JPWO2016104607A1 - 新規ω3不飽和脂肪酸酵素およびエイコサペンタエン酸の製造方法 - Google Patents
新規ω3不飽和脂肪酸酵素およびエイコサペンタエン酸の製造方法 Download PDFInfo
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Abstract
Description
また本発明は、上記ポリペプチドをコードするポリヌクレオチドを提供する。
また本発明は、上記ポリヌクレオチドを含むベクターを提供する。
また本発明は、上記ポリヌクレオチドが導入された形質転換細胞を提供する。
さらに本発明は、上記ポリペプチドを発現する細胞を培養することを含む、エイコサペンタエン酸含有脂質の生産方法を提供する。
さらに本発明は、上記方法により生産されたエイコサペンタエン酸含有脂質を精製することを含む、エイコサペンタエン酸の生産方法を提供する。
(A)配列番号2に示されるアミノ酸配列;
(B)配列番号2で示されるアミノ酸配列と90%以上、好ましくは95%以上、より好ましくは98%以上、さらに好ましくは99%以上の同一性を有するアミノ酸配列;
(C)配列番号2に示されるアミノ酸配列において、1個または複数個のアミノ酸の欠失、置換、挿入および付加から選択される変異を施されたアミノ酸配列。
(D)配列番号4に示されるアミノ酸配列;
(E)配列番号4で示されるアミノ酸配列と90%以上、好ましくは95%以上、より好ましくは98%以上、さらに好ましくは99%以上の同一性を有するアミノ酸配列;
(F)配列番号4に示されるアミノ酸配列において、1個または複数個のアミノ酸の欠失、置換、挿入および付加から選択される変異を施されたアミノ酸配列。
上記アミノ酸配列におけるアミノ酸の欠失、置換、挿入および付加の位置は、変異後のポリペプチドに常温下での炭素数20の脂肪酸に対するω3不飽和化活性が保持される限り、特に限定されない。
(a)配列番号1に示されるヌクレオチド配列;
(b)配列番号1に示されるヌクレオチド配列と90%以上、好ましくは95%以上、より好ましくは98%以上、さらに好ましくは99%以上の同一性を有するヌクレオチド配列;
(c)配列番号1に示されるヌクレオチド配列において、1個または複数個のヌクレオチドの欠失、置換、挿入および付加から選択される変異を施されたヌクレオチド配列;
(d)配列番号1に示されるヌクレオチド配列とストリンジェントな条件でハイブリダイズするヌクレオチド配列;
(e)配列番号3に示されるヌクレオチド配列;
(f)配列番号3に示されるヌクレオチド配列と90%以上、好ましくは95%以上、より好ましくは98%以上、さらに好ましくは99%以上の同一性を有するヌクレオチド配列;
(g)配列番号3に示されるヌクレオチド配列において、1個または複数個のヌクレオチドの欠失、置換、挿入および付加から選択される変異を施されたヌクレオチド配列;
または、
(h)配列番号3に示されるヌクレオチド配列とストリンジェントな条件でハイブリダイズするヌクレオチド配列。
上記ヌクレオチド配列におけるヌクレオチドの欠失、置換、挿入および付加の位置は、変異後のポリヌクレオチドにコードされるポリペプチドが常温下で炭素数20の脂肪酸に対するω3不飽和化活性を保持する限り、特に限定されない。
GY培地:2%(w/v)グルコース、1%酵母エキス。
Czapek−Dox寒天培地:3%スクロース、0.2%NaNO3、0.1%KH2PO4、0.05%KCl、0.05%MgSO4・7H2O、0.001%FeSO4・7H2O、2%寒天、pH6.0。
YPD培地:ポリペプトン20g、酵母抽出物10g、アデニン0.4g、寒天20gおよびグルコース20gを1000mLの水に希釈。
LB−Mg寒天培地:1%トリプトン、0.5%酵母エキス、85mM NaCl、0.5mM MgSO4・7H2O、0.5mM NaOH、1.5%寒天、pH7.0。
最少培地(MM):10mM K2HPO4、10mM KH2PO4、2.5mM NaCl、2mM MgSO4・7H2O、0.7mM CaCl2、9μM FeSO4・7H2O、4mM (NH4)2SO4、10mMグルコース、pH7.0。
誘導培地(IM):MMに0.5%(w/v)グリセロール、200μMアセトシリンゴン、40mM 2−(N−モルホリノ)エタンスルホン酸(MES)を加えて、pH5.3に調製。
SC培地:5.0g Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate(Difco)、1.7g (NH4)2SO4、20gグルコース、20g寒天、20mgアデニン、30mgチロシン、1.0mgメチオニン、2.0mgアルギニン、2.0mgヒスチジン、4.0mgリジン、4.0mgトリプトファン、5.0mgスレオニン、6.0mgイソロイシン、6.0mgロイシン、6.0mg Lフェニルアラニン。
プレクトスピラ・ミリアンドラをGY培地10mL中にて28℃で5日間振とう培養し、菌体を回収した。集菌した菌体を2mLチューブに入れ、ビーズショッカー(Yasui Kikai)を用いて1700rpm、10秒×2回の条件にて破壊した。破壊した菌体から、ISOGEN(Bio−Rad)を用いて、製品プロトコールに従いmRNAを抽出した。抽出したmRNAを、Prime ScriptTMII High Fidelity RT−PCR Kit(TaKaRa)およびプライマー〔5’−GAAATGGCCGACGTGAACACCTCCTCGC−3’(配列番号7)、および5’−CTATGCGCGCTTGGTGAGCACCTCGC−3’(配列番号8)〕を用いて逆転写し、配列番号3で示されるcDNAを調製した。このcDNAは、配列番号4で示されるアミノ酸配列のポリペプチドをコードしていた。
配列番号1のヌクレオチド配列を、M.alpinaにあわせてコドン至適化し、配列番号5で示されるポリヌクレオチドを得た。この配列番号5で示されるポリヌクレオチドのCDSの上流および下流にSpeIおよびBamHIサイトを構築し、SpMA−RQ(ampR)プラスミドにクローニングした。調製したプラスミドを、SpeIおよびBamHI制限酵素で処理し、得られた遺伝子の断片を、恒常的高発現プロモーターであるSSA2プロモーターを含むプラスミドpBIG35(京都府立大学から提供されたpBIG2RHPH2を改変、Appl.Environ.Microbiol.,2009,75:5529−5535に記載)に連結し、発現カセットを構築した。当該発現カセットを、さらに、ウラシル要求性のマーカー遺伝子(ura5)とタンデムに連結させ、形質転換用バイナリーベクター、pBIGSSA2pPmD17genome−intron modを構築した(図2)。
M.alpina(ウラシル要求性株)を0.05mg/mLウラシル含有Czapek−Dox寒天培地で培養し、培養物を集菌し、次いでMiracloth(Calbiochem)でろ過することで、M.alpinaの胞子懸濁液を調製した。当該M.alpina(ウラシル要求性株)に、実施例2で構築したpBIGSSA2pPmD17genome−intron modベクターを以下に説明するATMT法(Appl.Environ.Microbiol.,2009,75:5529−5535)により導入し、ω3不飽和化酵素導入株を作製した。
配列番号3のヌクレオチド配列を、M.alpinaにあわせてコドン至適化し、配列番号6で示されるポリヌクレオチドを得た。この配列番号6で示されるポリヌクレオチドを用いた以外は、実施例2と同様にして、形質転換用バイナリーベクターpBIGSSA2pPmD17cDNAmodを構築した(図2)。
遺伝子導入用ベクターとしてpBIGSSA2pPmD17cDNAmodを用いた以外は、実施例3と同様の手順でω3不飽和化酵素導入株を作製した。
実施例3および5で得られたω3不飽和化酵素遺伝子導入M.alpina株を、それぞれ4mLのGY培地にて、28℃で3、7、および10日間、120rpmで好気的に培養した。対照として、当該ω3不飽和化酵素遺伝子を導入していないM.alpina株を同様に培養した。各培養液から吸引ろ過にて菌体を回収し、120℃で3時間乾燥した。乾燥菌体に、0.5mg/mLの内部標準(M.alpinaが生合成できない炭素数23の飽和脂肪酸)を含むジクロロメタン溶液1mLおよび塩酸メタノール2mLを加え、55℃、2時間の温浴にて脂肪酸をメチルエステル化した。反応液に蒸留水1mLとヘキサン4mLを加えてヘキサン層を抽出し、減圧遠心して脂肪酸メチルエステルを回収した。
回収したサンプルをクロロホルムに溶解し、ガス液体クロマトグラフィー(GLC)にてサンプル中の脂肪酸組成を測定した。GLCは、島津社製GC−2010を用い、GLサイエンス社製キャピラリーカラムTC70(0.25mm×60m)を用い、カラム温度180℃、気化室温度250℃、検出器温度250℃、キャリアガスHe、メイクアップガスN2、H2流量40mL/min、Air流量400mL/min、スプリット比50、分析時間30minの条件にて行った。抽出された各脂肪酸の量を、GLCのチャートのピーク面積値から内部標準の脂肪酸量を基準として定量し、培養液1mL当たりおよび乾燥菌体1mgあたりの各脂肪酸の量を算出した。さらに、総脂肪酸量に対する各脂肪酸の割合を求めた。
その結果、実施例3および実施例5のω3不飽和化酵素遺伝子導入株では、それぞれ、最大40.8%および39.6%のEPAの蓄積がみられた(図3)。一方、対照株ではEPAは産生されなかった(蓄積を測定できず)。
本発明のω3不飽和化酵素を保有するプレクトスピラ・ミリアンドラと、Δ17不飽和化酵素を保有することが報告されているサプロレグニア・ディクリナ(例えば、特許文献4)との間で、炭素数20の脂肪酸に対するω3不飽和化活性を比較した。さらに、他の8種のサプロレグニア・ディクリナ類縁菌についてもω3不飽和化活性を調べた。
表2に記載のプレクトスピラ・ミリアンドラ(NBRC No.32548)、サプロレグニア・ディクリナ(NBRC No.32710)、および他の8種の菌株を、それぞれ5mLのGY培地にて、28℃で7日間培養し、培養後の培地における、ω3不飽和化酵素の産物であるEPA(20:5n−3)と、その基質となるARA(20:4n−6)の量を、ガス液体クロマトグラフィー(GLC)により測定した。
その結果、表2に示すとおり、プレクトスピラ・ミリアンドラでは、サプロレグニア・ディクリナおよびその類縁菌と比べて、ARAに対するEPAの含有比が高かった。これらの結果から、プレクトスピラ・ミリアンドラのω3不飽和化酵素が、ARAからEPAへの変換効率が高く、効率よくEPAを産生することができる酵素であることが示唆された。
Claims (8)
- 配列番号2に示されるアミノ酸配列と80%以上の同一性を有するアミノ酸配列からなり、かつ炭素数20の脂肪酸に対するω3不飽和化活性を有するポリペプチド。
- 配列番号2に示されるアミノ酸配列と80%以上の同一性を有するアミノ酸配列が、以下のアミノ酸配列である、請求項1記載のポリペプチド:
(A)配列番号2に示されるアミノ酸配列;
(B)配列番号2に示されるアミノ酸配列と90%以上の同一性を有するアミノ酸配列;
(C)配列番号2に示されるアミノ酸配列において、1個または複数個のアミノ酸の欠失、置換、挿入および付加から選択される変異を施されたアミノ酸配列;
(D)配列番号4に示されるアミノ酸配列;
(E)配列番号4に示されるアミノ酸配列と90%以上の同一性を有するアミノ酸配列;
または、
(F)配列番号4に示されるアミノ酸配列において、1個または複数個のアミノ酸の欠失、置換、挿入および付加から選択される変異を施されたアミノ酸配列。 - 請求項1又は2記載のポリペプチドをコードするポリヌクレオチド。
- 下記に示されるヌクレオチド配列からなる請求項3記載のポリヌクレオチド:
(a)配列番号1に示されるヌクレオチド配列;
(b)配列番号1に示されるヌクレオチド配列と90%以上の同一性を有するヌクレオチド配列;
(c)配列番号1に示されるヌクレオチド配列において、1個または複数個のヌクレオチドの欠失、置換、挿入および付加から選択される変異を施されたヌクレオチド配列;
(d)配列番号1に示されるヌクレオチド配列とストリンジェントな条件でハイブリダイズするヌクレオチド配列;
(e)配列番号3に示されるヌクレオチド配列;
(f)配列番号3に示されるヌクレオチド配列と90%以上の同一性を有するヌクレオチド配列;
(g)配列番号3に示されるヌクレオチド配列において、1個または複数個のヌクレオチドの欠失、置換、挿入および付加から選択される変異を施されたヌクレオチド配列;
(h)配列番号3に示されるヌクレオチド配列とストリンジェントな条件でハイブリダイズするヌクレオチド配列;または、
(i)該(a)〜(h)に示されるヌクレオチド配列をコドン至適化したヌクレオチド配列。 - 請求項3又は4記載のポリヌクレオチドを含むベクター。
- 請求項3又は4記載のポリヌクレオチドが導入された形質転換細胞。
- 請求項1又は2記載のポリペプチドを発現する細胞を培養することを含む、エイコサペンタエン酸含有脂質の生産方法。
- 請求項7記載の方法により生産されたエイコサペンタエン酸含有脂質を精製することを含む、エイコサペンタエン酸の生産方法。
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