KR101666266B1 - 13- 13- - a method for the production of 13-hydroxy-fatty acid by using lactobacillus acidophilus 13-linoleate hydratase and -decalactone from the hydroxy fatty acid by using waltomyces lipofer and a composition therefor - Google Patents

Abstract

The present invention relates to the production of 13-hydroxycarboxylic acid by 13-linolenic acid hydrolase derived from Lactobacillus acidophilus and to a process for preparing delta-decaractone using the wortomyces lipophor and a composition thereof,
According to the present invention, by using a high specificity and environmentally friendly method, 13-hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid, 13- -Octadecadienoic acid can be produced with a high yield. The 13-hydroxy-9-octadecenoic acid thus produced is usefully used as a starting material for the synthesis of lactone, and the lactone production method using yeast Is a more environmentally friendly and highly specific method than the chemical method of production. In order to produce delta-lactone, it is possible to mass-produce coconut flavor, which is a raw material for food, or delta-decaractone, which indicates the flavor of dairy product, with high yield by a production method through cell self-enzyme reaction.

Description

[0001] The present invention relates to the production of 13-hydroxycarboxylic acid by 13-linolenic acid hydratase derived from Lactobacillus acidophilus, and to a process for preparing delta-decaractone using woltomyces lipophor, ACID BY USING LACTOBACILLUS ACIDOPHILUS 13-LINOLEATE HYDRATASE AND δ-DECALACTONE FROM THE HYDROXY FATTY ACID BY USING WALTOMYCES LIPOFER AND A COMPOSITION THEREFOR}

The present invention relates to the production of 13-hydroxycarboxylic acid by 13-linolenic acid hydrolase derived from Lactobacillus acidophilus and to a process for preparing delta-decaractone using the wortomyces lipophor and a composition thereof,

According to the present invention, by using a high specificity and environmentally friendly method, 13-hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid, 13- -Octadecadienoic acid can be produced with a high yield. The 13-hydroxy-9-octadecenoic acid thus produced is usefully used as a starting material for the synthesis of lactone, and the lactone production method using yeast Is a more environmentally friendly and highly specific method than the chemical method of production. In order to produce delta-lactone, it is possible to mass-produce coconut flavor, which is a raw material for food, or delta-decaractone, which indicates the flavor of dairy product, with high yield by a production method through cell self-enzyme reaction.

Hydroxylated fatty acids are found in nature, such as triglycerol, wax, cervicoside, and other lipids present in plants, insects and microorganisms. There are three types of hydroxylated fatty acids that microorganisms produce through bioconversion of unsaturated fatty acids: monohydroxy, dihydroxy, and tri-hydroxycarboxylic acids. Hydroxylated fatty acid is an important industrial substance as a precursor of lactone which constitutes spice. It is used as a starting material of resin, wax, nylon, plastic, lubricant and coating because it has better reactivity than fatty acid which is not other fatty acid. .

13-Hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid and 13-hydroxy-6,9-octadecadienoic acid are used as lubricants and synthesize plasticizers and resins It is an important material for producing sebacic acid used. Lactones derived from these fatty acids are found in plants, yeasts, molds, etc., and are a major component of fruits, milk, and butter. Yeast biologically convert hydrolytic fatty acids in various positions to produce two types of lactones, mainly gamma (?) Or delta (delta). Hydroxylated fatty acids such as 13-hydroxy-9-octadecenoic acid are converted to delta-decalactone by yeast. Delta-decaractone is characterized by coconut and cheese flavors, is an essential ingredient in food processing, and is increasingly used in the food industry. However, this substance is synthesized by organic synthesis because it exists in a small amount in a few fruits. However, the increase of the national income, the demand of the natural food and the flour material, There is an absolute need for a bioconversion-based approach to preference.

Microorganisms that produce 13-hydroxy-9-octadecenoic acid in the past include Enterococcus faecalis), Vorbis Streptococcus (Streptococcus bovis JB1), Lactobacillus ( Lactobacillus acidophilus IFO 13951), Pediococcus sp. AKU 1080 But these microorganisms not only produce 13-hydroxy-9-octadecenoic acid in low yields, but also produce by-products such as 10-hydroxy-12-octadecenoic acid or 10,13- Noriaki Kishimoto et al., Lipids (2003), 38, 12, 1269-1274), which is uneconomical in industrialization.

The use of recombinant microorganisms containing enzymes and related enzymes has not been reported so far. In addition, yeasts that previously produced delta-lactones include Saccharomyces cerevisiae), Yalova Li Wia poly Utica (Yarrowia lipolytica), Rio Deva drink your device century (Debaryomyces hansenii , Hansenula anomala ). However, the production yield of the delta-lactones is low, and the production yield of the delta-lactones is low. The production of the delta-lactones through the fermentation conventionally consumes a large amount of the consumed medium and time.

[Prior Patent Literature]

Korean Patent Publication No. 1020130001039

DISCLOSURE OF THE INVENTION The object of the present invention is to solve the above-mentioned problems and to solve the above-mentioned problems, and it is an object of the present invention to provide a process for the production of 13-hydroxynaphthalic acid, 13- , 13-hydroxy-6,9-octadecadienoic acid at a high yield, an enzyme thereof, and a recombinant microorganism.

Another object of the present invention is to provide a microorganism capable of producing delta-decaractone in a high yield through 13-hydroxy-9-octadecenoic acid.

It is still another object of the present invention to provide a biological method capable of producing delta-decaractone in high yield.

It is another object of the present invention to provide a composition capable of producing delta-decaractone in high yield.

In order to achieve the above object, the present invention provides a method for producing Lactobacillus acidophilus Lactobacillus acidophilus strain is subjected to polymerase chain reaction (PCR) using a primer to amplify the 13-linolenic acid hydrolase gene to prepare a 13-linolenic hydrolase gene.

In one embodiment of the present invention, the 13-linoleic acid hydrolase gene is preferably composed of the nucleotide sequence of SEQ ID NO: 1. However, the present invention can be applied to the 13-linoleic acid hydrolase gene through mutation such as substitution, deletion, inversion, Are included within the scope of the present invention.

In another embodiment of the present invention, the primer sequence is preferably composed of the nucleotide sequences of SEQ ID NOS: 2 and 3, but is not limited thereto.

The present invention also provides a recombinant expression vector comprising a 13-linolenic hydrazinase gene comprising the nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a transformant in which the expression vector of the present invention is transformed into a host cell.

In one embodiment of the present invention, the host cell is preferably E. coli but is not limited thereto.

The present invention also provides a method for producing 13-hydroxy-9-octadecenoic acid by adding linoleic acid as a substrate to the transformant of the present invention.

The present invention also provides a method for producing 13-hydroxy-9,15-octadecadienoic acid by adding alpha linolenic acid as a substrate to the transformant of the present invention.

The present invention also provides a method for producing 13-hydroxy-6,9-octadecadienoic acid by adding gamma linolenic acid to the transformant of the present invention.

The present invention also provides a composition for producing 13-hydroxy-9-octadecenoic acid comprising a 13-linolenic hydrazinase gene comprising the nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a method for producing delta-decaractone by reacting a hydroxylated fatty acid with a wortomyces reporter.

In one embodiment of the present invention, there is provided a process for producing delta-decaractone, wherein the fatty acid is 13-hydroxy-9-octadecenoic acid.

The present invention also provides a composition for the production of delta-decaractone, which comprises a hydroxylated fatty acid and a wortomyces reporter as an active ingredient.

The present invention also provides a composition comprising the delta-decaractone produced by the method of the present invention.

Hereinafter, the present invention will be described.

The present inventors have investigated an enzyme capable of converting linoleic acid, which is a rich unsaturated fatty acid of plant, into a hydroxylated fatty acid, and found that 13 -hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid , 13-hydroxy-6,9-octadecadienoic acid in high yields, and the enzyme and the recombinant microorganism were selected, and through the 13-hydroxy-9-octadecenoic acid obtained using this enzyme, Yeasts were selected which can produce decaractone in high yield.

The inventors of the present invention have found that by using 13-linolenic acid hydrolase more effectively, 13-hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid, In order to develop a method for producing novel acid, a large amount of 13-linolenic acid hydrolase and recombinant strain derived from Lactobacillus acidophilus was produced, and this environmentally friendly process was used to produce 13- -9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid and 13-hydroxy-6,9-octadecadienoic acid can be produced in high yields, Hydroxy- 9-octadecenoic acid to delta- decaractone by using a Waltomyces lipofer , thereby completing the present invention.

The present invention relates to Lactobacillus < RTI ID = 0.0 > The present invention relates to a recombinant expression vector comprising 13-linolenic acid hydrolase derived from a plant , an acidophilus , and a microorganism transformed with the same. Also disclosed is a method for producing 13-linolenic acid hydrolase using the same, Octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid, 13-hydroxy-6,9-octadecadienoic acid, and yeast Waltomyces lipofer And a method for producing delta-decaractone using the same.

13-linolenic acid hydratase (C-13 positional specific linoleate hydratase) was synthesized from 13-hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid (13-hydroxy-9,15-octadecadienoic acid), and 13-hydroxy-6,9-octadecadienoic acid.

The present invention also relates to the use of 13-hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid, (C-13 positional specific linoleate hydratase), which is used for the production of 13-hydroxy-6,9-octadecadienoic acid, Lt; / RTI > microorganism.

Specifically, the present invention relates to a method for producing 13-hydroxy-9-octadecenoic acid by using linoleate, alpha-linoleate and gamma-linolenoleate as substrates, acid, 13-hydroxy-9,15-octadecadienoic acid, 13-hydroxy-6,9-octadecadienoic acid (13-hydroxy-6,9 Lactobacillus < / RTI > ( Lactobacillus < RTI ID = 0.0 > acidophilus strain. < / RTI >

The present invention also provides a recombinant expression vector comprising 13-linolenic acid hydrolase.

Specifically, the present invention provides It provides a recombinant expression vector pET 15b / C-13 positional specific linoleate hydratase derived from Lactobacillus acidophilus strain and containing oleic acid hydrolase gene.

The present invention also provides a microorganism transformed with a recombinant expression vector comprising 13-linolenic acid hydrolase. Lactobacillus < RTI ID = 0.0 > acidophilus ) (ER) 2566 pET 15b / C-13 positional specific linoleate hydratase transformed with the recombinant expression vector containing the 13-linolenic hydrazinase gene derived from the strain.

In the present invention, any recombinant expression vector can be used as long as it can be used in the production of 13-hydroxy-9-octadecenoic acid. Any vector can be used as long as it is used in a gene recombinant method including expression vector pET-15b. As the microorganism transformed with the expression vector, it is preferable to use Escherichia coli ER 2566. However, by using the cultured 13-linolenic acid hydrolase capable of producing an enzyme protein over-expressing the desired gene and transformed with the recombinant expression vector 13-hydroxy-9-octadecenoic acid in high yield.

More specifically, the present invention relates to a process for the preparation of 13-hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid and 13- 13-Hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid, 13-hydroxy-6,9-octadecanoic acid Decadienic acid. ≪ / RTI >

As described above, the 13-linolenic acid hydrolase is preferably cultured in E. coli transformed with the recombinant expression vector containing 13-linolenic acid hydrolase to induce the expression of the recombinant enzyme gene and recover the expressed protein.

In addition, it is preferable to use a fatty acid as a substrate, and more preferably to use linoleate, alpha-linoleate, gamma-linolenoleate. In addition, the time of the enzyme reaction can be appropriately controlled by a conventional method.

The present invention provides a method for producing delta-decaractone by bioconversion reaction using yeast.

Specifically, the present invention provides a process for producing delta-decaractone using a Walnut Meisser reporter, which is a new yeast capable of converting 13-hydroxy-9-octadecenoic acid into delta-decaractone.

In the present invention, the yeast culture medium of yeast Wortomyces lipofer can be YM broth, and the reaction medium for converting 13-hydroxy-9-octadecenoic acid into delta-decaractone is a new method A citrate-phosphate reaction medium containing 3.4 g / l nitrogen source is preferred.

The concentration of yeast is preferably 5 g / l.

The biotransformation reaction temperature is preferably in the range of 20 ° C to 40 ° C, more preferably 28 ° C to 35 ° C.

In addition, the bioconversion reaction is preferably performed in a pH range of from 6.0 to 7.0, more preferably in a range of pH of 6.5 to 7.0. The reaction medium is preferably citrate-phosphate reaction medium.

In addition, the bioconversion reaction is preferably performed in the range of 150 rpm to 250 rpm, more preferably 200 rpm to 250 rpm.

Preferably, the substrate is 13-hydroxy-9-octadecenoic acid. The concentration of the oleic acid is preferably adjusted in the range of 40 g / l to 80 g / l, more preferably 50 g / l to 70 g / lt; / RTI > range. In addition, various types of hydroxide substrates for the production of gamma or delta-lactones may be used.

The methods of the present invention comprise the step of introducing a microorganism, e. G., A recombinant microorganism, preferably a vector or gene described herein (e. G., A wild type and / or mutant gene) and / It is characterized by cultured recombinant microorganisms.

The term "recombinant" microorganism refers to a microorganism that has been genetically altered, modified or manipulated (e. G., Genetically engineered) to produce a modified or different genotype and / or phenotype (For example, bacteria, yeast cells, fungal cells, and the like) that exhibit a change in the gene affects the coding nucleic acid sequence of the microorganism.

Expression vectors according to the invention are theoretically available, for example, in Sambrook et al. (1989). ≪ / RTI >

The literature also describes functional components of the vector, such as suitable promoters, enhancers, termination and polyadenylation signals, antibiotic resistance genes, selectable markers, cloning start points and splicing signals. Conventional cloning vectors can be used to amplify components, e. G., Plasmids, bacteriophages, phagemids, cosmids or baculoviruses, retroviruses, adenoviruses, adeno-associated viruses, Artificial chromosome / microchromosome as well as the same viral vector can be produced.

The Lactobacillus acidophilus ( Lactobacillus < RTI ID = 0.0 > acidophilus ) 13-linolenic acid hydratase, which is a high-specificity and eco-friendly method, is a raw material for industrial products, 13-hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid, Octadecadienoic acid can be produced with high yield, and the 13-hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid, 13-hydroxide -6,9-octadecadienoic acid can be usefully used as a starting material for the synthesis of various lactones. Also, the above reaction product, 13-hydroxy-9-octadecenoic acid, can be used as a substrate and a yeast Wortomyces reporter can be used to produce coconut flavored delta-decaractone as a raw material for food additives, Unlike conventional chemical methods, environmentally friendly biotransformation can produce a flavor close to nature.

Figures 1a, 1b and 1c show Lactobacillus < RTI ID = 0.0 > acidophilus ), 13-hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid and 13-hydroxy-6,9-octadecadienoic acid using a recombinant strain derived from KCTC 3164 Hourly production. (1): 13-Hydroxy-9-octadecenoic acid O: Linoleic acid, 1b: 13-Hydroxy-9,15-octadecadienes acid O: Alpha linolenic acid, -Hydroxy-6,9-octadecadienoic acid O: gamma linolenic acid)
2 shows the hourly production amount of delta- decaractone using yeast Waltomyces lipofer (o: 13-hydroxy-9-octadecenoic acid, l: delta-decaractone)

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are for illustrative purposes only and are not to be construed as limiting the scope of the invention to those skilled in the local arts.

Example 1 Preparation of Recombinant Expression Vectors and Transforming Microorganisms Containing 13-Linoleic Acid Hydrase Gene

In order to prepare the oleosin hydrolase gene of the present invention, Lactobacillus < RTI ID = 0.0 > acidophilus strains were first isolated.

Specifically, the gene sequence and the amino acid sequence are not known Lactobacillus < RTI ID = 0.0 > acidophilus) screening the KCTC 3164 strain, and Lactobacillus Ecija of another strain also on the basis of the DNA nucleotide sequence of the pillar's (Lactobacillus acidophilus) designed primers (primer) ((5'-TTCATATGTATTATTCCAATGGTAATTACGA -3 '( SEQ ID NO: 2) , 5'-GCCTCGAGTTAGACTAAATTTGCTTCTTTAAGTA-3 '(SEQ ID NO: 3)) and subjected to polymerase chain reaction (PCR) to amplify the nucleotide sequence of the corresponding gene.

The 13-linoleic acid hydrolase gene, which is obtained in large quantities, Nde I and Xho I were used to insert pET 15b / 13-linoleic acid hydrolase into the plasmid vector pET 15b (Novagen).

The recombinant expression vector thus obtained was transformed into Escherichia coli strain ER 2566 by a conventional transformation method (Sambrook et al. (1989)), and the transformed microorganism was added with 20% glycerine solution Hydroxynaphthalenic acid, 13-hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid and 13-hydroxy-6,9-octadecadienoic acid, Respectively.

Example  2. 13- Linoleic acid  Production and recovery of hydration enzymes

Recombinant E. coli for protein expression of the enzyme was cultured at 37 ° C. in a 500 ml LB (Difco, Sparks, MD, USA) medium and a flask with 50 μg / ml of ampicillin under 200 rpm aeration conditions. When the absorbance of the bacteria reached 0.6 at 600 nm, IPTG was added to the final concentration of 0.1 mM to induce expression of 13-linolenic hydrazinase, and the culture was incubated for 6 hours at 37 ° C with stirring at 200 rpm.

Escherichia coli cells containing the cultured 13-linolenic acid hydrolase were collected and used. The 13-linolenic acid hydrolase produced as above was centrifuged at 6,000 x g for 30 minutes at 4 ° C, washed twice with 0.85% NaCl, 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole, and 0.1 mM phenylmethylsulfonyl fluoride, and the cell solution was disrupted with a sonicator. The cell lysate was further centrifuged at 13,000 x g for 20 minutes at 4 ° C. Cell pellet was removed, and only the supernatant was obtained. Fast protein liquid chromatography (Bio-Rad Laboratories, Hercules, Calif., USA ) Was loaded with a Histrap HP adsorption column using a His-tag and separated into an enzyme solution used for producing 13-hydroxy-9-octadecenoic acid.

Example  3. 13-Hydroxy-9- Octadecenoic  Acid, 13-hydroxy-9,15- jade Tadecadienoic acid, 13-hydroxy-6,9- Octadecadien  Production of acid

The productivity of 13-hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid and 13-hydroxy-6,9-octadecadienoic acid using the recombinant strain of the present invention , A solution of 13-hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid (manufactured by Takara Shuzo Co., Ltd.) using linolenic acid at a concentration of 2.8 g / , And 13-hydroxy-6,9-octadecadienoic acid were measured.

As a result, Lactobacillus acidophilus- derived 13-linolenic acid hydrolase produced 2.24 g / l of 13-hydroxy-9-octadecenoic acid at 2.8 g / l of linoleic acid after 7 hours of reaction to yield 0.32 g / Conversion yield. (Fig. 1a) In the case of alpha-linolenic acid at 2.8 g / l, it produced 1.20 g / l of 13-hydroxy-9,15-octadecadienoic acid after 16 hours of reaction, yielding a productivity of 0.08 g / Respectively. (Fig. 1b). In the case of gamma linolenic acid at 2.8 g / l, 1.82 g / l of 13-hydroxy-6,9-octadecadienoic acid was produced after 7 hours of reaction to yield 0.26 g / l productivity and 65% Respectively. (Fig. 1c), the strains showing the highest productivity in 13-hydroxynaphthalene-9-octadecenoic acid to date are Pediococcus sp. After 4 days of reaction with AKU 1080, 2.3 g / l of 13-hydroxy-9-octadecenoic acid was produced at 12.3 g / l linoleic acid, yielding 0.02 g / l hourly yield and 18.7% conversion yield.

Example  4. Investigation of bioconversion activity and yeast selection according to yeast type

In the present invention, the expression of the beta-oxidative peroxidase that converts 13 -hydroxy-9-octadecenoic acid into delta-decaractone is increased without using a conventional strain medium to obtain an increased yeast and wortomyces reporter, A reaction medium containing 13-hydroxy-9-octadecenoic acid as a substrate was used. Yeast and Wortomyces reporter were inoculated into a test tube containing 15 ml of YM broth and incubated for 12 hours. Then, the beta-oxidation-inducing enzyme converting 13-hydroxy-9-octadecenoic acid to delta-decaractone Of citrate-phosphate reaction medium containing 1.0 to 4.0 g / l of nitrogen source, 0.1 to 0.5 ml / l of tween 80 and 1.0 to 3.0 g / l of 13-hydroxy-9-octadecenoic acid And then cultured in a 2-liter baffled flask. At this time, the stirring speed was set to 200 rpm, the incubation temperature was maintained at 28 ° C, and the expression of the enzyme was induced by culturing for 24 hours. The culture was centrifuged at 10,000 × g for 20 minutes at 4 ° C. and then washed twice with 50 mM citrate-phosphate reaction medium (pH 7.5).

The bioconversion reaction was carried out in a reaction medium of 10 g / l citrate-phosphate (pH 7.0) containing 8 mM 13-hydroxy-9-octadecenoic acid, 5 g / l yeast and 0.5 ml / The reaction was continued until time. The reaction temperature was 28 ° C and stirring speed was 200 rpm. 1 ml was recovered from the bead flask according to time, and the reaction was stopped and extracted with the same ratio of ethyl acetate. After completion of the reaction, the amount of reduction of 10-hydroxystearic acid and the yield of gamma-dodecaractone were measured, and the results are shown in FIG.

FIG. 2 is a graph showing that, when the yeast wortomyces lipophor is cultured by reacting 5 g / L yeast amount with 2.5 g / L 13-hydroxy-9-octadecenoic acid in the present invention, 13 -hydroxy-9-octadeceno (?) And the production of delta-decaractone (?) Resulting therefrom.

As a result, as shown in Fig. 2, when 15 hours passed after reaction of 13-hydroxy-9-octadecenoic acid and yeast wortomyces reporter, the delta-decaractone showed the maximum production, About 50%. To date, the yeast with the highest productivity in delta-decaractone has been reported to be Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) at 0.09 g / l (Maria de laat et al. 1992. Natural delta-lactones and process of the production thereof. United States Patent US005128261). (Fig. 2)

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific descriptions are only for the preferred embodiments and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> KONKUK UNIVERSITY INDUSTRIAL COOPERATION CORP <120> A METHOD FOR THE PRODUCTION OF 13-HYDROXY-FATTY ACID BY USING          LACTOBACILLUS ACIDOPHILUS 13-LINOLEATE HYDRATASE AND          DELTA-DECALACTONE FROM THE HYDROXY FATTY ACID BY USING          WALTOMYCES LIPOFER AND A COMPOSITION THEREFOR <130> HY140100 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1773 <212> DNA <213> Lactobacillus acidophilus <400> 1 atgcattata gtagtggtaa ttatgaagct tttgtaaacg caagtaaacc taaggatgtc 60 gatcagaagt ccgcatatct tgttggttca ggtttggcat cgcttgctag tgctgtattt 120 ttaattcgtg atggtcacat gaagggtgat agaattcata tccttgaaga attgagcctt 180 ccaggtggtt caatggatgg gatctataat aagcaaaaag aaagctacat cattcgtggt 240 ggtcgtgaaa tggaagccca ttttgaatgc ttgtgggact tgtttagatc gattccatca 300 gctgaaaata aagatgaatc ggtcctggat gaattttacc gtttaaatag aaaagatcca 360 agtttcgcaa agactcgtgt cattgttaac cgcggacatg aacttccaac tgacggtcaa 420 ttacttctta ctcccaaggc tgttaaagaa attattgatc tttgcttaac tcctgaaaaa 480 gatttacaaa ataaaaaaat taatgaagtc tttagtaaag aattttttga atcaaacttc 540 tggctttact ggtcaacgat gtttgccttt gagccatggg caagtgcgat ggaaatgcgt 600 cgttacttaa tgcgttttgt tcaacacgtt tctacactta agaatttatc atcactacgc 660 tttactaagt ataaccaata tgaatcatta attttaccaa tggttaaata cttgaaagat 720 cgcggcgtgc aattccatta caacaccgtt gttgataata tctttgttaa ccgttcaaat 780 ggtgaaaaga ttgctaagca aattctttta actgaaaacg gtgaaaaaaa gagcatcgat 840 ttaacagaaa atgacctcgt cttcgttact aacggttcaa ttactgaaag tacaacttat 900 ggtgataact tgcacccagc ttctgaggaa cataaattag gtgctacttg gaaattatgg 960 caaaacttgg cagcgcaaga tgatgacttc ggtcacccag atgtcttctg caaggatatt 1020 ccaaaggcta actgggtaat gtctgctaca attactttta agaataatga tattgtgcca 1080 ttcattgaag cagttaataa gaaggatcca cacagcggct caattgtaac tagtgggcct 1140 actacgatta aggattctaa ctggctactt ggttattcaa tcagtcgtca gcctcacttt 1200 gaagcacaaa agcctaacga attgattgta tggctttatg gtttgttctc agacaccaaa 1260 ggtaactatg ttgaaaagac tatgcctgac tgtaacggta ttgaattatg tgaagaatgg 1320 ctttaccaca tgggtgttcc tgaagaaaga atcccagaaa tggcttcagc tgctacgact 1380 attccagcac acatgccata tattacttca tacttcatgc caagagcatt aggcgacaga 1440 cccaaggttg tgccagacca ctcaaagaac ttggccttca ttggtaactt tgctgaaacg 1500 ccaagagaca ctgtctttac cactgaatac tctgtcagaa ctgcgatgga agctgtatac 1560 accttgctta acattgatcg tggtgtgcca gaagtatttg catctgcctt cgatgtcaga 1620 atgctcatga acgcaatgta ctacttgaat gatcaaaaga agcttgaaga tcttgatttg 1680 cctattgctg aaaagttggc aattaagggg atgctcaaga aagttaaggg cacttatata 1740 gaggaattgc ttaagaagta taagttggtt tag 1773 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 ttcatatgta ttattccaat ggtaattacg a 31 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 3 gcctcgagtt agactaaatt tgcttcttta agta 34

Claims (15)

delete delete delete 13-hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid or 13-hydroxy-6,9-dihydroxynaphthoic acid, which comprises a 13-linolenic acid hydrolase gene consisting of the nucleotide sequence of SEQ ID NO: Recombinant expression vector for the production of octadecadienoic acid. A transformant wherein the expression vector of claim 4 is transformed into a host cell. 6. The transformant according to claim 5, wherein the host cell is Escherichia coli. A process for producing 13-hydroxy-9-octadecenoic acid by adding linoleic acid as a substrate to the transformant of claim 5. A method for producing 13-hydroxy-9,15-octadecadienoic acid by adding alpha linolenic acid as a substrate to the transformant of claim 5. A method for producing 13-hydroxy-6,9-octadecadienoic acid by adding gamma linolenic acid to the transformant of claim 5. 13-hydroxy-9-octadecenoic acid, 13-hydroxy-9,15-octadecadienoic acid or 13-hydroxy-6,9-dihydroxynaphthoic acid, which comprises a 13-linolenic acid hydrolase gene consisting of the nucleotide sequence of SEQ ID NO: Composition for the production of octadecadienoic acid. delete delete delete delete delete

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