JP4405627B2 - Novel polypeptide, DNA encoding the polypeptide, and uses thereof - Google Patents

Description

【００１９】
表１から明らかなようにｐＩＺ３１１インサート領域は全体にわたり種々のフェノールヒドロキシラーゼ（Phenolhydroxylase）と相同性が高く、特にPseudomonas sp. Strain CF600のフェノールヒドロキシラーゼ（Phenolhydroxylase）との相同性が高い。
【００２０】
これらの結果から、インドール−７−ヒドロキシラーゼ活性に関与するＯＲＦはＩｏｘＡ，Ｂ，Ｃ，Ｄ，Ｅ，Ｆ，ＧおよびＩｏｘＲであると考えられた。ＩｏｘＡの上流には種々のフェノールヒドロキシラーゼと同様にσ５４依存−２４／−１２タイププロモ−タ−のコンセンサス配列ＴＧＧＣ−Ｎ８−ＴＧＣＡが存在しており（配列番号１中、2548〜2563番）、転写調節タンパク質ＩｏｘＲ、ＤＮＡ結合タンパク質ＩＨＦ、およびσ５４によりＩｏｘオペロンの転写制御がなされているものと考えられる。
【００２１】
６）各種デレーションミュータントを用いたインドールの７位水酸化に必要な遺伝子（ＯＲＦ）領域の検討
(i)Ｉｏｘデレーションミュータント（deletion mutant）の作成
まず、種々のＯＲＦに沿って特異的な配列番号３〜４のセンスプライマー（Sense primer）と配列番号５〜７のアンチセンスプライマー（Antisense primer）（各々２８ｍｅｒ）を作製した（下線部は制限酵素ＸｂａＩの認識配列である。）。
【００２２】
ｉｏｘＲＲＦ：ＣＴＴＣＴＡＧＡＧＧＣＡＴＴＣＧＧＴＧＡＡＧＧＣＡＴＧＣ（配列番号３）、
ｉｏｘＡＦ：ＴＴＴＣＴＡＧＡＣＴＴＧＴＣＴＣＣＴＣＣＴＧＣＧＴＧＣＣ（配列番号４）、
ｃｏｍｐ．Ｒ：ＧＡＴＣＴＡＧＡＴＣＣＡＧＴＧＧＣＣＧＣＡＴＴＴＧＣＣＧ（配列番号５）
ｉｏｘＧＲ：ＧＡＴＣＴＡＧＡＴＴＡＴＧＣＴＴＧＴＴＴＧＧＴＡＣＡＧＣ（配列番号６）、
ｉｏｘＦＲ：ＧＴＴＣＴＡＧＡＣＴＡＧＡＴＧＣＧＣＴＴＧＡＡＣＡＧＣＧ（配列番号７）。
【００２３】
上記プライマーとTaKaRa LA Taq（TaKaRa製）を用い、ｐＩＺ３１１を鋳型としてＰＣＲ反応（反応条件：９６℃で３分間、その後９８℃で２０秒−６０℃で２０秒−６８℃で１０分間の２５サイクル）を行なった。アガロースゲル電気泳動にてＰＣＲ産物ができていることを確認し、反応溶液をキアクイック・ＰＣＲピュリフィケーション・キット（QIAquick PCR Purification Kit，QIAGE製）を用いて精製した。精製産物をＤＮＡライゲーション・キット・ヴァージョン２（Ligation Kit Ver.2，TaKaRa製）を用いて大腸菌ベクターｐＴ７−Ｂｌｕｅ（Novagen製）に連結し、大腸菌ＪＭ１０９株（TaKaRa製）を形質転換し、アンピシリン５０μｇ／ｍｌを添加したＬＢ寒天培地（ポリヘプトン 1.0％，酵母エキス 0.5％，ＮａＣｌ 0.5％，グルコース 0.1％，寒天 1.5％）にプレーテイングを行なった。翌朝、コロニーができていることを確認し、ＬＢ培地（ポリペプトン 1.0％、酵母エキス 0.5％、ＮａＣｌ 0.5％、グルコース 0.1％）に接種後、プラスミドの抽出を行なった。制限酵素ＸｂａＩ（TaKaRa製）にて処理後、アガロースゲル電気泳動を行ない断片の有無を確認した。ｉｏｘＲＲＦプライマーとｃｏｍｐ．Ｒプライマーを用いてプラスミドｐＩＯＸＲＣｏｍｐ．を、ｉｏｘＲＲＦプライマーとｉｏｘＦＲプライマーを用いてプラスミドｐＩＯＸＲＦを、ｉｏｘＲＲＦプライマーとｉｏｘＧＲプライマーを用いてプラスミドｐＩＯＸＲＧを、ｉｏｘＡＦプライマーとｉｏｘＦＲプライマーを用いてプラスミドｐＩＯＸＡＦを、ｉｏｘＡＦプライマーとｉｏｘＧＲプライマーを用いてプラスミドｐＩＯＸＡＧを構築した。
【００２４】
(ii)デレーションミュータントの培養試験
常法によりプラスミドｐＩＯＸＲＣｏｍｐ．、ｐＩＯＸＲ、ｐＩＯＸＲＧ、ｐＩＯＸＡＦ、ｐＩＯＸＡＧで大腸菌ＪＭ１０９株を形質転換してそれぞれのクローンを得た。各クローンをアンピシリン５０μｇ／ｍｌを添加したＬＢ培地に植菌して３７℃で一晩震盪培養を行なった。培養液５００μｌをアンピシリン５０μｇ／ｍｌを添加したＬＢ培地（５０ｍｌ／２５０ｍｌ容のエルレンマイヤーフラスコ）に植菌し、３７℃でロータリーシェーカー上で震盪培養を開始し、培養８時間目で終濃度が１００μｇ／ｍｌになるようにインドールとＦｅＳＯ₄・７Ｈ₂Ｏをそれぞれ添加した。その後、経時的に培養液２ｍｌのサンプリングを行ない、培養液から遠心分離によって菌体を除きその上清中のインドールと７位水酸化インドールの定量をＨＰＬＣ（カラム：YMC-PackA-312ODS（4.6×１５０ｍｍ），移動相：５０％アセトニトリル，流速：0.5ｍｌ／ｍｉｎ，検出：２８０ｎｍ）を用いて行なった。その結果、それぞれの形質転換株の７位水酸化インドール蓄積量は、ｐＩＯＸＡＦ転換株で11.8μＭ、ｐＩＯＸＡＧ転換株で66.6μｍ、ｐＩＯＸＲＦ転換株で21.4μＭ、ｐＩＯＸＲＧ転換株で59.4μＭ、ｐＩＯＸＲＣｏｍｐ．転換株で９８μＭであった。このことからインドールの７位を水酸化するのに必要なＯＲＦはＩｏｘＡ、ＩｏｘＢ、ＩｏｘＣ、ＩｏｘＤ、ＩｏｘＥ、ＩｏｘＦであることが分かった。
【００２５】
以上の結果及び前記表１の結果から、配列番号１の塩基配列でコードされるポリペプチド群の予想される役割を図１に示す。
【００２６】
次に前記の方法にてクローニングされた遺伝子で形質転換された微生物もしくは該遺伝子を染色体上に有する微生物の培養方法および生産された７位水酸化インドールおよび７位水酸化トリプトファンの製造法について詳細に説明する。
本発明で使用する培地組成は、使用する微生物が良好に生育し、かつインドール誘導体の７位を水酸化する活性を発現するのに適当な炭素源、窒素源、無機塩及び天然有機栄養物等により成り立っている。炭素源としては、グルコース、フラクトース、グリセロール、ソルビトール、アルコール類、酢酸、澱粉等を単独に用いるかもしくは併用でき、その使用濃度は特に限定されず、おおよそ１〜１０％が適当である。窒素源としてはアンモニア、尿素、硫安、硝安、酢安等の化合物を一種または二種以上使用することができる。無機塩としては、燐酸一カリウム、燐酸二カリウム、硫酸マグネシウム、硫酸マンガン、硫酸第一鉄などの塩類を使用することが出きる。さらに使用菌の生育促進効果を持つ有機栄養源としては、ペプトン、肉エキス、酵母エキス、コーンスチーブリカー、カザミノ酸などが用いられ、さらにビタミン類、核酸類を少量培地に含有せしめることもできる。
【００２７】
インドールの７位を水酸化するためのインドールの添加時期は、菌体の生育の最初から及び生育後のいずれでもよく、また、培養液から菌体を回収し菌体を適当な水溶液に懸濁させた液中に添加してもよい。インドールは種々の濃度で用いることが出きるが、約0.1ｇ／リットルから１ｇ／リットルの濃度で用いることが望ましく、これを一括添加しても分割して添加してもよい。
培養は通気撹拌培養、振盪培養等の好気的条件下、あるいは静置培養でもよい。培養中のpHは中性ないし弱アルカリ性が好適であり、このためのｐＨ中和剤としては、アンモニア、水酸化ナトリウム、水酸化カリウム、炭酸カルシウム、塩酸等の公知のものが使用できる。培養温度は２０〜４０℃の範囲で使用菌の成育に最適な温度で培養できる。培養時間は使用菌株の相違、原料の添加方法、添加濃度等により差はあるが、おおよそ１〜４日で７−ヒドロキシインドール及び７−ヒドロキシトリプトファンが生成蓄積する。
【００２８】
生成蓄積した７−ヒドロキシインドール及び７−ヒドロキシトリプトファンは、それ自体公知の方法に準拠して単離・精製することができる。すなわち、疎水性の担体あるいはイオン吸着樹脂に吸着せしめ、有機溶媒あるいはアルカリ水、酸性水で溶出し、濃縮後、結晶化等によって回収される。
【００２９】
【実施例】
以下に実施例によって本発明を詳細に説明する。
実施例１：
酵母エキス 0.1％、ペプトン 0.1％、ＫＨ₂ＰＯ₄ 0.1％、グルコース 0.1％、寒天２％からなる斜面寒天培地（加熱殺菌前にｐＨ 6.8に調整）上で成育したキサントモナス・マルトヒィリアＭｅｒ−Ｈ１０８株を一白金耳取り、酵母エキス 0.1％、ペプトン 0.1％、ＫＨ₂ＰＯ₄ 0.1％からなる培地（加熱殺菌前にｐＨ 6.8に調整）５０ｍｌを含む２５０ｍｌ容のエルレンマイヤーフラスコに植菌し、２８℃で１９時間ロータリーシェーカー上で培養を行なった。この種母培養液１５ｍｌをインドール 0.01％、酵母エキス 0.1％、ペプトン 0.1％、ＫＨ₂ＰＯ₄ 0.1％からなる培地（加熱殺菌前にｐＨ 6.8に調整）1.5リットルを含む３リットル容のジャーファメンターに植菌し２８℃で２１時間、通気撹拌培養（通気量0.75リットル／分、撹拌４００ｒｐｍ）を行ない、培養液中に７−ヒドロキシインドールを２５μｇ／ｍｌ生成蓄積させた。培養液から菌体を遠心分離によって除いた後、培養上清1.4リットルを水で充填したダイヤイオンＨＰ−２０カラム（樹脂は三菱化成製、７φ×１２ｃｍ）に通塔し、７−ヒドロキシインドールを樹脂に吸着させ１リットルの水で洗浄後、５００ｍｌのメタノールでカラムから７−ヒドロキシインドールを溶出させた。溶出液から７−ヒドロキシインドール溶出画分を集めてエバポレーターで減圧下に濃縮して水およびメタノールを留去し７−ヒドロキシインドールを結晶として２３ｍｇ得た。こうして得た７−ヒドロキシインドールについてＦＡＢ−ＭＳおよび¹Ｈ−ＮＭＲを測定した。
【００３０】
ＦＡＢ−ＭＳ（マトリックス；グリセリン）：ｍ／ｚ １３３（Ｍ⁺）、
¹Ｈ−ＮＭＲ（４００ＭＨｚ、ＣＤ₃ＯＤ）：δ 6.39 (1H, d, J=3.3Hz), 6.49 (1H, d, J=7.7Hz), 6.79 (1H, dd, J=7.7Hz, J=7.7Hz), 7.03 (1H, d, J=7.7Hz), 7.15 (1H, d, J=3.3Hz)。
【００３１】
実施例２：
酵母エキス 0.1％、ペプトン 0.1％、ＫＨ₂ＰＯ₄ 0.1％、グルコース 0.1％、寒天２％からなる斜面寒天培地（加熱殺菌前にｐＨ 6.8に調整）上で成育したキサントモナス・マルトヒィリアＭｅｒ−Ｈ１０８株を一白金耳取り、酵母エキス 0.1％、ペプトン 0.1％、ＫＨ₂ＰＯ₄ 0.1％からなる培地（加熱殺菌前にｐＨ 6.8に調整）５０ｍｌを含む２５０ｍｌ容エルレンマイヤーフラスコに植菌し、２８℃で１９時間ロータリーシェーカー上で培養を行なった。この種母培養液１５０ｍｌをインドール 0.01％、酵母エキス 0.1％、ペプトン 0.1％、ＫＨ₂ＰＯ₄ 0.1％、0.01％ ＦｅＳＯ₄・７Ｈ₂Ｏからなる培地（加熱殺菌前にｐＨ 6.8に調整）１５リットルを含む３０リットル容ジャーファメンターに植菌し、２８℃で通気撹拌培養（通気量 7.5リットル／分、撹拌２００ｒｐｍ）を行なった。培養１５時間目にＬ−セリンを7.5ｇ添加し２６時間まで培養を行ない、培養液中に７−ヒドロキシトリプトファンを９μｇ／ｍｌ生成蓄積させた。培養終了後、培養液から菌体を遠心分離によって除き、得られた培養上清１４リットルを強塩基性イオン交換樹脂ダイアイオンＰＡ３０６ｓカラム（樹脂は三菱化成製、７φ×１５ｃｍ）に通塔し、７−ヒドロキシトリプトファンを樹脂に吸着させ１リットルの水で洗浄後、２リットルの0.1Ｎ塩酸で７−ヒドロキシトリプトファンを溶出させた。溶出液から７−ヒドロキシトリプトファンの溶出画分を合わせて１Ｎ ＮａＯＨでｐＨ 6.0にしてからエバポレーターで減圧下に濃縮後、凍結乾燥を行なって粗精製物を21.3ｇ得た。これを２０ｍＭリン酸緩衝液（ｐＨ 6.8）１０ｍｌに溶かし、分取ＨＰＬＣ（カラム：Inertsil PREP-ODS（２×２５ｃｍ、ガスクロ工業）、移動相：５％ ＭｅＯＨ ２０ｍＭリン酸緩衝液（ｐＨ 6.8）、流速：7.0ｍｌ／分、検出：ＵＶ２１０ｎｍ）にかけ保持時間34.6分の７−ヒドロキシトリプトファンのピークを分取した。分取した画分を脱塩するため移動相を水とした他は先と同じ条件の分取ＨＰＬＣで７−ヒドロキシトリプトファンのピークを分取し凍結乾燥して７−ヒドロキシトリプトファンをナトリウム塩として８６ｍｇ得た。こうして得た７−ヒドロキシトリプトファンのＦＡＢ−ＭＳおよび¹Ｈ−ＮＭＲを測定した。
【００３２】
ＦＡＢ−ＭＳ（マトリックス；ＮＢＡ）：ｍ／ｚ２２１（（Ｍ＋Ｈ）⁺）、
¹Ｈ−ＮＭＲ（４００ＭＨｚ、ＣＤ₃ＯＤ）：δ 3.05 (1H, dd, J=9.5Hz, J=15.0Hz), 3.45 (1H, dd, J=3.3Hz, J=15.0Hz), 3.79 (1H, dd, J=4.0Hz, J=9.5Hz), 6.57 (1H, d, J=7.7Hz), 6.85 (1H, dd, J=7.7Hz, J=7.7Hz), 7.13 (1H, s), 7.19 (1H, d, J=7.7Hz)。
【００３３】
実施例３：
ｐＩＺ３１１で形質転換したＥ.ｃｏｌｉ ＪＭ１０９をカナマイシン硫酸塩２０μｇ／ｍｌを含むＬ培地（ポリペプトン 1.0％，酵母抽出液（Yeast extract）0.5％，ＮａＣｌ 0.5％,グルコース 0.1％，ｐＨ 7.2）に植菌し、３２℃で一晩振盪培養した。この培養液から菌体を遠心分離によって除いた後、培養上清をＨＰＬＣ（カラム：ＯＤＳ−Ａ（0.6×１５ｃｍ）、ＹＭＣ）、移動相：５０％アセトニトリル、流速：0.5ｍｌ／分、検出：ＵＶ２８０ｎｍ）にかけ保持時間 10.08分の７−ヒドロキシインドールのピークを検出した。７−ヒドロキシインドールが約２０μｇ／ｍｌ検出された。
【配列表】

【図面の簡単な説明】
【図１】本発明による遺伝子でコードされるポリペプチド群の予想される役割を示す。図中太線矢印はプライマーを示し、細線矢印は遺伝子の大きさと向きを示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a polypeptide having an enzyme activity that hydroxylates 7-position of indole, DNA encoding the polypeptide, recombinant DNA obtained by incorporating the DNA, microorganisms holding the recombinant DNA, indole 7 The present invention relates to a method for producing 7-hydroxyindole and 7-hydroxytryptophan useful as a raw material for pharmaceuticals, dyes and reagents using a microorganism that produces an enzyme that hydroxylates the position or a microorganism that retains the recombinant DNA.
[0002]
[Prior art]
Conventionally, 7-hydroxyindole is produced by a synthesis method, but requires several steps and is expensive to produce. Further, as a production method by a microbial conversion method, a method (WO 9534657) is known in which 3-hydroxy-2-amino-benzoic acid is added to genetically engineered Escherichia coli and produced using a tryptophan-anthranilic acid cycle. There is no known production method by hydroxylating the 7-position of indole using a microorganism or an enzyme. On the other hand, as a method for producing 7-hydroxytryptophan, in addition to the synthesis method, as a microbial conversion method, in addition to the microbial conversion method described above, 7-hydroxyindole produced by the synthesis method is converted into 7-hydroxytryptophan by a microorganism. However, there is no known method for directly producing 7-hydroxytryptophan from indole using microorganisms.
[0003]
[Problems to be solved by the invention]
An object of the present invention is to provide a polypeptide having an enzyme activity for hydroxylating the 7-position of indole in order to produce 7-hydroxyindole and 7-hydroxytryptophan, which are regarded as important raw materials for pharmaceuticals, dyes and reagents, at low cost. It is to provide a method for producing 7-hydroxyindole and 7-hydroxytryptophan using a microorganism retaining the gene, as well as providing a group and a gene encoding the polypeptide.
[0004]
[Means for Solving the Problems]
In view of the above problems, the present inventors searched for a microorganism that hydroxylates 7th position of indole, and hydroxylated 7th position of indole added to the medium by microorganisms belonging to the genus Xanthomonas isolated from soil. It has been discovered that it has the ability to produce 7-hydroxyindole and also produces 7-hydroxytryptophan. Next, the present inventors attempted to clone a gene involved in hydroxylation at the 7-position of indole from the microorganism, and succeeded in cloning the gene group to complete the present invention.
[0005]
Hereinafter, the present invention will be described in detail.
As the polypeptide of the present invention, any polypeptide can be used as long as it has an activity of hydroxylating position 7 of indole. For example, in the nucleotide sequence represented by SEQ ID NO: 1, (1) from 2602 to 2796 A polypeptide having an amino acid sequence encoded by DNA having the second sequence, (2) a polypeptide having an amino acid sequence encoded by DNA having the sequence from 2825 to 3829, (3) from 3847 to 4116 A polypeptide having an amino acid sequence encoded by DNA having a sequence, (4) a polypeptide having an amino acid sequence encoded by DNA having a sequence from 4145th to 5704th, and (5) a sequence from 5740th to 6099th A polypeptide having an amino acid sequence encoded by DNA having (6) an amino acid sequence encoded by DNA having a sequence from 6126 to 7181 One amino acid sequence of a polypeptide, (7) a polypeptide having an amino acid sequence encoded by DNA having the 7190th to 7528th sequence, or a group of polypeptides having an amino acid sequence represented by (1) to (7) Mention may be made of a polypeptide having an amino acid sequence in which the above amino acids are deleted, substituted or added, and having an activity of hydroxylating position 7 of indole.
[0006]
The present invention includes (1) the DNA represented by SEQ ID NO: 1 and DNA that hybridizes with the DNA under stringent conditions, as well as (2) 2602 to 2796 in the base sequence represented by SEQ ID NO: 1. DNA having the second sequence or DNA that hybridizes with the DNA under stringent conditions, (3) DNA having the 2825th to 3829th sequences or DNA with the DNA that hybridizes under the stringent conditions ( 4) DNA having the 3847th to 4116th sequence or DNA hybridizing under stringent conditions, (5) DNA having the 4145th to 5704th sequence or DNA thereof under stringent conditions DNA that hybridizes, (6) DNA having the sequence from 5740th to 6099th, or stringent with the DNA DNA that hybridizes under conditions, (7) DNA having a sequence from 6126 to 7181 or DNA that hybridizes with the DNA under stringent conditions, (8) DNA having a sequence from 7190 to 7528, or DNA that hybridizes with the DNA under stringent conditions, (9) DNA represented by SEQ ID NO: 2 or DNA that hybridizes with the DNA under stringent conditions.
[0007]
The present invention also holds a recombinant DNA obtained by incorporating any combination of at least one of the DNAs of (1) to (9) and two or more and up to 8 types of DNA, and the recombinant DNA. Microorganisms and the microorganisms described above under conditions for expressing at least one of (1) to (7), any combination of two to seven, and a polypeptide (polypeptide group) containing all of them 7-hydroxyindole or 7-hydroxytryptophan is produced and accumulated by adding indole or an indole derivative to the culture solution or a solution containing cells separated from the culture solution, and collecting them. And a process for producing 7-hydroxyindole or 7-hydroxytryptophan.
[0008]
As a microorganism suitable for carrying out the present invention, for example, Xanthomonas maltophilia Mer-H108 strain can be mentioned. This strain is a strain isolated from the field soil of Fujisawa City, and its form is 0.4 μm wide gonococci with motility by polar flagella, yeast extract 0.1%, peptone 0.1%, KH ₂ PO _Four Grows aerobically on an agar medium (adjusted to pH 6.8 before heat sterilization) consisting of 0.1%, glucose 0.1%, and agar 2%. When cultured at 31 ° C for 1 day, it is a pale yellow flat with a spot of 0.5 mm or less Gram-stained negative bacteria that form a colony. Based on these properties, the strain was identified as Xanthomonas maltophilia based on Barjays Manual of Systematic Bacteriology, and named Xanthomonas maltophilia Mer-H108. However, the strain that can be used in the present invention is not limited to this strain, and any strain that can achieve its purpose can be used. This strain Mer-H108 has been deposited as Xanthomonas maltophilia H108 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (Accession No. FERM P-17359).
[0009]
Next, the cloning of the gene related to the 7-position hydroxylation of indole from Xanthomonas maltophilia Mer-H108 strain will be described in detail.
1) Acquisition of non-converted shares
Xanthomonas maltophilia Mer-H108 strain containing 100 μg / ml indole (Yeast Extract (Difco) 0.1%, Bacto Peptone (Difco) 0.1%, KH ₂ PO _Four 0.1%, FeSO _Four ・ 7H ₂ (O 0.01%, pH 6.8) 50 ml of a platinum ear was inoculated from the slant medium and cultured overnight at 28 ° C. with shaking. Using this as an inoculum, 500 μl of the culture solution was inoculated into 50 ml of a separation medium, and cultured with shaking at 28 ° C. for 6 hours. The cells were collected from this culture by centrifugation at 5000 × g for 10 minutes, washed once with 0.1 M phosphate buffer (pH 7.0), and suspended in 5 ml of 0.1 M phosphate buffer (pH 7.0). . NTG was added to this cell suspension at a final concentration of 1 mg / ml, and cultured with shaking at 28 ° C. for 30 minutes. The cells collected from this culture solution were washed once with 0.1 M phosphate buffer (pH 7.0), the entire amount was inoculated into 50 ml of a separation medium, and cultured with shaking at 28 ° C. overnight. 500 μl of this culture solution was dispensed and stored frozen at −70 ° C. as a mutant stock solution.
[0010]
This mutant stock solution is diluted 1000-fold with sterilized water, and an agar medium (Yeast Extract (Difco) 0.1%, Bacto Peptone (Difco) 0.1%, KH) containing 100 μg / ml indole of 8 cm petri dish. ₂ PO _Four 0.1%, FeSO _Four ・ 7H ₂ O 0.01%, agar 1.5%, pH 6.8) was applied in a volume of 100 μl and cultured at 28 ° C. for one week. Among the colonies that had grown, colonies showing white were inoculated into 1 ml of a separation medium, and cultured with shaking at 28 ° C. for 2 days. 20 μl of this culture solution was spotted on each lane of a silica gel TLC plate and dried. The plate was developed with a solvent system consisting of chloroform: diethyl ether (3: 2), Ehrlich color was developed, and the presence or absence of 7-hydroxyindole was examined for each lane. In this way, 18-9 mutant strains that did not convert indole to 7-hydroxyindole at all were isolated from the mutant strains. This mutant strain has been deposited as Xanthomonas maltilia mutant 18-9 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (Accession No. FERM P-17420).
[0011]
2) Construction of host-vector system and transformation system
Xanthomonas maltophilia Mer-H108 strain was inoculated into 3 ml of a separation medium and cultured overnight at 32 ° C. with shaking. Using this as an inoculum, 100 μl of the culture solution was inoculated into 50 ml of a separation medium and cultured with shaking at 32 ° C. for 5 hours. Cells were collected from this culture by centrifugation at 5000 × g for 10 minutes, washed once with a 10% glycerol solution, and suspended in 200 μl of a 10% glycerol solution as an electrocell. 1 μl of a 200 μg / ml TE solution of Broad Host Range Vector pBBR122 (Mo Bi Tec) was added to the electrocell. This was placed in a 0.2 cm Electrocuvette (BIORAD), and an electric pulse was applied once under the conditions of 2.5 KV, 200Ω, and 25 μF using Gene Pulser II (BIORAD). The cell was placed in a Falcon tube, 1 ml of ice-cooled separation medium was added, and the mixture was cultured with shaking at 32 ° C. for 2 hours. This culture solution was applied to a separated agar medium containing 20 μg / ml of kanamycin sulfate and cultured at 32 ° C. for 3 days. 1.0 × 10 ^Four Individual transformants were obtained.
[0012]
3) Construction of plasmid pCF704
Primer with EcoRI site and NcoI site attached to the end (Pharmacia), Taq polymerase (Pharmacia) and PCR thermal cycler (Thermal Cycler) PERSONAL (TaKaRa), cloning site of pUC18 And its peripheral region 95 bp were amplified. This DNA fragment was digested with restriction enzymes EcoRI and NcoI, and ligated to the EcoRI and NcoI sites of pBBR122 using Ligation Kit version 2 (TaKaRa). Plasmid pCF704 was prepared from a transformant obtained by transforming E. coli competent cells JM109 (TaKaRa) with this reaction solution using a QIAGEN Plasmid Midi Kit.
[0013]
4) Construction of plasmid pIZ311
Genomic DNA of Xanthomonas maltophilia Mer-H108 strain was extracted and purified according to QIAGEN Blood and Cell Culture DNA Kit. This genomic DNA was partially digested with the restriction enzyme SauIIIAI, the 4-8 Kbp fragment was excised from the agarose gel, the DNA was recovered and purified using Ultra Free C3 unit 0.45 μm (Millipore), and dissolved in TE solution. It was set as the solution. On the other hand, pCF704 was digested with the restriction enzyme BamHI and dephosphorylated with alkaline phosphatase. A DNA library was prepared by ligation reaction between this and the insert DNA solution using Ligation Kit version 2 (TaKaRa).
[0014]
This DNA library was added to the electrocell of the 18-9 mutant strain, and an electric pulse was applied. The cell was placed in a Falcon tube, 1 ml of ice-cooled separation medium was added, and the mixture was cultured with shaking at 32 ° C. for 2 hours. 100 μl of the whole culture was applied to an 8 cm petri dish-separated agar medium containing 20 μg / ml kanamycin sulfate and 100 μg / ml indole, and cultured at 32 ° C. for 3 weeks. Among the colonies that had grown, the colonies showing red color were spread on an 8 cm petri dish-separated agar medium containing 20 μg / ml of kanamycin and 100 μg / ml of indole, and cultured at 32 ° C. for 5 days. Grow the cells that have grown with a cork borer and extract with methanol, HPLC analysis (mobile phase: 50% CH _Three CN, flow rate: 0.5 mL / min, detection wavelength: 280 nm, column temperature: 35 ° C., column: YMC-Pack ODS A-312 S-5 120A, 6 × 150 mm), 7-hydroxyindole was detected, and the insert DNA was detected It was confirmed that the 7-position hydroxylation activity mutation of indole was complemented. A plasmid was prepared from this complementary strain using a QIAGEN Plasmid Midi Kit, E. coli JM109 was further transformed with this plasmid, and the re-prepared plasmid was designated pIZ311. An insert DNA of about 9.5 Kbp was inserted into pIZ311. Xanthomonas maltophilia H108 transformed with pIZ311 is deposited as Xanthomonas maltilia H108 (pIZ311) at the Biotechnology Institute of Industrial Science and Technology (Accession No. FERM P-17421).
[0015]
E. coli JM109 transformed with pIZ311 was inoculated into L medium (Polypepton 1.0%, Yeast extract 0.5%, NaCl 0.5%, Glucose 0.1%, pH 7.2) containing 20 μg / ml of kanamycin sulfate, and then inoculated at 32 ° C. The culture was shaken overnight. When this culture supernatant was analyzed by HPLC, 7-hydroxyindole was detected at about 20 μg / ml. The gene related to the 7-position hydroxylation of indole derived from the introduced Xanthomonas maltophilia Mer-H108 strain is expressed in E. coli JM109 and maintained in its activity, and is generated by the tryptophan synthesis or degradation pathway of E. coli. It is considered that the indole is hydroxylated at the 7-position. This indicates that the gene related to hydroxylation at the 7-position of indole exists on the cloned pIZ311.
[0016]
5) Determination of gene base sequence of pIZ311 insert region
The base sequence of the pIZ311 insert region was determined by the primer walking method using ABIPRISM 377 XL DNA Sequencer (Perkin Elmer) (SEQ ID NO: 1). Furthermore, the method of Bibb et al. (Gene, 30 157 (1984)), as a result of examining the open reading frame (ORF), IoxA (SEQ ID NO: 1, 2602-2796), IoxB (SEQ ID NO: 1, 2825-3829), IoxC (SEQ ID NO: 1) , 3847-4116), IoxD (SEQ ID NO: 1, 4145-5704), IoxE (SEQ ID NO: 1, 5740-6099), IoxF (SEQ ID NO: 1, 6126-7181), IoxG (SEQ ID NO: 1) ORF and a part of IoxI (SEQ ID NO: 1, 7190-7528), IoxH (SEQ ID NO: 1, 7258-8496), IoxR (SEQ ID NO: 1, 2287-580 (reverse)) 1) Nos. 8544-9320) were found. Amino acid sequences considered to be encoded by these base sequences are also shown. Since IoxR is encoded in the reverse direction, it is shown in SEQ ID NO: 2 from SacI site 2730 bases. In SEQ ID NO: 1, Shine-Dalgarno (Shine-) is included in 2591-2596, 2815-2820, 3837-3849, 4134-4139, 5739-5740, 6114-6118, 7182-7185, 7551-7555, 8553-8536. Dalgarno) The sequence that seems to be the sequence exists.
[0017]
Table 1 shows the results of FASTA search for the amino acid sequences considered to be encoded by the above IoxA, IoxB, IoxC, IoxD, IoxE, IoxF, IoxG, IoxH, IoxR and IoxI.
[0018]
[Table 1]

[0019]
As is apparent from Table 1, the pIZ311 insert region has a high homology with various phenol hydroxylases (Phenolhydroxylase) throughout, and particularly has a high homology with Pseudomonas sp. Strain CF600 phenol hydroxylase (Phenolhydroxylase).
[0020]
From these results, it was considered that ORFs involved in indole-7-hydroxylase activity were IoxA, B, C, D, E, F, G, and IoxR. As with various phenol hydroxylases, a consensus sequence TGGC-N8-TGCA of σ54-dependent -24 / -12 type promoter is present upstream of IoxA (SEQ ID NO: 1, 2548 to 2563), It is considered that transcription control of the Iox operon is performed by the transcriptional regulatory protein IoxR, the DNA binding protein IHF, and σ54.
[0021]
6) Examination of the gene (ORF) region necessary for 7-position hydroxylation of indole using various deletion mutants
(i) Creation of Iox deletion mutant
First, specific sense primers (Sense primer) of SEQ ID NOs: 3 to 4 and antisense primers (SEQ ID NO: 5 to 7) (each 28 mer) were prepared along various ORFs (underlined portions are restriction enzymes). XbaI recognition sequence.)
[0022]
ioxRRF: CT TCTAGA GGCATTCGGGTGAAGGGCATGC (SEQ ID NO: 3),
ioxAF: TT TCTAGA CTTGTCTCCCTCCTCGCGTGCC (SEQ ID NO: 4),
comp. R: GA TCTAGA TCCAGTGGCCCCATTTGCCG (SEQ ID NO: 5)
ioxGR: GA TCTAGA TTAGCTCTGTTTGGTACAGC (SEQ ID NO: 6),
ioxFR: GT TCTAGA CTAGATGCGCTTGAACAGCG (SEQ ID NO: 7).
[0023]
PCR reaction using the above primers and TaKaRa LA Taq (TaKaRa) and pIZ311 as a template (reaction conditions: 96 ° C. for 3 minutes, then 98 ° C. for 20 seconds to 60 ° C. for 20 seconds to 68 ° C. for 10 minutes for 25 cycles) ). It was confirmed that the PCR product was formed by agarose gel electrophoresis, and the reaction solution was purified using a QIAquick PCR Purification Kit (QIAquick PCR Purification Kit, manufactured by QIAGE). The purified product was ligated to Escherichia coli vector pT7-Blue (manufactured by Novagen) using DNA Ligation Kit Version 2 (manufactured by TaKaRa), transformed into Escherichia coli JM109 strain (manufactured by TaKaRa), and 50 μg of ampicillin. / LB was added to LB agar medium (polyheptone 1.0%, yeast extract 0.5%, NaCl 0.5%, glucose 0.1%, agar 1.5%). The next morning, it was confirmed that colonies were formed, and the plasmid was extracted after inoculating LB medium (polypeptone 1.0%, yeast extract 0.5%, NaCl 0.5%, glucose 0.1%). After treatment with restriction enzyme XbaI (TaKaRa), agarose gel electrophoresis was performed to confirm the presence or absence of fragments. ioxRRF primer and comp. The plasmid pIOXRComp. Construct plasmid pIOXRF using ioxRRF primer and ioxFR primer, plasmid pIOXRG using ioxRRF primer and ioxGR primer, plasmid pIOXAF using ioxAF primer and ioxFR primer, and plasmid pIOXAG using ioxAF primer and ioxGR primer did.
[0024]
(ii) Culture test of delation mutant
The plasmid pIOXRComp. PIOXR, pIOXRG, pIOXAF, and pIOXAG were used to transform Escherichia coli JM109 strain to obtain respective clones. Each clone was inoculated into LB medium supplemented with 50 μg / ml of ampicillin and cultured overnight at 37 ° C. with shaking. 500 μl of the culture solution was inoculated into LB medium (50 ml / 250 ml Erlenmeyer flask) supplemented with 50 μg / ml ampicillin, and shaking culture was started on a rotary shaker at 37 ° C., and the final concentration reached 8 hours after culturing. Indole and FeSO to 100 μg / ml _Four ・ 7H ₂ O was added respectively. Thereafter, 2 ml of the culture solution was sampled over time, the cells were removed from the culture solution by centrifugation, and the indole and 7-position hydroxylated indole in the supernatant were quantified by HPLC (column: YMC-PackA-312ODS (4.6 × 150 mm), mobile phase: 50% acetonitrile, flow rate: 0.5 ml / min, detection: 280 nm). As a result, the 7-position hydroxylated indole accumulated in each transformant was 11.8 μM for the pIOXAF transformant, 66.6 μm for the pIOXAG transformant, 21.4 μM for the pIOXRF transformant, 59.4 μM for the pIOXRG transformant, and pIOXRComp. The converted strain was 98 μM. From this, it was found that the ORFs required for hydroxylating the 7-position of indole were IoxA, IoxB, IoxC, IoxD, IoxE, and IoxF.
[0025]
From the above results and the results of Table 1, the expected role of the polypeptide group encoded by the nucleotide sequence of SEQ ID NO: 1 is shown in FIG.
[0026]
Next, a method for culturing a microorganism transformed with the gene cloned by the above method or a microorganism having the gene on the chromosome and a method for producing the produced 7-position hydroxylated indole and 7-position hydroxylated tryptophan are described in detail. explain.
The medium composition used in the present invention is a carbon source, nitrogen source, inorganic salt, natural organic nutrient, etc. suitable for the microorganism used to grow well and to express the activity of hydroxylating position 7 of the indole derivative. It consists of. As the carbon source, glucose, fructose, glycerol, sorbitol, alcohols, acetic acid, starch and the like can be used alone or in combination, and the concentration used is not particularly limited, and approximately 1 to 10% is suitable. As the nitrogen source, one or more compounds such as ammonia, urea, ammonium sulfate, ammonium nitrate, and ammonium acetate can be used. As the inorganic salt, salts such as monopotassium phosphate, dipotassium phosphate, magnesium sulfate, manganese sulfate, ferrous sulfate can be used. Furthermore, peptone, meat extract, yeast extract, corn steep liquor, casamino acid and the like are used as organic nutrient sources having an effect of promoting the growth of the used bacteria, and vitamins and nucleic acids can also be contained in a small amount of medium.
[0027]
The indole can be added to hydroxylate the 7-position of indole either from the beginning of the growth of the cells or after the growth. The cells are recovered from the culture and suspended in a suitable aqueous solution. You may add in the made liquid. Indole can be used at various concentrations, but it is desirable to use it at a concentration of about 0.1 g / liter to 1 g / liter, and it may be added all at once or dividedly.
The culture may be aerobic conditions such as aeration and agitation culture and shaking culture, or stationary culture. The pH during the culture is preferably neutral to weakly alkaline, and known pH neutralizing agents such as ammonia, sodium hydroxide, potassium hydroxide, calcium carbonate, hydrochloric acid and the like can be used. The culture temperature can be in the range of 20 to 40 ° C. at a temperature optimal for the growth of the bacteria used. Although the culture time varies depending on the strain used, the method of adding raw materials, the concentration of addition, etc., 7-hydroxyindole and 7-hydroxytryptophan are produced and accumulated in approximately 1 to 4 days.
[0028]
The produced and accumulated 7-hydroxyindole and 7-hydroxytryptophan can be isolated and purified according to a method known per se. That is, it is adsorbed on a hydrophobic carrier or ion-adsorbing resin, eluted with an organic solvent, alkaline water, or acidic water, concentrated, and then recovered by crystallization or the like.
[0029]
【Example】
Hereinafter, the present invention will be described in detail by way of examples.
Example 1:
Yeast extract 0.1%, peptone 0.1%, KH ₂ PO _Four One Xanthomonas maltophila Mer-H108 strain grown on a slope agar medium (adjusted to pH 6.8 before heat sterilization) consisting of 0.1%, glucose 0.1%, and agar 2%, yeast extract 0.1%, peptone 0.1% , KH ₂ PO _Four A 250 ml Erlenmeyer flask containing 50 ml of 0.1% medium (adjusted to pH 6.8 before heat sterilization) was inoculated and cultured on a rotary shaker at 28 ° C. for 19 hours. 15 ml of this seed culture solution is 0.01% indole, 0.1% yeast extract, 0.1% peptone, KH ₂ PO _Four Inoculate a 3 liter jar fermenter containing 1.5 liters of 0.1% culture medium (adjusted to pH 6.8 before heat sterilization) and aeration and agitation culture at 28 ° C. for 21 hours (aeration 0.75 liter / min, agitation 400 rpm) Then, 7-hydroxyindole was produced and accumulated in the culture solution at 25 μg / ml. After removing the cells from the culture solution by centrifugation, they were passed through a Diaion HP-20 column (resin made by Mitsubishi Kasei, 7φ × 12 cm) filled with 1.4 liters of the culture supernatant, and 7-hydroxyindole was added. After adsorption onto the resin and washing with 1 liter of water, 7-hydroxyindole was eluted from the column with 500 ml of methanol. The fraction eluted with 7-hydroxyindole was collected from the eluate and concentrated under reduced pressure with an evaporator to distill off water and methanol to obtain 23 mg of 7-hydroxyindole as crystals. The 7-hydroxyindole thus obtained was subjected to FAB-MS and ¹ 1 H-NMR was measured.
[0030]
FAB-MS (matrix; glycerin): m / z 133 (M ⁺ ),
¹ H-NMR (400 MHz, CD _Three OD): δ 6.39 (1H, d, J = 3.3Hz), 6.49 (1H, d, J = 7.7Hz), 6.79 (1H, dd, J = 7.7Hz, J = 7.7Hz), 7.03 (1H, d , J = 7.7Hz), 7.15 (1H, d, J = 3.3Hz).
[0031]
Example 2:
Yeast extract 0.1%, peptone 0.1%, KH ₂ PO _Four One Xanthomonas maltophila Mer-H108 strain grown on a slope agar medium (adjusted to pH 6.8 before heat sterilization) consisting of 0.1%, glucose 0.1%, and agar 2%, yeast extract 0.1%, peptone 0.1% , KH ₂ PO _Four A 250 ml Erlenmeyer flask containing 50 ml of a 0.1% medium (adjusted to pH 6.8 before heat sterilization) was inoculated and cultured on a rotary shaker at 28 ° C. for 19 hours. 150 ml of this seed culture solution is 0.01% indole, 0.1% yeast extract, 0.1% peptone, KH ₂ PO _Four 0.1%, 0.01% FeSO _Four ・ 7H ₂ A 30-liter jar fermenter containing 15 liters of a medium consisting of O (adjusted to pH 6.8 before heat sterilization) was inoculated, and aerated and agitated culture (aerated volume 7.5 liters / minute, agitated 200 rpm) was performed at 28 ° C. At 15 hours of culture, 7.5 g of L-serine was added, and the culture was continued for 26 hours. 7-hydroxytryptophan was produced and accumulated in the culture solution at 9 μg / ml. After completion of the culture, the cells were removed from the culture by centrifugation, and 14 liters of the obtained culture supernatant was passed through a strongly basic ion exchange resin Diaion PA306s column (resin is manufactured by Mitsubishi Kasei, 7φ × 15 cm). 7-Hydroxytryptophan was adsorbed on the resin, washed with 1 liter of water, and then 7-hydroxytryptophan was eluted with 2 liters of 0.1N hydrochloric acid. The elution fractions of 7-hydroxytryptophan were combined from the eluate, adjusted to pH 6.0 with 1N NaOH, concentrated under reduced pressure with an evaporator, and lyophilized to obtain 21.3 g of a crude product. This was dissolved in 10 ml of 20 mM phosphate buffer (pH 6.8), preparative HPLC (column: Inertsil PREP-ODS (2 × 25 cm, Gaschrom Industries), mobile phase: 5% MeOH 20 mM phosphate buffer (pH 6.8), The peak of 7-hydroxytryptophan with a retention time of 34.6 minutes was collected by applying a flow rate of 7.0 ml / min and detection of UV 210 nm. The desiccated fraction was desalted to remove the 7-hydroxytryptophan peak by preparative HPLC under the same conditions as above except that the mobile phase was water, and lyophilized to 86 mg of 7-hydroxytryptophan as a sodium salt. Obtained. FAB-MS of 7-hydroxytryptophan thus obtained and ¹ 1 H-NMR was measured.
[0032]
FAB-MS (matrix; NBA): m / z 221 ((M + H) ⁺ ),
¹ H-NMR (400 MHz, CD _Three OD): δ 3.05 (1H, dd, J = 9.5Hz, J = 15.0Hz), 3.45 (1H, dd, J = 3.3Hz, J = 15.0Hz), 3.79 (1H, dd, J = 4.0Hz, J = 9.5Hz), 6.57 (1H, d, J = 7.7Hz), 6.85 (1H, dd, J = 7.7Hz, J = 7.7Hz), 7.13 (1H, s), 7.19 (1H, d, J = 7.7 Hz).
[0033]
Example 3:
E. coli JM109 transformed with pIZ311 was inoculated into L medium (polypeptone 1.0%, yeast extract 0.5%, NaCl 0.5%, glucose 0.1%, pH 7.2) containing 20 μg / ml kanamycin sulfate. The culture was shaken overnight at 32 ° C. After microbial cells were removed from the culture by centrifugation, the culture supernatant was HPLC (column: ODS-A (0.6 × 15 cm), YMC), mobile phase: 50% acetonitrile, flow rate: 0.5 ml / min, detection: A peak of 7-hydroxyindole having a retention time of 10.08 minutes was detected by UV irradiation at 280 nm. About 20 μg / ml of 7-hydroxyindole was detected.
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the expected role of a group of polypeptides encoded by genes according to the invention. In the figure, bold arrows indicate primers, and thin arrows indicate the size and direction of genes.

Claims (12)

A DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a DNA that hybridizes with the DNA under stringent conditions.   A recombinant DNA obtained by incorporating the DNA of claim 1.   A microorganism holding the recombinant DNA according to claim 2. By culturing the microorganism according to the presence of indole in claim 3, 7-hydroxy India Lumpur was allowed to produce and accumulate method of 7-hydroxy Indian Lumpur and collecting it. Method for manufacturing 7-hydroxy Indian Lumpur according to claim 4, wherein the microorganism belonging to the microorganism Gae Ssherihia genus. Retaining the DNA of claim 1, by culturing a microorganism that produces an enzyme that hydroxide 7 position of the indole in the presence of indole, 7-hydroxy India Lumpur allowed production storage, collecting the fact method for manufacturing 7-hydroxy Indian Lumpur, wherein. Method for manufacturing 7-hydroxy Indian Lumpur according to claim 6 wherein the microorganism is a microorganism belonging to the genus Xanthomonas. The method for producing 7-hydroxyindole according to claim 6, wherein the microorganism is Xanthomonas maltophilia Mer-H108 strain (FERM P-17359). 7 -hydroxytryptophan is produced and accumulated by culturing a microorganism that retains the DNA according to claim 1 and produces an enzyme that hydroxylates position 7 of indole in the presence of indole and L-serine ; method for producing hydroxytryptophan - 7 taken that you characterized that. The method for producing 7 -hydroxytryptophan according to claim 9 , wherein the microorganism belongs to the genus Xanthomonas. The method for producing 7-hydroxytryptophan according to claim 9, wherein the microorganism is Xanthomonas maltophilia Mer-H108 strain (FERM P-17359). Xanthomonas maltophilia Mer-H108 strain (FERM P-17359).

Images