KR101883832B1 - Delta-7 desaturase gene from Phaeodactylum tricornutum, and uses thereof - Google Patents

Abstract

The present invention relates to the use of phaeodactylum tricornutum) derived delta -7 unsaturated enzyme (Delta-7 Desaturase, D7DES) gene, and by using this, cis-hexahydro-7-de sensan (cis-7-hexadecenoic acid) or trans-9-octadecyl sensan (trans-9-octadecenoic acid. < / RTI > As a result of inserting the above-mentioned delta-7 unsaturated enzyme (D7DES) into a transformation vector, an unsaturated fatty acid having a double bond introduced into the 7th carbon was produced. Therefore, the fatty acid composition of the microorganism or plant was changed by metabolic engineering Can be usefully used.

Description

Delta-7 desaturase gene and its use (Delta-7 desaturase gene from Phaeodactylum tricornutum, and uses thereof)

The present invention relates to the use of phaeodactylum tricornutum) derived delta -7 unsaturated enzyme (Delta-7 Desaturase, D7DES) gene, and by using this, cis-hexahydro-7-de sensan (cis-7-hexadecenoic acid) or trans-9-octadecyl sensan (trans-9-octadecenoic acid. < / RTI >

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 at least two 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 'saturated' 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. Unsaturated fatty acids are also known to have lower melting points and to increase the fluidity of cell membranes.

Fatty acids in plants are an important constituent of cell membranes and storage oils in seeds. Linolenic acid (18: 3), a type of unsaturated fatty acid, is synthesized by different substrates and enzymes in plant plastids and endoplasmic reticulum (Non-Patent Document 1). The microsomal linoleic acid unsaturated enzyme (hereinafter abbreviated as 'FAD3') exists in the intracellular endoplasmic reticulum and linolenic acid (18: 2), a double unsaturated fatty acid present at the sn-1 and sn-2 positions of phosphatidylcholine, It is an enzyme that catalyzes the conversion of linoleic acid (18: 3), an unsaturated fatty acid.

Oil produced from seeds of plants is used not only for edible use such as vegetable oil and margarine but also for various industrial raw materials such as soap, paint, surfactant, and biodiesel (Non-Patent Document 2). In particular, the degree of unsaturation of fatty acids in vegetable oils is an important indicator for the industrial application of vegetable oils. For example, soybean oil having a high content of oleic acid (18: 1) is known as a vegetable oil that is healthier than soybean oil having high linoleic acid content because of its high oxidative stability. On the other hand, soybean oil having a high content of linoleic acid is used as a drying oil in paints, inks and the like, by using the property of drying quickly in the air. The unsaturation of fatty acids in such vegetable oils is determined by the enzymatic activity of various unsaturated enzyme genes (Non-Patent Document 3).

On the other hand, phaeodactylum tricornutum ) is a kind of marine diatom, and it is known that EPA (Eicosapentaenoic acid), which is an unsaturated fatty acid, has a high content (Patent Document 1). However, studies on unsaturated fatty acid synthetase derived from Phaeodactylum tricornutum have not been performed so far.

Accordingly, the present inventors have isolated a novel fatty acid unsaturated enzyme gene from Phaeodactylum tricornutum and confirmed its unsaturated fatty acid synthesis function, thereby completing the present invention.

Korean Patent Laid-Open Publication No. 2014-0132226

 Somerville C, Browse J, Jaworski JG, Ohlrogge J. Lipids. In B Buchanan, W Gruissem, R Jones, eds., Biochemistry & Molecular Biology of Plants. 2000, pp. 456-527, American Society of Plant Physiologists, Rockville.  Ohlrogge JB. Design of new plant products: engineering of fatty acid metabolism. Plant Physiol., 1994, 104: 821-826.  Kinney AJ, Cahoon EB, Hitz WD. Manupulating desaturase acrivities in transgenic crop plants. Biochem Soc Trans., 2002, 30: 1099-1103.

An object of the present invention is to provide a Delta-7 desaturase (D7DES) gene consisting of the nucleotide sequence of SEQ ID NO: 1.

Another object of the present invention is to provide a delta-7 unsaturated protein protein consisting of the amino acid sequence of SEQ ID NO: 2 which is encoded from the gene.

It is still another object of the present invention to provide a recombinant vector containing the gene.

It is still another object of the present invention to provide a host cell transformed with the recombinant vector.

It is another object of the present invention to provide a method for producing cis-7-hexadecenoic acid or trans-9-octadecenoic acid in a host cell using the gene .

In order to achieve the above object, one embodiment of the present invention provides a Delta-7 Desaturase (D7DES) gene comprising the nucleotide sequence of SEQ ID NO: 1.

The gene is expressed in phaeodactylum tricornutum ). The above-mentioned " Phaeodactylum tricornutum "is a kind of diatom of marine microalgae, and contains various lipids at a weight of about 20-30% of dry cell weight.

As used herein, the term " Delta-7 Desaturase (D7DES) "refers to an enzyme that produces unsaturated fatty acids by introducing a double bond at the carbon position 7 of the saturated fatty acid.

The gene consisting of the nucleotide sequence of SEQ ID NO: 1 has a nucleotide sequence of 1,362 bp.

The delta-7 desaturase gene includes a homologous gene having homology with the gene consisting of the nucleotide sequence of SEQ ID NO: 1.

 The "homologous gene" is a gene having a sequence homology of preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 99% or more, with the nucleotide sequence of SEQ ID NO: Quot; refers to a gene that exhibits a function substantially equivalent to the delta-7 unsaturated enzyme gene of the present invention. Sequence homology can be analyzed by methods known in the art.

As used herein, the term "substantially homogenous function" refers to the production of an unsaturated fatty acid by introducing a double bond at the carbon position 7 of the saturated fatty acid. Such functional equivalents include, for example, amino acid sequence variants in which some of the amino acids of the amino acid sequence of SEQ ID NO: 2 are substituted, deleted or added. Substitution of amino acids is preferably conservative substitution. Examples of conservative substitutions of amino acids present in nature are as follows: (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of the amino acid is preferably located at a site that is not directly involved in the activity of the delta-7 unsaturated enzyme of the present invention. Also included within the scope of such functional equivalents are protein derivatives in which the basic skeleton of the delta-7 unsaturated enzyme and its chemical structure has been modified while maintaining its physiological activity. These include, for example, fusion proteins made by fusion with other proteins such as GFP, while retaining the structural modification and physiological activity to change the stability, storage stability, volatility or solubility of the protein of the present invention.

The transformant according to the present invention is not only a gene having the nucleotide sequence of SEQ ID NO: 1 but also a modified sequence in which some nucleotides of SEQ ID NO: 1 are substituted, deleted or added, A nucleotide sequence encoding a protein exhibiting an activity equivalent to that of a protein expressed from the expression vector, i.e., the delta-7 unsaturated enzyme, can be used.

Another embodiment of the present invention provides a delta-7 unsaturated protein protein consisting of the amino acid sequence of SEQ ID NO: 2 encoded from the gene.

The protein consisting of the amino acid sequence of SEQ ID NO: 2 consists of 453 amino acids.

The delta-7 desaturase protein comprises a homologous protein having homology to a protein consisting of the amino acid sequence of SEQ ID NO: 2.

The "homologous protein" refers to a protein having a sequence homology of preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 99% or more homologous to the amino acid sequence of SEQ ID NO: Quot; refers to a protein that exhibits substantially the same function as the delta-7 desaturase protein of the present invention.

The delta-7 desaturase protein may comprise a cytochrome b5-domain and a histidine box.

Another embodiment of the present invention provides a recombinant vector comprising the delta-7 desaturase gene.

Accordingly, the recombinant vector may include a gene encoding a delta-7 unsaturated enzyme gene comprising the nucleotide sequence of SEQ ID NO: 1 or a gene encoding the delta-7 unsaturated enzyme protein comprising the amino acid sequence of SEQ ID NO: 2.

The recombinant vector may be for promoting an unsaturated fatty acid into which a double bond is introduced to carbon number 7, and more specifically, cis-7-hexadecenoic acid or trans-9-octadecenoic acid (trans- octadecenoic acid.

As used herein, 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.

As used herein, the term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is delivered into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "expression vector" is often used interchangeably with a "recombinant vector ". The term "recombinant 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 recombinant vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter (for example, pL? Promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.) , Ribosome binding sites for initiation of detoxification, and transcription / translation termination sequences.

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 recombinant vector of the present invention is an expression vector and a 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 (e.g., Adenovirus 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.

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. Since the selection of the transformant can be carried out by various tissues at various stages, the expression promoter is always preferable in the present invention. Thus, the constant expression promoter does not limit the selectivity.

The expression vector may 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 Bar, glyphosate, glufosinate ammonium or phosphinothricin, kanamycin, G418, Bleomycin, But are not limited to, antibiotic resistance genes such as hygromycin and chloramphenicol.

Another embodiment of the present invention provides a host cell transformed with said recombinant vector.

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) and the like insect cells, human cells (e.g., CHO cells (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines), plant cells and plants can be used. Specifically, the host cell may be a yeast or a plant cell. More specifically, the plant cell may be a cell of a vegetable oil plant. More specifically, the above-mentioned maintenance plant may be a sesame, rapeseed, sunflower, Peanuts, beans, coco, oil palm, olive, soy, corn, jojoba, and the like.

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 .

Another embodiment of the present invention provides a method of producing cis-7-hexadecenoic acid or trans-9-octadecenoic acid in a host cell, Specifically, the method comprises the step of amplifying a phaeodactylum ( Phaeodactylum tricornutum) may comprise the step of transforming a host cell with a recombinant vector comprising the delta -7 unsaturated enzyme (Delta-7 Desaturase, D7DES) gene of origin.

Specifically, the host cell may be a yeast or a plant cell. More specifically, the plant cell may be a cell of a vegetable oil plant. More specifically, the above-mentioned maintenance plant may be a sesame, rapeseed, sunflower, Peanuts, beans, coco, oil palm, olive, soy, corn, jojoba, and the like.

The above-mentioned delta-7 unsaturated enzyme (D7DES) can be converted to hexadecenoic acid (C16: 1 ^{? 7} ) by unsaturation of the 7th carbon of palmitic acid (C16: 0), stearic acid , C18: 0) is desirably converted to elaidic acid (9-octadecenoic acid (E), C18: 1 trans-9) by unsaturation but not limited thereto.

As a result of induction of expression by inserting a diatomaceous delta-7 unsaturated enzyme (D7DES) according to one embodiment of the present invention into a transformation vector, an unsaturated fatty acid having a double bond introduced into the 7th carbon was produced. It can be usefully used to change microbes or the fatty acid composition of plants.

Also, cis-7-hexadecenoic acid or trans-9-octadecenoic acid produced by the delta-7 unsaturated enzyme of the present invention may be utilized as a functional substance in the fields of medicine and cosmetics.

Figure 1 shows the phaeodactylum < RTI ID = 0.0 > tricornutum ).
Figure 2 is a graph showing the effect of Phaeodactylum < RTI ID = 0.0 > 7 shows the conserved domains and phylogeny of delta-7 unsaturated enzyme ( PtD7DES ) derived from tricornutum: Klebsormidium delta-5 fatty acid unsaturation enzyme derived from flaccidum (KfD5DES); Dictyostelium delta-5 fatty acid unsaturated enzyme derived from fasciculatum (DfD5DES); Euglena gracilis ) -derived delta-4 fatty acid unsaturation enzyme (EgD4DES); Delta-4-unsaturated enzyme (TaD4DES) derived from Thraustochytrium aureum ; Microsaturated Delta 5 unsaturated enzyme (SrD5DES) derived from Salpingoeca rosetta ; Thalassiosira pseudonana) CCMP1335 derived from unsaturated delta 5 enzyme (TpD5DES).
Figure 3 is a graph showing the effect of Phaeodactylum < RTI ID = 0.0 > tricornutum) derived delta -7 unsaturated enzyme (PtD7DES) amino acid sequence analysis is: keulrep sorbitan Medium flat raksi Doom (Klebsormidium flaccidum) derived from unsaturated fatty acids, delta-5 enzyme (KfD5DES); Dictyostelium delta-5 fatty acid unsaturated enzyme derived from fasciculatum (DfD5DES); Euglena gracilis ) -derived delta-4 fatty acid unsaturation enzyme (EgD4DES); Thraustochytrium < / RTI > aureum- derived delta-4-unsaturated enzyme (TaD4DES); Microsaturated Delta 5 unsaturated enzyme (SrD5DES) derived from Salpingoeca rosetta ; Delta 5 unsaturated enzyme (TpD5DES) derived from Thalassiosira pseudonana CCMP1335.
Figure 4 is a graph showing the effect of Phaeodactylum < RTI ID = 0.0 > tricornutum) is TMHMM results derived delta -7 unsaturated enzyme (PtD7DES) protein.
FIG. 5 is a graph showing the effect of Phaeodactylum tricornutum) are derived from unsaturated enzyme delta -7 (PtD7DES) a topology model, based on the TMHMM results of protein (model topology).
Figure 6 is a graph showing the effect of Phaeodactylum < RTI ID = 0.0 > (PtD7DES) gene derived from a tricornutum- derived delta-7 unsaturated enzyme (PtD7DES) gene.
Fig. 7 shows results of fatty acid GC-MS analysis of pYES2-PtD7DES-transformed yeast.
Fig. 8 shows the result of identification of cis-7-hexadecenoic acid newly produced in pYES2-PtD7DES-transformed yeast.
Fig. 9 shows the result of identification of trans-9-octadecenoic acid newly produced in the pYES2-PtD7DES transgenic yeast.
FIG. 10 is a view showing a biosynthetic pathway of an isomer according to the double bond position of the unsaturated fatty acid C16: 1 family. FIG.

Hereinafter, the present invention will be described in more detail in the following Examples. It should be noted, however, that the following examples are illustrative only and do not limit or limit the scope of the present invention. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

< Example  1> Paedo Til Room Tricornutum  ( Phaeodactylum tricornutum ) As a result, Isolation and analysis of fatty acid biosynthesis genes

Phaeodactylum tricornutum ) and the structure of the fatty acid biosynthesis gene was analyzed.

<1-1> Paedo Til Room Tricornutum  ( Phaeodactylum tricornutum ) Derived gene Pt Isolation of D7DES

Using a RACE-PCR in marine diatoms par ohdak estimation needed for useful fatty acid biosynthesis from tilrum tree cor nutum (Phaeodactylum tricornutum) an unsaturated fatty acid was isolated enzymes (putative fatty acid desaturase) ORF ( Open Reading Frame).

Specifically, RACE-PCR was performed on the basis of the fatty acid unsaturation enzyme of Thalassiosira pseudonana , a marine diatomite whose base sequence is known. Phaeodactylum RACE-PCR was performed using the cDNA obtained from the total RNA of tricornutum as a template. In the RACE-PCR, the primers were PtD7D-F (5'-ATGGCTACGACTACCCCGAG-3 ': SEQ ID NO: 3) and PtD7D-R (5'-TCAGTCCAGACCAACCGACAATTG-3': SEQ ID NO: 4). At this time, RACE-PCR was performed under the conditions of PCR amplification for 3 minutes at 94 ° C, followed by heating at 94 ° C for 30 seconds, heating at 58 ° C for 30 seconds, and heating at 72 ° C for 1 minute and 30 seconds. After repeating 40 cycles (40 cycles), the cells were further heated at 72 ° C for 5 minutes. RACE-PCR was performed using Applied Biosystems' Gene Amp ^® PCR System 9700, and specific PCR products amplified under the above conditions were loaded on 1% agarose gel and subjected to HiYield ™ Gel / PCR DNA Extraction Kit (RBC), followed by sequencing.

The resultant specific PCR product was cloned into pGEM and the nucleotide sequence was analyzed (Fig. 1). As a result, 1,362 bp of the predicted fatty acid desaturase nucleotide sequence was confirmed and the amino acid sequence thereof was shown in SEQ ID NO: 2 Respectively. The present inventors deduced this estimated fatty acid unsaturated enzyme as Delta-7 Desaturase ( D7DES ) and named Pt D7DES ( Phaeodactylum tricornutum Delta-7 Desaturase).

<1-2> Pt Of D7DES  Structure analysis

NCBI Blast analysis of an amino acid 453 codon encoded by SEQ ID NO: 1 confirmed the N-terminal cytochrome b5-domain and FAD-like domain characteristic of the fatty acid biosynthetic polymerase. In addition, phylogenetic tree showing the biosynthetic relationship of Pt D7DES was confirmed. As a result, Delta 5 fatty acid unsaturated enzyme (KfD5DES) derived from Klebsormidium flaccidum ; Dictyostelium delta-5 fatty acid unsaturated enzyme derived from fasciculatum (DfD5DES); Euglena gracilis ) -derived delta-4 fatty acid unsaturation enzyme (EgD4DES); Thraustochytrium &lt; / RTI &gt; aureum- derived delta-4-unsaturated enzyme (TaD4DES); Microsaturated Delta 5 unsaturated enzyme (SrD5DES) derived from Salpingoeca rosetta ; Thalassiosira homologous to delta-5-unsaturated enzymes or delta-4-unsaturated enzymes of various species such as pseudonana CCMP1335-derived delta penta- desaturase (TpD5DES) (FIG. 2).

As a result of the amino acid sequence analysis with the above-mentioned unsaturated enzymes of various species, it was found that the cytochrome b5 heme-binding domain, which is a characteristic of the fatty acid unsaturated enzyme, and homology in the histidine box 1, (Fig. 3).

Drawing its topology model, there are four membrane-spanning helices. This predicted protein model was drawn based on the TMHMM algorithm (http://www.cgs.dtu.dk/services/TMHMM/) (FIGS. 4 and 5).

< Example  2> Pt Of D7DES  Function analysis

<2-1> On PtD7DES  Confirmation of the synthesis of new unsaturated fatty acids

The delta 7 unsaturated enzyme ( Pt D7DES) gene isolated from Phaeodactylum tricornutum according to Example 1 was cloned into pYES2 , a yeast ( S. cerevisiae ) expression carrier. The yeast (pYES2- PtD7DES ) into which the delta-7 desaturase gene was inserted was used to transform Pt D7DES In order to confirm the function of the gene, seed culture was carried out at 28 ° C in a medium containing uracil deficiency and raffinose / galactose. The seed culture was added to the new medium to a final concentration of OD600 = 0.4, and co-cultured at 20 ° C for 3 days and at 15 ° C for 3 days to induce the conversion to a new product. This was analyzed by gas chromatography.

As a result, a new product (C16: 1) in which one double bond was formed by fatty acid polymerase from palmitic acid (C16: 0) was found in the pYES2- PtD7DES transgenic yeast compared to the control (pYES2) A new product with one double bond (C18: 1) was seen from the acid (Stearic acid, C18: 0) (Figure 6).

Therefore, it was confirmed that two kinds of unsaturated fatty acids having one double bond were synthesized by PtD7DES.

<2-2> Identification of unsaturated fatty acids produced

The new products produced in Example 2-1 were analyzed by GC-MS (gass chromatograph-mass spectrometer) (Fig. 7). Peaks in the time zone shown in FIG. 7 were analyzed through a fatty acid standard library, and the results are shown in Table 1.

A new peak (RT 31.5) with one double bond formed by fatty acid polymerase from palmitic acid (C16: 0) was identified as hexadecenoic acid , C16: 1 ^{△ 7)} was, stearic acid (stearic acid, C18: 0) new product (RT 35.2) occurred one double bond from the El fluoride acid (elaidic acid, 9-octadecenoic acid (E), C18: 1 trans-9). The results of identification of the ionized molecular weight values for these two materials are shown in FIGS. 8 and 9. FIG.

The isomer according to the double bond position of C16: 1 with one double bond in 16 carbon chains is palmitoleic acid (9cis-16: 1) and sapienic acid (6cis-16: 1) . Therefore, the biosynthetic pathway for the two previously known unsaturated fatty acids and hexadecenoic acid (7cis-16: 1) newly found in this experiment is schematically shown in FIG.

<110> Republic of Korea <120> Delta-7 desaturase gene from Phaeodactylum tricornutum, and uses          the <130> P16R12D1122 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 1362 <212> DNA <213> Artificial Sequence <220> <223> D7DES gene <400> 1 atggctacga ctaccccgag ccagtccaag acactcgacc cattgctatt gtggatgatt 60 cacggaaact actatgacct ccacacgtat gtatcgcggc atcccggtgg aaaggaagct 120 attctgttgg gacgcggtcg cgactgcacc gccttgttcg aatcctacca tcccttcact 180 tcgcagcacc gtcgtgtatt ggaaaaacat cgagtgacaa ttgacacctc gttttgcagc 240 aaacagcaag acagaaaagt atcgaaacaa acagtttcat ccagcgaaca agccagtgac 300 gtcttttaca acatgttgtg tcagcgcgtc gcgcacgcac tgcaatctca aggagtggat 360 ccgatccgtg atcgaggtgc cacatggaca cgcagtatct actacgtctt tctgttcgcc 420 gcattgctag ctagcggata cgctcactgc acgggaagcc tactcggtag ccttctcttt 480 ggtgtggctg gttggtttat tggtgctttg ggacacgatg gcggccactt tgctgtctct 540 cgccgagcct ggctcaacga tttttccgtc tggggtattt cctggttgtg caatcctatc 600 atgtggcaac accagcacac gtacgcgcac catagtttca ccaacgaatt tgatcacgat 660 ccggatttgc accacttcac aacctttctc cgagtacatc gcaagtttca gcaaaactgt 720 atttatcgca accaagccaa ctgggtgtac gtgttttggg cctatacctt tgttaccttt 780 ggcgcatgct tttggattcc atggggcgtg ttgcgggagc agaccttgta cggactcgtc 840 gattggacgg atcggaagcg tccgtcacgg acggcggcct ttgtgttcca cttggtaacc 900 tactttggtc tcgtcatggt gctccccttt tggacgcacg gaacttggta caaggcgtgt 960 ttgggtgtca tcttgcacat gaccacctcg ggcttgatct ttgccttttt ctcgcaaatc 1020 aatcacatca acgaactatc gttggacgaa aaggtcgtga aactgcaccg atccacgttg 1080 gatcctaccg tgcgcgattc gtgggccgta gcgcaggtgg aagcctccaa caatttttgc 1140 accgactcgg cattttggca tctgttctcc aacggactta atttgcaaat tgaacaccat 1200 ctgtttccgg gaatcaacca ttgccacttg catcacatcg cgccggtcgt ccgcgaaacc 1260 tgcaatgagt acggtgtgcg gtacaagtcg tacgatagtt ggtccgatat aatgcgagca 1320 atgttgaagt ggctcgacca attgtcggtt ggtctggact ga 1362 <210> 2 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> D7DES protein <400> 2 Met Ala Thr Thr Thr Pro Ser Gln Ser Lys Thr Leu Asp Pro Leu Leu   1 5 10 15 Leu Trp Met Ile His Gly Asn Tyr Tyr Asp Leu His Thr Tyr Val Ser              20 25 30 Arg His Pro Gly Gly Lys Glu Ala Ile Leu Leu Gly Arg Gly Arg Asp          35 40 45 Cys Thr Ala Leu Phe Glu Ser Tyr His Pro Phe Thr Ser Gln His Arg      50 55 60 Arg Val Leu Glu Lys His Arg Val Thr Ile Asp Thr Ser Phe Cys Ser  65 70 75 80 Lys Gln Gln Asp Arg Lys Val Ser Lys Gln Thr Val Ser Ser Ser Glu                  85 90 95 Gln Ala Ser Asp Val Phe Tyr Asn Met Leu Cys Gln Arg Val Ala His             100 105 110 Ala Leu Gln Ser Gln Gly Val Asp Pro Ile Arg Asp Arg Gly Ala Thr         115 120 125 Trp Thr Arg Ser Ile Tyr Tyr Val Phe Leu Phe Ala Ala Leu Leu Ala     130 135 140 Ser Gly Tyr Ala His Cys Thr Gly Ser Leu Leu Gly Ser Leu Leu Phe 145 150 155 160 Gly Val Ala Gly Trp Phe Ile Gly Ala Leu Gly His Asp Gly Gly His                 165 170 175 Phe Ala Val Ser Arg Arg Ala Trp Leu Asn Asp Phe Ser Val Trp Gly             180 185 190 Ile Ser Trp Leu Cys Asn Pro Ile Met Trp Gln His Gln His Thr Tyr         195 200 205 Ala His His Ser Phe Thr Asn Glu Phe Asp His Asp Pro Asp Leu His     210 215 220 His Phe Thr Thr Phe Leu Arg Val His Arg Lys Phe Gln Gln Asn Cys 225 230 235 240 Ile Tyr Arg Asn Gln Ala Asn Trp Val Tyr Val Phe Trp Ala Tyr Thr                 245 250 255 Phe Val Thr Phe Gly Ala Cys Phe Trp Ile Pro Trp Gly Val Leu Arg             260 265 270 Glu Gln Thr Leu Tyr Gly Leu Val Asp Trp Thr Asp Arg Lys Arg Pro         275 280 285 Ser Arg Thr Ala Phe Val Phe His Leu Val Thr Tyr Phe Gly Leu     290 295 300 Val Met Val Leu Pro Phe Trp Thr His Gly Thr Trp Tyr Lys Ala Cys 305 310 315 320 Leu Gly Val Ile Leu His Met Thr Thr Ser Gly Leu Ile Phe Ala Phe                 325 330 335 Phe Ser Gln Ile Asn His Ile Asn Glu Leu Ser Leu Asp Glu Lys Val             340 345 350 Val Lys Leu His Arg Ser Thr Leu Asp Pro Thr Val Arg Asp Ser Trp         355 360 365 Ala Val Ala Gln Val Glu Ala Ser Asn Asn Phe Cys Thr Asp Ser Ala     370 375 380 Phe Trp His Leu Phe Ser Asn Gly Leu Asn Leu Gln Ile Glu His His 385 390 395 400 Leu Phe Pro Gly Ile Asn His Cys His Leu His His Ile Ala Pro Val                 405 410 415 Val Arg Glu Thr Cys Asn Glu Tyr Gly Val Arg Tyr Lys Ser Tyr Asp             420 425 430 Ser Trp Ser Asp Ile Met Arg Ala Met Leu Lys Trp Leu Asp Gln Leu         435 440 445 Ser Val Gly Leu Asp     450 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PtD7D_F <400> 3 atggctacga ctaccccgag 20 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PtD7D_R <400> 4 tcagtccaga ccaaccgaca attg 24

Claims (11)

delete delete delete delete delete delete delete delete Comprising the step of transforming a host cell with a recombinant vector comprising a Delta-7 Desaturase (D7DES) gene derived from Phaeodactylum tricornutum comprising the nucleotide sequence of SEQ ID NO: 1 , A method for producing cis-7-hexadecenoic acid or trans-9-octadecenoic acid in a host cell.
10. The method according to claim 9, wherein the host cell is a yeast or a plant cell, wherein the host cell is produced from cis-7-hexadecene or trans-9-octadecenoic acid.
The plant cell of claim 10, wherein the plant cell is a cell of a vegetable oil plant selected from the group consisting of sesame, rapeseed, sunflower, castor, peanut, bean, coconut, oil palm, olive, soybean, corn and jojoba Gt; cis-7-hexadecene or trans-9-octadecenoic acid in a host cell.

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