JP3694928B2 - DNA fragment having promoter activity derived from basidiomycete Coriolus genus - Google Patents

Description

を³²Ｐで放射能標識したものである。
【００１９】
この結果、約４０，０００個のプラークの中から４個の陽性クローンを選抜することができた。陽性クローンから常法に従って調製した組換え体ファージＤＮＡを各種制限酵素で消化し、上記２種の合成ＤＮＡを用いてサザンハイブリダイゼーションを行なった。その結果、制限酵素ＥｃｏＲＩ（宝酒造社製）、且つＢａｍＨＩ（宝酒造社製）で消化して得られた断片中に単一のＤＮＡバンドとして３．８ｋｂｐにハイブリダイズするクローンが認められた。
【００２０】
上記ＤＮＡ断片３．８ｋｂｐをアガロースゲル電気泳動法により切り出し、大腸菌ベクターｐＵＣ１８（宝酒造社製）のＥｃｏＲＩ、ＢａｍＨＩサイトにサブクローン化し、大腸菌ＪＭ１０９株（宝酒造社製）へ形質転換した。サブクローン化したＤＮＡを大量に調製し、超遠心操作（５０，０００ｒｐｍ，１６ｈｒｓ，１５℃）で精製し、塩基配列を決定した。塩基配列の決定はシーケナーゼキット（Ｕｎｉｔｅｄ Ｓｔａｔｅｓ Ｂｉｏｃｈｅｍｉｃａｌ社製）を用いて行った。
【００２１】
翻訳開始点から下流に８５０番目までの塩基配列を配列番号２に示す。その結果、上記塩基配列の範囲内においてアラゲカワラタケ由来ＧＰＤ遺伝子は６つのイントロンにより分断されていた。イントロンを除いた塩基配列から推定されるアミノ酸配列を、配列番号３に示す。このアミノ酸配列は、これまで報告されているＧＰＤ遺伝子と高い類似性が認められた。
【００２２】
なお、得られたアラゲカワラタケ由来ＧＰＤ遺伝子を含む大腸菌形質転換株Ｅ．ｃｏｌｉ ＪＭ１０９／ｐＣＨＧＰＤは工業技術院生命工学工業技術研究所にＦＥＲＭ Ｐ−１５０１５として寄託されている。
実施例３．ＧＰＤ遺伝子制御領域に支配されるＯＣＴ構造遺伝子の連結
担子菌アラゲカワラタケ用のＯＣＴ発現選択マーカー用プラスミドｐＧＰＲＧはＧＰＤ遺伝子のプロモーター領域の下流にＯＣＴ染色体遺伝子（特開平６−０５４６９１；ＦＥＲＭ Ｐ−１２６７９）の構造遺伝子領域を連結し、本来のＯＣＴ遺伝子からプロモーター領域を置換した選択マーカー遺伝子とした。
【００２３】
具体的にはＧＰＤ染色体遺伝子ＥｃｏＲＩ且つＢａｍＨＩ消化により得られる３．８ｋｂｐのＤＮＡ断片をファージベクターＭ１３ｍｐ１８のＥｃｏＲＩ、ＢａｍＨＩサイトにＴ４ＤＮＡリガーゼを用いて連結し、これを大腸菌ＪＭ１０９株に形質転換し、一本鎖ファージＤＮＡを調製した。次に、図２断片１に示すＤＮＡプライマーをフォスフォアミダイド法により合成し、上記一本鎖ファージＤＮＡにアニーリングを行い、プライマー伸長法によりＧＰＤ遺伝子のプロモーター領域だけを合成し、制限酵素ＥｃｏＲＩ（宝酒造社製）で切断することにより０．９ｋｂｐのＤＮＡ断片を調製した（断片１）。この塩基配列は配列番号１である。
【００２４】
一方、ＯＣＴの成熟型酵素をコードしている遺伝子領域を取り出すため、プラスミドｐＵＣＲ１（特開平６−０５４６９１号公報；ＦＥＲＭ Ｐ−１２６７７）を制限酵素ＮｃｏＩ（宝酒造社製）で切断し、次にＭｕｎｇ ｂｅａｎ ｎｕｃｌｅａｓｅ（宝酒造社製）により突出部分を削り込み平滑末端とし、約２．５ｋｂｐのＤＮＡ断片を得た（断片２）。
【００２５】
大腸菌ベクターｐＵＣ１８を制限酵素ＥｃｏＲＩとＳｍａＩ（宝酒造社製）で切断し、上記２種類のＤＮＡ断片を混合して、Ｔ４ＤＮＡリガーゼで連結した後、大腸菌ＪＭ１０９株の形質転換を行い、アンピシリン耐性の形質転換株の中から上記断片１及び断片２の２種類のＤＮＡ断片が同時に挿入されているプラスミドを単離し、これをｐＧＰＲＧと命名した（図２）。
実施例４．アラゲカワラタケ（Ｃｏｒｉｏｌｕｓ ｈｉｒｓｕｔｕｓ）形質転換法
ａ．一核菌糸培養
直径６mm前後のガラスビーズを約３０個入れた５００ｍｌ容の三角フラスコにＳＭＹ培地（シュークロース１％、麦芽エキス１％、酵母エキス０．４％）１００ｍｌを分注して滅菌後、アラゲカワラタケ（Ｃｏｒｉｏｌｕｓ ｈｉｒｓｕｔｕｓ）ＯＪＩ−１０７８の平板寒天培地から直径５mmの寒天片をコルクボーラーで打ち抜きＳＭＹ培地に植菌し、２８℃で７日間静置培養した（前培養）。
【００２６】
ただし、菌糸を細分化するために、１日に１〜２回振り混ぜた。次に、１Ｌ容の三角フラスコにＳＭＹ培地２００ｍｌを分注し、さらに回転子を入れ、滅菌後、前培養菌糸をナイロンメッシュ（孔径３０μｍ）で濾集し、全量を植菌し、２８℃で培養した。なお、スターラーで１日２時間撹拌することにより菌糸を細分化した。この培養を４日間行なった。
【００２７】
ｂ．プロトプラストの調製
上記液体培養菌糸をナイロンメッシュ（孔径３０μｍ）で濾集し、浸透圧調節溶液（０．５Ｍ ＭｇＳＯ₄、５０ｍＭマレイン酸バッファー（ｐＨ５．６））で洗浄した。次に湿菌体１００ｍｇあたり１ｍｌの細胞壁分解酵素液に懸濁し、緩やかに振盪しながら２８℃で４時間インキュベートしてプロトプラストを遊離させた。細胞壁溶解酵素として、次の市販酵素製剤を組み合わせて使用した。即ち、セルラーゼ・オノズカ（ｃｅｌｌｕｌａｓｅ ＯＮＯＺＵＫＡ ＲＳ）（ヤクルト社製）５ｍｇ、ノボザイム（Ｎｏｖｏｚｙｍｅ２３４）（ノボ社製）１０ｍｇを上記浸透圧調節溶液１ｍｌに溶解して酵素液として用いた。
【００２８】
ｃ．プロトプラストの精製
上記酵素反応液からナイロンメッシュ（孔径３０μｍ）で菌糸断片を除いた後、プロトプラストの回収率を高めるため、ナイロンメッシュ上に残存する菌糸断片とプロトプラストを上記浸透圧調節溶液で１回洗浄した。得られたプロトプラスト懸濁液を遠心分離（１，０００ｘｇ、５分間）し、上清を除去し、４ｍｌの１Ｍシュークロース（２０ｍＭ ＭＯＰＳ緩衝液、ｐＨ６．３）で再懸濁後、遠心操作を繰り返し、上記１Ｍシュークロース溶液で２回洗浄した。沈澱物に１Ｍソルビトール溶液（２０ｍＭ ＭＥＳ、ｐＨ６．４）に４０ｍＭ塩化カルシウムを加えた溶液５００μｌに懸濁し、プロトプラスト溶液とした。この溶液を４℃で保存した。
【００２９】
プロトプラスト濃度は血球計算盤を用いて直接検鏡により求めた。全ての遠心操作はスウィングローターで１，０００ｘｇ、５分間、室温下で行った。
ｄ．形質転換
１０⁶個／１００μｌ濃度のプロトプラスト溶液１００μｌに対し、実施例３で作製したプラスミドｐＧＰＲＧ２μｇを添加し、３０分間氷冷した。つぎに、液量に対し等量のＰＥＧ溶液（５０％ ＰＥＧ３４００、２０ｍＭ ＭＯＰＳ（ｐＨ６．４））を加え、３０分間氷冷した。つぎに、０．５Ｍシュークロースおよびロイシンを含む最少軟寒天培地（寒天１％）に混合してプレートに撒いた。上記プレートを２８℃で４日間培養を行ない、形質転換体を得た。この時における形質転換効率は３００コロニー／μｇ形質転換ＤＮＡであった。なお、プラスミドベクターｐＵＣ１８だけのＤＮＡ供与体を用いた対照実験では形質転換体は全く得られなかった。
実施例５．リグニンパーオキシダーゼ発現プラスミドの構築
上記実施例３で作製したＤＮＡ断片１（担子菌アラゲカワラタケ用のＧＰＤ遺伝子のプロモーター）の下流にリグニンパーオキシダーゼ染色体遺伝子（特開平５−２６０９７８号；ｐＢＳＬＰＯＧ７；ＦＥＲＭ Ｐ−１２６８３）の構造遺伝子領域を連結し、発現プラスミドとした。
【００３０】
具体的にはリグニンパーオキシダーゼの構造遺伝子部分を取り出しはプラスミドｐＢＳＬＰＯＧ７を基礎として図３の断片３に示すプライマーを用いて実施例３と同様にプライマー伸長法によりＤＮＡ断片を作製し、制限酵素ＨｉｎｄＩＩＩで切断することによりリグニンパーオキシダーゼ構造遺伝子部分からなる１．７ｋｂｐのＤＮＡ断片を得た（断片３）。
【００３１】
さらに大腸菌ベクターｐＵＣ１８を制限酵素ＥｃｏＲＩとＨｉｎｄＩＩＩで切断し、実施例３記載のＤＮＡ断片（断片１）と上記ＤＮＡ断片（断片３）の２種類のＤＮＡ断片を混合し、Ｔ４ＤＮＡリガーゼで連結した後、大腸菌ＪＭ１０９株の形質転換を行い、アンピシリン耐性の形質転換株の中から断片１及び断片３の２種類のＤＮＡ断片が同時に挿入されているプラスミドを単離し、これをｐＧＰＨＬＧと命名した（図３）。
実施例６．リグニンパーオキシダーゼを高分泌生産するアラゲカワラタケ形質転換体の作製
実施例５で得たｐＧＰＨＬＧを用いてアルギニン要求性アラゲカワラタケ（ＯＪＩ−１０７８）を形質転換する場合、選択マーカーとしてアラゲカワラタケ由来のＯＣＴ遺伝子保持するプラスミド（ｐＵＣＲ１）を同時に導入すること（ＰＥＧ法、もしくはエレクトロポーレーション法など）により形質転換体ｐＧＰＨＬＧ／ＯＪＩ−１０７８を得た。ここで、形質転換に供与することができるＤＮＡは環状、もしくは直鎖状にかかわらず、目的とする形質転換体を得ることができた。以下に形質転換条件を記す。
【００３２】
約１０⁶個／１００μｌ濃度のプロトプラスト溶液を１００μｌに対し上記実施例５で作製したプラスミド２μｇを環状、もしくは直鎖状で添加し、さらに選択マーカーとしてｐＵＣＲ１を０．２μｇ添加し、３０分間氷冷した。
次に、等量のＰＥＧ溶液（５０％ ＰＥＧ３４００、２０ｍＭ ＭＯＰＳ（ｐＨ６．４））を加え、３０分間氷冷した。０．５Ｍシュークロースおよびロイシンを含む最少軟寒天培地（寒天１％）に混合してプレートに撒いた。上記プレートを２８℃で４日間培養を行ない、形質転換体を得た。さらに上記形質転換株からＤＮＡを調製し、目的とするリグニンパーオキシダーゼ発現プラスミドが組み込まれていることをサザンハイブリダイゼーションにより確認した。
実施例７．形質転換株の培養およびリグニンパーオキシダーゼの活性測定
上記実施例６で得られた形質転換株（ｐＧＰＨＬＧ／ＯＪＩ−１０７８）を５００ｍｌ容の三角フラスコに５０ｍｌのグルコースペプトン液体培地（グルコース２０ｇ／ｌ、ペプトン５ｇ／ｌ、酵母エキス２ｇ／ｌ、ＫＨ₂ＰＯ₄１ｇ／ｌ、ＭｇＳＯ₄・７Ｈ₂Ｏ０．５ｇ／ｌ、りん酸でｐＨ４．５に調製）に５ｍｍの寒天片を５個接種し、２８℃で６日間、振盪下で培養した（前前培養）。その後、菌体をワーリングブレンダーで破砕し、さらに２日間グルコースペプトン培地で２８℃で振盪培養を継続した（前培養）。次に、菌体を集菌し、菌体を新鮮なグルコースペプトン液体培地５０ｍｌに再懸濁し、新しい５００ｍｌ容三角フラスコへ移し、２８℃で静置培養を実施した。得られる培養液を遠心分離し、その上清を得た。
【００３３】
活性測定法は８ｍＭベラトリルアルコール２５μｌ、０．５Ｍ酒石酸ナトリウム緩衝液（ｐＨ３．０）５０μｌ、酵素液４００μｌ、２．７ｍＭ過酸化水素２５μｌを充分に混合し、反応の結果生じるベラトルアルデヒドの３１０ｎｍの吸光度を時間を追って記録することにより行い、上記培養上清中にベラトリルアルコールをベラトルアルデヒドへ変換する酵素活性が静置培養開始５日目で３００〜５００ユニット／ｍｌの範囲で認められた。ここで酵素活性単位は１分間にベラトルアルデヒド１μｍｏｌ増加させる活性を１ユニットとした。一方、同条件下で培養した供与ＤＮＡを含まないＯＪＩ−１０７８株の培養上清には本活性は認められなかった。
実施例．８ マンガンパーオキシダーゼ発現プラスミドの構築
担子菌アラゲカワラタケ用のＧＰＤ遺伝子のプロモーターの支配下にさらされたマンガンパーオキシダーゼ発現用プラスミドｐＧＰＭＰＧは実施例３で作製したＤＮＡ断片１の下流にマンガンパーオキシダーゼ染色体遺伝子（特開平７−１２３５４０；ｐＢＳＭＰＯＧ１；微工研菌寄第１４９３３号）の構造遺伝子を連結し、発現プラスミドとした。
【００３４】
具体的にはマンガンパーオキシダーゼの構造遺伝子部分の取り出しはプラスミドｐＢＳＭＰＧ１を基礎として図４の断片４に示すプライマーを用いて実施例３と同様にプライマー伸長法によりＤＮＡ断片を作製し、制限酵素ＨｉｎｄＩＩＩで切断することによりマンガンパーオキシダーゼ構造遺伝子部分からなる２．８ｋｂｐのＤＮＡ断片を得た（断片４）。
【００３５】
さらに大腸菌ベクターｐＵＣ１８を制限酵素ＥｃｏＲＩとＨｉｎｄＩＩＩで切断し、上記２種類のＤＮＡ断片を混合し、Ｔ４ＤＮＡリガーゼで連結した後、大腸菌ＪＭ１０９株の形質転換を行い、アンピシリン耐性の形質転換株の中から２種類のＤＮＡ断片が同時に挿入されているプラスミドを単離し、これをｐＧＰＭＰＧと命名した（図４）。
【００３６】
実施例９．マンガンパーオキシダーゼを高分泌生産するアラゲカワラタケ形質転換体の作製
実施例８で得たｐＧＰＭＰＧを用いてアルギニン要求性アラゲカワラタケ（ＯＪＩ−１０７８）を形質転換する場合、選択マーカーとしてアラゲカワラタケ由来のＯＣＴ遺伝子を保持するプラスミド（ｐＵＣＲ１）を同時に導入すること（ＰＥＧ法、もしくはエレクトロポーレーション法など）により形質転換体ｐＧＰＭＰＧ／ＯＪＩ−１０７８を得た。ここで、形質転換に供与することができるＤＮＡは環状、もしくは直鎖状にかかわらず、目的とする形質転換体を得ることができた。以下に形質転換条件を記す。
【００３７】
約１０⁶ 個／１００μｌ濃度のプロトプラスト溶液を１００μｌに対し実施例５で作製したプラスミド２μｇを環状、もしくは直鎖状で添加し、さらに選択マーカーとしてｐＵＣＲ１を０．２μｇ添加し、３０分間氷冷した。
次に、等量のＰＥＧ溶液（５０％ ＰＥＧ３４００、２０ｍＭ ＭＯＰＳ（ｐＨ６．４））を加え、３０分間氷冷した。０．５Ｍシュークロースおよびロイシンを含む最小軟寒天培地（寒天１％）に混合してプレートに撒いた。上記プレートを２８℃で４日間培養を行い、形質転換体を得た。さらに上記形質転換株からＤＮＡを調製し、目的とするリグニンパーオキシダーゼ発現プラスミドが組み込まれていることをサザンハイブリダイゼーションにより確認した。
実施例１０．形質転換株の培養およびマンガンパーオキシダーゼの活性測定
上記実施例９で得られた形質転換株（ｐＧＰＭＰＧ／ＯＪＩ−１０７８）を５００ｍｌ容の三角フラスコに５０ｍｌのグルコース・ペプトン液体培地（グルコース２０ｇ／ｌ、ペプトン５ｇ／ｌ、酵母エキス２ｇ／ｌ、ＫＨ₂ ＰＯ₄ １ｇ／ｌ、ＭｇＳＯ₄ ・７Ｈ₂ Ｏ０．５ｇ／ｌ、りん酸でｐＨ５．０に調製）に５０ｍｍ² の寒天片を５個接種し、２８℃で６日間、振盪下で培養した（前前培養）。その後、菌体をワーリングブレンダーで破砕し、菌体液１０ｍｌを新鮮なグルコースペプトン培地１０ｍｌを含む直径９ｃｍのシャーレへ植菌し、２８℃で１０日間静置培養を行い菌体マットを形成させた。次に、菌体を集菌し、ワーリングブレンダーにより菌体を破砕洗浄後、新鮮な改変グルコースペプトン液体培地（グルコース２０ｇ／ｌ、ペプトン５ｇ／ｌ、酵母エキス２ｇ／ｌ、ＫＨ₂ ＰＯ₄ １ｇ／ｌ、ＭｇＳＯ₄ ・７Ｈ₂ Ｏ０．５ｇ／ｌ、０．２ｍＭ ＭｎＳＯ₄ りん酸でｐＨ５．０に調製）２０ｍｌに再懸濁し、新しい２００ｍｌ容三角フラスコへ移し、２８℃で静置培養を実施した。得られる培養液を遠心分離し、その上清を得た。
【００３８】
活性測定法は０．５Ｍマロン酸ナトリウム緩衝液（ｐＨ５．５）５０μｌ、酵素液４４５μｌ、１０ｍＭ過酸化水素５μｌ、１ｍＭ ＭｎＳＯ₄ １００μｌを充分に混合し、反応の結果生じるＭｎ（ＩＩＩ）マロン酸複合体の２７０ｎｍの吸光度増加を時間を追って記録することにより行い、上記培養上清中にＭｎ（ＩＩＩ）マロン酸複合体を生成する酵素活性が静置培養開始７日目で５μｍｏｌ／ｍｌ／ｍｉｎで認められた。ここで酵素活性単位は１分間にＭｎ（ＩＩＩ）マロン酸複合体１μｍｏｌ増加させる活性を１ユニットとした。一方、同条件下で培養した供与ＤＮＡを含まないＯＪＩ−１０７８株の培養上清には本活性は認められなかった。
【００３９】
【発明の効果】
本発明は、これまで遺伝子組換え技術による酵素の大量生産が困難とされていたリグニンパーオキシダーゼ又はマンガンパーオキシダーゼを、アラゲカワラタケの形質転換系において活性のある形で、さらに高生産させるための新規なＤＮＡ断片を提供するものである。
【００４０】
【配列表】
配列番号：１
配列の長さ：９２２
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：genomic DNA
起源
生物名：アラゲカワラタケ（Ｃｏｒｉｏｌｕｓ ｈｉｒｓｕｔｕｓ）
株名：ＩＦＯ４９１７
配列の特徴
860-866 TFIID site
配列：

【００４１】
配列番号：２
配列の長さ：８５０
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：genomic DNA
起源
生物名：アラゲカワラタケ（Ｃｏｒｉｏｌｕｓ ｈｉｒｓｕｔｕｓ）
株名：ＩＦＯ４９１７
配列の特徴
1-850 P CDS
7-63 intron
87-153 intron
159-216 intron
287-350 intron
567-630 intron
749-813 intron
配列：

【００４２】
配列番号：３
配列の長さ：１６０
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：ペプチド
起源
生物名：アラゲカワラタケ（Ｃｏｒｉｏｌｕｓ ｈｉｒｓｕｔｕｓ）
株名：ＩＦＯ４９１７
配列の特徴
1-160 P mat peptide
配列：

【図面の簡単な説明】
【図１】図１はアラゲカワラタケのグリセルアルデヒド−３−りん酸脱水素酵素遺伝子の染色体上の制限酵素地図である。
【図２】図２は実施例３に示したアルギニン要求性アラゲカワラタケ変異株へのために使用されるプラスミドｐＧＰＲＧの構築図である。
【図３】図３は実施例５に示したリグニンパーオキシダーゼ発現プラスミドｐＧＰＨＬＧの構築図である。
【図４】図４は実施例８に示したマンガンパーオキシダーゼ発現プラスミドｐＧＰＭＰＧの構築図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel DNA fragment having promoter activity obtained from a basidiomycete, Arakawakawatake, a recombinant DNA containing the same, a transformant containing the recombinant DNA, and use thereof.
[0002]
[Prior art]
To date, a system for producing various polypeptides using recombinant DNA technology has been described in E. coli (E.coli). However, E. coli (E.coli) Is often not suitable as a host. For example, E. coli (E.coli), When a useful polypeptide derived from a higher organism such as human is produced, it is often not produced as an active enzyme protein. In addition to the target polypeptide, many toxic substances are produced, which makes it difficult to purify the target product. As a means for solving the above problems, production methods using lower eukaryotic yeast as a host have been actively studied. In this case, however, there is a problem that the productivity of the target polypeptide is low. Therefore, for the production of polypeptides, Aspergillus (Aspergillus) Genus and other fungi, and funerocaate (Phanerochaete) Transformation systems using basidiomycetes such as the genus and Coriolus have been developed, and enzyme protein production has been studied.
[0003]
Basidiomycetes belong to eukaryotes, but are considered more closely related to animal cells than yeast [T. L. Smith, Proc. Natl. Acad. Sci. USA,867063 (1989)]. Alagekawaratake, which has strong lignin-degrading ability, is a basidiomycete belonging to the genus Coriolus. The host and vector system has been developed by the present inventors and has been used to produce lignin peroxidase, which has been difficult until now, using recombinant DNA technology. Succeeded [see JP-A-6-054691]. However, since the promoter region used is the promoter region of the ornithine carbamoyltransferase gene, which is an amino acid synthesis enzyme gene, and the phenol oxidase gene involved in lignin degradation, it is low in productivity or is a secondary metabolite until the gene is expressed. The production amount was similar to that obtained when the wild strain IFO4917 was produced in a lignin peroxidase production medium (low carbon / low nitrogen source). Moreover, when lignin peroxidase production was attempted in a transformation system using Escherichia coli or yeast, it was not produced in an active state [R. L. Farrell, European Patent Office No. 0, 261, 080, (1989)]. In addition, the fungus Trichoderma reesei (Tricoderma  reesei) Using the host / vector system of Ferrebia radiata (Phlebia  radiata) -Derived lignin peroxidase gene was ligated under the promoter of cellobiohydrase gene, and enzyme production was attempted, but active lignin peroxidase was not produced [M. Saloheimo, et al. , Gene,85343 (1989)]. Furthermore, it has been reported that promoters used in enzyme protein production systems by genetic recombination of other species such as filamentous fungi do not function in basidiomycetes [A. Lorna, et al. Curr. Genet. ,16, 35 (1989)]. In addition, the ArgB gene derived from Aspergillus nidulans did not function in Arakawaaratake [A. Tsukamoto, et al. , US Patent Appln. S. N. 08 / 027,986 (1993)]. Then, although interest is increasing about the promoter which has more powerful transcriptional activity which functions in a basidiomycete Aragekawaratake, there is no report that such a promoter was acquired until now.
[0004]
[Problems to be solved by the invention]
An object of the present invention is to provide a promoter for mass production of a target protein using a basidiomycete, Arakawakawatake host / vector system, a protein gene system using the promoter, and mass expression of a protein using the gene system It is to provide a production method.
[0005]
[Means for Solving the Problems]
The present inventors paid attention to the glyceraldehyde-3-phosphate dehydrogenase (hereinafter abbreviated as GPD) gene of Pseudomonas aeruginosa, which is constitutively expressed, and DNA encoding GPD from the chromosomal DNA restriction enzyme fragment of Pseudomonas aeruginosa A signal peptide of a high-temperature-inducible lignin peroxidase gene [JP-A-5-260978] or a manganese peroxidase gene [JP-A-7-123540] cloned from an Arakawa kawatake, downstream of the promoter region of this gene. And obtained a lignin peroxidase high-producing strain and a manganese peroxidase high-producing strain by transferring to an ornithine carbamoyltransferase (hereinafter abbreviated as OCT) -deficient Alagekawa aratake mutant. Departure It came to complete Ming.
[0006]
That is, the present invention relates to a coriolus genus Arakawaratake that controls expression of a target gene (Coriolus  hirsutusA DNA fragment comprising the promoter DNA sequence derived from Examples of the target gene include ornithine carbamoyltransferase gene, lignin peroxidase gene, manganese peroxidase gene and the like. Examples of the promoter DNA sequence include substantially the base sequence of SEQ ID NO: 1. Here, “substantially the base sequence of SEQ ID NO: 1” is the same as the base sequence of SEQ ID NO: 1, although the base sequence of SEQ ID NO: 1 and a part of the base sequence are substituted, inserted or deleted It is meant to indicate all of the base sequences having the following actions / effects.
[0007]
Furthermore, the present invention is a recombinant vector comprising the above DNA fragment and a target gene.
Examples of the target gene include ornithine carbamoyltransferase gene, lignin peroxidase gene, manganese peroxidase and the like. However, the target gene is not limited to the above three types, and any target gene can be used.
[0008]
Furthermore, the present invention is a basidiomycete Aragekawaratake transformant transformed with the above recombinant vector.
Furthermore, the present invention is a method for producing lignin peroxidase or manganese peroxidase, comprising culturing the above-mentioned Coriolas basidiomycetous transformant and collecting lignin peroxidase or manganese peroxidase from the culture.
[0009]
Hereinafter, the present invention will be described in detail.
The donor of chromosomal DNA used in the present invention is Aragekawaratake.Coriorus hirsutusIFO4917 strain.
In order to prepare chromosomal DNA from this strain, the method of Yelton et al. [Proc. Natl. Acad. Sci. USA,81, 1470 (1984)], etc., a normal chromosomal DNA extraction method is used.
[0010]
Next, the chromosomal DNA obtained isSauAfter treatment with an appropriate restriction enzyme such as 3AI and partial decomposition, fractionation is performed by sucrose density gradient ultracentrifugation to obtain a DNA fragment of 10 kbp to 25 kbp. A chromosomal gene library is prepared by inserting the DNA fragment obtained above into phage DNA treated with a restriction enzyme that produces the same cohesive ends. Examples of the phage DNA include EMBL3 [AM, Frischauf, et al. , J .; Mol. Biol.170, 827 (1983)]. After insertion, packaging is performed in vitro to obtain a chromosomal DNA library. For subcloning, a conventional cloning vector, preferably an E. coli cloning vector, such as a pUC vector, such as pUC18 [C. Yanish-Perron, et al. , Gene,33, 103 (1985)].
[0011]
In isolation of the GPD gene from the chromosomal gene library obtained above, plaque hybridization was performed using a synthetic DNA prepared based on the DNA sequence of the GPD gene isolated from other species. Do. The isolated fragment containing the GPD chromosomal gene consisted of the restriction enzyme map shown in FIG.
[0012]
The fragment containing the GPD chromosomal gene was inserted into an appropriate cloning vector, and the nucleotide sequence was determined by the method of Sanger et al. [Proc. Natl. Acad. Sci. USA,74, 5463 (1977)]. As the cloning vector, a bacterial cloning vector, particularly a pUC cloning vector such as an E. coli cloning vector, for example, pUC18 is used.
[0013]
Next, as described in detail in Example 3 below, the structural gene portion of the OCT gene is ligated as a reporter gene, and the necessary region having the promoter activity of the GPD gene is determined by transferring it to the OCT-deficient Arakawa aratake mutant. be able to.
As the OCT structural gene, for example, those described in JP-A-6-054691 are used. In addition, JM109 / pUCR1, which is Escherichia coli transformed with the plasmid pUCR1 containing this structural gene, is deposited as FERM P-12679 at the Institute of Biotechnology, Industrial Technology Institute.
[0014]
Further, the OCT cDNA gene of Aragekawaratake is described in JP-A-6-054691. Escherichia coli JM109 / pUCRM transformed with the plasmid pUCRM containing this structural gene is deposited as FERM P-13424 at the Institute of Biotechnology, Industrial Technology Institute.
Next, as described in detail in Example 5 below, a lignin peroxidase structural gene portion or a manganese peroxidase structural gene portion is linked downstream of the GPD promoter region, and a large amount of lignin peroxidase or manganese peroxidase is produced by genetic recombination. Can be produced.
[0015]
In addition, an auxotrophic mutant OJI-1078 lacking OCT activity of Pseudomonas aeruginosa, an E. coli recombinant strain containing a high-temperature-induced lignin peroxidase geneE.coli  XL-1 blue / pBSLPOOG7, an E. coli recombinant strain containing a manganese peroxidase geneE.coli  E. coli recombinant strain containing promoter region of GPD gene derived from JM109 / pBSLPOOG1 and Arakawa KawatakeE.coli  JM109 / pCHGPD has been deposited at the National Institute of Advanced Industrial Science and Technology as FERM P-12777, FERM P-12683, FERM P-14933 and FERM P-15015, respectively.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in more detail with reference to examples. However, the technical scope of the present invention is not limited by these examples.
Example 1. Chromosomal gene library preparation
An agar piece of 5 mm in diameter was punched out from a plate agar culture of Alage Kawaratake (IFO4917 strain) with a cork borer, and glucose peptone medium (glucose 2%, polypeptone 0.5%, yeast extract 0.2%, KH₂PO_Four0.1% MgSO_Four・ 7H₂(O 0.05%, adjusted to pH 4.5 with phosphoric acid) Inoculated into 200 ml, and subjected to rotary shaking culture at 28 ° C. for 7 days. After collecting the cells, the cells were washed with 1 L of sterilized water and frozen with liquid nitrogen.
[0017]
5 g of this frozen cell was pulverized using a mortar. The crushed cells were transferred to a centrifuge tube, 10 ml of Lysis buffer (100 mM Tris (pH 8), 100 mM EDTA, 100 mM NaCl, and proteinase K added to 100 μg / ml) was added, and the mixture was incubated at 55 ° C. for 3 hours. After incubation, phenol extraction and chloroform extraction were performed. Ethanol was gradually added to the extracted aqueous layer portion, and when DNA was precipitated, the chromosomal DNA was wound and suspended in TE solution (10 mM Tris (pH 8), 1 mM EDTA).
[0018]
100 μg of the obtained chromosomal DNA was used as a restriction enzyme.SauPartially digested with 3AI (Takara Shuzo) and fractionated by 5-20% sucrose density gradient ultracentrifugation (30,000 rpm, 18 hours) to collect 10-25 kbp fragment sections. This fragment classification was classified into phage λEMBL3- manufactured by Toyobo Co., Ltd.BamAfter ligation to the HI arm using T4 DNA ligase (Takara Shuzo Co., Ltd.), packaging of the obtained phage DNA using STRATAGENE Gigapack Gold, infection with Escherichia coli P2392 (STRATAGENE), a chromosomal DNA library did.
Example 2 Isolation of GPD gene from chromosomal gene library
A clone containing the GPD gene was selected from the chromosomal DNA library by plaque hybridization. This series of operations was carried out by a conventional method [Sambrook et al., “Molecular Cloning A Laboratory Manual / 2nd Edition (1989)].
The probe used for plaque hybridization is a synthetic oligomer with the following sequence:

The³²Radiolabeled with P.
[0019]
As a result, 4 positive clones could be selected from about 40,000 plaques. Recombinant phage DNA prepared from a positive clone according to a conventional method was digested with various restriction enzymes, and Southern hybridization was performed using the above two kinds of synthetic DNAs. As a result, restriction enzymesEcoRI (Takara Shuzo) andBamIn the fragment obtained by digestion with HI (Takara Shuzo), a clone that hybridized to 3.8 kbp as a single DNA band was observed.
[0020]
The DNA fragment 3.8 kbp was excised by agarose gel electrophoresis, and the E. coli vector pUC18 (Takara Shuzo Co., Ltd.)EcoRI,BamSubcloned into the HI site and transformed into Escherichia coli JM109 strain (Takara Shuzo). A large amount of subcloned DNA was prepared, purified by ultracentrifugation (50,000 rpm, 16 hrs, 15 ° C.), and the nucleotide sequence was determined. The base sequence was determined using a Sequenase kit (manufactured by United States Biochemical).
[0021]
The base sequence from the translation start point to the 850th downstream is shown in SEQ ID NO: 2. As a result, in the range of the above-mentioned base sequence, the GPD gene derived from Aragekawaratake was divided by 6 introns. The amino acid sequence deduced from the base sequence excluding introns is shown in SEQ ID NO: 3. This amino acid sequence was found to be highly similar to the GPD gene reported so far.
[0022]
In addition, the obtained Escherichia coli transformed strain containing the GPD gene derived from MushroomE.coli  JM109 / pCHGPD is deposited as FERM P-15015 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology.
Example 3 Linkage of OCT structural genes controlled by GPD gene regulatory region
The plasmid pGPRG for the OCT expression selection marker for basidiomycete Alagekawaratake is linked to the structural gene region of the OCT chromosomal gene (JP-A-6-054691; FERM P-12679) downstream of the promoter region of the GPD gene, and from the original OCT gene. A selectable marker gene in which the promoter region was replaced was used.
[0023]
Specifically, GPD chromosome geneEcoRI andBamA DNA fragment of 3.8 kbp obtained by HI digestion was added to the phage vector M13mp18.EcoRI,BamThis was ligated to the HI site using T4 DNA ligase and transformed into E. coli JM109 strain to prepare single-stranded phage DNA. Next, the DNA primer shown in FIG. 2 fragment 1 was synthesized by the phosphoramidide method, annealed to the single-stranded phage DNA, and only the promoter region of the GPD gene was synthesized by the primer extension method, and the restriction enzyme EcoRI ( A 0.9-kbp DNA fragment was prepared by cutting with Takara Shuzo Co., Ltd. (fragment 1). This base sequence is SEQ ID NO: 1.
[0024]
On the other hand, in order to extract a gene region encoding the mature enzyme of OCT, plasmid pUCR1 (Japanese Patent Laid-Open No. 6-054691; FERM P-12777) is cleaved with restriction enzyme NcoI (Takara Shuzo), and then Mung. By using bean nuclease (manufactured by Takara Shuzo Co., Ltd.), the protruding portion was scraped to make a blunt end to obtain a DNA fragment of about 2.5 kbp (fragment 2).
[0025]
E. coli vector pUC18EcoRI andSmaAfter cutting with I (Takara Shuzo Co., Ltd.), mixing the above two types of DNA fragments and ligating with T4 DNA ligase, transformation of E. coli JM109 strain was carried out. Among the ampicillin resistant transformants, the above fragment 1 and A plasmid in which two types of DNA fragments of fragment 2 were simultaneously inserted was isolated and named pGPRG (FIG. 2).
Example 4 Method of transformation of coralus hirutus
a. Mononuclear mycelium culture
Disperse 100 ml of SMY medium (1% sucrose, 1% malt extract, 0.4% yeast extract) into a 500 ml Erlenmeyer flask containing about 30 glass beads with a diameter of about 6 mm.Coriolus  hirsutus) An agar piece having a diameter of 5 mm was punched out of a plate agar medium of OJI-1078 with a cork borer, inoculated into an SMY medium, and cultured at 28 ° C. for 7 days (preculture).
[0026]
However, in order to subdivide the mycelium, it was shaken once or twice a day. Next, 200 ml of SMY medium was dispensed into a 1 L Erlenmeyer flask, and a rotor was added. After sterilization, the precultured mycelium was collected by filtration with a nylon mesh (pore size 30 μm), and the whole amount was inoculated at 28 ° C. Cultured. The mycelium was subdivided by stirring for 2 hours a day with a stirrer. This culture was carried out for 4 days.
[0027]
b. Protoplast preparation
The liquid culture mycelium was collected by filtration with a nylon mesh (pore size 30 μm), and an osmotic pressure adjusting solution (0.5M MgSO_FourAnd 50 mM maleate buffer (pH 5.6)). Next, the suspension was suspended in 1 ml of cell wall degrading enzyme solution per 100 mg of wet cells and incubated at 28 ° C. for 4 hours with gentle shaking to release protoplasts. The following commercially available enzyme preparations were used in combination as cell wall lytic enzymes. That is, 5 mg of cellulase ONOZUKA RS (manufactured by Yakult) and 10 mg of Novozyme 234 (manufactured by Novo) were dissolved in 1 ml of the osmotic pressure adjusting solution and used as an enzyme solution.
[0028]
c. Protoplast purification
After removing mycelial fragments from the enzyme reaction solution with a nylon mesh (pore size 30 μm), the hyphal fragments remaining on the nylon mesh and the protoplasts were washed once with the osmotic pressure adjusting solution in order to increase the recovery rate of protoplasts. The obtained protoplast suspension was centrifuged (1,000 × g, 5 minutes), the supernatant was removed, and the suspension was resuspended with 4 ml of 1M sucrose (20 mM MOPS buffer, pH 6.3), followed by centrifugation. Repeatedly washed twice with the 1M sucrose solution. The precipitate was suspended in 500 μl of a 1M sorbitol solution (20 mM MES, pH 6.4) and 40 mM calcium chloride added to form a protoplast solution. This solution was stored at 4 ° C.
[0029]
The protoplast concentration was determined by direct microscopy using a hemocytometer. All centrifugation operations were performed at 1,000 × g for 5 minutes at room temperature using a swing rotor.
d. Transformation
10⁶2 μg of the plasmid pGPRG prepared in Example 3 was added to 100 μl of the protoplast solution having a concentration of 100/100 μl, and ice-cooled for 30 minutes. Next, an equal amount of PEG solution (50% PEG3400, 20 mM MOPS (pH 6.4)) was added to the liquid volume, and the mixture was cooled on ice for 30 minutes. Next, the mixture was mixed with a minimal soft agar medium (1% agar) containing 0.5M sucrose and leucine and plated on a plate. The plate was cultured at 28 ° C. for 4 days to obtain a transformant. The transformation efficiency at this time was 300 colonies / μg transformed DNA. In the control experiment using a DNA donor consisting of only the plasmid vector pUC18, no transformants were obtained.
Embodiment 5 FIG. Construction of lignin peroxidase expression plasmid
The structural gene region of the lignin peroxidase chromosomal gene (Japanese Patent Laid-Open No. 5-260978; pBSLPOG7; FERM P-12683) is downstream of the DNA fragment 1 (GPD gene promoter for basidiomycete larvae) produced in Example 3 above. Ligated to form an expression plasmid.
[0030]
Specifically, the structural gene portion of lignin peroxidase was taken out and a DNA fragment was prepared by the primer extension method in the same manner as in Example 3 using the primer shown in fragment 3 of FIG. 3 on the basis of plasmid pBSLPOOG7.HinBy cleaving with dIII, a DNA fragment of 1.7 kbp consisting of a lignin peroxidase structural gene portion was obtained (fragment 3).
[0031]
In addition, the E. coli vector pUC18 isEcoRI andHinAfter digestion with dIII, two kinds of DNA fragments (fragment 1) described in Example 3 and the above DNA fragment (fragment 3) were mixed, ligated with T4 DNA ligase, and then transformed into E. coli strain JM109, From the ampicillin resistant transformant, a plasmid into which two types of DNA fragments, Fragment 1 and Fragment 3, were simultaneously inserted was isolated and named pGPLG (FIG. 3).
Example 6 Preparation of the transformant of Arakawaratake with high secretory production of lignin peroxidase
When transforming arginine auxotrophic larvae (OJI-1078) using the pGPHLG obtained in Example 5, a plasmid (pUCR1) carrying the OCT gene derived from larvae lucidum (pUCR1) is simultaneously introduced as a selection marker (PEG method, Alternatively, a transformant pGPLG / OJI-1078 was obtained by an electroporation method or the like. Here, regardless of whether the DNA that can be donated for transformation is circular or linear, the desired transformant could be obtained. The transformation conditions are described below.
[0032]
About 10⁶Per 100 μl of protoplast solutionthe above2 μg of the plasmid prepared in Example 5 was added in a circular or linear form, 0.2 μg of pUCR1 was further added as a selection marker, and the mixture was ice-cooled for 30 minutes.
Next, an equal amount of PEG solution (50% PEG3400, 20 mM MOPS (pH 6.4)) was added and cooled on ice for 30 minutes. The mixture was mixed with a minimal soft agar medium (1% agar) containing 0.5M sucrose and leucine and plated on a plate. The plate was cultured at 28 ° C. for 4 days to obtain a transformant. Furthermore, DNA was prepared from the transformant, and it was confirmed by Southern hybridization that the target lignin peroxidase expression plasmid was incorporated.
Example 7 Culture of transformed strains and measurement of lignin peroxidase activity
The transformed strain (pGPLG / OJI-1078) obtained in Example 6 was placed in a 500 ml Erlenmeyer flask with 50 ml of glucose peptone liquid medium (glucose 20 g / l, peptone 5 g / l, yeast extract 2 g / l, KH₂PO_Four1g / l, MgSO_Four・ 7H₂O 0.5 g / l, adjusted to pH 4.5 with phosphoric acid) were inoculated with 5 agar pieces of 5 mm and cultured at 28 ° C. for 6 days under shaking (pre-culture). Thereafter, the cells were crushed with a Waring blender, and the shaking culture was continued for 2 days at 28 ° C. in a glucose peptone medium (preculture). Next, the cells were collected, the cells were resuspended in 50 ml of fresh glucose peptone liquid medium, transferred to a new 500 ml Erlenmeyer flask, and static culture was performed at 28 ° C. The resulting culture broth was centrifuged to obtain the supernatant.
[0033]
The activity was measured by mixing 25 μl of 8 mM veratryl alcohol, 50 μl of 0.5 M sodium tartrate buffer (pH 3.0), 400 μl of enzyme solution and 25 μl of 2.7 mM hydrogen peroxide, and mixing 310 nm of veratraldehyde resulting from the reaction. The enzyme activity for converting veratryl alcohol to veratraldehyde was observed in the culture supernatant in the range of 300 to 500 units / ml on the 5th day from the start of stationary culture. It was. Here, the enzyme activity unit was defined as 1 unit of activity for increasing 1 μmol of veratraldehyde per minute. On the other hand, this activity was not observed in the culture supernatant of OJI-1078 strain which did not contain donor DNA cultured under the same conditions.
Example. 8 Construction of manganese peroxidase expression plasmid
Manganese peroxidase expression plasmid pGPMPG exposed under the control of the promoter of the GPD gene for basidiomycete Aragekawaratake is a manganese peroxidase chromosomal gene (JP-A-7-123540; pBSMPOOG1) downstream of DNA fragment 1 prepared in Example 3. A structural gene of Mikoken Bacterium No. 14933) was ligated to obtain an expression plasmid.
[0034]
Specifically, the structural gene part of manganese peroxidase was extracted by preparing a DNA fragment by the primer extension method in the same manner as in Example 3 using the primer shown in fragment 4 of FIG. By cleaving, a 2.8 kbp DNA fragment comprising a manganese peroxidase structural gene portion was obtained (fragment 4).
[0035]
Further, the E. coli vector pUC18 was cleaved with restriction enzymes EcoRI and HindIII, the above two kinds of DNA fragments were mixed, ligated with T4 DNA ligase, E. coli JM109 strain was transformed, and 2 ampicillin resistant transformants were selected. A plasmid in which different types of DNA fragments were simultaneously inserted was isolated and named pGPPMPG (FIG. 4).
[0036]
Example 9 Preparation of a transformant of Arakawakawara that produces high secretion of manganese peroxidase
When transforming arginine auxotrophic jellyfish (OJI-1078) using pGPPMPG obtained in Example 8, a plasmid (pUCR1) carrying the OCT gene derived from jellyfish moth (PUCR1) is simultaneously introduced as a selection marker (PEG method) Alternatively, a transformant pGPPMG / OJI-1078 was obtained by an electroporation method or the like. Here, regardless of whether the DNA that can be donated for transformation is circular or linear, the desired transformant could be obtained. The transformation conditions are described below.
[0037]
About 10⁶2 μg of the plasmid prepared in Example 5 was added in a circular or linear form to 100 μl of a protoplast solution having a concentration of 100 / l / 100 μl, and 0.2 μg of pUCR1 was further added as a selection marker, followed by ice cooling for 30 minutes.
Next, an equal amount of PEG solution (50% PEG3400, 20 mM MOPS (pH 6.4)) was added and cooled on ice for 30 minutes. The mixture was mixed with a minimum soft agar medium (1% agar) containing 0.5M sucrose and leucine and plated on a plate. The plate was cultured at 28 ° C. for 4 days to obtain a transformant. Furthermore, DNA was prepared from the transformant, and it was confirmed by Southern hybridization that the target lignin peroxidase expression plasmid was incorporated.
Example 10 Culture of transformed strains and measurement of manganese peroxidase activity
The transformed strain (pGPPMG / OJI-1078) obtained in Example 9 was placed in a 500 ml Erlenmeyer flask with 50 ml of glucose peptone liquid medium (glucose 20 g / l, peptone 5 g / l, yeast extract 2 g / l, KH₂PO_Four1g / l, MgSO_Four・ 7H₂O 0.5g / l, adjusted to pH 5.0 with phosphoric acid) 50mm²Five agar pieces were inoculated and cultured at 28 ° C. for 6 days under shaking (pre-culture). Thereafter, the cells were crushed with a Waring blender, and 10 ml of the cell solution was inoculated into a petri dish having a diameter of 9 cm containing 10 ml of fresh glucose peptone medium, followed by static culture at 28 ° C. for 10 days to form a cell mat. Next, after collecting the cells and crushing and washing them with a Waring blender, a fresh modified glucose peptone liquid medium (glucose 20 g / l, peptone 5 g / l, yeast extract 2 g / l, KH₂PO_Four1g / l, MgSO_Four・ 7H₂O 0.5 g / l, 0.2 mM MnSO_Four(Adjusted to pH 5.0 with phosphoric acid) Resuspended in 20 ml, transferred to a new 200 ml Erlenmeyer flask, and statically cultured at 28 ° C. The resulting culture broth was centrifuged to obtain the supernatant.
[0038]
The activity was measured by 50 μl of 0.5 M sodium malonate buffer (pH 5.5), 445 μl of enzyme solution, 5 μl of 10 mM hydrogen peroxide, 1 mM MnSO._Four100 μl was mixed well, and the increase in absorbance at 270 nm of the Mn (III) malonic acid complex resulting from the reaction was recorded over time to form the Mn (III) malonic acid complex in the culture supernatant. Enzyme activity was observed at 5 μmol / ml / min on the 7th day from the start of stationary culture. Here, the enzyme activity unit was defined as 1 unit of activity for increasing 1 μmol of Mn (III) malonic acid complex per minute. On the other hand, this activity was not observed in the culture supernatant of OJI-1078 strain which did not contain donor DNA cultured under the same conditions.
[0039]
【The invention's effect】
The present invention provides a novel method for producing lignin peroxidase or manganese peroxidase, which has heretofore been difficult to mass-produce enzymes by genetic recombination technology, in an active form in the transformation system of the larvae of Arakawakawara. DNA fragments are provided.
[0040]
[Sequence Listing]
SEQ ID NO: 1
Sequence length: 922
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: genomic DNA
origin
Name of organism: Alage Kawaratake (Coriolus  hirsutus)
Stock name: IFO4917
Sequence features
860-866 TFIID site
Array:

[0041]
SEQ ID NO: 2
Sequence length: 850
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: genomic DNA
origin
Name of organism: Alage Kawaratake (Coriolus  hirsutus)
Stock name: IFO4917
Sequence features
1-850 P CDS
7-63 intron
87-153 intron
159-216 intron
287-350 intron
567-630 intron
749-813 intron
Array:

[0042]
SEQ ID NO: 3
Sequence length: 160
Sequence type: amino acid
Topology: Linear
Sequence type: Peptide
origin
Name of organism: Alage Kawaratake (Coriolus  hirsutus)
Stock name: IFO4917
Sequence features
1-160 P mat peptide
Array:

[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a restriction map on the chromosome of the glyceraldehyde-3-phosphate dehydrogenase gene of Arakawakawatake.
FIG. 2 is a construction diagram of a plasmid pGPRG used for the arginine-requiring Aragekawa aratake mutant shown in Example 3.
FIG. 3 is a construction diagram of the lignin peroxidase expression plasmid pGPHLG shown in Example 5.
4 is a construction diagram of the manganese peroxidase expression plasmid pGPMPG shown in Example 8. FIG.

Claims (3)

A recombinant vector comprising a DNA fragment comprising a promoter DNA consisting essentially of the base sequence of SEQ ID NO: 1, and a lignin peroxidase gene or a manganese peroxidase gene derived from Pseudomonas aeruginosa . A basidiomycete Aragekawaratake transformant transformed with the recombinant vector according to claim 1 . A method for producing lignin peroxidase or manganese peroxidase, comprising culturing the transformant according to claim 2 and collecting lignin peroxidase or manganese peroxidase from the culture.

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