JP2003116566A - METHOD FOR PRODUCING n-3-BASED DOCOSAPENTAENOIC ACID - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an industrial method for producing an n-3-based docosapentaenoic acid which is 1 kind of n-3-based highly unsaturated fatty acids. SOLUTION: This method for producing the n-3-based docosapentaenoic acid is provided by accumulating the n-3-based docosapentaenoic acid in transformed cells or at the outside of the cells by the transformed cells obtained by introducing a chain-extension enzyme gene derived from a mammalian animal as an vector of an extrachromosomal expressing or intrachromosomal expressing plasmid into a host cells to perform the chain extension of eicosapentaenoic acid.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing n-3 docosapentaenoic acid.

[0002]

2. Description of the Related Art In n-3 polyunsaturated fatty acids, there are three or more double bonds including a methylene group, and the position of the first double bond is present at the third carbon from the methyl end. A generic term for fatty acids having 18 to 22 carbon atoms. This n-3 polyunsaturated fatty acid is
By ingesting α-linolenic acid (ALA, C18: 3n-3), which is an essential fatty acid produced in plants such as soybean, the introduction of double bonds by desaturase and chain extension enzyme and carbon chain extension Repeatedly, Icosapentaenoic acid (EP, a direct precursor fatty acid of prostaglandin III group)
A, C20: 5n-3) and the final product, docosahexaenoic acid (DHA, C22: 6n-), which is abundantly present in the retina or cranial nervous system and is known to have an extremely important function.
Converted to 3) to maintain homeostasis in the body (homeostasis)
Play an extremely large role in.

As n-3 polyunsaturated fatty acids, EPA and DHA are already well known, and materials extracted and purified from fish oils such as tuna, sardines, mackerel, and saury are enriched in various foods. Used as an additive. Among the n-3 polyunsaturated fatty acids, the existence thereof is known, but the research on its physiological action has not been advanced in practice. n-3 docosapentaenoic acid (C2
2: 5n-3, which may hereinafter be abbreviated as DPA (n-3)), is newly reported to have 10 to 20 times the physiological activity as a coronary arteriosclerosis-suppressing factor than EPA. (Docosapentaenoic acid: another key n-
3 player? INFORM, 8,426-447 (1997)).

DPA (n-3) is an n-3 polyunsaturated fatty acid having 22 carbon atoms and having cis type unsaturated bonds at the 7, 10, 13, 16 and 19th carbons from the carboxylic acid terminal. , EPA is an n-3 polyunsaturated fatty acid with an extended carbon chain length, and is a fatty acid intermediate that is converted to DHA. Its presence in the natural world is rarely found in fish oil, and its presence is found only slightly in marine mammals. Existence is only recognized,
It is 1% or less in the whale (Sea whale) (Japan Oil Chemistry Association edition: Oil Chemistry Handbook 3rd revised edition pp.131, 1990, Maruzen Co., Ltd.).

In the labyrinthine marine fungi, D
Bacteria producing about 1 to 3% of PA (n-3) have been obtained, but their fatty acid content is low (Kendrick, A. et al.
and C. Ratledge, Lipids, 27, 15-10 (1992)). The biosynthetic pathway of DHA in the marine bacterium Schizochytrium sp. Is completely different from the DHA biosynthetic pathway in mammals, and DPA (n-6) is used as a precursor fatty acid, and DPA (n-3) is used. It is reported that the large accumulation of DPA (n-3) is unlikely due to its biosynthetic pathway, since () is not its precursor fatty acid (of highly unsaturated fatty acids in labyrinthula). Biosynthesis: Yutaka Aki, Osamu Suzuki, Ocean and Life, 23 (1) 46-
51 (2001). On the other hand, Traustochytrium s also belongs to the Labyrinthula family.
It was recently reported that a Δ4 fatty acid desaturase gene that converts DPA (n-3) as a precursor fatty acid into DHA was obtained from p.) (Identificaton of a Δ4 desatu
rase from Traustchytrium sp. involved in biosynthe
sis of docosahexaenoic acid by heterologous expres
sion in Saccharomyces cerevisiae and Brasixa junca
(2001), Xiao Quis, Haping Hong Samuel L. and L. Mack
enzie, J. Biological Chemistry).

[0006] There are various biosynthetic pathways of DHA in marine bacteria, and although the possibility of the presence of a strain that accumulates DPA (n-3) in a large amount cannot be ruled out, DPA
Attempts were made to screen a large number of samples of (n-3) for a large number of samples, but they have not been obtained at this stage. Even in marine bacteria, DPA (n-3) is a precursor fatty acid of DHA, so that it is promptly converted to DHA, and it is presumed that few strains accumulate in the cells.

[0007] DPA (n-3) is difficult to obtain from oils and fats of marine mammals due to its small content, and has not been marketed as a new n-3 polyunsaturated fatty acid material. In addition, since it is not easy to obtain the material, it cannot be said that the study on its physiological function has been sufficiently conducted. Further, EPA is abundant in fish oil, and its isolation has already been technically established.
Therefore, if EPA can be converted to DPA (n-3) by some method, it is possible to establish a high production technique of n-3 docosapentaenoic acid.

[0008]

SUMMARY OF THE INVENTION It is an object of the present invention to provide an industrial method for producing n-3 docosapentaenoic acid by extending the chain length of EPA.

[0009]

The present inventors have completed the present invention as a result of intensive studies to solve the above problems. According to the present invention, the following method is provided. (1) When culturing a transformant obtained by introducing a mammalian-derived fatty acid chain elongation enzyme gene in a medium, by adding icosapentaenoic acid to the medium, the carbon chain length of the icosapentaenoic acid is extended. And then converting into n-3 docosapentaenoic acid. (2) The method for producing n-3 docosapentaenoic acid according to (1) above, wherein the fatty acid chain extender gene is derived from a mammal. (3) The fatty acid chain elongation enzyme gene has a rat liver cDNA
The method for producing n-3 docosapentaenoic acid according to (2) above, which is a fatty acid chain length-extending enzyme gene cloned from the A library. (4) The n-3 docosapentaene according to (3) above, wherein the fatty acid chain length-extending enzyme gene is a gene encoding the amino acid sequence of 299 set forth in SEQ ID NO: 1 in Sequence Listing. Method for producing acid. (5) The fatty acid chain elongation enzyme gene is a gene having a 900 bp nucleotide sequence set forth in SEQ ID NO: 1 of the Sequence Listing, wherein the n-3 docosapentaenoic acid is set forth in (4) above. Manufacturing method. (6) Any of the above (1) to (5), wherein the transformant is transformed with an extrachromosomally integrated expression plasmid in the introduction of the fatty acid chain elongation enzyme gene into a host cell. The method for producing the n-3 docosapentaenoic acid according to Crab. (7) Any of the above-mentioned (1) to (5), wherein the transformant is transformed with an intrachromosomally integrated expression plasmid in the introduction of the fatty acid chain extender gene into a host cell. The method for producing the n-3 docosapentaenoic acid according to Crab. (8) The method for producing n-3 docosapentaenoic acid according to (6) or (7), wherein the host cell is a eukaryotic cell. (9) The method for producing n-3 docosapentaenoic acid according to (8) above, wherein the eukaryotic cell is yeast. (10) The yeast is Saccharomyces cerevisiae (Sacc
haromyces cerevisiae) or Pichia pastoris (Pich
ia pastoris), The method for producing n-3 docosapentaenoic acid according to (9) above. (11) GY medium, YM medium or medium composition, glucose 1-10%, yeast extract 0.1-3%,
NaNO ₃ 0.05-0.4%, KH ₂ PO ₄ 0.01
_{~0.2%, MgSO 4 · 7H 2} O 0.01~0.2%
The method for producing n-3 docosapentaenoic acid according to any one of (1) to (10) above, which is carried out at a culture temperature of 20 to 35 ° C. using a medium in which is dissolved in water. (12) The n-3 docosapentaenoic acid according to any one of (1) to (11) above, wherein the icosapentaenoic acid added to the medium is a free fatty acid or its methyl or ethyl ester. Manufacturing method. (13) n- as described in any of (1) to (12) above.
N-obtained by the method for producing 3-system docosapentaenoic acid
Type 3 docosapentaenoic acid or its ester.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The reaction raw material used in the present invention is icosapentaenoic acid or its ester. In this case, as the ester, a linear alkyl ester having 1 to 6 carbon atoms, preferably methyl and ethyl ester is used.

In the present invention, a transformant in which a mammalian-derived fatty acid chain elongation enzyme gene (hereinafter, also simply referred to as enzyme gene) expression plasmid (hereinafter, simply referred to as plasmid) is introduced as a vector into a host cell is cultured. At this time, icosapentaenoic acid or its ester is added to the medium.

The enzyme gene used in the present invention may be of mammalian origin. In this case, mammals include humans, mice, rats, monkeys, rabbits, guinea pigs and the like.

As the enzyme gene, rat liver cDNA
The fatty acid chain extender enzyme (rELO1) having an amino acid sequence deduced from the nucleotide sequence of the enzyme gene obtained from the library and cloned has a high homology (93.0%) with the human-derived enzyme (HELO1). ), But has low homology with nematodes (Cele; F56H11.4) and filamentous fungi (Malp; GLELO) (2
7.3% and 31.4%) are clear (Cloning of
a human cDNA encoding a novel enzyme involved in
the elongation of long-chain polyunsaturated fatty
acid (2000), Amenda E. Lonard, Emile G. Bobink, Jos
eph Dorad, Paul E. Kroeger, Lu-Te Chuang, Jennifer
M. Thurmond, Jennifer M. Parker-Branes, Tapas Da
s, Yung-Sheng Huang and Pradip Mukerji, Biochem.
J., 350,756-770)). Moreover, in the fatty acid chain length-extending enzymes of nematodes and filamentous fungi, the chain length of the fatty acid has 1 carbon atom.
It is recognized that 8 fatty acids have a function of extending to a carbon number of 20, but do not have a function of extending a fatty acid having 20 carbon atoms to a fatty acid having 22 carbon atoms (Heterologus reconstituti
on in yeast of the polyunsaturated fatty acid bios
ynthetic pathway (2000), Frederic Beaudoin, Louis
V. Michaelson, Sandra J. Hey, Mervyn J. Lewis, Pet
er R. Shewry, Olga Sayanova and Johnathan A. Napie
r, PANS, 97,6421-6426, Identification and characte
rization of an enzyme involved in the elongation o
f n-6 and n-3 polyunsaturated fatty acids (2000), Je
nnifer M. Paker-Barnes, Tapas Das, Emile Bobik, Am
anda E. Leonard, Jennifer M. Thurmond, Lu-Te Chaun
g, Yung-Sheng Huang, and Pradip Mukerji, PNAS, 97,8
284-8289). In order to extend the carbon chain length of icosapentaenoic acid having 20 carbon atoms to docosapentaenoic acid having 22 carbon atoms, a mammalian-derived fatty acid chain elongation enzyme is required, and n-
It is shown that the gene encoding the fatty acid chain length-extending enzyme for fulfilling the purpose of producing 3 system docosapentaenoic acid is of mammalian origin.

The cloning of the rat liver fatty acid chain extender gene was carried out by referring to the entire amino acid sequence (299 residues) of the fatty acid chain extender HELO1 identified in human, and using mouse EST clones homologous thereto as NCB clones.
A search in the I database gave AU079897 and BE535118 as those showing high homology. These overlap with each other and each contain a 5'side containing a start codon and a 3'region containing a stop codon. The sequence connecting them is HELO1.
Encoded 299 amino acids with high homology to. The primers used for PCR were designed based on the gene information of this mouse. As an example of primers used for PCR,
Primer F1 and primer R1 (for the respective base sequences, see SEQ ID NOs: 2 and 3 in the sequence listing) can be used. The primer F1 contains AAGCTT restriction enzyme Hi.
It contains the ndIII cleavage site and the start codon of ATG, and the primer R1 contains the cleavage site of TCTAGA by the restriction enzyme XbaI and the stop codon TCA. PCR using primers F1 and R1 as a template for rat liver cDNA library (Takara)
The amplification product (rELO1) sandwiched between the primers F1 and R1 was obtained, and the nucleotide sequence was determined.

The gene (rELO1) contains an open reading frame of 900 bp and encodes 299 amino acids, and its entire base sequence and deduced amino acid sequence are shown in SEQ ID NO: 1 of the sequence listing, respectively. Regarding the deduced amino acid sequence of the gene (rELO1), human (HELO1) and nematode (Caenohabditis elegans, Cele;
F56H11.4) and filamentous fungi (Mortierella alpina, Malp; GLE
(LO) was compared with fatty acid chain extender. In each amino acid sequence, a histidine cluster (HXXHH) of the type found in desaturase was conserved in one place.

A DNA fragment containing the open reading frame of the gene (rELO1) was inserted into the yeast expression vector pYES2 (manufactured by INVITROGEN), and r
A plasmid for extrachromosomal expression of ELO1 in yeast was constructed. By using an expression vector that integrates the enzyme gene into the chromosome, Saccharomyces cere
visiae) INVSc1 (his3, leu2, trp1, ura3) (INVITR
The yeast which has been introduced into yeast (eg, OGEN) and recombined is cultured by a conventional method. That is, using YPD medium, 28
After culturing with shaking at ℃ for 2 days and adding icosapentaenoic acid to the culture medium, DP
It was confirmed that A (n-3) was accumulated. Final concentration 2
Galactose was added to induce the expression so that the ratio became 0.1%, and after culturing for 28 hours, the cells were collected. After washing the cells with distilled water, chloroform-methanol (2: 1, v / v) was added to extract lipids. Add 10% hydrochloric acid-methanol to the chloroform layer 60
The fatty acid was methyl-esterified by treating at 3 ° C. for 3 hours.

The fatty acid methyl ester was extracted with hexane and then subjected to gas chromatography (Shimadzu G).
The fatty acid composition was analyzed by C-17A, column TC-70). The GC-MS analysis is based on the GC material (JEO
L) was used for EI-MS at 70 eV. As a result, a DNA fragment containing the open reading frame of the gene (rELO1) was inserted into an expression vector in yeast, and first subjected to yeast expression under the condition that no substrate fatty acid was added, a chain extension product of palmitooleic acid The formation of certain C18: 1n-7 was observed. γ-
In the presence of linolenic acid (GLA) or arachidonic acid, generation of peaks corresponding to dihomo-γ-linolenic acid (DGLA) and docosatetraenoic acid was observed, respectively.

As a result of expression of the gene (rELO1) in yeast in the presence of icosapentaenoic acid, the target substance n-3 docosapentaenoic acid could be produced at a conversion rate of 51.6%. (Figure 1). In addition, α-linolenic acid, n-3 octadecatetraenoic acid, and DGLA were also found to have chain length extension, and the conversion rates of these substrate fatty acids are shown in Table 1. Regarding mead acid (C20: 3n-9) and n-6 docosatetraenoic acid (C22: 4n-6), no chain extension was observed even when added as a substrate fatty acid.

Table 1 summarizes the substrate specificity of the gene (rELO1) expressing enzyme.

[0020]

[Table 1]

* Conversion rate =% produced fatty acid / (% produced fatty acid +% substrate fatty acid) × 100, the average value of 3 times is shown.

The gene (rELO1) cloned from a rat liver cDNA library encodes a fatty acid chain extension enzyme, and the enzyme produced by the gene (rELO1) extends the chain length of a fatty acid from It is an enzyme having a function of converting into n-3 type docosapentaenoic acid.

In the present invention, as an expression vector in a host cell for active expression of the enzyme gene, for example, the gene (rELO1) is incorporated into the host cell, and an extrachromosomal integration plasmid such as yeast expression plasmid pYES2 is used. A transformant transformed with / rELO1 is used. That is, for the active expression of the enzyme gene, the yeast Saccharomyces cerevisiae (Sa
The extrachromosomal expression plasmid pYES2 / rELO1 (6.8 kb) of ccharomyces cerevisiae) is shown in FIG. The integration of the plasmid into the yeast Saccharomyces cerevisiae is URA3
Selected by marker. Gene expression is. It is controlled by the GAL1 promoter (PGAL1) and the CYC1 terminator (TCYC1) and is induced by galactose.

The plasmid (pYES2 / rELO1)
Transformation of yeast into yeast is preferably performed as follows. Saccharomyces cerevisiae
myces cerevisiae) INVSc1 (his3, leu2, trp1, ura3) (I
Yeast (such as NVITROGEN) is cultured by a conventional method. That is, permeation culture was performed at 28 ° C. for 2 days using YPD medium. The obtained culture solution is purified by repeated centrifugation, and then the lithium acetate / PEG solution and the above plasmid vector pYES2 / rELO1 solution are added, mixed well, and then cultured again. The culture is preferably carried out in a sufficiently dried SD medium at 28 ° C. for 3 to 4 days.

The gene of the present invention (rELO1)
For integration into a host cell, an intrachromosomal integration plasmid such as yeast expression plasmid pMO3 / rELO
Transformed with 1. That is, for the active expression of the enzyme gene, the yeast Saccharomyces cerevisiae (Sacc) constructed as an example of an expression vector in a host cell was used.
haromyces cerevisiae) plasmid p for intrachromosomal expression
MO3 / rELO1 (6.8 kb) is shown in FIG.
When the plasmid is introduced into yeast after digested with a restriction enzyme at the XhoI site, it is randomly integrated into chromosomal DNA by the δ sequence (transposon). Transformation of the yeast expression plasmid vector pMO3 / rELO1 into yeast is carried out using the plasmid vector pYES2 /
Performed similarly to rELO1 and selected by the URA3 marker, but does not require selection after chromosomal integration.

In the present invention, the host cell for expressing the gene may be a eukaryotic cell. That is, the above Saccharomyces cerevisiae
other yeast or Mucor s (Mucor s)
p.), Mortierella sp., and other filamentous fungi, as well as plants and animals as host cells, can be transformed by selecting appropriate conditions for each. When these transformants are used, by appropriately supplying icosapentaenoic acid and adjusting the expression conditions of the gene, it is possible to convert icosapentaenoic acid to n-3 docosapentaenoic acid by extending the carbon chain length. .

In the present invention, among the eukaryotic cells,
Yeast is preferably used as a host cell for preparing a transformant involved in the production of the n-3 docosapentaenoic acid. Yeast is a eukaryotic cell, and in particular, it has an excellent function for expression of a mammalian-derived fatty acid desaturase and fatty acid chain elongation enzyme gene, and moreover, a transformant into which the gene has been introduced, As described in the embodiment of the production method, many conventional knowledge such as the construction of vector plasmids can be utilized for the production, and in the functional expression of the produced transformant, it can be easily cultured. In setting culture conditions for promoting expression, it has an excellent function in terms of simplicity in setting conditions for adding icosapentaenoic acid as a raw material for producing n-3 docosapentaenoic acid. ing.

In the present invention, among the yeasts, the n
It is preferable to use Saccharomyces cerevisiae or Pichia pastoris as a host cell for producing a transformant involved in the production of -3 system docosapentaenoic acid. The extrachromosomal expression plasmid pYES shown in FIG. 2 for incorporating into the yeast Saccharomyces cerevisiae constructed as an example of an expression vector in a host cell for active expression of the long enzyme gene.
2 / rELO1 (6.8 kb) is Saccharomyces cerevisiae INVSc1
(His3, leu2, trp1, ura3) (manufactured by INVITROGEN) and incorporated into yeast. A transformant production system using yeast Pichia pastoris as a host cell is excellent in transformant expression in the host and vector systems, and has excellent bacterial growth performance. It can be used as a desirable host cell for industrial production of n-3 docosapentaenoic acid.

In the present invention, the transformant, for example, the transformed yeast YEL-5 strain (FERM), is mixed with GY medium,
YM medium or medium composition, 1-10% preferably 2 to 8% glucose, 0.1% to 3% preferably 0.5-2% yeast extract, NaNO ₃ 0.05 to 0.4% preferably 0.1 ~0.3%, KH ₂ PO ₄ 0.01~0.2
% Preferably _{0.02~0.1%, MgSO 4 · 7H 2} O
The culture temperature is 20 to 35 ° C., preferably 2 to 0.01 to 0.2%, preferably 0.02 to 0.1% dissolved in water.
Cultivated at 5 to 32 ° C. and icosapentaenoic acid 0.1 to 20
It is characterized by adding 0 mM, preferably 0.5 to 100 mM, to accumulate the fatty acid inside or outside the cell. That is, when culturing the transformant, the culture conditions such as the type of carbon source and nitrogen source of the medium, the culture temperature, and the culture time are optimal for producing a large amount of n-3 docosapentaenoic acid. The conditions can be appropriately selected.

Hereinafter, the present invention will be described in detail with reference to Examples. The present invention is not limited to the embodiments. Unless otherwise specified, the enzymes and reagents used in the examples were those at the analytical and biochemical levels purchased from Takara Shuzo, Sigma, Nacalai Tesque, Katayama Kagaku or Difco.

Example 1 Cloning of rat fatty acid chain extender gene.

A mouse EST clone homologous to the entire amino acid sequence (299 residues) of the fatty acid chain extender HELO1 identified in human was searched in the NCBI database, and AU079897 and BE535118 was obtained. These overlapped each other and each contained a 5'side containing a start codon and a 3'region containing a stop codon, and the concatenated sequence encoded 299 amino acids highly homologous to HELO1. From the gene information of this mouse, the following primer F1 and primer R
Created 1. The primer F1 contains the restriction enzyme HindIII cleavage site of AAGCTT and the start codon of ATG, and the primer R1 contains the restriction enzyme Xba of TCTAGA.
It contains a cleavage site by I and the stop codon TCA.
F1,5'-AGGTAAGCTT ATG GAACAT
TTCGATGTCATCT-3 ′ (SEQ ID NO: 2) (Italic type is HindIII site, underline is start codon) R1,5′-CTCTCTAGA TCA ATCCCTTT
CGCTGCTTCCTGGG-3 '(SEQ ID NO: 3) (Italic type indicates Xba I site, underline indicates stop codon)

Using these, PCR was carried out using a rat liver cDNA library (Takara) as a template. At this time, TdT with high fidelity as a polymerase
(Terminal nucleic acid-adding enzyme) KOD-plus (T
OYOBO) was used. The composition of the reaction mixture is as follows.

[0034]

[Table 2]

The reaction condition of PCR is 94 ° C. × 2 sec, then 94 ° C. × 15 sec / 60 ° C. × 30 sec.
/ 68 ° C x 1 min was set as one cycle and repeated 35 times. The reaction solution was subjected to agarose gel electrophoresis, stained, and then irradiated with ultraviolet rays to confirm one band.
The amplified fragment (rELO1) obtained as a result was designated as pYES2.
(Invitrogen) was inserted into the corresponding site to prepare a transformant, and the nucleotide sequence was determined by the dye terminator method.

The specific method of carrying out the method will be described below. (1) Extraction of DNA fragment from agarose gel The DNA fragment of interest confirmed by UV transilluminator was cut out from the agarose gel and subjected to CONCERT.
Rapid Gel Extraction System (GIBCOBRL) and QIAEX II Gel Extraction Kit (QI
Purification was performed using each purification kit manufactured by AGEN).
The method was according to the attached protocol. (2) Ligation into vector The fragment obtained and purified in (1) above was transformed into pGE
M-T easy vector system (PROMEGA)
Were mixed so that the insert: vector ratio was 1:10. The same amount of Ligation High (TOYOBO
(Manufactured by the company) was added, and the mixture was incubated at 16 ° C for 1 hour or more.

(3) Preparation of competent cells E. E. coli DH5α was streaked on M9 plate and cultured at 37 ° C. until colonies could be confirmed. Scrap a single colony, 100 mL (1 L flask)
Was suspended in SOB medium and cultured with shaking at 18 ° C. OD
When 600 = 0.4 to 0.8 was reached, the culture was terminated and the culture solution was immediately cooled in ice water for 10 minutes. The culture was centrifuged at 4 ° C. at 3000 rpm for 15 minutes and the supernatant was discarded. 33 mL of ice-cold Transformation buffer
After suspending in (TB), the mixture was ice-cooled for 10 minutes. Then, the cells were collected by centrifugation at 3000 rpm for 15 minutes at 4 ° C., and suspended in 4 mL of ice-cold TB. 1 mL of 5
0% glycerol was added, dispensed in 400 μL aliquots, frozen with liquid nitrogen and stored at −80 ° C. The TB used here was 10 mM PIPES, 15 mM CaCl.
₂ · 2H ₂ O, 250mM KCl and 55 mM Mn
It is composed of Cl ₂ .4H ₂ O.

(4) Transformation into E. coli E. coli prepared as competent cells E. coli DH5α was thawed on ice and mixed with the ligated vector solution. After incubating on ice for 5 minutes, heat shock was applied at 37 ° C. for 2 minutes, and then incubation was again performed on ice for 5 minutes. Next, add 900 μL of sterilized water to this and 1200
It was centrifuged at 0 rpm for 2 minutes. Discard the supernatant and discard the precipitate
It was suspended in L 100 mM IPTG and seeded on an LB plate medium containing X-Gal and ampicillin. After incubating the plate at 37 ° C. for 12 hours, the presence or absence of colonies was confirmed.

(5) Preparation of plasmid on mini scale An appropriate amount of Escherichia coli solution that had been cultured overnight was sampled, and 9000 rpm
The cells were recovered by centrifugation at 2 minutes. To Soluti
100 μL of onI was added and vortex-suspended, and 200 μL of Solution II was further added to lyse the cells. Incubate on ice for 5 minutes, then Solution II
After adding I of 200 μL and vortexing, 12000r
It was centrifuged at pm for 10 minutes. The obtained supernatant was collected in another tube, and an equal amount of isopropanol was added to the mixture to purify it by alcohol precipitation. The Solution used here
I to III have the following compositions. Solution I: 50 mM glucose, 25 mM Tris-HCl (pH 8.
0), 10 mM EDTA SolutionII: 0.2N NaOH, 1% SDSSolutionIII: 3M K-
acetate, 11.5% acetate

(6) Confirmation of insert by digestion with restriction enzymes Each enzyme, buffer and sample DNA were mixed in the following composition.

[Table 3] Depending on the type of restriction enzyme, selection of the type of buffer and addition of Triton X-100 and BSA were appropriately performed. The reaction was carried out at 37 ° C.

(7) Incorporation into yeast expression plasmid vector In the above, pGEM-T easy vector.
The rat liver fatty acid chain elongation enzyme gene (rELO1) cloned in (PROMEGA) was HindII
The insert was purified by digestion with restriction enzymes I and XbaI, agarose gel electrophoresis and gel extraction, and the insert was purified. This insert was also digested with HindIII and XbaI and then purified by dephosphorylation to obtain p
Ligation into the HindIII and XbaI sites of YES2, E. coli DH5α. The colonies were subjected to liquid culture overnight, and a small amount of plasmid was purified, insert check by restriction enzyme digestion, and forward / reverse determination were performed. The constructed plasmid vector was designated as pYES2 / r.
It was named ELO1.

The base sequence was determined by the dye terminator method as follows. DYEnamic ET Terminator Cyc
Samples were prepared using le Sequencing Kit (Amersham Pharmacia) and 310 Genetic Analyzer (Applied B
The sequence was determined by iosystems. as a result,
The obtained rat liver fatty acid chain length-extending enzyme gene (rELO
1) contains an open reading frame of 900 bp and encodes 299 amino acids, and its entire base sequence and its deduced amino acid sequence are shown in SEQ ID NO: 1 of the sequence listing, respectively. Regarding the deduced amino acid sequence of the rat liver fatty acid chain elongation enzyme gene (rELO1) (SEQ ID NO: 1 in the sequence listing), human (HELO1) and nematode (Caen)
ohabditis elegans, Cele; F56H11.4) and filamentous fungi (Mortier
ella alpina, Malp; GLELO) and a fatty acid chain extender.

Although rELO1 has a very high homology (93.0%) with HELO1, the nematode (Cele;
F56H11.4) and filamentous fungus (Malp; GLEL)
Low homology with O) (27.3% and 31.4)
%) Has been revealed. In each amino acid sequence, a histidine cluster (HXXHH) of the type found in desaturase was conserved in one place. From these results, it is clear that the fragment consisting of the nucleotide sequence set forth in SEQ ID NO: 1 in this sequence listing is a gene encoding a fatty acid chain length-extending enzyme.

Example 2 Construction of a plasmid vector for extrachromosomal expression of rat fatty acid chain elongation enzyme gene (rELO1).

Yeast Saccharomyces cerevisiae (Saccha
romyces cerevisiae) extrachromosomal expression vector pY
Using ES2 (manufactured by INVITROGEN), the rat fatty acid chain elongation enzyme gene (rELO1) in yeast
A plasmid vector for expressing the activity of was constructed as follows. The plasmid pYES2 / rELO1 (6.8) for extrachromosomal expression of the yeast Saccharomyces cerevisiae constructed in Example 1.
kb) is shown in FIG. Integration of this plasmid is selected by the URA3 marker. Gene expression is GA
It is regulated by the L1 promoter (PGAL1) and the CYC1 terminator (TCYC1) and is induced by galactose.

Example 3 A plasmid vector for intrachromosomal integration expression of rat fatty acid chain elongation enzyme gene (rELO1).

Yeast Saccharomyces cerevisiae (Saccha
romyces cerevisiae) plasmid vector pMO3 (manufactured by INVITROGEN) for expression in the chromosome.
Was used to construct a plasmid vector for expressing the activity of rat fatty acid chain elongation enzyme gene (rELO1) in yeast as follows. The yeast Saccharomyces cerevisiae constructed in the same manner as in Example 1 (Saccharo
The plasmid vector pMO3 / rELO1 (7.8 kb) for intrachromosomal integration expression of myces cerevisiae) is shown in FIG. When this plasmid is introduced into yeast after digestion with a restriction enzyme at the XhoI site, it is randomly integrated into chromosomal DNA by the δ sequence (transposon). It is selected by the URA3 marker but does not require selection after it has been integrated into the chromosome. Gene expression is GA
It is regulated by the P promoter (PGAP) and GAP terminator (TGAP), and is constitutively expressed.

Example 4 Transformation of a rat fatty acid chain elongase gene (rELO1) into yeast and expression of activity of the gene in yeast.

The rELO1 gene extrachromosomal expression plasmid vector (pYES2 / rELO1, FIG. 2) was transformed into the yeast Saccharomyces cerevisiae (Saccha) by the lithium acetate method.
romyces cerevisiae) INVSc1 (α / a, his3, leu2, ura3, trp
1) (manufactured by INVITROGEN) and transformed. The procedure is as follows. Saccharomyces cerevisiae (Saccharomyce) was added to 5 mL of YPD liquid medium.
S. cerevisiae) INVSc1 strain was inoculated at 28 ° C for 2
Shaking culture was performed for a day. The composition of the YPD liquid medium is glucose 2%, polypeptone 2% and yeast extract 1%.
Is included. Take 1 mL of the culture solution into a tube and 2
After centrifugation at 000 rpm for 5 minutes, the supernatant was discarded. After adding 1 mL of lithium acetate solution to the precipitate and mixing well, 200
After centrifugation at 0 rpm for 5 minutes, the supernatant was discarded.

The lithium acetate solution used here is
100 mM lithium acetate, 10 mM Tris HC
1 (pH 7.5) and 1 mM EDTA. Next, 200 μ of lithium acetate / PEG solution for precipitation
L and 5 μL of plasmid solution (1 μg / μL) were added,
After mixing well, the mixture was incubated in a 30 ° C. water bath for 1 hour. Then, it was further incubated in a water bath at 42 ° C for 10 minutes. Lithium acetate / PE used here
G solution is 100 mM lithium acetate, 10 mM Tr
is HCl (pH 7.5), 1 mM EDTA and 40% PEG4000. Subsequently, this culture was plated on well-dried SD medium and cultured at 28 ° C. for 3 or 4 days. The composition of SD medium is glucose 2
%, Yeast nitrogen-base w / o amino acid 0.7%,
Histidine 20 μg / mL, Leucine 60 μg / m
L, tryptophan 40 μg / mL.

The expression of the activity of the fatty acid chain extender gene in yeast will be described below. The colonies that appeared on the SD medium were scraped off, and 5 mL of SCT medium (selection medium)
Preculture was performed overnight at 28 ° C. The composition of SCT medium was raffinose 4%, yeast nitrogen-base w / o.
amino acid 0.7%, surfactant (trade name: terg
Itol) 1%, histidine 20 μg / mL, leucine 60 μg / mL, tryptophan 40 μg / mL. SCT medium, linoleic acid (C18: 2n-6),
α-linolenic acid (C18: 3n-3), γ-linolenic acid (C18: 3n-6), n-3 octadecatetraenoic acid (C18: 4n-3), dihomo-γ-linolenic acid (C2)
0: 3n-6), mead acid (C20: 3n-9), arachidonic acid (C20: 4n-6), icosapentaenoic acid (C20: 5n-3) and n-6 docosatetraenoic acid (C20: 4n-6). 1) as a substrate
45 mL of each was prepared by adding mM. To each of these 9 SCT media and the media without the addition of fatty acids,
The preculture solution was inoculated so that the number of cells was 2 × 10 ⁵ / mL, galactose was added while shaking at 28 ° C., and the cells were further cultured for 16 hours.

After the completion of the culture, the culture solution was transferred to a 50 mL conical tube, centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. Next, 5 mL of sterilized water was added to the cells, vortexed, centrifuged again at 2000 rpm for 5 minutes, and the supernatant was discarded. Extraction of lipids from the cells was performed as follows.
Suspend the cells in 1 mL of sterilized water, transfer to a screw cap test tube, and equal volume of chloroform-methanol (2: 1, v / v)
Was added, vortexed, and centrifuged at 2000 rpm for 2 minutes. Take the lower chloroform layer into a test tube, add 10% hydrochloric acid-methanol to the chloroform layer, and add 60%.
Methyl esterification was carried out by treating at 3 ° C for 3 hours.
After extracting the fatty acid methyl ester with hexane, the fatty acid composition was analyzed by gas chromatography (Shimadzu GC-17A, column TC-70). GC-MS
Analysis is performed using GC material (JEOL), 70 eV
EI-MS in.

As a result, a DNA fragment containing the open reading frame of the rat liver fatty acid chain elongase gene (rELO1) was inserted into the yeast expression vector, and first,
When subjected to yeast expression without the addition of substrate fatty acid, the chain extension product of palmitooleic acid, C18: 1n
Formation of -7 was observed. At this time, a peak, which is a trace amount and seems to correspond to C20: 1n-7 in which the chain length was further extended, was observed at 9.6 min, and the chain length extension products derived from these endogenous fatty acids were added with the substrate fatty acid. If also detected. In the presence of γ-linolenic acid (GLA) or arachidonic acid, generation of peaks corresponding to dihomo-γ-linolenic acid (DGLA) and docosatetraenoic acid was observed, respectively. In addition, when the expression analysis was performed in the presence of linoleic acid, the production of a chain length extension product, C20: 2Δ11,14, was confirmed although it was a trace amount. As a result of expressing rELO1 in yeast in the presence of icosapentaenoic acid, the target substance n-3 docosapentaenoic acid could be produced at a conversion rate of 51.6% (FIG. 1).

In addition, α-linolenic acid, n-3 octadecatetraenoic acid and DGLA were also found to have chain length extension, and the conversion rates of these substrate fatty acids are shown in Table 1. For mead acid (C20: 3n-9) and n-6 docosatetraenoic acid (C22: 4n-6), chain length extension was not observed even when the substrate fatty acid was added (Table 1). From this, in the yeast YEL-5 strain transformed with the rat liver fatty acid chain elongation enzyme gene, it was converted to n-3 docosapentaenoic acid by culturing in the presence of icosapentaenoic acid, and n-3 docosapentaenoic acid was converted. It was confirmed that enoic acid was produced.

[0055]

[Table 4]

[0056]

[Table 5]

[0057]

[Table 6]

[0058]

[Table 7]

[0059]

INDUSTRIAL APPLICABILITY The method for producing n-3 docosapentaenoic acid according to the present invention has no conventional production technique for n-3.
It enables the factory production of docosapentaenoic acid. In addition, n-3 docosapentaenoic acid is expected to have high physiological activity, and its product is expected to have a wide range of uses such as pharmaceuticals, health foods, and specified health foods.

[Brief description of drawings]

FIG. 1 is a diagram showing the results of gas chromatography (GC) of fatty acids generated by yeast expression of the fatty acid chain length-extending enzyme gene (rELO1) introduced into the transformed yeast of the present invention. That is, pYES2 or pYE
The plasmid in which the rELO1 gene was inserted into S2 was used for the yeast Saccharomyces cerevisiae.
e) to induce expression by galactose,
The intracellular fatty acid was analyzed by GC. The production of DPA (n-3) was confirmed by adding EPA as a substrate. EPA is icosapentaenoic acid (C20: 5n-3), DPA is n-
3 shows docosapentaenoic acid (C22: 5n-3), respectively.

[Fig. 2] Plasmid for extrachromosomal expression of rat fatty acid chain elongation enzyme (rELO1) (pYES2 / rELO1)
It is the figure which showed. In the figure, PGAL1 is GAL1 promoter, TCYC1 is CYC1 terminator, URA
3 indicates a marker.

[Fig. 3] A plasmid for expression in the chromosome of rat fatty acid chain elongase rELO1 (pMO3 / rELO1).
It is the figure which showed. In the figure, PGAP is a GAP promoter, TGAP is a GAP terminator, δ is a transposon δ sequence, and URA3 is a marker.

[Procedure amendment]

[Submission date] September 30, 2002 (2002.9.3)
0)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction content]

[Document name] Statement

Title of the invention Method for producing n-3 docosapentaenoic acid

[Claims]

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing n-3 docosapentaenoic acid.

[0002]

2. Description of the Related Art In n-3 polyunsaturated fatty acids, there are three or more double bonds including a methylene group, and the position of the first double bond is present at the third carbon from the methyl end. A generic term for fatty acids having 18 to 22 carbon atoms. This n-3 polyunsaturated fatty acid is
By ingesting α-linolenic acid (ALA, C18: 3n-3), which is an essential fatty acid produced in plants such as soybean, the introduction of double bonds by desaturase and chain extension enzyme and carbon chain extension Repeatedly, Icosapentaenoic acid (EP, a direct precursor fatty acid of prostaglandin III group)
A, C20: 5n-3) and the final product, docosahexaenoic acid (DHA, C22: 6n-), which is abundantly present in the retina or cranial nervous system and is known to have an extremely important function.
Converted to 3) to maintain homeostasis in the body (homeostasis)
Play an extremely large role in.

As n-3 polyunsaturated fatty acids, EPA and DHA are already well known, and materials extracted and purified from fish oils such as tuna, sardines, mackerel, and saury are enriched in various foods. Used as an additive. Among the n-3 polyunsaturated fatty acids, the existence thereof is known, but the research on its physiological action has not been advanced in practice. n-3 docosapentaenoic acid (C2
2: 5n-3, which may hereinafter be abbreviated as DPA (n-3)), is newly reported to have 10 to 20 times the physiological activity as a coronary arteriosclerosis-suppressing factor than EPA. (Docosapentaenoic acid: another key n-
3 player? INFORM, 8,426-447 (1997)).

DPA (n-3) is an n-3 polyunsaturated fatty acid having 22 carbon atoms and having cis type unsaturated bonds at the 7, 10, 13, 16 and 19th carbons from the carboxylic acid terminal. , EPA is an n-3 polyunsaturated fatty acid with an extended carbon chain length, and is a fatty acid intermediate that is converted to DHA. Its presence in the natural world is rarely found in fish oil, and its presence is found only slightly in marine mammals. Existence is only recognized,
It is 1% or less in the whale (Sea whale) (Japan Oil Chemistry Association edition: Oil Chemistry Handbook 3rd revised edition pp.131, 1990, Maruzen Co., Ltd.).

In the labyrinthine marine fungi, D
Bacteria producing about 1 to 3% of PA (n-3) have been obtained, but their fatty acid content is low (Kendrick, A. et al.
and C. Ratledge, Lipids, 27, 15-10 (1992)). The biosynthetic pathway of DHA in the marine bacterium Schizochytrium sp. Is completely different from the DHA biosynthetic pathway in mammals, and DPA (n-6) is used as a precursor fatty acid, and DPA (n-3) is used. It is reported that the large accumulation of DPA (n-3) is unlikely due to its biosynthetic pathway, since () is not its precursor fatty acid (of highly unsaturated fatty acids in labyrinthula). Biosynthesis: Yutaka Aki, Osamu Suzuki, Ocean and Life, 23 (1) 46-
51 (2001). On the other hand, Traustochytrium s also belongs to the Labyrinthula family.
It was recently reported that a Δ4 fatty acid desaturase gene that converts DPA (n-3) as a precursor fatty acid into DHA was obtained from p.) (Identificaton of a Δ4 desatu
rase from Traustchytrium sp. involved in biosynthe
sis of docosahexaenoic acid by heterologous expres
sion in Saccharomyces cerevisiae and Brasixa junc
a (2001), Xiao Quis, Haping Hong Samuel L. and L. Mac
kenzie, J. Biological Chemistry).

[0006] There are various biosynthetic pathways of DHA in marine bacteria, and although the possibility of the presence of a strain that accumulates DPA (n-3) in a large amount cannot be ruled out, DPA
Attempts were made to screen a large number of samples of (n-3) for a large number of samples, but they have not been obtained at this stage. Even in marine bacteria, DPA (n-3) is a precursor fatty acid of DHA, so that it is promptly converted to DHA, and it is presumed that few strains accumulate in the cells.

[0007] DPA (n-3) is difficult to obtain from oils and fats of marine mammals due to its small content, and has not been marketed as a new n-3 polyunsaturated fatty acid material. In addition, since it is not easy to obtain the material, it cannot be said that the study on its physiological function has been sufficiently conducted. Further, EPA is abundant in fish oil, and its isolation has already been technically established.
Therefore, if EPA can be converted to DPA (n-3) by some method, it is possible to establish a high production technique of n-3 docosapentaenoic acid.

[0008]

SUMMARY OF THE INVENTION It is an object of the present invention to provide an industrial method for producing n-3 docosapentaenoic acid by extending the chain length of EPA.

[0009]

The present inventors have completed the present invention as a result of intensive studies to solve the above problems. According to the present invention, the following method is provided. (1) When culturing a transformant obtained by introducing a mammalian-derived fatty acid chain elongation enzyme gene in a medium, by adding icosapentaenoic acid to the medium, the carbon chain length of the icosapentaenoic acid is extended. And then converting into n-3 docosapentaenoic acid. (2) The method for producing n-3 docosapentaenoic acid according to (1) above, wherein the fatty acid chain extender gene is derived from a mammal. (3) The fatty acid chain elongation enzyme gene has a rat liver cDNA
The method for producing n-3 docosapentaenoic acid according to (2) above, which is a fatty acid chain length-extending enzyme gene cloned from the A library. (4) The n-3 docosapentaene according to (3) above, wherein the fatty acid chain length-extending enzyme gene is a gene encoding the amino acid sequence of 299 set forth in SEQ ID NO: 1 in Sequence Listing. Method for producing acid. (5) The fatty acid chain elongation enzyme gene is a gene having a 900 bp nucleotide sequence set forth in SEQ ID NO: 1 of the Sequence Listing, wherein the n-3 docosapentaenoic acid is set forth in (4) above. Manufacturing method. (6) Any of the above (1) to (5), wherein the transformant is transformed with an extrachromosomally integrated expression plasmid in the introduction of the fatty acid chain elongation enzyme gene into a host cell. The method for producing the n-3 docosapentaenoic acid according to Crab. (7) Any of the above-mentioned (1) to (5), wherein the transformant is transformed with an intrachromosomally integrated expression plasmid in the introduction of the fatty acid chain extender gene into a host cell. The method for producing the n-3 docosapentaenoic acid according to Crab. (8) The method for producing n-3 docosapentaenoic acid according to (6) or (7), wherein the host cell is a eukaryotic cell. (9) The method for producing n-3 docosapentaenoic acid according to (8) above, wherein the eukaryotic cell is yeast. (10) The yeast is Saccharomyces cerevisiae (Sacc
haromyces cerevisiae) or Pichia pastoris (Pich
ia pastoris), The method for producing n-3 docosapentaenoic acid according to (9) above. (11) GY medium, YM medium or medium composition, glucose 1-10%, yeast extract 0.1-3%,
NaNO ₃ 0.05-0.4%, KH ₂ PO ₄ 0.01
_{~0.2%, MgSO 4 · 7H 2} O 0.01~0.2%
The method for producing n-3 docosapentaenoic acid according to any one of (1) to (10) above, which is carried out at a culture temperature of 20 to 35 ° C. using a medium in which is dissolved in water. (12) The n-3 docosapentaenoic acid according to any one of (1) to (11) above, wherein the icosapentaenoic acid added to the medium is a free fatty acid or its methyl or ethyl ester. Manufacturing method. (13) n- as described in any of (1) to (12) above.
N-obtained by the method for producing 3-system docosapentaenoic acid
Type 3 docosapentaenoic acid or its ester.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The reaction raw material used in the present invention is icosapentaenoic acid or its ester. In this case, as the ester, a linear alkyl ester having 1 to 6 carbon atoms, preferably methyl and ethyl ester is used.

In the present invention, a transformant in which a mammalian-derived fatty acid chain elongation enzyme gene (hereinafter, also simply referred to as enzyme gene) expression plasmid (hereinafter, simply referred to as plasmid) is introduced as a vector into a host cell is cultured. At this time, icosapentaenoic acid or its ester is added to the medium.

The enzyme gene used in the present invention may be of mammalian origin. In this case, mammals include humans, mice, rats, monkeys, rabbits, guinea pigs and the like.

As the enzyme gene, rat liver cDNA
The fatty acid chain extender enzyme (rELO1) having an amino acid sequence deduced from the nucleotide sequence of the enzyme gene obtained from the library and cloned has a high homology (93.0%) with the human-derived enzyme (HELO1). ), But has low homology with nematodes (Cele; F56H11.4) and filamentous fungi (Malp; GLELO) (2
7.3% and 31.4%) are clear (Cloning of
a human cDNA encoding a novel enzyme involved in
the elongation of long-chain polyunsaturated fatty
acid (2000), Amenda E. Lonard, Emile G. Bobink, Jos
eph Dorad, Paul E. Kroeger, Lu-Te Chuang, Jennifer
M. Thurmond, Jennifer M. Parker-Branes, Tapas Da
s, Yung-Sheng Huang and Pradip Mukerji, Biochem.
J., 350,756-770)). Moreover, in the fatty acid chain length-extending enzymes of nematodes and filamentous fungi, the chain length of the fatty acid has 1 carbon atom.
It is recognized that 8 fatty acids have a function of extending to a carbon number of 20, but do not have a function of extending a fatty acid having 20 carbon atoms to a fatty acid having 22 carbon atoms (Heterologus reconstituti
on in yeast of the polyunsaturated fatty acid bios
ynthetic pathway (2000), Frederic Beaudoin, Louis
V. Michaelson, Sandra J. Hey, Mervyn J. Lewis, Pet
er R. Shewry, Olga Sayanova and Johnathan A. Napie
r, PANS, 97,6421-6426, Identification and characte
rization of an enzyme involved in the elongation o
f n-6 and n-3 polyunsaturated fatty acids (2000), Je
nnifer M. Paker-Barnes, Tapas Das, Emile Bobik, Am
anda E. Leonard, Jennifer M. Thurmond, Lu-Te Chaun
g, Yung-Sheng Huang, and Pradip Mukerji, PNAS, 97,8
284-8289). In order to extend the carbon chain length of icosapentaenoic acid having 20 carbon atoms to docosapentaenoic acid having 22 carbon atoms, a mammalian-derived fatty acid chain elongation enzyme is required, and n-
It is shown that the gene encoding the fatty acid chain length-extending enzyme for fulfilling the purpose of producing 3 system docosapentaenoic acid is of mammalian origin.

The cloning of the rat liver fatty acid chain extender gene was carried out by referring to the entire amino acid sequence (299 residues) of the fatty acid chain extender HELO1 identified in human, and using mouse EST clones homologous thereto as NCB clones.
A search in the I database gave AU079897 and BE535118 as those showing high homology. These overlap with each other and each contain a 5'side containing a start codon and a 3'region containing a stop codon. The sequence connecting them is HELO1.
Encoded 299 amino acids with high homology to. The primers used for PCR were designed based on the gene information of this mouse. As an example of primers used for PCR,
Primer F1 and primer R1 (for the respective base sequences, see SEQ ID NOs: 2 and 3 in the sequence listing) can be used. The primer F1 contains AAGCTT restriction enzyme Hi.
It contains the ndIII cleavage site and the start codon of ATG, and the primer R1 contains the cleavage site of TCTAGA by the restriction enzyme XbaI and the stop codon TCA. PCR using primers F1 and R1 as a template for rat liver cDNA library (Takara)
The amplification product (rELO1) sandwiched between the primers F1 and R1 was obtained, and the nucleotide sequence was determined.

The gene (rELO1) contains an open reading frame of 900 bp and encodes 299 amino acids, and its entire base sequence and deduced amino acid sequence are shown in SEQ ID NO: 1 of the sequence listing, respectively. Regarding the deduced amino acid sequence of the gene (rELO1), human (HELO1) and nematode (Caenohabditis elegans, Cele;
F56H11.4) and filamentous fungi (Mortierella alpina, Malp; GLE
(LO) was compared with fatty acid chain extender. In each amino acid sequence, a histidine cluster (HXXHH) of the type found in desaturase was conserved in one place.

A DNA fragment containing the open reading frame of the gene (rELO1) was inserted into the yeast expression vector pYES2 (manufactured by INVITROGEN), and r
A plasmid for extrachromosomal expression of ELO1 in yeast was constructed. By using an expression vector that integrates the enzyme gene into the chromosome, Saccharomyces cere
visiae) INVSc1 (his3, leu2, trp1, ura3) (INVITR
The yeast which has been introduced into yeast (eg, OGEN) and recombined is cultured by a conventional method. That is, using YPD medium, 28
After culturing with shaking at ℃ for 2 days and adding icosapentaenoic acid to the culture medium, DP
It was confirmed that A (n-3) was accumulated. Final concentration 2
Galactose was added to induce the expression so that the ratio became 0.1%, and after culturing for 28 hours, the cells were collected. After washing the cells with distilled water, chloroform-methanol (2: 1, v / v) was added to extract lipids. Add 10% hydrochloric acid-methanol to the chloroform layer 60
The fatty acid was methyl-esterified by treating at 3 ° C. for 3 hours.

The fatty acid methyl ester was extracted with hexane and then subjected to gas chromatography (Shimadzu G).
The fatty acid composition was analyzed by C-17A, column TC-70). The GC-MS analysis is based on the GC material (JEO
L) was used for EI-MS at 70 eV. As a result, a DNA fragment containing the open reading frame of the gene (rELO1) was inserted into an expression vector in yeast, and first subjected to yeast expression under the condition that no substrate fatty acid was added, a chain extension product of palmitooleic acid The formation of certain C18: 1n-7 was observed. γ-
In the presence of linolenic acid (GLA) or arachidonic acid, generation of peaks corresponding to dihomo-γ-linolenic acid (DGLA) and docosatetraenoic acid was observed, respectively.

As a result of expression of the gene (rELO1) in yeast in the presence of icosapentaenoic acid, the target substance n-3 docosapentaenoic acid could be produced at a conversion rate of 51.6%. (Figure 1). In addition, α-linolenic acid, n-3 octadecatetraenoic acid, and DGLA were also found to have chain length extension, and the conversion rates of these substrate fatty acids are shown in Table 1. Regarding mead acid (C20: 3n-9) and n-6 docosatetraenoic acid (C22: 4n-6), no chain extension was observed even when added as a substrate fatty acid.

Table 1 summarizes the substrate specificity of the gene (rELO1) expressing enzyme.

[0020]

[Table 1]

* Conversion rate =% produced fatty acid / (% produced fatty acid +% substrate fatty acid) × 100, the average value of 3 times is shown.

The gene (rELO1) cloned from a rat liver cDNA library encodes a fatty acid chain extension enzyme, and the enzyme produced by the gene (rELO1) extends the chain length of a fatty acid from It is an enzyme having a function of converting into n-3 type docosapentaenoic acid.

In the present invention, as an expression vector in a host cell for active expression of the enzyme gene, for example, the gene (rELO1) is incorporated into the host cell, and an extrachromosomal integration plasmid such as yeast expression plasmid pYES2 is used. A transformant transformed with / rELO1 is used. That is, for the active expression of the enzyme gene, the yeast Saccharomyces cerevisiae (Sa
The extrachromosomal expression plasmid pYES2 / rELO1 (6.8 kb) of ccharomyces cerevisiae) is shown in FIG. The integration of the plasmid into the yeast Saccharomyces cerevisiae is URA3
Selected by marker. Gene expression is. It is controlled by the GAL1 promoter (PGAL1) and the CYC1 terminator (TCYC1) and is induced by galactose.

The plasmid (pYES2 / rELO1)
Transformation of yeast into yeast is preferably performed as follows. Saccharomyces cerevisiae
myces cerevisiae) INVSc1 (his3, leu2, trp1, ura3) (I
Yeast (such as NVITROGEN) is cultured by a conventional method. That is, permeation culture was performed at 28 ° C. for 2 days using YPD medium. The obtained culture solution is purified by repeated centrifugation, and then the lithium acetate / PEG solution and the above plasmid vector pYES2 / rELO1 solution are added, mixed well, and then cultured again. The culture is preferably carried out in a sufficiently dried SD medium at 28 ° C. for 3 to 4 days.

The gene of the present invention (rELO1)
For integration into a host cell, an intrachromosomal integration plasmid such as yeast expression plasmid pMO3 / rELO
Transformed with 1. That is, for the active expression of the enzyme gene, the yeast Saccharomyces cerevisiae (Sacc) constructed as an example of an expression vector in a host cell was used.
haromyces cerevisiae) plasmid p for intrachromosomal expression
MO3 / rELO1 (6.8 kb) is shown in FIG.
When the plasmid is introduced into yeast after digested with a restriction enzyme at the XhoI site, it is randomly integrated into chromosomal DNA by the δ sequence (transposon). Transformation of the yeast expression plasmid vector pMO3 / rELO1 into yeast is carried out using the plasmid vector pYES2 /
Performed similarly to rELO1 and selected by the URA3 marker, but does not require selection after chromosomal integration.

In the present invention, the host cell for expressing the gene may be a eukaryotic cell. That is, the above Saccharomyces cerevisiae
other yeast or Mucor s (Mucor s)
p.), Mortierella sp., and other filamentous fungi, as well as plants and animals as host cells, can be transformed by selecting appropriate conditions for each. When these transformants are used, by appropriately supplying icosapentaenoic acid and adjusting the expression conditions of the gene, it is possible to convert icosapentaenoic acid to n-3 docosapentaenoic acid by extending the carbon chain length. .

In the present invention, among the eukaryotic cells,
Yeast is preferably used as a host cell for preparing a transformant involved in the production of the n-3 docosapentaenoic acid. Yeast is a eukaryotic cell, and in particular, it has an excellent function for expression of a mammalian-derived fatty acid desaturase and fatty acid chain elongation enzyme gene, and moreover, a transformant into which the gene has been introduced, As described in the embodiment of the production method, many conventional knowledge such as the construction of vector plasmids can be utilized for the production, and in the functional expression of the produced transformant, it can be easily cultured. In setting culture conditions for promoting expression, it has an excellent function in terms of simplicity in setting conditions for adding icosapentaenoic acid as a raw material for producing n-3 docosapentaenoic acid. ing.

In the present invention, among the yeasts, the n
It is preferable to use Saccharomyces cerevisiae or Pichia pastoris as a host cell for producing a transformant involved in the production of -3 system docosapentaenoic acid. The extrachromosomal expression plasmid pYES shown in FIG. 2 for incorporating into the yeast Saccharomyces cerevisiae constructed as an example of an expression vector in a host cell for active expression of the long enzyme gene.
2 / rELO1 (6.8 kb) is Saccharomyces cerevisiae INVSc1
(His3, leu2, trp1, ura3) (manufactured by INVITROGEN) and incorporated into yeast. A transformant production system using yeast Pichia pastoris as a host cell is excellent in transformant expression in the host and vector systems, and has excellent bacterial growth performance. It can be used as a desirable host cell for industrial production of n-3 docosapentaenoic acid.

In the present invention, the transformant, for example, the transformed yeast YEL-5 strain (FERM), is mixed with GY medium,
YM medium or medium composition, 1-10% preferably 2 to 8% glucose, 0.1% to 3% preferably 0.5-2% yeast extract, NaNO ₃ 0.05 to 0.4% preferably 0.1 ~0.3%, KH ₂ PO ₄ 0.01~0.2
% Preferably _{0.02~0.1%, MgSO 4 · 7H 2} O
The culture temperature is 20 to 35 ° C., preferably 2 to 0.01 to 0.2%, preferably 0.02 to 0.1% dissolved in water.
Cultivated at 5 to 32 ° C. and icosapentaenoic acid 0.1 to 20
It is characterized by adding 0 mM, preferably 0.5 to 100 mM, to accumulate the fatty acid inside or outside the cell. That is, when culturing the transformant, the culture conditions such as the type of carbon source and nitrogen source of the medium, the culture temperature, and the culture time are optimal for producing a large amount of n-3 docosapentaenoic acid. The conditions can be appropriately selected.

Hereinafter, the present invention will be described in detail with reference to Examples. The present invention is not limited to the embodiments. Unless otherwise specified, the enzymes and reagents used in the examples were those at the analytical and biochemical levels purchased from Takara Shuzo, Sigma, Nacalai Tesque, Katayama Kagaku or Difco.

Example 1 Cloning of rat fatty acid chain extender gene.

A mouse EST clone homologous to the entire amino acid sequence (299 residues) of the fatty acid chain extender HELO1 identified in human was searched in the NCBI database, and AU079897 and BE535118 was obtained. These overlapped each other and each contained a 5'side containing a start codon and a 3'region containing a stop codon, and the concatenated sequence encoded 299 amino acids highly homologous to HELO1. From the gene information of this mouse, the following primer F1 and primer R
Created 1. The primer F1 contains the restriction enzyme HindIII cleavage site of AAGCTT and the start codon of ATG, and the primer R1 contains the restriction enzyme Xba of TCTAGA.
It contains a cleavage site by I and the stop codon TCA. F1,5'-AGGTAAGCTT ATG GAACAT
TTCGATGTCATCT-3 ′ (SEQ ID NO: 2) (Italic type is HindIII site, underline is start codon) R1,5′-CTCTCTAGA TCA ATCCCTTT
CGCTGCTTCCTGGG-3 '(SEQ ID NO: 3) (Italic type indicates Xba I site, underline indicates stop codon)

Using these, PCR was carried out using a rat liver cDNA library (Takara) as a template. At this time, TdT with high fidelity as a polymerase
(Terminal nucleic acid-adding enzyme) KOD-plus (T
OYOBO) was used. The composition of the reaction mixture is as follows.

[0034]

[Table 2]

The reaction condition of PCR is 94 ° C. × 2 sec, then 94 ° C. × 15 sec / 60 ° C. × 30 sec.
/ 68 ° C x 1 min was set as one cycle and repeated 35 times. The reaction solution was subjected to agarose gel electrophoresis, stained, and then irradiated with ultraviolet rays to confirm one band.
The amplified fragment (rELO1) obtained as a result was designated as pYES2.
(Invitrogen) was inserted into the corresponding site to prepare a transformant, and the nucleotide sequence was determined by the dye terminator method.

The specific method of carrying out the method will be described below. (1) Extraction of DNA fragment from agarose gel The DNA fragment of interest confirmed by UV transilluminator was cut out from the agarose gel and subjected to CONCERT.
Rapid Gel Extraction System (GIBCOBRL) and QIAEX II Gel Extraction Kit (QI
Purification was performed using each purification kit manufactured by AGEN).
The method was according to the attached protocol. (2) Ligation into vector The fragment obtained and purified in (1) above was transformed into pGE
M-T easy vector system (PROMEGA)
Were mixed so that the insert: vector ratio was 1:10. The same amount of Ligation High (TOYOBO
(Manufactured by the company) was added, and the mixture was incubated at 16 ° C for 1 hour or more.

(3) Preparation of competent cells E. E. coli DH5α was streaked on M9 plate and cultured at 37 ° C. until colonies could be confirmed. Scrap a single colony, 100 mL (1 L flask)
Was suspended in SOB medium and cultured with shaking at 18 ° C. OD
When 600 = 0.4 to 0.8 was reached, the culture was terminated and the culture solution was immediately cooled in ice water for 10 minutes. The culture was centrifuged at 4 ° C. at 3000 rpm for 15 minutes and the supernatant was discarded. 33 mL of ice-cold Transformation buffer
After suspending in (TB), the mixture was ice-cooled for 10 minutes. Then, the cells were collected by centrifugation at 3000 rpm for 15 minutes at 4 ° C., and suspended in 4 mL of ice-cold TB. 1 mL of 5
0% glycerol was added, dispensed in 400 μL aliquots, frozen with liquid nitrogen and stored at −80 ° C. The TB used here was 10 mM PIPES, 15 mM CaCl.
₂ · 2H ₂ O, 250mM KCl and 55 mM Mn
It is composed of Cl ₂ .4H ₂ O.

(4) Transformation into E. coli E. coli prepared as competent cells E. coli DH5α was thawed on ice and mixed with the ligated vector solution. After incubating on ice for 5 minutes, heat shock was applied at 37 ° C. for 2 minutes, and then incubation was again performed on ice for 5 minutes. Next, add 900 μL of sterilized water to this and 1200
It was centrifuged at 0 rpm for 2 minutes. Discard the supernatant and discard the precipitate
It was suspended in L 100 mM IPTG and seeded on an LB plate medium containing X-Gal and ampicillin. After incubating the plate at 37 ° C. for 12 hours, the presence or absence of colonies was confirmed.

(5) Preparation of plasmid on mini scale An appropriate amount of Escherichia coli solution that had been cultured overnight was sampled, and 9000 rpm
The cells were recovered by centrifugation at 2 minutes. To Soluti
100 μL of onI was added and vortex-suspended, and 200 μL of Solution II was further added to lyse the cells. Incubate on ice for 5 minutes, then Solution II
After adding I of 200 μL and vortexing, 12000r
It was centrifuged at pm for 10 minutes. The obtained supernatant was collected in another tube, and an equal amount of isopropanol was added to the mixture to purify it by alcohol precipitation. The Solution used here
I to III have the following compositions. Solution I: 50 mM glucose, 25 mM Tris-HCl (pH 8.
0), 10 mM EDTA SolutionII: 0.2N NaOH, 1% SDSSolutionIII: 3M K-
acetate, 11.5% acetate

(6) Confirmation of insert by digestion with restriction enzymes Each enzyme, buffer and sample DNA were mixed in the following composition.

[Table 3] Depending on the type of restriction enzyme, selection of the type of buffer and addition of Triton X-100 and BSA were appropriately performed. The reaction was carried out at 37 ° C.

(7) Incorporation into yeast expression plasmid vector In the above, pGEM-T easy vector.
The rat liver fatty acid chain elongation enzyme gene (rELO1) cloned in (PROMEGA) was HindII
The insert was purified by digestion with restriction enzymes I and XbaI, agarose gel electrophoresis and gel extraction, and the insert was purified. This insert was also digested with HindIII and XbaI and then purified by dephosphorylation to obtain p
Ligation into the HindIII and XbaI sites of YES2, E. coli DH5α. The colonies were subjected to liquid culture overnight, and a small amount of plasmid was purified, insert check by restriction enzyme digestion, and forward / reverse determination were performed. The constructed plasmid vector was designated as pYES2 / r.
It was named ELO1.

The base sequence was determined by the dye terminator method as follows. DYEnamic ET Terminator Cyc
Samples were prepared using le Sequencing Kit (Amersham Pharmacia) and 310 Genetic Analyzer (Applied B
The sequence was determined by iosystems. as a result,
The obtained rat liver fatty acid chain length-extending enzyme gene (rELO
1) contains an open reading frame of 900 bp and encodes 299 amino acids, and its entire base sequence and its deduced amino acid sequence are shown in SEQ ID NO: 1 of the sequence listing, respectively. Regarding the deduced amino acid sequence of the rat liver fatty acid chain elongation enzyme gene (rELO1) (SEQ ID NO: 1 in the sequence listing), human (HELO1) and nematode (Caen)
ohabditis elegans, Cele; F56H11.4) and filamentous fungi (Mortier
ella alpina, Malp; GLELO) and a fatty acid chain extender.

Although rELO1 has a very high homology (93.0%) with HELO1, the nematode (Cele;
F56H11.4) and filamentous fungus (Malp; GLEL)
Low homology with O) (27.3% and 31.4)
%) Has been revealed. In each amino acid sequence, a histidine cluster (HXXHH) of the type found in desaturase was conserved in one place. From these results, it is clear that the fragment consisting of the nucleotide sequence set forth in SEQ ID NO: 1 in this sequence listing is a gene encoding a fatty acid chain length-extending enzyme.

Example 2 Construction of a plasmid vector for extrachromosomal expression of rat fatty acid chain elongation enzyme gene (rELO1).

Yeast Saccharomyces cerevisiae (Saccha
romyces cerevisiae) extrachromosomal expression vector pY
Using ES2 (manufactured by INVITROGEN), the rat fatty acid chain elongation enzyme gene (rELO1) in yeast
A plasmid vector for expressing the activity of was constructed as follows. The plasmid pYES2 / rELO1 (6.8) for extrachromosomal expression of the yeast Saccharomyces cerevisiae constructed in Example 1.
kb) is shown in FIG. Integration of this plasmid is selected by the URA3 marker. Gene expression is GA
It is regulated by the L1 promoter (PGAL1) and the CYC1 terminator (TCYC1) and is induced by galactose.

Example 3 A plasmid vector for intrachromosomal integration expression of rat fatty acid chain elongation enzyme gene (rELO1).

Yeast Saccharomyces cerevisiae (Saccha
romyces cerevisiae) plasmid vector pMO3 (manufactured by INVITROGEN) for expression in the chromosome.
Was used to construct a plasmid vector for expressing the activity of rat fatty acid chain elongation enzyme gene (rELO1) in yeast as follows. The yeast Saccharomyces cerevisiae constructed in the same manner as in Example 1 (Saccharo
The plasmid vector pMO3 / rELO1 (7.8 kb) for intrachromosomal integration expression of myces cerevisiae) is shown in FIG. When this plasmid is introduced into yeast after digestion with a restriction enzyme at the XhoI site, it is randomly integrated into chromosomal DNA by the δ sequence (transposon). It is selected by the URA3 marker but does not require selection after it has been integrated into the chromosome. Gene expression is GA
It is regulated by the P promoter (PGAP) and GAP terminator (TGAP), and is constitutively expressed.

Example 4 Transformation of a rat fatty acid chain elongase gene (rELO1) into yeast and expression of activity of the gene in yeast.

The rELO1 gene extrachromosomal expression plasmid vector (pYES2 / rELO1, FIG. 2) was transformed into the yeast Saccharomyces cerevisiae (Saccha) by the lithium acetate method.
romyces cerevisiae) INVSc1 (α / a, his3, leu2, ura3, trp
1) (manufactured by INVITROGEN) and transformed. The procedure is as follows. Saccharomyces cerevisiae (Saccharomyce) was added to 5 mL of YPD liquid medium.
S. cerevisiae) INVSc1 strain was inoculated at 28 ° C for 2
Shaking culture was performed for a day. The composition of the YPD liquid medium is glucose 2%, polypeptone 2% and yeast extract 1%.
Is included. Take 1 mL of the culture solution into a tube and 2
After centrifugation at 000 rpm for 5 minutes, the supernatant was discarded. After adding 1 mL of lithium acetate solution to the precipitate and mixing well, 200
After centrifugation at 0 rpm for 5 minutes, the supernatant was discarded.

The lithium acetate solution used here is
100 mM lithium acetate, 10 mM Tris HC
1 (pH 7.5) and 1 mM EDTA. Next, 200 μ of lithium acetate / PEG solution for precipitation
L and 5 μL of plasmid solution (1 μg / μL) were added,
After mixing well, the mixture was incubated in a 30 ° C. water bath for 1 hour. Then, it was further incubated in a water bath at 42 ° C for 10 minutes. Lithium acetate / PE used here
G solution is 100 mM lithium acetate, 10 mM Tr
is HCl (pH 7.5), 1 mM EDTA and 40% PEG4000. Subsequently, this culture was plated on well-dried SD medium and cultured at 28 ° C. for 3 or 4 days. The composition of SD medium is glucose 2
%, Yeast nitrogen-base w / o amino acid 0.7%,
Histidine 20 μg / mL, Leucine 60 μg / m
L, tryptophan 40 μg / mL.

The expression of the activity of the fatty acid chain extender gene in yeast will be described below. The colonies that appeared on the SD medium were scraped off, and 5 mL of SCT medium (selection medium)
Preculture was performed overnight at 28 ° C. The composition of SCT medium was raffinose 4%, yeast nitrogen-base w / o.
amino acid 0.7%, surfactant (trade name: terg
Itol) 1%, histidine 20 μg / mL, leucine 60 μg / mL, tryptophan 40 μg / mL. SCT medium, linoleic acid (C18: 2n-6),
α-linolenic acid (C18: 3n-3), γ-linolenic acid (C18: 3n-6), n-3 octadecatetraenoic acid (C18: 4n-3), dihomo-γ-linolenic acid (C2)
0: 3n-6), mead acid (C20: 3n-9), arachidonic acid (C20: 4n-6), icosapentaenoic acid (C20: 5n-3) and n-6 docosatetraenoic acid (C20: 4n-6). 1) as a substrate
45 mL of each was prepared by adding mM. To each of these 9 SCT media and the media without the addition of fatty acids,
The preculture solution was inoculated so that the number of cells was 2 × 10 ⁵ / mL, galactose was added while shaking at 28 ° C., and the cells were further cultured for 16 hours.

After the completion of the culture, the culture solution was transferred to a 50 mL conical tube, centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. Next, 5 mL of sterilized water was added to the cells, vortexed, centrifuged again at 2000 rpm for 5 minutes, and the supernatant was discarded. Extraction of lipids from the cells was performed as follows.
Suspend the cells in 1 mL of sterilized water, transfer to a screw cap test tube, and equal volume of chloroform-methanol (2: 1, v / v)
Was added, vortexed, and centrifuged at 2000 rpm for 2 minutes. Take the lower chloroform layer into a test tube, add 10% hydrochloric acid-methanol to the chloroform layer, and add 60%.
Methyl esterification was carried out by treating at 3 ° C for 3 hours.
After extracting the fatty acid methyl ester with hexane, the fatty acid composition was analyzed by gas chromatography (Shimadzu GC-17A, column TC-70). GC-MS
Analysis is performed using GC material (JEOL), 70 eV
EI-MS in.

As a result, a DNA fragment containing the open reading frame of the rat liver fatty acid chain elongase gene (rELO1) was inserted into the yeast expression vector, and first,
When subjected to yeast expression without the addition of substrate fatty acid, the chain extension product of palmitooleic acid, C18: 1n
Formation of -7 was observed. At this time, a peak, which is a trace amount and seems to correspond to C20: 1n-7 in which the chain length was further extended, was observed at 9.6 min, and the chain length extension products derived from these endogenous fatty acids were added with the substrate fatty acid. If also detected. In the presence of γ-linolenic acid (GLA) or arachidonic acid, generation of peaks corresponding to dihomo-γ-linolenic acid (DGLA) and docosatetraenoic acid was observed, respectively. In addition, when the expression analysis was performed in the presence of linoleic acid, the production of a chain length extension product, C20: 2Δ11,14, was confirmed although it was a trace amount. As a result of expressing rELO1 in yeast in the presence of icosapentaenoic acid, the target substance n-3 docosapentaenoic acid could be produced at a conversion rate of 51.6% (FIG. 1).

In addition, α-linolenic acid, n-3 octadecatetraenoic acid and DGLA were also found to have chain length extension, and the conversion rates of these substrate fatty acids are shown in Table 1. For mead acid (C20: 3n-9) and n-6 docosatetraenoic acid (C22: 4n-6), chain length extension was not observed even when the substrate fatty acid was added (Table 1). From this, in the yeast YEL-5 strain transformed with the rat liver fatty acid chain elongation enzyme gene, it was converted to n-3 docosapentaenoic acid by culturing in the presence of icosapentaenoic acid, and n-3 docosapentaenoic acid was converted. It was confirmed that enoic acid was produced.

[0055]

INDUSTRIAL APPLICABILITY The method for producing n-3 docosapentaenoic acid according to the present invention has no conventional production technique for n-3.
It enables the factory production of docosapentaenoic acid. In addition, n-3 docosapentaenoic acid is expected to have high physiological activity, and its product is expected to have a wide range of uses such as pharmaceuticals, health foods, and specified health foods.

[0056]

[Brief description of drawings]

FIG. 1 is a diagram showing the results of gas chromatography (GC) of fatty acids generated by yeast expression of the fatty acid chain length-extending enzyme gene (rELO1) introduced into the transformed yeast of the present invention. That is, pYES2 or pYE
The plasmid in which the rELO1 gene was inserted into S2 was used for the yeast Saccharomyces cerevisiae.
e) to induce expression by galactose,
The intracellular fatty acid was analyzed by GC. The production of DPA (n-3) was confirmed by adding EPA as a substrate. EPA is icosapentaenoic acid (C20: 5n-3), DPA is n-
3 shows docosapentaenoic acid (C22: 5n-3), respectively.

[Fig. 2] Plasmid for extrachromosomal expression of rat fatty acid chain elongation enzyme (rELO1) (pYES2 / rELO1)
It is the figure which showed. In the figure, PGAL1 is GAL1 promoter, TCYC1 is CYC1 terminator, URA
3 indicates a marker.

[Fig. 3] A plasmid for expression in the chromosome of rat fatty acid chain elongase rELO1 (pMO3 / rELO1).
It is the figure which showed. In the figure, PGAP is a GAP promoter, TGAP is a GAP terminator, δ is a transposon δ sequence, and URA3 is a marker.

[0057]

[Sequence list]   SEQUENCE LISTNG <110> Osamu Suzuki <130> 51371 <160> 3 <210> 1 <211> 900 <212> DNA <213> rat liver <400> 1 Array ATG GAA CAT TTC GAT GCG TCA CTC AGT ACC TAT TTC AGG GCA TTA CTG 48 Met Glu His Phe Asp Ala Ser Leu Ser Thr Tyr Phe Arg Ala Leu Leu          5 10 15 GGC CCC CGA GAC ACA CGA GTC AAA GGA TGG TTT CTT CTG GAC AAT TAC 96 Gly Pro Arg Asp Thr Arg Val Lys Gly Trp Phe Leu Leu Asp Asn Tyr             20 25 30 ATC CCT ACG TTT GTC TGC TCT GCC ATT TAT TTA CTC ATC GTG TGG CTG 144 Ile Pro Thr Phe Val Cys Ser Ala Ile Tyr Leu Leu Ile Val Trp Leu         35 40 45 GGA CCA AAA TAC ATG AAA AAC AGG CAG CCG TTC TCT TGC CGA GGC ATC 192 Gly Pro Lys Tyr Met Lys Asp Arg Gln Pro Phe Ser Cys Arg Gly Ile     50 55 60 CTG GTG GTG TAT AAT CTT GGG CTC ACC CTG CTG TCT CTC TAC ATG TTC 240 Leu Val Val Tyr Asn Leu Gly Leu Thr Leu Leu Ser Leu Tyr Met Phe 65 70 75 80 TAT GAG TTG GTG ACA GGT GTG TGG GAA GGC AAA TAC AAC TTC TTC TGT 288 Tyr Glu Leu Val Thr Gly Val Trp Glu Gly Lys Tyr Asn Phe Phe Cys                 85 90 95 CAG GGA ACA CGC AGC GCA GGA GAA TCA GAT ATG AAG GTT ATT CGT GTC 336 Gln Gly Thr Arg Ser Ala Gly Glu Ser Asp Met Lys Val Ile Arg Val             100 105 110 CTC TGG TGG TAC TAC TTT TCC AAA CTC ATA GAA TTC ATG GAC ACC TTT 384 Leu Trp Trp Tyr Tyr Phe Ser Lys Leu Ile Glu Phe Met Asp Thr Phe         115 120 125 TTC TTC ATT CTT CGC AAG AAC AAC CAC CAG ATC ACA GTC CTG CAC GTC 432 Phe Phe Ile Leu Arg Lys Asn Asn His Gln Ile Thr Val Leu His Val     130 135 140 TAC CAC CAT GCC ACT ATG CTC AAC ATC TGG TGG TTT GTC ATG AAC TGG 480 Tyr His His Ala Thr Met Leu Asn Ile Trp Trp Phe Val Met Asn Trp 145 150 155 160 GTT CCC TGC GGC CAT TCG TAC TTC GGT GCG ACG CTC AAC AGC TTC ATC 528 Val Pro Cys Gly His Ser Tyr Phe Gly Ala Thr Leu Asn Ser Phe Ile                 165 170 175 CAC GTC CTC ATG TAC TCG TAC TAT GGC CTG TCC TCT GTC CCT TCC ATG 576 His Val Leu Met Tyr Ser Tyr Tyr Gly Leu Ser Ser Val Pro Ser Met             180 185 190 CGT CCC TAC CTC TGG TGG AAA AAG TAC ATC ACT CAG GGG CAG CTG GTC 624 Arg Pro Tyr Leu Trp Trp Lys Lys Tyr Ile Thr Gln Gly Gln Leu Val         195 200 205 CAG TTT GTG CTG ACA ATC ATC CAG ACC AGC TGC GGG GTC ATC TGG CCG 672 Gln Phe Val Leu Thr Ile Ile Gln Thr Ser Cys Gly Val Ile Trp Pro     210 215 220 TGC TCC TTC CCT CTC GGG TGG CTG TAC TTC CAG ATC GGA TAC ATG ATT 720 Cys Ser Phe Pro Leu Gly Trp Leu Tyr Phe Gln Ile Gly Tyr Met Ile 225 230 235 240 TCC CTG ATT GCT CTC TTC ACA AAC TTC TAC ATT CAG ACT TAC AAC AAG 768 Ser Leu Ile Ala Leu Phe Thr Asn Phe Tyr Ile Gln Thr Tyr Asn Lys                 245 250 255 AAA GGG GCC TCT CGG AGG AAA GAG CAC CTG AAG GGC CAC CAG AAC GGG 816 Lys Gly Ala Ser Arg Arg Lys Glu His Leu Lys Gly His Gln Asn Gly             260 265 270 TCT ATG ACT GCC GTC AAT GGA CAC ACC AAC AAC TTT GCT TCC CTG GAG 864 Ser Met Thr Ala Val Asn Gly His Thr Asn Asn Phe Ala Ser Leu Glu         275 280 285 AAC AGT GTG ACG TCA AGG AAG CAG CGG AAG GAT TGA 900 Asn Ser Val Thr Ser Arg Lys Gln Arg Lys Asp *       290 295 299 <210> 2 <211> 32 <212> DNA <213> artificial sequence <400> 2 AGGTAAGCTT ATGGAACATT TCGATGTCAT CT 32 <210> 3 <211> 33 <212> DNA <213> artificial sequence <400> 3 CTCTCTAGAT CAATCCTTTC GCTGCTTCCT GGG 33

Front page continuation (51) Int.Cl. ⁷ identification code FI theme code (reference) C12R 1: 865 C12N 15/00 ZNAA (C12N 1/19 C12R 1:84) (C12P 7/64 C12R 1: 865) (C12P 7/64 C12R 1:84) F term (reference) 4B024 AA05 BA80 CA04 DA11 EA04 GA11 4B064 AD90 CA19 CC24 DA01 DA10 4B065 AA77X AA80X AA91Y AB01 AC14 BA02 BB10 CA13 CA41 CA44 4H059 BA26 BB05 BB07 BC48

Claims (13)

[Claims] 1. When culturing a transformant obtained by introducing a mammal-derived fatty acid chain length-extending enzyme gene in a medium,
A method for producing n-3 docosapentaenoic acid, which comprises adding icosapentaenoic acid to the medium to extend the carbon chain length of the icosapentaenoic acid to convert it into n-3 docosapentaenoic acid. 2. The n according to claim 1, wherein the fatty acid chain elongation enzyme gene is a gene of mammalian origin.
-3 Method for producing docosapentaenoic acid. 3. The n-3 docosapentaenoic acid according to claim 2, wherein the fatty acid chain length-extending enzyme gene is a fatty acid chain length-extending enzyme gene cloned from a rat liver cDNA library. Manufacturing method. 4. The n- according to claim 3, wherein the fatty acid chain elongation enzyme gene is a gene encoding the amino acid sequence of 299 set forth in SEQ ID NO: 1 in the sequence listing.
Method for producing 3-system docosapentaenoic acid. 5. The n-3 docosapentaene according to claim 4, wherein the fatty acid chain length-extending enzyme gene is a gene having a 900 bp nucleotide sequence set forth in SEQ ID NO: 1 in the sequence listing. Method for producing acid. 6. The transformant according to any one of claims 1 to 5, wherein the transformant is transformed with an extrachromosomally integrated expression plasmid when the fatty acid chain extender gene is introduced into a host cell. The method for producing n-3 docosapentaenoic acid according to 1. 7. The transformant according to any one of claims 1 to 5, wherein the transformant is transformed with an intrachromosomal integration expression plasmid in the introduction of the fatty acid chain extender gene into a host cell. The method for producing n-3 docosapentaenoic acid according to 1. 8. The method for producing n-3 docosapentaenoic acid according to claim 6, wherein the host cell is a eukaryotic cell. 9. The method for producing n-3 docosapentaenoic acid according to claim 8, wherein the eukaryotic cell is yeast. 10. The method for producing n-3 docosapentaenoic acid according to claim 9, wherein the yeast is Saccharomyces cerevisiae or Pichia pastoris. 11. GY medium, YM medium or medium composition, glucose 1-10%, yeast extract 0.1.
_{~3%, NaNO 3 0.05~0.4%,} KH 2 PO 4
_{0.01~0.2%, MgSO 4 · 7H 2} O 0.01
~ Culture temperature of 20 ~ using a medium in which 0.2% is dissolved in water
The method for producing n-3 docosapentaenoic acid according to any one of claims 1 to 10, wherein the method is performed at 35 ° C. 12. The n-3 docosapentaenoic acid according to claim 1, wherein the icosapentaenoic acid added to the medium is a free fatty acid or its methyl or ethyl ester. Production method. 13. The n according to claim 1.
N obtained by the method for producing -3 series docosapentaenoic acid
-3 series docosapentaenoic acid or its ester.