JP2018527944A - O−アセチルホモセリンスルフヒドリラーゼ変異体及びそれを用いたl−メチオニン製造方法 - Google Patents
O−アセチルホモセリンスルフヒドリラーゼ変異体及びそれを用いたl−メチオニン製造方法 Download PDFInfo
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- JP2018527944A JP2018527944A JP2018515858A JP2018515858A JP2018527944A JP 2018527944 A JP2018527944 A JP 2018527944A JP 2018515858 A JP2018515858 A JP 2018515858A JP 2018515858 A JP2018515858 A JP 2018515858A JP 2018527944 A JP2018527944 A JP 2018527944A
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- acetylhomoserine sulfhydrylase
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Abstract
Description
本出願の他の目的は、前記変異体をコードするポリヌクレオチドを提供することにある。
本出願のもう一つの目的は、前記O−アセチルホモセリンスルフヒドリラーゼ変異体を生産する微生物を提供することにある。
前記配列番号1のアミノ酸配列で記載されたポリペプチドのN末端から196番目のアミノ酸であるバリンが他のアミノ酸に置換された、O−アセチルホモセリンスルフヒドリラーゼの活性を有する変異型ポリペプチドをコードするポリヌクレオチドであってもよい。その例として、前記ポリヌクレオチドは、配列番号4の塩基配列を有するポリヌクレオチドであってもよく、これに制限されるものではない。また、配列番号4の塩基配列と少なくとも80%、90%、95%、97%または99%の相同性を有するポリヌクレオチドであってもよい。また、コドン縮退性(codon degeneracy)により、前記変異型ポリペプチドと翻訳されうるポリヌクレオチド及び配列番号4の塩基配列に対して、相補的な塩基配列からなる核酸と厳格な条件下で混成化し、前記O−アセチルホモセリンスルフヒドリラーゼの活性を有するポリペプチドをコードする塩基配列も含まれうることは自明である。
本出願で用語、「抗生剤抵抗性遺伝子」とは、抗生剤に対して抵抗性を有する遺伝子であって、これを有している細胞は該当抗生剤を処理した環境でも生存するため、大腸菌で大量にプラスミドを得る過程で選別マーカーとして有用に使用される。本出願で抗生剤抵抗性遺伝子は、遺伝子は本出願の核心的な技術であるベクターの最適な組み合わせによる発現効率に多く影響を与える要素がないため、選別マーカーとして一般的に用いられる抗生剤抵抗性遺伝子を制限なく用いてもよい。具体的な例として、アンピシリン(ampicilin)、テトラサイクリン(tetracyclin)、カナマイシン(kanamycin)、クロラムフェニコール(chloroamphenicol)、ストレプトマイシン(streptomycin)またはネオマイシン(neomycin)に対する抵抗遺伝子などを用いてもよい。
(実施例)
以下、実施例を挙げて本出願をさらに詳細に説明する。これらの実施例は、単に本出願を例示するためのもので、本出願の範囲がこれらの実施例により制限されるものと解釈されない。
実施例1−1:ロドバクター属に由来のO−アセチルホモセリンスルフヒドリラーゼの発現
公知の代表的なロドバクター属微生物であるロドバクター・スフェロイデス(Rhodobacter sphaeroides)に由来のO−アセチルホモセリンスルフヒドリラーゼがクローニングされたベクターpCL−PCJ1:metZ−rsp(特許文献2)を大腸菌(Escherichia coli)K12 W3110(ATCC 27325)細胞に熱ショック法を用いて形質転換させた。その後、25μg/mlのクロラムフェニコール(chloramphenicol)を含有しているLB培地で培養した後、形質転換されたコロニーを選別した。選別されたコロニーを25μg/mlクロラムフェニコールを含むLB培地3mlに接種して33℃、200rpmの条件で6時間培養した。これを再び250μlを取り、新しい25μg/mlクロラムフェニコールが含まれた25mlLB培地(250ml容量のフラスコ)に再接種した後、33℃、200rpmの条件で15時間培養し、野生型であるO−アセチルホモセリンスルフヒドリラーゼを発現させた。
前記野生型O−アセチルホモセリンスルフヒドリラーゼの活性を高めうる変異の位置として196番のアミノ酸であるバリンを選定した。
前記酵素変異体及び野生型酵素の培養液に2%(v/v)キシレン(p−Xylene)を添加して33℃、1,150rpmで1時間処理した。その後、一部を取り50mMリン酸カリウム(potassium phosphate)(pH7.4、pH6.4)、15g/L O−アセチルホモセリン、0.05mM PLP(pyridoxal5’−phosphate)、30mMナトリウムメチルメルカプタン(sodium methyl mercaptan)が含まれた緩衝液に入れて、33℃、1,150rpmで5分間反応させた後、反応液をHPLC分析で生成されたL−メチオニンの量を測定した。
実施例2−1:O−アセチルホモセリンスルフヒドリラーゼの精製
前記実施例1で得られたロドバクター・スフェロイデスに由来のO−アセチルホモセリンスルフヒドリラーゼ野生型及びV196T変異体の培養液25mlを遠心分離して細胞を回収した。O−アセチルホモセリンスルフヒドリラーゼを含有する無細胞抽出物を製造するために、細胞を150mM NaCl、5%グリセロール(glycerol)を含有する15mlの50mMリン酸カリウム(potassium phosphate)(pH7.8)に懸濁させて、懸濁液の温度を10℃未満に維持しつつ、ソニケーター(sonicator)を用いて破砕した。その後、細胞懸濁液を15,000×gで25分間遠心分離して細胞破片を除去した。無細胞抽出物15mlの分取量を、先に50mMリン酸カリウム(potassium phosphate)(pH7.8)で平衡化された10mlのGEヘルスケアモノキュー(MonoQ)カラムにサンプルをロードした後、800mM NaClを含有する50mMリン酸カリウム(pH7.8)に10カラム体積で勾配をかけてカラムを洗浄/溶出させた。O−アセチルホモセリンスルフヒドリラーゼは、電気伝導度(conductivity)15〜20(mS/cm)で溶離された。溶離液をミリポア/アミコンセントリコン(Millipore/Amicon Centricon)遠心分離機式フィルター装置(MWCO 3kDa)を用いて、2〜3mlで濃縮させた。濃縮液を150mM NaCl、5%グリセロールを含有する50mMリン酸カリウム(pH7.8)に先に平衡化されたスーパーデックスカラム(HiLoad 16/600 Superdex 75pg、GEヘルスケア)にロードして、分子量サイズ別に分離した。
前記図1の結果は、本出願の変異体は、大腸菌で過量に発現するだけでなく、高純度に精製しうることを示唆する。
精製された前記ロドバクター・スフェロイデスに由来のO−アセチルホモセリンスルフヒドリラーゼ野生型及びV196T変異体を1.3mg/mlの濃度で同量50mMリン酸カリウム(potassium phosphate)(pH7.4及びpH6.4)、15g/L O−アセチルホモセリン、0.05mM PLP(pyridoxal5’−phosphate)、30mMナトリウムメチルメルカプタン(sodium methyl mercaptan)が含まれている緩衝液に入れて、33℃、1,150rpmで5分間反応させた。その後、反応液をHPLC分析して生成されたL−メチオニンの量を測定した。変異体と野生型の活性を下記表3に示した。
実施例3−1:V196S、V196L、V196D、V196K変異体の作製
実施例1で位置選択的突然変異(site−directed mutagenesis)方法を用いてロドバクター・スフェロイデス(Rhodobacter sphaeroides)に由来の野生型O−アセチルホモセリンスルフヒドリラーゼのアミノ酸配列196番目のアミノ酸であるバリン(Val、V)をセリン(Ser、S)、ロイシン(Leu、L)、アスパラギン酸(Asp、D)、リジン(Lys、K)にそれぞれ置換した酵素変異体を用いたことを除いて実施例1−2と同様に行い前記遺伝子を含むベクター及び前記ベクターで形質転換されたE.coli K12 W3110菌株を製造した。
前記実施例3−1で得られた変異体の培養液を実施例1−3と同様に評価し、表4に示した。
このような結果は、本出願の新規な変異体であるO−アセチルホモセリンスルフヒドリラーゼ変異体であるV196T変異体の酵素活性が野生型に比べて有意的に改善され、L−メチオニンを過量に製造しうるだけでなく、低pHであるほど増大されたL−メチオニンの転換活性を示して、過量のL−メチオニンを生産するため、最終産物である酢酸が高濃度で蓄積された時点でも活性阻害が低く、過量のL−メチオニンを生産しうることを示唆する。また、細胞内培養液の形態だけでなく、無細胞抽出物の形態で精製した場合にも、L−メチオニンを過量に転換して製造しうることを示唆する。
Claims (7)
- 配列番号1のアミノ酸配列で記載されたポリペプチドのN末端から196番目のアミノ酸であるバリンがトレオニンに置換された、O−アセチルホモセリンスルフヒドリラーゼの活性を有する、変異型ポリペプチド。
- 請求項1に記載の変異型ポリペプチドをコードする、ポリヌクレオチド。
- 請求項2に記載のポリヌクレオチドを含む、ベクター。
- 配列番号1のアミノ酸配列で記載されたポリペプチドのN末端から196番目のアミノ酸であるバリンがトレオニンに置換された、O−アセチルホモセリンスルフヒドリラーゼの活性を有する変異型ポリペプチドを発現したり、請求項3に記載のベクターを含みうる、O−アセチルホモセリンスルフヒドリラーゼを生産するエシェリキア属微生物。
- 前記エシェリキア属微生物が、大腸菌(Escherichia coli)である、請求項4に記載のエシェリキア属微生物。
- 請求項1に記載の変異型ポリペプチド、またはそれを生産する微生物またはその培養物を、O−アセチルホモセリン及びメチルメルカプタンと反応させる段階を含む、メチオニンを製造する方法。
- 前記反応で生成されたメチオニンを回収する段階をさらに含む、請求項6に記載のメチオニンを製造する方法。
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