JP4016948B2 - DNA fragment having promoter activity - Google Patents

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その結果、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、上記の合成ＤＮＡを用いてサザンハイブリダイゼーションを行った。その結果、制限酵素ＸｈｏＩで消化して得られた断片中に７．２ｋｂｐ及び５．３ｋｂｐのＤＮＡバンドとしてプローブにハイブリダイズするクローンが認められた。
上記ＤＮＡ断片７．２ｋｂｐ及び５．３ｋｂｐ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ｐＢｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ＋（東洋紡社製）のＸｈｏＩサイトにサブクローニング化し、プラスミドｐＣＨＣＤＨ１及びｐＣＨＣＤＨ２を得た。これらのプラスミドをそれぞれ大腸菌ＪＭ１０９株へ形質転換した。なお、得られたコリオラス・ヒルスタス由来セロビオースデヒドロゲナーゼ遺伝子のプロモーター領域を含む大腸菌形質転換株Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＪＭ１０９／ｐＣＨＣＤＨ１及びＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＪＭ１０９／ｐＣＨＣＤＨ２は、２００２年２月８日付けで独立行政法人産業技術総合研究所特許生物寄託センター（日本国茨城県つくば市東１丁目１番地１ 中央第６）に寄託され、それぞれ受託番号ＦＥＲＭ ＢＰ−８２７８、ＦＥＲＭ ＢＰ−８２７９が付与されている。次いで、サブクローニング化したＤＮＡを大量に調製し、超遠心操作（５０，０００ｒｐｍ，１６ｈ，１５℃）で精製し、塩基配列を決定した。塩基配列の決定はＵｎｉｔｅｄ Ｓｔａｔｅｓ Ｂｉｏｃｈｅｍｉｃａｌ社製のシーケンシングキットを用いて行った。セロビオースデヒドロゲナーゼ遺伝子のプロモーター配列を含むＤＮＡ断片の塩基配列を配列番号１又は２に示す。
［実施例３］ セロビオースデヒドロゲナーゼ遺伝子プロモーターによるマンガンペルオキシダーゼ遺伝子の発現ベクターの構築
実施例２で得られたコリオラス・ヒルスタス由来のセロビオースデヒドロゲナーゼ遺伝子プロモーター領域を含むプラスミドｐＣＨＣＤＨ１から下記の配列番号４及び５に示す２つのプライマーを用いてＰＣＲ法を行うことにより、セロビオースデヒドロゲナーゼプロモーター領域のＢａｍＨＩ〜ブラントエンドを有する２．２ｋｂｐ断片（断片１）を増幅した。

次に、コリオラス・ヒルスタス由来マンガンペルオキシダーゼ遺伝子を含むプラスミドｐＢＳＭＰＯＧ１（ＦＥＲＭ Ｐ−１４９３３）を以下の配列番号６及び７に示す２つのプロモーターを用いてＰＣＲ法を行うことにより、マンガンペルオキシダーゼ構造遺伝子部分ブラントエンド〜ＥｃｏＲＩ約２．２ｋｂｐ（断片２）を増幅した。
大腸菌ベクターｐＵＣ１８を制限酵素ＢａｍＨＩ〜ＥｃｏＲＩで切断し、上記の断片１及び２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断片１及び断片２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ｐＣＤＨＰＭＮと命名した。

［実施例４］ マンガンペルオキシダーゼを高分泌生産するコリオラス・ヒルスタス形質転換体の作製
以下の方法により、コリオラス・ヒルスタスのプロトプラスト溶液を得た。
ａ．一核菌糸体培養
直径６ｍｍ前後のガラスビーズを約３０個入れた５００ｍｌ容三角フラスコに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、コリオラス・ヒルスタスＯＪＩ−１０７８株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）。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時間程度撹拌することにより菌糸を細分化した。この培養を４日間行った。
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 ＭｇＳＯ_４、５０ｍｌ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、緩やかに震盪しながら２８℃で３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、ヤタラーゼ（Ｙａｔａｌａｓｅ）（宝酒造社製）１０ｍｇを上記浸透圧調節溶液１ｍｇに溶解して酵素液として用いた。
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（１，０００ｋ×ｇ、５分間）し、上静を除去し、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。沈殿物に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。
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。すべての遠心操作はスウィングローターで１，０００×ｇ、５分間、室温下で行った。
ｄ．形質転換
約１０^６個／１００μｌのプロトプラスト溶液に対し実施例３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た。さらに選択マーカーとして、コリオラス・ヒルスタス由来のオルニチンカルバモイルトランスフェラーゼ遺伝子を保持するプラスミド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シュークロース及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捲いた。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、目的とするマンガンペルオキシダーゼ遺伝子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５］ 形質転換体によるマンガンペルオキシダーゼの生産
酸素漂白後広葉樹パルプ（ＬＯＫＰ）・ペプトン培地（ＬＯＫＰ１％、ポリペプトン０．５％、酵母エキス０．２％、ＫＨ_２ＰＯ_４、ＭｇＳＯ_４０．０５％、リン酸でｐＨ４．５に調製）を１００ｍｌずつ含む３００ｍｌ容三角フラスコに、上記実施例４で得られた形質転換株５０ｍｍ^２の寒天片を５個接種し、２８℃、１００ｒｐｍで回転振盪培養した。６日後、得られた培養液を遠心分離し、その上清を得た。
酵素活性は０．５Ｍマロン酸ナトリウム緩衝液（ｐＨ５．５）５０μｌ、酵素液３４５μｌ、１０ｍＭ過酸化水素５μｌ、１ｍＭ ＭｎＳＯ_４１００μｌを十分混合し、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）マロン酸複合体を酵素活性が、培養開始６日目で６μｍｏｌ／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。
［実施例６］ セロビオースデヒドロゲナーゼ遺伝子プロモーターによるリグニンペルオキシダーゼ遺伝子の発現ベクターの構築
コリオラス・ヒルスタス由来セロビオースデヒドロゲナーゼ遺伝子のプロモーターの下流にリグニンペル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。
具体的には、実施例２で得られたコリオラス・ヒルスタス由来のセロビオースデヒドロゲナーゼ遺伝子プロモーター領域を含むプラスミドｐＣＨＣＤＨ１から実施例３において配列番号４及び５に示す２つのプライマーを用いてＰＣＲ法を行うことにより、プロモーター領域のＢａｍＨＩ〜ブラントエンドを有する２．２ｋｂｐ断片（断片３）を増幅した。また、リグニンペル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ｐＢＳＬＰＯＧ７をテンプレートにして以下の配列番号８に示すプライマーを用いて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ペルオキシダーゼ構造遺伝子部分からなる１．７ｋｂｐのＤＮＡ断片（断片４）を得た。

さらに、大腸菌ベクターをｐＵＣ１８を制限酵素ＥｃｏＲＩとＨｉｎｄＩＩＩで切断し、上記断片５及び６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上記断片３及び４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ｐＣＤＨＰＬＰと命名した。
［実施例７］ リグニンペルオキシダーゼを高分泌生産するコリオラス・ヒルスタス形質転換体の作製
実施例４に記載の方法により、コリオラス・ヒルスタスのプロトプラスト溶液を得た。約１０^６個／１００μｌのプロトプラスト溶液に対し実施例６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た。さらに選択マーカーとして、コリオラス・ヒルスタス由来のオルニチンカルバモイルトランスフェラーゼ遺伝子を保持するプラスミド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シュークロース及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捲いた。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ペルオキシダーゼ遺伝子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８］ 形質転換体によるリグニンペルオキシダーゼの生産
酸素漂白後広葉樹パルプ（ＬＯＫＰ）・ペプトン培地（ＬＯＫＰ１％、ポリペプトン０．５％、酵母エキス０．２％、ＫＨ_２ＰＯ_４、ＭｇＳＯ_４０．０５％、リン酸でｐＨ４．５に調製）を１００ｍｌずつ含む３００ｍｌ容三角フラスコに、上記実施例７で得られた形質転換株５０ｍｍ^２の寒天片を５個接種し、２８℃、１００ｒｐｍで回転振盪培養した。６日後、得られた培養液を遠心分離し、その上清を得た。
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を十分混合し、反応の結果生じるベラトルムアルデヒドの３１０ｎｍの吸光度を経時的に行い、上記培養上清中にベラトリルアルコールをベラトルムアルデヒドへと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分間にベラトルムアルデヒド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９］ セロビオースデヒドロゲナーゼ遺伝子プロモーターによるラッカーゼ遺伝子の発現ベクターの構築
実施例２で得られたプラスミドｐＣＨＣＤＨ１をテンプレートにして以下の配列番号９及び１０に示す２つのプライマーを用いたＰＣＲを実施し、ＢａｍＨＩ処理を行うことによりＢａｍＨＩ〜ブラントエンドを有するＤＮＡ断片（断片７）を得た。

一方、コリオラス・ヒルスタス由来ラッカーゼ遺伝子を含むプラスミドＯＪ−ＰＯＧ−Ｅ１（寄託番号ＢＰ−２７９３）を以下の配列番号１１及び１２に示す２つのプライマーを用いてＰＣＲ法により増幅した。

得られたＰＣＲ断片を上記のＴＡ−Ｃｌｏｎｏｉｎｇベクターへ導入し、プラスミドｐＴＡＬＡＣを得た。プラスミドｐＴＡＬＡＣを制限酵素ＸｈｏＩで消化後、修飾酵素Ｋｌｅｎｏｗ ｆｒａｇｍｅｎｔを用いて平滑化し、その後、制限酵素ＥｃｏＲＩで消化し、ラッカーゼ構造遺伝子部分（断片６）を得た。
大腸菌ベクターｐＵＣ１８をＢａｍＨＩとＥｃｏＲＩで消化したのち、上記断片７及び８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上記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ｐＣＤＨＰＬＡＣと命名した。
［実施例１０］ ラッカーゼを高分泌生産するコリオラス・ヒルスタス形質転換体の作製
実施例４に記載の方法により、コリオラス・ヒルスタスのプロトプラスト溶液を得た。約１０^６個／１００μｌのプロトプラスト溶液に対し実施例９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た。さらに選択マーカーとして、コリオラス・ヒルスタス由来のオルニチンカルバモイルトランスフェラーゼ遺伝子を保持するプラスミド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シュークロース及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捲いた。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、目的とするラッカ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１１］ 形質転換体によるラッカーゼの生産
酸素漂白後広葉樹パルプ（ＬＯＫＰ）・ペプトン培地（ＬＯＫＰ１％、ポリペプトン０．５％、酵母エキス０．２％、ＫＨ_２ＰＯ_４、ＭｇＳＯ_４０．０５％、リン酸でｐＨ４．５に調製）を１００ｍｌずつ含む３００ｍｌ容三角フラスコに、上記実施例１０で得られた形質転換株を５０ｍｍ^２の寒天片を５個接種し、２８℃、１００ｒｐｍで回転振盪培養した。６日後、得られた培養液を遠心分離し、その上清を得た。
酵素活性は１Ｍ酢酸ナトリウム緩衝液（ｐＨ４．０）５０μｌ、５ｍＭ ２，２’−ａｚｉｎｏ−ｂｉｓ（３−ｅｔｈｉｌｂｅｎｚｔｈｉａｚｏｌｉｎｅ−６−ｓｕｌｆｏｎａｔｅ）（ＡＢＴＳ）５０μｌ、酵素液４００μｌを加え、反応の結果生じるＡＢＴＳの酸化物を４２０ｎｍの吸光度増加を時間を追って記録することにより行い、上記培養上清中に酵素活性が培養開始５日目で４０ユニット／ｍＬで認められた。ここで、酵素活性単位は１分間に１μｍｏｌのＡＢＴＳを酸化するのに必要とされる酵素量と定義する。一方、同条件下で培養した供与ＤＮＡを含まないＯＪＩ−１０７８株の培養上清にはわずか５ユニット／ｍｌしか認められなかった。
２．セロビオヒドロラーゼＩ遺伝子のプロモーター配列を含む第１のＤＮＡ断片を用いたリグニン分解酵素の生産と利用
［実施例１２］ 染色体遺伝子ライブラリーからのセロビオヒドロラーゼＩ遺伝子プロモーター配列を含むＤＮＡ断片の単離
実施例１と同様にして、染色体遺伝子ライブラリーを得た。得られた該ライブラリーからプラークハイブリダイゼーションによりセロビオヒドロラーゼＩ遺伝子を含むクローンの選抜を行った。この一連の操作は常法［Ｓａｍｂｒｏｏｋら著、”Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ Ａ ＬａｂｏｒａｔｏｒｙＭａｎｕａｌ／２ｎｄ Ｅｄｉｔｉｏｎ（１９８９）］によった。
プラークハイブリダイゼーションに用いたプローブは下記配列番号１６の配列を持つ合成オリゴマーをアマシャム社製のオリゴＤＮＡ標識キットを用い３’末端をフルオレセインで標識したものである。

ただし、配列番号１６で塩基Ｙは塩基ＴまたはＣを示し、塩基Ｒは塩基ＧまたはＡを示す。
その結果、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、上記の合成ＤＮＡを用いてサザンハイブリダイゼーションを行った。その結果、制限酵素ＥｃｏＲＩならびにＢａｍＨＩで消化して得られた断片中に単一のＤＮＡバンドとして４．６ｋｂｐにハイブリダイズするクローンが認められた。
上記ＤＮＡ断片４．６ｋｂｐ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ｐＵＣ１９のＥｃＯＲＩ−ＢａｍＨＩサイトにサブクローン化し、大腸菌ＪＭ１０９株へ形質転換し、プラスミドｐＣＨＣＢＨＩ３１を得た。サブクローン化したＤＮＡ断片の塩基配列を決定した。塩基配列の決定はＡｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製のＢｉｇＤｙｅ Ｔｅｒｍｉｎａｔｏｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇキットを用いて反応を行い、Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製ＤＮＡシークエンサーＰＲＩＳＭ ３１０を用いて泳動を行い、塩基配列を解析した。
塩基配列を配列番号１４に示す。その結果、上記塩基配列の範囲内においてコリオラス・ヒルスタス由来セロビオヒドロラーゼＩ遺伝子は２つのイントロンにより分断されていた。また、塩基配列から推定されるアミノ酸配列を配列番号１５に示す。
［実施例１３］ セロビオヒドロラーゼＩ遺伝子プロモーターによるマンガンペルオキシダーゼ遺伝子の発現ベクターの構築
コリオラス・ヒルスタス由来セロビオヒドロラーゼＩ遺伝子プロモーター領域を含むプラスミドｐＣＨＣＢＨＩ３１を制限酵素ＮｃｏＩで消化した後、修飾酵素Ｋｌｅｎｏｗ ｆｒａｇｍｅｎｔを用いて平滑化を行い、自己連結を行い、ＮｃｏＩサイトを消失させる。得られたＮｃｏＩサイト消失プラスミドから以下に示す２つのプライマー（１，２）を用いてＰＣＲ法により、プロモーター領域２．３ｋｂｐとして増幅し、ｐＵＣ１８のＳｍａＩサイトへＴ４ＤＮＡリガーゼを用いて連結し、プラスミドｐＣＨＣＢＨＩ３１Ｐを得た。

次に、コリオラス・ヒルスタス由来マンガンペルオキシダーゼ遺伝子を含むプラスミドｐＢＳＭＰＯＧ１（寄託番号ＦＥＲＭ Ｐ−１４９３３）を以下に示す２つのプライマー（３，４）を用いてＰＣＲ法によりマンガンペルオキシダーゼ構造遺伝子部分ＮｃｏＩ約２．２ｋｂ断片を増幅する。

得られたＰＣＲ断片をＩｎ Ｖｉｔｒｏｇｅｎ社製のＴＡ ｃｌｏｎｉｎｇ ｋｉｔを用いて挿入し、ｐＴＡＭＰとした。次に、得られたｐＴＡＭＰを制限酵素ＮｃｏＩで消化し、約２．２ｋｂを切り出し、上記ｐＣＨＣＢＨＩ３１ＰのＮｃｏＩサイトへ挿入し、プラスミドｐＣＨＣＢＨＩ３１ＰＭＰを得た（図１）。
［実施例１４］ マンガンペルオキシダーゼを高生産する形質転換体の作製
実施例４と同様にして、プロトプラストを調製・精製した。次いで、以下のようにして形質転換を行った。
１０^６個／１００μｌ濃度のプロトプラスト溶液１００μｌに対し、実施例１３で作製したプラスミドｐＣＨＣＢＨＩ３１ＰＭＰ 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コリオラス・ヒルスタス由来のオルニチンカルバモイルトランスフェラーゼ遺伝子を保持するｐＵＣＲ１を０．２μｇ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、目的とするマンガンペル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１５］ 形質転換体によるマンガンペルオキシダーゼの生産
上記実施例１４で得られた形質転換株を５００ｍｌ容の三角フラスコに５０ｍｌのパルプ・ペプトン液体培地（晒パルプ３０ｇ／ｌ、ペプトン１０ｇ／ｌ、ＫＨ_２ＰＯ_４１．５ｇ／ｌ、ＭｇＳＯ_４・７Ｈ_２Ｏ０．５ｇ／ｌ、塩酸チアミン２ｍｇ／ｌ、ＭｎＳＯ_４・５Ｈ_２Ｏ４８ｍｇ／ｌ、リン酸でｐＨ５．０に調整）に５０ｍｍ^２の寒天片を５個接種し、２８℃で６日間、振盪下で培養した。６日後、得られる培養液を遠心分離し、その上清を得た。
酵素活性は０．５Ｍマロン酸ナトリウム緩衝液（ｐＨ５．５）５０μｌ、酵素液３４５μｌ、１０ｍＭ過酸化水素５μｌ、１ｍＭ ＭｎＳＯ_４１００μｌ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）マロン酸複合体を酵素活性が培養開始６日目で３．５μｍｏｌ／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。
［実施例１６］ セロビオヒドロラーゼＩ遺伝子プロモーターによるラッカーゼ遺伝子の発現ベクターの構築
実施例１３で得られたプラスミドｐＣＨＣＢＨＩ３１Ｐを制限酵素ＮｃｏＩ（宝酒造社製）で消化後、Ｋｌｅｎｏｗ ｆｒａｇｍｅｎｔを用いて平滑化した。その後、制限酵素ＥｃｏＲＩで消化し、発現ベクター部分とした。
次に、コリオラス・ヒルスタス由来ラッカーゼ遺伝子を含むプラスミドＯＪ−ＰＯＧ−Ｅ１（ＦＥＲＭ ＢＰ−２７９３）を以下に示す２つのプライマー（５，６）を用いてＰＣＲ法により増幅した。

得られたＰＣＲ断片を上記のＴＡ−ｃｌｏｎｉｎｇベクターへ導入し、プラスミドｐＴＡＬＡＣを得た。
プラスミドｐＴＡＬＡＣを制限酵素ＸｈｏＩで消化後、修飾酵素ｋｌｅｎｏｗ ｆｒａｇｍｅｎｔを用いて平滑化する。その後、制限酵素ＥｃｏＲＩで消化し、ラッカーゼ構造遺伝子部分を得、上記処理したセロビオヒドロラーゼＩ発現ベクターｐＣＨＣＢＨＩ３１Ｐへ導入した。
得られたプラスミドをｐＣＨＣＢＨＩ３１ＰＬＡＣと命名した（図２）。
［実施例１７］ ラッカーゼを高分泌生産するコリオラス・ヒルスタス形質転換体の作製
実施例１６で得たｐＣＨＣＢＨＩ３１ＰＬＡＣを用いてアルギニン要求性コリオラス・ヒルスタスＯＪＩ−１０７８株（ＦＥＲＭ ＢＰ−４２１０）を形質転換する場合、選択マーカーとしてコリオラス・ヒルスタス由来の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、もしくは直鎖状に拘らず、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。
約１０^６個／１００μｌ濃度のプロトプラスト溶液を１００μｌに対し実施例１６で作製したプラスミドｐＣＨＣＢＨＩ３１ＰＬＡＣ 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、目的とするラッカ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１８］ 形質転換体によるラッカーゼの生産
上記実施例１７で得られた形質転換株を５００ｍｌ容の三角フラスコに５０ｍｌのパルプ・ペプトン液体培地（晒パルプ３０ｇ／ｌ、ペプトン１０ｇ／ｌ、ＫＨ_２ＰＯ_４１．５ｇ／ｌ、ＭｇＳＯ_４・７Ｈ_２Ｏ０．５ｇ／ｌ、塩酸チアミン２ｍｇ／ｌ、ＣｕＳＯ_４・５Ｈ_２Ｏ１００ｍｇ／ｌ、リン酸でｐＨ５．０に調整）に５０ｍｍ^２の寒天片を５個接種し、２８℃で６日間、振盪下で培養した。６日後、得られる培養液を遠心分離し、その上清を得た。
酵素活性は１Ｍ酢酸ナトリウム緩衝液（ｐＨ４．０）５０μｌ、５ｍＭ ＡＢＴＳ（２，２’−ａｚｉｎｏ−ｂｉｓ（３−ｅｔｈｉｌｂｅｎｚｔｈｉａｚｏｌｉｎｅ−６−ｓｕｌｆｏｎａｔｅ）５０μｌ、酵素液４００μｌを加え、反応の結果生じるＡＢＴＳの酸化物を４２０ｎｍの吸光度増加を時間を追って記録することにより行い、上記培養上清中に酵素活性が培養開始５日目で３５ユニット／ｍｌ認められた。ここで酵素活性単位は１分間に１μｍｏｌのＡＢＴＳを酸化するのに必要とされる酵素量と定義する。一方、同条件下で培養した供与ＤＮＡを含まないコリオラス・ヒルスタスＯＪＩ−１０７８株（ＦＥＲＭ ＢＰ−４２１０）の培養上清にはわずか５ユニット／ｍｌしか認められなかった。
［実施例１９］ セロビオヒドロラーゼＩ遺伝子プロモーターによるリグニンペルオキシダーゼ遺伝子の発現ベクターの構築
実施例１３で得られたプラスミドｐＣＨＣＢＨＩ３１Ｐを制限酵素ＮｃｏＩ（宝酒造社製）で消化し、発現ベクター部分とした。
次に、コリオラス・ヒルスタス由来高温誘導リグニンペルオキシダーゼ遺伝子を含むプラスミドｐＢＳＬＰＯＧ７／Ｅ．ｃｏｌｉ ＪＭ１０９（ＦＥＲＭ Ｐ−１２６８３）を以下に示す２つのプライマー（７，８）を用いてＰＣＲ法により増幅した。

得られたＰＣＲ断片を上記のＴＡ−ｃｌｏｎｉｎｇベクターへ導入し、プラスミドｐＴＡＬｉＰを得た。
プラスミドｐＴＡＬｉＰを制限酵素ＮｃｏＩで消化し、リグニンペルオキシダーゼ構造遺伝子部分を得、上記処理したセロビオヒドロラーゼＩ発現ベクターｐＣＨＣＢＨＩ３１Ｐへ導入した。
得られたプラスミドをｐＣＨＣＢＨＩ３１ＰＬｉＰと命名した（図３）。
［実施例２０］ リグニンペルオキシダーゼを高分泌生産するコリオラス・ヒルスタス形質転換体の作製
実施例１９で得たｐＣＨＣＢＨＩ３１ＰＬｉＰを用いてアルギニン要求性コリオラス・ヒルスタスＯＪＩ−１０７８株（ＦＥＲＭ ＢＰ−４２１０）を形質転換する場合、選択マーカーとしてコリオラス・ヒルスタス由来のオルニチンカルバモイルトランスフェラーゼ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、もしくは直鎖状に拘らず、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。
約１０^６個／１００μｌ濃度のプロトプラスト溶液を１００μｌに対し実施例９で作製したプラスミドｐＣＨＣＢＨＩ３１ＰＬｉＰ 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ペル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２１］ 形質転換体によるリグニンペルオキシダーゼの生産
上記実施例２０で得られた形質転換株を５００ｍｌ容の三角フラスコに５０ｍｌのパルプ・ペプトン液体培地（晒パルプ３０ｇ／ｌ、ペプトン１０ｇ／ｌ、ＫＨ_２ＰＯ_４１．５ｇ／ｌ、ＭｇＳＯ_４・７Ｈ_２Ｏ０．５ｇ／ｌ、塩酸チアミン２ｍｇ／ｌ、５ｍＭベラトリルアルコール、リン酸でｐＨ４．５に調整）に５０ｍｍ^２の寒天片を５個接種し、２８℃で６日間、振盪下で培養した。６日後、得られる培養液を遠心分離し、その上清を得た。
酵素活性は１Ｍ酒石酸ナトリウム緩衝液（ｐＨ３．０）１００μｌ、８ｍＭベラトリルアルコール２５μｌ、酵素液３５０μｌ、５．４ｍＭ過酸化水素２５μｌを加え、反応の結果生じるベラトルアルデヒドを３１０ｎｍの吸光度増加を時間を追って記録することにより行い、上記培養上清中に酵素活性が培養開始５日目で０．８ユニット／ｍｌ認められた。ここで酵素活性単位は１分間に１μｍｏｌのベラトルアルデヒドを生成するのに必要とされる酵素量と定義する。一方、同条件下で培養した供与ＤＮＡを含まないコリオラス・ヒルスタスＯＪＩ−１０７８株（ＦＥＲＭ ＢＰ−４２１０）の培養上清にはリグニンペルオキシダーゼ活性は認められなかった。
３．セロビオヒドロラーゼＩ遺伝子のプロモーター配列を含む第２のＤＮＡ断片を用いたリグニン分解酵素の生産と利用
［実施例２２］ 染色体遺伝子ライブラリーからのセロビオヒドロラーゼＩ遺伝子の単離
実施例１２と同様にして、染色体遺伝子ライブラリーからクローンの選抜を行った。プラークハイブリダイゼーションに用いたプローブは下記配列番号２８の配列を持つ合成オリゴマーをアマシャム社製のオリゴＤＮＡ標識キットを用い３’末端をフルオレセインで標識したものである。

ただし、配列番号２８で塩基Ｙは塩基ＴまたはＣを示し、塩基Ｒは塩基ＧまたはＡを示す。
その結果、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、上記の合成ＤＮＡを用いてサザンハイブリダイゼーションを行った。その結果、制限酵素ＳａｌＩで消化して得られた断片中に単一のＤＮＡバンドとして４．２ｋｂｐにハイブリダイズするクローンが認められた。
上記ＤＮＡ断片４．２ｋｂｐ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ｐＵＣ１９のＳａｌＩサイトにサブクローン化し、大腸菌ＪＭ１０９株へ形質転換し、プラスミドｐＣＨＣＢＨＩ２７を得た。サブクローン化したＤＮＡ断片の塩基配列を決定した。塩基配列の決定はＡｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製のＢｉｇＤｙｅ Ｔｅｒｍｉｎａｔｏｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇキットを用いて反応を行い、Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製ＤＮＡシークエンサーＰＲＩＳＭ ３１０を用いて泳動を行い、塩基配列を解析した。
塩基配列を配列番号２６に示す。その結果、上記塩基配列の範囲内においてコリオラス・ヒルスタス由来セロビオヒドロラーゼＩ遺伝子は２つのイントロンにより分断されていた。また、塩基配列から推定されるアミノ酸配列を配列番号２７に示す。
［実施例２３］ セロビオヒドロラーゼＩ遺伝子プロモーターによるマンガンペルオキシダーゼ遺伝子の発現ベクターの構築
コリオラス・ヒルスタス由来セロビオヒドロラーゼＩ遺伝子プロモーター領域を含むプラスミドｐＣＨＣＢＨＩ２７から以下に示す２つのプライマー（１，２）を用いてＰＣＲ法により、プロモーター領域２．１ｋｂｐとして増幅し、ｐＵＣ１８のＳｍａＩサイトへＴ４ＤＮＡリガーゼを用いて連結し、プラスミドｐＣＨＣＢＨＩ２７Ｐを得た。

次に、コリオラス・ヒルスタス由来マンガンペルオキシダーゼ遺伝子を含むプラスミドｐＢＳＭＰＯＧ１（寄託番号ＦＥＲＭ Ｐ−１４９３３）を以下に示す２つのプライマー（３，４）を用いてＰＣＲ法によりマンガンペルオキシダーゼ構造遺伝子部分ＮｃｏＩ約２．２ｋｂ断片を増幅する。

得られたＰＣＲ断片をＩｎ Ｖｉｔｒｏｇｅｎ社製のＴＡ ｃｌｏｎｉｎｇ ｋｉｔを用いて挿入し、ｐＴＡＭＰとした。次に、得られたｐＴＡＭＰを制限酵素ＮｃｏＩで消化し、約２．２ｋｂを切り出し、上記ｐＣＨＣＢＨＩ２７ＰのＮｃｏＩサイトへ挿入し、プラスミドｐＣＨＣＢＨＩ２７ＰＭＰを得た（図４）。
［実施例２４］ 形質転換体
実施例４と同様にして、プロトプラストを調製・精製した。次いで、以下のようにして形質転換を行った。
１０^６個／１００μｌ濃度のプロトプラスト溶液１００μｌに対し、実施例２３で作製したプラスミドｐＣＨＣＢＨＩ２７ＰＭＰ 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コリオラス・ヒルスタス由来のオルニチンカルバモイルトランスフェラーゼ遺伝子を保持するｐＵＣＲ１を０．２μｇ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、目的とするマンガンペル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２５］ 形質転換体によるマンガンペルオキシダーゼの生産
上記実施例２４で得られた形質転換株を５００ｍｌ容の三角フラスコに５０ｍｌのパルプ・ペプトン液体培地（晒パルプ３０ｇ／ｌ、ペプトン１０ｇ／ｌ、ＫＨ_２ＰＯ_４１．５ｇ／ｌ、ＭｇＳＯ_４・７Ｈ_２Ｏ０．５ｇ／ｌ、塩酸チアミン２ｍｇ／ｌ、ＭｎＳＯ_４・５Ｈ_２Ｏ４８ｍｇ／ｌ、リン酸でｐＨ５．０に調整）に５０ｍｍ^２の寒天片を５個接種し、２８℃で６日間、振盪下で培養した。６日後、得られる培養液を遠心分離し、その上清を得た。
酵素活性は０．５Ｍマロン酸ナトリウム緩衝液（ｐＨ５．５）５０μｌ、酵素液３４５μｌ、１０ｍＭ過酸化水素５μｌ、１ｍＭ ＭｎＳＯ_４１００μｌ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）マロン酸複合体を酵素活性が培養開始６日目で８．５μｍｏｌ／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を含まないコリオラス・ヒルスタスＯＪＩ−１０７８株（ＦＥＲＭ ＢＰ−４２１０）の培養上清には本活性は認められなかった。
［実施例２６］ セロビオヒドロラーゼＩ遺伝子プロモーターによるラッカーゼ遺伝子の発現ベクターの構築
実施例２３で得られたプラスミドｐＣＨＣＢＨＩ２７Ｐを制限酵素ＮｃｏＩ（宝酒造社製）で消化後、Ｋｌｅｎｏｗ ｆｒａｇｍｅｎｔを用いて平滑化した。その後、制限酵素ＥｃｏＲＩで消化し、発現ベクター部分とした。
次に、コリオラス・ヒルスタス由来ラッカーゼ遺伝子を含むプラスミドＯＪ−ＰＯＧ−Ｅ１（ＦＥＲＭ ＢＰ−２７９３）を以下に示す２つのプライマー（５，６）を用いてＰＣＲ法により増幅した。

得られたＰＣＲ断片を上記のＴＡ−ｃｌｏｎｉｎｇベクターへ導入し、プラスミドｐＴＡＬＡＣを得た。
プラスミドｐＴＡＬＡＣを制限酵素ＸｈｏＩで消化後、修飾酵素ｋｌｅｎｏｗ ｆｒａｇｍｅｎｔを用いて平滑化する。その後、制限酵素ＥｃｏＲＩで消化し、ラッカーゼ構造遺伝子部分を得、上記処理したセロビオヒドロラーゼＩ発現ベクターｐＣＨＣＢＨＩ２７Ｐへ導入した。
得られたプラスミドをｐＣＨＣＢＨＩ２７ＰＬＡＣと命名した（図５）。
［実施例２７］ ラッカーゼを高分泌生産するコリオラス・ヒルスタス形質転換体の作製
実施例２６で得たｐＣＨＣＢＨＩ２７ＰＬＡＣを用いてアルギニン要求性コリオラス・ヒルスタスＯＪＩ−１０７８株（ＦＥＲＭ ＢＰ−４２１０）を形質転換する場合、選択マーカーとしてコリオラス・ヒルスタス由来の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、もしくは直鎖状に拘らず、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。
約１０^６個／１００μｌ濃度のプロトプラスト溶液を１００μｌに対し実施例２６で作製したプラスミドｐＣＨＣＢＨＩ２７ＰＬＡＣ 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、目的とするラッカ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２８］ 形質転換体によるラッカーゼの生産
上記実施例２７で得られた形質転換株を５００ｍｌ容の三角フラスコに５０ｍｌのパルプ・ペプトン液体培地（晒パルプ３０ｇ／ｌ、ペプトン１０ｇ／ｌ、ＫＨ_２ＰＯ_４１．５ｇ／ｌ、ＭｇＳＯ_４・７Ｈ_２Ｏ０．５ｇ／ｌ、塩酸チアミン２ｍｇ／ｌ、ＣｕＳＯ_４・５Ｈ_２Ｏ１００ｍｇ／ｌ、リン酸でｐＨ５．０に調整）に５０ｍｍ^２の寒天片を５個接種し、２８℃で６日間、振盪下で培養した。６日後、得られる培養液を遠心分離し、その上清を得た。
酵素活性は１Ｍ酢酸ナトリウム緩衝液（ｐＨ４．０）５０μｌ、５ｍＭ ＡＢＴＳ（２，２’−ａｚｉｎｏ−ｂｉｓ（３−ｅｔｈｉｌｂｅｎｚｔｈｉａｚｏｌｉｎｅ−６−ｓｕｌｆｏｎａｔｅ）５０μｌ、酵素液４００μｌを加え、反応の結果生じるＡＢＴＳの酸化物を４２０ｎｍの吸光度増加を時間を追って記録することにより行い、上記培養上清中に酵素活性が培養開始５日目で５７ユニット／ｍｌ認められた。ここで酵素活性単位は１分間に１μｍｏｌのＡＢＴＳを酸化するのに必要とされる酵素量と定義する。一方、同条件下で培養した供与ＤＮＡを含まないコリオラス・ヒルスタスＯＪＩ−１０７８株（ＦＥＲＭ ＢＰ−４２１０）の培養上清にはわずか５ユニット／ｍｌしか認められなかった。
［実施例２９］ セロビオヒドロラーゼＩ遺伝子プロモーターによるリグニンペルオキシダーゼ遺伝子の発現ベクターの構築
実施例２３で得られたプラスミドｐＣＨＣＢＨＩ２７Ｐを制限酵素ＮｃｏＩ（宝酒造社製）で消化し、発現ベクター部分とした。
次に、コリオラス・ヒルスタス由来高温誘導リグニンペルオキシダーゼ遺伝子を含むプラスミドｐＢＳＬＰＯＧ７／Ｅ．ｃｏｌｉ ＪＭ１０９（ＦＥＲＭ Ｐ−１２６８３）を以下に示す２つのプライマー（７，８）を用いてＰＣＲ法により増幅した。

得られたＰＣＲ断片を上記のＴＡ−ｃｌｏｎｉｎｇベクターへ導入し、プラスミドｐＴＡＬｉＰを得た。
プラスミドｐＴＡＬｉＰを制限酵素ＮｃｏＩで消化し、リグニンペルオキシダーゼ構造遺伝子部分を得、上記処理したセロビオヒドロラーゼＩ発現ベクターｐＣＨＣＢＨＩ２７Ｐへ導入した。
得られたプラスミドをｐＣＨＣＢＨＩ２７ＰＬｉＰと命名した（図６）。
［実施例３０］ リグニンペルオキシダーゼを高分泌生産するコリオラス・ヒルスタス形質転換体の作製
実施例２９で得たｐＣＨＣＢＨＩ２７ＰＬｉＰを用いてアルギニン要求性コリオラス・ヒルスタスＯＪＩ−１０７８株（ＦＥＲＭ ＢＰ−４２１０）を形質転換する場合、選択マーカーとしてコリオラス・ヒルスタス由来のオルニチンカルバモイルトランスフェラーゼ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、もしくは直鎖状に拘らず、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。
約１０^６個／１００μｌ濃度のプロトプラスト溶液を１００μｌに対し実施例２９で作製したプラスミドｐＣＨＣＢＨＩ２７ＰＬｉＰ 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ペル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３１］ 形質転換体によるリグニンペルオキシダーゼの生産
上記実施例３０で得られた形質転換株を５００ｍｌ容の三角フラスコに５０ｍｌのパルプ・ペプトン液体培地（晒パルプ３０ｇ／ｌ、ペプトン１０ｇ／ｌ、ＫＨ_２ＰＯ_４１．５ｇ／ｌ、ＭｇＳＯ_４・７Ｈ_２Ｏ０．５ｇ／ｌ、塩酸チアミン２ｍｇ／ｌ、５ｍＭベラトリルアルコール、リン酸でｐＨ４．５に調整）に５０ｍｍ^２の寒天片を５個接種し、２８℃で６日間、振盪下で培養した。６日後、得られる培養液を遠心分離し、その上清を得た。
酵素活性は１Ｍ酒石酸ナトリウム緩衝液（ｐＨ３．０）１００μｌ、８ｍＭベラトリルアルコール２５μｌ、酵素液３５０μｌ、５．４ｍＭ過酸化水素２５μｌを加え、反応の結果生じるベラトルアルデヒドを３１０ｎｍの吸光度増加を時間を追って記録することにより行い、上記培養上清中に酵素活性が培養開始５日目で１．５ユニット／ｍｌ認められた。ここで酵素活性単位は１分間に１μｍｏｌのベラトルアルデヒドを生成するのに必要とされる酵素量と定義する。一方、同条件下で培養した供与ＤＮＡを含まないコリオラス・ヒルスタスＯＪＩ−１０７８株（ＦＥＲＭ ＢＰ−４２１０）の培養上清にはリグニンペルオキシダーゼ活性は認められなかった。
４．セロビオヒドロラーゼＩ遺伝子のプロモーター配列を含む第３のＤＮＡ断片を用いたリグニン分解酵素の生産と利用
［実施例３２］ 染色体遺伝子ライブラリーからのセロビオヒドラーゼＩ遺伝子の単離
実施例１２と同様にして、染色体遺伝子ライブラリーからクローンの選抜を行った。プラークハイブリダイゼーションに用いたプローブは下記配列番号４０の配列を持つ合成オリゴマーをアマシャム社製のオリゴＤＮＡ標識キットを用い３’末端をフルオレセインで標識したものである。

ただし、配列番号４０で塩基Ｙは塩基ＴまたはＣを示し、塩基Ｒは塩基ＧまたはＡを示す。
その結果、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、上記の合成ＤＮＡを用いてサザンハイブリダイゼーションを行った。その結果、制限酵素ＰｓｔＩならびにＮｈｅＩで消化して得られた断片中に単一のＤＮＡバンドとして３．９ｋｂｐにハイブリダイズするクローンが認められた。
上記ＤＮＡ断片３．９ｋｂｐ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ｐＢｌｕｅｓｃｒｉｐｔｓＩＩ ＳＫ−のＰｓｔＩ−ＳｐｅＩサイトにサブクローン化し、大腸菌ＪＭ１０９株へ形質転換し、プラスミドｐＣＨＣＢＨＩ２６を得た。サブクローン化したＤＮＡ断片の塩基配列を決定した。塩基配列の決定はＡｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製のＢｉｇＤｙｅ Ｔｅｒｍｉｎａｔｏｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇキットを用いて反応を行い、Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製ＤＮＡシークエンサーＰＲＩＳＭ ３１０を用いて泳動を行い、塩基配列を解析した。
塩基配列を配列番号３８に示す。その結果、上記塩基配列の範囲内においてコリオラス・ヒルスタス由来セロビオヒドロラーゼＩ遺伝子は２つのイントロンにより分断されていた。また、塩基配列から推定されるアミノ酸配列を配列番号３９に示す。
［実施例３３］ セロビオヒドロラーゼＩ遺伝子プロモーターによるマンガンペルオキシダーゼ遺伝子の発現ベクターの構築
コリオラス・ヒルスタス由来セロビオヒドロラーゼＩ遺伝子プロモーター領域を含むプラスミドｐＣＨＣＢＨＩ２６を制限酵素ＮｃｏＩで消化し、ｋｌｅｎｏｗ ｆｒａｇｍｅｎｔを用いて平滑末端とした後、Ｔ４ＤＮＡリガーゼを用いて自己連結を生じさせ、ＮｃｏＩサイトが消失したプラスミドｐＣＢＣＢＨＩ２６−ＮｃｏＩを得た。次に上記プラスミドを以下に示す２つのプライマー（１，２）を用いてＰＣＲ法により、プロモーター領域１．１ｋｂｐとして増幅し、ｐＵＣ１８のＳｍａＩサイトへＴ４ＤＮＡリガーゼを用いて連結し、プラスミドｐＣＨＣＢＨＩ２６Ｐを得た。

次に、コリオラス・ヒルスタス由来マンガンペルオキシダーゼ遺伝子を含むプラスミドｐＢＳＭＰＯＧ１（寄託番号ＦＥＲＭ Ｐ−１４９３３）を以下に示す２つのプライマー（３，４）を用いてＰＣＲ法によりマンガンペルオキシダーゼ構造遺伝子部分ＮｃｏＩ約２．２ｋｂ断片を増幅する。

得られたＰＣＲ断片をＩｎ Ｖｉｔｒｏｇｅｎ社製のＴＡ ｃｌｏｎｉｎｇ ｋｉｔを用いて挿入し、ｐＴＡＭＰとした。次に、得られたｐＴＡＭＰを制限酵素ＮｃｏＩで消化し、約２．２ｋｂを切り出し、上記ｐＣＨＣＢＨＩ２６ＰのＮｃｏＩサイトへ挿入し、プラスミドｐＣＨＣＢＨＩ２６ＰＭＰを得た（図７）。［実施例３４］ コリオラス・ヒルスタス（Ｃｏｒｉｏｌｕｓ ｈｉｒｓｕｔｕｓ）形質転換法
実施例４と同様にして、プロトプラストを調製・精製した。次いで、以下のようにして形質転換を行った。
１０^６個／１００μｌ濃度のプロトプラスト溶液１００μｌに対し、実施例３３で作製したプラスミドｐＣＨＣＢＨＩ２６ＰＭＰ 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コリオラス・ヒルスタス由来のオルニチンカルバモイルトランスフェラーゼ遺伝子を保持するｐＵＣＲ１を０．２μｇ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、目的とするマンガンペル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３５］ 形質転換体によるマンガンペルオキシダーゼの生産
上記実施例３４で得られた形質転換株を５００ｍｌ容の三角フラスコに５０ｍｌのパルプ・ペプトン液体培地（晒パルプ３０ｇ／ｌ、ペプトン１０ｇ／ｌ、ＫＨ_２ＰＯ_４１．５ｇ／ｌ、ＭｇＳＯ_４・７Ｈ_２Ｏ０．５ｇ／ｌ、塩酸チアミン２ｍｇ／ｌ、ＭｎＳＯ_４・５Ｈ_２Ｏ４８ｍｇ／ｌ、リン酸でｐＨ５．０に調整）に５０ｍｍ^２の寒天片を５個接種し、２８℃で６日間、振盪下で培養した。６日後、得られる培養液を遠心分離し、その上清を得た。
酵素活性は０．５Ｍマロン酸ナトリウム緩衝液（ｐＨ５．５）５０μｌ、酵素液３４５μｌ、１０ｍＭ過酸化水素５μｌ、１ｍＭ ＭｎＳＯ_４１００μｌ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）マロン酸複合体を酵素活性が培養開始６日目で５．５μｍｏｌ／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を含まないコリオラス・ヒルスタスＯＪＩ−１０７８株（ＦＥＲＭ ＢＰ−４２１０）の培養上清には本活性は認められなかった。
［実施例３６］ セロビオヒドロラーゼＩ遺伝子プロモーターによるラッカーゼ遺伝子の発現ベクターの構築
実施例３３で得られたプラスミドｐＣＨＣＢＨＩ２６Ｐを制限酵素ＮｃｏＩ（宝酒造社製）で消化後、Ｋｌｅｎｏｗ ｆｒａｇｍｅｎｔを用いて平滑化した。その後、制限酵素ＥｃｏＲＩで消化し、発現ベクター部分とした。
次に、コリオラス・ヒルスタス由来ラッカーゼ遺伝子を含むプラスミドＯＪ−ＰＯＧ−Ｅ１（寄託番号ＢＰ−２７９３）を以下に示す２つのプライマー（５，６）を用いてＰＣＲ法により増幅した。

得られたＰＣＲ断片を上記のＴＡ−ｃｌｏｎｉｎｇベクターへ導入し、プラスミドｐＴＡＬＡＣを得た。
プラスミドｐＴＡＬＡＣを制限酵素ＸｈｏＩで消化後、修飾酵素ｋｌｅｎｏｗ ｆｒａｇｍｅｎｔを用いて平滑化した。その後、制限酵素ＥｃｏＲＩで消化し、ラッカーゼ構造遺伝子部分を得、上記処理したセロビオヒドロラーゼＩ発現ベクターｐＣＨＣＢＨＩ２６Ｐへ導入した。
得られたプラスミドをｐＣＨＣＢＨＩ２６ＰＬＡＣと命名した（図８）。
［実施例３７］ ラッカーゼを高分泌生産するコリオラス・ヒルスタス形質転換体の作製
実施例３６で得たｐＣＨＣＢＨＩ２６ＰＬＡＣを用いてアルギニン要求性コリオラス・ヒルスタスＯＪＩ−１０７８株（ＦＥＲＭ ＢＰ−４２１０）を形質転換する場合、選択マーカーとしてコリオラス・ヒルスタス由来の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、もしくは直鎖状に拘らず、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。
約１０^６個／１００μｌ濃度のプロトプラスト溶液を１００μｌに対し実施例３６で作製したプラスミドｐＣＨＣＢＨＩ２６ＰＬＡＣ 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、目的とするラッカ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３８］ 形質転換体によるラッカーゼの生産
上記実施例３７で得られた形質転換株を５００ｍｌ容の三角フラスコに５０ｍｌのパルプ・ペプトン液体培地（晒パルプ３０ｇ／ｌ、ペプトン１０ｇ／ｌ、ＫＨ_２ＰＯ_４１．５ｇ／ｌ、ＭｇＳＯ_４・７Ｈ_２Ｏ０．５ｇ／ｌ、塩酸チアミン２ｍｇ／ｌ、ＣｕＳＯ_４・５Ｈ_２Ｏ１００ｍｇ／ｌ、リン酸でｐＨ５．０に調整）に５０ｍｍ^２の寒天片を５個接種し、２８℃で６日間、振盪下で培養した。６日後、得られる培養液を遠心分離し、その上清を得た。
酵素活性は１Ｍ酢酸ナトリウム緩衝液（ｐＨ４．０）５０μｌ、５ｍＭ ＡＢＴＳ（２，２’−ａｚｉｎｏ−ｂｉｓ（３−ｅｔｈｉｌｂｅｎｚｔｈｉａｚｏｌｉｎｅ−６−ｓｕｌｆｏｎａｔｅ）５０μｌ、酵素液４００μｌを加え、反応の結果生じるＡＢＴＳの酸化物を４２０ｎｍの吸光度増加を時間を追って記録することにより行い、上記培養上清中に酵素活性が培養開始５日目で５２ユニット／ｍｌ認められた。ここで酵素活性単位は１分間に１μｍｏｌのＡＢＴＳを酸化するのに必要とされる酵素量と定義する。一方、同条件下で培養した供与ＤＮＡを含まないコリオラス・ヒルスタスＯＪＩ−１０７８株（ＦＥＲＭ ＢＰ−４２１０）の培養上清にはわずか５ユニット／ｍｌしか認められなかった。
［実施例３９］ セロビオヒドロラーゼＩ遺伝子プロモーターによるリグニンペルオキシダーゼ遺伝子の発現ベクターの構築
実施例３３で得られたプラスミドｐＣＨＣＢＨＩ２６Ｐを制限酵素ＮｃｏＩ（宝酒造社製）で消化し、発現ベクター部分とした。
次に、コリオラス・ヒルスタス由来高温誘導リグニンペルオキシダーゼ遺伝子を含むプラスミドｐＢＳＬＰＯＧ７／Ｅ．ｃｏｌｉ ＪＭ１０９（ＦＥＲＭ Ｐ−１２６８３）を以下に示す２つのプライマー（７，８）を用いてＰＣＲ法により増幅した。

得られたＰＣＲ断片を上記のＴＡ−ｃｌｏｎｉｎｇベクターへ導入し、プラスミドｐＴＡＬｉＰを得た。
プラスミドｐＴＡＬｉＰを制限酵素ＮｃｏＩで消化し、リグニンペルオキシダーゼ構造遺伝子部分を得、上記処理したセロビオヒドロラーゼＩ発現ベクターｐＣＨＣＢＨＩ２６Ｐへ導入した。
得られたプラスミドをｐＣＨＣＢＨＩ２６ＰＬｉＰと命名した（図９）。
［実施例４０］ リグニンペルオキシダーゼを高分泌生産するコリオラス・ヒルスタス形質転換体の作製；
実施例３９で得たｐＣＨＣＢＨＩ２６ＰＬｉＰを用いてアルギニン要求性コリオラス・ヒルスタスＯＪＩ−１０７８株（ＦＥＲＭ ＢＰ−４２１０）を形質転換する場合、選択マーカーとしてコリオラス・ヒルスタス由来のオルニチンカルバモイルトランスフェラーゼ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、もしくは直鎖状に拘らず、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。
約１０^６個／１００μｌ濃度のプロトプラスト溶液を１００μｌに対し実施例３９で作製したプラスミドｐＣＨＣＢＨＩ２６ＰＬｉＰ 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ペル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４１］ 形質転換体によるリグニンペルオキシダーゼの生産
上記実施例４０で得られた形質転換株を５００ｍｌ容の三角フラスコに５０ｍｌのパルプ・ペプトン液体培地（晒パルプ３０ｇ／ｌ、ペプトン１０ｇ／ｌ、ＫＨ_２ＰＯ_４１．５ｇ／ｌ、ＭｇＳＯ_４・７Ｈ_２Ｏ０．５ｇ／ｌ、塩酸チアミン２ｍｇ／ｌ、５ｍＭベラトリルアルコール、リン酸でｐＨ４．５に調整）に５０ｍｍ^２の寒天片を５個接種し、２８℃で６日間、振盪下で培養した。６日後、得られる培養液を遠心分離し、その上清を得た。
酵素活性は１Ｍ酒石酸ナトリウム緩衝液（ｐＨ３．０）１００μｌ、８ｍＭベラトリルアルコール２５μｌ、酵素液３５０μｌ、５．４ｍＭ過酸化水素２５μｌを加え、反応の結果生じるベラトルアルデヒドを３１０ｎｍの吸光度増加を時間を追って記録することにより行い、上記培養上清中に酵素活性が培養開始５日目で１．０ユニット／ｍｌ認められた。ここで酵素活性単位は１分間に１μｍｏｌのベラトルアルデヒドを生成するのに必要とされる酵素量と定義する。一方、同条件下で培養した供与ＤＮＡを含まないコリオラス・ヒルスタスＯＪＩ−１０７８株（ＦＥＲＭ ＢＰ−４２１０）の培養上清にはリグニンペルオキシダーゼ活性は認められなかった。
５．セロビオヒドロラーゼＩＩ遺伝子のプロモーター配列を含むＤＮＡ断片を用いたリグニン分解酵素の生産と利用
［実施例４２］ 染色体遺伝子ライブラリーからのセロビオヒドロラーゼＩＩ遺伝子のプロモーター配列を含むＤＮＡ断片の単離
実施例１と同様にして、染色体遺伝子ライブラリーを得た。得られた該ライブラリーからプラークハイブリダイゼーションによりセロビオヒドロラーゼＩＩ遺伝子のプロモーター領域を含むクローンの選抜を行った。この一連の操作は常法（Ｓａｍｂｒｏｏｋら著、Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ／２ｎｄ Ｅｄｉｔｉｏｎ（１９８９））によった。プラークハイブリダイショーンに用いたプローブは、ファネロケエテ・クリソスポリウムのセロビオヒドロラーゼＩＩの塩基配列に基づいて作製した、以下の配列番号５２に示す合成オリゴマーをアマシャム社製のオリゴＤＮＡ標識キットを用い３’末端をフルオレセインで標識したものである。

その結果、約１００，０００個のプラークの中から８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、上記の合成ＤＮＡを用いてサザンハイブリダイゼーションを行った。その結果、制限酵素ＥｃｏＲＶならびにＮｃｏＩで消化して得られた断片中に単一のＤＮＡバンドとして５．０ｋｂｐにハイブリダイズするクローンが認められた。
上記ＤＮＡ断片を回収するため、制限酵素ＮｃｏＩで消化後Ｋｌｅｎｏｗ断片により平滑化し、さらにＥｃｏＲＶで消化することにより得られる５．０ｋｂｐのＤＮＡ断片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ｐＵＣ１９のＳｍａＩサイトにサブクローン化し、プラスミドｐＣＨＣＢＨＩＩを得た。このプラスミドを大腸菌ＪＭ１０９株へ形質転換した。また、前記のサブクローン化したＤＮＡ断片の塩基配列を決定した。塩基配列の決定はＡｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製のＢｉｇＤｙｅ Ｔｅｒｍｉｎａｔｏｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇキットを用いて反応を行い、Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製ＤＮＡシークエンサーＰＲＩＳＭ ３１０を用いて泳動を行い、塩基配列を解析した。その塩基配列を配列番号５０に示す。その結果、上記塩基配列の範囲内において、コリオラス・ヒルスタス由来のセロビオヒドロラーゼＩＩ遺伝子は６つのイントロンにより分断されていた。また、塩基配列から推定されるアミノ酸配列を配列番号５１に示した。
［実施例４３］ セロビオヒドロラーゼＩＩ遺伝子プロモーターによるマンガンペルオキシダーゼ遺伝子の発現ベクターの構築
コリオラス・ヒルスタス由来セロビオヒドロラーゼＩＩ遺伝子のプロモーター配列を含む、実施例４２で得られたプラスミドｐＣＨＣＢＨＩＩから、以下の配列番号５３及び５４に示す２つのプライマーを用い、ＰＣＲ法により増幅し、プロモーター領域３．１ｋｂｐを得た。

このＰＣＲにより得られたＤＮＡ断片を大腸菌クローニングベクターｐＵＣ１８のＳｍａＩサイトへＴ４ＤＮＡリガーゼを用いて連結し、プラスミドｐＣＨＣＢＨＩＩＰを得た。
次に、コリオラス・ヒルスタス由来マンガンペルオキシダーゼ遺伝子を含むプラスミドｐＢＳＭＰＯＧ１（ＦＥＲＭ Ｐ−１４９３３）を、以下の配列番号５５及び５６に示す２つのプライマーを用い、ＰＣＲ法により増幅し、マンガンペルオキシダーゼ構造遺伝子ＮｃｏＩ切断部分約２．２ｋｂ断片を得た。

このＰＣＲにより得られたＤＮＡ断片をＩｎ Ｖｉｔｒｏｇｅｎ社製のＴＡ ｃｌｏｎｉｎｇ ｋｉｔを用いてＴＡ ｃｌｏｎｉｎｇベクターに挿入し、ｐＴＡＭＰとした。次に、得られたｐＴＡＭＰを制限酵素ＮｃｏＩで消化し、約２．２ｋｂを切り出し、上記のプラスミドｐＣＨＣＢＨＩＩＰのＮｃｏＩサイトへ挿入し、コリオラス・ヒルスタス由来のセロビオヒドロラーゼＩＩ遺伝子プロモーター配列とマンガンペルオキシダーゼ構造遺伝子を含むプラスミドｐＣＨＣＢＨＩＩＰＭＰを得た（図１０）。
［実施例４４］ コリオラス・ヒルスタス形質転換体の作製
実施例４と同様にしてプロトプラストの調製・精製を行った。次いで、以下のようにして形質転換を行った。
１０^６個／１００μｌ濃度のプロトプラスト溶液１００μｌに対し、実施例３３で作製したプラスミドｐＣＨＣＢＨＩＩＰＭＰ ２μｇを環状、もしくは直鎖状で添加した。さらに選択マーカーとして、コリオラス・ヒルスタス由来のオルニチンカルバモイルトランスフェラーゼ遺伝子を保持するプラスミドｐＵＣＲ１を０．２μｇ添加し、３０分間氷冷した。次に、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。次に、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、目的とするマンガンペルオキシダーゼ発現プラスミドｐＣＨＣＢＨＩＩＰＭＰ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４５］ 形質転換体によるマンガンペルオキシダーゼの生産
５００ｍｌ容の三角フラスコに５０ｍｌのパルプ・ペプトン液体培地（晒パルプ３０ｇ／ｌ、ペプトン１０ｇ／ｌ、ＫＨ_２ＰＯ_４１．５ｇ／ｌ、ＭｇＳＯ_４・７Ｈ_２Ｏ０．５ｇ／ｌ、塩酸チアミン２ｍｇ／ｌ、ＭｎＳＯ_４・５Ｈ_２Ｏ４８ｍｇ／ｌ、リン酸でｐＨ５．０に調整）に、上記実施例４４で得られた形質転換株の５０ｍｍ^２の寒天片を５個接種し、２８℃で６日間、振盪下で培養した。６日後、得られる培養液を遠心分離し、その上清を得た。
酵素活性は０．５Ｍマロン酸ナトリウム緩衝液（ｐＨ５．５）５０μｌ、酵素液３４５μｌ、１０ｍＭ過酸化水素５μｌ、１ｍＭ ＭｎＳＯ_４１００μｌ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った。
その結果、上記培養上清中には、マンガンペルオキシダーゼの酵素活性によって生成されるＭｎ（ＩＩＩ）マロン酸複合体が培養開始６日目で６μｍｏｌ／ｍｌ／ｍｉｎ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。
［実施例４６］ セロビオヒドロラーゼＩＩ遺伝子プロモーターによるラッカーゼ遺伝子の発現ベクターの構築
実施例４３で得られたプラスミドｐＣＨＣＢＨＩＩＰを制限酵素ＮｃｏＩ（宝酒造社製）で消化後、Ｋｌｅｎｏｗ ｆｒａｇｍｅｎｔを用いて平滑化した。その後、制限酵素ＥｃｏＲＩで消化し、セロビオヒドロラーゼＩＩ遺伝子プロモーター発現ベクター部分とした。
次に、コリオラス・ヒルスタス由来ラッカーゼ遺伝子を含むプラスミドＯＪ−ＰＯＧ−Ｅ１（ＢＰ−２７９３）を以下に示す配列番号５７及び５８のプライマーを用いてＰＣＲ法により増幅した。

得られたＰＣＲによるＤＮＡ断片を実施例３と同様にＴＡ−ｃｌｏｎｉｎｇベクターへ導入し、プラスミドｐＴＡＬＡＣを得た。
プラスミドｐＴＡＬＡＣを制限酵素ＸｈｏＩで消化後、修飾酵素ｋｌｅｎｏｗ ｆｒａｇｍｅｎｔを用いて平滑化した。その後、制限酵素ＥｃｏＲＩで消化し、ラッカーゼ構造遺伝子部分を得た。
実施例４３で得られたセロビオヒドロラーゼＩＩ発現ベクターｐＣＨＣＢＨＩＩＰのＮｃｏＩサイトへラッカーゼ構造遺伝子部分を導入し、プラスミドｐＣＨＣＢＨＩＩＰＬＡＣを得た（図１１）。
［実施例４７］ ラッカーゼを高分泌生産するコリオラス・ヒルスタス形質転換体の作製
実施例４４記載の方法により、コリオラス・ヒルスタスのプロトプラスト溶液を得た。約１０^６個／１００μｌのプロトプラスト溶液に対し実施例４６で作製したプラスミドｐＣＨＣＢＨＩＩＰＬＡＣ ２μｇを環状もしくは直鎖状で添加した。さらに選択マーカーとして、コリオラス・ヒルスタス由来のオルニチンカルバモイルトランスフェラーゼ遺伝子を保持するプラスミド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シュークロース及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捲いた。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、目的とするラッカーゼ発現プラスミドｐＣＨＣＢＨＩＩＰＬＡＣ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４８］ 形質転換体によるラッカーゼの生産
５００ｍｌ容の三角フラスコに５０ｍｌのパルプ・ペプトン液体培地（晒パルプ３０ｇ／ｌ、ペプトン１０ｇ／ｌ、ＫＨ_２ＰＯ_４１．５ｇ／ｌ、ＭｇＳＯ_４・７Ｈ_２Ｏ０．５ｇ／ｌ、塩酸チアミン２ｍｇ／ｌ、ＣｕＳＯ_４・５Ｈ_２Ｏ１００ｍｇ／ｌ、リン酸でｐＨ５．０に調整）に、上記実施例４７で得られた形質転換株の５０ｍｍ^２の寒天片を５個接種し、２８℃で６日間、振盪下で培養した。６日後、得られる培養液を遠心分離し、その上清を得た。
酵素活性は１Ｍ酢酸ナトリウム緩衝液（ｐＨ４．０）５０μｌ、５ｍＭ ＡＢＴＳ（２，２’−ａｚｉｎｏ−ｂｉｓ（３−ｅｔｈｉｌｂｅｎｚｔｈｉａｚｏｌｉｎｅ−６−ｓｕｌｆｏｎａｔｅ）５０μｌ、酵素液４００μｌを加え、反応の結果生じるＡＢＴＳの酸化物を４２０ｎｍの吸光度増加を時間を追って記録することにより行った。その結果、上記培養上清中に酵素活性が培養開始５日目で４０ユニット／ｍｌ認められた。ここで酵素活性単位（ユニット）は、１分間に１μｍｏｌのＡＢＴＳを酸化するのに必要とされる酵素量とした。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、酵素活性はわずか５ユニット／ｍｌしか認められなかった。
［実施例４９］ セロビオヒドロラーゼＩＩ遺伝子プロモーターによるリグニンペルオキシダーゼ遺伝子の発現ベクターの構築
実施例４３で得られたプラスミドｐＣＨＣＢＨＩＩＰを制限酵素ＮｃｏＩ（宝酒造社製）で消化し、セロビオヒドロラーゼＩＩ発現ベクター部分とした。
次に、コリオラス・ヒルスタス由来高温誘導リグニンペルオキシダーゼ遺伝子を含むプラスミドｐＢＳＬＰＯＧ７／Ｅ．ｃｏｌｉ ＪＭ１０９（ＦＥＲＭ Ｐ−１２６８３）を以下に示す配列番号５９及び６０の２つのプライマーを用いてＰＣＲ法により増幅した。

得られたＰＣＲ断片を実施例４３と同様にＴＡ−ｃｌｏｎｉｎｇベクターへ導入し、プラスミドｐＴＡＬｉＰを得た。次いで、プラスミドｐＴＡＬｉＰを制限酵素ＮｃｏＩで消化し、リグニンペルオキシダーゼ構造遺伝子部分を得た。
実施例４３で得られたセロビオヒドロラーゼＩＩ発現ベクターｐＣＨＣＢＨＩＩＰのＮｃｏＩサイトへリグニンペルオキシダーゼ構造遺伝子部分を導入し、プラスミドｐＣＨＣＢＨＨＰＬｉＰを得た（図１２）。
［実施例５０］ リグニンペルオキシダーゼを高分泌生産するコリオラス・ヒルスタス形質転換体の作製
実施例４４に記載の方法により、コリオラス・ヒルスタスのプロトプラスト溶液を得た。約１０^６個／１００μｌのプロトプラスト溶液に対し実施例４９で作製したプラスミドｐＣＨＣＢＨＩＩＰＬｉＰ ２μｇを環状もしくは直鎖状で添加した。さらに選択マーカーとして、コリオラス・ヒルスタス由来のオルニチンカルバモイルトランスフェラーゼ遺伝子を保持するプラスミド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ペルオキシダーゼ発現プラスミドｐＣＨＣＢＨＩＩＰＬｉＰ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５１］ 形質転換体によるリグニンペルオキシダーゼの生産
５００ｍｌ容の三角フラスコに５０ｍｌのパルプ・ペプトン液体培地（晒パルプ３０ｇ／ｌ、ペプトン１０ｇ／ｌ、ＫＨ_２ＰＯ_４１．５ｇ／ｌ、ＭｇＳＯ_４・７Ｈ_２Ｏ０．５ｇ／ｌ、塩酸チアミン２ｍｇ／ｌ、５ｍＭベラトリルアルコール、リン酸でｐＨ４．５に調整）に上記実施例１０で得られた形質転換株の５０ｍｍ^２の寒天片を５個接種し、２８℃で６日間、振盪下で培養した。６日後、得られる培養液を遠心分離し、その上清を得た。
酵素活性は１Ｍ酒石酸ナトリウム緩衝液（ｐＨ３．０）１００μｌ、８ｍＭベラトリルアルコール２５μｌ、酵素液３５０μｌ、５．４ｍＭ過酸化水素２５μｌを加え、反応の結果生じるベラトルアルデヒドを３１０ｎｍの吸光度増加を時間を追って記録することにより行った。上記培養上清中に、酵素活性が培養開始５日目で１．２ユニット／ｍｌ認められた。ここで酵素活性単位（ユニット）は、１分間に１μｍｏｌのベラトルアルデヒドを生成するのに必要とされる酵素量とした。一方、同条件下で培養した供与ＤＮＡを含まないコリオラス・ヒルスタスＯＪＩ−１０７８株（ＦＥＲＭ ＢＰ−４２１０）の培養上清にはリグニンペルオキシダーゼ活性は認められなかった。
［実施例５２］ マンガンペルオキシダーゼを高分泌生産する形質転換体を用いたチップ処理
実施例４４により選抜したマンガンペルオキシダーゼ高分泌生産する形質転換株をポテトデキストロース寒天培地上で２８℃にて培養した後、４℃で保存した。このプレートから直径５ｍｍのコルクボーラーで打ち抜いた切片を５つずつ、グルコース・ペプトン培地（グルコース２％、ポリペプトン０．５％、酵母エキス０．２％、ＫＨ_２ＰＯ_４、ＭｇＳＯ_４０．０５％、リン酸でｐＨ４．５に調製）を１００ｍｌずつ含む３００ｍｌ容三角フラスコに植菌し、２８℃、１００ｒｐｍで１週間震盪培養した。培養後、菌体をろ別し、菌体に残存した培地を滅菌水で洗浄した。菌体は滅菌水と共に、ワーリングブレンダーで１０ｓｅｃ粉砕し、絶乾重量１ｋｇのユーカリ材に対し、菌体の乾燥重量が１０ｍｇになるように植菌した。植菌後は菌が全体に行き渡るようによく撹拌した。培養は２８℃で通気をしながら１週間静置培養を行った。チップ含水率が４０〜６５％になるように随時飽和水蒸気を通気させた。通気する際の通気量は対チップ当り、０．０１ｖｖｍになるように行った。
［実施例５３］ マンガンペルオキシダーゼ高分泌生産する形質転換体による機械パルプの製造
ラジアータパイン材を用い実施例５２に準じてチップ処理を行った。処理後の木材チップをラボ用リファイナー（熊谷理機社製）を用いて叩解して、カナディアンスタンダードフリネスを２００ｍｌとした後、パルプ物理用試験用手抄きシートの調製はＴａｐｐｉ試験法Ｔ２０５ｏｍ−８１に準拠して、パルプ手抄きシートの物理試験はＴａｐｐｉ法Ｔ２２０ｏｍ−８３に準拠して行った。使用電力量はワットメーター（Ｈｉｏｋｉｄｅｎｋｉ ｍｏｄｅｌ３１３３）と積分計（ｍｏｄｅｌ３１４１）を用いた。チップ収率測定は水分を含んだ木材チップを容器に絶乾重量で１ｋｇ分取し、処理前後のチップ絶乾重量を測定し、以下の式を用いてチップ収率を算出した。
（処理後の絶乾重量）／（処理前の絶乾重量）×１００
表１に示すようにセロビオースデヒドロゲナーゼを抑制した形質転換株は収率減を抑制することができ、解繊エネルギーを削減できた。また、引裂き強さ、破裂強さ共に増加した。野生株で処理すると解繊エネルギーの削減効果は得られたものの、紙力は低下した。

［実施例５４］ マンガンペルオキシダーゼ高分泌生産する形質転換体により処理したチップの蒸解
実施例５２に準じてユーカリ材のチップ処理を行った後、絶乾重量４００ｇのチップを測りとりオートクレーブ内で液比５、硫化度３０％、有効アルカリ１７％（Ｎａ_２Ｏとして）となるように蒸解白液を加え、蒸解温度を１５０℃にてクラフト蒸解を行った。クラフト蒸解終了後、黒液を分離し、得られたチップを高濃度離解機によって解繊後、濾布で遠心脱水と水洗浄を３回繰り返した。次いでスクリーンにより、未蒸解物を除き、遠心脱水し蒸解未漂白パルプを得た。
上記のクラフト蒸解して得られたパルプに対して、ＮａＯＨを２．０質量％添加し、酸素ガスを注入し、１００℃、酸素ゲージ圧０．４９ＭＰａ（５ｋｇ／ｃｍ^２）で６０分間処理を行った。
上記で得たパルプを下記に示すように、Ｄ−Ｅ−Ｐ−Ｄの４段漂白処理を行った。最初の二酸化塩素処理（Ｄ）は、パルプ濃度が１０質量％となるように調製し、二酸化塩素を０．４質量％添加し、７０℃、４０分間処理を行った。次いで、イオン交換水にて洗浄、脱水後、パルプ濃度を１０質量％に調製し、苛性ソーダを１質量％添加し、７０℃、９０分間のアルカリ抽出処理（Ｅ）を行った。次いで、イオン交換水にて洗浄、脱水後、パルプ濃度を１０質量％に調製し、過酸化水素０．５質量％、苛性ソーダ０．５質量％を順次添加し、７０℃、１２０分間の過酸化水素処理（Ｐ）を行った。次いで、イオン交換水にて洗浄、脱水後、パルプ濃度を１０％に調製し、二酸化塩素０．２５質量％を添加し、７０℃、１８０分間二酸化塩素処理（Ｄ）を行った。最後にイオン交換水にて洗浄、脱水後、ＪＩＳ Ｐ ８１２３に準じた白色度８６．０％の漂白パルプを得た。
上記で得たパルプ濃度が４質量％のパルプスラリーをリファイナーによりフリーネスが４１０ｍｌ（ＣＳＦ）となるように叩解した。
［実施例５５］ Ｋａ価の測定、パルプ物理用試験用手抄きシートの調製、パルプ手抄きシートの物理試験
カッパー価の測定は、ＪＩＳ Ｐ ８２１１に準じて行った。パルプ物理用試験用手抄きシートの調製はＴａｐｐｉ試験法Ｔ２０５−ｏｍ８１に準拠して、パルプ手抄きシートの物理試験はＴａｐｐｉ法Ｔ２２０ｏｍ−８３に準拠して行った。表２に示すように、形質転換体、野生株で木材チップを処理すると蒸解後のＫａ価は減少し、精選収率は増加し、粕率は減少した。また、表３に示すように野生株と比較すると、形質転換体は紙力低下を引き起こさなかった。

産業上の利用性
本発明は、宿主の担子菌、特にコリオラス・ヒルスタスで機能し、かつ該宿主においてリグニン分解酵素を大量に生産させるためのプロモーター配列を提供するものであり、遺伝子組換えによる酵素の大量生産が困難とされてきた、マンガンペルオキシダーゼ、リグニンペルオキシダーゼおよびラッカーゼ等のリグニン分解酵素の効率的な生産を可能とし、さらに該酵素を高生産する宿主細胞を用いて木材チップを処理することができる。
本明細書で引用した全ての刊行物、特許及び特許出願をそのまま参考として本明細書にとり入れるものとする。
【配列表】

【図面の簡単な説明】
図１は、実施例１３に記載されるマンガンペルオキシダーゼ発現プラスミドｐＣＨＣＢＨＩ３１ＰＭＰの構造を示す図である。
図２は、実施例１６に記載されるラッカーゼ発現プラスミドｐＣＨＣＢＨＩ３１ＰＬＡＣの構造を示す図である。
図３は、実施例１９に記載されるリグニンペルオキシダーゼ発現ベクターｐＣＨＣＢＨＩ３１ＰＬｉＰの構造を示す図である。
図４は、実施例２３に記載されるマンガンペルオキシダーゼ発現プラスミドｐＣＨＣＢＨＩ２７ＰＭＰの構造を示す図である。
図５は、実施例２６に記載されるラッカーゼ発現プラスミドｐＣＨＣＢＨＩ２７ＰＬＡＣの構造を示す図である。
図６は、実施例２９に記載されるリグニンペルオキシダーゼ発現ベクターｐＣＨＣＢＨＩ２７ＰＬｉＰの構造を示す図である。
図７は、実施例３３に記載されるマンガンペルオキシダーゼ発現プラスミドｐＣＨＣＢＨＩ２６ＰＭＰの構造を示す図である。
図８は、実施例３６に記載されるラッカーゼ発現プラスミドｐＣＨＣＢＨＩ２６ＰＬＡＣの構造を示す図である。
図９は、実施例３９に記載されるリグニンペルオキシダーゼ発現ベクターｐＣＨＣＢＨＩ２６ＰＬｉＰの構造を示す図である。
図１０は、実施例４３に記載されるマンガンペルオキシダーゼ発現プラスミドｐＣＨＣＢＨＩＩＰＭＰの構造を示す図である。
図１１は、実施例４６に記載されるラッカーゼ発現プラスミドｐＣＨＣＢＨＩＩＰＬＡＣの構造を示す図である。
図１２は、実施例４９に記載されるリグニンペルオキシダーゼ発現プラスミドｐＣＨＣＢＨＩＩＰＬｉＰの構造を示す図である。Technical field
The present invention relates to a novel DNA fragment having promoter activity obtained from a basidiomycete, particularly Coriolus hirsutus, a recombinant DNA containing the same, a vector containing the DNA fragment or the recombinant DNA, and a host comprising the vector The present invention relates to a cell, a method for producing lignin-degrading enzyme using the host cell, and a wood chip treatment method using the enzyme.
Background art
To date, systems that produce various polypeptides using recombinant DNA technology have been performed using host systems centered on E. coli. However, E. coli is often not suitable as a host. For example, in Escherichia coli, when a useful polypeptide derived from a higher organism such as human is produced, it is not produced as an active enzyme protein, and many toxic substances other than the target polypeptide are produced, and the target product is highly purified. There are problems such as difficulty. As a means for solving these problems, methods for producing lower eukaryotic yeast as a host have been actively studied, but a new problem of low productivity has arisen. Therefore, for the production of polypeptides, transformation systems using higher organisms, eukaryotes, such as filamentous fungi such as Aspergillus genus, and basidiomycetes such as Phanerocheet genus and Coriolus genus, as hosts. Has been developed and enzyme protein production is being studied.
Basidiomycetes belong to eukaryotes, but are considered more closely related to animal cells than yeast (TL Smith, Proc. Natl. Acad. Sci. USA, 86, 7063 (1989)). Coriolus hirsutus, which has strong lignin-degrading ability, is a basidiomycete belonging to the genus Coriolus. It has been successful (Japanese Patent Laid-Open No. 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, the productivity of lignin peroxidase is low, and the wild strain IFO 4917 is produced from lignin peroxidase. It was the same level as that produced in the production medium (low carbon / low nitrogen source), and lignin peroxidase was obtained as a secondary metabolite, and thus it took time for gene expression. 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)).
Therefore, interest in a promoter that functions in basidiomycetes and has a stronger transcriptional activity has increased, and the present applicant has previously explained the nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gene derived from Coriolus hirsutus. A DNA fragment comprising a ras gene promoter region or a priA gene promoter region derived from Coriolus hirsutus or Shiitake, and a host / vector system using the same (Japanese Patent Laid-Open No. 9-47289) A host / vector system (Japanese Patent Laid-Open No. 2000-342275) using the same has been found.
However, to date, further promoter DNA fragments that can effectively produce large amounts of useful polypeptides, particularly lignin-degrading enzymes in basidiomycetes, using a cellulolytic enzyme promoter, and host / vector systems using the fragments, Development of a high production method of lignin-degrading enzyme using the host / vector system and a wood chip treatment method using the enzyme has not been disclosed yet.
Disclosure of the invention
An object of the present invention is to provide a promoter for highly producing a useful polypeptide, particularly a lignin degrading enzyme, in a basidiomycete host. Another object of the present invention is to provide a host / vector system containing the promoter, a high production method of lignin degrading enzyme using the system, and a wood chip treatment method using the enzyme.
As a result of intensive studies to solve the above problems, the present inventors have focused on basidiomycetes expressed in the presence of woody biomass components such as cellulose, particularly the cellulolytic enzyme gene of Coriolus hirsutus, DNA fragments encoding various cellulolytic enzyme genes are cloned from the Hilstas chromosomal DNA restriction enzyme fragment, and the promoter region upstream of this gene is sequenced, and this promoter region encodes a useful polypeptide in the host / vector system. It was found to be effective for the expression of genes.
Further, downstream of the promoter region, a structural gene containing a signal peptide of a manganese peroxidase gene, a high temperature-inducible lignin peroxidase gene (Japanese Patent Laid-Open No. 5-260978) or a laccase gene cloned from Coriolus hirsutus is ligated in a host cell. By transferring to a certain ornithine carbamoyltransferase-deficient Coriolus hirsutus mutant, a manganese peroxidase, lignin peroxidase or laccase high-producing strain was successfully obtained, and the present invention was completed.
Therefore, the present invention provides the following (1) to (12).
(1) A recombinant DNA comprising a DNA fragment comprising a promoter sequence of a cellulose-degrading enzyme gene derived from a basidiomycete and a gene encoding a lignin degrading enzyme, wherein the gene is linked to the DNA fragment so that the gene can be transcribed And a step of preparing a vector containing high production of lignin-degrading enzyme by culturing the host cell producing high lignin-degrading enzyme in the presence of cellulose. A method for producing a lignin degrading enzyme, comprising a step of producing a degrading enzyme.
(2) Recombinant DNA comprising a DNA fragment containing a promoter sequence of a cellulose-degrading enzyme gene derived from a basidiomycete and a gene encoding lignin-degrading enzyme, wherein the gene is linked to the DNA fragment so that it can be transcribed A step of preparing a vector containing, a step of performing transformation using the vector to prepare a host cell with high production of lignin-degrading enzyme, and inoculating the host cell with high production of lignin-degrading enzyme into a wood chip for treatment A method for processing wood chips, comprising a process.
(3) The promoter sequence of the cellulolytic enzyme gene is a promoter sequence of cellobiose dehydrogenase gene, cellobiohydrolase I gene, cellobiohydrolase II gene, endoglucanase gene, or β-glucosidase gene (1) or (2) The method described in 1.
(4) The DNA fragment containing the cellulolytic enzyme gene promoter sequence is an isolated DNA fragment containing the cellobiose dehydrogenase gene promoter sequence having any of the following base sequences (a) to (c): (1) The method according to (2) or (3).
(A) The base sequence represented by SEQ ID NO: 1 or 2.
(B) A base sequence having a base sequence that hybridizes under stringent conditions with a base sequence having a base sequence complementary to the base sequence of (a), and having cellobiose dehydrogenase gene promoter activity.
(C) A base sequence having a base sequence in which one or more bases are deleted, substituted or added in SEQ ID NO: 1 or 2, and having cellobiose dehydrogenase gene promoter activity.
(5) An isolated DNA fragment containing a cellobiohydrolase I gene promoter sequence, wherein the DNA fragment containing a cellulolytic enzyme gene promoter sequence has any one of the following base sequences (a) to (c): The method according to (1), (2) or (3).
(A) The base sequence represented by SEQ ID NO: 13, 25 or 37.
(B) A base sequence having a base sequence that hybridizes under stringent conditions with a base sequence having a base sequence complementary to the base sequence of (a) and having cellobiohydrolase I gene promoter activity.
(C) A base sequence having a base sequence in which one or more bases are deleted, substituted or added in SEQ ID NO: 13, 25 or 37 and having cellobiohydrolase I gene promoter activity.
(6) An isolated DNA fragment containing a cellobiohydrolase II gene promoter sequence, wherein the DNA fragment containing a cellulolytic enzyme gene promoter sequence has any of the following base sequences (a) to (c): The method according to (1), (2) or (3).
(A) The base sequence represented by SEQ ID NO: 49.
(B) A base sequence having a base sequence that hybridizes under stringent conditions with a base sequence having a base sequence complementary to the base sequence of (a) and having cellobiohydrolase II gene promoter activity.
(C) A base sequence having a base sequence in which one or more bases are deleted, substituted or added in SEQ ID NO: 49, and having cellobiohydrolase II gene promoter activity.
(7) The method according to any one of (1) to (6), wherein the gene encoding lignin degrading enzyme is selected from the group consisting of a manganese peroxidase gene, a lignin peroxidase gene, and a laccase gene.
(8) The method according to any one of (1) to (7), wherein the basidiomycete is Coriolus hirsutus.
(9) The method according to any one of (1) to (8), wherein the host cell is Coriolus hirsutus.
(10) A wood chip obtained by the method according to any one of (1) to (9).
(11) A method for producing pulp using the wood chip according to (10).
(12) Pulp obtained by the method according to (11).
This specification is described in the specification and / or drawings of Japanese Patent Application Nos. 2002-048774, 2002-206762, 2002-251720, 2002-251923, and 2002-255205 which are the basis of the priority of the present application. It includes the contents.
Description of sequence listing
SEQ ID NO: 3 shows a probe for screening a clone containing the cellobiose dehydrogenase gene derived from Coriolus hirsutus IFO 4917.
SEQ ID NO: 4 shows a sense primer for synthesizing a 2.3 kbp DNA fragment containing the cellobiose dehydrogenase gene promoter region derived from Coriolus hirsutus IFO 4917 strain by PCR.
SEQ ID NO: 5 shows an antisense primer for synthesizing a 2.3 kbp DNA fragment containing the cellobiose dehydrogenase gene promoter region derived from Coriolus hirsutus IFO 4917 strain by PCR.
SEQ ID NO: 6 shows a sense primer for synthesizing a 2.2 kbp DNA fragment containing a manganese peroxidase structural gene portion derived from Coriolus hirsutus IFO 4917 strain by PCR.
SEQ ID NO: 7 shows an antisense primer for synthesizing a 2.2 kbp DNA fragment containing a manganese peroxidase structural gene portion derived from Coriolus hirsutus IFO 4917 strain by PCR.
SEQ ID NO: 8 shows a sense primer for synthesizing a 1.7 kbp DNA fragment containing a lignin peroxidase structural gene portion derived from Coriolus hirsutus IFO 4917 strain by PCR.
SEQ ID NO: 9 shows a sense primer for synthesizing a DNA fragment containing the cellobiose dehydrogenase gene promoter region derived from Coriolus hirsutus IFO 4917 strain by PCR.
SEQ ID NO: 10 shows an antisense primer for synthesizing a DNA fragment containing the cellobiose dehydrogenase gene promoter region derived from Coriolus hirsutus IFO 4917 strain by PCR.
SEQ ID NO: 11 shows a sense primer for synthesizing a DNA fragment containing a laccase structural gene portion derived from Coriolus hirsutus IFO 4917 strain by PCR.
SEQ ID NO: 12 shows an antisense primer for synthesizing a DNA fragment containing the laccase structural gene portion derived from Coriolus hirsutus IFO 4917 strain by PCR.
SEQ ID NO: 16 shows a probe for screening a clone containing the plasmid pCHCBHI31 gene derived from Coriolus hirsutus IFO 4917 strain. The base “y” represents thymidine or cytidine, and the base “r” represents guanosine or adenosine.
SEQ ID NO: 17 comprises 2.3 kbp containing the plasmid pCHCBHI31 gene promoter region derived from the Coriolus genus Coriolus hirsutus IFO 4917 strain Nco A sense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 18 comprises 2.3 kbp containing the plasmid pCHCBHI31 gene promoter region from the Coriolus genus Coriolus hirsutus strain IFO 4917 Nco An antisense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 19 contains 2.2 kbp containing a manganese peroxidase structural gene portion derived from the Coriolus genus Coriolus hirsutus IFO 4917 Nco A sense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 20 contains 2.2 kbp containing a manganese peroxidase structural gene portion derived from Coriolus genus Coriolus hirsutus IFO 4917 Nco An antisense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 21 contains 2.4 kbp containing a laccase structural gene portion derived from the Coriolus genus Coriolus hirsutus IFO 4917 strain Xho I- Eco The sense primer for synthesizing the RI fragment by PCR is shown.
SEQ ID NO: 22 contains 2.4 kbp containing a laccase structural gene portion derived from the Coriolus genus Coriolus hirsutus IFO 4917 strain Xho I- Eco An antisense primer for synthesizing the RI fragment by PCR is shown.
SEQ ID NO: 23 is 2.2 kbp containing a lignin peroxidase structural gene portion derived from the Coriolus genus Coriolus hirsutus IFO 4917 Nco A sense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 24 contains 2.2 kbp containing a lignin peroxidase structural gene portion derived from Coriolus genus Coriolus hirsutus IFO 4917 Nco An antisense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 28 shows a probe for screening a clone containing the cellobiohydrolase II gene derived from Coriolus hirsutus IFO 4917 strain. The base “i” represents inosine.
SEQ ID NO: 29 shows a sense primer for synthesizing a 3.1 kbp NcoI fragment containing the cellobiohydrolase II (CBHII) gene promoter region from Coriolus hirsutus IFO 4917 strain by PCR.
SEQ ID NO: 30 shows an antisense primer for PCR synthesis of a 3.1 kbp NcoI fragment containing the cellobiohydrolase II gene promoter region from Coriolus hirsutus IFO 4917 strain.
SEQ ID NO: 31 shows a sense primer for synthesizing a 2.2 kbp NcoI fragment containing a manganese peroxidase structural gene portion derived from Coriolus hirsutus IFO 4917 strain by PCR.
SEQ ID NO: 32 shows an antisense primer for synthesizing a 2.2 kbp NcoI fragment containing the manganese peroxidase structural gene portion derived from Coriolus hirsutus IFO 4917 strain by PCR.
SEQ ID NO: 33 shows a sense primer for synthesizing a 2.4 kbp XhoI-EcoRI fragment containing a laccase structural gene portion derived from Coriolus hirsutus IFO 4917 strain by PCR.
SEQ ID NO: 34 shows an antisense primer for synthesizing a 2.4 kbp XhoI-EcoRI fragment containing a laccase structural gene portion derived from Coriolus hirsutus IFO 4917 strain by PCR.
SEQ ID NO: 35 is a sense primer for synthesizing a 2.2 kbp NcoI fragment containing the lignin peroxidase structural gene portion derived from Coriolus hirsutus IFO 4917 strain by PCR.
SEQ ID NO: 36 is an antisense primer for PCR synthesis of a 2.2 kbp NcoI fragment containing the lignin peroxidase structural gene portion derived from Coriolus hirsutus IFO 4917.
SEQ ID NO: 40 shows a probe for screening a clone containing the cellobiohydrolase II gene derived from Coriolus hirsutus IFO 4917. The base “y” represents thymidine or cytidine, and the base “r” represents guanosine or adenosine.
SEQ ID NO: 41 is 1.1 kbp containing the cellobiohydrolase II (CBHI26) gene promoter region from Coriolus hirsutus IFO 4917 Nco A sense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 42 is 1.1 kbp containing the cellobiohydrolase II (CBHI26) gene promoter region from Coriolus hirsutus IFO 4917 strain Nco An antisense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 43 contains 2.2 kbp containing a manganese peroxidase structural gene portion derived from Coriolus hirsutus IFO 4917 strain Nco A sense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 44 contains 2.2 kbp containing a manganese peroxidase structural gene portion from Coriolus hirsutus IFO 4917 strain Nco An antisense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 45 contains 2.4 kbp containing a laccase structural gene portion derived from Coriolus hirsutus IFO 4917 strain Xho I- Eco The sense primer for synthesizing the RI fragment by PCR is shown.
SEQ ID NO: 46 contains 2.4 kbp containing a laccase structural gene portion derived from Coriolus hirsutus IFO 4917 strain Xho I- Eco An antisense primer for synthesizing the RI fragment by PCR is shown.
SEQ ID NO: 47 is 2.2 kbp containing a lignin peroxidase structural gene portion derived from Coriolus hirsutus IFO 4917 Nco A sense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 48 contains 2.2 kbp containing a lignin peroxidase structural gene portion from Coriolus hirsutus IFO 4917 strain Nco An antisense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 52 shows a probe for screening a clone containing the cellobiohydrolase II gene derived from Coriolus hirsutus IFO 4917. The base “i” represents inosine.
SEQ ID NO: 53 contains 3.1 kbp containing the cellobiohydrolase II gene promoter region from Coriolus hirsutus IFO 4917 strain Nco A sense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 54 contains 3.1 kbp containing the cellobiohydrolase II gene promoter region from Coriolus hirsutus IFO 4917 Nco An antisense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 55 contains 2.2 kbp containing a manganese peroxidase structural gene portion derived from Coriolus hirsutus IFO 4917 strain Nco A sense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 56 contains 2.2 kbp containing a manganese peroxidase structural gene portion from Coriolus hirsutus IFO 4917 strain Nco An antisense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 57 contains 2.4 kbp containing a laccase structural gene portion derived from Coriolus hirsutus IFO 4917 strain Xho I- Eco The sense primer for synthesizing the RI fragment by PCR is shown.
SEQ ID NO: 58 contains 2.4 kb of laccase structural gene derived from Coriolus hirsutus strain IFO 4917 Xho I- Eco An antisense primer for synthesizing the RI fragment by PCR is shown.
SEQ ID NO: 59 contains 2.2 kbp containing a lignin peroxidase structural gene portion from Coriolus hirsutus strain IFO 4917 Nco A sense primer for synthesizing the I fragment by PCR is shown.
SEQ ID NO: 60 contains 2.2 kb containing a lignin peroxidase structural gene portion from Coriolus hirsutus IFO 4917 strain Nco An antisense primer for synthesizing the I fragment by PCR is shown.
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in more detail.
The present invention, in the first aspect, comprises a DNA fragment comprising a promoter sequence of a cellulose-degrading enzyme gene derived from a basidiomycete and a gene encoding a lignin degrading enzyme. A step of producing a vector containing the combined recombinant DNA, a step of performing transformation using the vector to prepare a host cell producing high lignin-degrading enzyme, and converting the host cell producing high lignin-degrading enzyme into cellulose A method for producing a lignin-degrading enzyme comprising the steps of culturing in the presence and producing a lignin-degrading enzyme.
As a first step of the method, a DNA fragment containing a promoter sequence of a basidiomycete-derived cellulolytic enzyme gene and a gene encoding a lignin degrading enzyme are bound to the DNA fragment so that the gene can be transcribed. A vector containing the recombinant DNA is prepared.
In the present invention, the promoter sequence of the cellulose degrading enzyme gene is not particularly limited as long as it is a promoter sequence of an enzyme capable of degrading cellulose, but cellobiose dehydrogenase gene, cellobiohydrolase I gene, cellobiohydrolase II gene, endoglucanase gene Or the promoter sequence of the β-glucosidase gene.
As the basidiomycete, any bacteria can be used as long as it is a genus having cellulose-degrading ability, and in particular, Coriolus hirsutus, whose Japanese name is called Arakawakawatake, is preferable.
The DNA fragment containing the promoter sequence of the cellulolytic enzyme gene is, for example, ~ 3. A DNA fragment as shown in FIG.
1. An isolated DNA fragment comprising a cellobiose dehydrogenase gene promoter sequence having any one of the following base sequences (a) to (c):
(A) The base sequence represented by SEQ ID NO: 1 or 2.
(B) A base sequence having a base sequence that hybridizes under stringent conditions with a base sequence having a base sequence complementary to the base sequence of (a), and having cellobiose dehydrogenase gene promoter activity.
(C) A base sequence having a base sequence in which one or more bases are deleted, substituted or added in SEQ ID NO: 1 or 2, and having cellobiose dehydrogenase gene promoter activity.
2. An isolated DNA fragment comprising the cellobiohydrolase I gene promoter sequence, which has any of the following base sequences (a) to (c):
(A) The base sequence represented by SEQ ID NO: 13, 25 or 37.
(B) A base sequence having a base sequence that hybridizes under stringent conditions with a base sequence having a base sequence complementary to the base sequence of (a) and having cellobiohydrolase I gene promoter activity.
(C) A base sequence having a base sequence in which one or more bases are deleted, substituted or added in SEQ ID NO: 13, 25 or 37 and having cellobiohydrolase I gene promoter activity.
3. An isolated DNA fragment comprising a cellobiohydrolase II gene promoter sequence having any one of the following base sequences (a) to (c):
(A) The base sequence represented by SEQ ID NO: 49.
(B) A base sequence having a base sequence that hybridizes under stringent conditions with a base sequence having a base sequence complementary to the base sequence of (a) and having cellobiohydrolase II gene promoter activity.
(C) A base sequence having a base sequence in which one or more bases are deleted, substituted or added in SEQ ID NO: 49, and having cellobiohydrolase II gene promoter activity.
Above 1. An isolated DNA fragment containing the cellobiose dehydrogenase gene promoter sequence described in 1. can be obtained by the following procedure.
To prepare chromosomal DNA from Coriolus hirsutus, the method of Yelton et al. [Proc. Natl. Acad. Sci. USA, 81, 1470 (1984)] and the like, and usual chromosomal DNA extraction methods can be used. Next, the obtained chromosomal DNA is treated with an appropriate restriction enzyme such as Sau3AI, subjected to partial degradation, and then fractionated by sucrose density gradient ultracentrifugation to obtain a fragment of 10 kbp to 25 kbp. The DNA fragment obtained above is ligated to phage DNA treated with a restriction enzyme that produces the same sticky end. Examples of the phage DNA include EMBL3 [AM, Frischauf et al., J. MoI. Mol. Biol. 170, 827 (1983)] λ phage DNA is used. The obtained DNA fragment-linked phage is packaged in vitro to obtain a chromosomal DNA library. For subcloning, a conventional cloning vector, preferably an E. coli cloning vector such as pUC18 [C. Yanish-Perron et al. Gene, 33, 103 (1985)]. Cloning vectors are not limited to those exemplified above, and commercially available or known ones described in literatures can be used.
In the isolation of the cellobiose dehydrogenase gene from the chromosomal gene library obtained above, the purified Coriolus hirsutus cellobiose dehydrogenase was completely digested with lysyl endopeptidase, and the amino acid sequence was deduced. A clone containing the promoter region of the cellobiose dehydrogenase gene is selected by plaque hybridization using a synthetic DNA probe prepared on the basis of the nucleotide sequence. A DNA fragment containing the promoter sequence of the cellobiose dehydrogenase gene is isolated from the selected clone, and a restriction enzyme map is prepared and sequenced. For sequencing, the above-mentioned fragment containing the cellobiose dehydrogenase gene was inserted into an appropriate cloning vector (for example, a pUC vector such as pUC19), and the method of Sanger et al. (Proc. Natl. Acad. Sci. USA, 74, 5463 (1977). )).
By the above procedure, the base sequence of the DNA fragment containing the cellobiose dehydrogenase gene promoter sequence derived from Coriolus hirsutus represented by SEQ ID NO: 1 or 2 was determined.
A DNA fragment containing the cellobiose dehydrogenase gene promoter sequence can be obtained by PCR (polymerase chain reaction) from a fragment containing the cellobiose dehydrogenase gene. As a primer for PCR, a sequence consisting of about 10 to 50 bases, preferably about 15 to 30 bases based on the base sequence represented by SEQ ID NO: 1 and its complementary sequence is used as a sense primer or an antisense primer. it can. For example, the sense primer shown in SEQ ID NO: 4 and the antisense primer shown in SEQ ID NO: 5 can be used. (See Example 3).
It can also be obtained by chemical synthesis according to conventional methods.
The promoter sequence or promoter region in the present invention has a function of regulating transcription of a structural gene, and a functional base sequence substantially conserved in a eukaryotic promoter is expressed by a motif (TATA, CCAA, GC). Box). Accordingly, the base sequence represented by SEQ ID NO: 1 is one specific example of such a promoter sequence, and a sequence that hybridizes under stringent conditions to a sequence complementary to the base sequence as long as it has promoter activity. Are also within the scope of the present invention. Here, “stringent conditions” refers to, for example, conditions in which the sodium concentration is 15 to 900 mM, preferably 15 to 150 mM, and the temperature is 37 to 80 ° C., preferably 50 to 65 ° C.
In order to mutate or modify a gene, a well-known site-specific mutation technique (for example, oligonucleotide site-directed mutagenesis or cassette mutagenesis) (for example, based on the nucleotide sequence represented by SEQ ID NO: 1 or 2) , Short protocols in Molecular Biology, Third Edition, John Wiley & Sons, Inc.).
In addition, Escherichia coli JM109 / pCHCDH1 and Escherichia coli JM109 / pCHCDH2 having a genomic DNA fragment containing a cellobiose dehydrogenase gene promoter sequence derived from Coriolus hirsutus are incorporated by reference on February 8, 2002. Deposited at the Research Center for Biological Biology (1st, 1st, 1st East, Tsukuba City, Ibaraki, Japan), and assigned with the accession numbers FERM BP-8278 and FERM BP-8279, respectively. DNA having the base sequence represented by SEQ ID NO: 1 contained in these deposited strains is also encompassed in the present invention.
In addition, the above 2. The three isolated DNA fragments containing the cellobiohydrolase I gene promoter sequence described in 1) can be obtained by the following procedure.
1) An isolated first DNA fragment containing the cellobiohydrolase I gene promoter sequence
In the same manner as described above, a chromosomal gene library was prepared from Coriolus hirsutus. Next, a synthetic DNA probe prepared from the obtained chromosomal gene library based on the base sequence of the cellobiohydrolase I gene isolated from other species (for example, Phanerochaete chrysosporium, etc.) A clone containing both the cellobiohydrolase I gene and the promoter region is selected according to the plaque hybridization used. A DNA fragment containing the gene of interest is isolated from the selected clone and sequenced. For sequencing, a fragment containing the above-mentioned cellobiohydrolase I chromosomal gene was inserted into an appropriate cloning vector (for example, a pUC vector such as pUC19), and the method of Sanger et al. (Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)).
The base sequence shown in SEQ ID NO: 14 was determined by the above procedure. The gene having this sequence is a genomic gene (SEQ ID NO: 14) consisting of 4624 base pairs including a cellobiohydrolase I gene derived from the genus Coriolus and a cellobiohydrolase I gene promoter region upstream thereof. In the sequence, the promoter region is present at base numbers 1 to 2293 (SEQ ID NO: 13), and TATAAA is observed at base numbers 2202 to 2207. On the other hand, the cellobiohydrolase I structural gene exists at base numbers 2294 to 3775, and is composed of three exons and two introns (intervening sequence). Specifically, intron 1 exists in 2586 to 2637, and intron 2 exists in 2965 to 3022, respectively. Furthermore, base numbers 3776-4624 are 3 ′ untranslated regions including a terminator. The amino acid sequence deduced from the sequence analysis was found to be the amino acid sequence shown in SEQ ID NO: 15 consisting of 457 amino acid residues.
A DNA fragment containing the cellobiohydrolase I gene promoter region can be obtained by PCR (polymerase chain reaction) from the above-mentioned fragment containing the cellobiohydrolase I chromosomal gene. As a primer for PCR, a sequence consisting of about 10 to 50 bases, preferably about 15 to 30 bases based on the base sequence shown in SEQ ID NO: 13 and its complementary sequence is used as a sense primer and an antisense primer. it can. For example, the sense primer shown in SEQ ID NO: 17 and the antisense primer shown in SEQ ID NO: 18 can be used (see Example 15).
It can also be obtained by chemical synthesis according to conventional methods.
The base sequence shown in SEQ ID NO: 13 is one specific example of such a promoter sequence. As long as the promoter sequence has promoter activity, the base sequence has one or more base mutations such as deletion, substitution, addition, etc. Sequences containing modifications are also within the scope of the present invention. Such a mutation or modification is based on a well-known site-directed mutagenesis technique such as oligonucleotide site-directed mutagenesis or cassette mutagenesis based on the base sequence shown in SEQ ID NO: 13 (for example, Short protocol In Molecular Biology, Third Edition). , John Wiley & Sons, Inc.).
2) An isolated second DNA fragment containing the cellobiohydrolase I gene promoter sequence
The second DNA fragment was sequenced in the same manner as in the case of the first DNA fragment. As a result, the base sequence shown in SEQ ID NO: 26 was determined. The gene having this sequence is a genomic gene consisting of 4170 base pairs including the cellobiohydrolase I gene derived from the genus Coriolus and the cellobiohydrolase I gene promoter region upstream thereof. In the sequence, the promoter region is present at base numbers 1 to 2182 (SEQ ID NO: 25), and TATAAA is observed at base numbers 2099 to 2104. On the other hand, the cellobiohydrolase I gene exists at base numbers 2183 to 3667, and is composed of three exons and two introns (intervening sequence). Specifically, intron 1 exists at 2475 to 2535 and intron 2 exists at 2862 to 2917, respectively. Furthermore, base numbers 3668 to 4170 are 3 ′ untranslated regions including a terminator. The amino acid sequence deduced from the sequence analysis was found to be the amino acid sequence shown in SEQ ID NO: 27 consisting of 457 amino acid residues.
The second DNA fragment containing the cellobiohydrolase I gene promoter region can be obtained from the above fragment containing the cellobiohydrolase I chromosomal gene by PCR (polymerase chain reaction). As a primer for PCR, a sequence consisting of about 10 to 50 bases, preferably about 15 to 30 bases based on the base sequence shown in SEQ ID NO: 25 and its complementary sequence is used as a sense primer or an antisense primer. it can. For example, the sense primer shown in SEQ ID NO: 29 and the antisense primer shown in SEQ ID NO: 30 can be used (see Example 25).
It can also be obtained by chemical synthesis according to conventional methods.
The base sequence shown in SEQ ID NO: 25 is one specific example of such a promoter sequence, and as long as it has promoter activity, mutations such as deletion, substitution, addition, etc. of one or more nucleotides in the base sequence Sequences containing modifications are also within the scope of the present invention. Such a mutation or modification is based on a well-known site-directed mutagenesis technique such as oligonucleotide site-directed mutagenesis or cassette mutagenesis based on the nucleotide sequence shown in SEQ ID NO: 1 (for example, Short protocol In Molecular Biology, Third Edition). , John Wiley & Sons, Inc.).
3) An isolated third DNA fragment containing the cellobiohydrolase I gene promoter sequence
The third DNA fragment was sequenced in the same manner as in the case of the first DNA fragment. As a result, the base sequence shown in SEQ ID NO: 38 was determined. The gene having this sequence is a genomic gene consisting of 3916 base pairs including the cellobiohydrolase I gene derived from the genus Coriolus and the cellobiohydrolase I gene promoter region upstream thereof. In the sequence, the promoter region is present at base numbers 1 to 1086 (SEQ ID NO: 37), and TATAAA is observed at base numbers 1001 to 1006. On the other hand, the cellobiohydrolase I structural gene exists at base numbers 1087 to 2579, and is composed of three exons and two introns (intervening sequence). Specifically, intron 1 exists in 1379 to 1444, and intron 2 exists in 1771 to 1829, respectively. Furthermore, base numbers 2580 to 3916 are 3 ′ untranslated regions including a terminator. The amino acid sequence deduced from the analysis of the sequence was found to be the amino acid sequence shown in SEQ ID NO: 39 consisting of 456 amino acid residues.
The third DNA fragment containing the cellobiohydrolase I gene promoter region can be obtained by PCR (polymerase chain reaction) from the above-mentioned fragment containing the cellobiohydrolase I chromosomal gene. As a primer for PCR, a sequence comprising about 10 to 50 bases, preferably about 15 to 30 bases based on the base sequence shown in SEQ ID NO: 37 and its complementary sequence, is used as a sense primer or an antisense primer. it can. For example, the sense primer shown in SEQ ID NO: 41 and the antisense primer shown in SEQ ID NO: 42 can be used (see Example 25).
It can also be obtained by chemical synthesis according to conventional methods.
The base sequence shown in SEQ ID NO: 37 is one specific example of such a promoter sequence, and as long as it has promoter activity, the base sequence has one or more base mutations such as deletion, substitution, addition, etc. Sequences containing modifications are also within the scope of the present invention. Such mutation or modification is based on a well-known site-specific mutation technique such as oligonucleotide site-directed mutagenesis or cassette mutagenesis based on the nucleotide sequence shown in SEQ ID NO: 37 (for example, Short protocol In Molecular Biology, Third Edition). , John Wiley & Sons, Inc.).
3. above. An isolated DNA fragment containing the cellobiohydrolase II gene promoter sequence described in 1. can be obtained by the following procedure.
Above 1. A chromosomal DNA library was obtained in the same manner as described above. Next, a synthetic DNA probe prepared from the obtained chromosomal DNA library based on the base sequence of the cellobiohydrolase II gene isolated from other species (for example, Phanerochete chrysosporium, etc.) A clone containing the cellobiohydrolase II gene is selected by performing plaque hybridization. A DNA fragment containing the cellobiohydrolase II gene is isolated from the selected clone and subjected to sequencing.
For sequencing, a DNA fragment containing the above-mentioned cellobiohydrolase II gene was inserted into an appropriate cloning vector (for example, a pUC vector such as pUC19), and the method of Sanger et al. (Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)).
The base sequence shown in SEQ ID NO: 50 was determined by the above procedure. The gene having this sequence is a genomic gene consisting of 4933 base pairs including the 3 ′ untranslated region including the structural gene of the cellobiohydrolase II gene derived from Coriolus hirsutus, the promoter region upstream of it, and the terminator.
In the sequence, the promoter region is present at nucleotide numbers 1 to 3121 (SEQ ID NO: 49), and TATAAA is observed at nucleotide numbers 3059 to 3064. On the other hand, the structural gene of cellobiohydrolase II exists at base numbers 3122 to 4822, and is composed of 7 exons and 6 introns (intervening sequence). Specifically, exon 1 is 3122-3221, exon 2 is 3280-3346, exon 3 is 3402-3549, exon 4 is 3605-4220, exon 5 is 4275-4363, exon 6 is 4425-4676, exon 7 is 4736 to 4822, respectively. Intron 1 is present in 3222-3279, intron 2 is present in 3347-3401, intron 3 is present in 3550-3604, intron 4 is present in 4221-4274, intron 5 is present in 4364-4424, and intron 6 is present in 4677-4735. Furthermore, base numbers 4823 to 4933 are 3 ′ untranslated regions including a terminator. The amino acid sequence deduced from the sequence analysis was found to be the amino acid sequence represented by SEQ ID NO: 51 consisting of 453 amino acid residues.
A DNA fragment containing the base sequence of the promoter region of the cellobiohydrolase II gene can be obtained from the fragment containing the cellobiohydrolase II gene by PCR (polymerase chain reaction). As a primer for PCR, a sequence consisting of about 10 to 50 bases, preferably about 15 to 30 bases based on the base sequence represented by SEQ ID NO: 49 and its complementary sequence is used as a sense primer or an antisense primer. it can. For example, the sense primer shown in SEQ ID NO: 53 and the antisense primer shown in SEQ ID NO: 55 can be used (see Example 3).
It can also be obtained by chemical synthesis according to conventional methods.
The base sequence represented by SEQ ID NO: 49 is one specific example of such a promoter sequence, and a sequence that hybridizes under stringent conditions to a sequence complementary to the base sequence as long as it has promoter activity. Are also within the scope of the present invention. Here, the stringent condition is represented by SEQ ID NO: 49, wherein the homology with the nucleotide sequence shown in SEQ ID NO: 49 is 70% or more, preferably 80% or more, more preferably 90% or more, particularly 95% or more. It means a condition that allows hybridization with a sequence containing a mutation or modification such as deletion, substitution, addition, etc. of one or more bases in the sequence. For example, the sodium concentration is 15 to 900 mM, preferably 15 to 150 mM. And the temperature is from 37 to 80 ° C, preferably from 50 to 65 ° C.
In order to mutate or modify a gene, based on the nucleotide sequence represented by SEQ ID NO: 49, a well-known site-specific mutation technique such as oligonucleotide site-specific mutagenesis or cassette mutagenesis (for example, Short protocol In Molecular Biology, Third Edition, John Wiley & Sons, Inc.).
The gene encoding a lignin degrading enzyme is not particularly limited as long as it is a gene encoding an enzyme capable of degrading lignin, and examples thereof include a manganese peroxidase gene, a lignin peroxidase gene, and a laccase gene. These genes can also be obtained by performing a known genomic cloning, cDNA cloning method or PCR method based on sequences registered in Genebank, sequences described in literatures, and the like. Or about the gene deposited, what can be obtained by the request for distribution can be utilized.
Further, in the first step of the present method, that transcription is possible means that transcription of a gene encoding a lignin-degrading enzyme into mRNA occurs in the host under the action of a promoter. A gene encoding a lignin degrading enzyme is bound downstream of a DNA fragment having a promoter sequence of a cellulose degrading enzyme gene, and is transcribed into mRNA by the operation of the promoter.
The DNA fragment containing the cellulolytic enzyme gene promoter sequence and the gene encoding lignin degrading enzyme can be ligated using an appropriate DNA ligase after introduction of restriction sites, blunting or cohesive termination, if necessary. it can. Recombinant DNA techniques including cloning, ligation, PCR, etc. are described in, for example, J. Sambrook et al. , Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989 and Short Protocols In Molecular Biology, Third Edition, A Compendium of Methods from Current Protocols in Molecular Biology, John Wiley & Sons, Inc. Can be used.
The type of vector is not particularly limited, but is selected according to the type of host transformed with this vector. As the vector, a vector that can autonomously replicate in a prokaryotic or eukaryotic host cell or can be homologously recombined into a chromosome can be used. Examples include plasmids, phage-containing viruses, and cosmids. The vector can appropriately include a selection marker, a replication origin, a terminator, a polylinker, an enhancer, a ribosome binding site, and the like. Various vectors for prokaryotic and eukaryotic organisms such as bacteria, fungi, yeast, animals, plants, etc. are commercially available, or are described in literatures, etc. DNA can be introduced into a vector. For example, DNA introduction is described in J. It can be performed using the techniques described in Sambrook et al. (Supra).
In the second step of this method, a host cell producing high lignin-degrading enzyme transformed with the vector defined above is prepared.
Here, the host cells are not only fungi including basidiomycetes, fungi and yeasts, but also other eukaryotic cells (animal cells, plant cells, insect cells, algae, etc.) and prokaryotic cells (bacteria, cyanobacteria, etc.). However, any host cell can be used as long as it can exert promoter activity in the expression of a gene encoding lignin-degrading enzyme. Among these, a preferred host cell is a basidiomycete, and particularly preferably Coriolus hirsutus. For example, specifically, an auxotrophic mutant OJI-1078 (FERM BP-4210) lacking ornithine carbamoyltransferase activity of Coriolus hirsutus described in Examples below can be used as a host. Further, as a selection marker gene, E. coli recombinant strain JM109 / pUCR1 (JP-A-6-054691; FERM BP-4201) containing ornithine carbamoyltransferase gene isolated from Coriolus hirsutus can be used.
Examples of transformation methods include, but are not limited to, calcium chloride / PEG method, calcium phosphate method, lithium acetate method, electroporation method, protoplast method, spheroplast method, lipofection method, and Agrobacterium method.
In the third step of this method, the transformed lignin-degrading enzyme high-producing host cell is cultured in the presence of cellulose to produce lignin-degrading enzyme.
Ligninase is produced in a secreted form when expressed and translated in a fusion form with a signal peptide, in which case it can be isolated directly from the medium, while a polypeptide is produced in a non-secreted form. When the cells are separated, the cells are disrupted by sonication, homogenizing, or the like to obtain an extract, and the polypeptide can be isolated from the extract. For isolation and purification, methods such as solvent extraction, salting out, desalting, organic solvent precipitation, ultrafiltration, ion exchange, hydrophobic interaction, HPLC, gel filtration and affinity chromatography, electrophoresis, and chromatofocusing are used alone. Or in combination.
For example, a large amount of manganese peroxidase, lignin peroxidase or laccase can be obtained by culturing host cells transformed with a recombinant vector in which a manganese peroxidase gene portion, a lignin peroxidase gene portion or a laccase gene portion is linked downstream of the promoter region of the present invention. Can be produced. Each gene is not particularly limited, and various genes having known sequences can be used. For example, an Escherichia coli group containing a manganese peroxidase cDNA gene deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center. Recombinant strain JM109 / pBSMPOC1 (FERM P-14932), E. coli recombinant strain JM109 / pBSMPOOG1 (FERM P-14933) containing manganese peroxidase chromosomal gene, E. coli recombinant E. coli containing high temperature inducible lignin peroxidase gene. E. coli XL-1 blue / pBSLPOOG7 (FERM P-12683), and laccase (also known as phenol oxidase) deposited as FERM BP-2793 (chromosomal gene), FERM P-10055 (cDNA) and FERM P-10061 (cDNA) ) E. coli recombinant strain containing the gene (Japanese Patent Publication No. 7-46995; Japanese Patent Publication No. 6-85717).
As a second aspect of the present invention, a DNA fragment comprising a promoter sequence of a basidiomycete-derived cellulolytic enzyme gene and a gene encoding lignin degrading enzyme are bound to the DNA fragment so that the gene can be transcribed. A step of preparing a vector containing the recombinant DNA, a step of performing transformation using the vector to prepare a host cell producing high lignin-degrading enzyme, and converting the host cell producing high lignin-degrading enzyme into a wood chip And a method for treating wood chips, comprising the step of inoculating and treating the wood chip.
Including a DNA fragment containing a promoter sequence of a basidiomycete-derived cellulolytic enzyme gene and a recombinant DNA containing a gene encoding a lignin-degrading enzyme and bound to the DNA fragment so that the gene can be transcribed The step of producing a vector and the step of preparing a host cell producing high lignin degrading enzyme by performing transformation using the vector are as defined in the first aspect.
Lignin-degrading enzyme high-producing host cells can be inoculated into wood chips and processed to save energy by reducing beating energy and shortening cooking time in the pulp production process.
The processing can be performed as follows, for example.
The obtained lignin-degrading enzyme high-producing host cells were cultured on a potato dextrose agar medium, stored at a low temperature (4 ° C.), and the sections punched out from this plate were treated with glucose peptone medium (glucose 2%, polypeptone 0. 5%, yeast extract 0.2%, KH ₂ PO ₄ , MgSO ₄ Inoculate into a 300 ml Erlenmeyer flask containing 100 ml of 0.05%, adjusted to pH 4.5 with phosphoric acid), and shake culture at 28 ° C. and 100 rpm for 1 week. After culturing, the host cells are filtered off, washed with sterilized water, and the remaining medium is removed.
Next, the obtained lignin-degrading enzyme high-producing host cells were pulverized with sterilized water in a Waring blender for 15 seconds, and inoculated so that the dry weight of the host cells was 1 g against eucalyptus wood chips of 1 kg of completely dry weight. To do. After inoculation, agitate well so that the host cells are distributed throughout. The culture is performed at 28 ° C. with aeration for 1 week. Moreover, saturated water vapor | steam is ventilated as needed so that a wood chip moisture content may be 40 to 65%. The ventilation rate when venting is set to 0.01 vvm per chip.
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
1. Production and utilization of lignin degrading enzymes using DNA fragments containing the promoter sequence of cellobiose dehydrogenase gene
[Example 1] Preparation of chromosome gene library
A 5 mm diameter agar piece was punched out from a plate agar culture of Coriolus hirsutus (IFO 4917 strain) with a cork borer, and glucose peptone medium (glucose 2%, polypeptone 0.5%, yeast extract 0.2%, KH ₂ PO ₄ 0.1% MgSO ₄ ・ 7H ₂ 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.
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 incubated at 55 ° C. for 3 hours. . After incubation, phenol treatment and chloroform treatment were performed, and ethanol was gradually added to the aqueous layer portion. When DNA was precipitated, the chromosomal DNA was wound and suspended in the TE solution.
100 μg of the obtained chromosomal DNA was partially digested with the restriction enzyme Sau3AI and fractionated by 5-20% sucrose density gradient ultracentrifugation (30,000 rpm, 18 hours) to collect 20-40 kbp fragment sections. This fragment was ligated to a Toyobo phage λEMBL3-Bam arm using T4 DNA ligase, and the resulting phage DNA was packaged using STRATAGENE Gigapack Gold, and then Escherichia coli P2392 (included in the Gigapack Gold). And a chromosomal DNA library.
[Example 2] Isolation of a DNA fragment containing the promoter sequence of cellobiose dehydrogenase gene from a chromosomal gene library
A clone containing the promoter region of the cellobiose dehydrogenase gene was selected from the chromosomal DNA library by plaque hybridization. This series of operations was performed according to a conventional method (Sambrook et al., Molecular Cloning A Laboratory Manual / 2nd Edition (1989)). The probe used for the plaque hybrid was prepared by labeling a synthetic oligomer having the following sequence with fluorescein at the 3 ′ end using an oligo DNA labeling kit manufactured by Amersham.

As a result, 4 positive clones could be selected from about 40,000 plaques. Recombinant phage DNA prepared from positive clones according to a conventional method was digested with various restriction enzymes, and Southern hybridization was performed using the above synthetic DNA. As a result, clones that hybridized to the probe as DNA bands of 7.2 kbp and 5.3 kbp were found in the fragments obtained by digestion with the restriction enzyme XhoI.
The above-mentioned DNA fragments 7.2 kbp and 5.3 kbp were excised by agarose gel electrophoresis and subcloned into the XhoI site of E. coli vector pBluescript II SK + (manufactured by Toyobo Co., Ltd.) to obtain plasmids pCHCDH1 and pCHCDH2. Each of these plasmids was transformed into E. coli strain JM109. In addition, the obtained Escherichia coli JM109 / pCHCDH1 and Escherichia coli JM109 / pCHCDH2 containing the promoter region of the cellobiose dehydrogenase gene derived from Coriolus hirsutus were incorporated by the National Institute of Advanced Industrial Science and Technology as of February 8, 2002. Deposited at the Patent Biological Depositary Center (1st, 1st, 1st East, Tsukuba City, Ibaraki, Japan), and assigned with accession numbers FERM BP-8278 and FERM BP-8279, respectively. Next, a large amount of subcloned DNA was prepared and purified by ultracentrifugation (50,000 rpm, 16 h, 15 ° C.), and the nucleotide sequence was determined. The nucleotide sequence was determined using a sequencing kit manufactured by United States Biochemical. SEQ ID NO: 1 or 2 shows the nucleotide sequence of a DNA fragment containing the promoter sequence of the cellobiose dehydrogenase gene.
[Example 3] Construction of an expression vector for a manganese peroxidase gene using a cellobiose dehydrogenase gene promoter
By performing PCR using the two primers shown in SEQ ID NOs: 4 and 5 below from the plasmid pCHCDH1 containing the cellobiose dehydrogenase gene promoter region derived from Coriolus hirsutus obtained in Example 2, BamHI of the cellobiose dehydrogenase promoter region A 2.2 kbp fragment (fragment 1) with a blunt end was amplified.

Next, a plasmid pBSMPOOG1 (FERM P-14933) containing a Corriolus hirsutus-derived manganese peroxidase gene is subjected to PCR using the two promoters shown in SEQ ID NOs: 6 and 7 below, whereby a manganese peroxidase structural gene partial blunt end is obtained. ~ EcoRI ~ 2.2 kbp (fragment 2) was amplified.
E. coli vector pUC18 was cleaved with restriction enzymes BamHI to EcoRI, the above DNA fragments 1 and 2 were mixed, ligated with T4 DNA ligase, E. coli JM109 strain was transformed, and ampicillin resistant transformant A plasmid in which the DNA fragments of fragment 1 and fragment 2 were inserted at the same time was isolated from the inside and named pCDHPMN.

[Example 4] Preparation of Coriolus hirsutus transformant producing high secretion of manganese peroxidase
A protoplast solution of Coriolus hirsutus was obtained by the following method.
a. Mononuclear mycelium culture
After sterilizing 100 ml of SMY medium (1% sucrose, 1% malt extract, 0.4% yeast extract) in a 500 ml Erlenmeyer flask containing about 30 glass beads with a diameter of about 6 mm, Coriolus hirsutus OJI An agar piece having a diameter of 5 mm was punched out of a plate agar medium of strain −1078 with a cork borer, inoculated into an SMY medium, and cultured at 28 ° C. for 7 days (preculture). 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. In addition, the mycelium was subdivided by stirring with a stirrer for about 2 hours a day. This culture was carried out for 4 days.
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 ₄ And 50 ml maleic acid 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 3 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 (Yakult) and 10 mg of Yatalase (Takara Shuzo) were dissolved in 1 mg of the osmotic pressure adjusting solution and used as an enzyme solution.
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 k × g, 5 minutes), the top was removed, resuspended in 4 ml of 1M sucrose (20 mM MOPS buffer, pH 6.3), and then centrifuged. The operation was repeated and 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) added with 40 mM calcium chloride to obtain a protoplast solution. This solution was stored at 4 ° C.
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
About 10 ⁶ 2 μg of the plasmid prepared in Example 3 was added in a circular or linear form to a protoplast solution of pcs / 100 μl. Further, 0.2 μg of plasmid pUCR1 carrying the ornithine carbamoyltransferase gene derived from Coriolus hirsutus was added as a selection marker, and the mixture was ice-cooled for 30 minutes. Next, an equal amount of PEG solution (50% PEG 3400, 20 mM MOPS (pH 6.4)) was added, and the mixture was ice-cooled 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 manganese peroxidase gene expression plasmid was incorporated.
[Example 5] Production of manganese peroxidase by transformant
Oxygen bleached hardwood pulp (LOKP) / peptone medium (LOKP 1%, polypeptone 0.5%, yeast extract 0.2%, KH ₂ PO ₄ , MgSO ₄ In a 300 ml Erlenmeyer flask containing 100 ml of 0.05%, adjusted to pH 4.5 with phosphoric acid), transformant 50 mm obtained in Example 4 above. ² Five agar pieces were inoculated and cultured at 28 ° C. and 100 rpm with rotary shaking. After 6 days, the resulting culture was centrifuged to obtain the supernatant.
The enzyme activity was 50 μl of 0.5 M sodium malonate buffer (pH 5.5), 345 μl of enzyme solution, 5 μl of 10 mM hydrogen peroxide, 1 mM MnSO. ₄ 100 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, and the Mn (III) malonic acid complex was enzyme-activated in the culture supernatant. Was observed at 6 μmol / ml / min on the 6th day from the start of the 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.
[Example 6] Construction of expression vector of lignin peroxidase gene by cellobiose dehydrogenase gene promoter
A structural gene region of a lignin peroxidase chromosomal gene (Japanese Patent Laid-Open No. 5-260978; pBSLPOOG7; FERM P-12683) was linked downstream of the promoter of the cellobiose dehydrogenase gene derived from Coriolus hirsutus to obtain an expression plasmid.
Specifically, by performing PCR using the two primers shown in SEQ ID NOS: 4 and 5 in Example 3 from the plasmid pCHCDH1 containing the cellobiose dehydrogenase gene promoter region derived from Coriolus hirsutus obtained in Example 2 A 2.2 kbp fragment (fragment 3) having the promoter region BamHI to blunt end was amplified. The structural gene portion of lignin peroxidase was extracted by preparing a DNA fragment by the primer extension method using the plasmid pBSLPOOG7 as a template and the primer shown in SEQ ID NO: 8 below, and cleaving it with the restriction enzyme HindIII. A 1.7 kbp DNA fragment (fragment 4) consisting of a portion was obtained.

Further, the E. coli vector was cleaved with pUC18 with restriction enzymes EcoRI and HindIII, the two kinds of DNA fragments of the above fragments 5 and 6 were mixed, ligated with T4 DNA ligase, E. coli JM109 strain was transformed, and ampicillin resistant A plasmid in which the DNA fragments of the above fragments 3 and 4 were simultaneously inserted was isolated from the transformant and named pCDHPLP.
[Example 7] Preparation of a Coriolus hirsutus transformant producing lignin peroxidase with high secretion
According to the method described in Example 4, a protoplast solution of Coriolus hirsutus was obtained. About 10 ⁶ 2 μg of the plasmid prepared in Example 6 was added in a circular or linear form to a protoplast solution of pcs / 100 μl. Further, 0.2 μg of plasmid pUCR1 carrying the ornithine carbamoyltransferase gene derived from Coriolus hirsutus was added as a selection marker, and the mixture was ice-cooled for 30 minutes. Next, an equal amount of PEG solution (50% PEG 3400, 20 mM MOPS (pH 6.4)) was added, and the mixture was ice-cooled 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 gene expression plasmid was incorporated.
[Example 8] Production of lignin peroxidase by transformant
Oxygen bleached hardwood pulp (LOKP) / peptone medium (LOKP 1%, polypeptone 0.5%, yeast extract 0.2%, KH ₂ PO ₄ , MgSO ₄ In a 300 ml Erlenmeyer flask containing 100 ml of 0.05%, adjusted to pH 4.5 with phosphoric acid), transformant 50 mm obtained in Example 7 above. ² Five agar pieces were inoculated and cultured at 28 ° C. and 100 rpm with rotary shaking. After 6 days, the resulting culture was centrifuged to obtain the supernatant.
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. Absorbance was measured over time, and the enzyme activity for converting veratryl alcohol to veratrum aldehyde 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. Here, the enzyme activity unit was defined as 1 unit of activity that increases 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 9] Construction of expression vector of laccase gene by cellobiose dehydrogenase gene promoter
Using the plasmid pCHCDH1 obtained in Example 2 as a template, PCR was carried out using the two primers shown in SEQ ID NOs: 9 and 10 below, and BamHI treatment was performed to obtain a DNA fragment having BamHI to blunt end (fragment 7 )

On the other hand, a plasmid OJ-POG-E1 (deposit number BP-2793) containing a laccase gene derived from Coriolus hirsutus was amplified by PCR using the two primers shown in SEQ ID NOs: 11 and 12 below.

The obtained PCR fragment was introduced into the above TA-Cloning vector to obtain a plasmid pTALAC. The plasmid pTALAC was digested with the restriction enzyme XhoI, smoothed using the modification enzyme Klenow fragment, and then digested with the restriction enzyme EcoRI to obtain a laccase structural gene portion (fragment 6).
After digesting the E. coli vector pUC18 with BamHI and EcoRI, the DNA fragments of the above fragments 7 and 8 are mixed, ligated with T4 DNA ligase, and then transformed into E. coli JM109 strain. From the ampicillin resistant transformants, A plasmid in which two types of DNA fragments were simultaneously inserted was isolated and named pCDHPLAC.
[Example 10] Production of a Coriolus hirsutus transformant producing laccase with high secretion
According to the method described in Example 4, a protoplast solution of Coriolus hirsutus was obtained. About 10 ⁶ 2 μg of the plasmid prepared in Example 9 was added in a circular or linear form to a protoplast solution of pcs / 100 μl. Further, 0.2 μg of plasmid pUCR1 carrying the ornithine carbamoyltransferase gene derived from Coriolus hirsutus was added as a selection marker, and the mixture was ice-cooled for 30 minutes. Next, an equal amount of PEG solution (50% PEG 3400, 20 mM MOPS (pH 6.4)) was added, and the mixture was ice-cooled 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. Further, DNA was prepared from the transformed strain, and it was confirmed by Southern hybridization that the target laccase expression plasmid was incorporated.
[Example 11] Production of laccase by transformant
Oxygen bleached hardwood pulp (LOKP) / peptone medium (LOKP 1%, polypeptone 0.5%, yeast extract 0.2%, KH ₂ PO ₄ , MgSO ₄ 0.05%, adjusted to pH 4.5 with phosphoric acid) in a 300 ml Erlenmeyer flask containing 100 ml each of the transformant obtained in Example 10 above, 50 mm ² Five agar pieces were inoculated and cultured at 28 ° C. and 100 rpm with rotary shaking. After 6 days, the resulting culture was centrifuged to obtain the supernatant.
Enzyme activity was determined by adding 50 μl of 1 M sodium acetate buffer (pH 4.0), 50 μl of 5 mM 2,2′-azino-bis (3-ethylbenzthiazine-6-sulfate) (ABTS), 400 μl of enzyme solution, and ABTS of the resulting ABTS The increase in absorbance at 420 nm was recorded over time for the oxide, and enzyme activity was observed in the culture supernatant at 40 units / mL on the fifth day of the culture. Here, the enzyme activity unit is defined as the amount of enzyme required to oxidize 1 μmol of ABTS per minute. On the other hand, only 5 units / ml was observed in the culture supernatant of the OJI-1078 strain that did not contain donor DNA cultured under the same conditions.
2. Production and utilization of lignin degrading enzyme using the first DNA fragment containing the promoter sequence of cellobiohydrolase I gene
[Example 12] Isolation of DNA fragment containing cellobiohydrolase I gene promoter sequence from chromosomal gene library
In the same manner as in Example 1, a chromosomal gene library was obtained. A clone containing the cellobiohydrolase I gene was selected from the obtained library by plaque hybridization. This series of operations was carried out according to a conventional method [Sambrook et al., “Molecular Cloning A Laboratory Manual / 2nd Edition (1989)].
The probe used for plaque hybridization is a synthetic oligomer having the sequence of SEQ ID NO: 16 labeled at the 3 'end with fluorescein using an oligo DNA labeling kit manufactured by Amersham.

However, in SEQ ID NO: 16, base Y represents base T or C, and base R represents base G or A.
As a result, 4 positive clones could be selected from about 40,000 plaques. Recombinant phage DNA prepared from positive clones according to a conventional method was digested with various restriction enzymes, and Southern hybridization was performed using the above synthetic DNA. As a result, a clone hybridizing to 4.6 kbp as a single DNA band was found in the fragment obtained by digestion with restriction enzymes EcoRI and BamHI.
The above DNA fragment 4.6 kbp was excised by agarose gel electrophoresis, subcloned into the EcORI-BamHI site of the E. coli vector pUC19, and transformed into E. coli JM109 strain to obtain plasmid pCHCBHI31. The base sequence of the subcloned DNA fragment was determined. Determination of the base sequence was performed using a BigDye Terminator Cycle Sequencing kit manufactured by Applied Biosystems, followed by electrophoresis using a DNA sequencer PRISM 310 manufactured by Applied Biosystems, and the base sequence was analyzed.
The base sequence is shown in SEQ ID NO: 14. As a result, the celliobiohydrolase I gene derived from Coriolus hirsutus was separated by two introns within the range of the base sequence. The amino acid sequence deduced from the base sequence is shown in SEQ ID NO: 15.
[Example 13] Construction of expression vector for manganese peroxidase gene by cellobiohydrolase I gene promoter
After digesting the plasmid pCHCBHI31 containing the celliobiohydrolase I gene promoter region derived from Coriolus hirsutus with the restriction enzyme NcoI, smoothing is performed using the modification enzyme Klenow fragment, self-ligation is performed, and the NcoI site is eliminated. The obtained NcoI site disappearance plasmid was amplified as a promoter region 2.3 kbp by PCR using the following two primers (1, 2), ligated to the SmaI site of pUC18 using T4 DNA ligase, and plasmid pCHCBHI31P Got.

Next, the plasmid pBSMPOOG1 (deposit number FERM P-14933) containing the Coriolus hirsutus-derived manganese peroxidase gene was deposited by PCR using the two primers (3,4) shown below, and the manganese peroxidase structural gene portion NcoI was about 2.2 kb. Amplify the fragment.

The obtained PCR fragment was inserted using TA cloning kit manufactured by In Vitrogen to obtain pTAMP. Next, the obtained pTAMP was digested with the restriction enzyme NcoI, and about 2.2 kb was excised and inserted into the NcoI site of the pCHCBHI31P to obtain a plasmid pCHCBHI31PMP (FIG. 1).
[Example 14] Production of transformant producing manganese peroxidase with high production
Protoplasts were prepared and purified in the same manner as in Example 4. Subsequently, transformation was performed as follows.
10 ⁶ 2 μg of the plasmid pCHCBHI31PMP prepared in Example 13 was added to 100 μl of a protoplast solution at a concentration of 100 μl / 100 μl in a circular or linear form, and pUCR1 holding an ornithine carbamoyltransferase gene derived from Coriolus hirsutus as a selection marker was added to 0 .2 μg was added and ice-cooled for 30 minutes. Next, an equal amount of PEG solution (50% PEG 3400, 20 mM MOPS (pH 6.4)) was added to the liquid volume, and the mixture was ice-cooled 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. Furthermore, DNA was prepared from the transformant, and it was confirmed by Southern hybridization that the target manganese peroxidase expression plasmid was incorporated.
[Example 15] Production of manganese peroxidase by a transformant
The transformed strain obtained in Example 14 was placed in a 500 ml Erlenmeyer flask with 50 ml of pulp / peptone liquid medium (bleached pulp 30 g / l, peptone 10 g / l, KH). ₂ PO ₄ 1.5 g / l, MgSO ₄ ・ 7H ₂ O 0.5 g / l, thiamine hydrochloride 2 mg / l, MnSO ₄ ・ 5H ₂ O48mg / l, adjusted to pH 5.0 with phosphoric acid) 50mm ² Five agar pieces were inoculated and cultured at 28 ° C. for 6 days under shaking. After 6 days, the resulting culture was centrifuged to obtain the supernatant.
The enzyme activity was 50 μl of 0.5 M sodium malonate buffer (pH 5.5), 345 μl of enzyme solution, 5 μl of 10 mM hydrogen peroxide, 1 mM MnSO. ₄ 100 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, and the Mn (III) malonic acid complex was added to the culture supernatant in the enzyme supernatant. The activity was observed at 3.5 μmol / ml / min on the 6th day from the start of the 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.
[Example 16] Construction of expression vector of laccase gene by cellobiohydrolase I gene promoter
The plasmid pCHCBHI31P obtained in Example 13 was digested with the restriction enzyme NcoI (manufactured by Takara Shuzo Co., Ltd.) and then smoothed using Klenow fragment. Thereafter, it was digested with the restriction enzyme EcoRI to obtain an expression vector portion.
Next, a plasmid OJ-POG-E1 (FERM BP-2793) containing a Coriolus hirsutus-derived laccase gene was amplified by PCR using the following two primers (5, 6).

The obtained PCR fragment was introduced into the above-mentioned TA-cloning vector to obtain a plasmid pTALAC.
The plasmid pTALAC is digested with the restriction enzyme XhoI and then blunted using the modification enzyme klenow fragment. Thereafter, digestion was performed with the restriction enzyme EcoRI to obtain a laccase structural gene portion, which was introduced into the above-treated cellobiohydrolase I expression vector pCHCBHI31P.
The resulting plasmid was named pCHCBHI31PLAC (FIG. 2).
[Example 17] Preparation of a Coriolus hirsutus transformant producing laccase with high secretion
When transforming the arginine-requiring Coriolus hirsutus strain OJI-1078 (FERM BP-4210) using the pCHCBHI31PLAC obtained in Example 16, a plasmid (pUCR1) carrying the OCT gene derived from Coriolus hirsutus as a selection marker is simultaneously used. A transformant was obtained by introduction (eg, PEG method or electroporation method). 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.
About 10 ⁶ 2 μg of the plasmid pCHCBHI31PLAC prepared in Example 16 was added to 100 μl of the protoplast solution at a concentration of 100 μl / 100 μl in a circular or linear form, 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% PEG 3400, 20 mM MOPS (pH 6.4)) was added, and the mixture was ice-cooled 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. Further, DNA was prepared from the transformed strain, and it was confirmed by Southern hybridization that the target laccase expression plasmid was incorporated.
[Example 18] Production of laccase by transformant
The transformed strain obtained in Example 17 was placed in a 500 ml Erlenmeyer flask with 50 ml of pulp / peptone liquid medium (bleached pulp 30 g / l, peptone 10 g / l, KH). ₂ PO ₄ 1.5g / l, MgSO ₄ ・ 7H ₂ O 0.5 g / l, thiamine hydrochloride 2 mg / l, CuSO ₄ ・ 5H ₂ O 100 mg / l, adjusted to pH 5.0 with phosphoric acid) 50 mm ² Five agar pieces were inoculated and cultured at 28 ° C. for 6 days under shaking. After 6 days, the resulting culture was centrifuged to obtain the supernatant.
The enzyme activity was 50 μl of 1 M sodium acetate buffer (pH 4.0), 5 μm ABTS (50 μl of 2,2′-azino-bis (3-ethylbenzthiazine-6-sulfate)), 400 μl of enzyme solution, and oxidation of ABTS resulting from the reaction. The increase in absorbance at 420 nm was recorded over time, and enzyme activity was observed in the culture supernatant at 35 units / ml on the 5th day of the culture, where the enzyme activity unit was 1 μmol per minute. It is defined as the amount of enzyme required to oxidize ABTS, while the culture supernatant of Coriolus hirsutus strain OJI-1078 (FERM BP-4210) without donor DNA cultured under the same conditions is only 5 Only units / ml were observed.
[Example 19] Construction of expression vector of lignin peroxidase gene by cellobiohydrolase I gene promoter
The plasmid pCHCBHI31P obtained in Example 13 was digested with the restriction enzyme NcoI (Takara Shuzo) to obtain an expression vector portion.
Next, a plasmid pBSLPOOG7 / E. Coli containing a high temperature inducible lignin peroxidase gene derived from Coriolus hirsutus. E. coli JM109 (FERM P-12683) was amplified by the PCR method using the following two primers (7, 8).

The obtained PCR fragment was introduced into the above TA-cloning vector to obtain a plasmid pTALiP.
The plasmid pTALiP was digested with the restriction enzyme NcoI to obtain a lignin peroxidase structural gene portion, which was introduced into the treated cellobiohydrolase I expression vector pCHCBHI31P.
The resulting plasmid was named pCHCBHI31PLiP (FIG. 3).
[Example 20] Preparation of a Coriolus hirsutus transformant producing lignin peroxidase with high secretion
When transforming the arginine-requiring Coriolus hirsutus strain OJI-1078 (FERM BP-4210) using the pCHCBHI31PLiP obtained in Example 19, the plasmid pUCR1 carrying the ornithine carbamoyltransferase gene derived from Coriolus hirsutus as a selection marker is used. A transformant was obtained by simultaneous introduction (eg, PEG method or electroporation method). 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.
About 10 ⁶ 2 μg of the plasmid pCHCBHI31PLiP prepared in Example 9 was added in a circular or linear form to 100 μl of the protoplast solution at a concentration of 100 μl / 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% PEG 3400, 20 mM MOPS (pH 6.4)) was added, and the mixture was ice-cooled 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. Further, DNA was prepared from the transformed strain, and it was confirmed by Southern hybridization that the target lignin peroxidase expression plasmid was incorporated.
[Example 21] Production of lignin peroxidase by a transformant
The transformed strain obtained in Example 20 was placed in a 500 ml Erlenmeyer flask with 50 ml of pulp / peptone liquid medium (bleached pulp 30 g / l, peptone 10 g / l, KH). ₂ PO ₄ 1.5g / l, MgSO ₄ ・ 7H ₂ O 0.5g / l, thiamine hydrochloride 2mg / l, 5mM veratryl alcohol, adjusted to pH 4.5 with phosphoric acid) 50mm ² Five agar pieces were inoculated and cultured at 28 ° C. for 6 days under shaking. After 6 days, the resulting culture was centrifuged to obtain the supernatant.
Enzyme activity was increased by adding 100 μl of 1 M sodium tartrate buffer (pH 3.0), 25 μl of 8 mM veratryl alcohol, 350 μl of enzyme solution, and 25 μl of 5.4 mM hydrogen peroxide, and increasing the absorbance of veratraldehyde resulting from the reaction over 310 nm. The enzyme activity was found to be 0.8 unit / ml in the culture supernatant on the 5th day from the start of the culture. Here, the enzyme activity unit is defined as the amount of enzyme required to produce 1 μmol of veratraldehyde per minute. On the other hand, lignin peroxidase activity was not observed in the culture supernatant of Coriolus hirsutus strain OJI-1078 (FERM BP-4210) that did not contain donor DNA cultured under the same conditions.
3. Production and utilization of lignin degrading enzyme using the second DNA fragment containing the promoter sequence of cellobiohydrolase I gene
[Example 22] Isolation of cellobiohydrolase I gene from a chromosomal gene library
In the same manner as in Example 12, clones were selected from the chromosomal gene library. The probe used for plaque hybridization is a synthetic oligomer having the sequence of SEQ ID NO: 28, labeled with fluorescein at the 3 ′ end using an oligo DNA labeling kit manufactured by Amersham.

However, in SEQ ID NO: 28, base Y represents base T or C, and base R represents base G or A.
As a result, 4 positive clones could be selected from about 40,000 plaques. Recombinant phage DNA prepared from positive clones according to a conventional method was digested with various restriction enzymes, and Southern hybridization was performed using the above synthetic DNA. As a result, a clone that hybridized to 4.2 kbp as a single DNA band was found in the fragment obtained by digestion with the restriction enzyme SalI.
The above DNA fragment 4.2 kbp was excised by agarose gel electrophoresis, subcloned into the SalI site of E. coli vector pUC19, and transformed into E. coli JM109 strain to obtain plasmid pCHCBHI27. The base sequence of the subcloned DNA fragment was determined. Determination of the base sequence was performed using a BigDye Terminator Cycle Sequencing kit manufactured by Applied Biosystems, followed by electrophoresis using a DNA sequencer PRISM 310 manufactured by Applied Biosystems, and the base sequence was analyzed.
The base sequence is shown in SEQ ID NO: 26. As a result, the celliobiohydrolase I gene derived from Coriolus hirsutus was separated by two introns within the range of the base sequence. The amino acid sequence deduced from the base sequence is shown in SEQ ID NO: 27.
[Example 23] Construction of expression vector for manganese peroxidase gene by cellobiohydrolase I gene promoter
The plasmid pCHCBHI27 containing the cellobiohydrolase I gene promoter region derived from Coriolus hirsutus is amplified as a promoter region of 2.1 kbp by PCR using the following two primers (1, 2), and T4 DNA ligase is added to the SmaI site of pUC18. Was used to obtain plasmid pCHCBHI27P.

Next, the plasmid pBSMPOOG1 (deposit number FERM P-14933) containing the Coriolus hirsutus-derived manganese peroxidase gene was deposited by PCR using the two primers (3,4) shown below, and the manganese peroxidase structural gene portion NcoI was about 2.2 kb. Amplify the fragment.

The obtained PCR fragment was inserted using TA cloning kit manufactured by In Vitrogen to obtain pTAMP. Next, the obtained pTAMP was digested with the restriction enzyme NcoI, and about 2.2 kb was excised and inserted into the NcoI site of the above pCHCBHI27P to obtain the plasmid pCHCBHI27PMP (FIG. 4).
[Example 24] Transformant
Protoplasts were prepared and purified in the same manner as in Example 4. Subsequently, transformation was performed as follows.
10 ⁶ 2 μg of the plasmid pCHCBHI27PMP prepared in Example 23 was added to 100 μl of a protoplast solution with a concentration of 100 μl / 100 μl in a circular or linear form, and pUCR1 carrying the ornithine carbamoyltransferase gene derived from Coriolus hirsutus as a selection marker was added to 0. 2 μg was added and ice-cooled for 30 minutes. Next, an equal amount of PEG solution (50% PEG 3400, 20 mM MOPS (pH 6.4)) was added to the liquid volume, and the mixture was ice-cooled 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. Furthermore, DNA was prepared from the transformant, and it was confirmed by Southern hybridization that the target manganese peroxidase expression plasmid was incorporated.
[Example 25] Manganese peroxidase production by transformants
The transformed strain obtained in Example 24 was placed in a 500 ml Erlenmeyer flask with 50 ml of pulp / peptone liquid medium (bleached pulp 30 g / l, peptone 10 g / l, KH). ₂ PO ₄ 1.5g / l, MgSO ₄ ・ 7H ₂ O 0.5 g / l, thiamine hydrochloride 2 mg / l, MnSO ₄ ・ 5H ₂ O48mg / l, adjusted to pH 5.0 with phosphoric acid) 50mm ² Five agar pieces were inoculated and cultured at 28 ° C. for 6 days under shaking. After 6 days, the resulting culture was centrifuged to obtain the supernatant.
The enzyme activity was 50 μl of 0.5 M sodium malonate buffer (pH 5.5), 345 μl of enzyme solution, 5 μl of 10 mM hydrogen peroxide, 1 mM MnSO. ₄ 100 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, and the Mn (III) malonic acid complex was added to the culture supernatant in the enzyme supernatant. The activity was observed at 8.5 μmol / ml / min on the 6th day from the start of the 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 Coriolus hirsutus strain OJI-1078 (FERM BP-4210) that did not contain donor DNA cultured under the same conditions.
[Example 26] Construction of expression vector for laccase gene by cellobiohydrolase I gene promoter
The plasmid pCHCBHI27P obtained in Example 23 was digested with the restriction enzyme NcoI (Takara Shuzo Co., Ltd.) and then smoothed using Klenow fragment. Thereafter, it was digested with the restriction enzyme EcoRI to obtain an expression vector portion.
Next, a plasmid OJ-POG-E1 (FERM BP-2793) containing a Coriolus hirsutus-derived laccase gene was amplified by PCR using the following two primers (5, 6).

The obtained PCR fragment was introduced into the above-mentioned TA-cloning vector to obtain a plasmid pTALAC.
The plasmid pTALAC is digested with the restriction enzyme XhoI and then blunted using the modification enzyme klenow fragment. Thereafter, digestion was performed with the restriction enzyme EcoRI to obtain a laccase structural gene portion, which was introduced into the above-treated cellobiohydrolase I expression vector pCHCBHI27P.
The resulting plasmid was named pCHCBHI27PLAC (FIG. 5).
[Example 27] Production of a Coriolus hirsutus transformant producing laccase with high secretion
When transforming the arginine-requiring Coriolus hirsutus strain OJI-1078 (FERM BP-4210) using the pCHCBHI27PLAC obtained in Example 26, a plasmid (pUCR1) carrying the OCT gene derived from Coriolus hirsutus as a selection marker is simultaneously used. A transformant was obtained by introduction (eg, PEG method or electroporation method). 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.
About 10 ⁶ 2 μg of the plasmid pCHCBHI27PLAC prepared in Example 26 was added to 100 μl of the protoplast solution having a concentration of 100 μl / 100 μl 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% PEG 3400, 20 mM MOPS (pH 6.4)) was added, and the mixture was ice-cooled 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. Further, DNA was prepared from the transformed strain, and it was confirmed by Southern hybridization that the target laccase expression plasmid was incorporated.
[Example 28] Production of laccase by transformant
The transformed strain obtained in Example 27 was placed in a 500 ml Erlenmeyer flask with 50 ml of pulp / peptone liquid medium (bleached pulp 30 g / l, peptone 10 g / l, KH). ₂ PO ₄ 1.5g / l, MgSO ₄ ・ 7H ₂ O 0.5 g / l, thiamine hydrochloride 2 mg / l, CuSO ₄ ・ 5H ₂ O 100 mg / l, adjusted to pH 5.0 with phosphoric acid) 50 mm ² Five agar pieces were inoculated and cultured at 28 ° C. for 6 days under shaking. After 6 days, the resulting culture was centrifuged to obtain the supernatant.
Enzyme activity was 50 μl of 1M sodium acetate buffer (pH 4.0), 5 mM ABTS (2,2′-azino-bis (3-ethilbenzthiazine-6-sulfate) 50 μl, enzyme solution 400 μl, and oxidation of ABTS resulting from the reaction. The increase in absorbance at 420 nm was recorded over time, and enzyme activity was found to be 57 units / ml in the culture supernatant on the 5th day from the start of the culture, where the enzyme activity unit was 1 μmol per minute. It is defined as the amount of enzyme required to oxidize ABTS, while the culture supernatant of Coriolus hirsutus strain OJI-1078 (FERM BP-4210) without donor DNA cultured under the same conditions is only 5 Only units / ml were observed.
[Example 29] Construction of expression vector of lignin peroxidase gene by cellobiohydrolase I gene promoter
The plasmid pCHCBHI27P obtained in Example 23 was digested with the restriction enzyme NcoI (Takara Shuzo) to obtain an expression vector portion.
Next, a plasmid pBSLPOOG7 / E. Coli containing a high temperature inducible lignin peroxidase gene derived from Coriolus hirsutus. E. coli JM109 (FERM P-12683) was amplified by the PCR method using the following two primers (7, 8).

The obtained PCR fragment was introduced into the above TA-cloning vector to obtain a plasmid pTALiP.
The plasmid pTALiP was digested with the restriction enzyme NcoI to obtain a lignin peroxidase structural gene portion, which was introduced into the treated cellobiohydrolase I expression vector pCHCBHI27P.
The resulting plasmid was named pCHCBHI27PLiP (FIG. 6).
[Example 30] Preparation of a Coriolus hirsutus transformant producing lignin peroxidase with high secretion
When transforming the arginine-requiring Coriolus hirsutus OJI-1078 strain (FERM BP-4210) using the pCHCBHI27PLiP obtained in Example 29, the plasmid pUCR1 carrying the ornithine carbamoyltransferase gene derived from Coriolus hirsutus as a selection marker is used. A transformant was obtained by simultaneous introduction (eg, PEG method or electroporation method). 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.
About 10 ⁶ 2 μg of the plasmid pCHCBHI27PLiP prepared in Example 29 was added to 100 μl of the protoplast solution at a concentration of 100 μl / 100 μl in a circular or linear form, 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% PEG 3400, 20 mM MOPS (pH 6.4)) was added, and the mixture was ice-cooled 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. Further, DNA was prepared from the transformed strain, and it was confirmed by Southern hybridization that the target lignin peroxidase expression plasmid was incorporated.
[Example 31] Production of lignin peroxidase by a transformant
The transformed strain obtained in Example 30 was placed in a 500 ml Erlenmeyer flask with 50 ml of pulp / peptone liquid medium (bleached pulp 30 g / l, peptone 10 g / l, KH). ₂ PO ₄ 1.5g / l, MgSO ₄ ・ 7H ₂ O 0.5g / l, thiamine hydrochloride 2mg / l, 5mM veratryl alcohol, adjusted to pH 4.5 with phosphoric acid) 50mm ² Five agar pieces were inoculated and cultured at 28 ° C. for 6 days under shaking. After 6 days, the resulting culture was centrifuged to obtain the supernatant.
Enzyme activity was increased by adding 100 μl of 1 M sodium tartrate buffer (pH 3.0), 25 μl of 8 mM veratryl alcohol, 350 μl of enzyme solution, and 25 μl of 5.4 mM hydrogen peroxide, and increasing the absorbance of veratraldehyde resulting from the reaction over 310 nm. The enzyme activity in the culture supernatant was found to be 1.5 units / ml on the 5th day from the start of the culture. Here, the enzyme activity unit is defined as the amount of enzyme required to produce 1 μmol of veratraldehyde per minute. On the other hand, lignin peroxidase activity was not observed in the culture supernatant of Coriolus hirsutus strain OJI-1078 (FERM BP-4210) that did not contain donor DNA cultured under the same conditions.
4). Production and utilization of lignin-degrading enzyme using a third DNA fragment containing the promoter sequence of cellobiohydrolase I gene
[Example 32] Isolation of cellobiohydrase I gene from a chromosomal gene library
In the same manner as in Example 12, clones were selected from the chromosomal gene library. The probe used for the plaque hybridization is a synthetic oligomer having the sequence of SEQ ID NO: 40 labeled with fluorescein at the 3 ′ end using an oligo DNA labeling kit manufactured by Amersham.

However, in SEQ ID NO: 40, base Y represents base T or C, and base R represents base G or A.
As a result, 4 positive clones could be selected from about 40,000 plaques. Recombinant phage DNA prepared from positive clones according to a conventional method was digested with various restriction enzymes, and Southern hybridization was performed using the above synthetic DNA. As a result, a clone that hybridized to 3.9 kbp as a single DNA band was found in the fragment obtained by digestion with restriction enzymes PstI and NheI.
The DNA fragment 3.9 kbp was excised by agarose gel electrophoresis, subcloned into the PstI-SpeI site of the E. coli vector pBluescriptsII SK-, and transformed into E. coli JM109 strain to obtain plasmid pCHCBHI26. The base sequence of the subcloned DNA fragment was determined. Determination of the base sequence was performed using a BigDye Terminator Cycle Sequencing kit manufactured by Applied Biosystems, followed by electrophoresis using a DNA sequencer PRISM 310 manufactured by Applied Biosystems, and the base sequence was analyzed.
The base sequence is shown in SEQ ID NO: 38. As a result, the celliobiohydrolase I gene derived from Coriolus hirsutus was separated by two introns within the range of the base sequence. The amino acid sequence deduced from the base sequence is shown in SEQ ID NO: 39.
[Example 33] Construction of manganese peroxidase gene expression vector by cellobiohydrolase I gene promoter
The plasmid pCHCBHI26 containing the celliobiohydrolase I gene promoter region derived from Coriolus hirsutus was digested with the restriction enzyme NcoI, made blunt using klenow fragment, then self-ligated using T4 DNA ligase, and the NcoI site disappeared Plasmid pCBCBHI26-NcoI was obtained. Next, the above plasmid was amplified as a promoter region of 1.1 kbp by PCR using the following two primers (1, 2), and ligated to the SmaI site of pUC18 using T4 DNA ligase to obtain plasmid pCHCBHI26P. .

Next, the plasmid pBSMPOOG1 (deposit number FERM P-14933) containing the Coriolus hirsutus-derived manganese peroxidase gene was deposited by PCR using the two primers (3,4) shown below, and the manganese peroxidase structural gene portion NcoI was about 2.2 kb. Amplify the fragment.

The obtained PCR fragment was inserted using TA cloning kit manufactured by In Vitrogen to obtain pTAMP. Next, the obtained pTAMP was digested with the restriction enzyme NcoI, and about 2.2 kb was excised and inserted into the NcoI site of the pCHCBHI26P to obtain the plasmid pCHCBHI26PMP (FIG. 7). [Example 34] Coriolus hirsutus transformation method
Protoplasts were prepared and purified in the same manner as in Example 4. Subsequently, transformation was performed as follows.
10 ⁶ 2 μg of the plasmid pCHCBHI26PMP prepared in Example 33 was added to 100 μl of a protoplast solution at a concentration of 100 μl / 100 μl in a circular or linear form, and pUCR1 retaining the ornithine carbamoyltransferase gene derived from Coriolus hirsutus as a selection marker .2 μg was added and ice-cooled for 30 minutes. Next, an equal amount of PEG solution (50% PEG 3400, 20 mM MOPS (pH 6.4)) was added to the liquid volume, and the mixture was ice-cooled 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. Furthermore, DNA was prepared from the transformant, and it was confirmed by Southern hybridization that the target manganese peroxidase expression plasmid was incorporated.
[Example 35] Manganese peroxidase production by transformant
The transformed strain obtained in Example 34 was placed in a 500 ml Erlenmeyer flask with 50 ml of pulp / peptone liquid medium (bleached pulp 30 g / l, peptone 10 g / l, KH). ₂ PO ₄ 1.5g / l, MgSO ₄ ・ 7H ₂ O 0.5 g / l, thiamine hydrochloride 2 mg / l, MnSO ₄ ・ 5H ₂ O48mg / l, adjusted to pH 5.0 with phosphoric acid) 50mm ² Five agar pieces were inoculated and cultured at 28 ° C. for 6 days under shaking. After 6 days, the resulting culture was centrifuged to obtain the supernatant.
The enzyme activity was 50 μl of 0.5 M sodium malonate buffer (pH 5.5), 345 μl of enzyme solution, 5 μl of 10 mM hydrogen peroxide, 1 mM MnSO. ₄ 100 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, and the Mn (III) malonic acid complex was added to the culture supernatant in the enzyme supernatant. The activity was observed at 5.5 μmol / ml / min on the 6th day from the start of the 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 Coriolus hirsutus strain OJI-1078 (FERM BP-4210) that did not contain donor DNA cultured under the same conditions.
[Example 36] Construction of expression vector of laccase gene by cellobiohydrolase I gene promoter
The plasmid pCHCBHI26P obtained in Example 33 was digested with the restriction enzyme NcoI (manufactured by Takara Shuzo Co., Ltd.) and then smoothed using Klenow fragment. Thereafter, it was digested with the restriction enzyme EcoRI to obtain an expression vector portion.
Next, the plasmid OJ-POG-E1 (deposit number BP-2793) containing the Coriolus hirsutus-derived laccase gene was amplified by PCR using the following two primers (5, 6).

The obtained PCR fragment was introduced into the above-mentioned TA-cloning vector to obtain a plasmid pTALAC.
The plasmid pTALAC was digested with the restriction enzyme XhoI and then blunted using the modification enzyme klenow fragment. Thereafter, digestion was performed with the restriction enzyme EcoRI to obtain a laccase structural gene portion, which was introduced into the above-treated cellobiohydrolase I expression vector pCHCBHI26P.
The resulting plasmid was named pCHCBHI26PLAC (FIG. 8).
[Example 37] Production of a Coriolus hirsutus transformant producing laccase with high secretion
When transforming the arginine-requiring Coriolus hirsutus OJI-1078 strain (FERM BP-4210) using the pCHCBHI26PLAC obtained in Example 36, a plasmid (pUCR1) carrying the OCT gene derived from Coriolus hirsutus as a selection marker is simultaneously used. A transformant was obtained by introduction (eg, PEG method or electroporation method). 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.
About 10 ⁶ 2 μg of the plasmid pCHCBHI26PLAC prepared in Example 36 was added to 100 μl of the protoplast solution at a concentration of 100 μl / 100 μl 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% PEG 3400, 20 mM MOPS (pH 6.4)) was added, and the mixture was ice-cooled 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. Further, DNA was prepared from the transformed strain, and it was confirmed by Southern hybridization that the target laccase expression plasmid was incorporated.
[Example 38] Production of laccase by transformant
The transformed strain obtained in Example 37 was placed in a 500 ml Erlenmeyer flask with 50 ml of pulp / peptone liquid medium (bleached pulp 30 g / l, peptone 10 g / l, KH). ₂ PO ₄ 1.5g / l, MgSO ₄ ・ 7H ₂ O 0.5 g / l, thiamine hydrochloride 2 mg / l, CuSO ₄ ・ 5H ₂ O 100 mg / l, adjusted to pH 5.0 with phosphoric acid) 50 mm ² Five agar pieces were inoculated and cultured at 28 ° C. for 6 days under shaking. After 6 days, the resulting culture was centrifuged to obtain the supernatant.
Enzyme activity was 50 μl of 1M sodium acetate buffer (pH 4.0), 5 mM ABTS (2,2′-azino-bis (3-ethilbenzthiazine-6-sulfate) 50 μl, enzyme solution 400 μl, and oxidation of ABTS resulting from the reaction. The increase in absorbance at 420 nm was recorded over time, and enzyme activity was found to be 52 units / ml in the culture supernatant on the 5th day of the culture, where the enzyme activity unit was 1 μmol per minute. It is defined as the amount of enzyme required to oxidize ABTS, while the culture supernatant of Coriolus hirsutus strain OJI-1078 (FERM BP-4210) without donor DNA cultured under the same conditions is only 5 Only units / ml were observed.
[Example 39] Construction of expression vector of lignin peroxidase gene by cellobiohydrolase I gene promoter
The plasmid pCHCBHI26P obtained in Example 33 was digested with the restriction enzyme NcoI (Takara Shuzo Co., Ltd.) to obtain an expression vector portion.
Next, a plasmid pBSLPOOG7 / E. Coli containing a high temperature inducible lignin peroxidase gene derived from Coriolus hirsutus. E. coli JM109 (FERM P-12683) was amplified by the PCR method using the following two primers (7, 8).

The obtained PCR fragment was introduced into the above TA-cloning vector to obtain a plasmid pTALiP.
The plasmid pTALiP was digested with the restriction enzyme NcoI to obtain a lignin peroxidase structural gene portion, which was introduced into the treated cellobiohydrolase I expression vector pCHCBHI26P.
The resulting plasmid was named pCHCBHI26PLiP (FIG. 9).
[Example 40] Preparation of a Coriolus hirsutus transformant that produces lignin peroxidase with high secretion;
When transforming the arginine-requiring Coriolus hirsutus strain OJI-1078 (FERM BP-4210) using the pCHCBHI26PLiP obtained in Example 39, the plasmid pUCR1 carrying the ornithine carbamoyltransferase gene derived from Coriolus hirsutus as a selection marker is used. A transformant was obtained by simultaneous introduction (eg, PEG method or electroporation method). 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.
About 10 ⁶ 2 μg of the plasmid pCHCBHI26PLiP prepared in Example 39 was added to 100 μl of the protoplast solution at a concentration of 100 μl / 100 μl 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% PEG 3400, 20 mM MOPS (pH 6.4)) was added, and the mixture was ice-cooled 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. Further, DNA was prepared from the transformed strain, and it was confirmed by Southern hybridization that the target lignin peroxidase expression plasmid was incorporated.
[Example 41] Production of lignin peroxidase by a transformant
The transformed strain obtained in Example 40 was placed in a 500 ml Erlenmeyer flask with 50 ml of pulp / peptone liquid medium (bleached pulp 30 g / l, peptone 10 g / l, KH). ₂ PO ₄ 1.5g / l, MgSO ₄ ・ 7H ₂ O 0.5g / l, thiamine hydrochloride 2mg / l, 5mM veratryl alcohol, adjusted to pH 4.5 with phosphoric acid) 50mm ² Five agar pieces were inoculated and cultured at 28 ° C. for 6 days under shaking. After 6 days, the resulting culture was centrifuged to obtain the supernatant.
Enzyme activity was increased by adding 100 μl of 1 M sodium tartrate buffer (pH 3.0), 25 μl of 8 mM veratryl alcohol, 350 μl of enzyme solution, and 25 μl of 5.4 mM hydrogen peroxide, and increasing the absorbance of veratraldehyde resulting from the reaction over 310 nm. The enzyme activity was found to be 1.0 unit / ml in the culture supernatant on the 5th day from the start of the culture. Here, the enzyme activity unit is defined as the amount of enzyme required to produce 1 μmol of veratraldehyde per minute. On the other hand, lignin peroxidase activity was not observed in the culture supernatant of Coriolus hirsutus strain OJI-1078 (FERM BP-4210) that did not contain donor DNA cultured under the same conditions.
5). Production and utilization of lignin-degrading enzymes using DNA fragments containing the promoter sequence of cellobiohydrolase II gene
[Example 42] Isolation of a DNA fragment containing the promoter sequence of cellobiohydrolase II gene from a chromosomal gene library
In the same manner as in Example 1, a chromosomal gene library was obtained. Clones containing the cellobiohydrolase II gene promoter region were selected from the obtained library by plaque hybridization. This series of operations was performed according to a conventional method (Sambrook et al., Molecular Cloning A Laboratory Manual / 2nd Edition (1989)). The probe used for the plaque hybrid was prepared by using an oligo DNA labeling kit manufactured by Amersham for the synthetic oligomer shown below in SEQ ID NO: 52, which was prepared based on the base sequence of cellobiohydrolase II of Fanerokeete chrysosporium 3 'The end is labeled with fluorescein.

As a result, 8 positive clones could be selected from about 100,000 plaques. Recombinant phage DNA prepared from positive clones according to a conventional method was digested with various restriction enzymes, and Southern hybridization was performed using the above synthetic DNA. As a result, a clone hybridizing to 5.0 kbp as a single DNA band was found in the fragments obtained by digestion with restriction enzymes EcoRV and NcoI.
In order to recover the above DNA fragment, a 5.0 kbp DNA fragment obtained by digestion with the restriction enzyme NcoI, smoothing with the Klenow fragment, and digestion with EcoRV was excised by agarose gel electrophoresis, and the SmaI site of the E. coli vector pUC19 Was subcloned into plasmid pCHCBHII. This plasmid was transformed into Escherichia coli JM109 strain. The base sequence of the subcloned DNA fragment was determined. Determination of the base sequence was performed using a BigDye Terminator Cycle Sequencing kit manufactured by Applied Biosystems, followed by electrophoresis using a DNA sequencer PRISM 310 manufactured by Applied Biosystems, and the base sequence was analyzed. The base sequence is shown in SEQ ID NO: 50. As a result, the cellobiohydrolase II gene derived from Coriolus hirsutus was separated by six introns within the range of the above base sequence. The amino acid sequence deduced from the base sequence is shown in SEQ ID NO: 51.
[Example 43] Construction of expression vector for manganese peroxidase gene by cellobiohydrolase II gene promoter
From the plasmid pCHCBHII obtained in Example 42 containing the promoter sequence of the celliobiohydrolase II gene derived from Coriolus hirsutus, the two primers shown in SEQ ID NOs: 53 and 54 below were used for amplification by the PCR method, and the promoter region 3 Obtained 1 kbp.

The DNA fragment obtained by this PCR was ligated to the SmaI site of E. coli cloning vector pUC18 using T4 DNA ligase to obtain plasmid pCHCBHIIP.
Next, a plasmid pBSMPOOG1 (FERM P-14933) containing a Coriolus hirsutus-derived manganese peroxidase gene was amplified by PCR using the two primers shown in SEQ ID NOs: 55 and 56 below, and a manganese peroxidase structural gene NcoI cleavage site was amplified. An approximately 2.2 kb fragment was obtained.

The DNA fragment obtained by this PCR was inserted into a TA cloning vector using a TA cloning kit manufactured by In Vitrogen to obtain pTAMP. Next, the obtained pTAMP is digested with the restriction enzyme NcoI, and about 2.2 kb is excised, inserted into the NcoI site of the above-described plasmid pCHCBHIIP, and the celliobiohydrolase II gene promoter sequence derived from Coriolus hirsutus and the manganese peroxidase structural gene Plasmid pCHCBHIIPMP containing was obtained (FIG. 10).
[Example 44] Production of Coriolus hirsutus transformant
Protoplasts were prepared and purified in the same manner as in Example 4. Subsequently, transformation was performed as follows.
10 ⁶ 2 μg of the plasmid pCHCBHIIPMP prepared in Example 33 was added to 100 μl of a protoplast solution at a concentration of 100 / l / 100 μl in a circular or linear form. Further, 0.2 μg of plasmid pUCR1 carrying the ornithine carbamoyltransferase gene derived from Coriolus hirsutus was added as a selection marker, and the mixture was ice-cooled for 30 minutes. Next, an equivalent amount of PEG solution (50% PEG 3400, 20 mM MOPS (pH 6.4)) was added to the liquid volume, and the mixture was ice-cooled 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.
Furthermore, DNA was prepared from the transformant, and it was confirmed by Southern hybridization that the target manganese peroxidase expression plasmid pCHCBHIIPMP was incorporated.
[Example 45] Production of manganese peroxidase by a transformant
In a 500 ml Erlenmeyer flask, 50 ml of pulp-peptone liquid medium (bleached pulp 30 g / l, peptone 10 g / l, KH ₂ PO ₄ 1.5g / l, MgSO ₄ ・ 7H ₂ O 0.5 g / l, thiamine hydrochloride 2 mg / l, MnSO ₄ ・ 5H ₂ O48 mg / l, adjusted to pH 5.0 with phosphoric acid) to 50 mm of the transformant obtained in Example 44 above. ² Five agar pieces were inoculated and cultured at 28 ° C. for 6 days under shaking. After 6 days, the resulting culture was centrifuged to obtain the supernatant.
The enzyme activity was 50 μl of 0.5 M sodium malonate buffer (pH 5.5), 345 μl of enzyme solution, 5 μl of 10 mM hydrogen peroxide, 1 mM MnSO. ₄ 100 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.
As a result, Mn (III) malonic acid complex produced by the enzyme activity of manganese peroxidase was found in the culture supernatant at 6 μmol / ml / min on the 6th day from the start of the 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.
[Example 46] Construction of expression vector of laccase gene by cellobiohydrolase II gene promoter
The plasmid pCHCBHIIP obtained in Example 43 was digested with the restriction enzyme NcoI (Takara Shuzo) and then smoothed using Klenow fragment. Thereafter, it was digested with the restriction enzyme EcoRI to obtain a cellobiohydrolase II gene promoter expression vector portion.
Next, the plasmid OJ-POG-E1 (BP-2793) containing the Coriolus hirsutus-derived laccase gene was amplified by PCR using the primers of SEQ ID NOs: 57 and 58 shown below.

The obtained DNA fragment by PCR was introduced into a TA-cloning vector in the same manner as in Example 3 to obtain a plasmid pTALAC.
The plasmid pTALAC was digested with the restriction enzyme XhoI and then blunted using the modification enzyme klenow fragment. Thereafter, digestion was performed with the restriction enzyme EcoRI to obtain a laccase structural gene portion.
The laccase structural gene portion was introduced into the NcoI site of the cellobiohydrolase II expression vector pCHCBHIIP obtained in Example 43 to obtain plasmid pCHCBHIPLAC (FIG. 11).
Example 47 Production of a Coriolus hirsutus transformant producing laccase with high secretion
By the method described in Example 44, a protoplast solution of Coriolus hirsutus was obtained. About 10 ⁶ 2 μg of the plasmid pCHCBHIIPLAC prepared in Example 46 was added to each protoplast solution of 100 pcs / 100 μl in a circular or linear form. Further, 0.2 μg of plasmid pUCR1 carrying the ornithine carbamoyltransferase gene derived from Coriolus hirsutus was added as a selection marker, and the mixture was ice-cooled for 30 minutes. Next, an equal amount of PEG solution (50% PEG 3400, 20 mM MOPS (pH 6.4)) was added, and the mixture was ice-cooled 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. Further, DNA was prepared from the transformant, and it was confirmed by Southern hybridization that the target laccase expression plasmid pCHCBHIIPLAC was incorporated.
[Example 48] Production of laccase by transformant
In a 500 ml Erlenmeyer flask, 50 ml of pulp-peptone liquid medium (bleached pulp 30 g / l, peptone 10 g / l, KH ₂ PO ₄ 1.5g / l, MgSO ₄ ・ 7H ₂ O 0.5 g / l, thiamine hydrochloride 2 mg / l, CuSO ₄ ・ 5H ₂ O 100 mg / l, adjusted to pH 5.0 with phosphoric acid) to 50 mm of the transformant obtained in Example 47 above. ² Five agar pieces were inoculated and cultured at 28 ° C. for 6 days under shaking. After 6 days, the resulting culture was centrifuged to obtain the supernatant.
The enzyme activity was 50 μl of 1 M sodium acetate buffer (pH 4.0), 5 μm ABTS (50 μl of 2,2′-azino-bis (3-ethylbenzthiazine-6-sulfate)), 400 μl of enzyme solution, and oxidation of ABTS resulting from the reaction. The increase in absorbance at 420 nm was recorded over time, and as a result, 40 units / ml of enzyme activity was observed in the culture supernatant on day 5 after the start of the culture. ) Is the amount of enzyme required to oxidize 1 μmol of ABTS per minute, whereas the culture supernatant of OJI-1078 strain that does not contain donor DNA cultured under the same conditions has enzyme activity Only 5 units / ml were observed.
[Example 49] Construction of expression vector of lignin peroxidase gene by cellobiohydrolase II gene promoter
The plasmid pCHCBHIIP obtained in Example 43 was digested with the restriction enzyme NcoI (Takara Shuzo) to obtain a cellobiohydrolase II expression vector portion.
Next, a plasmid pBSLPOOG7 / E. Coli containing a high temperature inducible lignin peroxidase gene derived from Coriolus hirsutus. E. coli JM109 (FERM P-12683) was amplified by the PCR method using two primers of SEQ ID NOs: 59 and 60 shown below.

The obtained PCR fragment was introduced into a TA-cloning vector in the same manner as in Example 43 to obtain a plasmid pTALiP. Subsequently, plasmid pTALiP was digested with restriction enzyme NcoI to obtain a lignin peroxidase structural gene portion.
The lignin peroxidase structural gene part was introduced into the NcoI site of the cellobiohydrolase II expression vector pCHCBHIIP obtained in Example 43 to obtain the plasmid pCHCBHHPLiP (FIG. 12).
[Example 50] Preparation of a Coriolus hirsutus transformant producing lignin peroxidase with high secretion
A protoplast solution of Coriolus hirsutus was obtained by the method described in Example 44. About 10 ⁶ 2 μg of the plasmid pCHCBHIIPLiP prepared in Example 49 was added to each protoplast solution of 100 cells / 100 μl in a circular or linear form. Further, 0.2 μg of plasmid pUCR1 carrying the ornithine carbamoyltransferase gene derived from Coriolus hirsutus was added as a selection marker, and the mixture was ice-cooled for 30 minutes.
Next, an equal amount of PEG solution (50% PEG 3400, 20 mM MOPS (pH 6.4)) was added, and the mixture was ice-cooled 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.
Further, DNA was prepared from the transformant, and it was confirmed by Southern hybridization that the target lignin peroxidase expression plasmid pCHCBHIIPLiP was incorporated.
[Example 51] Production of lignin peroxidase by a transformant
In a 500 ml Erlenmeyer flask, 50 ml of pulp-peptone liquid medium (bleached pulp 30 g / l, peptone 10 g / l, KH ₂ PO ₄ 1.5g / l, MgSO ₄ ・ 7H ₂ O 0.5 g / l, thiamine hydrochloride 2 mg / l, 5 mM veratryl alcohol, adjusted to pH 4.5 with phosphoric acid) 50 mm of the transformant obtained in Example 10 above ² Five agar pieces were inoculated and cultured at 28 ° C. for 6 days under shaking. After 6 days, the resulting culture was centrifuged to obtain the supernatant.
Enzyme activity was increased by adding 100 μl of 1M sodium tartrate buffer (pH 3.0), 25 μl of 8 mM veratryl alcohol, 350 μl of enzyme solution, 25 μl of 5.4 mM hydrogen peroxide, and increasing the absorbance of veratraldehyde resulting from the reaction over 310 nm. This was done by recording later. Enzyme activity was found to be 1.2 units / ml in the culture supernatant on the 5th day from the start of the culture. Here, the enzyme activity unit (unit) was the amount of enzyme required to produce 1 μmol of veratraldehyde per minute. On the other hand, lignin peroxidase activity was not observed in the culture supernatant of Coriolus hirsutus strain OJI-1078 (FERM BP-4210) that did not contain donor DNA cultured under the same conditions.
[Example 52] Chip treatment using a transformant producing highly secreted manganese peroxidase
The transformed strain producing high secretion of manganese peroxidase selected in Example 44 was cultured at 28 ° C. on potato dextrose agar medium, and stored at 4 ° C. Five sections of this plate punched out with a cork borer with a diameter of 5 mm were added to a glucose peptone medium (glucose 2%, polypeptone 0.5%, yeast extract 0.2%, KH ₂ PO ₄ , MgSO ₄ 0.05%, adjusted to pH 4.5 with phosphoric acid) was inoculated into a 300 ml Erlenmeyer flask containing 100 ml each, and cultured with shaking at 28 ° C. and 100 rpm for 1 week. After the cultivation, the cells were filtered off, and the medium remaining in the cells was washed with sterilized water. The microbial cells were pulverized with sterilized water for 10 seconds by a Waring blender, and inoculated so that the dry weight of the microbial cells was 10 mg against 1 kg of the eucalyptus wood having an absolutely dry weight. After inoculation, the mixture was stirred well so that the bacteria spread throughout. The culture was performed by static culture for 1 week with aeration at 28 ° C. Saturated water vapor was aerated as needed so that the moisture content of the chip was 40 to 65%. The amount of ventilation when venting was set to 0.01 vvm per chip.
[Example 53] Production of mechanical pulp by a transformant producing high secretion of manganese peroxidase
Chip treatment was performed in accordance with Example 52 using a radiata pine material. The treated wood chips are beaten using a lab refiner (manufactured by Kumagai Riki Co., Ltd.) to make the Canadian standard freeness 200 ml, and then the preparation of the handsheet for pulp physics is prepared using the Tappi test method T205 om- 81, the physical testing of the handmade pulp sheet was performed in accordance with the Tappi method T220om-83. As the amount of power used, a wattmeter (Hiokidenki model 3133) and an integrator (model 3141) were used. The chip yield was measured by taking 1 kg of moisture-containing wood chips into a container at an absolute dry weight, measuring the absolute dry weight of the chips before and after treatment, and calculating the chip yield using the following formula.
(Absolute dry weight after treatment) / (absolute dry weight before treatment) × 100
As shown in Table 1, the transformed strains that suppressed cellobiose dehydrogenase were able to suppress the yield loss and reduce the fibrillation energy. In addition, both tear strength and burst strength increased. When treated with wild strains, the fibrillation energy was reduced, but the paper strength decreased.

[Example 54] Cooking of chips treated with transformants producing high secretion of manganese peroxidase
After performing eucalyptus chip treatment according to Example 52, a chip with an absolute dry weight of 400 g was measured, and the liquid ratio was 5, the sulfidity was 30%, and the effective alkali was 17% (Na ₂ The cooking white liquor was added so as to obtain O), and kraft cooking was performed at a cooking temperature of 150 ° C. After completion of the kraft cooking, the black liquor was separated, and the obtained chips were defibrated with a high-concentration disintegrator, and then centrifugal dehydration and water washing were repeated three times with a filter cloth. Next, undistilled material was removed by a screen, and centrifugal dehydration was performed to obtain a digested unbleached pulp.
To the pulp obtained by the above kraft cooking, 2.0% by mass of NaOH is added, oxygen gas is injected, 100 ° C., oxygen gauge pressure 0.49 MPa (5 kg / cm ² ) For 60 minutes.
The pulp obtained above was subjected to a four-stage bleaching process of D-E-P-D as shown below. The first chlorine dioxide treatment (D) was prepared such that the pulp concentration was 10% by mass, 0.4% by mass of chlorine dioxide was added, and the treatment was performed at 70 ° C. for 40 minutes. Next, after washing with deionized water and dehydration, the pulp concentration was adjusted to 10% by mass, 1% by mass of caustic soda was added, and alkali extraction treatment (E) at 70 ° C. for 90 minutes was performed. Next, after washing with deionized water and dehydration, the pulp concentration is adjusted to 10% by mass, 0.5% by mass of hydrogen peroxide and 0.5% by mass of caustic soda are sequentially added, and the peroxide is oxidized at 70 ° C. for 120 minutes. Hydrogen treatment (P) was performed. Next, after washing with deionized water and dehydration, the pulp concentration was adjusted to 10%, 0.25% by mass of chlorine dioxide was added, and chlorine dioxide treatment (D) was performed at 70 ° C. for 180 minutes. Finally, after washing with deionized water and dehydration, a bleached pulp having a whiteness of 86.0% according to JIS P 8123 was obtained.
The pulp slurry having a pulp concentration of 4% by mass obtained above was beaten with a refiner so that the freeness was 410 ml (CSF).
[Example 55] Measurement of Ka number, preparation of handmade sheet for pulp physics test, physical test of pulp handmade sheet
The kappa number was measured according to JIS P 8211. The preparation of the handsheet for pulp physics test was performed according to Tappi test method T205-om81, and the physical test of the pulp handsheet was performed according to Tappi method T220 om-83. As shown in Table 2, when wood chips were treated with transformants and wild strains, the Ka value after cooking decreased, the yield of selection increased, and the drought rate decreased. Moreover, as shown in Table 3, the transformant did not cause a decrease in paper strength when compared with the wild type.

Industrial availability
The present invention provides a promoter sequence that functions in a basidiomycete of a host, particularly Coriolus hirsutus, and produces a large amount of lignin-degrading enzyme in the host, so that mass production of the enzyme by genetic recombination is difficult Thus, it is possible to efficiently produce lignin-degrading enzymes such as manganese peroxidase, lignin peroxidase and laccase, and to process wood chips using host cells that produce the enzyme in high amounts.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the structure of the manganese peroxidase expression plasmid pCHCBHI31PMP described in Example 13.
FIG. 2 shows the structure of the laccase expression plasmid pCHCBHI31PLAC described in Example 16.
FIG. 3 shows the structure of the lignin peroxidase expression vector pCHCBHI31PLiP described in Example 19.
FIG. 4 is a diagram showing the structure of the manganese peroxidase expression plasmid pCHCBHI27PMP described in Example 23.
FIG. 5 shows the structure of the laccase expression plasmid pCHCBHI27PLAC described in Example 26.
FIG. 6 shows the structure of the lignin peroxidase expression vector pCHCBHI27PLiP described in Example 29.
FIG. 7 shows the structure of the manganese peroxidase expression plasmid pCHCBHI26PMP described in Example 33.
FIG. 8 shows the structure of the laccase expression plasmid pCHCBHI26PLAC described in Example 36.
FIG. 9 shows the structure of the lignin peroxidase expression vector pCHCBHI26PLiP described in Example 39.
FIG. 10 shows the structure of the manganese peroxidase expression plasmid pCHCBHIIPMP described in Example 43.
FIG. 11 shows the structure of the laccase expression plasmid pCHCBHIIPLAC described in Example 46.
FIG. 12 shows the structure of the lignin peroxidase expression plasmid pCHCBHIIPLiP described in Example 49.

Claims (10)

Coriolus Hirstas (Coriolus) a DNA fragment containing a promoter sequence of cellobiose dehydrogenase gene, cellobiohydrolase I gene, cellobiohydrolase II gene, endoglucanase gene, or β-glucosidase gene derived from hirsutus) , and a gene encoding lignin degrading enzyme, Producing a vector comprising recombinant DNA joined to the DNA fragment so that the gene can be transcribed;
Performing a transformation using the vector to prepare a host cell producing high lignin-degrading enzyme;
A method for producing a lignin-degrading enzyme, comprising a step of culturing the lignin-degrading enzyme high-producing host cell in the presence of cellulose to produce a lignin-degrading enzyme. Coriolus Hirstas (Coriolus) a DNA fragment containing a promoter sequence of cellobiose dehydrogenase gene, cellobiohydrolase I gene, cellobiohydrolase II gene, endoglucanase gene, or β-glucosidase gene derived from hirsutus) , and a gene encoding lignin degrading enzyme, Producing a vector comprising recombinant DNA joined to the DNA fragment so that the gene can be transcribed;
Performing a transformation using the vector to prepare a host cell producing high lignin-degrading enzyme;
A method for treating wood chips, comprising the step of inoculating the wood chips with the lignin-degrading enzyme high-producing host cells. DNA fragment containing the promoter sequence has any one of the nucleotide sequences of the following (a) ~ (c), is an isolated DNA fragment containing the cellobiose dehydrogenase gene promoter sequence, according to claim 1 or 2 Method.
(A) The base sequence represented by SEQ ID NO: 1 or 2.
(B) A base sequence having a base sequence that hybridizes under stringent conditions with a base sequence having a base sequence complementary to the base sequence of (a), and having cellobiose dehydrogenase gene promoter activity.
(C) A base sequence having a base sequence in which one or more bases are deleted, substituted or added in SEQ ID NO: 1 or 2, and having cellobiose dehydrogenase gene promoter activity. DNA fragment containing the promoter sequence is the following (a) having any of the nucleotide sequences of ~ (c), isolated DNA fragment comprising a cellobiohydrolase I gene promoter sequence, to claim 1 or 2 The method described.
(A) The base sequence represented by SEQ ID NO: 13, 25 or 37.
(B) A base sequence having a base sequence that hybridizes under stringent conditions with a base sequence having a base sequence complementary to the base sequence of (a) and having cellobiohydrolase I gene promoter activity.
(C) A base sequence having a base sequence in which one or more bases are deleted, substituted or added in SEQ ID NO: 13, 25 or 37 and having cellobiohydrolase I gene promoter activity. DNA fragment containing the promoter sequence is the following (a) having any of the nucleotide sequences of ~ (c), isolated DNA fragment comprising a cellobiohydrolase II gene promoter sequence, to claim 1 or 2 The method described.
(A) The base sequence represented by SEQ ID NO: 49.
(B) A base sequence having a base sequence that hybridizes under stringent conditions with a base sequence having a base sequence complementary to the base sequence of (a) and having cellobiohydrolase II gene promoter activity.
(C) A base sequence having a base sequence in which one or more bases are deleted, substituted or added in SEQ ID NO: 49, and having cellobiohydrolase II gene promoter activity. The method according to any one of claims 1 to 5 , wherein the gene encoding the lignin degrading enzyme is selected from the group consisting of a manganese peroxidase gene, a lignin peroxidase gene and a laccase gene. The method according to any one of claims 1 to 6 , wherein the host cell is Coriolus hirsutus. An isolated DNA fragment comprising a cellobiose dehydrogenase gene promoter sequence having any one of the following base sequences (a) to (c):
(A) The base sequence represented by SEQ ID NO: 1 or 2.
(B) A base sequence having a base sequence that hybridizes under stringent conditions with a base sequence having a base sequence complementary to the base sequence of (a), and having cellobiose dehydrogenase gene promoter activity.
(C) A base sequence having a base sequence in which one or more bases are deleted, substituted or added in SEQ ID NO: 1 or 2, and having cellobiose dehydrogenase gene promoter activity. An isolated DNA fragment comprising the cellobiohydrolase I gene promoter sequence, which has any of the following base sequences (a) to (c):
(A) The base sequence represented by SEQ ID NO: 13, 25 or 37.
(B) A base sequence having a base sequence that hybridizes under stringent conditions with a base sequence having a base sequence complementary to the base sequence of (a) and having cellobiohydrolase I gene promoter activity.
(C) A base sequence having a base sequence in which one or more bases are deleted, substituted or added in SEQ ID NO: 13, 25 or 37 and having cellobiohydrolase I gene promoter activity. An isolated DNA fragment comprising a cellobiohydrolase II gene promoter sequence having any one of the following base sequences (a) to (c):
(A) The base sequence represented by SEQ ID NO: 49.
(B) A base sequence having a base sequence that hybridizes under stringent conditions with a base sequence having a base sequence complementary to the base sequence of (a) and having cellobiohydrolase II gene promoter activity.
(C) A base sequence having a base sequence in which one or more bases are deleted, substituted or added in SEQ ID NO: 49, and having cellobiohydrolase II gene promoter activity.

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