KR101491777B1 - Fatty acid chain elongase gene from muraenesox cinereus and uses thereof - Google Patents

Abstract

The present invention relates to a method for producing (DHA), docosatetraenoic acid (DTA), and docosa-pentaenoic acid (DHA), using the fatty acid chain prolongation enzyme protein and the same. docosapentaenoic acid, DPA). < / RTI >

Description

FATTY ACID CHAIN ELONGASE GENE FROM MURAENESOX CINEREUS AND USES THEREOF <br> <br> <br> Patents - stay tuned to the technology FATTY ACID CHAIN ELONGASE GENE FROM MURAENESOX CINEREUS AND USES THEREOF

The present invention relates to a fatty acid chain extension enzyme gene derived from mackerel ( Muraenesox cinereus) and a use thereof, and more particularly to a fatty acid chain extension enzyme gene derived from Muraenesox cinereus by converting a fatty acid through a fatty acid chain extension using a fatty acid chain extension enzyme gene, The present invention relates to a process for producing docosapentaenoic acid (DPA) and docosatetraenoic acid (DTA), di-homo-gamma linolenic acid (DHLA)

Unsaturated fatty acids are chain-like compounds having one or more double bonds in a molecule and are widely distributed in plants and animals, and are generally composed of even numbers of about 12 to 20 carbon atoms. When a fatty acid molecule has only one double bond, it is called a monounsaturated fatty acid and has two or more double bonds and is called a polyunsaturated fatty acid. Unsaturated fatty acids are divided into a cis configuration and a trans configuration depending on the type of double bond followed by the hydrogen atom. The cis type means that the adjacent hydrogen atom is in the same direction as the double bond, and the trans type means that the next two hydrogen atoms are bonded in the opposite direction to the double bond. When a double bond is formed in a fatty acid chain, the hydrogen atom has to be removed, so the term &quot; saturated &quot; in saturated fatty acids means that the hydrogen atom is saturated. In intracellular metabolism, hydrogen-carbon bonds are broken or oxidized for energy production. Thus, unsaturated fatty acids contain less energy than saturated fatty acids of the same size. It is also known that unsaturated fatty acids have a lower melting point, which also increases the fluidity of the cell membrane.

Saturated fatty acid intake is associated with the risk of cardiovascular diseases such as hypercholesterolemia and atherosclerosis while the intake of unsaturated fatty acids is associated with a decrease in blood cholesterol and a reduced risk of developing atherosclerosis Is known to be associated with. Examples of unsaturated fatty acids include palmitoleic acid, oleic acid, myristoleic acid, linoleic acid, arachidonic acid, eicosapentaenoic acid, Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and the like. Foods containing such unsaturated fatty acids include avocados, nuts, vegetable oils such as canola oil and olive oil. Although unsaturated fatty acids are healthier than saturated fatty acids, the Food and Drug Administration (FDA) recommends that the intake of unsaturated fatty acids should not exceed 30% of the daily calorie intake.

Arachidonic acid (ARA, C20: 4 ω6), which is a polyunsaturated fatty acid (PUFA) based on omega 6 (ω6) and omega 3 (ω3), and eicosapentaenoic acid , Docosahexaenoic acid (DHA, C22: 6 ω3) is an essential component of the animal cell membrane, and it is bound to phosphatidylcholine and inserted into the phospholipid layer of the cell membrane Thereby improving the fluidity of the cell membrane, reducing blood cholesterol, and inhibiting cancer proliferation. In particular, DHA has been shown to have important functions such as enhancement of brain function and memory, improvement of brain function, improvement of learning function, and hormone regulating action as a precursor of hormones such as prostaglandin. Recently, it has formed a big market in the fields of food additives, cosmetic raw materials and pharmaceuticals. In mammals, linoleic acid (LA, C18: 2 ω6) and ω6 / δ12 and ω3 / Δ15 desaturase were not expressed and thus, de novo synthesis of PUFA was not possible. Alpha-linolenic acid (ALA, C18: 3 ω3) must be ingested as food (see Figure 1).

DHA is mainly produced from blue fish such as tuna. However, if a large amount of DHA is added to food, it becomes problematic in terms of merchantability due to fish-specific fishy smell, and pure separation and purification for removing the odor are the key. DHA production can also be achieved through marine algae, but due to the low growth rate, high-volume equipment is required for the mass production of DHA. In order to solve these problems and produce low-cost and high-efficiency DHA, interest in the production of PUFAs using expression systems in plant seeds is increasing. PUFAs are biosynthesized through a series of desaturation and 2-carbon chain elongation steps from the precursors LA and ALA (Fig. 1). Plants do not biosynthesize PUFAs and the introduction of Δ6 desaturase (Δ6 desaturase, Δ6DES), Δ6 chain extension enzyme (Δ6 elongase, Δ6ELS), and Δ5 desaturase (Δ5 DES) to biosynthesize PUFAs from LA and ALA Is required. Therefore, it can be obtained by metabolically engineering a plant seed-specific expression of a marine microorganism, algae or fish-derived fatty acid unsaturation enzyme and a 2-carbon chain extension enzyme gene accumulating a high content of PUFAs.

To date, three pathways of PUFA biosynthesis have been known in nature [Qiu X (2003) Biosynthesis of docosahexaenoic acid (DHA, 22: 6-4, 7, 10, 13, 16, 19): two distinct pathways. Prostaglandins Leukot Essent Fatty Acids 68, 181-186.]. (1) Saprolegnia belonging to the water mold family uses a pathway for synthesizing EPA through Δ6 unsaturation, prolongation and Δ5 unsaturation on the precursor ALA [Pereira SL et al. (2004a)], while novel omega 3-fatty acid desaturase involved in the biosynthesis of eicosapentaenoic acid. Biochem J 378, 665-671.), (2) protoplasts such as Euglena biosynthes EPA by Δ9 extension, Δ8 unsaturation and Δ5 unsaturation [Wallis and Browse, (1999) The Delta8-desaturase of Euglena gracills: an alternate pathway for synthesis of 20-carbon polyunsaturated fatty acids. Arch Biochem Biophys 365, 307-316.] (Fig. 1). EPA biosynthesized through the above two pathways ultimately biosynthesize DHA by Δ5 extension and Δ4 unsaturation [Pereira et al., 2004b]. Mammalian biosynthesizes DHA via a more complex four-step pathway from EPA, that is, retro-conversion by β-oxidation (Sprecher et al., 1995). . J Lipid Res 36, 2471-2477., 1995; Wallis et al., Polyunsaturated fatty acid synthesis: what will they think of next Trends Biochem Sci 27, 467-473. (3) Schizochytrium and marine microorganism (Shewanella) biosynthesize DHA using PKSe (polyketide synthase) pathway [Metz et al., (2001) Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science 293,290-293.]. The PKS pathway is composed of 3-ketoacyl-synthase, 3-ketoacyl reductase, 3-ketoacyl reductase, 3-ketoacyl reductase, The synthesis domain of PKS such as acyl carrier protein, chain length factor and acyl transferase is constituted as one gene cluster to reduce the number of reactions required for PUFA biosynthesis have. When these marine organisms are used as useful genetic resources for restructuring the PUFA biosynthetic pathway of land plants, it is possible to establish a genetic engineering base for EPA and DHA, which are mainly consumed only through fish, through plants.

Accordingly, the present inventors have isolated a fatty acid chain extension enzyme gene ( McELOVL5 ), which is involved in the synthesis of polyunsaturated fatty acids having various physiological activities such as blood circulation system, hormone secretion system, and immune system, in a high organism, from the sea anemone ( Muraenesox cinereus ) And confirmed its function and completed the present invention.

Korea Patent No. 0316068 Korea Patent No. 0606612

An object of the present invention is to provide a fatty acid chain extension enzyme protein derived from the mushroom ( Muraenesox cinereus ) comprising the amino acid sequence of SEQ ID NO: 2, a gene coding therefor, a recombinant vector containing the gene and a host cell transformed with the recombinant vector .

It is another object of the present invention to provide a method for producing polyunsaturated fatty acids by using the above-mentioned mugwort-derived fatty acid chain extension enzyme gene.

Still another object of the present invention is to provide a plant producing the polyunsaturated fatty acid and a method for producing the same.

Yet another object of the present invention is to provide a composition comprising the above-mentioned mugwort-derived fatty acid chain extension enzyme gene.

The present invention provides a fatty acid chain elongase protein derived from the marsh eel.

The present invention provides a gene encoding the protein.

The present invention provides a recombinant vector comprising the gene.

The present invention provides a host cell transformed with said recombinant vector.

The present invention also provides a method for producing a polyunsaturated fatty acid in a host cell comprising transforming a host cell with the recombinant vector.

The present invention also provides a method for producing a plant producing a highly unsaturated fatty acid comprising the step of transforming a plant cell with the recombinant vector.

In addition, the present invention provides a plant producing the highly unsaturated fatty acid produced by the above method and a seed thereof.

The present invention also provides a composition for producing a polyunsaturated fatty acid containing the gene.

The present invention provides a conger eel-derived fatty acid chain extension enzyme protein McELOVL5 S consisting of the amino acid sequence shown in SEQ ID NO: 2.

The range of the McELOVL5 protein according to the present invention includes a protein having the amino acid sequence shown in SEQ ID NO: 2 isolated from dome and a functional equivalent of the protein. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2. "Substantially homogenous physiological activity" refers to the presence of polyunsaturated fatty acids, preferably di-homo-gamma linolenic acid (DHLA), docosatetraenoic acid (DTA) ) And docosapentaenoic acid (DPA), respectively. The term " polyunsaturated fatty acid "

The present invention also provides a gene encoding the McELOVL5 protein. The gene of the present invention includes both genomic DNA and cDNA encoding McELOVL5 protein. Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1.

Variants of the above base sequences are also included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the &lt; / RTI &gt;

The present invention also provides a recombinant vector comprising the McELOVL5 gene according to the present invention. The recombinant vector may preferably be a yeast recombinant vector, and more preferably, the pYES2- McELOVL5 vector described in Fig. 6, but is not limited thereto.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

The vector of the present invention can typically be constructed as a vector for cloning or expression. In addition, the vector of the present invention can be constructed by using prokaryotic cells or eukaryotic cells as hosts. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., pL? Promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.) It is common to include a ribosome binding site and a transcription / translation termination sequence for initiation of translation. When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthesis pathway, and the left promoter of the phage lambda (pL 貫 promoter) can be used as the regulatory region.

The vectors that can be used in the present invention include plasmids such as pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19 which are frequently used in the art, phages such as λgt4 · λB ,? -charon,?? z1, and M13), or a virus (e.g., SV40, etc.).

On the other hand, when the vector of the present invention is an expression vector and the eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a mammalian virus Virus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and tk promoter of HSV) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

The vector of the present invention may be a selection marker and may include an antibiotic resistance gene commonly used in the art, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, And resistance genes for tetracycline.

The present invention also provides a host cell transformed with the recombinant vector of the present invention. Any host cell known in the art may be used as the host cell capable of continuously cloning and expressing the vector of the present invention in a stable and prokaryotic cell, for example, E. coli JM109, E. coli BL21, E. coli RR1 , E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus tulifene, and Salmonella typhimurium, Serratia marcesensus and various Pseudomonas species And enterobacteria and strains such as the species.

When the vector of the present invention is transformed into eukaryotic cells, yeast ( Saccharomyce cerevisiae ), insect cells, human cells (for example, Chinese hamster ovary, W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and plant cells. The host cell is preferably yeast.

The method of delivering the vector of the present invention into a host cell can be carried out by the CaCl ₂ method, one method (Hanahan, D., J. MoI. Biol., 166: 557-580 (1983)) when the host cell is a prokaryotic cell, And an electric drilling method or the like. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment .

The present invention also relates to a method for producing di-homo-gamma linolenic acid (D-homo-gamma-linolenic acid) in a host cell, which comprises transforming a host cell with a recombinant vector comprising a fatty acid chain- The present invention provides a method for producing polyunsaturated fatty acids selected from the group consisting of DHA, DHLA, docosatetraenoic acid (DTA), and docosapentaenoic acid (DPA).

The unsaturated fatty acid may be di-homo-gamma linolenic acid (DHLA) produced by extending the carbon chain of gamma-linolenic acid (GLA).

The unsaturated fatty acid may be docosatetraenoic acid (DTA, C22: 4) produced by extending the carbon chain of arachidonic acid (ARA, C20: 4).

The unsaturated fatty acid may be docosapentaenoic acid (DPA, C22: 5) produced by extending the carbon chain of eicosapentaenoic acid (EPA, C20: 5).

The extension of the carbon chain is activated by activating the fatty acid chain extension enzyme gene derived from the mussels of the present invention to convert arachidonic acid having C20: 4 to docosatetraenoic acid having 2 units of C22: 4, 5 eicosapentaenoic acid is also converted to docosapentaenoic acid, which is a C22: 5 carbon unit extended by two carbon atoms.

The term " polyunsaturated fatty acid "refers to a fatty acid containing four or more double bonds. Omega-6 and omega-3 fatty acids, and the class of polyunsaturated fatty acids in the art is well known. In the present invention, it is particularly preferable to use di-homo-gamma linolenic acid (DHLA), docosatetraenoic acid (DTA) and docosapentaenoic acid (DPA) And the like.

In the method of the present invention, the fatty acid chain extension gene and the recombinant vector are as described above. In addition, the host cell is preferably yeast.

In one embodiment of the present invention, the fatty acid chain extension enzyme gene obtained from P. pneumoniae was activated to convert from gamma-linolenic acid (GLA) to di-homo-gamma-linolenic acid (DGLA, C20: 3n-6) The product DGLA produced 3.97% of total fatty acids and showed a high substrate conversion of 64%. In the case of arachidonic acid (ARA, C20: 4n-6) or eicosapentaenoic acid (EPA, C20: 5n-3), ARA was replaced with docosatetraenoic acid (DTA, C22: , And EPA was converted to docosapentaenoic acid (DPA, C22: 5n-3). DTA accounted for 2.03% of total fatty acids and showed 15.5% fatty acid chain extension conversion. DPA accounted for 4.41% of total fatty acids and showed fatty acid chain extension conversion of 47.5%. From this result, it can be seen that the mature conger eel-derived fatty acid chain extension enzyme gene McELOVL5 has a higher substrate activity for the omega-3 pathway than the omega-6 pathway.

The present invention also relates to a method for the production of di-homo-gamma linolenic acid (DHLA), which comprises the step of transforming a plant cell with a recombinant vector comprising a fatty acid chain- There is provided a method of producing a plant producing a highly unsaturated fatty acid selected from the group consisting of docosatetraenoic acid (DTA) and docosapentaenoic acid (DPA). In the method of the present invention, the fatty acid chain extension enzyme gene is as described above.

A preferred example of a recombinant vector is a Ti-plasmid vector which is capable of transferring a so-called T-region to a plant cell when it is present in a suitable host such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant genes, but are not limited thereto.

In the recombinant vector of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS, or histone promoter, but is not limited thereto. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, constitutive promoters do not limit selectivity.

In the recombinant vector of the present invention, conventional terminators can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens ) Octopine gene terminator, but the present invention is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. The method is based on the calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373) (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202,179-185 (Klein et al., 1987, Nature 327, 70), the infiltration of plants or the transformation of mature pollen or vesicles into Agrobacterium tumefaciens Infection by viruses (non-integrative) in virus-mediated gene transfer (EP 0 301 316), and the like. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Particularly preferred is the use of so-called binary vector techniques as described in EP A 120 516 and U.S. Pat. No. 4,940,838.

The present invention also provides a plant producing gamma-linolenic acid or stearidic acid produced by the above method and seeds thereof.

The plant according to the present invention is selected from the group consisting of food crops selected from the group consisting of rice, wheat, barley, corn, soybean, potato, wheat, red bean, oats and millet; Vegetable crops selected from the group consisting of Arabidopsis, cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions and carrots; Ginseng, tobacco, cotton, sesame, sugar cane, beet, perilla, peanut and rapeseed; Apple trees, pears, jujubes, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots and banana; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops selected from the group consisting of Ryegrass, Red Clover, Orchardgrass, Alpha Alpha, Tall Fescue, and Fereniallaigrus.

The present invention also relates to a method for the production of a composition comprising di-homo-gamma linolenic acid (DHLA), docosatetraenoic acid (DTA) and the like, which comprises a fatty acid chain- Wherein the composition is selected from the group consisting of docosapentaenoic acid (DPA) and a polyunsaturated fatty acid. In the composition of the present invention, the fatty acid chain extension enzyme gene is as described above. The composition of the present invention contains a fatty acid chain extension enzyme gene derived from an eel of mackerel as an active ingredient, and the polyunsaturated fatty acid can be produced in a plant by transforming the fatty acid chain extension enzyme gene into a plant.

The polyunsaturated fatty acid can be synthesized using the fatty acid chain extension enzyme gene ( McELOVL5 ) according to the present invention. Further, the present invention has the effect of providing a microorganism or a plant producing the highly unsaturated fatty acid such as omega fatty acid using the gene. Specifically, the present invention can be applied to the development of high value-added agricultural income crops by applying the fatty acid chain-extending enzyme gene derived from the mussels of the present invention to the transformation of oilseed crops.

Figure 1 shows an alternative pathway for biosynthesis of polyunsaturated fatty acids in eukaryotic cells.
Figure 2 shows the bi-directional fragmentation of the fatty acid chain extension enzyme gene by 5'-RACE PCR.
Fig. 3 shows the gene insertion and its size through fatty acid chain extension enzyme gene isolation and restriction enzyme cleavage of full size.
Fig. 4 shows a result of comparing the nucleotide sequence similarity of the fatty acid chain extension enzyme gene derived from mugwort ( Muraenesox cinereus) .
Fig. 5 shows the result of the analysis of the similarity of the amino acid sequence of the fatty acid chain extension enzyme gene derived from the mackerel ( Muraenesox cinereus) .
6 shows the production and cloning confirmation of a yeast transformation carrier (pYES2- McELOVL5 ) for expression of a fatty acid chain extension enzyme gene.
FIG. 7 shows the yeast transformation of the fatty acid chain extension enzyme gene expression carrier (pYES2- McELOVL5 ) selected in the minimal medium.
FIG. 8 is a fatty acid gas chromatogram obtained by analyzing the conversion of pYES2 and pYES2- McELOVL5 to DGLA for the substrate GLA.
9 is a fatty acid gas chromatogram obtained by analyzing the conversion of pYES2 and pYES2- McELOVL5 transformed yeast into DTA for substrate ARA.
10 is a fatty acid gas chromatogram obtained by analyzing the conversion of pYES2 and pYES2- McELOVL5 transformed yeast into DPA for substrate EPA.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1: mackerel ( Muraenesox cinereus ) Isolation and Sequence Analysis of Fatty Acid Chain Extension Enzymes from

The partial sequence of the fatty acid chain extension gene ( McELOVL5 ) was isolated from the mackerel ( Muraenesox cinereus) using a 5'- and 3'-RACE method (SMART RACE cDNA Amplification kit) A primer capable of securing a full-length clone of the fatty acid chain extension gene ( McELOVL5 ) gene was rewritten and then subjected to RACE-PCR to obtain McELOVL5 Gene. Obtained McELOVL5 The gene was inserted into the pGEM T-Easy vector, and its size was confirmed by restriction enzyme digestion ( EcoRI ) (FIG. 3) and sequenced (SEQ ID NO: 1). Sequence analysis revealed that McELOVL5 gene was composed of ORF (Open reading frame) of 885 bp size.

The fatty acid chain elongase gene obtained from the P. japonicus brain showed a difference in four places indicated by red in the DNA sequence (McELO-12 in FIG. 4). In the amino acid sequence, only the difference between the 102nd and 160th positions of the previously known AsELOVL5 (Acanthopagrus schlegelii, GenBank accession no gb1218654) and SsElovl5b (Salmo salar, GenBank Accession No. gb | FG237531 | The remainder consisted of five transmembrane-spanning domains (designated 1-5 in FIG. 5) with the characteristics of a chain extension enzyme and a single histidine box promoting chain extension enzyme activity (Fig. 5, McELO-12). As shown in Fig.

Example 2 Yeast of Fatty Acid Chain Extension Gene ( Saccharomyces cerevisiae ) Construction of transgenic expression vectors and confirmation of transformation

The chain extension enzyme gene ( McELOVL5) isolated from mackerel was inserted into pYES2 ( Saccharomyces cerevisiae ) expressing carrier using restriction enzymes BamH I and EcoR I. Confirmation of insertion was confirmed by the same restriction enzyme cleavage of pYES- McELOVL5 carrier (FIG. 6).

The constructed vector was transformed into yeast using lithium acetate method and selected in the minimal medium lacking uracil. The selected clones were confirmed to contain the inserted McELOVL5 gene by comparison with pYES2 containing only the control vector, and the clones were reverse-transformed into E. coli to confirm that they were correct clones (FIG. 7).

Example 3: Confirmation of chain extension from carbon chain C18 to C22 of a fatty acid chain extension enzyme gene-transforming yeast

In order to confirm the function of McELOVL5 gene using yeast (pYES2- McELOVL5 ) inserted with fatty acid chain extension enzyme gene ( McELOVL5) , seed culture was carried out at 28 ° C in a medium supplemented with uracil deficiency and raffinose / galactose. (GLA, C18: 3 n-6) or arachidonic acid (ARA, C20: 4, n-6) and Eikosha Co., Ltd. were added to the new medium to a final concentration of OD600 = 0.5. (EPA, C20: 5 n-3) were respectively added to a final concentration of 0.5 mM and 0.1% (v / v) tergitol NP-40 was added so that the substrate fused well in the medium. Co-cultivation for 3 days at 20 &lt; 0 &gt; C and for 3 days at 15 &lt; 0 &gt; C resulted in the conversion of the added substrate to the new product. For the control, only yeast transformed with pYES2 carrier was used.

A feeding test was conducted to confirm the activity of fatty acid chain extension enzymes obtained from P. gingivalis. Yeast transformants containing pYES2-McELOVL5 (12-4) and control pYES2 were transformed with gamma-linolenic acid (GLA, C18: 3n-6) with 18 carbon chains in a culture medium containing 2% Chain extension of fatty acids added. As shown in FIG. 8 and Table 1, the result was that pYES2-McELOVL5 (11-7) was converted from GLA to di-homo-gum marinolenic acid (DGLA, C20: 3n-6) 3.97% of fatty acids were produced and showed 64% higher substrate conversion.

A substrate test for 20 carbon chains against pYES2-McELOVL5 (12-4) was performed. When the substrate arachidonic acid (ARA, C20: 4n-6) or eicosapentaenoic acid (EPA, C20: 5n-3) was added, the yeast transformant pYES2-McELOVL5 (12-4) (DPA, C22: 5n-3) (FIG. 10) with the substrate ARA as docosatetraenoic acid (DTA, C22: 4n-6) (FIG. 9) and the EPA to the docosapentaenoic acid . Again, Table 1 shows that DTA accounts for 2.03% of total fatty acids and 15.5% fatty acid chain extension conversion. DPA accounted for 4.41% of total fatty acids and 47.5% fatty acid chain extension conversion. Therefore, it can be seen that the marshmallow- derived fatty acid chain extension enzyme gene McELOVL5 of the present invention shows a higher substrate activity for the omega-3 pathway than the omega-6 pathway.

(In the table, "16: 0" is taken as an example in the table, the number 16 represents the number of carbon chains, and the numbers after the unsaturation indicate unsaturated fatty acids.

As described above, a carrier (pYES2-McELOVL5) was prepared so that the fatty acid chain extension enzyme gene was regulated by the galactose-inducible promoter of pYES2, which is a yeast expression carrier, and transformed into yeast and cultured by adding GLA, ARA and EPA as substrates DGLA, DTA and DPA were synthesized in the control.

<110> REPUBLIC OF KOREA <120> FATTY ACID CHAIN ELONGASE GENE FROM MURAENESOX CINEREUS AND USES          THEREOF <130> P120988 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 885 <212> DNA <213> Muraenesox cinereus <400> 1 atggaaatgt ttaaccacag gctaaataca tatatcgatt catggatggg acccagagat 60 cagagggtca gaggctggct ccttttggat gactaccctc caacgtttgc actcacggta 120 gcgtacctgc tgatcgtttg gatgggacca aagtacatga aaaacagaca gcccttctcc 180 tgcagaggtc tgctggtggt ctacaacctc gggctcaccc tgctgtccct gtacatgttc 240 tgcgagctgg tgaatggtat gtggcagggg aactacaact tcttctgcca gaacacacac 300 agcgttggag aggctgacac aaagatcata aacgtcctct ggtggtacta cttctccaag 360 ctcatcgagt tcatggacac cttcttcttc atcctgcgga agaacaacca ccagatcacc 420 ttcctgcacg tgtaccacca cgccagcatg ctcaacatct ggtggttcgt catgaactgg 480 gtgccgtgcg gacactcata ttttggcgcc tccctcaaca gcttcatcca cgtcctgatg 540 tactcgtact acggcctgtc cgccatcccc gccctgcggc cttacctctg gtggaagaaa 600 tacatcactc agggtcagct gatccagttt gttatgacca tgactcagac tagctgtgct 660 gtggtctggc cttgtgggtt ccctatgggc tggctgtact tccagatcag ctacatggtc 720 acgctaatcg cacttttctc aaacttctac atacagacct atcagaagca gggagccttc 780 agaaggaaag agcatcagaa cggctctgcg gcggccatga acgggcattc caacggggtg 840 tcgcccgctg aagacctaac gcgtaggaaa ctgagggtag actga 885 <210> 2 <211> 294 <212> PRT <213> Muraenesox cinereus <400> 2 Met Glu Met Phe Asn His Arg Leu Asn Thr Tyr Ile Asp Ser Trp Met   1 5 10 15 Gly Pro Arg Asp Gln Arg Val Val Gly Trp Leu Leu Leu Asp Asp Tyr              20 25 30 Pro Pro Thr Phe Ala Leu Thr Val Ala Tyr Leu Leu Ile Val Trp Met          35 40 45 Gly Pro Lys Tyr Met Lys Asn Arg Gln Pro Phe Ser Cys Arg Gly Leu      50 55 60 Leu Val Val Tyr Asn Leu Gly Leu Thr Leu Leu Ser Leu Tyr Met Phe  65 70 75 80 Cys Glu Leu Val Asn Gly Met Trp Gln Gly Asn Tyr Asn Phe Phe Cys                  85 90 95 Gln Asn Thr His Ser Val Gly Glu Ala Asp Thr Lys Ile Ile Asn Val             100 105 110 Leu Trp Trp Tyr Tyr Phe Ser Lys Leu Ile Glu Phe Met Asp Thr Phe         115 120 125 Phe Phe Ile Leu Arg Lys Asn Asn His Gln Ile Thr Phe Leu His Val     130 135 140 Tyr His His Ala Ser Met Leu Asn Ile Trp Trp Phe Val Met Asn Trp 145 150 155 160 Val Pro Cys Gly His Ser Tyr Phe Gly Ala Ser Leu Asn Ser Ser Phe Ile                 165 170 175 His Val Leu Met Tyr Ser Tyr Tyr Gly Leu Ser Ala Ile Pro Ala Leu             180 185 190 Arg Pro Tyr Leu Trp Trp Lys Lys Tyr Ile Thr Gln Gly Gln Leu Ile         195 200 205 Gln Phe Val Met Thr Met Thr Gln Thr Ser Cys Ala Val Val Trp Pro     210 215 220 Cys Gly Phe Pro Met Gly Trp Leu Tyr Phe Gln Ile Ser Tyr Met Val 225 230 235 240 Thr Leu Ile Ala Leu Phe Ser Asn Phe Tyr Ile Gln Thr Tyr Gln Lys                 245 250 255 Gln Gly Ala Phe Arg Arg Lys Glu His Gln Asn Gly Ser Ala Ala Ala             260 265 270 Met Asn Gly His Ser Asn Gly Val Ser Pro Ala Glu Asp Leu Thr Arg         275 280 285 Arg Lys Leu Arg Val Asp     290

Claims (17)

A fatty acid chain extension enzyme protein derived from the myrtle ( Muraenesox cinereus ) comprising the amino acid sequence of SEQ ID NO: 2.
A gene encoding the protein of claim 1.
3. The method of claim 2,
Wherein the gene is a nucleotide sequence of SEQ ID NO: 1.
A recombinant vector comprising the gene of claim 2.
5. The method of claim 4,
The recombinant vector is the pYES2- McELOVL5 vector described in Fig.
A host cell transformed with the recombinant vector of claim 4.
The method according to claim 6,
Wherein the host cell is a yeast.
Homo-gamma (D-homo-gamma) gene in a host cell, which comprises transforming a host cell with a recombinant vector comprising a mackerel-derived fatty acid chain extension gene consisting of the nucleotide sequence of SEQ ID NO: wherein the polyunsaturated fatty acid is selected from the group consisting of linolenic acid, DHLA, docosatetraenoic acid (DTA), and docosapentaenoic acid (DPA).
9. The method of claim 8,
The polyunsaturated fatty acid is a polyunsaturated fatty acid which is a di-homo-gamma linolenic acid (DHLA) produced by extending the carbon chain of gamma-linolenic acid (GLA) Production method.
9. The method of claim 8,
Wherein said polyunsaturated fatty acid is docosatetraenoic acid (DTA) produced by extending the carbon chain of arachidonic acid (ARA).
9. The method of claim 8,
Wherein said polyunsaturated fatty acid is docosapentaenoic acid (DPA) produced by extending the carbon chain of eicosapentaenoic acid (EPA).
delete 9. The method of claim 8,
Wherein said host cell is yeast.
A step of transforming a plant cell with a recombinant vector comprising a myrtle-derived fatty acid chain extension enzyme gene comprising the nucleotide sequence of SEQ ID NO: 1, wherein the di-homo-gamma linolenic acid, DHLA, docosatetraenoic acid (DTA), and docosapentaenoic acid (DPA). &Lt; Desc / Clms Page number 2 &gt;
A pharmaceutical composition comprising a di-homo-gamma linolenic acid (DHLA), docosatetraenoic acid (DTA), and docosapentaenoic acid (DTA) prepared by the method of claim 14, acid, DPA). &lt; / RTI &gt;
15. A plant seed according to claim 15.
(DHLA), docosatetraenoic acid (DHA), or the like, which comprises the mature codfish-derived fatty acid chain extension enzyme gene comprising the nucleotide sequence of SEQ ID NO: DTA) and docosapentaenoic acid (DPA). &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt;

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