JP4182192B2 - Genes encoding enzymes involved in biosynthesis of icosapentaenoic acid and / or docosahexaenoic acid - Google Patents

Description

【００３０】
前記種々のORFの塩基配列より推定されるアミノ酸配列（配列番号３、５、７、９）と、ＥＰＡ生合成酵素群をコードする遺伝子群の各ORFの塩基配列より推定されるアミノ酸配列（特開平8-242867に記載）とを比較したところ、ある程度の相同性を有することが見出された。その結果を表２に示す。
【００３１】

【００３２】
この結果より、配列番号２、４、６、８（コスミド10C7に含まれるORFa,b,c,d）のコードするタンパクは、ＥＰＡ生合成酵素群をコードするORF6,7,8,9の産物とそれぞれ機能的に類似していることが予想された。
【００３３】
実施例２−１． コスミド10C7のゲノムDNA挿入部分の制限酵素断片のサブクローニング
コスミド10C7のゲノムDNA挿入部分のORFbを含むEcoRI(2391)-EcoRI(14761)断片をプラスミドpSTV28（宝酒造社製）のEcoRI部位に挿入して、プラスミドEco-Eco/pSTV28を作成した。また、ORFdを含むPstI(16802)-PstI(22263)断片をプラスミドpSTV28のPstI部位に挿入して、プラスミドPst-Pst/pSTV28を作成した。また、ORFcを含むEcoRV(11172)-PvuI(17457)断片を末端平滑化後プラスミドpSTV29のSmaI部位に挿入して、プラスミドEco-Pvu/pSTV29を作成した。各サブクローンの挿入断片の位置を図２に示す。
【００３４】
実施例２−２． ＥＰＡ生合成系遺伝子群のORF9,10欠失クローンの作成
ＥＰＡ生合成酵素群をコードする遺伝子群を含むプラスミドpEPA（特開平8-242867に記載）のXhoI(5660)-SpeI(31446)断片（ORF2〜8を含む）をプラスミドpBluescript（STRATAGENE社製）のXhoI-SpeI部位に連結して、pXS-BS2を作成した。pXS-BS2の挿入断片の位置を図３に示す。本プラスミドはＥＰＡの生合成に必須な遺伝子のうちのORF9を欠失している。
【００３５】
実施例２−３． ＥＰＡ生合成系遺伝子群のORF2,4,5,8,10欠失クローンの作成ＥＰＡ産生能を有するプラスミドΔ2,4,5,10/pNEB（WO98/01565に記載）から PstI(24813)-EcoT22I(28946)（ORF8の一部）部分を欠失させて、Δ2,4,5,8,10/pNEBを作成した。Δ2,4,5,8,10/pNEBの挿入断片の位置を図３に示す。本プラスミドはＥＰＡの生合成に必須な遺伝子のうちのORF8を欠失している。
【００３６】
実施例２−４． ORF7またはORF9欠失ＥＰＡ生合成系遺伝子群の相補によるＥＰＡ生産能の回復
実施例２−１で作成したプラスミドのうちの１種を導入した大腸菌JM109株を、さらにpEPAΔ7（pEPAからORF7の一部であるSalI(22260)-NruI(23845)部分を欠失させたプラスミド、特開平8-242867に記載）または実施例２−２で作成したpXS-BS2を用いて形質転換した。これらの形質転換株と、pEPAΔ7またはpXS-BS2のみを有する形質転換株を、50μg/mlのアンピシリン及びクロラムフェニコールを含むLB培地5mlに接種し、25℃で培養した。遠心分離により菌体を得、１回洗浄したのち凍結乾燥し、塩化水素飽和のメタノール2mlに溶解して密閉容器に入れ、80℃で１時間保温して脂肪酸をメチルエステル化した。放冷後、ヘキサン2mlで３回抽出し、ヘキサン層を乾固して10mlのメタノールに溶解した。この溶解物の一部をガスクロマトグラフィーにより分析したところ、ＥＰＡのピークが観察された。それぞれの形質転換株の保有するプラスミドの組み合わせと、ピークの面積比から算出した菌体の全脂肪酸エステル中におけるＥＰＡメチルエステルの割合を表３に示す。
【００３７】

【００３８】
上記の結果より、ＥＰＡ生合成系遺伝子群のうちのORF7またはORF9を欠失させたクローンのＥＰＡ産生能が、コスミドp10C7由来のDNA断片の存在によって回復することが示された。すなわち、コスミドp10C7中にはORF7及びORF9と同様の機能を有する、高度不飽和脂肪酸の生合成に関与する遺伝子が含まれていることが示された。
【００３９】
実施例２−５． ORF8欠失ＥＰＡ生合成系遺伝子群の相補によるＥＰＡ生産能の回復及びＤＨＡ生産
実施例２−１で作成したプラスミドEco-Pvu/pSTV29を導入した大腸菌JM109株を、さらに実施例２−３で作成したプラスミドΔ2,4,5,8,10/pNEBを用いて形質転換し、アンピシリン及びクロラムフェニコールを含む寒天平板培地を用いて選別を行って、２種のプラスミドを有する組み換え体JM109/p3,6,7,9/pcを得た。この形質転換株を、実施例２−４と同様にして18℃で培養し、菌体の脂肪酸組成をガスクロマトグラフィーにより分析した。その結果、ＥＰＡのピークが観察され、その全脂肪酸エステル中における割合は、ピークの面積比から約22.0%と算出された。一方、ガスクロマトグラフィー上でEPAメチルエステルよりも高分子側に新たなピークが観察された。このピークの物質の全脂肪酸エステル中における割合は、ピークの面積比から約1.5%と算出された。GC-MS分析の結果、このピークの物質の分子量は342であり、ＤＨＡ(22:6)メチルエステルの値と一致し、各フラグメントのピークは標品のそれと一致したことから、本物質はＤＨＡメチルエステルと同定された。なお、MSフラグメントピークは以下の通りであった。Mass : 342(M+), 327, 311, 299, 286, 273, 241, 215, 206, 173, 159, 145, 131, 119, 105, 91, 79, 67, 55, 41, 27.
【００４０】
上記の結果より、ORF8を欠失させたＥＰＡ生合成系遺伝子群を有するクローンのＥＰＡ産生能が、コスミドp10C7由来のDNA断片の存在によって回復し、さらにＤＨＡの生産能も獲得したことが示された。すなわち、コスミドp10C7中にはORF8と同様の機能を有し、かつＤＨＡの生合成に関与する遺伝子が含まれていることが示された。
【００４１】
【配列表】
【００４２】

【００４３】

【００４４】

【００４５】

【００４６】

【００４７】

【００４８】

【００４９】

【００５０】

【００５１】

【００５２】
【図面の簡単な説明】
【図１】コスミドp10C7の制限酵素地図を示す。図面の太線部分はドコサヘキサエン酸産生菌の染色体由来の遺伝子断片を示す。太線内の白色部分は塩基配列を決定した部分を示す。
【図２】コスミドp10C7のサブクローンの挿入断片の位置を示す。
【図３】ＥＰＡ生合成系遺伝子群のサブクローンの挿入断片の位置を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention contains a gene encoding a group of enzymes involved in biosynthesis of icosapentaenoic acid (hereinafter referred to as EPA) and / or docosahexaenoic acid (hereinafter referred to as DHA) useful as raw materials for pharmaceuticals, foods, feeds, etc. And a microorganism transformed with the plasmid.
[0002]
[Prior art]
Conventionally, attempts have been made to produce DHA by the action of microorganisms. For example, marine microalgae {Phytochemistry, 30, 2963-2967 (1991), JP 7-8221, JP 7-95875} and flagella fungi {J. Am. Oil Chem. Soc., 68, 509-514 ( 1991), JP-A-1-199588}, there is a report on a method for producing a DHA-containing triglyceride. In addition, a report on the production of DHA by deep-sea microorganisms under high pressure conditions or under a low temperature of 5 ° C. or lower {Appl. Environ. Microbiol., 51, 730-737 (1986) (Vibrio marinas ATCC15381); Lipids, 29, 527 -528 (1994)}, there is a report of a method for producing DHA and DHA-containing phospholipids using marine bacteria (Japanese Patent Laid-Open No. 10-191993). However, a gene involved in DHA biosynthesis of these microorganisms has not been reported.
[0003]
On the other hand, EPA biosynthetic genes have been obtained from the chromosomal DNA of marine bacteria producing EPA, which is a highly unsaturated fatty acid similar to DHA, and a method for producing EPA using recombinant Escherichia coli into which this gene has been introduced [JP-A-6-46864 {WO93 / 23545-A (EPO594868-A)}, JP-A-8-242867, WO98 / 01565]. However, these reports do not mention DHA production.
[0004]
[Problems to be solved by the invention]
The productivity of such useful substances in wild strains that produce useful substances is generally low, and when the ability of these microorganisms is to be used industrially, the microorganisms are improved by various methods, that is, the productivity is improved. . The present invention uses a genetic recombination method to find a DHA biosynthetic enzyme group gene unknown in the literature and introduce it into other organisms, thereby improving DHA productivity and an organism having no DHA biosynthetic ability. The purpose is to provide DHA biosynthetic ability to the target, and thus to establish an advantageous method for producing DHA. At the same time, another object is to find a new gene group that can be used for more efficient production of EPA.
[0005]
[Means for Solving the Problems]
As a result of intensive studies, the present inventors have recovered the EPA production ability of deletion mutants of EPA biosynthesis genes from the chromosomal DNA of the DHA-producing marine bacterium Shiwanella sp. SCRC-21406 strain (FERM BP-5979). The gene group which has the ability to make it discovered was discovered. Furthermore, it discovered that DHA production was possible by combining this with an EPA biosynthesis system gene group, and completed this invention.
Accordingly, the present invention provides EPA and / or DHA biosynthetic enzyme group genes, expression plasmids containing the same, and microorganisms transformed with the plasmids.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, a gene encoding an enzyme group involved in EPA and / or DHA biosynthesis by extracting DNA from a microorganism (gene source microorganism) having the ability to produce DHA and cleaving this DNA with a restriction enzyme Is excised and introduced into a suitable vector to prepare an expression plasmid.
[0007]
In the present invention, the case where Shiwanella sp. SCRC-21406 strain (FERM BP-5979) is used as an example of the gene source of the DHA biosynthetic enzyme group has been specifically described. However, DHA can be produced. Any material may be used, and the genus, species or strain is not particularly limited. These microorganisms can be easily obtained at a public microorganism depository and can be newly separated from the natural world.
[0008]
By using the gene of the present invention, for example, a bacterium that is a heterologous host such as Escherichia coli or Bacillus subtilis or a homologous host such as Shivanella genus, or yeast, filamentous fungi, etc. By transformation, EPA and DHA production strains can be artificially created. Furthermore, EPA and DHA producing plants can be produced by introducing this gene into higher plants such as soybean, sunflower, rape and sesame. As a region for the expression of EPA and / or DHA biosynthetic enzyme group genes, it is possible to use a control region inherently associated with the genes of these enzymes, but the expression level is improved or expressed. It is preferable to prepare a separate promoter / operator system in order to enable induction of. When Escherichia coli is used as a host, as such promoter / operator system, T7, lac, bla, trp , tac, lavUV5, P L, P R, can be used promoter / operator system, such as l _PP As the SD sequence, an SD sequence such as trp leader peptide, lacZ, metapyrocatecase or cII gene can be used. Furthermore, a transcription terminator such as the rrnBT ₁ T ₂ terminator of the ribosomal gene of E. coli can be provided downstream of the coding region. Further, in the gene expression, a host / vector system of Saccharomyces cerevisiae can be used. In this case, the promoter of yeast alcohol dehydrogenase gene, the promoter of acid phosphatase gene, The promoter of seraldehyde-3-phosphate dehydrogenase, the promoter of enolase gene, etc. can be used. In this case, the plasmid is selected for the sequence for replication in yeast, and the yeast containing the plasmid is selected. It is preferable to contain an auxotrophic marker as a selectable marker, for example, a auxotrophic sequence such as Leu, Trp, His.
[0009]
In gene introduction into higher plants, there are a method using a vector and a direct introduction method. Vectors include Ti plasmids, DNA viruses such as cauliflower mosaic virus (CaMV), gemini virus, cassava tent virus, tomato golden mosaic virus, RNA viruses such as brom mosaic virus (BMV) and tobacco mosaic virus (TMV). In this case, examples of the promoter include CaMV 35S promoter. Examples of the direct introduction method into protoplasts include calcium phosphate method, polyethylene glycol method, microinjection, electroporation, liposome method and the like. Moreover, the particle gun method is also mentioned as a direct introduction method to a plant cell.
[0010]
In general, the expression level of a specific protein in E. coli is affected by the number of gene copies, transcription efficiency, mRNA stability, translation efficiency, protein stability, and the like. In order to modify the promoter, SD region, terminator and the like, which are these control regions, using genetic engineering techniques, the smaller the plasmid, the easier it is to handle. Further, the copy number of a gene is related to the size of the plasmid encoding the gene, and the copy number tends to increase when the size is small. For this reason, a DNA fragment containing an enzyme group gene involved in the biosynthesis of EPA and / or DHA described in the examples of the present invention is inserted into a plasmid, and subcloning of this plasmid DNA is repeated to repeat It is also possible to obtain a smaller plasmid by excising unnecessary portions. Smaller EPA and / or DHA biosynthetic enzyme group genes obtained by this method are also included in the present invention. In addition, in order to increase the stability and activity of the enzyme, it is possible to convert a partial base sequence (amino acid sequence) of a gene using known methods, or insert several bases at the stage of gene replication. Although deletions or substitutions may occur, these mutant genes are also included in the present invention.
In the present invention, a gene encoding an enzyme group involved in the biosynthesis of EPA and / or DHA is under stringent conditions with those encoded by the nucleotide sequences represented by SEQ ID NOs: 2, 4, 6 and 8. Includes those that hybridize.
[0011]
As the host Escherichia coli of the present invention, any bacterial strain derived from Escherichia coli K12 strain can be used. For example, JM83, JM101, JM103, JM105, JM109, JM109 (DE3), RR1, RB791, W3110, C600, HB101, DH1, AG1, NM554, BL21 (DE3), etc. can be used. Examples of the host yeast include AH22, DC5, D-13-1A, YNN140 and the like.
[0012]
The enzyme group encoded by the gene of the present invention can induce higher fatty acids synthesized by the biosynthetic system inherent in the host organism to EPA and / or DHA. Furthermore, it is possible to change the fatty acid composition of the host organism by using a part of this gene group.
[0013]
Next, the present invention will be described more specifically with reference to examples.
[0014]
【Example】
[0015]
Example 1-1. Preparation of genomic DNA of Shiwanella sp. SCRC-21406 Peptone 1.0%, yeast extract 0.5% dissolved in 1/2 concentration artificial seawater, 100 ml of medium adjusted to pH 7.0 was heat sterilized at 121 ° C for 20 minutes, Shewanella sp. SCRC-21406 (FERM BP-5979) was inoculated and cultured aerobically at 15 ° C for 24 hours. After culturing, the cells were collected with a centrifugal separator and washed once with 20 ml of SET buffer (50 mM EDTA, 20% Sucrose, 50 mM Tris-HCl, pH 7.6). The obtained cells were frozen in dry ice / acetone for 2 minutes, dissolved in water, and suspended in 2 ml of the same buffer. 1 mg of lysozyme and 1 mg of RNase A were added thereto, mixed and then left on ice for 15 minutes. Further, 125 ml of 10% SDS was added and shaken at 37 ° C. for 6 hours, then 0.6 mg proteinase K and 1.5 ml of chloroform were added, and the mixture was shaken overnight at 37 ° C. Distilled water (1 ml) and chloroform (10 ml) were added thereto and mixed gently for 5 minutes, and then phenol (10 ml) was added and further mixed for 5 minutes, followed by centrifugation to collect the upper layer. 10 ml of chloroform was added thereto, mixed gently for 5 minutes, and then centrifuged again to obtain an upper layer. This was repeated once more, and 0.2 ml of 5 M saline solution and 12 ml of ethanol were added to the obtained upper layer and mixed gently, and the precipitated DNA was wound up with a glass rod. This was washed in ethanol and dissolved in 3 ml of TEN buffer (1 mM EDTA, 10 mM NaCl, 10 mM Tris-HCl, pH 7.6). The amount of DNA obtained was about 4.7 mg. 157 μg of this DNA was partially decomposed using the restriction enzyme Sau3AI, then electrophoresed on a 0.3% agarose gel, and a DNA fragment of about 20 kbp or more was isolated by electroelution. This was extracted with phenol / chloroform, precipitated with ethanol, and dissolved in 500 ml of TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 7.6). Of these, 100 ml was mixed with 100 ml of 20% PEG / 2.5M NaCl, and centrifuged to obtain a precipitate. This precipitate was dissolved in 100 ml of TE buffer, extracted with phenol, phenol / chloroform and chloroform, ethanol precipitated and dissolved in 10 ml of TE buffer.
[0016]
Example 1-2. Cosmid pWE15 (manufactured by STRATAGENE) was used as a genomic DNA library production vector. 10 μg of pWE15 was completely digested with the restriction enzyme BamHI, and terminal dephosphorylation was performed with calf intestinal alkaline phosphatase, followed by extraction with phenol and chloroform. This was ethanol precipitated and dissolved in 10 μl of TE buffer. Mix 1.5 μg of the vector DNA obtained by the above method and 1.3 μg of the restriction enzyme Sau3AI partial degradation product of genomic DNA prepared in Example 1, add T4DNA ligase 175U, and react at 4 ° C. overnight. The DNA strands were ligated. 2/5 of this reaction product was packaged by a conventional method to obtain a phage, which was infected with E. coli K12 / XL1-Blue MR strain. This phage-infected E. coli solution is applied to LB agar plate medium (tryptone 1%, yeast extract 0.5%, salt 1%, agar 2%) containing 50 μg / ml ampicillin and kanamycin, and cultured at 37 ° C. overnight. did. 1687 colonies that emerged were fished in a 96-well plate for storage of the same composition medium to prepare a library.
[0017]
Example 1-3. Colony hybridization 1-3-1. Preparation of Membrane Approximately 1687 colonies of the Shiwanella sp. SCRC-21406 genomic library prepared in Example 1-2 were picked up in 96 LB agar medium containing 100 mM ampicillin and 50 mM kanamycin and cultured overnight at 37 ° C. . After growth, colonies were copied onto a nylon membrane (Hybond ^™ N, Amersham). This membrane was treated twice with denaturing solution (0.5NNaOH, 1.5MNaCl) for 7 minutes and neutralizing solution (1.5MNaCl, 0.5MTris-HCl, pH7.2, 1mMNa ₂ EDTA) for 3 minutes, then 2xSSC (30mM citric acid) Sodium, 300 mM sodium chloride) and air-dried.
[0018]
1-3-2. DNA probe preparation
As a template DNA for DNA probe preparation, the SpeI (31447) -NheI (32522) fragment of ORF9 of the EPA biosynthesis gene group (described in JP-A-8-242867) possessed by the EPA-producing marine bacterium Shiwanella sp. used. Using the random primer DNA labeling kit Ver.2 (manufactured by Takara), 25 ng of the above DNA fragment was [a- ³² P] labeled according to the specifications. Free [a- ³² P] dCTP that was not incorporated into DNA was removed using a MicroSpin ^™ S-300HR (Pharmacia) column and purified to obtain a DNA probe.
[0019]
1-3-3. Hybridization Membrane prepared in 1-3-1 above was sonicated with 50% formamide, 5xSSCP (0.1 M sodium phosphate buffer, pH 6.8, 75 mM sodium citrate, 0.5 M sodium chloride), 5 mg / ml skim milk. Prehybridization was performed by immersing in a prehybridization solution containing 250 μg / ml of sperm sperm DNA and 0.5% SDS at 42 ° C. for 3 hours or more. Then, discard the prehybridization solution and immerse the membrane in a hybridization solution with the probe prepared in 1-3-2 (10% dextransulfate added to the prehybridization solution) at 42 ° C overnight. Hybridization was performed. The membrane treated as described above was replaced with a fresh 2 × SSC solution containing 0.1% SDS for 5 minutes and washed at room temperature with shaking for 10 minutes. Thereafter, it was washed with a 0.1 × SSC solution containing 0.1% SDS at 65 ° C. for 10 minutes. The washed membrane was wrapped in a wrap film and image analysis was performed by autoradiography or BAS-1500 (Fuji Photo Film) to examine the distribution of isotopes. As a result, 40 positive clones were obtained.
[0020]
1-3-4. Analysis of cosmid of positive clone A cosmid was prepared from the positive clone obtained in 1-3-3 above, and digested with a restriction enzyme EcoRI and subjected to agarose electrophoresis. After electrophoresis, agarose gel was stained with ethidium bromide to detect the DNA band, and 29 clones containing the inserted fragment were selected.
[0021]
Example 1-4. Dot hybridization 1-4-1. Preparation of membrane Prepare 55.5 μl of 2x SSC solution containing 1 μl each of the 29 cosmid DNA solutions of 29 positive clones obtained in Example 1-3, heat-treat at 95 ° C. for 5 minutes, and then ice-cool for 2 minutes. 1 μl each was spotted on a nylon membrane. The membrane was treated with a denaturing solution for 1 minute and a neutralizing solution for 1 minute, then air-dried, wrapped in a wrap film, and irradiated with a UV lamp for 4 minutes to immobilize DNA on the membrane.
[0022]
1-4-2. DNA probe preparation
In addition to the SpeI-NheI fragment of ORF9 of Example 1-3-2, as a template DNA for preparing a DNA probe, a DraIII (13887) -DraIII (16031) fragment, EcoRI (17922)-in ORF6 of the EPA biosynthesis gene An EcoRI (18252) fragment, an ORF7 EcoRI-XbaI fragment (about 1 kb), and an ORF8 PstI-PstI fragment (about 1.2 kb) were used. Labeling and purification of DNA fragments were performed in the same manner as in Example 1-3-2.
[0023]
1-4-3. Hybridization hybridization was performed in the same manner as in Example 1-3-3. The membrane was washed by the same method as in Example 1-3-3 for a 2 × SSC solution containing 0.1% SDS. Subsequent washing was performed as follows. If the DNA probe is the DraIII-DraIII fragment of ORF6, the PstI-PstI fragment of ORF8, and the SpeI-NheI fragment of ORF9, the ORF7 EcoRI-XbaI fragment twice in a 0.1x SSC solution containing 0.1% SDS at 65 ° C for 10 minutes In this case, the plate was washed with a 0.2x SSC solution containing 0.1% SDS at 55 ° C for 10 minutes. The washed membrane was processed and analyzed in the same manner as in Example 1-3-3. As a result, cosmids obtained from 21 clones hybridized with the SpeI-NheI fragment of ORF9, 4 of which were very strong. Hybridized. Of these four clones, only clone 10C7 hybridized with all three other DNA probes.
[0024]
Example 1-5. Southern blot hybridization 1-5-1. Preparation of Membrane A positive clone, 10C7 cosmid DNA, which was found to hybridize with all DNA probes in Example 1-4-3, was named p10C7. This p10C7 was cleaved with the restriction enzyme EcoRI, and electrophoresed at 100 V for about 4 hours using a 0.7% agarose gel (SeaKem GTG, manufactured by FMC). After electrophoresis, ethidium bromide staining was performed to detect DNA bands, and six bands were observed. These bands were named E1 to E6 in descending order of molecular weight. The gel after observation is washed with distilled water, shaken in 0.25N HCl for 15 minutes, washed with distilled water, shaken in denaturing solution for 30 minutes, then immersed in a blotting solution (0.25NNaOH, 1.5MNaCl) and placed on a nylon membrane. A vacuum blot was performed for 1 hour. The membrane was washed with 2 x SSC and then air dried.
[0025]
1-5-2. Hybridization
The DNA probe prepared in Example 1-4-2 was used. Hybridization and membrane washing were performed in the same manner as in Example 1-4-3. The washed membrane was processed and analyzed in the same manner as in Example 1-4-3. As a result, the restriction enzyme EcoRI cleaved fragment of cosmid p10C7 was observed to hybridize with DNA probes of ORF6, 8, and 9 of the EPA biosynthesis gene. That is, cosmid p10C7 was shown to have homology with the base sequence of the EPA biosynthesis gene group of Shiwanella sp. SCRC-2874 strain.
[0026]
Example 1-6. Preparation of restriction enzyme map of cosmid p10C7 The genomic DNA insertion part of cosmid p10C7 prepared from the transformed strain XL1-Blue MR / p10C7 was cleaved with various restriction enzymes to prepare restriction enzyme cleavage maps (FIG. 1).
[0027]
Example 1-7. Preparation of transformant JM109 / p10C7 Using cosmid p10C7, E. coli JM109 was transformed to obtain JM109 / p10C7. This strain was deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology as FERM P-17058 on November 18, 1998.
[0028]
Example 1-8. Sequence Analysis The entire base sequence of the white part in FIG. 1 in the genomic DNA insertion part of cosmid p10C7 is shown in SEQ ID NO: 1. In this base sequence, four open reading frames (ORF): a to d can be specified, and their respective base sequences and corresponding amino acid sequences are shown in SEQ ID NOs: 2, 4, 6, 8. . Table 1 shows the relationship between the entire nucleotide sequence (SEQ ID NO: 1) and ORFa to d (SEQ ID NOs: 2, 4, 6, 8).
[0029]

[0030]
Amino acid sequences deduced from the base sequences of the various ORFs (SEQ ID NOs: 3, 5, 7, 9) and amino acid sequences deduced from the base sequences of the ORFs of the genes encoding the EPA biosynthesis enzyme group (Described in Kaihei 8-242867) was found to have some degree of homology. The results are shown in Table 2.
[0031]

[0032]
From this result, the protein encoded by SEQ ID NO: 2, 4, 6, 8 (ORFa, b, c, d contained in cosmid 10C7) is the product of ORF6, 7, 8, 9 encoding EPA biosynthetic enzymes. Were expected to be functionally similar to each other.
[0033]
Example 2-1. Subcloning of restriction enzyme fragment of genomic DNA insertion part of cosmid 10C7 Inserting EcoRI (2391) -EcoRI (14761) fragment containing ORFb of genomic DNA insertion part of cosmid 10C7 into the EcoRI site of plasmid pSTV28 (Takara Shuzo) Plasmid Eco-Eco / pSTV28 was created. In addition, a PstI (16802) -PstI (22263) fragment containing ORFd was inserted into the PstI site of plasmid pSTV28 to create plasmid Pst-Pst / pSTV28. In addition, the EcoRV (11172) -PvuI (17457) fragment containing ORFc was blunt-ended and inserted into the SmaI site of plasmid pSTV29 to prepare plasmid Eco-Pvu / pSTV29. The position of the inserted fragment of each subclone is shown in FIG.
[0034]
Example 2-2. Preparation of ORF9,10 deletion clone of EPA biosynthesis gene group XhoI (5660) -SpeI (31446) fragment of plasmid pEPA (described in JP-A-8-242867) containing gene group encoding EPA biosynthesis enzyme group ( (Including ORF2-8) was ligated to the XhoI-SpeI site of plasmid pBluescript (manufactured by STRATAGENE) to create pXS-BS2. The position of the insert fragment of pXS-BS2 is shown in FIG. This plasmid lacks ORF9, a gene essential for EPA biosynthesis.
[0035]
Example 2-3. Generation of ORF2,4,5,8,10 deletion clone of EPA biosynthesis gene group From plasmid Δ2,4,5,10 / pNEB (described in WO98 / 01565) having EPA production ability to PstI (24813) -EcoT22I The (28946) (part of ORF8) portion was deleted to create Δ2,4,5,8,10 / pNEB. The position of the inserted fragment of Δ2, 4, 5, 8, 10 / pNEB is shown in FIG. This plasmid lacks ORF8, a gene essential for EPA biosynthesis.
[0036]
Example 2-4. Recovery of EPA production ability by complementation of ORF7 or ORF9-deficient EPA biosynthetic genes The E. coli JM109 strain into which one of the plasmids prepared in Example 2-1 was introduced was further transformed into pEPAΔ7 (part of ORF7 from pEPA The plasmid was deleted from the SalI (22260) -NruI (23845) portion described in JP-A-8-242867) or pXS-BS2 prepared in Example 2-2. These transformants and transformants having only pEPAΔ7 or pXS-BS2 were inoculated into 5 ml of LB medium containing 50 μg / ml ampicillin and chloramphenicol and cultured at 25 ° C. Bacteria were obtained by centrifugation, washed once, lyophilized, dissolved in 2 ml of methanol saturated with hydrogen chloride, placed in a sealed container, and kept at 80 ° C. for 1 hour to methylate the fatty acid. After allowing to cool, extraction was performed 3 times with 2 ml of hexane, and the hexane layer was dried and dissolved in 10 ml of methanol. When a part of the lysate was analyzed by gas chromatography, an EPA peak was observed. Table 3 shows the combination of plasmids possessed by each transformant and the ratio of EPA methyl ester in the total fatty acid ester of the microbial cells calculated from the peak area ratio.
[0037]

[0038]
From the above results, it was shown that the EPA production ability of clones lacking ORF7 or ORF9 in the EPA biosynthesis system gene group was recovered by the presence of a DNA fragment derived from cosmid p10C7. That is, it was shown that cosmid p10C7 contains a gene involved in biosynthesis of highly unsaturated fatty acids having the same function as ORF7 and ORF9.
[0039]
Example 2-5. Recovery of EPA production ability by complementation of ORF8-deficient EPA biosynthetic genes and DHA production The Escherichia coli JM109 strain into which the plasmid Eco-Pvu / pSTV29 prepared in Example 2-1 was introduced was further prepared in Example 2-3. The transformed plasmid Δ2,4,5,8,10 / pNEB was selected using an agar plate medium containing ampicillin and chloramphenicol, and recombinant JM109 / p3 having two plasmids was obtained. 6,7,9 / pc were obtained. This transformed strain was cultured at 18 ° C. in the same manner as in Example 2-4, and the fatty acid composition of the bacterial cells was analyzed by gas chromatography. As a result, an EPA peak was observed, and the ratio in the total fatty acid ester was calculated to be about 22.0% from the peak area ratio. On the other hand, a new peak was observed on the polymer side of EPA methyl ester on gas chromatography. The ratio of the peak substance in the total fatty acid ester was calculated to be about 1.5% from the peak area ratio. As a result of GC-MS analysis, the molecular weight of the substance of this peak was 342, which was consistent with the value of DHA (22: 6) methyl ester, and the peak of each fragment was consistent with that of the standard. Identified as methyl ester. The MS fragment peak was as follows. Mass: 342 (M +), 327, 311, 299, 286, 273, 241, 215, 206, 173, 159, 145, 131, 119, 105, 91, 79, 67, 55, 41, 27.
[0040]
From the above results, it was shown that the EPA production ability of clones having the EPA biosynthetic gene group lacking ORF8 was restored by the presence of the DNA fragment derived from cosmid p10C7, and the DHA production ability was also acquired. It was. That is, it was shown that cosmid p10C7 has a function similar to that of ORF8 and includes a gene involved in DHA biosynthesis.
[0041]
[Sequence Listing]
[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]
[Brief description of the drawings]
FIG. 1 shows a restriction enzyme map of cosmid p10C7. The bold line part of a drawing shows the gene fragment derived from the chromosome of docosahexaenoic acid producing bacteria. The white part in the bold line indicates the part for which the base sequence was determined.
FIG. 2 shows the position of the insert fragment of the cosmid p10C7 subclone.
FIG. 3 shows the position of an inserted fragment of a subclone of the EPA biosynthesis gene group.

Claims (7)

Enzyme group involved in biosynthesis of icosapentaenoic acid and / or docosahexaenoic acid, which is contained in an approximately 37 kb gene fragment having the restriction enzyme map shown in FIG. A gene encoding The gene according to claim 1, wherein the docosahexaenoic acid-producing bacterium is Shiwanella sp. SCRC-21406 (FERM BP-5979). A gene encoding an enzyme group involved in biosynthesis of icosapentaenoic acid and / or docosahexaenoic acid encoded by the base sequence represented by SEQ ID NO: 1. A gene encoding an enzyme group involved in biosynthesis of icosapentaenoic acid and / or docosahexaenoic acid, comprising at least one base sequence selected from the base sequence group represented by SEQ ID NOs: 2, 4, 6 and 8. A gene encoding an enzyme group involved in biosynthesis of icosapentaenoic acid and / or docosahexaenoic acid, comprising at least one amino acid sequence selected from the amino acid sequence groups represented by SEQ ID NOs: 3, 5, 7, and 9. A plasmid comprising the gene according to claim 3 or 4. A microorganism transformed with the plasmid according to claim 6.

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