JP4002833B2 - Method for producing polypeptide, polynucleotide, recombinant nucleic acid, recombinant, prenyl alcohol - Google Patents

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ホスファターゼの基質特異性
「ホスファターゼ活性の発現」の項で得られた形質転換体を５００ｍＬのＳＤ選択培地でＯＤ_６００が約１になるまで培養し、遠心分離により菌体を集菌した。得られた菌体ペレットから、Ｗｕ Ｗ．Ｉ．らの方法（（１９９８）Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２７１（４），１８６８−１８７６）を参考にして、発現したホスファターゼの部分精製を行った。部分精製酵素を用いて、ホスファターゼの基質特異性を以下の方法で調べた。
５μＭ ＧＧＰＰ（２．１ｋＢｑの［^３Ｈ］ＧＧＰＰを含む）および部分精製酵素を含む反応バッファー〔最終濃度０．１Ｍ ｃｉｔｒａｔｅ（ｐＨ６．０）、５ｍＭ ＥＤＴＡ〕に対して、ＧＧＰＰ以外の追加基質として１００μＭ ＡＴＰ、１００μＭグルコース６リン酸（Ｇ６Ｐ）、１００μＭピロリン酸ナトリウム（ＮａＰＰ）、１００μＭ β−グリセロリン酸（β−ＧＰ）、１００μＭ ｐ−ニトロフェニルリン酸（ｐＮＰＰ）、１００μＭ ＭＶＰ、１００μＭ ＭＶＰＰ、１００μＭ ＩＰＰ、１００μＭ ＤＭＡＰＰ、１００μＭ ＧＰＰ、５μＭ ＦＰＰ又は５μＭ ＧＧＰＰのいずれかを添加し、３０分間３７°Ｃで反応を行なった。そして１００μＬアルコールＫＯＨ液を添加して反応を停止し、ヘキサン５００μＬを加えた後、１分間ボルテックスミキサーにて激しく撹拌し、生成した［^３Ｈ］ＧＧＯＨを抽出した。このヘキサン層３００μＬを分取して液体シンチレーションカウンターで放射活性を測定することにより、ＧＧＰＰａｓｅ比活性を求めた。［^３Ｈ］ＧＧＰＰ以外の追加基質を加えなかったとき（＋ｎｏｎｅ）のＧＧＰＰａｓｅ比活性を１００％とした場合の相対活性を表２にまとめた。
【表２】

表２の結果から、プレニル二リン酸生合成経路に関係しないリン酸化合物（ＡＴＰ、Ｇ６Ｐ、ＮａＰＰ、β−ＧＰ又はｐＮＰＰ）を、追加基質としてＧＧＰＰの２０倍量加えても、追加基質を加えなかった場合と比較してＧＧＰＰに対するホスファターゼ活性は９０％以上保持していた。即ち、実験に供したホスファターゼのプレニル二リン酸生合成経路に関係しないリン酸化合物に対するホスファターゼ活性は、ＧＧＰＰに対する酵素活性に比べて低いことが確かめられた。次に、プレニルリン酸前駆体（ＭＶＰ，ＭＶＰＰ）、炭素数５のＩＰＰ又はＤＭＡＰＰ、あるいは炭素数１０のＧＰＰを、追加基質として、ＧＧＰＰの２０倍量加えても追加基質を加えなかった場合と比較してＧＧＰＰに対するホスファターゼ活性は９０％以上保持していた。即ち、実験に供したホスファターゼのプレニルリン酸前駆体及び炭素数１０以下のプレニルリン酸化合物に対するホスファターゼ活性は、ＧＧＰＰに対するホスファターゼ活性に比べて低いことが確かめられた。一方、炭素数１５のｃｏｌｄ ＦＰＰを、追加基質として、ＧＧＰＰと等モル（５μＭ）加えた場合には、基質競合により［^３Ｈ］ＧＧＰＰに対する見かけ上のホスファターゼ活性が約４０〜６０％減少した。これはｃｏｌｄ ＧＧＰＰを、追加基質として、ＧＧＰＰと等モル（５μＭ）加えた場合の見かけ上のホスファターゼ活性の減少度合いと同程度であった。即ち、実験に供したホスファターゼの炭素数１５のＦＰＰに対するホスファターゼ活性と炭素数２０のＧＧＰＰに対する酵素活性は同等であることが確かめられた。以上のことから、実験に供したホスファターゼは次のような活性を示すことが明らかとなった。即ち、炭素数１５以上のプレニルリン酸（Ａ）を基質とした酵素活性測定系において、過剰量（モル比で２０倍）のプレニル二リン酸生合成経路に関係しないリン酸化合物又は炭素数１０以下のプレニルリン酸又はプレニルリン酸前駆体（Ｂ）を共存させても、（Ａ）に対する酵素活性が（Ｂ）を共存させなかった場合と比べて９０％以上の活性を示す。
酵母によるプレニルアルコールの生産
本発明に係るホスファターゼ遺伝子を用いて生体内でプレニルアルコールを大量生産させるためには、この遺伝子を適当なベクターあるいはＤＮＡ断片に組んで宿主に導入し、生体内で発現させればよい。
更に、プレニル二リン酸生合成経路（メバロン酸経路、非メバロン酸経路を含む）に関わる幾つかの酵素の遺伝子を用いてプレニルリン酸の供給能力を向上む）に関わる幾つかの酵素の遺伝子を用いてプレニルリン酸の供給能力を向上させたもとで上記ホスファターゼ遺伝子を導入すれば、より高いプレニルアルコール生産性を期待できる。例えば、ヒドロキシメチルグルタリルＣｏＡ（ＨＭＧ−ＣｏＡ）還元酵素は、メバロン酸経路の中で律速酵素であることが知られている（Ｄｉｍｓｔｅｒ−Ｄｅｎｋ，Ｄ．ｅｔ ａｌ（１９９４）Ｍｏｌ．Ｂｉｏｌ．Ｃｅｌｌ．５，６５５−６６５．）。又、イソペンテニル二リン酸イソメラーゼ（ＩＰＰイソメラーゼ）遺伝子の発現を増強したＥ． ｃｏｌｉでカロテノイドの生産量が増加することが報告されている（三沢ら、特開平８−２４２８６１）。ＧＧＰＰ合成酵素遺伝子（例えば、Ｓ． ｃｅｒｅｖｉａｅ ＢＴＳ１）は、ＧＧＯＨの前駆体であるＧＧＰＰを合成する酵素である。かかるＧＧＰＰ合成酵素遺伝子の発現の増強により、ＧＧＰＰ生産能力の強化を期待できる。よって、これらの遺伝子の導入又は発現強化と、本発明に係るホスファターゼ遺伝子の導入又は発現強化とを組合わせることにより、より高いプレニルアルコール生産性の実現を期待できる。
ホスファターゼ遺伝子を導入したＷ３０３−１Ｂによるプレニルアルコールの生産
ｐＲＳ４３５ＧＡＰ−ＢＴＳ１をＳ． ｃｅｒｅｖｉｓｉａｅ Ｗ３０３−１Ｂに導入し、得られた組換え体に対して更に、本発明に係るホスファターゼ遺伝子を組込んだ発現ベクターｐＲＳ４３６ＧＡＰ−ＤＰＰ１、ｐＲＳ４３６ＧＡＰ−ＬＰＰ１、ｐＲＳ４３６ＧＡＰ−Ｄｒｉ４２、ｐＲＳ４３６ＧＡＰ−ＰＡＰ２、ｐＲＳ４３６ＧＡＰ−ＡｔＰＡＰ１又はｐＲＳ４３６ＧＡＰ−ＡｔＰＡＰ２を導入した。各組換え体及び宿主のＧＧＯＨ生産性を測定した結果を表３に示す。
【表３】

Ｗ３０３−１ＢではＧＧＯＨの生産は検出されなかったが、ＧＧＰＰ合成酵素遺伝子を導入することによりＧＧＯＨが０．２ｍｇ／Ｌ生産されるようになった。これは、ＧＧＰＰ合成酵活性上昇によりＧＧＰＰが生産され、このＧＧＰＰが酵母菌体内在性の脱リン酸化活性によりＧＧＯＨに変換されたことを示唆している。更に、この株に本発明に係るホスファターゼ遺伝子を導入することにより、ＧＧＰＰ合成酵素遺伝子のみを導入した場合に比べて、５．７〜１７倍にＧＧＯＨの生産性が向上した。即ち、ＧＧＰＰ合成酵素遺伝子の導入によりある程度のＧＧＯＨ生産性向上が認められるが、ＧＧＯＨを更に効率的に大量生産させるためには、本発明に係るホスファターゼ遺伝子の導入が重要であることが判った。
ホスファターゼ遺伝子を導入したＹＰＨ４９９によるプレニルアルコールの生産
ｐＲＳ４３４ＴＥＦ−ＨＭＧ１、ｐＲＳ４４５ＧＡＰ−ＢＴＳ１又はｐＲＳ４３５ＧＡＰ−ＩＤＩ１を、Ｓ． ｃｅｒｅｖｉｓｉａｅ ＹＰＨ４９９に導入した。又、別途に、ｐＲＳ４３４ＴＥＦ−ＨＭＧ１及びｐＲＳ４４５ＧＡＰ−ＢＴＳ１の２種のプラスミドをＹＰＨ４９９に導入した。上記２種のプラスミドを導入した組換え体に対して、更に本発明のホスファターゼ遺伝子を組込んだ発現ベクターｐＲＳ４３６ＧＡＰ−ＤＰＰ１、ｐＲＳ４３６ＧＡＰ−ＬＰＰ１、ｐＲＳ４３６ＧＡＰ−Ｄｒｉ４２、ｐＲＳ４３６ＧＡＰ−ＰＡＰ２、ｐＲＳ４３６ＧＡＰ−ＡｔＰＡＰ１又はｐＲＳ４３６ＧＡＰ−ＡｔＰＡＰ２の各プラスミドを導入した。各組換え体及び宿主のＦＯＨ、ＧＧＯＨ生産性を測定した結果を表４に示す。
【表４】

ＹＰＨ４９９ではＧＧＯＨは検出されなかった。ＩＰＰ−△イソメラーゼ遺伝子のみの導入ではＧＧＯＨは検出されなかったが、ＨＭＧ−ＣｏＡ還元酵素遺伝子のみの導入によってＧＧＯＨの生産量がある程度増大し（ＧＧＯＨ ０．０５ｍｇ／Ｌ）、ＧＧＰＰ合成酵素遺伝子の導入によってもＧＧＯＨの生産量がある程度増大した（ＧＧＯＨ ０．２ｍｇ／Ｌ）。
ＨＭＧ−ＣｏＡ還元酵素遺伝子とＧＧＰＰ合成酵素遺伝子の２種の遺伝子を導入することによりプレニルアルコール生産性向上の相乗効果が認められた。但しその場合に比較して、上記の２種の遺伝子に加えて更に本発明のホスファターゼ遺伝子を導入することにより、１．３倍〜５．９倍にＧＧＯＨ生産性が向上した。
ホスファターゼ遺伝子を導入したＡ４５１によるプレニルアルコールの生産
Ａ４５１のＡＵＲ１遺伝子がＡＵＲ１−Ｃ遺伝子に置換変異した株をＡＵＲＧＧ１０１（ＡＵＲ１：：ＡＵＲ１−Ｃ）と呼ぶ。ＡＵＲＧＧ１０１にｐＹＨＭＧ０４４を導入し（１５−２と呼ぶ）、１５−２に更にｐＲＳ４３５ＧＡＰ−ＢＴＳ１を導入し、ｐＲＳ４３５ＧＡＰ−ＢＴＳ１／１５−２を得た。ｐＲＳ４３５ＧＡＰ−ＢＴＳ１／１５−２にｐＲＳ４３４ＧＡＰ−ＤＰＰ１，ｐＲＳ４３４ＧＡＰ−ＬＰＰ１，ｐＲＳ４３４ＧＡＰ−Ｄｒｉ４２，ｐＲＳ４３４ＧＡＰ−ＰＡＰ２，ｐＲＳ４３６ＧＡＰ−ＡｔＰＡＰ１又はｐＲＳ４３４ＧＡＰ−ＡｔＰＡＰ２を導入した。各組換え体及び宿主のプレニルアルコール生産性を測定した結果を表５に示す。
【表５】

Ａ４５１ではプレニルアルコールの生産は検出されなかった。ＨＭＧ−ＣｏＡ還元酵素遺伝子とＧＧＰＰ合成酵素遺伝子の２種の遺伝子を導入した場合、ＦＯＨ、ＧＧＯＨがある程度生産されるようになった（ＦＯＨ １．４ｍｇ／Ｌ，ＧＧＯＨ ０．４ｍｇ／Ｌ）。更にこの株に本発明に係るホスファターゼ遺伝子を導入すると、上記２種の遺伝子を導入した場合に比較してＦＯＨで２．６〜２０倍、ＧＧＯＨで８．８倍〜３１倍に生産性が向上した。
この結果は、ＨＭＧ−ＣｏＡ還元酵素遺伝子やＧＧＰＰ合成酵素遺伝子が導入されている場合、ＦＯＨやＧＧＯＨの生産の増量に対しては、ホスファターゼ遺伝子の導入が決定的であることを示している。
ＧＧＰＰ合成酵素遺伝子ＢＴＳ１とプレニルリン酸特異的ホスファターゼ遺伝子ＤＰＰ１の連結
Ｎｉｋａｗａらの方法（Ｎｉｋａｗａ，Ｊ．ａｎｄ Ｋａｗａｂａｔａ，Ｍ．（１９９８）Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ．２６．８６０−８６１．）を参考にして、ＰＣＲ法により、プレニルリン酸特異的ホスファタゼ遺伝子ＤＰＰ１とＧＧＰＰ合成酵素遺伝子ＢＴＳ１の連結遺伝子（以後、「ＤＰＧＧ」と呼ぶ）を作製した。
１ｓｔＰＣＲの条件を以下に示す。１ＸＫＯＤ ｂｕｆｆｅｒ（ＴＯＹＯＢＯ），０．２ｍＭ ｄＮＴＰｓ，１ｍＭ ＭｇＣｌ_２，２Ｕ ＫＯＤ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（ＴＯＹＯＢＯ），２０ｐｍｏｌプライマー１，２０ｐｍｏｌプイマー２，６０ｎｇ ＹＰＨ４９９ ゲノムＤＮＡを含む４０μＬの反応液を調製し、次のように１ｓｔ ＰＣＲを行った。
９８°Ｃ−１５秒，４４°Ｃ−２秒，７４°Ｃ−３０秒；２５サイクル
プライマー１として配列番号４３に示す塩基配列を持つＳ−ＤＰＰ１−１、プライマー２として配列番号４４に示す塩基配列を持つＤＰＰ−２を用いたときのＰＣＲ産物を＃１とした。また、プライマー１として配列番号４５に示す塩基配列を持つＢＴＳ−３、プライマー２として配列番号４６に示す塩基配列を持つＸ−ＢＴＳ−４を用いたときのＰＣＲ産物を＃２とした。得られたＰＣＲ産物をアガロースゲル電気泳動により精製した。次に、Ａｎｎｅａｌｉｎｇ ａｎｄ ｅｘｔｅｎｓｉｏｎ反応を以下の条件で行った。１ＸＫＯＤ ｂｕｆｆｅｒ，０．２ｍＭ ｄＮＴＰｓ，１ｍＭ ＭｇＣｌ_２，０．２５Ｕ ＫＯＤ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ，０．５μＬ ＰＣＲ 産物＃１，０．５μＬ ＰＣＲ 産物＃２を含む１０μＬの反応液を調製し、次のように行った。
９８°Ｃ−１５秒，５４°Ｃ−２秒，７４°Ｃ−３０秒；５サイクル
得られたＰＣＲ産物の一部についてアガロースゲル電気泳動を行い、ＰＣＲ産物＃１および＃２の連結遺伝子（約１．９ｋｂ）が合成されていることを確認した。
続いて２ｎｄ ＰＣＲを以下の条件で行った。１ＸＫＯＤ ｂｕｆｆｅｒ（ＴＯＹＯＢＯ），０．２ｍＭ ｄＮＴＰｓ，１ｍＭ ＭｇＣｌ_２，０．５Ｕ ＫＯＤ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（ＴＯＹＯＢＯ），２０ｐｍｏｌ Ｓ−ＤＰＰ−１，２０ｏｌ Ｘ−ＢＴＳ−４，２μＬ Ａｎｎｅａｌｉｎｇ ａｎｄ ｅｘｔｅｎｓｉｏｎ反応液を含む１０μＬの反応液を調製し、次のように２ｎｄ ＰＣＲ反応を行った。
９８°Ｃ−１５秒，５８°Ｃ−２秒，７４°Ｃ−３０秒；２５サイクル
得られたＰＣＲ産物をアガロースゲル電気泳動により精製した（これをＳＸ−ＤＰＧＧと呼ぶ）。ＳＸ−ＤＰＧＧを制限酵素ＳａｃＩＩおよびＸｈｏＩで消化した後、ｐＲＳ４３４ＧＡＰのＳａｃＩＩ−ＸｈｏＩ切断部位に挿入し、ｐＲＳ４３４ＧＡＰ−ＤＰＧＧを得た。ｐＲＳ４３４ＧＡＰ−ＤＰＧＧの構造遺伝子部分と、制限酵素サイト周辺とのＤＮＡシークエンシングを行い、設計通りの塩基配列を持っていることを確認した。以上の方法により、ＤＰＰ１とＢＴＳ１の結合領域にリンカー配列（５’ ｇｇｔｇｇｔｇｇｔｔｃｔ ３’）を導入したホスファターゼ連結遺伝子ＤＰＧＧの発現ベクターを作製した。
ファルネシル二リン酸（ＦＰＰ）合成酵素遺伝子ＥＲＧ２０とプレニルリン酸特異的ホスファターゼ遺伝子ＤＰＰ１の連結
前記ＢＴＳ１とＤＰＰ１の連結の場合と同様にして、ＦＰＰ合成酵素遺伝子ＥＲＧ２０（Ｇｅｎ Ｂａｎｋ ａｃｃｅｓｓｉｏｎ Ｎｏ．ＮＣ００１１４２，１０５００６．．１０６０６４）とプレニルリン酸特異的ホスファターゼ遺伝子ＤＰＰ１を連結した遺伝子（ＤＰＦと呼ぶ）を作製した。
プライマー１として配列番号４７に示す塩基配列を持つＳ−ＤＰＰ１３Ａ、プライマー２としてＤＰＰ−２を用いて１ｓｔ ＰＣＲを行い、ＰＣＲ産物＃３を得た。又、プライマー１として配列番号４８に示す塩基配列を持つＤＰＦ３、プライマー２として配列番号４９に示す塩基配列を持つＸ−ＦＰＳ４を用いて１ｓｔ ＰＣＲを行い、ＰＣＲ産物＃４を得た。これらのＰＣＲ産物をアガロースゲル電気泳動により精製した。
ＰＣＲ産物＃３および＃４を用いてＡｎｎｅａｌｉｎｇ ａｎｄ ｅｘｔｅｎｓｉｏｎ反応を行い、得られたＰＣＲ産物の一部についてアガロースゲル電気泳動を行い、ＰＣＲ産物＃３および＃４の連結遺伝子（約１．９ｋｂ）が合成されていることを確認した。Ａｎｎｅａｌｉｎｇ ａｎｄ ｅｘｔｅｎｓｉｏｎ反応液、Ｓ−ＤＰＰ１３ＡおよびＸ−ＦＰＳ４を用いて２ｎｄ ＰＣＲを行い、得られたＰＣＲ産物をアガロースゲル電気泳動により精製した（これをＳＸ−ＤＰＦと呼ぶ）。ＳＸ−ＤＰＦを制限酵素ＳａｃＩＩおよびＸｈｏＩで消化した後、ｐＲＳ４３４ＧＡＰおよびｐＲＳ４３５ＧＡＰのＳａｃＩＩ−ＸｈｏＩ切断部位に挿入し、それぞれｐＲＳ４３４ＧＡＰ−ＤＰＦ、ｐＲＳ４３５ＧＡＰ−ＤＰＦを得た。ｐＲＳ４３４ＧＡＰ−ＤＰＦ、ｐＲＳ４３５ＧＡＰ−ＤＰＦの構造遺伝子部分と制限酵素サイト周辺とのＤＮＡシークエンシングを行い、設計通りの塩基配列を持っていることを確認した。以上の方法により、ＤＰＰ１とＥＲＧ２０の結合領域にリンカー配列（５’ ｇｇｔｇｇｔｇｇｔｔｃｔ ３’）を導入したホスファターゼ連結遺伝子ＤＰＦの発現ベクターを作製した。
ホスファターゼ連結遺伝子ＤＰＦおよびＤＰＧＧを導入したＹＰＨ４９９のＧＧＰＰａｓｅ活性の発現
ＤＰＦを発現した場合はＤＰＰ１がコードするホスファターゼとＥＲＧ２０がコードするファルネシル二リン酸合成酵素との融合酵素が発現され、ＤＰＧＧを発現した場合はＤＰＰ１がコードするホスファターゼとＢＴＳ１がコードするゲラニルゲラニル二リン酸合成酵素との融合酵素が発現されると考えられる。そこで、連結遺伝子ＤＰＦおよびＤＰＧＧを発現した場合でもＧＧＰＰａｓｅ活性が発現されるかどうかを調べた。
ＰＲＳ４３４ＧＡＰ−ＤＰＰ１、ｐＲＳ４３４ＧＡＰ−ＤＰＦ及びｐＲＳ４３４ＧＡＰ−ＤＰＧＧをそれぞれＳ． ｃｅｒｅｖｉｓｉａｅ ＹＰＨ４９９へ導入して形質転換を行い、得られた組換え体のＧＧＰＰａｓｅ比活性を、前記「ホスファターゼ活性の発現」の項で記載した方法と同様にして測定した。その結果を表６に示す。
【表６】

ＤＰＰ１を単独で発現させた株のＧＧＰＰａｓｅ比活性は野生株の約１０倍であった。一方、ＤＰＦ及びＤＰＧＧを発現させた株のＧＧＰＰａｓｅ比活性は野生株の約４倍であった。即ち、ＤＰＦ及びＤＰＧＧを発現させた株においては、ＤＰＰ１を単独で発現させた場合に比べるとＧＧＰＰａｓｅ活性の発現量は少ないものの、野生株に比べると有意にＧＧＰＰａｓｅ比活性が増大した。この結果から、連結遺伝子ＤＰＦおよびＤＰＧＧを発現した場合でもＧＧＰＰａｓｅ活性が発現されることが明らかとなった。
ホスファターゼ連結遺伝子ＤＰＦおよびＤＰＧＧを導入したＡ４５１によるプレニルアルコール生産
ＨＭＧ−ＣｏＡ還元酵素遺伝子ＨＭＧ１の発現ベクターであるｐＲＳ４３４ＧＡＰ−ＨＭＧ１をＡ４５１に導入し（これをＡＨ１と呼ぶ）、ＡＨ１に更にｐＲＳ４３５ＧＡＰ−ＤＰＦを導入した。こうして得られた組換え体、及びＡ４５１、ＡＨ１をＹＭ培地で培養し、ＦＯＨ生産性を測定した。その結果を表７に示す。
【表７】

Ａ４５１ではＦＯＨの生産は検出されなかった。ＨＭＧ１を発現したＡＨ１ではＦＯＨがある程度生産されるようになった（ＦＯＨ ０．３ｍｇ／Ｌ）。ＡＨ１にホスファターゼ連結遺伝子ＤＰＦを導入すると、ＡＨ１に比べてＦＯＨの生産性が１１．３倍に向上した（ＦＯＨ ３．４ｍｇ／Ｌ）。この結果から、ホスファターゼ遺伝子とプレニル二リン酸生合成系路に関与する酵素の遺伝子（特にプレニル二リン酸合成酵素遺伝子）とを連結した連結遺伝子の導入が、プレニルアルコールの生産性向上に有効であることが分かった。
ホスファターゼ連結遺伝子ＤＰＦおよびＤＰＧＧを導入したＡＵＲＧＧ１０１によるプレニルアルコール生産
前記した１５−２に対して、それぞれｐＲＳ４３４ＧＡＰ−ＤＰＦおよびｐＲＳ４３４ＧＡＰ−ＤＰＧＧを導入した。又、別途に１５−２に対してｐＲＳ４３５ＧＡＰ−ＢＴＳ１とｐＲＳ４３４ＧＡＰ−ＤＰＰ１の２種のプラスミドを導入した。各組換え体および宿主をＳＧ選択培地で培養し、プレニルアルコール生産性を測定した結果を表８に示す。
【表８】

Ａ４５１ではプレニルアルコールの生産は検出されず、ＡＵＲＧＧ１０１のプレニルアルコール生産量は微量であった。ＡＵＲＧＧ１０１にＨＭＧ０４４遺伝子を導入した１５−２では、ＦＯＨ，ＧＧＯＨがある程度生産されるようになった（ＦＯＨ ８．５ｍｇ／Ｌ，ＧＧＯＨ ２．２ｍｇ／Ｌ）。１５−２にホスファターゼ連結遺伝子ＤＰＦを導入すると、１５−２に比べてＦＯＨでは７．８倍、ＧＧＯＨでは２．６倍に生産性が向上した（ＦＯＨ ６６．７ｍｇ／Ｌ，ＧＧＯＨ ５．７ｍｇ／Ｌ）。次に、１５−２にＧＧＰＰ合成酵素遺伝子ＢＴＳ１とプレニルリン酸特異的ホスファターゼ遺伝子ＤＰＰ１の２種の遺伝子を別々のプラスミド上に組み込んで導入したところ、１５−２と比べてＦＯＨ，ＧＧＯＨの生産性が向上した（ＦＯＨ ２８．８ｍｇ／Ｌ，ＧＧＯＨ １３．７ｍｇ／Ｌ）。一方、１５−２に連結遺伝子ＤＰＧＧを導入すると、ＢＴＳ１とＤＰＰ１を別々のプラスミド上に組み込んで導入した場合と比べて、ＦＯＨでは２．４倍、ＧＧＯＨでは５．５倍に生産性が向上した（ＦＯＨ ６８．７ｍｇ／Ｌ，ＧＧＯＨ ７５．５ｍｇ／Ｌ）。
この結果から、ホスファターゼ遺伝子とプレニル二リン酸生合成系路に関与する酵素の遺伝子（特にプレニル二リン酸合成酵素遺伝子）とを連結した連結遺伝子の導入が、プレニルアルコールの生産性向上に非常に有効であることが分かった。
ジャー培養
１５−２に連結遺伝子ＤＰＧＧを導入した株（ｐＲＳ４３４ＧＡＰ−ＤＰＧＧ／１５−２）を５Ｌ容ジャーファーメンタ（ＭＳＪ−Ｕ２Ｗ；丸菱バイオエンジ）で培養して、その生産性を測定し、連結遺伝子ＤＰＧＧを導入することによる生産性向上への効果を更に確認することとした。培地は、５０ｇ ガラクトース（ナカライ）、１００ｇ パクトペプトン（ＤＩＦＣＯ）、５０ｇ 酵母エキス（ＤＩＦＣＯ）、５０ｍｌ 大豆油（ナカライ）、５ｍｌ アデカノールＬＧ１０９、水道水５１を混合して作製し、ジャーファーメンタで滅菌（１２１℃、２０分）して培養に用いた。
ジャー培養条件は次の通りである。植菌量２％、温度３３℃、通気量１ＶＶＭ、かくはん速度３００ｒｐｍ、ガラクトース・フィード・レート＝３．４ｇ／Ｌ、ｐＨ７．０（４Ｎ ＮａＯＨで制御）。経時的に培養液を採取してプレニルアルコール生産量を定量した結果を図２に示す。
培養１３４時間目にＦＯＨおよびＧＧＯＨの生産量は極大に達し、それぞれ０．３８ｇ／Ｌ、０．２４ｇ／Ｌであった。
酸性ホスファターゼ遺伝子ＰＨＯ３およびアルカリ性ホスファターゼ遺伝子ＰＨＯ８のクローニングおよび発現ベクターの作製
リン酸エステル化合物に対する加水分解活性において基質特異性のない（非特異的な）ホスファターゼをコードする遺伝子のクローニング及び発現ベクターの作製を、以下の方法で行った。非特異的ホスファターゼとしてＳ． ｃｅｒｅｖｉｓｉａｅの酸性ホスファターゼ遺伝子ＰＨＯ３（ＧｅｎＢａｎｋ ａｃｃｅｓｓｉｏｎ Ｎｏ．ＮＣ００１１３４，ｃｏｍｐｌｅｍｅｎｔ（４２７６５７．．４２９０６０））及びアルカリ性ホスファターゼ遺伝子ＰＨＯ８（ＧｅｎＢａｎｋ ａｃｃｅｓｓｉｏｎ Ｎｏ．ＮＣ００１１３６，ｃｏｍｐｌｅｍｅｎｔ（１４１８５３７．．１４２０２３７））を用いた。
ＰＣＲ法により、Ｓ． ｃｅｒｅｖｉｓｉａｅ ＹＰＨ４９９ゲノムＤＮＡを鋳型として、Ｓ． ｃｅｒｅｖｉｓｉａｅの遺伝子ＰＨＯ３約１．４ｋｂｐ断片を増幅した。ＰＣＲプライマーは、配列番号５０に示す塩基配列を持つプライマーと、配列番号５１に示す塩基配列を持つプライマーである。ＰＣＲ断片をアガロースゲル電気泳動によって精製した後、ｐＣＲ２．１−ＴＯＰＯへＴ／Ａライゲーションによりクローニングした。クローニングした断片の塩基配列決定を行い、ＳＧＤの配列と比較したところ、ＰＣＲエラーは無かった。作成したプラスミドＤＮＡをｐＣＲ−ＰＨＯ３と呼ぶ。ｐＣＲ−ＰＨＯ３からＳａｃＩＩ−ＸｈｏＩによって切出される１．４ｋｂｐ断片を調製し、ｐＲＳ４３４ＧＡＰのＳａｃＩＩ−ＸｈｏＩ切断部位に挿入した。作製したプラスミドをｐＲＳ４３４ＧＡＰ−ＰＨＯ３と呼ぶ。
ＰＣＲ法により、Ｓ． ｃｅｒｅｖｉｓｉａｅ ＹＰＨ４９９ゲノムＤＮＡを鋳型として、Ｓ． ｃｅｒｅｖｉｓｉａｅの遺伝子ＰＨＯ８約１．７ｋｂｐ断片を増幅した。ＰＣＲプライマーは、配列番号５２に示す塩基配列を持つプライマーと、配列番号５３に示す塩基配列を持つプライマーである。ＰＣＲ断片をアガロースゲル電気泳動により精製した後ｐＣＲ２．１−ＴＯＰＯへＴ／Ａライゲーションによりクローニングした。クローニングした断片の塩基配列決定を行い、ＳＧＤの配列と比較したところ、ＰＣＲエラーは無かった。作成したプラスミドＤＮＡをｐＣＲ−ＰＨＯ８と呼ぶ。ｐＣＲ−ＰＨＯ８からＳａｃＩＩ−ＳｍａＩによって切出される１．７ｋｂｐ断片を調製し、ｐＲＳ４３４ＧＡＰのＳａｃＩＩ−ＳｍａＩ切断部位に挿入した。作製したプラスミドをｐＲＳ４３４ＧＡＰ−ＰＨＯ８と呼ぶ。
非特異的ホスファターゼ活性の測定
酸性ホスファターゼ活性はＦｅｒｍｉｎａｎらの方法（Ｅ．Ｆｅｒｍｉｎａｎ，ｅｔ ａｌ．（１９９７）Ｍｉｃｒｏｂｉｏｌ．１４３，２６１５−２６２５）を参考にして以下のようにして測定した。ｐＲＳ４３４ＧＡＰ−ＰＨＯ３を前記１５−２に導入し、得られた組換え体および宿主を５ｍＬのＳＤ選択培地で１日間培養し、遠心分離により菌体を集菌した。菌体を生理食塩水で２度洗い、３００μＬの抽出バッファー（１０ｍＭ ｃｉｔｒａｔｅ（ｐＨ４．３）、１０ｍＭ β−ｍｅｒｃａｐｔｏｅｔｈａｎｏｌ、１ｍＭ ＰＭＳＦ、１０μｇ／Ｌ ａｐｒｏｔｉｎｉｎ、１０μｇ／Ｌ ｌｅｕｐｅｐｔｉｎ、１μｇ／Ｌ ｐｅｐｓｔａｔｉｎ）に懸濁した。そして、等量のガラスビーズ（φ＝０．４５−０．５ｍｍ）を加え、ボルテックスミキサーにて１５分間強く攪拌（４°Ｃ）して菌体を破砕した。更に３００μＬの抽出バッファーを加え、遠心分離（１８，０００×ｇ、１５分、４°Ｃ）した後、上清を分取して粗酵素画分とした。１０ｍＭ ｃｉｔｒａｔｅ（ｐＨ４．３）で適当に希釈した粗酵素画分１００μＬに、１００μＬのｐ−ニトロフェニルリン酸（ｐＮＰＰ）溶液（１０ｍＭ ｐＮＰＰ、２００ｍＭ ｃｉｔｒａｔｅ（ｐＨ４．３））を添加し、３０°Ｃで１５分間反応を行った。５０μＬの１Ｍ ＮａＯＨ水溶液を添加して反応を停止し、４０５ｎｍの吸光度を測定した。０〜０．０３Ｕ／ｍＬの酸性ホスファターゼ（Ｒｏｃｈｅ Ｄｉａｇｎｏｓｔｉｃｓ）を用いて検量線を作成し、前記で求めた４０５ｎｍの吸光度を酵素活性値（Ｕｎｉｔ）に換算した。粗酵素画分中のタンパク質濃度は、Ｂｉｏ−Ｒａｄ Ｐｒｏｔｅｉｎ Ａｓｓａｙで、ＢＳＡを標準タンパク質として測定し、粗酵素画分中の酸性ホスファターゼ比活性（Ｕ／ｍｇ ｐｒｏｔｅｉｎ）を求めた。また、ここで得られた粗酵素画分のＧＧＰＰａｓｅ比活性は、反応バッファーとして１２０ｍＭ ｃｉｔｒａｔｅ（ｐＨ４．３）を用いて、前記「ホスファターゼ活性の発現」の項で記載した酵素活性測定法と同様の方法で測定した。
アルカリ性ホスファターゼ活性はＤｈａｍｉｊａらの方法（Ｓ．Ｓ．Ｄｈａｍｉｊａ，ｅｔ ａｌ．（１９８７）Ｃｕｒｒｅｎｔ Ｇｅｎｅｔｉｃｓ，１１，４６７−４７３）を参考にして以下のようにして測定した。ｐＲＳ４３４ＧＡＰ−ＰＨＯ８を前記１５−２に導入し、得られた組換え体および宿主を５ｍＬのＳＤ選択培地で１日間培養し、遠心分離により菌体を集菌した。菌体を生理食塩水で２度洗い、３００μＬの抽出バッファー（１００ｍＭ Ｔｒｉｓ・ＨＣｌ（ｐＨ８．５）、１ｍＭ ＭｇＣｌ_２、１０ｍＭ β−ｍｅｒｃａｐｔｏｅｔｈａｎｏｌ、１ｍＭ ＰＭＳＦ、１０μｇ／Ｌ ａｐｒｏｔｉｎｉｎ、１０μｇ／Ｌ ｌｅｕｐｅｐｔｉｎ、１μｇ／Ｌ ｐｅｐｓｔａｔｉｎ）に懸濁した。そして、等量のガラスビーズ（φ＝０．４５−０．５ｍｍ）を加え、ボルテックスミキサーにて１５分間強く攪拌（４°Ｃ）して菌体を破砕した。更に３００μＬの抽出バッファーを加え、遠心分離（１８，０００×ｇ、１５分、４°Ｃ）により可溶性画分（上清）を分取して粗酵素画分とした。１００ｍＭ Ｔｒｉｓ・ＨＣｌ（ｐＨ８．５）で適当に希釈した粗酵素画分１００μＬに、１００μＬのｐ−ニトロフェニルリン酸（ｐＮＰＰ）溶液（１０ｍＭ ｐＮＰＰ、１００ｍＭ Ｔｒｉｓ・ＨＣｌ（ｐＨ８．５））を添加し、３０°Ｃで１５分間反応を行った。５０μＬの１Ｍ ＮａＯＨ水溶液を添加して反応を停止し、４０５ｎｍの吸光度を測定した。０〜０．３Ｕ／ｍＬのアルカリ性ホスファターゼ（ｃａｌｆ ｉｎｔｅｓｔｉｎｅ，Ｒｏｃｈｅ Ｄｉａｇｎｏｓｔｉｃｓ）を用いて検量線を作成し、前記で求めた４０５ｎｍの吸光度を酵素活性値（Ｕｎｉｔ）に換算した。アルカリ性ホスファターゼ比活性（Ｕ／ｍｇ ｐｒｏｔｅｉｎ）は酸性ホスファターゼの場合と同様にして求めた。又、ここで得られた粗酵素画分のＧＧＰＰａｓｅ比活性は、反応バッファーとして６０ｍＭ Ｔｒｉｓ・ＨＣｌ（ｐＨ８．５）、１ｍＭ ＭｇＣｌ_２を用いて、前記「ホスファターゼ活性の発現」の項で記載した酵素活性測定法と同様の方法で測定した。
以上の方法で測定した非特異的ホスファターゼ比活性（酸性ホスファターゼ比活性およびアルカリ性ホスファターゼ比活性）を以下ｐＮＰＰａｓｅ比活性と呼ぶ。
非特異的ホスファターゼ遺伝子を導入した１５−２のホスファターゼ活性の発現とプレニルアルコールの生産
酸性ホスファターゼ遺伝子ＰＨＯ３及びアルカリ性ホスファターゼ遺伝子ＰＨＯ８をそれぞれ組込んだ発現ベクターを導入した１５−２のｐＮＰＰａｓｅ比活性、ＧＧＰＰａｓｅ比活性及びプレニルアルコール生産性（ＳＧ選択培地で培養）を測定した。その結果を表９に示す。表９では、宿主を１とした相対値で表示している。表中のＧＧＰＰａｓｅ比活性の項で、＋ｐＮＰＰの列は、前記「ホスファターゼの基質特異性」の項で記載したのと同様に、ＧＧＰＰ以外の追加基質として１００μＭのｐＮＰＰを添加した場合のＧＧＰＰａｓｅ比活性値を示し、−ｐＮＰＰの列は、ｐＮＰＰを添加せずにＧＧＰＰａｓｅ比活性を測定した値を示す。
【表９】

酸性ホスファターゼ遺伝子又はアルカリ性ホスファターゼ遺伝子の導入により、ｐＮＰＰａｓｅ比活性は宿主に比べて２８〜３５倍に増大した。ＧＧＰＰａｓｅ比活性は、追加基質としてｐＮＰＰを加えない場合は宿主に比べて３〜６倍に増大した。しかし、追加基質としてｐＮＰＰを加えると［^３Ｈ］ＧＧＰＰに対する見かけ上のホスファターゼ活性はゼロとなった。このことから、酸性ホスファターゼ遺伝子およびアルカリ性ホスファターゼ遺伝子の導入により発現されたホスファターゼは、ＧＧＰＰとｐＮＰＰのいずれとも反応する基質特異性の低いホスファターゼであることが示された。この結果は、炭素数１５以上のプレニルリン酸に基質特異性のあるホスファターゼ遺伝子を導入した場合と、対照的である（表２参照）。一方、プレニルアルコールの生産性は、酸性ホスファターゼ遺伝子又はアルカリ性ホスファターゼ遺伝子を導入しても向上しなかった。以上の結果から、非特異的ホスファターゼ遺伝子を宿主に導入して、ｐＮＰＰａｓｅ活性又はＧＧＰＰａｓｅ活性を上昇させても、プレニルアルコールの生産性の向上への効果は無いことが分かった。
実施例の評価
以上の実施例は、次のように評価される。
即ち、炭素数１５以上のプレニルリン酸に基質特異性のあるホスファターゼをコードする６種類の遺伝子を、それぞれＦＯＨおよびＧＧＯＨ産生酵母に導入することにより、６種類いずれの遺伝子を導入した場合においても、ＧＧＰＰａｓｅ比活性が上昇した。更に、６種類いずれの遺伝子を導入した場合においても、ＦＯＨおよびＧＧＯＨの生産性が実際に数倍〜数十倍に向上することを示した。
本願発明者らは、ＦＯＨおよびＧＧＯＨの生産性を向上させることのできるホスファターゼのアミノ酸配列には、第１図に示すホスファターゼモチーフが含まれていることを見出した。このホスファターゼモチーフは、炭素数１５以上のプレニルリン酸に対する基質特異性に関係があると推測している。
更に、炭素数１５以上のプレニルリン酸に基質特異性のあるホスファターゼをコードする遺伝子とプレニル二リン酸生合成系路に関与する酵素をコードする遺伝子を連結してなる遺伝子を、ＦＯＨおよびＧＧＯＨ産生酵母に導入することにより、ＦＯＨおよびＧＧＯＨの生産性が更に向上した。上記連結遺伝子を導入した場合、連結せずに別々のプラスミド上に組み込んで導入した場合と比べてＦＯＨおよびＧＧＯＨの生産性が、数倍に向上することも示した。
一方、リン酸エステル化合物に対する加水分解活性において基質特異性のない（非特異的な）ホスファターゼをコードする遺伝子（ＰＨＯ３，ＰＨＯ８）を、ＦＯＨおよびＧＧＯＨ産生酵母に導入した場合、ＧＧＰＰａｓｅ比活性は上昇したが、ＦＯＨおよびＧＧＯＨの生産性は向上しなかった。非特異的ホスファターゼは、基質特異性がないために菌体中のあらゆるリン酸エステル化合物と反応すると考えられる。そのため、菌体中のプレニルリン酸以外のリン酸エステル化合物（例えば菌体中に多量に存在すると考えられるＡＴＰ等）とも反応してしまう。そして、実質的にプレニルリン酸と反応してプレニルアルコールを産生できる非特異的ホスファターゼの数は非常に限られてしまうため、プレニルアルコールの生産性が向上しなかったと推定される。
又、これらの非特異的なホスファターゼのアミノ酸配列を詳細に検討した結果、第１図に示したようなホスファターゼモチーフは含まれていないことが明らかとなった。このことは、上記ホスファターゼモチーフを含み炭素数１５以上のプレニルリン酸に基質特異性のあるホスファターゼ遺伝子の導入又は発現強化が、プレニルアルコールの生産性の向上に対して決定的であることを示している。
産業上の利用分野
以上のように、本発明は、プレニルアルコール、特に生物学的に活性な全トランス型で、しかも炭素数１５以上であるプレニルアルコールの効率的な大量生産を可能とする。
【配列表】

【図面の簡単な説明】
第１図は本発明に係る各ポリペプチドの保存配列を示す図である。
第２図はプレニルアルコール生産量の定量結果を示す図である。Technical field
The present invention relates to a method for producing a polypeptide, polynucleotide, recombinant nucleic acid, recombinant and prenyl alcohol. More specifically, the present invention relates to a phosphatase that is effective in selectively mass-producing prenyl alcohol having 15 or more carbon atoms, a polynucleotide encoding such a phosphatase, a recombinant nucleic acid containing such a polynucleotide, Recombinants into which various polynucleotides have been introduced, and prenyl alcohol (particularly having a biologically active trans-isomer structure by controlling the expression of enzymes involved in biosynthesis of prenyl alcohol in living cells, The present invention relates to a method for efficiently mass-producing, for example, FOH or GGOH having 15 or more carbon atoms.
Background art
The isoprenoid biosynthetic pathway is thought to be universally provided for various species. The biosynthetic pathway of isopentenyl diphosphate (IPP), the basic skeletal unit of isoprenoid compounds, is the mevalonate pathway that exists mainly in eukaryotes and the non-mevalonate pathway (DXP pathway) that exists mainly in prokaryotes. Two routes are known.
In the mevalonate pathway, acetyl CoA as a starting material, acetoacetyl CoA, hydroxymethylglutaryl CoA (HMG-CoA), mevalonate, mevalonate 5-phosphate (MVP), mevalonate 5-diphosphate (MVPP) Then, IPP having 5 carbon atoms, which is an active isoprene unit, is biosynthesized. In the non-mevalonic acid pathway, IPP is biosynthesized via 1-deoxy-D-xylulose 5-phosphate produced by condensation of pyruvic acid and glyceraldehyde 3-phosphate. Although knowledge about the non-mevalonate pathway is gradually accumulating, some of the enzymes involved and the genes encoding them are still unclear.
IPP is converted to dimethylallyl diphosphate (DMAPP) by an isomerization reaction. DMAPP is subjected to sequential polycondensation with IPP and trans-type (E-type), so that carbon number 10 geranyl diphosphate (GPP), carbon number 15 farnesyl diphosphate (FPP), carbon number 20 geranylgeranyl diphosphate (GGPP)... In addition, undecaprenyl diphosphate, dehydrodrisyl diphosphate, and the like are biosynthesized by sequential polycondensation of IPP to cis-type (Z-type) with respect to all-trans type FPP and GGPP.
The above IPP, DMAPP, GPP, FPP, GGPP, undecaprenyl diphosphate, dehydrodosyl diphosphate, etc. are collectively referred to as prenyl diphosphate. Among these prenyl diphosphates, highly useful (biologically active) all-trans pure products are suitable for production by biosynthesis and are difficult to control by chemical synthesis.
In addition, these prenyl diphosphates are separated by diphosphite by the action of phosphatase (or by relaying prenyl monophosphate from which one phosphate is separated from prenyl diphosphate, Of the phosphoric acid is separated) to produce the corresponding prenyl alcohol. In the present specification, hereinafter, prenyl diphosphate and prenyl monophosphate are collectively referred to as “prenyl phosphate”.
Among the above-mentioned prenyl phosphoric acid and prenyl alcohol, those having 15 or more carbon atoms are particularly useful. For example, FPP and GGPP, which are prenyl diphosphates having 15 or more carbon atoms, farnesol (FOH) having 15 carbon atoms, and geranylgeraniol (GGOH) having 20 carbon atoms, which are dephosphorylated oxides, include phytol, vitamin Synthetic substrates such as E, vitamin K, ubiquinone (CoQ). Heptaprenyl alcohol having 35 carbon atoms, decaprenyl alcohol having 50 carbon atoms, and the like are also useful as pharmaceutical intermediates, and FPP, GGPP, FOH, GGOH and the like can be used as synthesis starting materials.
Further, when comparing prenyl phosphate and prenyl alcohol, prenyl phosphate generally has a relatively high water solubility, but it has a non-polar group and is difficult to extract and purify. It is advantageous to produce prenyl alcohol that can be easily extracted and purified with a non-polar organic solvent such as Furthermore, prenyl alcohol is used for chemical synthesis of a compound having a farnesyl group or a geranylgeranyl group.
In view of the above, using biosynthetic pathways, prenyl alcohol having a biologically active trans-type isomer structure, particularly having 15 or more carbon atoms (for example, geranyl farnesol, hexa It is desired to provide a method for efficiently mass-producing prenyl alcohol, heptaprenyl alcohol, octaprenyl alcohol, nonaprenyl alcohol, decaprenyl alcohol, undecaprenyl alcohol, FOH, GGOH and the like.
Japanese Patent Publication No. 63-17437 discloses a method for producing polyprenyl alcohol by culturing a Pseudomonas strain, but only 3 mg / L of all-trans-type undecaprenyl alcohol is produced. And production of GGOH is not disclosed. Japanese Patent Application Laid-Open No. 9-238692 discloses a method for producing GGOH and GGPP by culturing plant cells of Euphorbiaceae, but only a maximum of 3.25 mg / L GGOH production is reported. Even if natural microorganisms and plant and animal cells are cultured as they are, efficient mass production of prenyl alcohol cannot be expected.
JP-A-10-248575 discloses an HMG-CoA (hydroxymethylglutaryl-CoA) reductase gene involved in the process of converting acetyl CoA to MVP or MVPP in yeast (Candida  utilis) To produce carotenoids. Japanese Patent Application Laid-Open No. 5-115298 discloses a method for increasing sterol accumulation in plants by increasing the copy number of the HMG-CoA reductase gene. JP-A-5-192184 discloses a method for increasing the accumulation of squalene and sterol by increasing the expression level of the HMG-CoA reductase gene in a mutant yeast having a deficiency in the expression of sterol biosynthesis enzyme. Has been. Japanese Patent Application Laid-Open No. 10-155497 discloses a carotenoid production method using a fermentation method using a microorganism into which a GGPP synthase gene (crtE) has been introduced. However, the methods disclosed in JP-A-10-248575, JP-A-5-115298, JP-A-5-192184 or JP-A-10-155497 are effective in mass production of prenyl alcohol. It is not tied to. Japanese Patent Laid-Open No. 53-127889 discloses a method for producing prenyl alcohol in vitro using phosphatase isolated from potato roots. However, this method is not a method using biosynthesis, and there is no disclosure about the use of a phosphatase gene.
On the other hand, regarding phosphatase having prenyl phosphate dephosphorylation activity, rat (Rattus  norvegicus) Report of prenyl pyrophosphatase (EC 3.1.7.1) present in liver microsomes (Bansal, Vs. et. Al (1994) Arch. Biochem. Biophys. 315 (2), 393 -399) but not isolated. yeast(S.  cerevisiae) Report that the diacylglycerol diphosphate phosphatase encoded by the DPP1 gene acts on IPP, GPP, FPP, and GGPP (Faulkner, A. et. Al (1999) J. Biol. Chem. 274 (21), 14831-14837), but it has not been elucidated what selective substrate specificity this phosphatase shows between prenyl phosphate and its precursor phosphate ester compounds (MVP and MVPP). Furthermore, in the above report of Faulkner et al., FIG. According to No. 5, data on the relative substrate specificity of each of the IPP, GPP, FPP, and GGPP are not so selective with respect to each other. The above two reports by Bansal et al. And Faulkner et al. Are exclusively intended for enzymological studies and do not suggest any industrial use of phosphatases or their genes.
In addition, in USP 6,242,227, if a gene encoding a phosphatase having a high effect of dephosphorylating FPP and GGPP is overexpressed, productivity of FOH and GGOH will be improved. There is a description. However, no examples relating to the use of the phosphatase gene are disclosed, and there is no description of a phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms. Furthermore, as shown in the Examples, the nonspecific phosphatase having no substrate specificity as described above has the effect of dephosphorylating FPP and GGPP, but the gene encoding the nonspecific phosphatase is overexpressed. However, the productivity of FOH and GGOH could not be improved.
In the production of a target substance using living cells such as microorganisms, a method for improving the productivity of a target substance by enhancing a specific step of a metabolic pathway by enhancing an enzyme action or the like is known as a general theory.
However, the construction of an efficient mass production system for prenyl alcohol in which the biosynthesis process of prenyl alcohol in living cells is artificially controlled has not yet been reported. In addition, it has not yet been clarified which step of the biosynthesis process should be controlled in order to specifically synthesize prenyl alcohol in living cells. Furthermore, a phosphatase that is effective in selectively mass-producing prenyl alcohol having 15 or more carbon atoms has not been clarified.
It is an object of the present invention to provide a phosphatase that is effective in selectively mass-producing prenyl alcohol having 15 or more carbon atoms, a polynucleotide encoding such a phosphatase, a recombinant nucleic acid containing such a polynucleotide, Recombinant into which nucleotides have been introduced, prenyl alcohol (particularly having a biologically active trans-type isomer structure, particularly having 15 or more carbon atoms) by controlling the expression of enzymes involved in biosynthesis of prenyl alcohol in living cells For example, FOH, GGOH).
Disclosure of the invention
1st invention of this application is a polypeptide which has the amino acid sequence shown in either of sequence number 1-sequence number 6, and shows the phosphatase activity which has substrate specificity to C15 or more prenyl phosphate.
The inventor of the present application has discovered that the polypeptide of the first invention is a phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms. Therefore, the polypeptide of the first invention can efficiently select and produce prenyl alcohol having 15 or more carbon atoms, such as FOH, GGOH, heptaprenyl alcohol, decaprenyl alcohol, and the like.
The second invention of the present application has an amino acid sequence in which 5 or less amino acids in the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6 are substituted, deleted, or added, and a prenyl phosphate having 15 or more carbon atoms. A polypeptide that exhibits phosphatase activity with substrate specificity.
The polypeptide according to the second invention can also be expected to have the same action and effect as the first invention.
The third invention of the present application is a polypeptide that contains at least one phosphatase motif of the following formulas (1) to (3) and exhibits phosphatase activity having substrate specificity for prenyl phosphate having 15 or more carbon atoms.
(1) KXXXGXXXRP (wherein X is selected from any amino acid residue)
(2) 1XXSGH (wherein, 1 is selected from amino acid residues S or T, and X is selected from any amino acid residue)
(3) SR2XDXXHXXXDVXXXGXXX3 (wherein 2 is selected from amino acid residues V or T, 3 is selected from amino acid residues G or A, and X is selected from any amino acid residue).
The inventor of the present application examined in detail the amino acid sequence of a polypeptide having substrate specificity for prenyl phosphate having 15 or more carbon atoms shown in SEQ ID NO: 1 to SEQ ID NO: 6, and in the third invention, (1) to (3) Conserved sequences common to these polypeptides were found. Although the details will be described later with reference to FIG. 1, this conserved sequence is considered to be a sequence (phosphatase motif) necessary for expressing phosphatase activity having substrate specificity for prenyl phosphate having 15 or more carbon atoms. Therefore, the polypeptide according to the third invention is considered to be a phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms.
According to a fourth invention of the present application, in the phosphatase motif represented by the formulas (1) to (3) according to the third invention, any one or more amino acid residues whose types are specified are subjected to a conservative substitution. It is a peptide. Here, the “amino acid residue whose type is specified” refers to an amino acid residue represented by a symbol other than “X” in the above formulas (1) to (3). A “conservative substitution” is a substitution of a given amino acid residue with another amino acid residue that is chemically or functionally similar so that the function of the polypeptide remains substantially unchanged. Say to do. Examples of chemically or functionally similar amino acids include hydrophobic amino acids (Ala, Ile, Leu, Phe, Pro, Trp, Val, Met), polar but uncharged amino acids (Asn, Cys, Gln, Gly, Ser, Thr, Tyr), basic amino acids (Arg, His, Lys), acidic amino acids (Asp, Glu), etc. (translated by EE Cornn et al., Translated by Nobuo Tamiya et al., “Corn Stamp Biochemistry 5th edition "Tokyo Kagaku Doujin, p56-58, 1988).
In the polypeptide according to the fourth invention, since any one or more amino acid residues specified in the phosphatase motif have undergone a conservative substitution, like the polypeptide according to the third invention, the polypeptide having 15 or more carbon atoms It is thought to be a phosphatase with substrate specificity for prenyl phosphate.
5th invention of this application is a polynucleotide which has a base sequence shown in either of sequence number 7-sequence number 12, and encodes the phosphatase which has a substrate specificity to prenyl phosphate with 15 or more carbon atoms.
The present inventor has discovered that the polypeptide encoded by the polynucleotide according to the fifth invention is a phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms. Therefore, by introducing or enhancing expression of the polynucleotide of the fifth invention into a living cell or organism, a phosphatase having a substrate specificity for prenyl phosphate having 15 or more carbon atoms is produced in the living cell or organism, More than 15 prenyl alcohols can be produced effectively.
The sixth invention of the present application has a base sequence that hybridizes under a predetermined stringent condition to the base sequence shown in any one of SEQ ID NO: 7 to SEQ ID NO: 12 or a base sequence complementary thereto, and carbon It is a polynucleotide encoding a phosphatase having substrate specificity for several 15 or more prenyl phosphates.
Also in the polynucleotide according to the sixth invention, the same actions and effects as in the fifth invention can be expected.
The seventh invention of the present application is a polynucleotide encoding any of the polypeptides according to the first to fourth inventions.
In the polynucleotide according to the seventh invention, the same actions and effects as in the fifth invention can be expected.
The eighth invention of the present application is a recombinant nucleic acid comprising any one of the polynucleotides according to the fifth to seventh inventions.
The eighth invention provides an effective means for introducing the polynucleotide according to the fifth to seventh inventions into a living cell or organism.
A ninth invention of the present application is a recombinant into which any of the recombinant nucleic acids according to the eighth invention has been introduced.
By the recombinant according to the ninth invention, prenyl alcohol having 15 or more carbon atoms can be selected and produced more advantageously.
According to a tenth aspect of the present invention, the host cell of the recombinant according to the ninth aspect is a fungus (Eumycetes), Ascomycetes (Ascomycetes), A recombinant that is one of the unicellular eukaryotes.
That is, as a recombinant host according to the ninth invention, for example, a fungus (Eumycetes), Ascomycetes (Ascomycetes), Any of single cell eukaryotes can be preferably used.
The eleventh invention of the present application is a recombinant in which the host cell of the recombinant according to the ninth invention is yeast.
That is, as a recombinant host according to the ninth invention, for example, yeast can be preferably used.
According to a twelfth aspect of the present invention, the yeast according to the eleventh aspect is a genus Saccharomyces (Saccharomyces) A yeast recombinant.
That is, as the yeast according to the eleventh invention, for example, Saccharomyces genus (SaccharomycesYeast) can be preferably used.
According to a thirteenth aspect of the present invention, the yeast according to the eleventh aspect is Saccharomyces cerevisiae (Saccharomyces  cerevisiae) YPH499 strain, YPH500 strain, A451 strain, W303-1A strain, W303-1B strain or a recombinant derived therefrom.
That is, examples of the yeast according to the eleventh invention include Saccharomyces cerevisiae (S.  cerevisiae) YPH499 strain, YPH500 strain, A451 strain, W303-1A strain, W303-1B strain or strains derived therefrom can be preferably used.
14th invention of this application is a manufacturing method of prenyl alcohol which culture | cultivates the host cell which introduce | transduced or enhanced expression of the phosphatase gene, and extract | collects prenyl alcohol from the culture.
The inventor of the present application uses the mevalonic acid pathway starting from acetyl-CoA or the non-mevalonic acid pathway starting from pyruvic acid to pass the prenyl alcohol via the various prenyl diphosphates to the prenyl alcohol. As a result of detailed experimental studies on the biosynthetic reaction, biologically active isomeric structures (for example, all trans-type, for example, by introducing or enhancing expression of phosphatase gene in living cells) Geranyl farnesol, hexaprenyl alcohol, heptaprenyl alcohol, octaprenyl alcohol, nonaprenyl alcohol, decaprenyl alcohol, undecaprenyl alcohol, FOH, GGOH, etc.) It was. On the other hand, the method of the fourteenth invention is also useful when it is desired to increase the overall production amount of prenyl alcohol having 5 or more carbon atoms.
The inventor of the present application has reduced productivity of prenyl alcohol having 15 or more carbon atoms (for example, all-trans-type FOH, GGOH) at present by introducing or enhancing expression of a phosphatase gene in an example using yeast as a host. It has succeeded in raising it 40 times. That is, the fourteenth invention provides a method for efficiently mass-producing prenyl alcohol, particularly trans-type prenyl alcohol, particularly trans-type prenyl alcohol having 15 or more carbon atoms.
A fifteenth aspect of the present invention is a method for producing prenyl alcohol, comprising culturing a host cell into which a phosphatase gene and a gene for an enzyme involved in a prenyl diphosphate biosynthesis pathway have been introduced or enhanced, and collecting prenyl alcohol from the culture. It is.
When the phosphatase gene and the gene of the enzyme involved in the prenyl diphosphate biosynthesis pathway are introduced or enhanced in the host cell as in the fifteenth invention, that is, the phosphatase gene is introduced or enhanced and the prenyl diphosphate biosynthesis is When the gene for an enzyme involved in any other step in the synthesis pathway is introduced or enhanced, the productivity of prenyl alcohol may be further increased. Contributing to efficient mass production of prenyl alcohol (especially prenyl alcohol having 15 or more carbon atoms, such as FOH, GGOH) even if only a gene encoding an enzyme involved in the prenyl diphosphate biosynthetic pathway is introduced or enhanced. Is limited. Furthermore, in this case, it is particularly preferable that the phosphatase gene and the gene of the enzyme involved in the prenyl diphosphate biosynthetic pathway are linked to introduce or enhance expression.
The sixteenth invention of the present application is a method for producing prenyl alcohol, wherein the gene of an enzyme involved in the prenyl diphosphate biosynthesis pathway according to the fifteenth invention is the following gene (4) and / or (5): .
(4) At least one gene selected from a farnesyl diphosphate synthase gene and a geranylgeranyl diphosphate synthase gene.
(5) Acetyl CoA synthase gene, acetyl CoA-acetyltransferase gene, hydroxymethylglutaryl CoA synthase gene, hydroxymethylglutaryl CoA reductase gene, mevalonate kinase gene, mevalonate phosphate kinase gene, mevalonate diphosphate Acid decarboxylase gene, isopentenyl diphosphate isomerase gene, deoxyxylulose phosphate reductoisomerase gene, deoxyxylulose phosphate synthase gene, MEP (2-C-methyl-D-erythritol 4-phosphate) cytidylyl Transferase genes, CDP-ME (4- (cytidine 5'-diphospho) -2-C-methyl-D-erythritol) kinase genes and And at least one gene selected from MECDP (2-C-methyl-D-erythritol 2,4-cyclodiphosphate) synthase genes.
In the fifteenth aspect of the invention, the genes listed in (4) of the sixteenth aspect of the invention are particularly preferred as genes that are introduced or enhanced together with the phosphatase gene. The genes listed in (5) of the 16th invention are also preferred.
The seventeenth invention of the present application is a gene in which the phosphatase gene according to the fourteenth to sixteenth inventions encodes a phosphatase having a substrate specificity for a prenyl phosphate having 15 or more carbon atoms in the hydrolysis activity for a phosphate ester compound. This is a method for producing prenyl alcohol.
When the phosphatase gene to be introduced or expression-enhanced is a gene encoding a phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms as in the 17th invention, an efficient large amount of prenyl alcohol having 15 or more carbon atoms can be efficiently produced. The effect is particularly remarkable for the purpose of production. As these prenyl alcohols, prenyl alcohols having various biologically active isomer structures exemplified in the above-mentioned column of “Operation / Effect of 14th Invention” can be particularly preferably exemplified.
18th invention of this application is a manufacturing method of the prenyl alcohol whose phosphatase gene which concerns on the said 14th invention-16th invention is any polypeptide in the said 5th invention-8th invention.
That is, the phosphatase gene according to the fifth to eighth inventions can be preferably used as the phosphatase gene introduced or enhanced in the fourteenth to sixteenth inventions.
In the nineteenth invention of the present application, the phosphatase gene according to the fourteenth to sixteenth inventions encodes a phosphatase other than a phosphatase having a substrate specificity for a prenyl phosphate having 15 or more carbon atoms in the hydrolysis activity for a phosphate ester compound. It is a manufacturing method of prenyl alcohol which is a gene.
When the phosphatase gene to be introduced or enhanced in expression is a gene encoding a phosphatase other than a phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms as in the 19th invention, the carbon number is 5 to 15 Various prenyl alcohols over a range can be obtained effectively.
The twentieth invention of the present application is the method for producing prenyl alcohol, wherein the host cell according to the fourteenth to nineteenth inventions is any one of the host cells according to the tenth to thirteenth inventions.
That is, any of the host cells according to the tenth to thirteenth inventions can be preferably used as the host cell in the fourteenth to nineteenth inventions.
A twenty-first invention of the present application is the method for producing a prenyl alcohol, wherein the prenyl alcohol according to the fourteenth to twentieth inventions has 15 or more carbon atoms.
In the method for producing prenyl alcohol according to the fourteenth to twentieth inventions, prenyl alcohol having 5 or 10 carbon atoms can also be collected, but prenyl alcohol having 15 or more carbon atoms has a relatively large production amount. It is easy to extract and purify with a nonpolar organic solvent and has high utility as described above. Therefore, it is particularly effective to collect prenyl alcohol having 15 or more carbon atoms. All of the prenyl alcohols having 15 or more carbon atoms can be collected, or one or more of them can be selectively collected.
According to a twenty-second invention of the present application, the prenyl alcohol according to the twenty-first invention is farnesol, geranylgeraniol, geranylfarnesol, hexaprenyl alcohol, heptaprenyl alcohol, octaprenyl alcohol, nonaprenyl alcohol, decaprenyl alcohol, undecaprenyl alcohol or It is a manufacturing method of prenyl alcohol which is 1 type, or 2 or more types of prenyl alcohol chosen from dodecaprenyl alcohol.
That is, as the prenyl alcohol having 15 or more carbon atoms collected in the twenty-first invention, one or more prenyl alcohols selected from the above-mentioned prenyl alcohols are particularly preferably exemplified.
A twenty-third invention of the present application is a method for producing a prenyl alcohol in which the prenyl alcohol according to the twelfth to twenty-second inventions is an all-trans type.
That is, in the method for producing prenyl alcohol according to the twelfth to twenty-second inventions, all-trans-type prenyl alcohol having high utility can be produced because it is biosynthesis using host cells. It is more preferable to collect such all-trans prenyl alcohol.
BEST MODE FOR CARRYING OUT THE INVENTION
Polypeptide
The polypeptide according to the present invention is a phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms. An example of a preferable standard for such phosphatase can be defined as follows. That is, when the specific activity with respect to (A) shown in the enzyme activity measurement system using only prenyl phosphate (A) having 15 or more carbon atoms as a substrate is defined as 100, 20 times the amount of carbon in (A) and molar ratio. The specific activity with respect to (A) when coexisting with several dozens of prenyl phosphoric acid or its precursor (B) is 80 or more, more preferably 85 or more, and still more preferably 90 or more.
A representative example of a polypeptide having substrate specificity for prenyl phosphate having 15 or more carbon atoms according to the present invention is a polypeptide having the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6. It was first found by the present inventor that all of these polypeptides are phosphatases having substrate specificity for prenyl phosphate having 15 or more carbon atoms.
A polypeptide having the amino acid sequence shown in SEQ ID NO: 1 is an Arabidopsis thaliana (Arabidopsis  thalianaPhosphatidic acid phosphatase homolog gene [Gen Bank accession No. : AC006200 (complement (16778..17686)), this gene was named AtPAP1]. The nucleotide sequence of AtPAP1 is shown in SEQ ID NO: 7. A polypeptide having the amino acid sequence shown in SEQ ID NO: 2 isA.  thalianaPhosphatidic acid phosphatase homolog gene [Gen Bank accession No. : AC007591 (3610.5006), this gene was named “AtPAP2”). The nucleotide sequence of AtPAP2 is shown in SEQ ID NO: 8.
The polypeptide having the amino acid sequence shown in SEQ ID NO: 3 is Saccharomyces cerevisiae (S.  cerevisiae) Phosphatase encoded by the gene DPP1 (Toke DA et al (1998) J. Biol. Chem. 273 (6), 3278-3284). Gen Bank accession No. of DPP1. Is NC 00136 and its base sequence is shown in SEQ ID NO: 9.
The polypeptide having the amino acid sequence shown in SEQ ID NO: 4 is a phosphatase encoded by the yeast gene LPP1 (Toke DA et al (1998) J. Biol. Chem. 273 (23), 14331-14338). LPP1 Gen Bank accession No. Is NC001136, and its base sequence is shown in SEQ ID NO: 10.
The polypeptide having the amino acid sequence shown in SEQ ID NO: 5 is rat (R.  norvegicus) Phosphatase encoded by the gene PAP2 (Brindley DN et al (1998) J. Biol. Chem. 273 (38), 24281-24284). PAP2 Gen Bank accession No. Is U90556 and its base sequence is shown in SEQ ID NO: 11. A polypeptide having the amino acid sequence shown in SEQ ID NO: 6 isR.  norvegicusThe phosphatase encoded by the gene Dri42 (Barila D et al (1996) J. Biol. Chem. 271 (47), 29928-29936). Dri42's Gen Bank accession No. Is Y07778, and its base sequence is shown in SEQ ID NO: 12.
Furthermore, as a result of detailed examination of the amino acid sequence of the polypeptide having substrate specificity for prenyl phosphate having 15 or more carbon atoms, shown in SEQ ID NO: 1 to SEQ ID NO: 6, it was conserved in common as shown in FIG. The sequence was found.
In FIG. 1, the polypeptides according to SEQ ID NO: 1 to SEQ ID NO: 6 are distinguished by the notation of the genes encoding them (for example, the notation “DPP1” represents the polypeptide according to SEQ ID NO: 3). The conserved sequences found in each of these polypeptides are shown. In the bottom line of FIG. 1, the amino acid sequences common to these conserved sequences are shown by the sequence notation of the multiple alignment method. In this sequence expression of multiple alignment, 1 is selected from amino acid residues S or T, 2 is selected from amino acid residues V or T, 3 is selected from amino acid residues G or A, and X is any amino acid It represents being selected from residues. n1 represents an arbitrary number (for example, 34 to 39) of arbitrary amino acid residues, and n2 also represents an arbitrary number (for example, 38 to 46) of arbitrary amino acid residues.
Accordingly, in the conserved sequence represented by the multiple alignment sequence notation of FIG. 1, at least one or all of the sequence portions (1) to (3) defined in the third invention are all or It is considered that this is a sequence (phosphatase motif) necessary for expressing phosphatase activity having substrate specificity for prenyl phosphate having 15 or more carbon atoms.
In addition, for a conserved sequence represented by the multiple alignment sequence notation in FIG. 1, a polypeptide that is a variant in which one or more of the specified amino acid residues has undergone the “conservative substitution” according to the above-mentioned definition. It is considered that prenyl phosphate having 15 or more carbon atoms exhibits phosphatase activity having substrate specificity.
Another representative example of the polypeptide according to the present invention is an amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 6, having an amino acid sequence in which 5 or less amino acids are substituted, deleted or added, and having 15 or more carbon atoms A polypeptide exhibiting phosphatase activity with substrate specificity for prenyl phosphate.
In the above, 5 or less amino acids to be substituted, deleted or added may be amino acids that are all adjacent to each other in the amino acid sequence. In addition, the above five or less amino acids may be amino acids that are partially adjacent to each other in the amino acid sequence. Furthermore, the five or less amino acids may be amino acids that are not adjacent to each other in the amino acid sequence.
The polypeptides listed above can be obtained directly from the cells of each of the above-mentioned source organisms by conventional enzyme isolation means, or the same can be obtained by introducing a DNA encoding each polypeptide into the host. Can be obtained in large quantities.
In the method for producing prenyl alcohol according to the fourteenth invention, the phosphatase encoded by the phosphatase gene to be introduced or enhanced in expression is not limited as long as it acts on at least prenyl phosphate. That is, various levels of substrates ranging from acid phosphatase and alkaline phosphatase having low substrate specificity to phosphatase having substrate specificity to prenyl phosphate and further to phosphatase having substrate specificity to prenyl phosphate having 15 or more carbon atoms. Specific phosphatases are included without limitation. However, it is more preferable to use a phosphatase having a higher substrate specificity, particularly a phosphatase having a substrate specificity for prenyl phosphate having 15 or more carbon atoms. Preferred examples of the phosphatase having a high substrate specificity for prenyl phosphate include the polypeptides according to the first to fourth inventions. Preferable examples also include polypeptides derived from yeast, animals or plants having equivalent functions.
Polynucleotide
In the present invention, the polynucleotide refers to single-stranded, double-stranded or triple-stranded DNA and / or RNA. In the present invention, the recombinant nucleic acid refers to a polynucleotide prepared for recombination.
The polynucleotide according to the present invention is a polynucleotide encoding a phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms.
A representative example of the polynucleotide according to the present invention is a polynucleotide having the base sequence shown in any one of SEQ ID NO: 7 to SEQ ID NO: 12. It was first discovered by the present inventor that they encode polypeptides exhibiting phosphatase activity that is substrate-specific for prenyl phosphate having 15 or more carbon atoms.
The polynucleotide having the base sequence shown in SEQ ID NO: 7 is the AtPAP1. The polynucleotide having the base sequence shown in SEQ ID NO: 8 is the AtPAP2. It is known that AtPAP1 is located in the second section of chromosome II published in the Arabidopsis Genome Project (Lin et al (1997) Nature 402, p.767-768), and AtPAP2 is the chromosome 1 BAC library. It is known to be located at F9L1 (F9L1.2 (Accession No. AC007591)).
The polynucleotide having the base sequence shown in SEQ ID NO: 9 is DPP1. The polynucleotide having the base sequence shown in SEQ ID NO: 10 is the LPP1. The polynucleotide having the base sequence shown in SEQ ID NO: 11 is the PAP2. The polynucleotide having the base sequence shown in SEQ ID NO: 12 is the above-mentioned Dri42.
Another representative example of the polynucleotide according to the present invention is a part of a conserved sequence represented by the multiple alignment sequence notation shown in FIG. 1 (among the sequence parts (1) to (3) defined in the third invention). A polynucleotide that encodes a polypeptide having an amino acid sequence that includes at least one or more portions) or all, or that encodes a polypeptide in which one or more of the amino acid residues identified therein have undergone conservative substitutions.
Still another representative example of the polynucleotide according to the present invention has a base sequence that hybridizes with the base sequence shown in any one of SEQ ID NO: 7 to SEQ ID NO: 12 under predetermined stringent conditions, and has 15 or more carbon atoms A polynucleotide encoding a phosphatase having substrate specificity for prenyl phosphate.
In the above description, “hybridizes under stringent conditions” means that in a suitable hybridization method such as colony hybridization method, plaque hybridization method or Southern blot hybridization method, one polynucleotide ( DNA) or the other polynucleotide (DNA) can be hybridized to a fragment of the polynucleotide. That is, one polynucleotide or a fragment of the polynucleotide immobilized on the filter is hybridized with the other polynucleotide at a predetermined temperature (X ° C) in the presence of 0.7 to 1 M NaCl. After that, when the filter is washed under an X ° C condition using an SSC solution of about 0.1 to 2 times (the composition of the SSC solution of 1 times concentration is composed of 150 mM sodium chloride and 15 mM sodium citrate). In addition, it means that the other polynucleotide can be identified. “X ° C.” is at least 50 ° C. or more, more preferably 60 ° C. or more, and further preferably 65 ° C. or more.
The polynucleotides listed above can be obtained from each of the above-mentioned source organisms by conventional isolation means, and may be obtained from various unicellular organisms such as other prokaryotes and animal and plant cells. Hybridization techniques (Southern 1975 J. Mol. Biol. 98: 503, Maniatis et al. Molecular Cloning Cold Harbor Laboratory Press) and PCR techniques (Saiki et al. 487 (1988)). That is, for those skilled in the art, using a base sequence of a known gene or a part thereof as a probe, or using an oligonucleotide that hybridizes to a part of the base sequence as a primer, a highly homologous polynucleotide is isolated, Cloning the phosphatase gene from the polynucleotide is easy. It is also easy to isolate the phosphatase using the above characteristic phosphatase activity as an index and clone the phosphatase gene from the amino acid sequence.
The phosphatase gene introduced or enhanced in the method for producing prenyl alcohol according to the fourteenth invention is not limited as long as it is a gene encoding phosphatase that acts on prenyl phosphate. That is, various levels of substrates ranging from acid phosphatase and alkaline phosphatase having low substrate specificity to phosphatase having substrate specificity to prenyl phosphate and further to phosphatase having substrate specificity to prenyl phosphate having 15 or more carbon atoms. A gene encoding a specific phosphatase is included without limitation. However, it is more preferable to introduce or enhance expression of a phosphatase having a higher substrate specificity, in particular, a gene encoding a phosphatase having a substrate specificity in prenyl phosphate having 15 or more carbon atoms. Preferred examples of the gene encoding a phosphatase having a high substrate specificity for prenyl phosphate include the polynucleotides according to the fifth to seventh inventions. Preferred examples also include yeast, plant or animal-derived polynucleotides having equivalent functions.
Introduction or enhanced expression of phosphatase gene
A gene encoding a non-specific phosphatase, more preferably a gene encoding a phosphatase having a substrate specificity for prenyl phosphate, more preferably a gene encoding a phosphatase having a substrate specificity for a prenyl phosphate having 15 or more carbon atoms. , Introduced into or enhanced in the host cell.
In the present invention, “introducing or enhancing expression of a phosphatase gene” has the following meaning. That is, “introducing a phosphatase gene” includes all cases where the phosphatase gene is introduced into a host cell as a recombinant nucleic acid. At least the case where a phosphatase gene is introduced by a vector and the case where a phosphatase gene is introduced by homologous recombination of a polynucleotide using a PCR fragment or the like are included. More preferably, the phosphatase gene is introduced by an expression vector that enhances expression. Further, “enhancement of expression of phosphatase gene” includes all cases where the expression of the phosphatase gene is enhanced as a result. For example, when the phosphatase gene is introduced as a recombinant nucleic acid with a means for enhancing its expression, when the copy number of the phosphatase gene in the host cell is increased, the culture condition of the host cell is adjusted (inducer of gene expression) And the like) to enhance the expression of the phosphatase gene.
Moreover, at the same time as introducing or enhancing the expression of genes encoding the above-mentioned various phosphatases, at least one gene of enzymes involved in the prenyl diphosphate biosynthesis pathway can be introduced or enhanced in the host cell. The form in which the gene encoding the phosphatase and the gene of the enzyme involved in the prenyl diphosphate biosynthetic pathway are simultaneously introduced or expression-enhanced is arbitrary. It may be introduced or enhanced by using different means, or may be introduced or enhanced at the same time as a linked gene as described below.
As a gene for an enzyme involved in the prenyl diphosphate biosynthetic pathway, for example, at least one gene selected from a hydroxymethylglutaryl CoA reductase gene, a farnesyl diphosphate synthase gene and a geranylgeranyl diphosphate synthase gene is preferable. It can be illustrated. Alternatively, acetyl CoA synthase gene, acetyl CoA-acetyltransferase gene, hydroxymethylglutaryl CoA synthase gene, mevalonate kinase gene, mevalonate phosphate kinase gene, mevalonate diphosphate decarboxylase gene, isopentenyl diphosphate Preferred examples include at least one gene selected from an isomerase gene, deoxyxylulose phosphate reductoisomerase gene, deoxyxylulose phosphate synthase gene, MEP cytidylyltransferase gene, CDP-ME kinase gene and MECDP synthase gene. .
In addition, genes encoding various phosphatases can be linked to genes of enzymes involved in the prenyl diphosphate biosynthetic pathway and introduced into host cells or enhanced in expression as described above. As a gene for an enzyme involved in the prenyl diphosphate biosynthetic pathway, for example, at least one gene selected from a farnesyl diphosphate synthase gene and a geranylgeranyl diphosphate synthase gene can be preferably exemplified. Alternatively, acetyl CoA synthase gene, acetyl CoA-acetyltransferase gene, hydroxymethylglutaryl CoA synthase gene, hydroxymethylglutaryl CoA reductase gene, mevalonate kinase gene, mevalonate phosphate kinase gene, mevalonate diphosphate Selected from decarboxylase gene, isopentenyl diphosphate isomerase gene, deoxyxylulose phosphate reductoisomerase gene, deoxyxylulose phosphate synthase gene, MEP cytidylyltransferase gene, CDP-ME kinase gene or MECDP synthase gene Preferred examples include at least one gene. Such a linked gene is also referred to as a “phosphatase linked gene”.
The linked gene is a gene in which two or more genes are linked so that they can be expressed as one reading frame (ORF), and the expressed polypeptide becomes a fusion protein. It is also possible to freely insert an artificial nucleotide sequence (linker sequence) in the binding region between the gene to be linked and the gene so that a three-dimensional structure in which each gene translation product functions properly can be obtained. For example, a linker sequence encoding a peptide sequence such as Gly Gly Gly Ser or Gly Gly Gly Gly Ser can be used. These sequences were used as linker sequences for linking H and L chains when expressing antibodies in E. coli (Huston J. S. et al (1988) Proc. Natl. Acad. Sci. USA 85: 5879-. 5883).
An expression vector or a polynucleotide fragment having an expression function of a gene to be introduced is linked to various polynucleotide fragments that enhance the expression of the phosphatase gene to be introduced or the phosphatase-linked gene. Preferably, the expression vector may include a transcription promoter, a transcription terminator, a recombinant selection marker gene, and an enhancer. More preferably, the plant expression vector may contain a T-DNA region. As a general expression vector construction method, for example, a gene fragment prepared by PCR or the like can be incorporated into an expression vector by a known method using an appropriate restriction enzyme and ligase.
Examples of vectors includeS.  cerevisiae“YEp13”, “YEp24”, “YCp50”, “pYES2”, “pRS414”, “pRS415”, “pRS416”, “pRS413”, “pRS404”, “pRS405], “PRS406”, “pRS403”, “pAUR101”, etc.Escherichia coli) As a host, plasmids “pSC101”, “pBR322”, “pHSG298”, “pVC18”, “pVC19”, “pTrc99A”, “pMa1-c2],“ pGEX2T ”,“ pTV118N ”, “PTV119N” etc., Bacillus subtilis (Bacillus  subtilisThe plasmids “pUB110”, “pC194”, and the like that are often used when a host is used as a host can be preferably used, and “pBI1221”, “pBI1101”, and other various types can be used without limitation.
The expression vector may contain a transcription promoter for constitutively or inducibly expressing phosphatase. For example,S.  cerevisiaeAlcohol dehydrogenase gene (ADH1 and ADH2), triose phosphate dehydrogenase gene (TDH3), genes related to galactose catabolism (GAL1, GAL7, GAL10), acid phosphatase gene (PHO5) or metallothionein gene (CUP1) transcription promoter. E. coli (E.  coli) Transcription promoters such as trp, lac, trc, tac can be used.
In addition, examples of transcription promoters for constant expression include cauliflower mosaic virus 35S promoter (Odell et al. 1985 Nature 313: 810) and rice actin promoter (Zhang et al. 1991 Plant Cell 3: 1155). ), Corn ubiquitin promoter (Cornejo et al 1993 Plant Mol. Biol. 23: 567) and other various types can be used without limitation. As a promoter for inducible expression, transcription promoter known to be expressed by external factors such as infection and invasion of filamentous fungi, bacteria, viruses, low temperature, high temperature, drying, ultraviolet irradiation, spraying of specific compounds, etc. Etc.
A recombinant nucleic acid for introducing or enhancing expression of a phosphatase gene or a phosphatase-linked gene does not necessarily have a vector function, and it is sufficient if it can be integrated, for example.
A phosphatase gene or a phosphatase-linked gene linked to an expression vector or other recombinant nucleic acid can contain a nucleotide sequence region that adds a localization signal to the vicinity of the lipid membrane.
Prenyl phosphate (especially prenyl phosphate having 15 or more carbon atoms) is hydrophobic, and high in the vicinity of the cell membrane of the host cell and in various cell organ membranes such as the Golgi apparatus and endoplasmic reticulum when the host cell is a eukaryotic cell. There is a tendency to localize to the concentration. Therefore, by adding a localization signal such as a hydrophobic peptide to phosphatase, the contact probability between phosphatase and prenyl phosphate as a substrate can be improved, and the productivity of prenyl alcohol can be increased. In particular, when the phosphatase gene is originally a gene encoding a non-substrate-specific phosphatase, the addition of the above signal to phosphatase is effective.
Representative examples of the localization signal are various known hydrophobic signal peptides. As is well known, it is also possible to select the localization site of the phosphatase in the host cell by selecting the localization signal. As a general method for constructing an expression vector or other recombinant nucleic acid with the addition of a base sequence region for adding a localization signal to a phosphatase gene or a phosphatase-linked gene, for example, various known signal peptides A gene domain encoding phosphatase gene or a phosphatase ligation gene is prepared by PCR, etc., these genes are ligated by a known method using an appropriate restriction enzyme and ligase, and the ligated gene is added to an appropriate expression vector. Can be incorporated.
Introduction of recombinant nucleic acid into host
When introducing or enhancing expression of the phosphatase gene according to the present invention into an organism, the type of host cell is not limited. For example, prokaryotes, fungi (Eumycetes), Ascomycetes (Ascomycetes), Unicellular eukaryotes, or live cells of animals or plants. Living tissue culture cells of multicellular organisms including plant tissue culture cells and animal tissue culture cells can be arbitrarily selected.
In the above plant cells or plant tissue culture cells, “plants” include algae, bryophytes, ferns, gymnosperms and angiosperms. In the above animal cells or animal tissue culture cells, the “animal” includes sponges, coelenterates, linear animals, molluscs, arthropods, sessile animals, and various vertebrates. Examples of living cells or living tissue culture cells of animals or plants include suspension culture cells of animals and plants, tissue culture cells of animal and plant organs such as viscera, leaves and roots, and callus in plants.
Although it does not specifically limit as a host,S.  cerevisiae, Pichia Pastoris (Pichia  pastris) Yeast belonging to the genus Saccharomyces and Pichia and others,E.  coli, Bacillus subtilis (Bacillus  subtilis), Bacillus brevis (Bacillus  brevisBacteria belonging to the genus Escherichia or Bacillus such as Aspergillus oryzae (Aspergillus  oryzae), Aspergillus niger (Aspergillus  nigar) And other fungi belonging to the genus Aspergillus, silkworms (Bombyx  mori), Cultured cells, animal cells such as COS cells or CHO cells, or plant cells.
As the host, yeast of the genus Saccharomyces can be preferably exemplified. More specifically,S.  cerevisiaeYPH499 (ATCC76625, MATa, ura3-52, lys2-801, ade2-101, trp1-delta63, his3-delta200, leu2-delta1), YPH500 (ATCC76626, MATLAB, ura3-52, lys2-801, ade2-1101, trp1-delta63, his3-delta200, leu2-delta1), A451 (ATCC200509, MATalpha, can1, leu2, trp1, ura3, aro7), W303-1A (ATCC208353, MATalpha, leu2-3, leu2-112, his3-11, ade2-1, ura3-1, trp1-1, can1-100) or W303-1B MATa, leu2 3, leu2-112, his3-11, ade2-1, ura3-1, trp1-1, can1-100; haploid of W303 and (ATCC201239)), especially preferred examples.
In introducing the phosphatase gene according to the present invention into a host cell, a recombinant nucleic acid suitable for the host can be used in various aspects as described above. More specifically, several established methods such as Hanahan's method can be used as a method for introducing a foreign gene into E. coli. As a method for introducing a foreign gene into yeast, several established methods such as a lithium method can be used. In the introduction of the phosphatase gene, it can be incorporated into the chromosome of the host cell or can be retained in the cell as a plasmid or the like.
Method for producing prenyl alcohol
The method for producing prenyl alcohol according to the present invention is advantageous in improving the productivity of prenyl alcohol as a whole, but the productivity of prenyl alcohol having 10 or more carbon atoms, particularly prenyl alcohol having 15 or more carbon atoms, is particularly remarkable. When generally improving the productivity of prenyl alcohol having 15 or more carbon atoms, particularly FOH and GGOH, in the method for producing prenyl alcohol, the method of the fifteenth invention is particularly effective. The methods of the thirteenth and nineteenth inventions also improve prenyl alcohol productivity.
The method for producing prenyl alcohol according to the present invention is advantageous in that industrially useful trans-type prenyl alcohol can be produced. Here, the “trans type” means a trans type in a prenyl alcohol having 5 carbon atoms and an all trans type in a prenyl alcohol having 10 or more carbon atoms.
Generally, it is recognized in the production method of the target product using a recombinant, but also in the prenyl alcohol production method of the present invention, the selection of the transgene, the selection of the host to be introduced, the introduction of the expression vector, etc. Factors such as selection of the means and construction method of the polynucleotide suitable for it, selection of the type or concentration of the medium or its additive, selection of recombinant culture conditions or growth conditions, and the like affect the production of prenyl alcohol. There is a case.
When collecting prenyl alcohol from a culture, collect prenyl alcohol in general, for example, prenyl alcohol having 15 or more carbon atoms or one or two or more of them. Whether to selectively collect prenyl alcohol of more than one species can be arbitrarily determined depending on the purpose of carrying out the prenyl alcohol production method.
The prenyl alcohol produced can be extracted not only from a cell disruption usually performed, but also directly from a culture solution using an appropriate organic solvent. Depending on the type of host used, including when yeast is used as the host, at least part of the prenyl alcohol may remain in the host cell or on the cell surface, but the cell membrane or cell wall may be disrupted, or non-polar such as hexane or pentane. It can be easily extracted through various known operations such as extraction with an organic solvent.
Example
Host, vector, etc.
S.  cerevisiae=E.  coliPRS404, pRS405, pRS406 (Stratagene), pYES2 (Invitrogen) and pAUR101 (Takara) were used as the shuttle vectors. When introducing and cloning a DNA fragment into a pT7Blue vector or pCR2.1-TOPO vector,E.  coli  JM109 competent cells (Takara) were used as the host. When a DNA fragment is introduced into a pRS vector or pALTER vector and cloned,E.  coli  SURE2 supercompetent cells (Stratagene) orE.  coli  HB101 competent cells (Takara) were used as the host.
Hosts for gene transfer includeS.  cerevisiaeYPH499, A451, and W303-1B were used.
S.  cerevisiaeWhen cloning a gene fromS.  cerevisiae  The cDNA library “Quick-Clone cDNA” (Clontec) derived from DBY746 was used. In addition, according to the protocol attached to the yeast genomic DNA preparation kit “Gentori-kun” (Takara),S.  cerevisiae  Genomic DNA was prepared from YPH499.
E.  coliFor preparation of plasmid DNA from the above, Wizard PureFection Plasmid DNA Purification System (Promega) was used.
PCR
Gene cloning by PCR was performed under the following conditions unless otherwise specified. 5 μL of 10 × ExTaq buffer (Takara), 4 μL of 2.5 mM dNTP mixture, 1 μL of 5 U / μL ExTaq (Takara), 10 pmol of primer 1 (FW primer, the same below), 10 pmol of primer 2 (RV primer, same below) , And a 50 μL solution containing 0.5 ng of cDNA or genomic DNA was prepared, and PCR was performed as follows.
94 ° C-45 seconds, 55 ° C-1 min, 72 ° C-2 min; 30 cycles.
Insertion of CYC1 transcription terminator into pRS vector
The CYC1t fragment, which is a CYC1 transcription terminator, was prepared by PCR. PCR primer having the nucleotide sequence shown in SEQ ID NO: 13 (XhoI-Tcyc1FW), PCR primer having the nucleotide sequence shown in SEQ ID NO: 14 (KpnI-Tcyc1RV), PCR primer having the nucleotide sequence shown in SEQ ID NO: 15 (ApaI-Tcyc1RV) Was used by the combination of the XhoI-Tcyc1FW and KpnI-Tcyc1RV and the combination of the XhoI-Tcyc1FW and ApaI-Tcyc1RV, and pYES2 was used as a template. As a reaction solution, 0.1 μg pYES2, 50 pmol primer DNA, 1 × pfu buffer with MgSO₄A 50 μL solution containing (Promega), 10 nmol dNTP, 1.5 U Pfu DNA polymerase (Promega), and 1 μL perfect match polymerase enhancer (Stratagene) was prepared, and PCR was performed as follows.
95 ° C-2 minutes
95 ° C-45 seconds, 60 ° C-30 seconds, 72 ° C-1 minutes; 30 cycles
72 ° C-5 minutes, 4 ° C stock
The two amplified DNAs were cleaved using restriction enzymes XhoI and KpnI or XhoI and ApaI, respectively, and a 260 bp DNA fragment was purified by agarose gel electrophoresis. These DNA fragments are called CYC1t-XK and CYC1tXA.
CYC1t-XK was inserted into the XhoI-KpnI cleavage site of pRS405, and CYC1tXA was inserted into the XhoI-ApaI cleavage sites of pRS404 and pRS406. These are called pRS405Tcyc, pRS404Tcyc, and pRS406Tcyc, respectively.
Preparation of transcription promoter
S.  cerevisiaeA DNA fragment containing the TDH3 transcription promoter (TDH3p) and the TEF2 transcription promoter (TEF2p) was prepared by PCR using the genomic DNA as a template. As PCR primers, a primer having the base sequence shown in SEQ ID NO: 16 (SacI-Ptdh3FW), a primer having the base sequence shown in SEQ ID NO: 17 (SacII-Ptdh3RV), and a primer having the base sequence shown in SEQ ID NO: 18 (SacI- Ptef2FW) and a primer (SacII-Ptef2RV) having the base sequence shown in SEQ ID NO: 19 were used. For the amplification of TDH3p, SacI-Ptdh3FW and SacII-Ptdh3RV were used as primers, and for the amplification of TEF2p, SacI-Ptef2FW and SacII-Ptef2RV were used as primers. As a moldS.  cerevisiaeGenomic DNA was used.
As a reaction solution, 0.46 μgS.  cerevisiaeA 100 μL solution containing genomic DNA, 100 pmol DNA, 1 × ExTaq buffer (Takara), 20 nmol dNTP, 0.5 U ExTaq DNA polymerase (Takara), 1 μL perfect match polymerase enhancer was prepared as follows.
95 ° C-2 minutes
95 ° C-45 seconds, 60 ° C-1 min, 72 ° C-2 min; 30 cycles
72 ° C-4 minutes, 4 ° C stock
The amplified DNA was cleaved with restriction enzymes SacI and SacII, and 680 bp and 400 bp DNA fragments were purified by agarose gel electrophoresis. These are called TDH3p and TEF2p.
Preparation of 2μ DNA replication initiation region
After cutting the YEp vector pYES2 with restriction enzymes SspI and NheI, a 1.5 kbp fragment containing 2 μDNA replication origin (2 μori) was purified by agarose gel electrophoresis and blunt-ended with Klenow enzyme. This DNA fragment is called 2 μOriSN.
Preparation of YEp type expression vector
pRS404Tcyc, pRS405Tcyc, pRS406Tcyc was inserted into NaeI cleavage site treated with BAP (bacterial alkaline phosphatase, Takara), and 2 μOriSN was inserted,E.  coli  After introduction into SURE2 and transformation, plasmid DNA was prepared. These were cleaved with restriction enzymes DraIII and EcoRI, HpaI, or PstI and PvuII, and then subjected to agarose gel electrophoresis to check the presence and orientation of 2 μori. In the prepared pRS404Tcyc, pRS405Tcyc, and pRS406Tcyc, plasmids in which 2 μori was inserted in the same direction as pYES2 were called pRS434Tcyc 2 μOri and pRS435Tcyc 2 μOri, pRS436Tcyc 2 μOri and pRS405Tc2 Call.
The DNA fragment of T34 was obtained by inserting the T34 gene from T34 with the transcription of the T34 gene into the Tac3p and TEF2p containing the transcription promoter from the T34 gene containing the transcription promoter T34 from the T34 gene. However, pRS435GAP was obtained from pRS435Tcyc 2 μOri, pRS436GAP was obtained from pRS436Tcyc 2 μOri, and pRS445GAP was obtained from RS445Tcyc 2 μOri. The YEp type expression vectors derived from the pRS vectors prepared here are collectively referred to as pRS expression vectors.
Subcloning of hydroxymethylglutaryl CoA reductase gene HMG1 and preparation of expression vector
In PCR method,S.  cerevisiaeUsing the cDNA ofS.  cerevisiae  An about 3.2 kbp fragment of the HMG1 gene was amplified. The PCR primer is a primer having the base sequence shown in SEQ ID NO: 20 and a primer having the base sequence shown in SEQ ID NO: 21. The PCR fragment was purified by agarose gel electrophoresis and then cloned into pT7Blue-T by T / A ligation. The prepared plasmid is called pT7HMG1.
The base sequence was determined by 373A DNA sequencer (Perkin Elmer) using pT7HMG1 as a template. PCR error was detected. The notation method of these PCR errors is, for example, “c203t”, in the base sequence registered in SGD (Saccharomyces Genome Database, http://genome-www.stanford.edu/Saccharomyces). It is shown that the 203rd base “c” is a base “t” due to an error when a is the first base. In other cases, the same notation is used. Among these PCR errors, for c203t, t1820c, and a2726g, substitution mutations of S68F, L607S, and H909R were introduced into the encoded amino acid residues, respectively. The notation of these substitution mutations is, for example, “S68F”. In the amino acid sequence based on the base sequence registered in the SGD, the 68th amino acid residue “S” is changed to “F” due to an error. Indicates that In other cases, the same notation is used. The other PCR error was silent mutation with no change in the encoded amino acid residue.
The amino acid substitution mutation due to the PCR error was corrected as follows by site-directed mutation (site-directed mutation). Site-specific mutagenesis was performed by the method described in Promegas and applications guide, third edition, 1996 Promega, ISBN 1-882274-57-1 published by Promega. pT7HMG1 was cleaved with restriction enzymes SmaI, ApaL1, and SalI, and a 3.2 kbp HMG1 fragment was prepared by agarose gel electrophoresis. These fragments were inserted into the SmaI-SalI cleavage site of pALTER-1 to prepare pALHMG1. After pALHMG1 was alkali-denatured, three types of HMG1 (190-216), HMG1 (1807 to 1833) and HMG1 (2713-2739) were used as mutative oligos, Amp Repair oligo (Promega), Knocko as a repair oligo. Annealing Knock Out oligo (Promega)E.  coli  Introduced into ES1301 (Promega). Then, the transformant holding the plasmid introduced with the site-specific mutation was accumulated and cultured in a medium containing 125 μg / mL ampicillin to prepare plasmid DNA. The nucleotide sequence of the above-mentioned mutagenic oligo HMG1 (190-216) is SEQ ID NO: 22, the nucleotide sequence of HMG1 (1807-1833) is SEQ ID NO: 23, and the nucleotide sequence of HMG1 (2713-2939) is SEQ ID NO: 24. Each is shown.
When the nucleotide sequence of the plasmid DNA thus obtained was determined by 373A DNA sequencer, the three errors c203t, t1820c, and a2726g were corrected to the sequences registered in SGD. This plasmid whose sequence in HMG1 is corrected is called pALHMG106.
After pALHMG106 was cleaved with restriction enzymes SmaI and SalI, a 3.2 kbp HMG1 gene fragment was purified by agarose gel electrophoresis. This was inserted into the SmaI-SalI cleavage site of pRS434GAP and pRS434TE. The physical map of the subcloned plasmid was checked by mapping each restriction enzyme of XhoI, SpeI, NaeI and SphI and confirming the base sequence of the border region of the inserted 3.2 kbp HMG1 gene fragment. Then, plasmids that could be produced as planned were selected. The selected plasmids are called pRS434GAP-HMG1 and pRS434TEF-HMG1, respectively.
Construction of pYHMG044 expression vector
An expression vector for a gene in which a part of the HMG1 coding region was deleted was prepared by the following method. A BamHI-SalI fragment containing HMG1 was prepared from pT7HMG1 and inserted into the BamHI-XhoI cleavage site of pYES2 to prepare an expression vector pYES-HMG1. Using a PCR primer (primer HMG1 (1191-1165)) having the base sequence shown in SEQ ID NO: 25 and a PCR primer (primer HMG1 (1267-1293)) having the base sequence shown in SEQ ID NO: 26, pYES-HMG1 A PCR method using a template was performed. The PCR amplification product is made blunt with Klenow enzyme, then recirculated by self-ligation,E.coli  After introduction into JM109 and transformation, plasmid DNA was prepared. It was confirmed by 373A DNA sequencer that the reading frames of the upstream and downstream amino acids of HMG1 in the obtained plasmid DNA were not shifted and that amino acid substitution due to PCR error did not occur in the vicinity of the binding site. The prepared plasmid is called pYHMG044.
Cloning of isopentenyl diphosphate Δ-isomerase gene IDI1 and preparation of expression vector
By PCR methodS.cerevisiae  Using a cDNA library as a template,S.cerevisiaeAn about 0.9 kbp fragment of the IDI1 gene was amplified. The PCR primer is a primer having the base sequence shown in SEQ ID NO: 27 and a primer having the base sequence shown in SEQ ID NO: 28. The PCR fragment was purified by agarose gel electrophoresis and then cloned into pT7Blue-T by T / A ligation. IDI1 was inserted in the opposite direction to lacZ in pT7Blue-T. When the nucleotide sequence of the cloned fragment was determined and compared with the sequence of SGD, there was no PCR error. The prepared plasmid DNA is called pT7IDI1.
A 0.9 kbp fragment excised by BamHI-SalI from pT7IDI1 was prepared and inserted into the BamHI-SalI cleavage site of pRS435GAP. A plasmid obtained by subcloning IDI1 and selected as a result of NcoI and BamHI recognition site mapping was selected. The selected plasmid is called pRS435GAP-IDI1.
Cloning of geranylgeranyl diphosphate synthase gene BTS1 and preparation of expression vector
By PCR methodS.  cerevisiae  Using a cDNA library as a template,S.  cerevisiaeA BTS1 gene of about 1.0 kbp fragment was amplified. The PCR primer is a primer having the base sequence shown in SEQ ID NO: 29 and a primer having the base sequence shown in SEQ ID NO: 30. The PCR fragment was purified by agarose gel electrophoresis and then cloned into pT7Blue-T by T / A ligation. When the nucleotide sequence of the cloned fragment was determined and compared with the sequence of SGD, there was no PCR error. The prepared plasmid DNA is called pT7BTS1.
A 1.0 kbp fragment cut by BamHI-SalI was prepared from pT7BTS1 and inserted into the BamHI-SalI cleavage site of pYES2. The prepared plasmid is called pYESGGPS.
Next, pYESGGPS was cleaved with restriction enzymes BamHI and MluI, and a 1.0 kbp fragment was purified by agarose gel electrophoresis. This was inserted into the BamHI-MluI cleavage site of pRS435GAP and pRS445GAP. These plasmids are called pRS435GAP-BTS1 and pRS445GAP-BTS1.
Cloning of gene DPP1 and preparation of expression vector
By PCR methodS.  cerevisiaeUsing genomic DNA as a template,S.  cerevisiaeThe gene DPP1 of about 0.9 kbp fragment was amplified. The PCR primers are a primer having the base sequence shown in SEQ ID NO: 31 and a primer having the base sequence shown in SEQ ID NO: 32. The PCR fragment was purified by agarose gel electrophoresis and then cloned into pCR2.1-TOPO by T / A ligation. When the nucleotide sequence of the cloned fragment was determined and compared with the sequence of SGD, there was no PCR error. The prepared plasmid DNA is called pCR-DPP1.
A 0.9 kbp fragment excised by SacII-XhoI from pCR-DPP1 was prepared and inserted into the SacII-XhoI cleavage sites of pRS434GAP and pRS436GAP. The prepared plasmids are called pRS434GAP-DPP1 and pRS436GAP-DPP1.
Cloning of gene LPP1 and construction of pRS expression vector
By PCR methodS.  cerevisiaeUsing genomic DNA as a template,S.  cerevisiaeThe gene LPP1 of about 0.9 kbp fragment was amplified. The PCR primers are a primer having the base sequence shown in SEQ ID NO: 33 and a primer having the base sequence shown in SEQ ID NO: 34. The PCR fragment was purified by agarose gel electrophoresis and then cloned into pCR2.1-TOPO by T / A ligation. When the nucleotide sequence of the cloned fragment was determined and compared with the sequence of SGD, there was no PCR error. The prepared plasmid DNA is called pCR-LPP1.
A 0.9 kbp fragment excised from pCR-LPP1 by SacII-XhoI was prepared and inserted into the SacII-XhoI cleavage sites of pRS434GAP and pRS436GAP. The prepared plasmids are called pRS434GAP-LPP1 and pRS436GAP-LPP1.
Cloning of gene Dri42 and preparation of expression vector
By PCR method, rat (R.  norvegicus) Gene Dri42 about 0.9 kbp fragment was amplified. The PCR primer is a primer having the base sequence shown in SEQ ID NO: 35 and a primer having the base sequence shown in SEQ ID NO: 36. As a template DNA, a rat liver cDNA library was prepared from PolyA + RNA (CLONTECH) derived from rat liver using Marathon cDNA Amplification Kit (CLONTECH). The PCR fragment was purified by agarose gel electrophoresis and then cloned into pCR2.1-TOPO by T / A ligation. When the nucleotide sequence of the cloned fragment was determined and compared with the GenBank sequence, there was no PCR error. The prepared plasmid DNA is called pCR-Di42.
A 0.9 kbp fragment excised by BamHI-SalI from pCR-Dri42 was prepared and inserted into the BamHI-SalI cleavage site of pRS434GAP and pRS436GAP. The prepared plasmids are called pRS434GAP-Dri42 and pRS436GAP-Dri42.
Cloning of gene PAP2 and preparation of expression vector
In PCR method,R.  norvegicusAn about 0.9 kbp fragment of the gene PAP2 was amplified. The PCR primer is a primer having the base sequence shown in SEQ ID NO: 37 and a primer having the base sequence shown in SEQ ID NO: 38. As a template DNA, a rat liver cDNA library was prepared from the above-mentioned PolyA + RNA (CLONTECH) using a Marathon cDNA Amplification Kit (CLONTECH) and used. The PCR fragment was purified by agarose gel electrophoresis and then cloned into pCR2.1-TOPO by T / A ligation. When the nucleotide sequence of the cloned fragment was determined and compared with the sequence of GenBank, there was no PCR error. The prepared plasmid DNA is called pCR-PAP2.
A 0.9 kbp fragment excised from pCR-PAP2 by BamHI-EcoRI was prepared and inserted into the BamHI-EcoRI cleavage site of pRS434GAP and pRS436GAP. The prepared plasmids are called pRS434GAP-PAP2 and pRS436GAP-PAP2.
Cloning of gene AtPAP1 and preparation of expression vector
By PCR methodA.  thalianaAn about 0.9 kbp fragment of the gene AtPAP1 was amplified. The PCR primer is a primer having the base sequence shown in SEQ ID NO: 39 and a primer having the base sequence shown in SEQ ID NO: 40. As template DNA,A.  thaliana  40 μL of cDNA Uni-ZAP XR Library (STRATAGENE) was heat-treated at 100 ° C. for 10 minutes, phenol extracted, chloroform extracted and ethanol precipitated, and dissolved in 10 μL of TE buffer. The obtained PCR fragment was purified by agarose gel electrophoresis and then cloned into pCR2.1-TOPO by T / A ligation. The prepared plasmid DNA is called pCR-AtPAP1. When the nucleotide sequence of cloned AtPAP1 was determined, it was as shown in SEQ ID NO: 1.
A 0.9 kbp fragment excised by BamHI-XhoI from pCR-AtPAP1 was prepared and inserted into the BamHI-XhoI cleavage site of pRS434GAP and pRS436GAP. The prepared plasmids are called pRS434GAP-AtPAP1 and pRS436GAP-AtPAP1.
Cloning of gene AtPAP2 and construction of expression vector
By PCR methodA.  thalianaAn about 0.9 kbp fragment of the gene AtPAP2 was amplified. The PCR primer is a primer having the base sequence shown in SEQ ID NO: 41 and a primer having the base sequence shown in SEQ ID NO: 42. As template DNA,A.  thaliana  40 μL of cDNA Uni-ZAP XR Library was heat-treated at 100 ° C. for 10 minutes, phenol extracted, chloroform extracted and ethanol precipitated, and dissolved in 10 μL of TE buffer. The obtained PCR fragment was purified by agarose gel electrophoresis and then cloned into pCR2.1-TOPO by T / A ligation. The prepared plasmid DNA is called pCR-AtPAP2. When the base sequence of the cloned AtPAP2 was determined, it was as shown in SEQ ID NO: 2.
A 0.9 kbp fragment excised by BamHI-XhoI from pCR-AtPAP2 was prepared and inserted into the BamHI-XhoI cleavage sites of pRS434GAP and pRS436GAP. The prepared plasmids are called pRS434GAP-AtPAP2 and pRS436GAP-AtPAP2.
Production of recombinant yeast
Using Frozen EZ yeast transformation kit (Zymo Research)S.  cereviaeThe transformation was performed. For transformation, 1 μg of plasmid DNA was used. The transformed cells were transformed into an SD selective plate medium [1.7 g / l Yeast Nitrogen Base without amino acid (Difco), 20 g / l glucose (Wako), 0.77 g / l Complete Supplement Mixture (Bio 101) 20 / L Agar (Wako) from which amino acids and / or nucleotides corresponding to the selection marker gene are removed] and the grown colonies are further streaked into another SD selective plate medium to purify the recombinant. This was used for the subsequent experiments.
Recombinant yeast culture
TransformedS.  cereviaeIs selected from SD selection medium [1.7 g / l Yeast Nitrogen Base with amino acid (Difco), 20 g / l glucose, 0.77 g / l Complete Supplement Mixture (Bio 101) according to the marker gene] Pre-cultured at 30 ° C. for 2 days without amino acids and / or nucleotides, pH 7]. Then, 25 μL of the preculture solution was added to 2.5 mL of YM medium (0.3% Yeast extract, 0.3% malt extract, 0.5% peptone, 1.0% glucose, pH 7) or SG selection medium (SD selection) Medium in which the glucose component of the medium is replaced with galactose), and 130 r. p. m. Were cultured by rotary shaking culture. However, before adding the preculture solution to the SG selective medium, the cells were washed with physiological saline so that no glucose component was introduced. When YPH499 was used as a host, adenine hemisulfate was added to the medium so as to be 40 μg / mL.
Extraction of prenyl alcohol from culture broth
Remove 50 μL from the recombinant yeast culture solution and use it as a 30-fold dilution.₆₀₀Was measured. To the remaining culture solution, 2.5 mL of methanol was added and mixed. Thereafter, about 5 mL of pentane was further added, and the mixture was vigorously stirred with a vortex mixer for 15 seconds and allowed to stand. Then, the pentane layer was placed in a new glass test tube, and pentane was vaporized in a draft to concentrate the solute component. Then, 10 μL of 1.0 mL / L undecanol was added as an internal standard substance to prepare a sample for GC / MS.
GC / MS analysis
The pentane extracted fraction was separated, identified and quantified using HP6890 / 5973 GC / MS system (Hewlett-Packard, Wilmington, DE). The column used is HP-5MS (0.25 mm × 30 m, film thickness 0.25 μm), and the analysis conditions are as follows.
Temperature conditions: inlet temperature 250 ° C, detector temperature 260 ° C
MS zone temperature: MS Quad 150 ° C, MS source 230 ° C, mass scan range 35-200.
Injection conditions: automatic injection mode, sample volume 2 μL, split ratio 1/20, carrier gas; helium 1.0 mL / min, solvent delay 2 minutes.
Temperature rise setting: 115 ° C 90 seconds-raised to 250 ° C (70 ° C / minute)-250 ° C 2 minutes-raised to 300 ° C (70 ° C / minute)-300 ° C 7 minutes. Post time 0.
Standard substance: internal standard; 0.01 μL 1-undecanol. Reference materials; (E) -nerolidol (Eisai), (all-E) -farnesol (Sigma), (all-E) -geranygeraniol (Eisai), squalane (Tokyo Kasei Kogyo).
Expression of phosphatase activity
pRS436GAP-DPP1, pRS436GAP-LPP1, pRS436GAP-Di42, pRS436GAP-PAP2, pRS436GAP-AtPAP1 and pRS436GAP-AtPAP2 vectors, respectivelyS.  cerevisiae  It was introduced into A451 and transformed. The resulting recombinants are each OD with 5 mL of SD selective medium.₆₀₀Was cultured until about 1 and the cells were collected by centrifugation. The cells were washed twice with physiological saline, and 300 μL of extraction buffer (50 mM Tris-HCl (pH 7.5), 10 mM β-mercaptoethanol, 2 mM EDTA, 1 mM PMSF, 10 μg / L aprotinin, 10 μg / L leupeptin, 1 μg / L) suspended in pepstatin). Then, an equal amount of glass beads (φ = 0.45-0.5 mm) was added, and the cells were crushed by vigorous stirring (4 ° C.) for 15 minutes with a vortex mixer. Further, 300 μL of extraction buffer was added, and unbroken cells were removed by centrifugation (1,000 g, 5 minutes). The supernatant was used as a crude enzyme fraction for enzyme activity measurement.
The measurement of GGPP phosphatase activity (GGPPase activity) was performed using 5 μM GGPP (2.1 kBq [³A suitable amount of the crude enzyme fraction was added to a reaction buffer (final concentration 0.1 M citrate (pH 6.0), 5 mM EDTA) containing H] GGPP, and the reaction was performed at 37 ° C. for 30 minutes (total) 100 μL). After the reaction, 100 μL of an alcohol KOH solution (90% ethanol: 40% KOH (1: 1)) was added to stop the reaction. And after adding 500 microliters of n-hexane, it stirred vigorously with the vortex mixer for 1 minute, and produced | generated [³H] GGOH was extracted into the hexane layer. 300 μL of this hexane layer was collected and the radioactivity was measured with a liquid scintillation counter. A calibration curve was prepared using 0 to 500 mU / mL alkaline phosphatase (calf intestine, Boehringer), and the radioactivity determined above was converted into an enzyme activity value (Unit). The protein concentration in the crude enzyme fraction was measured using BSA as a standard protein with Bio-Rad Protein Assay (Bio-Rad), and the GGPPase specific activity (U / mg protein) in the crude enzyme solution was determined. Table 1 shows the relative activities when the GGPPase specific activity of the wild strain is 1. The phosphatase gene-introduced strain increased 2.4-34.8 times the specific activity of GGPPase compared to the wild-type strain, so that it was confirmed that GGPPase activity was expressed by introducing the phosphatase gene.
[Table 1]

Substrate specificity of phosphatase
The transformant obtained in the section “Expression of phosphatase activity” is ODed with 500 mL of SD selective medium.₆₀₀Was cultured until about 1 and the cells were collected by centrifugation. From the obtained bacterial cell pellet, Wu W.W. I. The purified phosphatase was partially purified with reference to their method ((1998) J. Biol. Chem. 271 (4), 1868-1876). Using partially purified enzyme, the substrate specificity of phosphatase was examined by the following method.
5 μM GGPP (2.1 kBq [³H] containing GGPP) and a reaction buffer containing a partially purified enzyme [final concentration 0.1 M citrate (pH 6.0), 5 mM EDTA], 100 μM ATP, 100 μM glucose 6-phosphate (G6P) as additional substrates other than GGPP ), 100 μM sodium pyrophosphate (NaPP), 100 μM β-glycerophosphate (β-GP), 100 μM p-nitrophenyl phosphate (pNPP), 100 μM MVP, 100 μM MVPP, 100 μM IPP, 100 μM DMAPP, 100 μM GPP, 5 μM FPP or One of 5 μM GGPP was added and the reaction was performed at 37 ° C. for 30 minutes. Then, 100 μL alcohol KOH solution was added to stop the reaction, 500 μL of hexane was added, and then vigorously stirred for 1 minute in a vortex mixer.³H] GGOH was extracted. 300 μL of this hexane layer was collected and the radioactivity was measured with a liquid scintillation counter to determine the GGPPase specific activity. [³H] The relative activities when no additional substrate other than GGPP was added (+ none) and the GGPPase specific activity was taken as 100% are summarized in Table 2.
[Table 2]

From the results shown in Table 2, even if a phosphate compound (ATP, G6P, NaPP, β-GP or pNPP) not related to the prenyl diphosphate biosynthetic pathway was added as an additional substrate in an amount 20 times that of GGPP, an additional substrate was added. The phosphatase activity with respect to GGPP retained 90% or more as compared with the case where it did not exist. That is, it was confirmed that the phosphatase activity for phosphate compounds not related to the prenyl diphosphate biosynthetic pathway of phosphatase used in the experiment was lower than the enzyme activity for GGPP. Next, compared with the case where prenyl phosphate precursor (MVP, MVPP), carbon number 5 IPP or DMAPP, or carbon number 10 GPP was added as an additional substrate even when 20 times the amount of GGPP was added, no additional substrate was added. Thus, the phosphatase activity against GGPP was maintained at 90% or more. That is, it was confirmed that the phosphatase activity for the prenyl phosphate precursor of phosphatase and the prenyl phosphate compound having 10 or less carbon atoms used in the experiment was lower than the phosphatase activity for GGPP. On the other hand, when cold FPP having 15 carbon atoms is added as an additional substrate in an equimolar amount (5 μM) with GGPP, [³The apparent phosphatase activity against H] GGPP was reduced by about 40-60%. This was similar to the apparent decrease in phosphatase activity when cold GGPP was added as an additional substrate in an equimolar amount (5 μM) with GGPP. That is, it was confirmed that the phosphatase activity for FPP having 15 carbon atoms and the enzyme activity for GGPP having 20 carbon atoms were equivalent to those of the phosphatase used in the experiment. From the above, it was revealed that the phosphatase used in the experiment showed the following activity. That is, in an enzyme activity measurement system using prenyl phosphate (A) having 15 or more carbon atoms as a substrate, an excessive amount (20 times in molar ratio) of a phosphate compound not related to the prenyl diphosphate biosynthesis pathway or 10 or less carbon atoms Even when the prenyl phosphate or prenyl phosphate precursor (B) is coexisted, the enzyme activity for (A) is 90% or more as compared with the case where (B) is not coexisted.
Production of prenyl alcohol by yeast.
In order to mass-produce prenyl alcohol in vivo using the phosphatase gene according to the present invention, this gene may be assembled into an appropriate vector or DNA fragment, introduced into a host, and expressed in vivo.
In addition, several enzyme genes related to prenyl diphosphate biosynthetic pathway (including mevalonate pathway and non-mevalonate pathway) are used to improve prenyl phosphate supply ability) When the phosphatase gene is introduced with the prenyl phosphate supply capability improved, higher prenyl alcohol productivity can be expected. For example, hydroxymethylglutaryl CoA (HMG-CoA) reductase is known to be the rate-limiting enzyme in the mevalonate pathway (Dimster-Denk, D. et al (1994) Mol. Biol. Cell. 5,655-665.). Moreover, the expression of the isopentenyl diphosphate isomerase (IPP isomerase) gene was enhanced.E.  coliHas been reported to increase carotenoid production (Misawa et al., JP-A-8-242861). GGPP synthase gene (eg,S.  cereviae  BTS1) is an enzyme that synthesizes GGPP, which is a precursor of GGOH. By enhancing the expression of the GGPP synthase gene, enhancement of GGPP production capability can be expected. Therefore, higher prenyl alcohol productivity can be expected by combining the introduction or expression enhancement of these genes with the introduction or expression enhancement of the phosphatase gene according to the present invention.
Production of prenyl alcohol by W303-1B introduced with phosphatase gene
pRS435GAP-BTS1S.  cerevisiae  The expression vector pRS436GAP-DPP1, pRS436GAP-LPP1, pRS436GAP-Dri42, pRS436GAP-PAP2, pRS436GAP-AtPAP1 or the expression vector incorporating the phosphatase gene according to the present invention into the recombinant obtained by introducing into W303-1B or pRS436GAP-AtPAP2 was introduced. Table 3 shows the results of measuring the GGOH productivity of each recombinant and host.
[Table 3]

No production of GGOH was detected in W303-1B, but 0.2 mg / L of GGOH was produced by introducing the GGPP synthase gene. This suggests that GGPP was produced by an increase in GGPP synthesizing fermentation activity, and this GGPP was converted to GGOH by the endogenous dephosphorylation activity in yeast. Furthermore, by introducing the phosphatase gene according to the present invention into this strain, the productivity of GGOH was improved by 5.7 to 17 times compared to the case where only the GGPP synthase gene was introduced. In other words, the introduction of the GGPP synthase gene shows some improvement in GGOH productivity, but it has been found that the introduction of the phosphatase gene according to the present invention is important in order to produce GGOH more efficiently and in large quantities.
Production of prenyl alcohol by YPH499 introduced with phosphatase gene
pRS434TEF-HMG1, pRS445GAP-BTS1, or pRS435GAP-IDI1,S.  cerevisiae  Introduced into YPH499. Separately, two types of plasmids, pRS434TEF-HMG1 and pRS445GAP-BTS1, were introduced into YPH499. Expression vectors pRS436GAP-DPP1, pRS436GAP-LPP1, pRS436GAP-Di42, pRS436GAP-PAP2, pRS436GAP-AtPAP1 or pRS436GAP-AtPAP2 into which the phosphatase gene of the present invention is further incorporated into the recombinant into which the above two plasmids are introduced. Each plasmid was introduced. Table 4 shows the measurement results of FOH and GGOH productivity of each recombinant and host.
[Table 4]

GGOH was not detected with YPH499. Although GGOH was not detected when only the IPP-Δ isomerase gene was introduced, the amount of GGOH produced increased to some extent by introducing only the HMG-CoA reductase gene (GGOH 0.05 mg / L), and the GGPP synthase gene was introduced. Also increased the production amount of GGOH to some extent (GGOH 0.2 mg / L).
A synergistic effect of improving prenyl alcohol productivity was observed by introducing two kinds of genes, HMG-CoA reductase gene and GGPP synthase gene. However, compared with that case, GGOH productivity was improved 1.3 to 5.9 times by introducing the phosphatase gene of the present invention in addition to the above two genes.
Production of prenyl alcohol by A451 introduced with phosphatase gene
A strain in which the AUR1 gene of A451 is substituted for the AUR1-C gene is referred to as AURGG101 (AUR1 :: AUR1-C). PYHMG044 was introduced into AURGG101 (referred to as 15-2), and pRS435GAP-BTS1 was further introduced into 15-2 to obtain pRS435GAP-BTS1 / 15-2. pRS434GAP-DPP1, pRS434GAP-LPP1, pRS434GAP-Dri42, pRS434GAP-PAP2, and pRS436GAP-AtPAP1 or pRS434GAP-AtPAP2 were introduced into pRS435GAP-BTS1 / 15-2. The results of measuring prenyl alcohol productivity of each recombinant and host are shown in Table 5.
[Table 5]

In A451, production of prenyl alcohol was not detected. When two kinds of genes, HMG-CoA reductase gene and GGPP synthase gene, were introduced, FOH and GGOH were produced to some extent (FOH 1.4 mg / L, GGOH 0.4 mg / L). Furthermore, when the phosphatase gene according to the present invention is introduced into this strain, productivity is improved 2.6 to 20 times with FOH and 8.8 to 31 times with GGOH, compared to the case where the above two genes are introduced. did.
This result indicates that when the HMG-CoA reductase gene or the GGPP synthase gene is introduced, the introduction of the phosphatase gene is crucial for the increase in production of FOH and GGOH.
Linkage between GGPP synthase gene BTS1 and prenyl phosphate-specific phosphatase gene DPP1
The prenyl phosphate-specific phosphatase gene DPP1 and the GGPP synthase gene BTS1 by the PCR method with reference to the method of Nikawa et al. (Nikawa, J. and Kawabata, M. (1998) Nucleic Acids Res. 26.860-861.). Of these genes (hereinafter referred to as “DPGG”).
The conditions for 1st PCR are shown below. 1XKOD buffer (TOYOBO), 0.2 mM dNTPs, 1 mM MgCl₂, 2U KOD DNA polymerase (TOYOBO), 20 pmol primer 1, 20 pmol primer 2, 60 ng YPH499 Genomic DNA-containing reaction solution was prepared, and 1st PCR was performed as follows.
98 ° C-15 seconds, 44 ° C-2 seconds, 74 ° C-30 seconds; 25 cycles
The PCR product when using S-DPP1-1 having the base sequence shown in SEQ ID NO: 43 as primer 1 and DPP-2 having the base sequence shown in SEQ ID NO: 44 as primer 2 was designated as # 1. The PCR product when using BTS-3 having the base sequence shown in SEQ ID NO: 45 as primer 1 and X-BTS-4 having the base sequence shown in SEQ ID NO: 46 as primer 2 was designated as # 2. The obtained PCR product was purified by agarose gel electrophoresis. Next, annealing and extension reaction was performed under the following conditions. 1X KOD buffer, 0.2 mM dNTPs, 1 mM MgCl₂, 0.25 U KOD DNA polymerase, 0.5 μL PCR product # 1, 0.5 μL PCR product # 2 containing 10 μL reaction solution was prepared and performed as follows.
98 ° C-15 seconds, 54 ° C-2 seconds, 74 ° C-30 seconds; 5 cycles
A part of the obtained PCR product was subjected to agarose gel electrophoresis, and it was confirmed that a ligated gene (about 1.9 kb) of PCR products # 1 and # 2 was synthesized.
Subsequently, 2nd PCR was performed under the following conditions. 1XKOD buffer (TOYOBO), 0.2 mM dNTPs, 1 mM MgCl₂, 0.5 U KOD DNA polymerase (TOYOBO), 20 pmol S-DPP-1, 20 ol X-BTS-4, 2 μL Annealing and extension reaction solution is prepared, and 2nd PCR reaction is performed as follows. It was.
98 ° C-15 seconds, 58 ° C-2 seconds, 74 ° C-30 seconds; 25 cycles
The obtained PCR product was purified by agarose gel electrophoresis (this is called SX-DPGG). SX-DPGG was digested with restriction enzymes SacII and XhoI and then inserted into the SacII-XhoI cleavage site of pRS434GAP to obtain pRS434GAP-DPGG. DNA sequencing was performed between the structural gene portion of pRS434GAP-DPGG and the vicinity of the restriction enzyme site, and it was confirmed that the nucleotide sequence was as designed. By the above method, an expression vector for the phosphatase-linked gene DPGG in which a linker sequence (5 'ggtgggtgttt 3') was introduced into the binding region between DPP1 and BTS1 was prepared.
Linkage of farnesyl diphosphate (FPP) synthase gene ERG20 and prenyl phosphate-specific phosphatase gene DPP1
In the same manner as in the case of linking BTS1 and DPP1, a gene (referred to as DPF) in which FPP synthase gene ERG20 (Gen Bank accession No. NC001142, 105006.1.106060) and prenyl phosphate-specific phosphatase gene DPP1 are linked is prepared. did.
1st PCR was performed using S-DPP13A having the base sequence shown in SEQ ID NO: 47 as primer 1 and DPP-2 as primer 2, and PCR product # 3 was obtained. Further, 1st PCR was carried out using DPF3 having the base sequence shown in SEQ ID NO: 48 as primer 1 and X-FPS4 having the base sequence shown in SEQ ID NO: 49 as primer 2, to obtain PCR product # 4. These PCR products were purified by agarose gel electrophoresis.
Annealing and extension reaction was performed using PCR products # 3 and # 4. A part of the obtained PCR product was subjected to agarose gel electrophoresis, and the ligated gene of PCR products # 3 and # 4 (about 1.9 kb) was obtained. It was confirmed that it was synthesized. 2nd PCR was performed using Annealing and extension reaction solution, S-DPP13A and X-FPS4, and the obtained PCR product was purified by agarose gel electrophoresis (this is referred to as SX-DPF). SX-DPF was digested with restriction enzymes SacII and XhoI and then inserted into the SacII-XhoI cleavage sites of pRS434GAP and pRS435GAP to obtain pRS434GAP-DPF and pRS435GAP-DPF, respectively. DNA sequencing was performed between the structural gene portion of pRS434GAP-DPF and pRS435GAP-DPF and the vicinity of the restriction enzyme site, and it was confirmed that the nucleotide sequence was as designed. By the above method, an expression vector for the phosphatase-linked gene DPF in which a linker sequence (5 ′ ggtgggtggttct 3 ′) was introduced into the binding region between DPP1 and ERG20 was prepared.
Expression of GGPPase activity of YPH499 introduced with phosphatase linking genes DPF and DPGG
When DPF is expressed, a fusion enzyme of phosphatase encoded by DPP1 and farnesyl diphosphate synthase encoded by ERG20 is expressed. When DPGG is expressed, phosphatase encoded by DPP1 and geranylgeranyl diphosphate encoded by BTS1 are expressed. It is thought that a fusion enzyme with a synthetic enzyme is expressed. Therefore, it was investigated whether or not GGPPase activity was expressed even when the linked genes DPF and DPGG were expressed.
PRS434GAP-DPP1, pRS434GAP-DPF and pRS434GAP-DPGG, respectivelyS.  cerevisiae  Transformation into YPH499 was carried out, and the GGPPase specific activity of the obtained recombinant was measured in the same manner as described in the above section “Expression of phosphatase activity”. The results are shown in Table 6.
[Table 6]

The GGPPase specific activity of the strain expressing DPP1 alone was about 10 times that of the wild strain. On the other hand, the GGPPase specific activity of the strain expressing DPF and DPGG was about 4 times that of the wild strain. That is, in the strain expressing DPF and DPGG, although the expression level of GGPPase activity was small compared to the case where DPP1 was expressed alone, the GGPPase specific activity was significantly increased compared to the wild strain. From this result, it was revealed that GGPPase activity was expressed even when the linked genes DPF and DPGG were expressed.
Prenyl alcohol production by A451 introduced with phosphatase linking genes DPF and DPGG
PRS434GAP-HMG1, which is an expression vector for the HMG-CoA reductase gene HMG1, was introduced into A451 (this is referred to as AH1), and pRS435GAP-DPF was further introduced into AH1. The recombinants thus obtained, A451 and AH1 were cultured in YM medium, and FOH productivity was measured. The results are shown in Table 7.
[Table 7]

In A451, production of FOH was not detected. AH1 expressing HMG1 produced FOH to some extent (FOH 0.3 mg / L). When the phosphatase-linked gene DPF was introduced into AH1, the productivity of FOH was improved 11.3 times compared to AH1 (FOH 3.4 mg / L). From this result, introduction of a linked gene that links the phosphatase gene and the gene of an enzyme involved in the prenyl diphosphate biosynthetic pathway (especially the prenyl diphosphate synthase gene) is effective in improving the productivity of prenyl alcohol. I found out.
Prenyl alcohol production by AURGG101 introduced with phosphatase linking genes DPF and DPGG
PRS434GAP-DPF and pRS434GAP-DPGG were introduced into the aforementioned 15-2, respectively. Separately, two types of plasmids, pRS435GAP-BTS1 and pRS434GAP-DPP1, were introduced into 15-2. Table 8 shows the results obtained by culturing each recombinant and host in an SG selective medium and measuring the prenyl alcohol productivity.
[Table 8]

In A451, the production of prenyl alcohol was not detected, and the amount of prenyl alcohol produced by AURGG101 was very small. In 15-2 in which the HMG044 gene was introduced into AURGG101, FOH and GGOH were produced to some extent (FOH 8.5 mg / L, GGOH 2.2 mg / L). When the phosphatase-linked gene DPF was introduced into 15-2, productivity was improved by 7.8 times for FOH and 2.6 times for GGOH compared to 15-2 (FOH 66.7 mg / L, GGOH 5.7 mg / L). Next, when two types of genes, GGPP synthase gene BTS1 and prenyl phosphate-specific phosphatase gene DPP1, were incorporated into 15-2 and introduced into separate plasmids, the productivity of FOH and GGOH was higher than that of 15-2. Improved (FOH 28.8 mg / L, GGOH 13.7 mg / L). On the other hand, when the ligated gene DPGG was introduced into 15-2, productivity was improved 2.4 times for FOH and 5.5 times for GGOH, compared to the case where BTS1 and DPP1 were introduced on separate plasmids. (FOH 68.7 mg / L, GGOH 75.5 mg / L).
From this result, the introduction of a linked gene that links the phosphatase gene and the gene of the enzyme involved in the prenyl diphosphate biosynthetic pathway (especially the prenyl diphosphate synthase gene) greatly improves the productivity of prenyl alcohol. It turns out to be effective.
Jar culture
A strain (pRS434GAP-DPGG / 15-2) in which the linking gene DPGG was introduced into 15-2 was cultured in a 5 L jar fermenter (MSJ-U2W; Maruhishi Bioengineer), and its productivity was measured. It was decided to further confirm the effect of improving the productivity by introducing DPGG. The medium was prepared by mixing 50 g galactose (Nacalai), 100 g pactopeptone (DIFCO), 50 g yeast extract (DIFCO), 50 ml soybean oil (Nacalai), 5 ml Adecanol LG109, and tap water 51 (sterilized with jar fermenter ( 121 ° C., 20 minutes) and used for culture.
The jar culture conditions are as follows. Amount of inoculation 2%, temperature 33 ° C., aeration rate 1 VVM, stirring speed 300 rpm, galactose feed rate = 3.4 g / L, pH 7.0 (controlled with 4N NaOH). The results of quantifying prenyl alcohol production by collecting the culture solution over time are shown in FIG.
At 134 hours after culturing, the production amounts of FOH and GGOH reached the maximum, which were 0.38 g / L and 0.24 g / L, respectively.
Cloning of acid phosphatase gene PHO3 and alkaline phosphatase gene PHO8 and construction of expression vector
Cloning of a gene encoding a phosphatase having no substrate specificity (non-specific) in hydrolysis activity for a phosphate ester compound and preparation of an expression vector were carried out by the following methods. As non-specific phosphataseS.  cerevisiaeAcid phosphatase gene PHO3 (GenBank accession No. NC001134, complement (427657.42929060)) and alkaline phosphatase gene PHO8 (GenBank accession No. NC001136, complement (1418537..14020237)) were used.
By PCR methodS.  cerevisiae  Using YPH499 genomic DNA as a template,S.  cerevisiaeAn approximately 1.4 kbp fragment of the gene PHO3 was amplified. The PCR primers are a primer having the base sequence shown in SEQ ID NO: 50 and a primer having the base sequence shown in SEQ ID NO: 51. The PCR fragment was purified by agarose gel electrophoresis and then cloned into pCR2.1-TOPO by T / A ligation. When the nucleotide sequence of the cloned fragment was determined and compared with the sequence of SGD, there was no PCR error. The prepared plasmid DNA is called pCR-PHO3. A 1.4 kbp fragment excised by SacII-XhoI from pCR-PHO3 was prepared and inserted into the SacII-XhoI cleavage site of pRS434GAP. The prepared plasmid is called pRS434GAP-PHO3.
By PCR methodS.  cerevisiae  Using YPH499 genomic DNA as a template,S.  cerevisiaeThe gene PHO8 fragment of about 1.7 kbp was amplified. The PCR primer is a primer having the base sequence shown in SEQ ID NO: 52 and a primer having the base sequence shown in SEQ ID NO: 53. The PCR fragment was purified by agarose gel electrophoresis and then cloned into pCR2.1-TOPO by T / A ligation. When the nucleotide sequence of the cloned fragment was determined and compared with the sequence of SGD, there was no PCR error. The prepared plasmid DNA is called pCR-PHO8. A 1.7 kbp fragment excised from pCR-PHO8 by SacII-SmaI was prepared and inserted into the SacII-SmaI cleavage site of pRS434GAP. The prepared plasmid is called pRS434GAP-PHO8.
Measurement of non-specific phosphatase activity
Acid phosphatase activity was measured as follows with reference to the method of Ferminan et al. (E. Ferminan, et al. (1997) Microbiol. 143, 2615-2625). pRS434GAP-PHO3 was introduced into 15-2, the obtained recombinant and host were cultured in 5 mL of SD selective medium for 1 day, and the cells were collected by centrifugation. The cells were washed twice with physiological saline and suspended in 300 μL of extraction buffer (10 mM citrate (pH 4.3), 10 mM β-mercaptoethanol, 1 mM PMSF, 10 μg / L aprotinin, 10 μg / L leupeptin, 1 μg / L pepstatin). did. Then, an equal amount of glass beads (φ = 0.45-0.5 mm) was added, and the cells were crushed by vigorous stirring (4 ° C.) for 15 minutes with a vortex mixer. Further, 300 μL of extraction buffer was added and centrifuged (18,000 × g, 15 minutes, 4 ° C.), and the supernatant was collected to obtain a crude enzyme fraction. To 100 μL of the crude enzyme fraction appropriately diluted with 10 mM citrate (pH 4.3), 100 μL of p-nitrophenyl phosphate (pNPP) solution (10 mM pNPP, 200 mM citrate (pH 4.3)) was added, and 30 ° C. The reaction was carried out for 15 minutes. 50 μL of 1 M NaOH aqueous solution was added to stop the reaction, and the absorbance at 405 nm was measured. A calibration curve was prepared using 0-0.03 U / mL acid phosphatase (Roche Diagnostics), and the absorbance at 405 nm determined above was converted to an enzyme activity value (Unit). The protein concentration in the crude enzyme fraction was measured by Bio-Rad Protein Assay using BSA as a standard protein, and the acid phosphatase specific activity (U / mg protein) in the crude enzyme fraction was determined. Further, the GGPPase specific activity of the crude enzyme fraction obtained here is the same as the enzyme activity measurement method described in the above section “Expression of phosphatase activity” using 120 mM citrate (pH 4.3) as a reaction buffer. Measured by the method.
Alkaline phosphatase activity was measured as follows with reference to the method of Dhamij et al. (SS Dhamija, et al. (1987) Current Genetics, 11, 467-473). pRS434GAP-PHO8 was introduced into 15-2, the obtained recombinant and host were cultured in 5 mL of SD selective medium for 1 day, and the cells were collected by centrifugation. The cells were washed twice with physiological saline, and 300 μL of extraction buffer (100 mM Tris · HCl (pH 8.5), 1 mM MgCl₂10 mM β-mercaptoethanol, 1 mM PMSF, 10 μg / L aprotinin, 10 μg / L leupeptin, 1 μg / L pepstatin). Then, an equal amount of glass beads (φ = 0.45-0.5 mm) was added, and the cells were crushed by vigorous stirring (4 ° C.) for 15 minutes with a vortex mixer. Further, 300 μL of extraction buffer was added, and the soluble fraction (supernatant) was collected by centrifugation (18,000 × g, 15 minutes, 4 ° C.) to obtain a crude enzyme fraction. To 100 μL of a crude enzyme fraction appropriately diluted with 100 mM Tris · HCl (pH 8.5), 100 μL of p-nitrophenyl phosphate (pNPP) solution (10 mM pNPP, 100 mM Tris · HCl (pH 8.5)) was added. The reaction was carried out at 30 ° C for 15 minutes. 50 μL of 1 M NaOH aqueous solution was added to stop the reaction, and the absorbance at 405 nm was measured. A calibration curve was prepared using 0 to 0.3 U / mL alkaline phosphatase (calf intestine, Roche Diagnostics), and the absorbance at 405 nm determined above was converted to an enzyme activity value (Unit). The alkaline phosphatase specific activity (U / mg protein) was determined in the same manner as in the case of acid phosphatase. Further, the GGPPase specific activity of the crude enzyme fraction obtained here was 60 mM Tris.HCl (pH 8.5), 1 mM MgCl as a reaction buffer.₂Was measured by the same method as the enzyme activity measurement method described in the above section “Expression of phosphatase activity”.
The non-specific phosphatase specific activity (acid phosphatase specific activity and alkaline phosphatase specific activity) measured by the above method is hereinafter referred to as pNPPase specific activity.
Expression of 15-2 phosphatase activity and prenyl alcohol production by introducing non-specific phosphatase gene
The pNPPase specific activity, GGPPase specific activity and prenyl alcohol productivity (cultured in SG selective medium) of 15-2 into which expression vectors incorporating the acid phosphatase gene PHO3 and the alkaline phosphatase gene PHO8 were respectively measured were measured. The results are shown in Table 9. Table 9 shows the relative values with the host as 1. In the section of GGPPase specific activity in the table, the column of + pNPP indicates the GGPPase specific activity when 100 μM pNPP is added as an additional substrate other than GGPP, as described in the above-mentioned “Substrate specificity of phosphatase” section. -PNPP column indicates values obtained by measuring GGPase specific activity without adding pNPP.
[Table 9]

With the introduction of the acid phosphatase gene or alkaline phosphatase gene, the pNPPase specific activity increased 28-35 times compared to the host. The GGPPase specific activity increased 3-6 times compared to the host when no pNPP was added as an additional substrate. However, when pNPP is added as an additional substrate,³The apparent phosphatase activity against H] GGPP was zero. This indicates that the phosphatase expressed by the introduction of the acid phosphatase gene and the alkaline phosphatase gene is a phosphatase with low substrate specificity that reacts with both GGPP and pNPP. This result is in contrast to the case where a substrate-specific phosphatase gene is introduced into prenyl phosphate having 15 or more carbon atoms (see Table 2). On the other hand, the productivity of prenyl alcohol was not improved even when an acid phosphatase gene or an alkaline phosphatase gene was introduced. From the above results, it was found that even when a non-specific phosphatase gene was introduced into a host to increase pNPPase activity or GGPPase activity, there was no effect on the improvement of prenyl alcohol productivity.
Example evaluation
The above examples are evaluated as follows.
That is, by introducing six types of genes encoding phosphatase having substrate specificity for prenyl phosphate having 15 or more carbon atoms into FOH and GGOH-producing yeast, respectively, when any of the six types of genes is introduced, GGPPase Specific activity increased. Furthermore, it was shown that the productivity of FOH and GGOH was actually improved several to several tens of times when any of the six genes was introduced.
The present inventors have found that the phosphatase motif shown in FIG. 1 is contained in the amino acid sequence of phosphatase that can improve the productivity of FOH and GGOH. It is speculated that this phosphatase motif is related to substrate specificity for prenyl phosphate having 15 or more carbon atoms.
Furthermore, a gene obtained by linking a gene encoding a phosphatase having a substrate specificity to prenyl phosphate having 15 or more carbon atoms and a gene encoding an enzyme involved in the prenyl diphosphate biosynthesis pathway is obtained by using FOH and GGOH-producing yeast. By introducing it into the product, the productivity of FOH and GGOH was further improved. It was also shown that when the ligated gene was introduced, the productivity of FOH and GGOH was improved several times as compared with the case where the ligated gene was introduced and incorporated on separate plasmids.
On the other hand, when a gene (PHO3, PHO8) encoding phosphatase having no substrate specificity (PHO3, PHO8) having no substrate specificity in the hydrolysis activity for the phosphate ester compound was introduced into FOH and GGOH-producing yeast, the specific activity of GPPase increased However, the productivity of FOH and GGOH was not improved. Non-specific phosphatase is thought to react with any phosphate compound in the microbial cell due to lack of substrate specificity. Therefore, it will also react with phosphate ester compounds other than prenyl phosphate in the cells (for example, ATP which is considered to be present in large amounts in the cells). And since the number of the non-specific phosphatases which can react with prenyl phosphate substantially and can produce prenyl alcohol will be very limited, it is estimated that the productivity of prenyl alcohol was not improved.
Moreover, as a result of examining the amino acid sequences of these non-specific phosphatases in detail, it was revealed that the phosphatase motif as shown in FIG. 1 was not included. This indicates that introduction or expression enhancement of a phosphatase gene having a substrate specificity for a prenyl phosphate having 15 or more carbon atoms and containing the phosphatase motif is crucial for improving the productivity of prenyl alcohol. .
Industrial application fields
As described above, the present invention enables efficient mass production of prenyl alcohol, particularly prenyl alcohol having a biologically active all-trans form and having 15 or more carbon atoms.
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the conserved sequence of each polypeptide according to the present invention.
FIG. 2 is a diagram showing the quantitative results of prenyl alcohol production.

Claims (12)

A method for producing prenyl alcohol, comprising culturing a host cell into which the phosphatase gene defined in any of (3) to (6) below is introduced or enhancing expression thereof, and collecting prenyl alcohol from the culture.
(3) It has the base sequence described in any one of SEQ ID NOs: 7, 8, and 10-12.
(4) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of any one of SEQ ID NOs: 7, 8, and 10-12 or a complementary nucleotide sequence thereof , and phosphoric acid In the hydrolysis activity with respect to the ester compound, when the specific activity with respect to (A) shown in the enzyme activity measurement system using only (A) as a substrate with respect to prenyl phosphate (A) having 15 or more carbon atoms is defined as ( It encodes a polypeptide exhibiting a substrate specificity that the specific activity relative to (A) is 80 or more in the presence of A) and a 20-fold amount of prenyl phosphate having a carbon number of 10 or less in molar ratio.
(5) encodes a polypeptide having the amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 4-6.
(6) 5 or less amino acids in the amino acid sequence shown in any of SEQ ID NO 1,2,4～6 is substitution, deletion or addition in the amino acid sequence, and hydrolytic activity towards phosphoric acid ester compound When the specific activity with respect to (A) shown in the enzyme activity measurement system using only (A) as a substrate is defined as 100 for prenyl phosphate (A) having 15 or more carbon atoms, (A) and molar ratio It encodes a polypeptide exhibiting substrate specificity that the specific activity relative to (A) is 80 or more in the presence of 20 times the amount of prenyl phosphate having 10 or less carbon atoms. A host cell into which the phosphatase gene defined in any of the following (3) to (6) and the gene of an enzyme involved in the prenyl diphosphate biosynthesis pathway are introduced or enhanced is cultured, and prenyl alcohol is obtained from the culture. A method for producing prenyl alcohol to be collected.
(3) It has the base sequence described in any one of SEQ ID NOs: 7, 8, and 10-12.
(4) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of any one of SEQ ID NOs: 7, 8, and 10-12 or a complementary nucleotide sequence thereof , and phosphoric acid In the hydrolysis activity with respect to the ester compound, when the specific activity with respect to (A) shown in the enzyme activity measurement system using only (A) as a substrate with respect to prenyl phosphate (A) having 15 or more carbon atoms is defined as ( It encodes a polypeptide exhibiting a substrate specificity that the specific activity relative to (A) is 80 or more in the presence of A) and a 20-fold amount of prenyl phosphate having a carbon number of 10 or less in molar ratio.
(5) encodes a polypeptide having the amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 4-6.
(6) 5 or less amino acids in the amino acid sequence shown in any of SEQ ID NO 1,2,4～6 is substitution, deletion or addition in the amino acid sequence, and hydrolytic activity towards phosphoric acid ester compound When the specific activity with respect to (A) shown in the enzyme activity measurement system using only (A) as a substrate is defined as 100 for prenyl phosphate (A) having 15 or more carbon atoms, (A) and molar ratio It encodes a polypeptide exhibiting substrate specificity that the specific activity relative to (A) is 80 or more in the presence of 20 times the amount of prenyl phosphate having 10 or less carbon atoms.   The method for producing prenyl alcohol according to claim 2, wherein the phosphatase gene and the gene of an enzyme involved in the prenyl diphosphate biosynthesis pathway are introduced or enhanced in expression as a linked gene. The method for producing prenyl alcohol according to claim 2, wherein the gene of the enzyme involved in the prenyl diphosphate biosynthesis pathway is the following gene (1) and / or (2).
(1) At least one gene selected from a farnesyl diphosphate synthase gene and a geranylgeranyl diphosphate synthase gene.
(2) Acetyl CoA synthase gene, acetyl CoA-acetyltransferase gene, hydroxymethylglutaryl CoA synthase gene, hydroxymethylglutaryl CoA reductase gene, mevalonate kinase gene, mevalonate phosphate kinase gene, mevalonate diphosphate From acid decarboxylase gene, isopentenyl diphosphate isomerase gene, deoxyxylulose phosphate reductoisomerase gene, deoxyxylulose phosphate synthase gene, MEP cytidylyltransferase gene, CDP-ME kinase gene and MECDP synthase gene At least one gene selected. The method for producing prenyl alcohol according to claim 3, wherein the gene of the enzyme involved in the prenyl diphosphate biosynthesis pathway is the following gene (1) and / or (2).
(1) At least one gene selected from a farnesyl diphosphate synthase gene and a geranylgeranyl diphosphate synthase gene.
(2) Acetyl CoA synthase gene, acetyl CoA-acetyltransferase gene, hydroxymethylglutaryl CoA synthase gene, hydroxymethylglutaryl CoA reductase gene, mevalonate kinase gene, mevalonate phosphate kinase gene, mevalonate diphosphate From acid decarboxylase gene, isopentenyl diphosphate isomerase gene, deoxyxylulose phosphate reductoisomerase gene, deoxyxylulose phosphate synthase gene, MEP cytidylyltransferase gene, CDP-ME kinase gene and MECDP synthase gene At least one gene selected. Specific activity for (A) shown in the enzyme activity measurement system using only (A) as a substrate for prenyl phosphate (A) having 15 or more carbon atoms in the hydrolysis activity of the phosphatase gene for phosphate compounds If the set to 100, shows the substrate specificity say is (a) and specific activity of 80 or more with respect to (a) in the case of the 20-fold amount Purenirurin acid having 10 or less carbon atoms are allowed to coexist in a molar ratio 請 Motomeko 1 production method of prenyl alcohol according to claim 5.   The prenyl phosphate is farnesyl phosphate, geranylgeranyl phosphate, geranyl farnesyl phosphate, hexaprenyl phosphate, heptaprenyl phosphate, octaprenyl phosphate, nonaprenyl phosphate, decaprenyl phosphate, undecaprenyl phosphate or dodecaprenyl phosphate The method for producing prenyl alcohol according to claim 6, which is one kind or two or more kinds. The method for producing prenyl alcohol according to any one of claims 1 to 5, wherein the host cell is any of the following host cells.
(11) Any of fungi (Eumycetes), ascomycetes (Ascomycetes), and unicellular eukaryotes.
(12) Yeast.
(13) Yeast of the genus Saccharomycetes.
(14) Saccharomyces cerevisiae YPH499 strain, YPH500 strain, A451 strain, W303-1A strain, W303-1B strain or a strain derived therefrom.   The said prenyl alcohol is a C15 or more thing, The manufacturing method of the prenyl alcohol in any one of Claims 1-5. The prenyl alcohol farnesol, geranylgeraniol, galley Nirufa Rene sole, hexamethylene prenyl alcohol, hepta prenyl alcohol, octaprenyl alcohol, nonaprenyl alcohol, decaprenyl alcohol, one or at which claims more undecaprenyl alcohol or dodecamethylene prenyl alcohol Item 10. A method for producing prenyl alcohol according to Item 9 .   The method for producing prenyl alcohol according to any one of claims 1 to 5, wherein the prenyl alcohol is an all-trans type. The method for producing prenyl alcohol according to claim 9 , wherein the prenyl alcohol is an all-trans type.

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