fungi. Alternatively, the microorganism is a yeast.

Alternatively, the organism is a plant. Preferably, the plant is selected from oilseed plants.

Preferably, the oilseed plants are rape, sunflower, cereals including corn, tobacco, legumes including peanuts and soybeans, safflower, oil palm, coconut and other palms, cotton, sesame, mustard, linseed, Selected from castor bean, borage, and primrose.

A tenth aspect of the present invention is to provide an organism-derived seed or other reproductive substance according to the ninth aspect of the present invention.

[0046] Preferably, the organism is a mammal.

An eleventh aspect of the present invention provides an isolated multienzyme biosynthetic pathway, wherein the biosynthetic pathway comprises the Δ5-fatty acid desaturase according to the first or second aspect of the present invention. It is.

The twelfth feature of the present invention is that the conversion is Δ5 according to the first or second feature of the present invention.
-To provide compounds produced by the conversion of a substrate, catalyzed by fatty acid desaturases.

A thirteenth aspect of the present invention is to provide an intermediate compound produced by a catalytic reaction with a Δ5-fatty acid desaturase according to the first or second aspect of the present invention.

A fourteenth aspect of the present invention is to provide a food or supplement comprising a polyunsaturated fatty acid produced by the method according to the sixth aspect of the present invention.

A fifteenth aspect of the present invention is to provide a pharmaceutical preparation comprising a polyunsaturated fatty acid produced by the method according to the sixth aspect of the present invention.

A sixteenth aspect of the present invention is to provide a prostaglandin synthesized by a biosynthetic pathway including the catalytic activity of a Δ5-fatty acid desaturase according to the first or second aspect of the present invention. .

A seventeenth aspect of the present invention is to provide a method for adjusting prostaglandin synthesis by adjusting the expression level of a DNA sequence according to the third aspect of the present invention.

An eighteenth aspect of the present invention is to provide a probe comprising all or part of the DNA sequence according to the third aspect of the present invention or an equivalent RNA sequence.

A nineteenth aspect of the present invention is to provide a probe comprising all or a part of the Δ5-fatty acid desaturase polypeptide according to the fourth aspect of the present invention.

A twentieth aspect of the present invention is to provide a method for isolating a Δ5-fatty acid desaturase using the probe according to the nineteenth aspect of the present invention.

The gene of the present invention is capable of transformation into human cells, and is applied to appropriate levels in vivo by gene therapy techniques to convert fatty acids into polyunsaturated fatty acids in a patient. It is possible to continuously supply the enzyme to be converted. this is,
For example, an effective prophylactic treatment for patients with high cholesterol levels or for patients with other medical disorders for which the administration of polyunsaturated fatty acids would be beneficial.

Further, all or part of the DNA sequence of the present invention or all or part of the polypeptide sequence of the present invention could be used as a search probe for research or diagnostic purposes.

The present invention will now be described, by way of example only, with reference to SEQ ID NO: 1 to SEQ ID NO: 4 and FIGS. 1 to 4 set forth below.

SEQ ID NO: 1 is a cDNA sequence encoding a Δ5-fatty acid desaturase from Mortierella alpina, SEQ ID NO: 2 is a cDNA sequence encoding a Δ5-fatty acid desaturase from Cenolabditis elegans, 3 is a peptide sequence obtained by translating the gene sequence of SEQ ID NO: 1, and SEQ ID NO: 4 is a peptide sequence obtained by translating the gene sequence of SEQ ID NO: 2.

Cloning and Sequencing of a Δ5-Fatty Acid Desaturase Gene from Mortierella alpina The DNA sequence of the present invention encodes a Δ5-fatty acid desaturase and combines a cDNA library template with a specifically designed primer.
Cloned using the technique. The function of the DNA sequence, namely the conversion of dihomo-γ-linolenic acid (DHL) to arachidonic acid (AA) and the conversion of eicosatetraenoic acid (ETA) to eicosapentaenoic acid (EPA), is determined by the corresponding c in yeast.
Proven by expressing DNA.

The Δ5-fatty acid desaturase gene from Mortierella alpina was
The first of the plant Δ12, Δ15 desaturases previously identified by nklin
And a third histidine base (Shanklin, J. Whittle, EJ & Fox, BG. Bioche
mistry. 33, 12787-12794, 1994), and a polymerase chain reaction using a cDNA derived from Mortierella alpina as a template and a specifically designed denatured oligonucleotide primer (DP) shown below. PCR) technique.

The modified oligonucleotide primers (DP) are as follows: 5′-GC GAATTA (A / T) TIGGICA (T / C) GA (T / C) T
G (T / C) GICA-3 ' (SEQ ID NO: 11) 5'-GC GAATTC ATTTT (G / T) IGG (A / G) AAIA (G / A
) (A / G) TG (A / G) TG-3 ′ (SEQ ID NO: 12) (In the sequence, I indicates inosine and the EcoRI site is underlined.)

The PCR amplification was thermocycled using a program that performed 32 cycles of 94 ° C. for 2 minutes, 94 ° C. for 45 seconds, 55 ° C. for 1 minute, 72 ° C. for 1 minute, followed by amplification at 72 ° C. for another 10 minutes. The procedure was carried out exactly as in the conventional method. PCR amplification products were separated on a 1% agarose gel.

[0065] The PCR products amplified from cDNA template of the Mortierella alpina is gel purified, cloned into pGEM-T (Promega ^RTM), was transformed into DH5α is E. coli expression host, the 660bp Product.

Primers (P) were designed against the 660 bp product sequence and fragment amplification was performed by PCR using the cloned 660 bp product fragment as a template. The sequence-specific primer (P) is 660 bp
Based on the product sequence.

ΔBfor is 5′-GATGCGTCTCACTTTTCA-3 ′ (SEQ ID NO: 13)
ΔBrev is 5′-GTGGTGCACACCCTGGTAGTT (SEQ ID NO: 14) .

The PCR amplification product was purified on a gel and used as a probe
The Alpina cDNA library was screened. The fragment probe hybridized to 25 of the 3.5 × 10 ⁵ phage clones screened, and one clone was shown by restriction enzyme analysis to have the expected size of 1.5 kb. This clone, designated L11, was selected for further analysis.

According to the sequence analysis of L11, 1 encoding a polypeptide of 446 amino acids
388 bp chain length open reading frame was revealed. GCG8
Analysis of protein and genomic databases using a program (Devereux J. et al., Nucleic Acids. Res., 12, 387-395, 1984) showed that L11 was a Δ6 desaturase from PCC6803 from Synechocystis sp. It showed a low level of identity of 20% with the gene (FIG. 1).

In FIG. 1, the sequences listed have the following access numbers: S54259 Δ12 Spirulina Access numbers: X86736, S54809 Δ6 Spirulina Access numbers: X87094, S68358 Putative sphingolipid desaturase Access numbers: X87143, S35157 Access number: L11421, PBOR6 Δ6 borage access number: U79010, FU2 Δ5 desaturase Access number: AF054824.

Furthermore, despite all three histidine boxes specific to the desaturase enzyme being present in the translated sequence, the third histidine box located at position 1159 bp in the sequence contains mutant QXXHH. Translated sequence also includes a heme-binding domain of cytochrome b ₅ like the N-terminus containing EHPGG (SEQ ID NO: 15) motif, this feature is observed only at the C-terminus of the desaturase other fungi previously ing.

Southern Blotting of Genomic DNA Sequence specific primers (P) designed for the L11 sequence between histidine boxes 1 and 3 of L11 were used in a PCR reaction to
The p region was amplified. The 660 bp PCR product was gel purified and Southern blots of restriction fragments of genomic DNA of Mortierella alpina and Mucor circinelloides were performed using the 660 bp fragment as a probe. The results suggest that the gene encoding the Δ5-fatty acid desaturase of the present invention is present only in the copy of Mortierella alpina and does not appear to be present in Muco sarcineroides. Furthermore, Mucor sarcineroides has no detectable Δ5-fatty acid desaturase activity.

Expression of Cloned Mortierella alpina Gene Encoding Δ5-Fatty Acid Desaturase To confirm that the L11 sequence encoded the Δ5-fatty acid desaturase enzyme, the cDNA was cloned from the GAL4 polymerase promoter producing plasmid pYES2 / L11. Under control, it was subcloned into the yeast expression vector pYES2, commercially available from Invitrogen ^™ . Expression of L11 was determined by Prome
using the transcription and translation system of ga ^TM pYES2 / L11 in vitro (in v
It was checked by in vitro) transcription-translation. ³⁵ S methionine labeled translation products were generated, run on SDS-PAGE, and visualized by exposure to autoradiographic film. The evaluated molecular weight of the product was 55-60 kD, and the control plasmid pYES2 without insert did not produce any labeled translation products.

The construction pYES2 / L11 was replaced with yeast (Saccharomyces cerevisiae: Saccha).
Romyces cerevisiae) and grown in uracil-deficient YCA medium. Transformants were selected by the presence of the URA3 selection marker carried by pYES2 / L11, and L11 expression was induced by the addition of a final concentration of 1% mM galactose. The medium was grown overnight in the presence of 0.5 mM dihomo-γ-linoleate, detergent (1% terditol NP-40) and 2% raffinose. Aliquots were collected at t = 0, t = 4 hours and t = 16 hours.

The total fatty acids of the yeast were analyzed by methyl ester GC. Methyl groups were transferred from lipids from induced and uninduced control samples with 1M HCL in methanol at 80 ° C. for 1 hour. Fatty acid methyl ester (F
AMES) was extracted in hexane. FAMES GC analysis was 25M x 0.
Hewlett Packard 58804 Series Ga equipped with a 32 mm RSL-500BP coupled capillary column and a flame photoionization detector
s Chromatograph was used.

The methyl ester of total fatty acids isolated from yeast harboring plasmid pYes2 / L11 and grown in the presence of galactose and dihomo-γ-linoleic acid was G
Analysis at C revealed additional peaks (see FIG. 3). This particular peak has the same retention time as authentic arachidonic acid standard (Sigma),
This indicates that the transgenic yeast can desaturate dihomo-γ-linolenic acid at the Δ5 position. Such a peak is expressed in the control sample (p
Yes2) (transformation using Yes2) (Fig. 3). The additional peak identity was due to the GC that this compound was previously identified as arachidonic acid.
Confirmed by MS (Kratos MS80RFA, scanning range 500-40 daltons, operated at ionization voltage of 70 eV).

This indicates that the DNA sequence from Mortierella alpina encodes a functional polypeptide involved in arachidonic acid synthesis in the presence of galactose and dihomo-γ-linolenate.

Cloning and Sequencing of the Δ5-Fatty Acid Desaturase Gene of Senolabuditis elegans Previously, the inventors have determined Δ5- and Δ6- of fungi from plant and animal species different from the previously identified microsomal desaturase. A fatty acid desaturase was identified. This difference is due to the presence of N-terminal extension which shows homology to the electron donor protein cytochrome b _5.

During the characterization of the fungus (Mortierella alpina) Δ5-fatty acid desaturase and Cenolabditis elegans Δ6-fatty acid desaturase (present in cosmid W08D2 (accession number Z70271)), we encoded the fatty acid desaturase. A related sequence of cosmid T13F2.1 (accession number Z81122), which also contains the DNA sequence of Cenolabditis elegans, which is believed to be, was identified.

Sequence analysis (Genefinder program (Wilson, R. et al., 1994, Nature, 36
8, 32-38)) revealed that cosmids W08D2 and T13F2 contain overlapping regions. Further definition, the cosmid T13F2 have been found to contain an open reading frame (ORF) (indicated as T13F2.1), which is the N-terminal cytochrome b ₅ domain (diagnostic His-Pro-Gly-G ly motif As well as three "histidine boxes" specific to all microsomal desaturases. In addition, this putative desaturase contained a mutated third histidine box in which the first histidine in the His-XX-His-His motif was replaced by H to Q. This substitution for glutamine has been shown in plant and animal Δ6-fatty acid desaturases and in fungal Δ5-fatty acid desaturases from Mortierella alpina.

The overlap between cosmids T13F2 and W08D2 is due to the putative desaturase ORF.
(T13F2.1) and the proximity of the Δ6-fatty acid desaturase allow identification of two sequences separated by 990 bases from the predicted stop codon of T13F2.1 to the methionine start triplet of the Δ6-fatty acid desaturase. It was revealed that they were arranged in a single line on cytochrome IV.

Heterofunctional expression of genomic DNA was not feasible, as sequence analysis predicts that the T13F2.1 ORF is dispersed in a large number of introns. Therefore, the polymerase chain reaction (PCR) using the following primers CEFOR and CEREV was used to amplify a cDNA clone corresponding to the putative long exon at the 5 'end of the T13F2.1 ORF: CEFOR: 5 '-ATGGTATTACGAGAGCAAGA-3' (SEQ ID NO 16) CEREV: 5'-TCTGGGATCTCTGGTTCTTG- 3 '( SEQ ID NO 17)

After initial denaturation at 94 ° C. for 2 minutes, amplification was performed as follows: 32 cycles of 94 ° C. for 45 seconds, 55 ° C. for 1 minute, 72 ° C. for 1 minute, and finally at 72 ° C. 1
Amplification was performed for 0 minutes.

The DNA fragment of the correct expected size was amplified (visualized on a 1% agarose gel), the gel band was excised, the DNA was purified and pGEM-T (Prom
ega) and the resulting plasmid was cloned into E. coli. coli DH5α cells. For sequencing, the plasmid DNA was converted to Qiagen.
The insert was purified using the QIAprep miniprep kit and the nucleotide sequence of the insert was identified by automated sequencing using an ABI-377 DNA sequencer.

The isolated 233 bp PCR amplified fragment was used to isolate the complete coding region corresponding to ORF T13F2.1 using Prof Yuji Kohara-M
The cDNA library of Cenolabditis elegans in a mixed state composed in λZapII provided by ishima, Japan was screened. The cloned PCR products were screened using standard techniques using probes (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual). Ready to Go DNL-Labelling reaction mix (Ph
DNA fragments were labeled with α [ ³² P] dCTP using A. marcia. Five plaques out of 1.4 × 10 ⁵ pfu screened for hybridization to the 233 bp fragment showed a positive signal, and cylindrical samples were withdrawn from the agar plate and eluted in SM buffer. The resulting phage suspension was screened for the presence of T13F2.1 by PCR amplification using CEFOR and CEREV. One clone (designated L4) was purified by hybridization screening at 65 ° C. with the 233 bp fragment isolated by two additional platings and PCR.
Plasmid L4 was released from lambda clone L4 by excision, and the cDNA inserts on both strands were sequenced using a Perkin Elmer ABI-377 DNA sequencer.

The obtained DNA sequence is shown in SEQ ID NO: 2, and the predicted amino acid sequence is shown in SEQ ID NO: 4.

Functional Analysis of L4 in Yeast The complete coding sequence of L4 (447 amino acid code) was amplified by PCR using primers YCEDFor and TCEDRev shown below (HindIII and BamHI flanking sites were also introduced on the side). ): YCEDFor: 5′-GCG AAGCTT AAAATGGTATTACGAG
AGCAAGAGC-3 ′ (SEQ ID NO: 18) (annealing to the starting methionine is shown in bold and the HindIII restriction site is underlined) YCEDRev: 5′-GCG GGATCC AATCTAGGCAATCTTTT
TTAGTCAA-3 ′ (SEQ ID NO: 19) (annealing of the stop codon to the complement is shown in bold and the BamHI restriction is underlined).

The amplified PCR product containing the complete coding region of L4 was prepared using HindIII and Bam
The yeast expression vector -pYES2 (Invitrogen) was ligated downstream of the GAL1 promoter using a III restriction site (enzyme from Boehringer Mannheim). The resulting configuration (denoted as pYES2 / L4)
E. FIG. E. coli and the fidelity of the insert generated by PCR in plasmid pYES2 / L4 was confirmed in vitro by transcription / translation using the TNT system (Promega). The resulting translation is labeled with ³⁵ S methionine,
Separated by SDS-PAGE and visualized by autoradiography.

The translation obtained from pYES2 / L4 has a molecular weight of about M _r 57,000, whereas the control vector pYES2 without insert does not produce a translation.

For functional analysis of the L4 coding region, the recombinant plasmid was transformed into Saccharomyces cerevisiae DBY746 by the lithium acetate method (Elble R., 19).
92, Bio Techniques, 13, 18-20). Cells were incubated with linoleic acid (18: 2Δ ^9,12 ) or di-homogenein in the presence of 1% terrigitol containing raffinose as a carbon source.
Cultured overnight in ^medium supplemented with either addition of γ-linolenic acid (C20: 3Δ8,11,14) (described in Napier et al., 1998, Biochem. J., 330, 611-614). These fatty acids are S.P. cerevisiae does not exist, but Δ6
Alternatively, it acts as a specific substrate for Δ5-desaturase. Vector GAL
Expression of the L4 coding region from one promoter was induced by the addition of up to 1% galactose. Culture growth was continued for 16 hours, after which an aliquot was removed for fatty acid analysis by GC. Total fatty acids extracted from yeast cultures were analyzed by gas chromatography of methyl esters (GC). 8 in methanol
The methyl group in the lipid was transferred using 1 M HCL at 0 ° C. for 1 hour, and then the fatty acid methyl ester (FAME) was extracted in hexane. FAMES GC analysis was performed on a Hewlett Packard 58 equipped with a 25M x 0.32 mm RSL-500BP coupled capillary column and a flame photoionization detector.
This was performed using an 80A Series Gas Chromatograph.
Fatty acids were identified by comparing to the retention time of a FAME standard (Sigma). The relative percentage of fatty acids was estimated from the peak area. Arachidonic acid was treated with Kra operating at a scanning range of 500-40 Daltons and an ionization voltage of 70 eV.
Identified by GC-MS using ts MS80RFA. FIG. 4 shows the results of GC analysis of fatty acid methyl esters of the transformed yeast strain. Induced pYE grown in the presence of di-homo-γ-linolenic acid compared to the empty vector control
It is clear that an additional peak was obtained in the trace from S2 / L4. This peak was also not from uninduced medium grown on di-homo-γ-linolenic acid, and pYES2 / L4 grown in the presence of linolenic acid did not accumulate new peaks, and It is important to note that this indicates that this fatty acid is not a substrate for the enzyme encoded by the Cenolabuditis elegans cDNA. The retention times of the additional peaks correspond to those of the authentic methyl arachidonic acid standard. Fatty acids produced from di-homo-γ-linolenic acid were converted to GCMS (Gas Chromatography Mass Spectr).
Ometry) and identified as arachidonic acid. Therefore, this result indicates that yeast cells transformed with plasmid pYES2 / L4 had a functional Δ5-
It shows that desaturase activity is obtained, at which point arachidonic acid can be synthesized from the substrate di-homo-γ-linolenic acid. Clearly, Δ5-desaturase in transformed yeast is an effective catalyst.

This indicates that the DNA sequence from Senolabuditis elegans encodes a functional polypeptide involved in the synthesis of arachidonic acid in the presence of galactose and di-homo-γ-linolenate. I have.

[Table 1]

[Table 2]

[Table 3]

[0092]

[Sequence List] <110> University of Bristol <120> Desaturase <130> P-7706F <160> 19 <210> 1 <400> 1 000 <210> 2 <211> 1344 <212> DNA <213> Seno Love di infantis elegans (Caenorhabditis elegans) <400> 2 atggtattac gagagcaaga gcatgagcca ttcttcatta aaattgatgg aaaatggtgt 60 caaattgacg atgctgtcct gagatcacat ccaggtggta gtgcaattac tacctataaa 120 aatatggatg ccactaccgt attccacaca ttccatactg gttctaaaga agcgtatcaa 180 tggctgacag aattgaaaaa agagtgccct acacaagaac cagagatccc agatattaag 240 gatgacccaa tcaaaggaat tgatgatgtg aacatgggaa ctttcaatat ttctgagaaa 300 cgatctgccc aaataaataa aagtttcact gatctacgta tgcgagttcg tgcagaagga 360 cttatggatg gatctccttt gttctacatt agaaaaattc ttgaaacaat cttcacaatt 420 ctttttgcat tctaccttca ataccacaca tattatcttc catcagctat tctaatggga 480 gttgcgtggc aacaattggg atggttaatc catgaattcg cacatcatca gttgttcaaa 540 aacagatact acaatgattt ggccagctat ttcgttggaa actttttaca aggattctca 600 tctggtggtt ggaa agagca gcacaatgtg catcacgcag ccacaaatgt tgttggacga 660 gacggagatc ttgatttagt cccattctat gctacagtgg cagaacatct caacaattat 720 tctcaggatt catgggttat gactctattc agatggcaac atgttcattg gacattcatg 780 ttaccattcc tccgtctctc gtggcttctt cagtcaatca tttttgttag tcagatgcca 840 actcattatt atgactatta cagaaatact gcgatttatg aacaggttgg tctctctttg 900 cactgggctt ggtcattggg tcaattgtat ttcctacccg attggtcaac tagaataatg 960 ttcttccttg tttctcatct tgttggaggt ttcctgctct ctcatgtagt tactttcaat 1020 cattattcag tggagaagtt tgcattgagc tcgaacatca tgtcaaatta cgcttgtctt 1080 caaatcatga ccacaagaaa tatgagacct ggaagattca ttgactggct ttggggaggt 1140 cttaactatc agattgagca ccatcttttc ccaacgatgc cacgacacaa cttgaacact 1200 gttatgccac ttgttaagga gtttgcagca gcaaatggtt taccatacat ggtcgacgat 1260 tatttcacag gattctggct tgaaattgag caattccgaa atattgcaaa tgttgctgct 1320 aaattgacta aaaagattgc ctag 1344 <210> 3 <211> 446 <212> PRT <213> Mortierella alpina <400> 3 Met Gly Thr Asp Gln Gly Lys Thr Phe T hr Trp Glu Glu Leu Ala Ala 1 5 10 15 His Asn Thr Lys Gly Asp Leu Phe Leu Ala Ile Arg Gly Arg Val Tyr 20 25 30 Asp Val Thr Lys Phe Leu Ser Arg His Pro Gly Gly Val Asp Thr Leu 35 40 45 Leu Leu Gly Ala Gly Arg Asp Val Thr Pro Val Phe Glu Met Tyr His 50 55 60 Ala Phe Gly Ala Ala Asp Ala Ile Met Lys Lys Tyr Tyr Val Gly Thr 65 70 75 80 Leu Val Ser Asn Glu Leu Pro Val Phe Pro Glu Pro Thr Val Phe His 85 90 95 Lys Thr Ile Lys Thr Arg Val Glu Gly Tyr Phe Thr Asp Arg Asp Ile 100 105 110 Asp Pro Lys Asn Arg Pro Glu Ile Trp Gly Arg Tyr Ala Leu Ile Phe 115 120 125 Gly Ser Leu Ile Ala Ser Tyr Tyr Ala Gln Leu Phe Val Pro Phe Val 130 135 140 Val Glu Arg Thr Trp Leu Gln Val Val Phe Ala Ile Ile Met Gly Phe 145 150 155 160 Ala Cys Ala Gln Val Gly Leu Asn Pro Leu His Asp Ala Ser His Phe 165 170 175 Ser Val Thr His Asn Pro Thr Val Trp Lys Ile Leu Gly Ala Thr His 180 185 190 Asp Phe Phe Asn Gly Ala Ser Tyr Leu Val Trp Met Tyr Gln His Met 195 200 205 Leu Gly His His Pro Tyr Thr Asn Ile Ala Gly Ala Asp Pro Asp Val 210 215 220 Ser Thr Phe Glu Pro Asp Val Arg Arg Ile Lys Pro Asn Gln Lys Trp 225 230 235 240 Phe Val Asn His Ile Asn Gln Asp Met Phe Val Pro Phe Leu Tyr Gly 245 250 255 Leu Leu Ala Phe Lys Val Arg Ile Gln Asp Ile Asn Ile Leu Tyr Phe 260 265 270 Val Lys Thr Asn Asp Ala Ile Arg Val Asn Pro Ile Ser Thr Trp His 275 280 285 Thr Val Met Phe Trp Gly Gly Lys Ala Phe Phe Val Trp Tyr Arg Leu 290 295 300 Ile Val Pro Leu Gln Tyr Leu Pro Leu Gly Lys Val Leu Leu Leu Phe 305 310 315 320 Thr Val Ala Asp Met Val Ser Ser Tyr Trp Leu Ala Leu Thr Phe Gln 325 330 335 Ala Asn His Val Val Glu Glu Val Gln Trp Pro Leu Pro Asp Glu Asn 340 345 350 Gly Ile Ile Gln Lys Asp Trp Ala Ala Met Gln Val Glu Thr Thr Gln 355 360 365 Asp Tyr Ala His Asp Ser His Leu Trp Thr Ser Ile Thr Gly Ser Leu 370 375 380 380 Asn Tyr Gln Ala Val His His Leu Phe Pro Asn Val Ser Gln His His 385 390 395 400 Tyr Pro Asp Ile Leu Ala Ile Ile Lys Asn Thr Cys Ser Glu Tyr Lys 405 410 415 Val Pro Tyr Leu Val Lys Asp Thr Phe Trp Gln Ala Phe Ala Ser His 420 425 430 Leu Glu His Leu Arg Val Leu Gly Leu Arg Pro Lys Glu Glu 435 440 445 <210> 4 <211> 447 <212> PRT <213> Caenorhabditis elegans <400> 4 Met Val Leu Arg Glu Gln Glu His Glu Pro Phe Phe Ile Lys Ile Asp 1 5 10 15 Gly Lys Trp Cys Gln Ile Asp Asp Ala Val Leu Arg Ser His Pro Gly 20 25 30 Gly Ser Ala Ile Thr Thr Tyr Lys Asn Met Asp Ala Thr Thr Val Phe 35 40 45 His Thr Phe His Thr Gly Ser Lys Glu Ala Tyr Gln Trp Leu Thr Glu 50 55 60 Leu Lys Lys Glu Cys Pro Thr Gln Glu Pro Glu Ile Pro Asp Ile Lys 65 70 75 80 Asp Asp Pro Ile Lys Gly Ile Asp Asp Val Asn Met Gly Thr Phe Asn 85 90 95 Ile Ser Glu Lys Arg Ser Ala Gln Ile Asn Lys Ser Phe Thr Asp Leu 100 105 110 Arg Met Arg Val Arg Ala Glu Gly Leu Met Asp Gly Ser Pro Leu Phe 115 120 125 Tyr Ile Arg Lys Ile Leu Glu Thr Ile Phe Thr Ile Leu Phe Ala Phe 130 135 140 Tyr Leu Gln Tyr His Thr Tyr Tyr Leu Pro Ser Ala Ile Leu Met Gly 145 150 155 160 Val Ala Trp Gln Gln Leu Gly Trp Leu Ile His Glu Phe Ala His His 165 170 175 Gln Leu Phe Lys Asn Arg Tyr Tyr Asn Asp Leu Ala Ser Tyr Phe Val 180 185 190 Gly Asn Phe Leu Gln Gly Phe Ser Ser Gly Gly Trp Lys Glu Gln His 195 200 205 Asn Val His His Ala Ala Thr Asn Val Val Gly Arg Asp Gly Asp Leu 210 215 220 Asp Leu Val Pro Phe Tyr Ala Thr Val Ala Glu His Leu Asn Asn Tyr 225 230 235 240 Ser Gln Asp Ser Trp Val Met Thr Leu Phe Arg Trp Gln His Val His 245 250 255 Trp Thr Phe Met Leu Pro Phe Leu Arg Leu Ser Trp Leu Leu Gln Ser 260 265 270 Ile Ile Phe Val Ser Gln Met Pro Thr His Tyr Tyr Asp Tyr Tyr Arg 275 280 285 Asn Thr Ala Ile Tyr Glu Gln Val Gly Leu Ser Leu His Trp Ala Trp 290 295 300 Ser Leu Gly Gln Leu Tyr Phe Leu Pro Asp Trp Ser Thr Arg Ile Met 305 310 315 320 Phe Phe Leu Val Ser His Leu Val Gly Gly Phe Leu Leu Ser His Val 325 330 335 Val Thr Phe Asn His Tyr Ser Val Glu Lys Phe Ala Leu Ser Ser Asn 340 345 350 Ile Met Ser Asn Tyr Ala Cys Leu Gln Ile Met Thr Thr Arg Asn Met 355 360 365 Arg Pro Gly Arg Phe Ile Asp Trp Leu Trp Gly Gly Leu Asn Tyr Gln 370 375 380 Ile Glu His His Leu Phe Pro Thr Met Pro Arg His Asn Leu Asn Thr 385 390 395 400 Val Met Pro Leu Val Lys Glu Phe Ala Ala Ala Asla Gly Leu Pro Tyr 405 410 415 Met Val Asp Asp Tyr Phe Thr Gly Phe Trp Leu Glu Ile Glu Gln Phe 420 425 430 Arg Asn Ile Ala Asn Val Ala Ala Lys Leu Thr Lys Lys Ile Ala 435 440 445 <210> 5 <211> 351 <212> PRT <213> Spirulina plastic tennis ( Spirulina platensis) <400> 5 Met Thr Leu Ser Ile Val Lys Ser Glu Asp Ser Ser Ser Arg Pro Ser 1 5 10 15 Ala Val Pro Ser Asp Leu Pro Leu Glu Glu Asp Ile Ile Asn Thr Leu 20 25 30 Pro Ser Gly Val Phe Val Gln Asp Arg Tyr Lys Ala Trp Met Thr Val 35 40 45 Ile Ile Asn Val Val Met Val Gly Leu Gly Trp Leu Gly Ile Ala Ile 50 55 60 Ala Pro Trp Phe Leu Leu Pro Val Val Trp Val Phe Thr Gly Thr Ala 65 70 75 80 Leu Thr Gly Phe Phe Val Ile Gly His Asp Cys Gly His Arg Ser Phe 85 90 95 Ser Arg Asn Val Trp Val Asn Asp Trp Val Gly His Ile Leu Phe Leu 100 105 110 Pro Ile Ile Tle Pro Phe His Ser Trp Arg Ile Gly His Asn G ln His 115 120 125 His Lys Tyr Thr Asn Arg Met Glu Leu Asp Asn Ala Trp Gln Pro Trp 130 135 140 Arg Lys Glu Glu Tyr Gln Asn Ala Gly Lys Phe Met Gln Val Thr Tyr 145 150 155 160 Asp Leu Phe Arg Gly Arg Ala Trp Trp Ile Gly Ser Ile Leu His Trp 165 170 175 Ala Ser Ile His Phe Asp Trp Thr Lys Phe Glu Gly Lys Gln Arg Gln 180 185 190 Gln Val Lys Phe Ser Ser Leu Leu Val Ile Gly Ala Ala Ala Ile Ala 195 200 205 Phe Pro Thr Met Ile Leu Thr Ile Gly Val Trp Gly Phe Val Lys Phe 210 215 220 Trp Val Ile Pro Trp Leu Val Phe His Phe Trp Met Ser Thr Phe Thr 225 230 235 240 Leu Leu His His Thr Ile Ala Asp Ile Pro Phe Arg Glu Pro Glu Gln 245 250 255 Trp His Glu Ala Glu Ser Gln Leu Ser Gly Thr Val His Cys Asn Tyr 260 265 270 Ser Arg Trp Gly Glu Phe Leu Cys His Asp Ile Asn Val His Ile Pro 275 280 285 His His Val Thr Thr Ala Ile Pro Trp Tyr Asn Leu Arg Thr Pro Thr 290 295 300 Pro Val Tyr Arg Lys Ile Gly Gly Glu Tyr Leu Tyr Pro Glu Cys Asp 305 310 315 320 Phe Ser Trp Gly Leu Met Lys Gln Val Val Asp His Ala Ile C ys Met 325 330 335 Met Arg Ile Thr Ile Ile Ser Gln Ser Leu Thr Thr Lys Arg Val 340 345 350 <210> 6 <211> 368 <212> PRT <213> Spirulina platensis <400> 6 Met Thr Ser Thr Thr Ser Lys Val Thr Phe Gly Lys Ser Ile Gly Phe 1 5 10 15 Arg Lys Glu Leu Asn Arg Arg Val Asn Ala Tyr Leu Glu Ala Glu Asn 20 25 30 Ile Ser Pro Arg Asp Asn Pro Pro Met Tyr Leu Lys Thr Ala Ile Ile 35 40 45 Leu Ala Trp Val Val Ser Ala Trp Thr Phe Val Val Phe Gly Pro Asp 50 55 60 Val Leu Trp Met Lys Leu Leu Gly Cys Ile Val Leu Gly Phe Gly Val 65 70 75 80 Ser Ala Val Gly Phe Asn Ile Ser His Asp Gly Asn His Gly Gly Tyr 85 90 95 Ser Lys Tyr Gln Trp Val Asn Tyr Leu Ser Gly Leu Thr His Asp Ala 100 105 110 Ile Gly Val Ser Ser Tyr Leu Trp Lys Phe Arg His Asn Val Leu His 115 120 125 His Thr Tyr Thr Asn Ile Leu Gly His Asp Val Glu Ile His Gly Asp 130 135 140 Glu Leu Val Arg Met Ser Pro Ser Met Glu Tyr Arg Trp Tyr His Arg 145 150 155 160 Tyr Gln His Trp Phe Ile Trp Phe Val Tyr Pro Phe Ile Pro Ty r Tyr 165 170 175 Trp Ser Ile Ala Asp Val Gln Thr Met Leu Phe Lys Arg Gln Tyr His 180 185 190 Asp His Glu Ile Pro Ser Pro Thr Trp Val Asp Ile Ala Thr Leu Leu 195 200 205 Ala Phe Lys Ala Phe Gly Val Ala Val Phe Leu Ile Ile Pro Ile Ala 210 215 220 Val Gly Tyr Ser Pro Leu Glu Ala Val Ile Gly Ala Ser Ile Val Tyr 225 230 235 240 Met Thr His Gly Leu Val Ala Cys Val Val Phe Met Leu Ala His Val 245 250 255 Ile Glu Pro Ala Glu Phe Leu Asp Pro Asp Asn Leu His Ile Asp Asp 260 265 270 Glu Trp Ala Ile Ala Gln Val Lys Thr Thr Val Asp Phe Ala Pro Asn 275 280 285 Asn Pro Ile Ile Asn Trp Tyr Val Gly Gly Leu Asn Tyr Gln Thr Val 290 295 300 His His Leu Phe Pro His Ile Cys His Ile His Tyr Pro Lys Ile Ala 305 310 315 320 Pro Ile Leu Ala Glu Val Cys Glu Glu Phe Gly Val Asn Tyr Ala Val 325 330 335 His Gln Thr Phe Phe Gly Ala Leu Ala Ala Asn Tyr Ser Trp Leu Lys 340 345 350 Lys Met Ser Ile Asn Pro Glu Thr Lys Ala Ile Glu Gln Leu Thr Val 355 360 365 <210> 7 <211> 458 <212> PRT <213> Helianthus annuus <400> 7 Me t Val Ser Pro Ser Ile Glu Val Leu Asn Ser Ile Ala Asp Gly Lys 1 5 10 15 Lys Tyr Ile Thr Ser Lys Glu Leu Lys Lys His Asn Asn Pro Asn Asp 20 25 30 Leu Trp Ile Ser Ile Leu Gly Lys Val Tyr Asn Val Thr Glu Trp Ala 35 40 45 Lys Glu His Pro Gly Gly Asp Ala Pro Leu Ile Asn Leu Ala Gly Gln 50 55 60 Asp Val Thr Asp Ala Phe Ile Ala Phe His Pro Gly Thr Ala Trp Lys 65 70 75 80 His Leu Asp Lys Leu Phe Thr Gly Tyr His Leu Lys Asp Tyr Gln Val 85 90 95 Ser Asp Ile Ser Arg Asp Tyr Arg Lys Leu Ala Ser Glu Phe Ala Lys 100 105 110 Ala Gly Met Phe Glu Lys Lys Gly His Gly Val Ile Tyr Ser Leu Cys 115 120 125 Phe Val Ser Leu Leu Leu Ser Ala Cys Val Tyr Gly Val Leu Tyr Ser 130 135 140 Gly Ser Phe Trp Ile His Met Leu Ser Gly Ala Ile Leu Gly Leu Ala 145 150 155 160 Trp Met Gln Ile Ala Tyr Leu Gly His Asp Ala Gly His Tyr Gln Met 165 170 175 Met Ala Thr Arg Gly Trp Asn Lys Phe Ala Gly Ile Phe Ile Gly Asn 180 185 190 Cys Ile Thr Gly Ile Ser Ile Ala Trp Trp Lys Trp Thr His Asn Ala 195 200 205 His His Ile Ala Cys Asn Ser Leu Asp Tyr Asp Pro Asp Leu Gln His 210 215 220 Leu Pro Met Leu Ala Val Ser Ser Lys Leu Phe Asn Ser Ile Thr Ser 225 230 235 240 Val Phe Tyr Gly Arg Gln Leu Thr Phe Asp Pro Leu Ala Arg Phe Phe 245 250 255 Val Ser Tyr Gln His Tyr Leu Tyr Tyr Pro Ile Met Cys Val Ala Arg 260 265 270 Val Asn Leu Tyr Leu Gln Thr Ile Leu Leu Leu Ile Ser Lys Arg Lys 275 280 285 Ile Pro Asp Arg Gly Leu Asn Ile Leu Gly Thr Leu Ile Phe Trp Thr 290 295 300 Trp Phe Pro Leu Leu Val Ser Arg Leu Pro Asn Trp Pro Glu Arg Val 305 310 315 320 Ala Phe Val Leu Val Ser Phe Cys Val Thr Gly Ile Gln His Ile Gln 325 330 335 Phe Thr Leu Asn His Phe Ser Gly Asp Val Tyr Val Gly Pro Pro Lys 340 345 350 350 Gly Asp Asn Trp Phe Glu Lys Gln Thr Arg Gly Thr Ile Asp Ile Ala 355 360 365 Cys Ser Ser Trp Met Asp Trp Phe Phe Gly Gly Leu Gln Phe Gln Leu 370 375 380 Glu His His Leu Phe Pro Arg Leu Pro Arg Cys His Leu Arg Ser Ile 385 390 395 400 Ser Pro Ile Cys Arg Glu Leu Cys Lys Lys Tyr Asn Leu Pro Tyr Val 405 410 410 415 Ser Leu Ser Phe Tyr Asp Ala Asn Val Thr Thr Leu Lys Thr Leu Arg 420 425 430 Thr Ala Ala Leu Gln Ala Arg Asp Leu Thr Asn Pro Ala Pro Gln Asn 435 440 445 Leu Ala Trp Glu Ala Phe Asn Thr His Gly 450 455 <210> 8 <211 > 359 <212> PRT <213> Synechocystis PCC6803 <400> 8 Met Leu Thr Ala Glu Arg Ile Lys Phe Thr Gln Lys Arg Gly Phe Arg 1 5 10 15 Arg Val Leu Asn Gln Arg Val Asp Ala Tyr Phe Ala Glu His Gly Leu 20 25 30 Thr Gln Arg Asp Asn Pro Ser Met Tyr Leu Lys Thr Leu Ile Ile Val 35 40 45 Leu Trp Leu Phe Ser Ala Trp Ala Phe Val Leu Phe Ala Pro Val Ile 50 55 60 Phe Pro Val Arg Leu Leu Gly Cys Met Val Leu Ala Ile Ala Leu Ala 65 70 75 80 Ala Phe Ser Phe Asn Val Gly His Asp Ala Asn His Asn Ala Tyr Ser 85 90 95 95 Ser Asn Pro His Ile Asn Arg Val Leu Gly Met Thr Tyr Asp Phe Val 100 105 110 Gly Leu Ser Ser Phe Leu Trp Arg Tyr Arg His Asn Tyr Leu His His 115 120 125 Thr Tyr Thr Asn Ile Leu Gly His Asp Val Glu Ile His Gly Asp Gly 130 135 140 Ala Val Arg Met Ser Pro Glu Gln Glu His Val Gly Ile Tyr Arg Phe 145 150 155 160 Gln Gln Phe Tyr Ile Trp Gly Leu Tyr Leu Phe Ile Pro Phe Tyr Trp 165 170 175 Phe Leu Tyr Asp Val Tyr Leu Val Leu Asn Lys Gly Lys Tyr His Asp 180 185 190 His Lys Ile Pro Pro Phe Gln Pro Leu Glu Leu Ala Ser Leu Leu Gly 195 200 205 Ile Lys Leu Leu Trp Leu Gly Tyr Val Phe Gly Leu Pro Leu Ala Leu 210 215 220 Gly Phe Ser Ile Pro Glu Val Leu Ile Gly Ala Ser Val Thr Tyr Met 225 230 235 240 Thr Tyr Gly Ile Val Val Cys Thr Ile Phe Met Leu Ala His Val Leu 245 250 255 Glu Ser Thr Glu Phe Leu Thr Pro Asp Gly Glu Ser Gly Ala Ile Asp 260 265 270 Asp Glu Trp Ala Ile Cys Gln Ile Arg Thr Thr Ala Asn Phe Ala Thr 275 280 285 Asn Asn Pro Phe Trp Asn Trp Phe Cys Gly Gly Leu Asn His Gln Val 290 295 300 Thr His His Leu Phe Pro Asn Ile Cys His Ile His Tyr Pro Gln Leu 305 310 315 320 Glu Asn Ile Ile Lys Asp Val Cys Gln Glu Phe Gly Val Glu Tyr Lys 325 330 335 Val Tyr Pro Thr Phe Lys Ala Ala Ile Ala Ser Asn Tyr Arg Trp Leu 340 345 350 Glu Ala Met Gly Lys Ala Ser 355 <210> 9 <211> 448 <212> PRT <213> Borago officinalis <400> 9 Met Ala Ala Gln Ile Lys Lys Tyr Ile Thr Ser Asp Glu Leu Lys Asn 1 5 10 15 His Asp Lys Pro Gly Asp Leu Trp Ile Ser Ile Gln Gly Lys Ala Tyr 20 25 30 Asp Val Ser Asp Trp Val Lys Asp His Pro Gly Gly Ser Phe Pro Leu 35 40 45 Lys Ser Leu Ala Gly Gln Glu Val Thr Asp Ala Phe Val Ala Phe His 50 55 60 Pro Ala Ser Thr Trp Lys Asn Leu Asp Lys Phe Phe Thr Gly Tyr Tyr 65 70 75 80 Leu Lys Asp Tyr Ser Val Ser Glu Val Ser Lys Asp Tyr Arg Lys Leu 85 90 95 Val Phe Glu Phe Ser Lys Met Gly Leu Tyr Asp Lys Lys Gly His Ile 100 105 110 Met Phe Ala Thr Leu Cys Phe Ile Ala Met Leu Phe Ala Met Ser Val 115 120 125 Tyr Gly Val Leu Phe Cys Glu Gly Val Leu Val His Leu Phe Ser Gly 130 135 140 Cys Leu Met Gly Phe Leu Trp Ile Gln Ser Gly Trp Ile Gly His Asp 145 150 155 160 Ala Gly His Tyr Met Val Val Ser Asp Ser Arg Leu Asn Lys Phe Met 165 170 175 Gly Ile Phe Ala Ala Asn Cys Leu Ser Gly Ile Ser Ile Gly Trp Trp 180 185 190 Lys Trp Asn His Asn Ala His His Ile Ala Cys Asn Ser Leu Glu Tyr 1 95 200 205 Asp Pro Asp Leu Gln Tyr Ile Pro Phe Leu Val Val Ser Ser Lys Phe 210 215 220 Phe Gly Ser Leu Thr Ser His Phe Tyr Glu Lys Arg Leu Thr Phe Asp 225 230 235 240 Ser Leu Ser Arg Phe Phe Val Ser Tyr Gln His Trp Thr Phe Tyr Pro 245 250 255 Ile Met Cys Ala Ala Arg Leu Asn Met Tyr Val Gln Ser Leu Ile Met 260 265 270 Leu Leu Thr Lys Arg Asn Val Ser Tyr Arg Ala His Glu Leu Leu Gly 275 280 285 Cys Leu Val Phe Ser Ile Trp Tyr Pro Leu Leu Val Ser Cys Leu Pro 290 295 300 Asn Trp Gly Glu Arg Ile Met Phe Val Ile Ala Ser Leu Ser Val Thr 305 310 315 320 Gly Met Gln Gln Val Gln Phe Ser Leu Asn His Phe Ser Ser Ser Val 325 330 335 Tyr Val Gly Lys Pro Lys Gly Asn Asn Trp Phe Glu Lys Gln Thr Asp 340 345 350 Gly Thr Leu Asp Ile Ser Cys Pro Pro Trp Met Asp Trp Phe His Gly 355 360 365 Gly Leu Gln Phe Gln Ile Glu His His Leu Phe Pro Lys Met Pro Arg 370 375 380 Cys Asn Leu Arg Lys Ile Ser Pro Tyr Val Ile Glu Leu Cys Lys Lys 385 390 395 400 His Asn Leu Pro Tyr Asn Tyr Ala Ser Phe Ser Lys Ala Asn Glu Met Four 05 410 415 Thr Leu Arg Thr Leu Arg Asn Thr Ala Leu Gln Ala Arg Asp Ile Thr 420 425 430 Lys Pro Leu Pro Lys Asn Leu Val Trp Glu Ala Leu His Thr His Gly 435 440 445 <210> 10 <211> 443 <212> PRT <213> Caenorhabditis elegans <400> 10 Met Val Val Asp Lys Asn Ala Ser Gly Leu Arg Met Lys Val Asp Gly 1 5 10 15 Lys Trp Leu Tyr Leu Ser Glu Glu Leu Val Lys Lys His Pro Gly Gly 20 25 30 Ala Val Ile Glu Gln Tyr Arg Asn Ser Asp Ala Thr His Ile Phe His 35 40 45 Ala Phe His Glu Gly Ser Ser Gln Ala Tyr Lys Gln Leu Asp Leu Leu 50 55 60 Lys Lys His Gly Glu His Asp Glu Phe Leu Glu Lys Gln Leu Glu Lys 65 70 75 80 Arg Leu Asp Lys Val Asp Ile Asn Val Ser Ala Tyr Asp Val Ser Val 85 90 95 Ala Gln Glu Lys Lys Met Val Glu Ser Phe Glu Lys Leu Arg Gln Lys 100 105 110 Leu His Asp Asp Gly Leu Met Lys Ala Asn Glu Thr Tyr Phe Leu Phe 115 120 125 Lys Ala Ile Ser Thr Leu Ser Ile Met Ala Phe Ala Phe Tyr Leu Gln 130 135 140 Tyr Leu Gly Trp Tyr Ile Thr Ser Ala Cys Leu Leu Ala Leu Ala Trp 145 150 155 160 Gln Gln Phe Gly Trp Leu Thr His Glu Phe Cys His Gln Gln Pro Thr 165 170 175 Lys Asn Arg Pro Leu Asn Asp Thr Ile Ser Leu Phe Phe Gly Asn Phe 180 185 190 Leu Gln Gly Phe Ser Arg Asp Trp Trp Lys Asp Lys His Asn Thr His 195 200 205 His Ala Ala Thr Asn Val Ile Asp His Asp Gly Asp Ile Asp Leu Ala 210 215 220 Pro Leu Phe Ala Phe Ile Pro Gly Asp Leu Cys Lys Tyr Lys Ala Ser 225 230 235 240 Phe Glu Lys Ala Ile Leu Lys Ile Val Pro Tyr Gln His Leu Tyr Phe 245 250 255 Thr Ala Met Leu Pro Met Leu Arg Phe Ser Trp Thr Gly Gln Ser Val 260 265 270 Gln Trp Val Phe Lys Glu Asn Gln Met Glu Tyr Lys Val Tyr Gln Arg 275 280 285 Asn Ala Phe Trp Glu Gln Ala Thr Ile Val Gly His Trp Ala Trp Val 290 295 300 Phe Tyr Gln Leu Phe Leu Leu Pro Thr Trp Pro Leu Arg Val Ala Tyr 305 310 315 320 Phe Ile Ile Ser Gln Met Gly Gly Gly Leu Leu Ile Ala His Val Val 325 330 335 Thr Phe Asn His Asn Ser Val Asp Lys Tyr Pro Ala Asn Ser Arg Ile 340 345 350 Leu Asn Asn Phe Ala Ala Leu Gln Ile Leu Thr Thr Arg Asn Met Thr 355 360 365 Pro Ser Pro Phe Ile Asp Trp Leu Trp Gly Gly Leu Asn Tyr Gln Ile 370 375 380 Glu His His Leu Phe Pro Thr Met Pro Arg Cys Asn Leu Asn Ala Cys 385 390 395 400 Val Lys Tyr Val Lys Glu Trp Cys Lys Glu Asn Asn Leu Pro Tyr Leu 405 410 415 Val Asp Asp Tyr Phe Asp Gly Tyr Ala Met Asn Leu Gln Gln Leu Lys 420 425 430 Asn Met Ala Glu His Ile Gln Ala Lys Ala Ala 435 440 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220><221> modified _ base <222> (11) <223> i <220><221> modified _ base <222> (14) <223> i <220><221> modified _ base <222> (25) <223> i <220><223> Description of Artificial Sequence: Primer <400> 11 gcgaattawt nggncaygay tgygnca 27 <210> 12 <211> 28 <212 > DNA <213> Artificial Sequence <220><221> modified_base <222> (11) <223> i <220><221> modified_base <222> (14) <223> i <220><221> modified_base <222 > (20) <223> i <220><223> Description of Artificial Sequence: Primer <400> 12 gcgaattcat ntknggraan arrtgrtg 28 <210> 13 <211> 18 <212> DNA <213> Art ificial Sequence <220><223> Description of Artificial Sequence: Primer <400> 13 gatgcgtctc acttttca 18 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: Primer <400> 14 gtggtgcaca gcctggtagt t 21 <210> 15 <211> 5 <212> PRT <213> Mortierella alpina <400> 15 Glu His Pro Gly Gly 1 5 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: Primer <400> 16 atggtattac gagagcaaga 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: Primer <400> 17 tctgggatct ctggttcttg 20 <210> 18 <211> 34 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: Primer <400> 18 gcgaagctta aaatggtatt acgagagcaa gagc 34 <210> 19 <211> 33 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: Primer <400> 19 gcgggatcca atctaggcaa tctttttagt caa 33

[Brief description of the drawings]

FIG. 1 is a list of various Δ6-desaturases and genes encoding Δ12-desaturase and Δ5-fatty acid desaturase of Mortierella alpina. In FIG. 1, the amino acid sequences of S54259, S54809, S68358, S35157 , PBOR6 and FU2 are shown in Sequence Listings 5 to 10, respectively.

FIG. 2 is a continuation of FIG. 1;

FIG. 3 is a continuation of FIG. 1;

FIG. 2 is a list of genes encoding Δ6-desaturase derived from Senolabuditis elegans, fungal Δ5-desaturase derived from Mortierella alpina, and Δ5-fatty acid desaturase.

FIG. 3 is a trace of gas chromatography of fatty acid methyl esters of an induced yeast cell transformant transformed with Mortierella alpina Δ5-fatty acid desaturase gene and a non-induced yeast cell transformant. .

FIG. 4 is a trace of gas chromatography of fatty acid methyl esters of an induced yeast cell transformant transformed with a Δ5-fatty acid desaturase gene of Senolabuditis elegans and a non-induced yeast cell transformant. is there.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C12N 1/15 C12N 1/19 4B064 1/19 1/21 4B065 1/21 9/02 4C206 5/10 C12P 7/64 9/02 C12Q 1/68 A C12P 7/64 C12N 15/00 ZNAA C12Q 1/68 5/00 A (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, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), L, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR , HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Invention Michelson, Louise United Kingdom Bristol BS 81 U Woodland Road School of Biological Sciences Bristol University (72) Inventor Stbert Keith UK Bristol BS 81 U Dorland Road School of Biological Sciences Bristol University F-term (reference) QA18 QQ01 QQ22 QQ42 QR32.

Claims (46)

[Claims] 1. An isolated animal-derived Δ5-fatty acid desaturase and a functional part thereof. 2. An isolated Δ5-fatty acid desaturase from Cenolabditis elegans. 3. A DNA sequence encoding the Δ5-fatty acid desaturase according to claim 1 or 2. 4. The DNA sequence according to claim 3, which encodes a Δ5-fatty acid desaturase capable of functioning due to the degeneracy of the genetic code. A DNA sequence comprising at least part of some equivalents. 5. The DNA sequence according to claim 4, which is a DNA sequence derived from Senolabditis elegans. 6. The DNA sequence according to claim 3, which encodes a functional Δ5-fatty acid desaturase, which sequence encodes a Δ5-fatty acid desaturase capable of functioning due to degeneracy of the genetic code. Or a DNA sequence comprising at least a portion of an equivalent of said sequence or a portion of said sequence. 7. The DNA sequence according to claim 6, which is a DNA sequence derived from Mortierella alpina. 8. The DNA sequence according to claim 3, wherein said DNA sequence can function in a mammal.
The DNA sequence according to any one of the above. 9. The DNA sequence of claim 8, wherein said DNA sequence is expressed in a mammal. 10. The D of claim 9, wherein said DNA sequence is expressed in humans.
NA sequence. 11. A DNA sequence obtained by modifying a naturally-occurring functional gene encoding the Δ5-fatty acid desaturase according to claim 1 or 2. 12. The DN of claim 11, wherein the modification comprises a modification by chemical, physical or biological means that does not remove the catalytic activity of the encoded enzyme.
A sequence. 13. The DNA sequence of claim 12, wherein said modification improves the catalytic activity of the encoded enzyme. 14. The DNA sequence according to claim 12, wherein the biological modification includes a recombinant DNA method and a forced mutation method. 15. The DNA sequence according to claim 14, wherein said forced mutation is DNA shuffling. A polypeptide encoded by the DNA sequence according to any one of claims 3 to 15. 17. The method of claim 16, wherein at least a portion of the polypeptide has the sequence set forth in SEQ ID NO: 3, a functional equivalent of the sequence, or a functional equivalent of a portion of the sequence. Polypeptide. 18. The method of claim 16, wherein at least a portion of the polypeptide has the sequence set forth in SEQ ID NO: 4, a functional equivalent of the sequence, or a functional equivalent of a portion of the sequence. Polypeptide. 19. The polypeptide according to any one of claims 16 to 18, wherein said polypeptide catalyzes the conversion of dihomo-γ-linolenic acid to arachidonic acid. 20. The polypeptide according to any one of claims 16 to 19, wherein said polypeptide has been modified without removing the catalytic activity of said encoded polypeptide. 21. The polypeptide of claim 20, wherein said polypeptide is modified in such a way as to induce a specific level of saturation with the substrate at a particular location within the molecular structure of said substrate. 22. A vector comprising the DNA sequence or a part of the DNA sequence according to any one of claims 3 to 15. 23. A polyunsaturated fatty acid comprising a step of contacting a suitable substrate with a Δ5-fatty acid desaturase according to claim 1 or 2 or a polypeptide according to any one of claims 16 to 21. Manufacturing method. 24. A method for converting dihomo-γ-linolenic acid to arachidonic acid, wherein the conversion is a Δ5-fatty acid desaturase according to claim 1 or 2 or any one of claims 16 to 21. A conversion method catalyzed by a polypeptide or modified polypeptide of the invention. 25. An organism that has been engineered to produce high levels of the polypeptide of any one of claims 16-21. 26. An engineered to produce high levels of a reaction product catalyzed by a Δ5-fatty acid desaturase according to claim 1 or 2 or a polypeptide according to any one of claims 16 to 21. Creature. 27. An organism that has been manipulated by the method of claim 23. 28. The organism according to claim 26, wherein the organism is a microorganism. 29. The organism according to claim 28, wherein said microorganism is selected from algae, bacteria and fungi. 30. The organism of claim 29, wherein said fungus comprises an algal fungus. 31. The organism according to claim 28, wherein said microorganism is yeast. 32. The organism according to any one of claims 25 to 27, wherein the organism is a plant. 33. The organism of claim 32, wherein said plant is selected from oilseed plants and tobacco. 34. The oilseed plants include rape including rape, sunflower, corn, legumes including tobacco, peanuts and soybeans, safflower, oil palm, coconut and other palms, cotton, sesame, mustard, linseed, and sesame. 34. The organism of claim 33, selected from, borage and primrose. 35. A seed or other reproductive substance derived from the organism of claim 33 or 34. 36. The organism according to any one of claims 25 to 27, wherein the organism is a mammal. 37. An isolated biosynthetic pathway of a multienzyme, wherein the pathway is a Δ5-desaturase according to claim 1 or 2 or a polypeptide according to any one of claims 16 to 21. The included route. 38. A compound produced by the conversion of a substrate, said conversion being catalyzed by a Δ5-desaturase according to claim 1 or 2 or a polypeptide according to any one of claims 16 to 21. Compound. 39. An intermediate compound produced by a catalytic reaction of the Δ5-desaturase according to claim 1 or 2 or the polypeptide according to any one of claims 16 to 21. 40. A food or supplement comprising the polyunsaturated fatty acid produced by the method according to claim 23 or 24. 41. A pharmaceutical preparation comprising a polyunsaturated fatty acid produced by the method according to claim 23. 42. A prostaglandin synthesized by a biosynthetic pathway including a catalytic activity of the Δ5-desaturase according to claim 1 or 2 or the polypeptide according to any one of claims 16 to 21. 43. A method for regulating prostaglandin synthesis by regulating the expression level of the DNA sequence according to any one of claims 3 to 15. 44. A probe comprising all or a part of the DNA sequence or the equivalent RNA sequence according to any one of claims 3 to 15. 45. A diagnostic or search probe comprising all or a part of the Δ5-desaturase according to claim 1 or 2 or the polypeptide according to any one of claims 16 to 21. 46. A method for isolating a Δ5-desaturase using the probe according to claim 44 or 45.