JP4943678B2 - 細胞の脂肪酸組成を改変する方法およびその利用 - Google Patents
細胞の脂肪酸組成を改変する方法およびその利用 Download PDFInfo
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Description
(1)不飽和脂肪酸合成能を有する細胞に対して、当該細胞が合成した不飽和脂肪酸の酸化を抑制することによって上記細胞の脂肪酸組成を改変する方法。
(2)上記不飽和脂肪酸合成能を有する細胞に対して、不飽和脂肪酸の酸化を抑制することのできるタンパク質をコードする遺伝子を発現可能に導入する工程を含む上記(1)に記載の方法。
(3)上記遺伝子は、カタラーゼ活性を有するタンパク質をコードする遺伝子である上記(2)に記載の方法。
(4)上記遺伝子は、ヴィブリオ・ルモイエンシスS−1株に由来する上記(3)に記載の方法。
(5)上記不飽和脂肪酸合成能を有する細胞は、不飽和脂肪酸合成能を有するタンパク質をコードする遺伝子を発現可能に導入してなる細胞である上記(1)〜(4)のいずれかに記載の方法。
(6)上記遺伝子は、EPA合成能を有するタンパク質をコードする遺伝子である上記(5)に記載の方法。
(7)不飽和脂肪酸合成能を有する細胞に対して、配列番号1に記載の配列を有するポリヌクレオチドを発現可能に導入することによって、上記細胞の脂肪酸組成を改変する方法。
(8)上記(1)〜(7)のいずれかに記載の方法を一工程として含む不飽和脂肪酸含有組成物の製造方法。
(9)上記(4)に記載の製造方法により製造される不飽和脂肪酸含有組成物。
(10)上記(1)〜(7)のいずれか1項に記載の方法によって脂肪酸組成が改変された細胞。
(11)不飽和脂肪酸合成能を有すると共に、不飽和脂肪酸の酸化を抑制することのできるタンパク質をコードした遺伝子が発現可能に導入されてなる細胞。
(12)宿主細胞に、不飽和脂肪酸合成酵素遺伝子と、不飽和脂肪酸の酸化を抑制することのできるタンパク質をコードした遺伝子とが共発現可能に導入されてなる上記(11)に記載の細胞。
<1.脂肪酸組成改変方法>
上記脂肪酸組成改変方法としては、不飽和脂肪酸合成能を有する細胞に対して、当該細胞が合成した不飽和脂肪酸の酸化を抑制することによって上記細胞の脂肪酸組成を改変することができればよく、対象となる細胞、不飽和脂肪酸、工程、試薬等は特に限定されない。
<2.利用可能な遺伝子>
以下に、上記<1>欄でのべた脂肪酸組成改変方法において利用可能な遺伝子について説明する。
(2−1)不飽和脂肪酸合成酵素遺伝子
本発明に利用可能な不飽和脂肪酸合成酵素遺伝子としては、宿主細胞内で不飽和脂肪酸を合成することができるものであればよい。例えば、EPAクラスター(EPA合成酵素遺伝子群)、DHA合成酵素遺伝子等、脂肪酸不飽和化酵素遺伝子など、目的とする不飽和脂肪酸によって、適宜変更可能である。
(2−2)不飽和脂肪酸の酸化を抑制するタンパク質およびそれをコードする遺伝子
「不飽和脂肪酸の酸化を抑制するタンパク質」とは、特に限定されるものではないが、例えば、過酸化水素等の活性酸素種を基質とする酸化還元酵素が挙げられる。より具体的には、過酸化水素を基質とするカタラーゼやペルオキシダーゼ等のヒドロペルオキシダーゼが挙げられる。
(2−3)遺伝子導入方法
上記<1>欄で述べたように、本発明に係る脂肪酸組成改変方法の例としては、遺伝子導入を利用する方法も含まれる。つまり、宿主細胞に酸化抑制遺伝子または不飽和脂肪酸合成酵素遺伝子のどちらか一方を発現可能に導入する方法、宿主細胞に、不飽和脂肪酸合成酵素遺伝子と、酸化抑制遺伝子とを共発現可能に導入する方法も、本発明に含まれる。
<3.細胞>
本発明には、上記<1>欄の方法(脂肪酸組成改変方法)によって脂肪酸組成が改変された細胞が含まれる。また、この他にも、不飽和脂肪酸合成能を有すると共に、酸化抑制遺伝子が発現可能に導入された細胞、酸化抑制タンパク質を発現すると共に、不飽和脂肪酸合成酵素をコードする遺伝子が発現可能に導入されてなる細胞、および、宿主細胞に、不飽和脂肪酸合成酵素遺伝子と、酸化抑制遺伝子とが共発現可能に導入されてなる細胞も含まれる。これらの細胞は、例えば、後述の不飽和脂肪酸含有組成物の製造に利用することができる。
<4.不飽和脂肪酸含有組成物およびその製造方法>
本発明に係る不飽和脂肪酸含有組成物の製造方法は、上述の脂肪酸組成改変方法を一工程として含めばよく、その他の工程、製造設備・器具等の諸条件については、特に限定されるものではない。
<ベクターおよび細胞>
以下、脂肪酸組成改変方法の対象となる細胞として、大腸菌を用い、酸化抑制遺伝子として、4.9kb断片を用いた。
上記条件で培養した大腸菌培養液から遠心分離によって細胞を回収した。この細胞に2Nメタノール性塩酸を加えて細胞を完全に懸濁した後、アルミブロックヒータを使用して懸濁液を80℃で60分間、または60℃で20分間保持することによって、脂質のメタノリシス反応を行った。こうして脂肪酸メチルエステル(FAME)に変換した脂肪酸成分を、ヘキサンにより抽出した。
〔実施例2および比較例2・3〕
EPAクラスターを有するベクターとしてΔ1349pNEBを、4.9kb断片を有するベクターとして実施例1と同様pKT230::vktAを導入し、実施例2とした。また、Δ1349pNEBのみを大腸菌に導入して比較例3とし、Δ1349pNEBとpKT230とを導入して比較例4とした。ベクター以外の他の操作は、実施例1と同様に行った。
〔実施例3および比較例5〕
EPAクラスターを有するベクターとしてpEPAΔ1を、4.9kb断片を有するベクターとしてプラスミドpGBM3::vktAを、実施例1と同様の方法で大腸菌に導入し、実施例3とした。また、4.9kb断片を持たない空のpGBM3とpEPAΔ1とを導入し、比較例5とした。
<ベクターおよび細胞>
細胞がもつカタラーゼ活性とEPA含量の関係を見るために、通常レベルのカタラーゼ活性をもつ大腸菌(DH5α)とカタラーゼ活性を全く持たない大腸菌の突然変異株(UM2:エール大学より分譲された)にEPA合成能を与えて、それぞれの細胞の全脂肪酸に占めるEPAの割合を調べた。
形質転換されたDH5α株またはUM2株は、LB培地により20℃、180rpmで48〜60時間、定常期まで振盪培養して、遠心により集菌した。細胞を1%NaCl溶液により3回洗浄後、湿細胞を前述の方法によりメタノリシスし、得られた脂肪酸メチルエステルをガスクロマトグラフィー(オーブン温度180℃、インジェジュクター温度240℃、ディテクター温度240℃)で分析した。
Claims (4)
- n−3系高度不飽和脂肪酸合成能を有する細胞に対して、カタラーゼをコードする遺伝子を発現可能に導入することによって、細胞のn−3系高度不飽和脂肪酸含量を増加させて、上記細胞のn−3系高度不飽和脂肪酸組成を改変する、方法。
- 上記遺伝子は、ヴィブリオ・ルモイエンシスS−1株に由来することを特徴とする請求項1に記載の方法。
- n−3系高度不飽和脂肪酸合成能を有する細胞に対して、配列番号1に記載の配列を有するポリヌクレオチドを発現可能に導入することによって、細胞のn−3系高度不飽和脂肪酸含量を増加させて、上記細胞中のn−3系高度不飽和脂肪酸組成を改変する、方法。
- 請求項1〜3のいずれか1項に記載の方法を一工程として含むことを特徴とするn−3系高度不飽和脂肪酸含有組成物の製造方法。
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