JP4487046B2 - 脂質含量の高い真核微生物及び該微生物を用いた脂質の製造方法 - Google Patents
脂質含量の高い真核微生物及び該微生物を用いた脂質の製造方法 Download PDFInfo
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Description
(1) サッカロミセス・セレビシェのIRA2遺伝子、PRE9遺伝子、SNF2遺伝子、SPT21遺伝子、PHO90遺伝子の一種以上が、破壊または機能低下しているサッカロミセス・セレビシェの変異株を培地に培養し、培養菌体から脂質を採取することを特徴とする、脂質の製造方法。
(2) 遺伝子の破壊が、栄養要求性を相補するマーカー遺伝子の挿入によるものであることを特徴とする、上記(1)に記載の製造方法。
(3) マーカー遺伝子がHIS3、URA3またはLEU2遺伝子であることを特徴とする、上記(2)に記載の製造方法。
(4) 窒素源欠乏培地で培養することを特徴とする、上記(3)に記載の製造方法。
本明細書にいう変異とは、上記5種の遺伝子あるいはこれらと相同性を有する遺伝子を破壊あるいは変異させて、その機能を欠失または低下させることをいう。したがって、変異株とはこれら遺伝子の機能が完全に欠失した菌株のみならず、遺伝子の機能が低下している菌株も含む。
脂質含量を高める変異遺伝子の同定
酵母サッカロミセス・セレビシェにトランスポゾンを用いた挿入ライブラリーを作用させて突然変異株を作成した。mTn-lacZ/LEU2トランスポゾン(Seifert, H.S. et al. Proc. Natl. Acad. Sci. U.S.A. 83, 735 (1986))を用いて作成されたライブラリー(Burns, N. et al. Genes Dev. 8, 1087 (1994))は、Yale Genome Analysis Centerより分与をうけた。このライブラリーをNotIで切断後、Gietz, D. et al. Nucleic Acids Res. 20, 1425 (1992) に記載された方法を用いて酵母サッカロミセス・セレビシェD451-3a株(MATa leu2-3 leu2-114 ura3 can1)(Hosaka, K. et al. J. Bacteriol. 172, 2005 (1990))を形質転換した。すなわち、100μl のこの株のコンピテントセルあたり10μl (100mg)のサーモンテスティスDNA、ライブラリー0.3μgを加えて、ロイシンを含まない選択培地で増殖してきたコロニーをmTn-lacZ/LEU2トランスポゾンが挿入された酵母変異株として集菌した。
変異遺伝子の破壊による脂質含量の高い酵母変異株の作成
酵母サッカロミセス・セレビシェのYPH499株(MATa ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1、ATCC76625 )を用い、該変異遺伝子の破壊株を作成した。
遺伝子の破壊は、Nikawa, J. and Kawabata, M. Nucleic Acids Res. 26, 860-861 (1998)に記載されたPCRを用いた方法に基づいて行った。
5つの遺伝子の破壊に用いたプライマーは以下の表2のとおりである。
Claims (4)
- サッカロミセス・セレビシェのIRA2遺伝子、PRE9遺伝子、SNF2遺伝子、SPT21遺伝子、PHO90遺伝子の一種以上が、破壊または機能低下しているサッカロミセス・セレビシェの変異株を培地に培養し、培養菌体から脂質を採取することを特徴とする、脂質の製造方法。
- 遺伝子の破壊が、栄養要求性を相補するマーカー遺伝子の挿入によるものであることを特徴とする、請求項1に記載の製造方法。
- マーカー遺伝子がHIS3、URA3またはLEU2遺伝子であることを特徴とする、請求項2に記載の製造方法。
- 窒素源欠乏培地で培養することを特徴とする、請求項3に記載の製造方法。
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