JP2003088368A - Method for producing prenyl alcohol - Google Patents

Method for producing prenyl alcohol

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Publication number
JP2003088368A
JP2003088368A JP2001282978A JP2001282978A JP2003088368A JP 2003088368 A JP2003088368 A JP 2003088368A JP 2001282978 A JP2001282978 A JP 2001282978A JP 2001282978 A JP2001282978 A JP 2001282978A JP 2003088368 A JP2003088368 A JP 2003088368A
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JP
Japan
Prior art keywords
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ala
ser
val
ile
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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JP2001282978A
Other languages
Japanese (ja)
Inventor
Toku Ooto
徳 大音
Atsuo Obata
充生 小畑
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Toyota Motor Corp
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Toyota Motor Corp
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Filing date
Publication date
Application filed by Toyota Motor Corp filed Critical Toyota Motor Corp
Priority to JP2001282978A priority Critical patent/JP2003088368A/en
Priority to CA002433534A priority patent/CA2433534A1/en
Priority to EP01272515A priority patent/EP1354956A4/en
Priority to US10/450,941 priority patent/US20040063182A1/en
Priority to PCT/JP2001/011215 priority patent/WO2002053747A1/en
Publication of JP2003088368A publication Critical patent/JP2003088368A/en
Priority to US12/060,434 priority patent/US8759046B2/en
Pending legal-status Critical Current

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing prenyl alcohol. SOLUTION: This method for producing prenyl alcohol is to culture mutant cells mutated so as to reduce the amount of a transcript of squalene synthetase gene having translational activity and collect prenyl alcohol from the culture products.

Description

【発明の詳細な説明】Detailed Description of the Invention

【０００１】[0001]

【発明の属する技術分野】本発明は、プレニルアルコー
ルの製造方法に関する。TECHNICAL FIELD The present invention relates to a method for producing prenyl alcohol.

【０００２】[0002]

【従来の技術】生体内でのテルペノイド（イソプレノイ
ド）合成は、アリル性二リン酸基質にイソペンテニル二
リン酸（isopentenyl diphosphate）(IPP, C5)を順次縮
合することによって直鎖プレニル二リン酸であるゲラニ
ル二リン酸（geranyl diphosphate (GPP, C10 )）、フ
ァルネシル二リン酸（farnesyl diphosphate (FPP,
C15)）、ゲラニルゲラニル二リン酸（geranylgeranyl d
iphosphate (GGPP, C20)）が生合成されるところから始
まる（図1）。図1中で、枠で囲まれた文字は酵素を表
し、hmgRはヒドロキシメチルグルタリルCo-A（hydroxym
ethyl glutaryl-CoA：HMG-CoA）還元酵素、GGPSはGGPP
合成酵素、FPSはFPP合成酵素を示す。2. Description of the Related Art Terpenoid (isoprenoid) synthesis in vivo is performed by linear condensation of linear prenyl diphosphate by sequentially condensing isopentenyl diphosphate (IPP, C 5 ) on an allyl diphosphate substrate. Geranyl diphosphate (GPP, C 10 ), farnesyl diphosphate (FPP,
C 15 )), geranylgeranyl diphosphate (geranylgeranyl d
It starts from the biosynthesis of iphosphate (GGPP, C 20 )) (Fig. 1). In Figure 1, the letters in boxes represent enzymes, and hmgR is hydroxymethylglutaryl Co-A (hydroxym
ethyl glutaryl-CoA: HMG-CoA) reductase, GGPS is GGPP
Synthetic enzyme, FPS means FPP synthase.

【０００３】プレニル二リン酸のなかでも、FPPは最も
重要な生合成中間体であり、膨大な種類のテルペノイド
類、例えば、エルゴステロール（プロビタミンD2）を含
むステロイド類、キノン（ビタミンK, VK）の側鎖、セ
スキテルペン類、スクアレン（SQ）、ファルネシル化タ
ンパク質のアンカー分子、ドリコール、バクトプレノー
ル、天然ゴムなどの合成前駆体である。Among prenyl diphosphates, FPP is the most important biosynthetic intermediate, and a huge variety of terpenoids such as steroids containing ergosterol (provitamin D 2 ) and quinone (vitamin K, VK) side chains, sesquiterpenes, squalene (SQ), farnesylated protein anchor molecules, dolichol, bactoprenol, natural rubber and other synthetic precursors.

【０００４】GGPPもまた、生体内では重要な生合成中間
体であり、フィトエン、リコペン、フィカプレノール、
レチノール（ビタミンA, VA）、β-カロテン（プロビタ
ミンA）、フィロキノン（ビタミンK1, VK1）、トコフェ
ロール類（ビタミンE, VE）、ゲラニルゲラニル化タン
パク質のアンカー分子、葉緑素の側鎖、ジベレリン、ア
ーキアのエーテル型脂質などを始めとする化合物の生合
成に必須である。GGPP is also an important biosynthetic intermediate in the living body, and includes phytoene, lycopene, ficaprenol,
Retinol (vitamin A, VA), β-carotene (provitamin A), phylloquinone (vitamin K 1 , VK 1 ), tocopherols (vitamin E, VE), geranylgeranylated protein anchor molecule, chlorophyll side chain, gibberellin, It is essential for the biosynthesis of compounds such as Archaeal ether lipids.

【０００５】これら炭素数20までのプレニル二リン酸は
生体内ではtrans型（（E）型）に縮合し、（E,E）-FP
P、(E,E,E)-GGPPであることが知られており、これら炭
素数15又は20までの全trans型((all-E)型)由来の幾何異
性を有するプレニル二リン酸又はプレニル基を前駆体と
して生理活性のある化合物の合成が行われる（K. Ogura
and T. Koyama, (1998) Chemical Reviews, 98, 1263-1
276; IUPAC-IUB JointCommission on Biochemical Nome
nclature (JCBN) Prenol nomenclature, Recommendatio
ns 1986, (http: //www. chem. qmw. ac. uk /iupac /m
isc /prenol. html)）。炭素数20又は15以下のプレニル
二リン酸でcis型（（Z）型）幾何異性を持つ唯一の例外
は、ネロールに代表されるモノテルペノイドの前駆体と
して知られる炭素数10のネリル二リン酸であるが、この
物質は、アリル性二リン酸基質としてジメチルアリル二
リン酸（DMAPP; 3,3-dimethylallyl diphosphate）にIP
Pが縮合して合成されるのか、炭素数10のtrans型((E)
型)幾何異性体であるゲラニル二リン酸（GPP）の異性化
によって合成されるのか明らかになっていない。cis型
（（Z）型）に縮合合成して合成されるイソプレノイ
ド、例えば、ドリコール、バクトプレノール（ウンデカ
プレノール）、フィカプレノール又は天然ゴムも、アリ
ル性プライマー基質として（E,E）-FPP又は(E,E,E)-GGP
Pから合成される（K. Ogura and T. Koyama, (1998) Ch
emical Reviews, 98, 1263-1276; IUPAC-IUB Joint Com
mission on Biochemical Nomenclature (JCBN) Prenol
nomenclature, Recommendations 1986, (http: //www.
chem. qmw. ac. uk /iupac /misc/prenol. html)）。[0005] These prenyl diphosphates having up to 20 carbon atoms are condensed to trans type (( E ) type) in the living body to give ( E, E ) -FP.
P, ( E, E, E ) -GGPP is known, and prenyl diphosphate having a geometric isomer derived from all trans type (( all-E ) type) having 15 or 20 carbon atoms or Synthesis of bioactive compounds using the prenyl group as a precursor (K. Ogura
and T. Koyama, (1998) Chemical Reviews, 98, 1263-1
276; IUPAC-IUB Joint Commission on Biochemical Nome
nclature (JCBN) Prenol nomenclature, Recommendatio
ns 1986, (http: // www. chem. qmw. ac. uk / iupac / m
isc /prenol.html)). The only exception to prenyl diphosphates having 20 or 15 carbon atoms and having cis- type (( Z ) -type) geometric isomerism is the neryl diphosphate having 10 carbon atoms, which is known as a precursor of monoterpenoids represented by nerol. However, this substance has an IP for dimethylallyl diphosphate (DMAPP; 3,3-dimethylallyl diphosphate) as an allyl diphosphate substrate.
Is it synthesized by condensation of P, trans type with 10 carbons (( E )
It is not clear whether it is synthesized by isomerization of the (type) geometric isomer geranyl diphosphate (GPP). Isoprenoids, such as dolichol, bactoprenol (undecaprenol), ficaprenol, or natural rubber, which are synthesized by condensation synthesis to the cis type (( Z ) type), also serve as allylic primer substrates ( E, E ). -FPP or ( E, E, E ) -GGP
Synthesized from P (K. Ogura and T. Koyama, (1998) Ch
emical Reviews, 98, 1263-1276; IUPAC-IUB Joint Com
mission on Biochemical Nomenclature (JCBN) Prenol
nomenclature, Recommendations 1986, (http: // www.
chem.qmw.ac.uk/iupac/misc/prenol.html)).

【０００６】(E,E)-FPPのアルコール誘導体である(E,E)
-ファルネソール(farnesol)(FOH, C 15)、その三級アル
コールの異性体である(E)-ネロリドール(nerolidol)(NO
H, C1 5)、又は(E,E,E)-GGPPのアルコール誘導体である
(E,E,E)-ゲラニルゲラニオール（geranylgeraniol)((E,
E,E)-GGOH, C20)等は、香料として用いられる精油中の
芳香物質として知られるが、薬理作用物質として有用な
上記ビタミン類をはじめとする化合物の合成出発物質と
してもまた重要な物質である（図1）。[0006]E, E) -Alcohol derivative of FPP (E, E)
-Farnesol (FOH, C 15), The third grade Al
Is an isomer of Cole (E) -Nero idol (nerolidol) (NO
H, C1 Five), Or (E, E, E) -Alcohol derivative of GGPP
(E, E, E) -Geranylgeraniol ((E,
E, E) -GGOH, C20), Etc. in the essential oil used as a fragrance
Known as an aroma substance, but useful as a pharmacological agent
Starting materials for the synthesis of compounds such as the above vitamins
However, it is still an important substance (Fig. 1).

【０００７】IPPの生体内での合成は、すべてメバロン
酸経路（アセチル補酵素A（acetyl-CoA）からメバロン
酸を経てIPPを合成する経路）によると考えられてきた
が、1980年代末より、M. Rohmerらがバクテリアを用い
て新しいIPP合成経路を明らかにした。これは、非メバ
ロン酸経路、DXP (1-deoxy-D-xylulose 5-phosphate)経
路、MEP （2-C-methyl-D-erythritol 4-phosphate）経
路又はRohmer経路と呼ばれており、グリセルアルデヒド
3-リン酸とピルビン酸から1-deoxy-D-xylulose 5-phosp
hateを経てIPPを合成する経路である。つまり、現在で
はIPP合成経路としてメバロン酸経路と非メバロン酸経
路の二つの主要経路が知られていることになる。[0007] It has been thought that all the in-vivo synthesis of IPP is based on the mevalonate pathway (a pathway for synthesizing IPP from acetyl coenzyme A (acetyl-CoA) via mevalonate), but since the end of the 1980s, M. Rohmer et al. Have revealed a new IPP synthetic pathway using bacteria. This is called the non-mevalonate pathway, the DXP (1-deoxy-D-xylulose 5-phosphate) pathway, the MEP (2- C- methyl-D-erythritol 4-phosphate) pathway, or the Rohmer pathway. aldehyde
3-phosphate and pyruvic acid from 1-deoxy-D-xylulose 5-phosp
It is a route to synthesize IPP via hate. In other words, the two major IPP synthetic pathways are now known, the mevalonate pathway and the non-mevalonate pathway.

【０００８】FOHやNOHは、精油等の天然物から少量調製
される以外には、現在化学合成法により合成されてい
る。また、GGOHも化学合成法により合成されている（特
開平8-133999号公報）。化学合成法で合成されるFOH、N
OH又はGGOHは、一般的には炭素骨格が同じであるが、二
重結合が(E)-型(trans型)と(Z)-型(cis型)の混合物とし
て得られる。(all-E)-型である(E,E)-FOH、(E)-NOH又は
(E,E,E)-GGOHは生物の代謝経路で合成される幾何異性を
持つ化合物であり、工業的利用価値を有する。(E,E)-FO
H、(E)-NOH又は(E,E,E)-GGOHを純粋な形で得るために
は、カラムクロマトグラフィーや精密蒸留などを利用し
た精製が必要である。しかし、熱的に不安定なアリルア
ルコール(allylalcohol)であるFOHとその異性体であるN
OHの精密蒸留、あるいはGGOHの精密蒸留は困難である。
また、カラムクロマトグラフィーによる精製は、多量の
溶媒充填剤を必要とし、順次溶出してくる各画分を分析
しながら回収し、さらに溶媒を除去しなければならない
など、操作が煩雑でコストも高く、工業実施には適さな
い。実際、実験用試薬として市販されている(E,E)-FO
H、(E,E,E)-GGOHは非常に高価である。そこで、(E)-、
(Z)-幾何異性体の生成制御や反応産物の繰り返し構造な
どの特徴から、(E,E)-FOH（以下「FOH」と記す）、(E)-
NOH（以下「NOH」と記す）、(E,E,E)-GGOH（以下「GGO
H」と記す）などの、cis-,trans-((Z)-, (E)-)異性体混
合物でないいわゆる活性型プレニルアルコールの純品を
大量に生産できる系の確立が望まれている。FOH and NOH are currently synthesized by a chemical synthesis method, except that they are prepared in small amounts from natural products such as essential oils. GGOH is also synthesized by a chemical synthesis method (Japanese Patent Laid-Open No. 8-133999). FOH and N synthesized by chemical synthesis method
OH or GGOH generally has the same carbon skeleton, but the double bond is obtained as a mixture of ( E ) -type ( trans type) and ( Z ) -type ( cis type). ( E, E ) -FOH, which is ( all-E ) -type, ( E ) -NOH or
( E, E, E ) -GGOH is a compound having geometrical isomerism that is synthesized by the metabolic pathway of organisms and has industrial utility value. ( E, E ) -FO
In order to obtain H, ( E ) -NOH or ( E, E, E ) -GGOH in a pure form, purification using column chromatography or precision distillation is necessary. However, the thermally labile allyl alcohol FOH and its isomer N
Precision distillation of OH or GGOH is difficult.
Further, the purification by column chromatography requires a large amount of a solvent filler, and it is necessary to collect and analyze the fractions that are sequentially eluted while further removing the solvent, which makes the operation complicated and the cost is high. , Not suitable for industrial implementation. In fact, it is commercially available as a laboratory reagent ( E, E ) -FO
H, ( E, E, E ) -GGOH is very expensive. So ( E )-,
(Z) - from features such as repeating structure of generating the control and reaction products of geometric isomers, (hereinafter referred to as "FOH") (E, E) -FOH, (E) -
NOH (hereinafter referred to as "NOH"), ( E, E, E ) -GGOH (hereinafter "GGO"
It is desired to establish a system capable of mass-producing a pure product of so-called activated prenyl alcohol which is not a mixture of cis- , trans -(( Z )-, ( E )-) isomers, such as "H").

【０００９】FOH、NOHやGGOHの生体内での合成基質は、
例えば出芽酵母であるサッカロマイセス・セレビシアエ
(Saccharomyces cerevisiae)の細胞内ではメバロン酸経
路を経て供給されるが、そのキー酵素と考えられるHMG-
CoA還元酵素を利用しても、FOHやGGOHに比べはるかに安
価に市販されている物質であるスクアレンの蓄積量が上
がることしか知られていない(特開平5-192184号公報；
Donald et al. (1997)Appl. Environ. Microbiol. 63,
3341-3344)。また、スクアレン合成酵素遺伝子ERG9の変
異導入によりスクアレン合成酵素遺伝子欠損株（ATCC64
031）を作製し、かつ、ステロール取り込み能を獲得し
た特別な出芽酵母を培養すると、培養液1リットルあた
り1.3mgのFOHを蓄積することが知られている (Chambon
et al.,(1990) Curr. Genet., 18, 41-46)。ATCC64031
は、我々がスクアレン合成酵素遺伝子ERG9の塩基配列決
定を行ったところ、ERG9遺伝子コード領域に置換変異が
導入された結果スクアレン合成酵素遺伝子欠損株である
erg9株になり、その結果1.3mg/lのFOH生産性を獲得して
いることが確認された。ATCC64031のスクアレン合成酵
素遺伝子コード領域に第745ヌクレオチドのCがTに、第7
97ヌクレオチドのTがGに2箇所置換変異しており、結果
としてコードするポリペプチドの第249アミノ酸残基のG
lnが終止コドンに（Q249STOP）、第266アミノ酸残基のI
leがArgに（I266R）置換変異し、コードするポリペプチ
ドの第249アミノ酸残基以降が欠失した酵素活性の無い
変異型スクアレン合成酵素を発現していたことがわかっ
ている。しかし、通常の株でERG9を欠損させても、生育
に必須なエルゴステロールが合成できず、かつ、通常培
養条件では外部からステロール類を取りこむ機能も無い
ため致死性となり、ERG9欠損株が得られず、FOH生産系
を構築することができない。また、(E)-NOH（以下「NO
H」と記す）の生合成法は知られていない。また、たと
え致死性を回避する新たな形質を付与できたとしても培
地中にエルゴステロール添加が必要になるなどの工業生
産には不利な条件が生じる。In vivo synthetic substrates for FOH, NOH and GGOH are
For example, Saccharomyces cerevisiae, a budding yeast
( Saccharomyces cerevisiae ) is supplied via the mevalonate pathway in the cell of HMG-, which is considered to be the key enzyme.
The use of CoA reductase is known only to increase the accumulated amount of squalene, which is a substance that is commercially available at a much lower cost than FOH and GGOH (JP-A-5-192184;
Donald et al . (1997) Appl. Environ. Microbiol. 63,
3341-3344). Also, squalene synthase gene-deficient strain (ATCC64 by mutagenesis of squalene synthase gene ERG9
It is known that 1.3 mg of FOH is accumulated per 1 liter of the culture medium when a special S. cerevisiae, which has acquired the ability to uptake sterols, is produced (C31).
et al. , (1990) Curr. Genet., 18, 41-46). ATCC64031
Is a squalene synthase gene-deficient strain as a result of the substitution mutation introduced into the ERG9 gene coding region when we sequenced the squalene synthase gene ERG9.
It was confirmed that the strain became erg9 strain, and as a result, acquired FOH productivity of 1.3 mg / l. In the squalene synthase gene coding region of ATCC64031, the C at the 745th nucleotide at the T and the 7th at the 7th nucleotide
The 97 nucleotide T is mutated in two substitutions with G, resulting in G of the 249th amino acid residue of the encoded polypeptide.
ln is the stop codon (Q249STOP), I of the 266th amino acid residue
It is known that le was mutated to Arg by (I266R) substitution mutation, and expressed a mutant squalene synthase having no enzymatic activity in which the 249th and later amino acid residues of the encoded polypeptide were deleted. However, even if ERG9 is deleted in a normal strain, ergosterol, which is essential for growth, cannot be synthesized, and under normal culture conditions, it is lethal because it does not have the function of taking in sterols from the outside, and an ERG9- deficient strain is obtained. Therefore, the FOH production system cannot be constructed. In addition, ( E ) -NOH (hereinafter "NO
H)) is not known. Further, even if a new trait that avoids lethality can be imparted, there are disadvantageous conditions for industrial production, such as the need to add ergosterol to the medium.

【００１０】GGOHの生合成に関しては、特開平9-238692
号公報において、植物細胞培養により1リットル培養液
あたり0.66-3.25mgの生産が報告されているが、工業化
には適さない高価な植物細胞培養用培地が必要であるう
え培養にも光が必要であり、従来の精油等の天然物から
のGGOH調製に比べても実用的でなく、より工業化に適し
た生合成法、例えば微生物培養による生合成法は全く知
られていない。Regarding the biosynthesis of GGOH, JP-A-9-238692
In the publication, production of 0.66-3.25 mg per 1 liter culture solution by plant cell culture is reported, but an expensive plant cell culture medium that is not suitable for industrialization is required and light is also required for culture. However, there is no known biosynthesis method that is not practical as compared with conventional preparation of GGOH from natural products such as essential oils and is more suitable for industrialization, for example, biosynthesis method by microbial culture.

【００１１】[0011]

【発明が解決しようとする課題】本発明は、翻訳活性の
あるスクアレン合成酵素遺伝子転写産物量を減少させる
ことによって、プレニルアルコールを製造するための方
法を提供することを目的とする。SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for producing prenyl alcohol by reducing the amount of squalene synthase gene transcript having translation activity.

【００１２】[0012]

【課題を解決するための手段】本発明者は、上記課題を
解決するため鋭意研究を行った結果、スクアレン合成酵
素遺伝子において翻訳活性を有する転写産物の量（「翻
訳活性のあるスクアレン合成酵素遺伝子転写産物量」と
いう）を減少させるために、スクアレン合成酵素遺伝子
の転写プロモーター領域を転写抑制型プロモーターで置
換してなる変異型細胞を転写抑制条件で培養することに
よってプレニルアルコール生産系の開発を行った。ま
た、HMG-CoA還元酵素遺伝子を代表とするIPP合成経路関
連酵素遺伝子又はそれらの変異型若しくは融合型遺伝子
を宿主細胞内で人為的に発現させる系を構築するため
に、恒常発現型又は誘導発現型転写プロモーターを含
み、各種栄養要求性マーカーを持った発現シャトルベク
ターの作製を行い、これに目的の遺伝子又はその変異型
遺伝子を組み込み、これを本発明の翻訳活性のあるスク
アレン合成酵素遺伝子転写産物量を減少できる変異型細
胞へ導入した。そして、その培養物からプレニルアルコ
ールを得、上記目的を達成することに成功し、本発明を
完成するに至った。Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has found that the amount of transcripts having a translation activity in the squalene synthase gene (“the squalene synthase gene having a translation activity”). In order to reduce the "transcript amount"), we developed a prenyl alcohol production system by culturing mutant cells in which the transcription promoter region of the squalene synthase gene was replaced with a transcription repressing promoter under transcription repressing conditions. It was In order to construct a system for artificially expressing an IPP synthesis pathway-related enzyme gene typified by the HMG-CoA reductase gene or a mutant or fusion type gene thereof in a host cell, constitutive expression type or inducible expression type is used. Of the squalene synthase gene transcription product having the translation activity of the present invention It was introduced into mutant cells that can reduce the amount. Then, prenyl alcohol was obtained from the culture, and the above objects were successfully achieved, and the present invention was completed.

【００１３】すなわち、本発明は以下の通りである。 (1)翻訳活性のあるスクアレン合成酵素遺伝子転写産物
量が減少できるように変異させた変異型細胞を培養し、
得られる培養物からプレニルアルコールを採取すること
を特徴とするプレニルアルコールの製造方法。 (2)翻訳活性のあるスクアレン合成酵素遺伝子転写産物
量が減少できるように変異させた変異型細胞に、IPP合
成経路関連酵素遺伝子を含む発現用組換えDNA又はゲノ
ムインテグレート用DNAを導入して組換え体を作製し、
該組換え体を培養し、得られる培養物からプレニルアル
コールを採取することを特徴とするプレニルアルコール
の製造方法。That is, the present invention is as follows. (1) culturing mutant cells mutated to reduce the amount of squalene synthase gene transcript with translation activity,
A method for producing prenyl alcohol, which comprises collecting prenyl alcohol from the obtained culture. (2) The recombinant cells for expression containing the IPP synthesis pathway-related enzyme gene or the DNA for genome integration are introduced into a mutant cell mutated to reduce the amount of squalene synthase gene transcript having translation activity, and assembled. Make a replacement,
A method for producing prenyl alcohol, which comprises culturing the recombinant and collecting prenyl alcohol from the resulting culture.

【００１４】(3)スクアレン合成酵素遺伝子の転写プロ
モーター領域を転写抑制型プロモーターで置換したプロ
モーター領域を含む変異型細胞を、転写抑制条件下で培
養し、翻訳活性のあるスクアレン合成酵素遺伝子転写産
物量を減少させることによって、得られる培養物からプ
レニルアルコールを採取することを特徴とするプレニル
アルコールの製造方法。 (4)スクアレン合成酵素遺伝子の転写プロモーター領域
を転写抑制型プロモーターで置換したプロモーター領域
を含み、翻訳活性のあるスクアレン合成酵素遺伝子転写
産物量を減少できるようにした変異型細胞に、IPP合成
経路関連酵素遺伝子を含む発現用組換えDNA又はゲノム
インテグレート用DNAを導入して組換え体を作製し、該
組換え体を転写抑制条件下で培養し、得られる培養物か
らプレニルアルコールを採取することを特徴とするプレ
ニルアルコールの製造方法。(3) Amount of squalene synthase gene transcript having translation activity, which is obtained by culturing mutant cells containing a promoter region in which the transcription promoter region of the squalene synthase gene is replaced with a transcription repressing promoter, under the conditions of transcription repression. A method for producing prenyl alcohol, which comprises collecting prenyl alcohol from the obtained culture by decreasing the amount of prenyl alcohol. (4) IPP synthesis pathway is related to mutant cells containing a promoter region in which the transcriptional promoter region of the squalene synthase gene is replaced with a transcription-repressing promoter, so that the amount of transcription products of the squalene synthase gene with translation activity can be reduced. To introduce a recombinant DNA for expression containing an enzyme gene or a DNA for genome integration to prepare a recombinant, culture the recombinant under transcriptional repressing conditions, and collect prenyl alcohol from the resulting culture. A method for producing prenyl alcohol, which is characterized.

【００１５】上記製造方法において、IPP合成経路関連
酵素遺伝子には、解糖系又はクエン酸回路に位置する化
合物からIPPが合成される反応にかかわる酵素遺伝子、
及び各種プレニル二リン酸合成酵素遺伝子が含まれる。
IPP合成経路関連遺伝子としてはメバロン酸経路関連酵
素遺伝子だけでなく、主に原核生物や、葉緑体などで近
年新たに見出されたIPP合成経路であるいわゆる非メバ
ロン酸経路（DXP(1-deoxy-D-xylulose 5-phosphate)経
路、MEP（2-C-methyl-D-erythritol 4-phosphate）経路
又はRohmer経路と呼ばれる場合もある）関連酵素遺伝子
も含まれる。In the above-mentioned production method, the IPP synthesis pathway-related enzyme gene is an enzyme gene involved in a reaction in which IPP is synthesized from a compound located in the glycolysis system or the citric acid cycle,
And various prenyl diphosphate synthase genes.
Not only the mevalonate pathway-related enzyme gene as an IPP synthesis pathway-related gene, but also the so-called non-mevalonate pathway (DXP (1- deoxy-D-xylulose 5-phosphate) pathway, MEP (2- C- methyl-D-erythritol 4-phosphate) pathway or Rohmer pathway) related enzyme genes are also included.

【００１６】また、上記製造方法において、転写抑制型
プロモーターとしては例えばGAL1プロモーターが挙げら
れ、GAL1プロモーター転写抑制条件としては例えばグル
コース含有培地を用いたものが挙げられ、IPP合成経路
関連酵素遺伝子として、例えば下記の(a) - (l)の遺伝
子からなる群から選ばれるいずれかの遺伝子が挙げられ
る。[0016] In the above production method, include, for example the GAL1 promoter as transcription repressor promoter, the GAL1 promoter transcriptional suppression condition include those with glucose-containing medium for example, as IPP synthesis pathway-related enzyme gene, Examples include any gene selected from the group consisting of the following genes (a)-(l).

【００１７】(a) ファルネシル二リン酸合成酵素遺伝子 (b) ゲラニルゲラニル二リン酸合成酵素遺伝子 (c) ヒドロキシメチルグルタリルCoA還元酵素遺伝子 (d) イソペンテニル二リン酸Δ-イソメラーゼ遺伝子 (e) メバロン酸キナーゼ遺伝子 (f) アセチルCoAアセチルトランスフェラーゼ遺伝子 (g) ヒドロキシメチルグルタリル-CoA合成酵素遺伝子 (h) メバロン酸リン酸キナーゼ遺伝子 (i) メバロン酸二リン酸脱炭酸酵素遺伝子 (j) 上記(a) - (i)の遺伝子の変異型遺伝子 (k) 上記(a) - (i)の遺伝子からなる群から選ばれる一
つの遺伝子又はその変異型遺伝子と、他の遺伝子又はそ
の変異型遺伝子との融合遺伝子（二つの遺伝子結合領域
に人工的な配列を挿入しても良い） (l) 上記(a) - (k)の遺伝子に、コードするポリペプチ
ドが小胞体シグナルを有するように、付加、置換又は挿
入変異を施した遺伝子(A) Farnesyl diphosphate synthase gene (b) Geranylgeranyl diphosphate synthase gene (c) Hydroxymethylglutaryl CoA reductase gene (d) Isopentenyl diphosphate Δ-isomerase gene (e) Mevalon Acid kinase gene (f) Acetyl CoA acetyltransferase gene (g) Hydroxymethylglutaryl-CoA synthase gene (h) Mevalonate phosphate kinase gene (i) Mevalonate diphosphate decarboxylase gene (j) Above (a )-(i) a mutant gene of the gene (k) (a)-(i) one gene or a mutant gene thereof selected from the group consisting of genes, and another gene or a mutant gene thereof Fusion gene (artificial sequence may be inserted into two gene binding regions) (l) Addition to the gene of (a)-(k) so that the encoding polypeptide has an endoplasmic reticulum signal, Replacement Gene which has been subjected to insertion mutation

【００１８】上記融合遺伝子中の遺伝子結合領域には、
融合前のそれぞれの遺伝子がコードするポリペプチドが
適切に機能するコンフォメーションがとれるような人工
的なアミノ酸配列をコードするヌクレオチド配列を自由
に挿入することもできる。たとえばGly Serをコードす
る5’GGGTCC3’などである。小胞体シグナルとしては、
例えば、サッカロマイセス・セレビシアエの場合C末端
に位置するHis Asp Glu Leu（配列番号30、「HDEL配
列」という）、又はAspAsp Glu Leu配列（配列番号31）
が知られ、真核生物一般では、C末端に位置するLys Asp
Glu Leu（配列番号32）が知られ、機能は全く同等であ
る小胞体保留シグナル（ER retension signal）として
働くことがわかっている（B.Lewin “Genes V” (199
4), Oxford University Press, New York, U.S.A., pp.
279-318;B. Alberts et al. “Molecular Biology of T
he Cell, third edtion” (1994), Garland Publishin
g. Inc., New York, U.S.A., §12−§13）（これらを
総称し「HDEL配列等」とする）。またこれらC末端4アミ
ノ酸残基による小胞体保留シグナル以外のシステムによ
る小胞体移行蛋白質の一部分のドメイン、たとえば、小
胞体への輸送シグナルとして機能するN末端側のシグナ
ルペプチド +H3N MetMet Ser Phe Val Ser Leu Leu Leu
Val Gly Ile Leu Phe Trp Ala Thr Glu AlaGlu Gln Le
u Thr Lys Cys Glu Val Phe Gln（配列番号33）などを
用いることもできる（B.Lewin “Genes V” (1994), Ox
ford University Press, New York, U.S.A., pp.279-31
8; B. Alberts et al. “Molecular Biology of The Ce
ll, third edtion” (1994), Garland Publishing. In
c., New York, U.S.A., §12−§13）。The gene binding region in the above fusion gene contains
It is also possible to freely insert a nucleotide sequence encoding an artificial amino acid sequence that allows the polypeptide encoded by each gene before fusion to have a conformation in which it functions properly. For example, a 5 'GGGTCC 3' encoding Gly Ser. As the endoplasmic reticulum signal,
For example, in the case of Saccharomyces cerevisiae, His Asp Glu Leu (SEQ ID NO: 30, referred to as “HDEL sequence”) located at the C-terminus, or AspAsp Glu Leu sequence (SEQ ID NO: 31)
Is known, and in eukaryotes in general, Lys Asp located at the C-terminus
Glu Leu (SEQ ID NO: 32) is known and is known to act as an ER retension signal, which has exactly the same function (B.Lewin “Genes V” (199
4), Oxford University Press, New York, USA, pp.
279-318; B. Alberts et al. “Molecular Biology of T
he Cell, third edtion ”(1994), Garland Publishin
g. Inc., New York, USA, §12-§13) (these are collectively referred to as "HDEL sequences etc."). In addition, a domain of a part of the endoplasmic reticulum translocation protein by a system other than the endoplasmic reticulum retention signal by these C-terminal 4 amino acid residues, for example, the N-terminal signal peptide that functions as a transport signal to the endoplasmic reticulum + H 3 N MetMet Ser Phe Val Ser Leu Leu Leu
Val Gly Ile Leu Phe Trp Ala Thr Glu AlaGlu Gln Le
u Thr Lys Cys Glu Val Phe Gln (SEQ ID NO: 33) can also be used (B. Lewin “Genes V” (1994), Ox
ford University Press, New York, USA, pp.279-31
8; B. Alberts et al. “Molecular Biology of The Ce
ll, third edtion ”(1994), Garland Publishing. In
c., New York, USA, §12-§13).

【００１９】遺伝子の付加、置換又は挿入変異により、
小胞体シグナルを付加、置換又は挿入したポリペプチド
は、野生型ポリペプチドにさらに4アミノ酸残基を付
加、置換又は挿入させた変異型ポリペプチドと見ること
もでき、ポリペプチドをコードする遺伝子は、野生型の
遺伝子にさらに1-12塩基程度のヌクレオチド配列を付
加、置換又は挿入した変異型遺伝子と見ることもでき
る。また、前記融合遺伝子がコードするポリペプチドも
また、野生型ポリペプチドに300アミノ酸残基以上のア
ミノ酸残基を付加、置換又は挿入させた変異型ポリペプ
チドと見ることもでき、融合遺伝子は野生型遺伝子に10
00塩基程度のヌクレオチド配列を付加、置換又は挿入さ
せた変異型遺伝子と見ることもできる。すなわち、シグ
ナルを新たに創出したアミノ酸残基をコードする遺伝子
又は融合遺伝子は、それ自体変異型遺伝子である。By the addition, substitution or insertion mutation of the gene,
A polypeptide having an endoplasmic reticulum signal added, substituted or inserted can also be seen as a mutant polypeptide in which 4 amino acid residues are further added, substituted or inserted into the wild-type polypeptide, and the gene encoding the polypeptide is It can also be considered as a mutant gene in which a nucleotide sequence of about 1-12 bases is further added, substituted or inserted into the wild-type gene. Further, the polypeptide encoded by the fusion gene can also be seen as a mutant polypeptide obtained by adding, substituting or inserting an amino acid residue of 300 amino acid residues or more into a wild-type polypeptide, and the fusion gene is a wild-type polypeptide. 10 for genes
It can also be regarded as a mutant gene in which a nucleotide sequence of about 00 bases is added, substituted or inserted. That is, the gene encoding the amino acid residue that newly created the signal or the fusion gene is itself a mutant gene.

【００２０】HDEL配列等をC末端に創出するためには置
換変異のほか、たとえば、１塩基の挿入によってフレー
ムがシフトしHDEL配列等を創出したり、2塩基の置換に
よってもともとのC末端（たとえばHDGIなど）からHDEL
配列等に変えることもできる。C末端付近にHDEL配列等
が存在していれば（例えばBTS1の場合）、ストップコド
ンを導入するだけでC末端にHDEL配列等を創出すること
もできる。さらに、C末端に新たな12ヌクレオチドを付
加してHDEL配列等をC末端に創出することもできる。In order to create an HDEL sequence or the like at the C terminus, in addition to substitution mutation, for example, the frame is shifted by the insertion of 1 base to create the HDEL sequence or the original C terminus (for example, by the replacement of 2 bases). HDGI) to HDEL
It can also be changed to an array or the like. If an HDEL sequence or the like exists near the C terminus (for example, in the case of BTS1), it is possible to create an HDEL sequence or the like at the C terminus simply by introducing a stop codon. Furthermore, a new 12 nucleotides can be added to the C-terminus to create an HDEL sequence or the like at the C-terminus.

【００２１】また、宿主細胞としては、例えば酵母、例
えば、子嚢菌類（Ascomycota）の子嚢菌酵母、担子菌類
（Basidiomycota）の担子菌酵母、又は不完全菌類（Fun
gi Imperfecti）の不完全菌酵母を用いることができ
る。好ましくは、子嚢菌酵母、特に出芽酵母であるサッ
カロマイセス・セレビシアエ、キャンディダ・ユーティ
リス(Candida utilis)又はピキア・パストリス(Pichia
pastris)等、***酵母であるシゾサッカロマイセス・ポ
ンベ(Shizosaccharomyces pombe)等が用いられる。種だ
けでなく株もプレニルアルコールを生産することができ
る限り特に限定されるものではなく、例えばサッカロマ
イセス・セレビシアエの株としては、A451、YPH499、YP
H500、W303-1A又はW303-1Bなどが挙げられる。また、プ
レニルアルコールとしては、FOH、NOH及び/又はGGOHを
例示することができる。The host cell may be, for example, yeast, for example, ascomycete yeast of Ascomycota, basidiomycete yeast of Basidiomycota, or incomplete fungus (Fun).
gi Imperfecti) imperfect yeast can be used. Preferably, ascomycete yeast, especially Saccharomyces cerevisiae, which is a budding yeast, Candida utilis or Pichia pastoris ( Pichia
pastris ) and other fission yeast such as Shizosaccharomyces pombe . Not only the species but also the strain is not particularly limited as long as it can produce prenyl alcohol, and for example, strains of Saccharomyces cerevisiae include A451, YPH499, YP.
Examples include H500, W303-1A, W303-1B and the like. Further, as prenyl alcohol, FOH, NOH and / or GGOH can be exemplified.

【００２２】また、上記製造方法において、転写抑制条
件下で培養する前に、非転写抑制条件下で培養すること
もできる。転写抑制条件下での培養はグルコース含有培
地での培養であり、非転写抑制条件下での培養はガラク
トース含有培地での培養である。ファルネシル二リン酸
合成酵素遺伝子は配列番号２又は４に示されるアミノ酸
配列をコードし、ゲラニルゲラニル二リン酸合成酵素遺
伝子は配列番号６に示されるアミノ酸配列をコードし、
ヒドロキシメチルグルタリルCoA還元酵素遺伝子は配列
番号８に示されるアミノ酸配列をコードし、メバロン酸
二リン酸脱炭酸酵素遺伝子は配列番号10に示されるアミ
ノ酸配列をコードするものを使用することができる。ま
た、ヒドロキシメチルグルタリルCoA還元酵素遺伝子の
変異型遺伝子は、配列番号11、13及び15-24に示される
いずれかの塩基配列を含むものである。In the above production method, it is also possible to culture under non-transcription suppressing conditions before culturing under transcription suppressing conditions. The culture under the transcription repressing condition is a culture in the glucose-containing medium, and the culture under the non-transcription repressing condition is a galactose-containing medium. The farnesyl diphosphate synthase gene encodes the amino acid sequence shown in SEQ ID NO: 2 or 4, and the geranylgeranyl diphosphate synthase gene encodes the amino acid sequence shown in SEQ ID NO: 6,
The hydroxymethylglutaryl CoA reductase gene can encode the amino acid sequence shown in SEQ ID NO: 8, and the mevalonate diphosphate decarboxylase gene can encode the amino acid sequence shown in SEQ ID NO: 10. Further, the mutant gene of the hydroxymethylglutaryl CoA reductase gene contains any of the nucleotide sequences shown in SEQ ID NOs: 11, 13 and 15-24.

【００２３】(5) 翻訳活性のあるスクアレン合成酵素遺
伝子転写産物量を減少できるように変異させた変異型細
胞。 (6) スクアレン合成酵素遺伝子の転写プロモーター領域
を転写抑制型プロモーターで置換したプロモーター領域
を含み、翻訳活性のあるスクアレン合成酵素遺伝子転写
産物量を減少できるように変異させた変異型細胞。以
下、本発明を詳細に説明する。(5) Mutant cells mutated to reduce the amount of squalene synthase gene transcript having translation activity. (6) A mutant cell which comprises a promoter region in which the transcription promoter region of the squalene synthase gene is replaced with a transcription repressing promoter, and is mutated so as to reduce the amount of the transcription product of the squalene synthase gene having translation activity. Hereinafter, the present invention will be described in detail.

【００２４】[0024]

【発明の実施の形態】本発明者は、代謝工学的な手法を
用い、生体内で活性型プレニルアルコール、特にFOH、N
OH又はGGOHを生産させる系の開発に臨んだ。本発明で
は、宿主細胞として酵母、特に出芽酵母サッカロマイセ
ス・セレビシアエを選び、その酵母において、スクアレ
ン合成酵素遺伝子ERG9の転写プロモーター領域と考えら
れるゲノムDNA部分を、グルコースで転写抑制できる転
写抑制型プロモーターのひとつであるGAL1遺伝子の転写
プロモーターで置き換えた株（EUG株）を作製した。そ
して、好気的培養条件下ではステロール取り込み能がな
い通常の組換え宿主株を特別な添加物の無い一般的なグ
ルコース含有培地（YM7培地、YPD培地、SD培地など）で
好気的に培養するだけでプレニルアルコールを培養液内
に蓄積できる系を開発した。BEST MODE FOR CARRYING OUT THE INVENTION The present inventor has used metabolic engineering techniques to produce active prenyl alcohols, particularly FOH and N, in vivo.
We set out to develop a system to produce OH or GGOH. In the present invention, yeast is selected as a host cell, particularly budding yeast Saccharomyces cerevisiae, and in the yeast, the genomic DNA portion considered to be the transcription promoter region of the squalene synthase gene ERG9 is one of transcription-repressible promoters capable of suppressing transcription with glucose. A strain (EUG strain) in which the transcription promoter of the GAL1 gene was replaced was prepared. Then, a normal recombinant host strain that does not have sterol uptake ability under aerobic culture conditions is aerobically cultured in a general glucose-containing medium (YM7 medium, YPD medium, SD medium, etc.) without any special additives. We have developed a system that can accumulate prenyl alcohol in the culture medium just by

【００２５】一般に、FPPは、FPP合成酵素の触媒によ
り、IPP及びDMAPPを基質として合成される。FPP以降の
経路としては、通常はFOHへの脱リン酸化反応は進行せ
ず、スクアレン合成酵素によるスクアレン・各種ステロ
ール合成、GGPP合成酵素によるGGPP・プレニル化蛋白質
合成、ヘキサプレニル二リン酸合成酵素、ヘプタプレニ
ル二リン酸合成酵素などの中鎖プレニル二リン酸合成酵
素によるヘキサプレニル二リン酸、ヘプタプレニル二リ
ン酸などの中鎖又は長鎖プレニル二リン酸・ユビキノン
合成、プレニル化蛋白質合成などに進行する（図1）(K.
Ogura and T. Koyama, (1998) Chemical Reviews, 98,
1263-1276）。つまり、翻訳活性のあるスクアレン合成
酵素遺伝子転写産物量を減少させ、細胞内のスクアレン
合成酵素活性を減少させると、FPPがある程度蓄積する
こと、FPPを基質とした反応経路の最終産物であるプレ
ニル化蛋白質やユビキノンが蓄積すること、又は生育必
須成分であるエルゴステロール合成ができなくなること
から生育阻害がかかることは予想できても、10mg/l又は
100mg/l以上の生産性を示すようなFOHの大量生産系を構
築できることは予想できない。さらにはNOHや、GGPP脱
リン酸化物のGGOHの生産系が得られることも予想できな
い（図1）。また、IPP合成経路関連酵素の酵素活性を増
大させることによってIPP合成を増強させても、FPP合成
の方向に効果が出るのかGGPP合成の方向に効果が出るの
か予想できないし、ましてや脱リン酸化物のFOH・GGOH
の高生産やFOH異性体のNOHの高生産は期待できない（図
1）。Generally, FPP is synthesized with IPP and DMAPP as substrates by the catalyst of FPP synthase. As the pathway after FPP, dephosphorylation reaction to FOH does not normally proceed, squalene / various sterol synthesis by squalene synthase, GGPP / prenylated protein synthesis by GGPP synthase, hexaprenyl diphosphate synthase, Heptaprenyl diphosphate synthase and other medium-chain prenyl diphosphate synthases progress to hexaprenyl diphosphate, heptaprenyl diphosphate and other medium- or long-chain prenyl diphosphate / ubiquinone synthesis, prenylated protein synthesis, etc. (Fig. 1) (K.
Ogura and T. Koyama, (1998) Chemical Reviews, 98,
1263-1276). In other words, when the amount of squalene synthase gene transcript with translation activity is reduced and the intracellular squalene synthase activity is reduced, FPP accumulates to some extent, and the final product of the reaction pathway, prenylation of FPP, using FPP as a substrate. Although growth inhibition can be expected due to the accumulation of proteins and ubiquinone, or the inability to synthesize ergosterol, which is an essential growth component, it is expected that 10 mg / l or
It is not possible to construct a mass production system of FOH that shows a productivity of 100 mg / l or more. Furthermore, it is not expected that NOH and GGPP dephosphorization GGOH production systems will be obtained (Fig. 1). Moreover, even if IPP synthesis is enhanced by increasing the enzyme activity of IPP synthesis pathway-related enzyme, it is not possible to predict whether the effect will be exerted in the direction of FPP synthesis or GGPP synthesis, let alone dephosphorylation. FOH / GGOH
High production of FOH and NOH of FOH isomer cannot be expected (Fig.
1).

【００２６】本発明は、翻訳活性のあるスクアレン合成
酵素遺伝子転写産物量を減少できるように当該細胞を変
異させ、その変異型細胞を培養し、翻訳活性を有するス
クアレン合成酵素遺伝子の転写産物量を減少させること
により、一般的な代謝経路マップ（Gerhard Michal編 B
iochemical Pathways; An Atlas of Biochemistry and
Molecular Biology (1999) John Wiley & Sons, Inc.,
New York）中にも明記されていないFOH、NOHやGGOH等の
プレニルアルコールを産生することができることを見出
し、プレニルアルコールの生物学的な大量生産系を開発
したものである。また、IPP合成経路関連酵素遺伝子、
その変異型遺伝子、それらの融合遺伝子等を転写ユニッ
トとして本発明で得られるプレニルアルコール生産型の
変異型細胞に導入し、さらなるプレニルアルコールの生
物学的な大量生産系を開発したものである。According to the present invention, the cells are mutated so that the amount of squalene synthase gene transcript having translation activity can be reduced, and the mutant cells are cultured to determine the amount of squalene synthase gene transcript having translation activity. By reducing the general metabolic pathway map (Gerhard Michal ed. B
iochemical Pathways; An Atlas of Biochemistry and
Molecular Biology (1999) John Wiley & Sons, Inc.,
New York) was found to be able to produce prenyl alcohol such as FOH, NOH and GGOH, which is not specified in New York, and a biological mass production system for prenyl alcohol was developed. In addition, IPP synthetic pathway related enzyme gene,
The mutant gene, a fusion gene thereof, or the like is introduced as a transcription unit into the prenyl alcohol-producing mutant cell obtained in the present invention to develop a further biological mass production system for prenyl alcohol.

【００２７】1．スクアレン合成酵素転写産物変異株の
作製 本発明において使用する変異型細胞は、宿主細胞のスク
アレン合成酵素遺伝子のコード領域には置換、挿入また
は付加のどの変異も導入しておらず、翻訳活性のあるス
クアレン合成酵素遺伝子転写産物量を減少させることが
できるように改変したスクアレン合成酵素遺伝子を含む
ものである。翻訳活性のある転写産物量を減少させるに
は、たとえば遺伝子の転写活性を抑制させ転写産物量自
体を減少させる方法や、アンチセンスRNAを利用するこ
とによって転写されたｍRNAの翻訳活性を抑制する方法
などがある。転写活性を抑えるには、例えば宿主ゲノム
に含まれるスクアレン合成酵素遺伝子の転写プロモータ
ー領域を転写抑制型プロモーターで置換したり、スクア
レン合成酵素遺伝子の転写に関わる領域に転写抑制活性
のある塩基配列を作出すればよい。また、翻訳活性を抑
制するには、たとえばスクアレン合成酵素遺伝子に対す
るアンチセンスRNAを転写する遺伝子を過剰発現させれ
ばよい。アンチセンスRNAとは標的の遺伝子から転写さ
れたRNAに対して相補的な塩基配列を有しているRNAのこ
とで、原核細胞、真核細胞のどちらの転写翻訳系システ
ムにおいても翻訳活性のあるmRNA量を低下させ、遺伝子
発現のリプレッサーとしての機能を持つRNAのことであ
る。例えば「遺伝子の発現と制御I（シリーズ分子生物
学の進歩）」日本分子生物学会 編・丸善株式会社1989
年6月30日発行・ISBN4-621-03375-1の第5章「細菌膜蛋
白質合成の制御」（水野猛 著）では、アンチセンスRNA
を用いてエシェリシア・コーライの遺伝子ompFの翻訳活
性のあるmRNA量を低下させOmpF蛋白質合成を抑制できる
ことが報告されている。水野がこの著書で述べているよ
うにある遺伝子のアンチセンスRNAは天然に存在し、タ
ーゲット遺伝子を決めれば遺伝子工学的手法によって発
現抑制活性のある人工アンチセンスRNAを作製すること
は当業者なら容易である。人工アンチセンスRNA技術
は、細菌であるエシェリシア・コーライだけでなく高等
生物、例えば哺乳類（マウス）、昆虫（ドロソフィラ・
メラノガスター（Drosophila melanogaster））や被子
植物（トマト）を宿主とした場合でも知られている（Mi
zunoet al. (1984) Proc. Natl. Acad. Sci. U.S.A., 8
1, 1966-1970; Aiba et al.(1987) J. Bacteriol, 169,
3007-3012; Green et al. (1986) Annu. Rev. Bioche
m., 55, 569-597; Harland et al. (1985) J. Cell. Bi
ol., 101, 1094-1099;Coleman et al. (1984) Cell, 3
7, 429-36; Han et al. (1991) Proc. Natl. Acad. Sc
i. U.S.A., 88, 4313-4317; Hackett et al. (2000) Pl
ant Physiol., 124, 1079-86）。1. Preparation of mutant strain of squalene synthase transcript The mutant cell used in the present invention has no translation, insertion or addition mutation in the coding region of the squalene synthase gene of the host cell, and has translation activity. The squalene synthase gene contains a squalene synthase gene modified so as to reduce the transcript amount. To reduce the amount of a transcription product having a translation activity, for example, a method of suppressing the transcription activity of a gene to decrease the amount of the transcription product itself, or a method of suppressing the translation activity of a transcribed mRNA by using antisense RNA and so on. To suppress the transcriptional activity, for example, the transcription promoter region of the squalene synthase gene contained in the host genome is replaced with a transcriptional repressive promoter, or a nucleotide sequence having a transcriptional repression activity is created in a region related to the transcription of the squalene synthase gene. do it. To suppress the translation activity, for example, a gene that transcribes an antisense RNA for the squalene synthase gene may be overexpressed. Antisense RNA is RNA that has a nucleotide sequence complementary to the RNA transcribed from the target gene, and has translation activity in both transcription systems of prokaryotic cells and eukaryotic cells. RNA that reduces the amount of mRNA and functions as a repressor of gene expression. For example, “Gene expression and regulation I (advanced series of molecular biology)” edited by Japan Molecular Biology Society, Maruzen Co., Ltd. 1989
Published June 30, 2013 ・ In ISBN4-621-03375-1, Chapter 5, "Regulation of bacterial membrane protein synthesis" (Takeshi Mizuno) describes antisense RNA.
It has been reported that the amount of mRNA having translational activity of the Escherichia coli gene ompF can be reduced by using Escherichia coli to suppress OmpF protein synthesis. As Mizuno describes in this book, the antisense RNA of a gene exists naturally, and it is easy for a person skilled in the art to produce an artificial antisense RNA having an expression suppressing activity by a genetic engineering method if the target gene is determined. Is. Artificial antisense RNA technology is used not only for bacteria such as Escherichia coli, but also for higher organisms such as mammals (mouse) and insects (drosophila
It is also known when melanogaster ( Drosophila melanogaster ) and angiosperms (tomatoes) are used as hosts (Mi
zuno et al. (1984) Proc. Natl. Acad. Sci. USA, 8
1, 1966-1970; Aiba et al. (1987) J. Bacteriol, 169,
3007-3012; Green et al. (1986) Annu. Rev. Bioche
m., 55, 569-597; Harland et al. (1985) J. Cell. Bi
ol., 101, 1094-1099; Coleman et al. (1984) Cell, 3
7, 429-36; Han et al. (1991) Proc. Natl. Acad. Sc
i. USA, 88, 4313-4317; Hackett et al. (2000) Pl
ant Physiol., 124, 1079-86).

【００２８】本発明において使用される宿主細胞とし
て、たとえば、発酵工業に古来から広く用いられ、IPP
合成経路としてメバロン酸経路を持ち、遺伝子組換えに
関する様々な手法を駆使できるもの、例えば出芽酵母等
の酵母を例示することができる。出芽酵母としては、例
えばサッカロマイセス・セレビシアエ（Saccharomycesc
erevisiae）を例示できるが、必ずしもサッカロマイセ
ス・セレビシアエなどの酵母に限定されるわけではな
く、スクアレン合成酵素遺伝子を保持している他の様々
な細胞を宿主に用いることができる。そして、スクアレ
ン合成酵素遺伝子コード領域に置換、挿入または欠失の
変異を導入する方法によってスクアレン合成酵素欠損を
導入しなくても、翻訳活性のあるスクアレン合成酵素遺
伝子の転写産物量を減少させれば、各種活性型プレニル
アルコールを生産させることができる。As the host cell used in the present invention, for example, IPP has been widely used since ancient times in the fermentation industry.
An example is one that has a mevalonate pathway as a synthetic pathway and can make full use of various techniques relating to gene recombination, for example, yeast such as budding yeast. The budding yeast, such as Saccharomyces cerevisiae (Saccharomycesc
erevisiae ), but the yeast is not necessarily limited to yeast such as Saccharomyces cerevisiae, and various other cells carrying a squalene synthase gene can be used as a host. And, even if the squalene synthase deficiency is not introduced by the method of introducing a substitution, insertion or deletion mutation in the squalene synthase gene coding region, if the transcript amount of the squalene synthase gene having translation activity is reduced, It is possible to produce various activated prenyl alcohols.

【００２９】翻訳活性のあるスクアレン合成酵素遺伝子
の転写産物量を減少させるためには、例えば転写抑制型
プロモーターを利用することができる。転写抑制型プロ
モーターとしては各種培養条件で転写制御できる転写プ
ロモーターであればどの転写プロモーターでも良い。例
えば、グルコース含有培地で転写抑制されるGAL1, GAL
2, GAL7, GAL10, GAL80, MEL1、グルコース枯渇条件で
転写抑制されるADH1, ENO2、高リン酸条件で転写抑制さ
れるPHO5, PHO81、細胞周期のG1期以外で転写抑制され
るHO、などの遺伝子の転写を司る転写プロモーターを挙
げることができる。他にも、L. Wodicka et al., (199
7) Nautre Biotechnology, 15, 1359-1367で報告されて
いるような、ある培養条件で転写産物が相当量検出され
るにもかかわらず別の培養条件では転写産物量が著しく
低下する遺伝子の転写を司る転写プロモーターも転写抑
制型プロモーターとして利用できる。例えば、(i)通常
栄養培地（rich medium）で転写産物が検出されないが
最小培地（minimal medium）で相当量検出される遺伝子
HSP12, INO1, YBR147W, YGR243Wなどの転写を司る転写
プロモーター、あるいは(ii)最小培地で転写産物が検出
されないが通常栄養培地で相当量検出される遺伝子YDR0
46C, GNP1, CHA1, PTR2などの転写を司る転写プロモー
ターなどが挙げられる。さらに、出芽酵母以外の転写抑
制型転写プロモーターを用いることも可能である。プロ
モーターは、必ずしも宿主細胞内で転写抑制活性を有す
るものである必要はなく、例えば、転写抑制因子遺伝子
を宿主細胞に導入し、その転写因子制御下で初めて転写
抑制されるタイプの転写プロモーターを用いることもで
きる。さらに、翻訳活性のあるスクアレン合成酵素転写
産物量を減少させることができれば、必ずしも宿主細胞
内で直接転写抑制制御、つまりmRNA合成制御が行える転
写プロモーターを利用する必要も無く、アンチセンスRN
Aなどの技術によって転写産物であるmRNAの翻訳活性を
不活性化させることによって、転写抑制型プロモーター
を利用した場合と同様に、スクアレン合成酵素遺伝子の
コード領域を改変せずに、プレニルアルコール生産させ
ることもできる。In order to reduce the amount of the transcription product of the squalene synthase gene having translation activity, for example, a transcription repressing promoter can be used. The transcription repressing promoter may be any transcription promoter as long as it can control transcription under various culture conditions. For example, GAL1 and GAL that are transcriptionally repressed in glucose-containing medium
2, GAL7, GAL10, GAL80, MEL1, ADH1 is transcriptional repression by glucose deprivation conditions, ENO2, PHO5 is transcriptional repression in high phosphate condition, PHO81, HO transcribed suppressed except G 1 phase of the cell cycle, such as A transcription promoter that controls the transcription of the gene can be mentioned. Besides, L. Wodicka et al ., (199
7) As reported in Nautre Biotechnology, 15, 1359-1367, transcription of a gene in which the amount of transcript is significantly reduced under one culture condition but the amount of transcript is significantly reduced under another culture condition The controlled transcription promoter can also be used as a transcription repressing promoter. For example, (i) Genes in which transcripts are not detected in normal nutrient medium (rich medium) but are detected in considerable amounts in minimal medium
Transcription promoter that controls transcription of HSP12 , INO1 , YBR147W, YGR243W, etc., or (ii) Gene YDR0, whose transcript is not detected in minimal medium but is detected in normal nutrient medium
46C, GNP1, CHA1, such as a transcriptional promoter which controls the transcription of such PTR2 the like. Furthermore, it is also possible to use a transcription repressing transcription promoter other than Saccharomyces cerevisiae. The promoter does not necessarily have to have a transcription repressing activity in the host cell. For example, a transcriptional promoter of a type in which a transcription repressor gene is introduced into a host cell and transcription is repressed for the first time under the control of the transcription factor is used. You can also Furthermore, if the amount of squalene synthase transcript having translation activity can be reduced, it is not always necessary to use a transcription promoter capable of direct transcription repression control, that is, mRNA synthesis control in the host cell.
By inactivating the translational activity of the mRNA that is a transcript by a technique such as A, prenyl alcohol is produced without modifying the coding region of the squalene synthase gene, as in the case of using a transcriptional repressor. You can also

【００３０】宿主細胞としてサッカロマイセス・セレビ
シアエを用い、細胞中のスクアレン合成酵素遺伝子転写
プロモーターを転写抑制型プロモーターで置換する方法
を以下に説明する。まず、酵母ゲノムデータベース（Sa
ccharomyces Genome Database (SGD); http://genome-w
ww.stanford.edu./Saccharomyces/）から、スクアレン
合成酵素遺伝子ERG9付近の遺伝子地図を引き出し、ERG9
転写プロモーター(ERG9p)置換用DNA断片増幅のためのPC
RプライマーDNAを設計した（図2）。A method for substituting a transcription repressing promoter for the squalene synthase gene transcription promoter in the cell using Saccharomyces cerevisiae as a host cell will be described below. First, the yeast genome database (Sa
ccharomyces Genome Database (SGD); http: // genome-w
From ww.stanford.edu./Saccharomyces/), a genetic map near the squalene synthase gene ERG9 was derived, and ERG9
PC for amplification of DNA fragment for replacement of transcription promoter ( ERG9 p)
The R primer DNA was designed (Fig. 2).

【００３１】次に、pYES2（図2C,図3）を制限酵素処理
した後、Klenow酵素で平滑末端化し、セルフライゲーシ
ョンにより2μ ori部分を削除したpYES2Δ（図2B）を鋳
型にしてPCR増幅することにより、形質転換体選択マー
カー遺伝子URA3と転写プロモーターGAL1pを含むDNA断片
を作製する。このDNAの一方の側に、ERG9遺伝子の一部
のDNAを、他方の側にERG9遺伝子の近傍に存在するYHR18
9WのDNAを連結して、形質転換用DNA断片を調製する（図
2A）。Next, pYES2 (FIG. 2C, FIG. 3) was treated with a restriction enzyme, blunt-ended with Klenow enzyme, and PCR-amplified using pYES2Δ (FIG. 2B) in which the 2 μ ori portion was deleted by self-ligation as a template. Thus, a DNA fragment containing the transformant selection marker gene URA3 and the transcription promoter GAL1 p is prepared. A part of the ERG9 gene DNA is present on one side of this DNA and the YHR18 gene present on the other side near the ERG9 gene.
9W DNA is ligated to prepare a DNA fragment for transformation (Fig.
2A).

【００３２】本発明において、変異型細胞を作製するた
めの宿主は特に限定されないが、工業上利用上重要な酵
母、例えば、子嚢菌類（Ascomycota）の子嚢菌酵母、担
子菌類（Basidiomycota）の担子菌酵母、又は不完全菌
類（Fungi Imperfecti）の不完全菌酵母を用いることが
できる。好ましくは、子嚢菌酵母、特に出芽酵母である
サッカロマイセス・セレビシアエ(S. cerevisiae)、キ
ャンディダ・ユーティリス(Candida utilis)又はピキア
・パストリス(Pichia pastris)等、***酵母であるシゾ
サッカロマイセス・ポンベ(Shizosaccharomyces pombe)
等が用いられる。サッカロマイセス・セレビシアエの場
合、変異導入のための宿主株として、例えば下記のA45
1、YPH499、YPH500、W303-1A、W303-1Bなどを使用する
ことができる。In the present invention, the host for producing the mutant cell is not particularly limited, but yeasts important for industrial use, for example, ascomycete yeast of Ascomycota and basidiomycete of Basidiomycota. Bacterial yeast or incomplete fungal yeast of Fungi Imperfecti can be used. Preferably, ascomycete yeast, particularly Saccharomyces cerevisiae which is a budding yeast ( S. cerevisiae ), Candida utilis ( Candida utilis ) or Pichia pastris ( Pichia pastris ) and the like, Schizosaccharomyces pombe which is a fission yeast. Shizosaccharomyces pombe ).
Etc. are used. In the case of Saccharomyces cerevisiae, as a host strain for mutation introduction, for example, the following A45
1, YPH499, YPH500, W303-1A, W303-1B etc. can be used.

【００３３】 A451 (ATCC200589, MATα can1 leu2 trp1 ura3 aro7) YPH499（ATCC76625, MATa ura3-52 lys2-801 ade2-101
trp1-Δ63 his3- Δ 200 leu2- Δ 1, Stratagene , La
Jolla, CA） YPH500（ATCC76626, MATα ura3-52 lys2-801 ade2-101
trp1-Δ63 his3- Δ200 leu2- Δ 1, Stratagene） W303-1A（ATCC208352, MATa leu2-3 leu2-112 his3-11
ade2-1 ura3-1 trp1-1can1-100） W303-1B（ATCC208353, MATα leu2-3 leu2-112 his3-11
ade2-1 ura3-1 trp1-1 can1-100）A451 (ATCC200589, MATα can1 leu2 trp1 ura3 aro7 ) YPH499 (ATCC76625, MATa ura3 -52 lys2 -801 ade2 -101
trp1 -Δ63 his3 - Δ 200 leu2 - Δ 1, Stratagene, La
Jolla, CA) YPH500 (ATCC76626, MATα ura3 -52 lys2 -801 ade2 -101
trp1 -Δ63 his3 -Δ200 leu2- Δ1 , Stratagene) W303-1A (ATCC208352, MATa leu2 -3 leu2 -112 his3 -11
ade2 -1 ura3 -1 trp1 -1 can1 -100) W303-1B (ATCC208353, MATα leu2 -3 leu2 -112 his3 -11
ade2 -1 ura3 -1 trp1 -1 can1 -100)

【００３４】DNA断片の形質転換は、公知の任意の手法
又は市販のキットを用いることができる。例えば、Zymo
Research (Orange, CA) より購入したFrozen EZ yeast
transformation II kitを用いて酵母にベクターの導入
を行う。宿主に導入した組換え用DNAは、相同組換えに
より宿主のゲノムDNAに組み込まれる。これを利用し
て、スクアレン合成酵素遺伝子ERG9の転写プロモーター
領域を転写抑制型プロモーターであるGAL1プロモーター
で置換し、変異株を得る。For transforming the DNA fragment, any known method or a commercially available kit can be used. For example, Zymo
Frozen EZ yeast purchased from Research (Orange, CA)
Introduce the vector into yeast using the transformation II kit. The recombinant DNA introduced into the host is integrated into the host genomic DNA by homologous recombination. Utilizing this, the transcription promoter region of the squalene synthase gene ERG9 is replaced with the transcriptional repressible promoter GAL1 promoter to obtain a mutant strain.

【００３５】翻訳活性のあるスクアレン合成酵素遺伝子
ERG9の転写産物量を減少させるために、ERG9プロモータ
ーを転写抑制型プロモーターであるGAL1プロモーターで
置換し、取得した組換え体をEUG (ERG9p::URA3-GAL1p)
株と名付けた。A451由来のクローンをEUG1-10、YPH499
由来のクローンをEUG11-20、YPH500由来のクローンをEU
G21-30、W303-1A由来のクローンをEUG31-50、W303-1B由
来のクローンをEUG51-70とする。Squalene synthase gene having translation activity
To reduce the amount of a transcript ERG9, replaced with the GAL1 promoter, a transcription inhibitory promoter ERG9 promoter, EUG the obtained recombinant (E RG9p :: U RA3- G AL1p )
I named it a stock. The clones derived from A451 are EUG1-10, YPH499
Clone derived from EU G11-20, YPH500 derived clone from EU
The clones derived from G21-30 and W303-1A are called EUG31-50 and the clones derived from W303-1B are called EUG51-70.

【００３６】EUG1-10 (A451, ERG9p::URA3-GAL1p)は、
本発明において樹立されたA451由来株であり、翻訳活性
のあるスクアレン合成酵素遺伝子ERG9転写産物量を減少
させることができる株である。この株は、スクアレン合
成酵素遺伝子ERG9のポリペプチドコード領域、形質転換
体選択マーカー遺伝子URA3及びERG9遺伝子座に転写プロ
モーターGAL1pを含む。EUG1-10 (A451, ERG9 p :: URA3 - GAL1 p) is
It is an A451-derived strain established in the present invention, and is a strain capable of decreasing the amount of squalene synthase gene ERG9 transcript having translation activity. This strain contains the transcriptional promoter GAL1 p at the polypeptide coding region of the squalene synthase gene ERG9 , the transformant selection marker gene URA3 and the ERG9 locus.

【００３７】EUG11-20 (YPH499, ERG9p::URA3-GAL1p)
は、本発明において樹立されたYPH499由来株であり、翻
訳活性のあるスクアレン合成酵素遺伝子ERG9転写産物量
を減少させることができる株である。この株は、ERG9の
ポリペプチドコード領域、URA3及びERG9遺伝子座にGAL1
pを含む。EUG11-20 (YPH499, ERG9 p :: URA3 - GAL1 p)
Is a YPH499-derived strain established in the present invention, and is a strain capable of reducing the amount of squalene synthase gene ERG9 transcript having translation activity. This strain polypeptide coding region of ERG9, the URA3 and ERG9 locus GAL1
Including p.

【００３８】EUG21-30 (YPH500, ERG9p::URA3-GAL1p)
は、本発明において樹立されたYPH500由来株であり、翻
訳活性のあるスクアレン合成酵素遺伝子ERG9転写産物量
を減少させることができる株である。この株は、ERG9の
ポリペプチドコード領域、URA3及びERG9遺伝子座にGAL1
pを含む。EUG21-30 (YPH500, ERG9 p :: URA3 - GAL1 p)
Is a YPH500-derived strain established in the present invention, and is a strain capable of decreasing the amount of squalene synthase gene ERG9 transcript having translation activity. This strain polypeptide coding region of ERG9, the URA3 and ERG9 locus GAL1
Including p.

【００３９】EUG31-50 (W303-1A, ERG9p::URA3-GAL1p)
は、本発明において樹立されたW303-1A由来株であり、
翻訳活性のあるスクアレン合成酵素遺伝子ERG9転写産物
量を減少させることができる株である。この株は、ERG9
のポリペプチドコード領域、URA3及びERG9遺伝子座にGA
L1pを含む。EUG31-50 (W303-1A, ERG9 p :: URA3 - GAL1 p)
Is a W303-1A-derived strain established in the present invention,
It is a strain that can reduce the amount of squalene synthase gene ERG9 transcript having translation activity. This strain is ERG9
GA at the URA3 and ERG9 loci
Including L1 p.

【００４０】EUG51-70 (W303-1B, ERG9p::URA3-GAL1p)
は、本発明において樹立されたW303-1B由来株であり、
翻訳活性のあるスクアレン合成酵素遺伝子ERG9転写産物
量を減少させることができる株である。この株は、ERG9
のポリペプチドコード領域、URA3及びERG9遺伝子座にGA
L1pを含む。EUG51-70 (W303-1B, ERG9 p :: URA3 - GAL1 p)
Is a W303-1B-derived strain established in the present invention,
It is a strain that can reduce the amount of squalene synthase gene ERG9 transcript having translation activity. This strain is ERG9
GA at the URA3 and ERG9 loci
Including L1 p.

【００４１】本発明において、このようにして得られた
EUG株をGAL1pの転写抑制条件であるグルコース含有培地
で培養し（培養法は後述）、ATCC64031で公知になって
いるようなスクアレン合成酵素遺伝子コード領域に置
換、挿入または欠失の変異を導入することをせずに、翻
訳活性のあるスクアレン合成酵素遺伝子転写産物量を減
少させることにより、エルゴステロールや界面活性剤な
どの特別な添加物無しに各種プレニルアルコールを生産
することができる。In the present invention, the thus obtained
The EUG strain was cultured in a glucose-containing medium which is a transcriptional repressing condition for GAL1 p (the culture method is described later), and a substitution, insertion or deletion mutation was introduced into the squalene synthase gene coding region known in ATCC64031. Without doing so, various prenyl alcohols can be produced without special additives such as ergosterol and a surfactant by reducing the amount of squalene synthase gene transcript having translation activity.

【００４２】2．発現用組換えDNA又はゲノムインテグレ
ート用DNA断片の作製 本発明において、上記EUG株を単独で培養するほか、IPP
合成経路関連酵素遺伝子を含む発現用組換えDNA又はゲ
ノムインテグレート用DNA断片を作製し、これを上記EUG
株に導入して培養してもよい。EUG株をさらに形質転換
するための発現用組換えDNAは、IPP合成経路関連酵素遺
伝子に、転写プロモーター及び転写ターミネーターDNA
を連結又は挿入することにより得ることができる。ま
た、IPP合成経路関連酵素遺伝子に、予め転写プロモー
ター及び転写ターミネーターが連結された遺伝子発現カ
セット（転写ユニット）を作製しておいて、これをベク
ターに組み込むこともできる。連結及び挿入の順序は任
意であるが、IPP合成経路関連酵素遺伝子の上流に転写
プロモーターを連結し、IPP合成経路関連酵素遺伝子の
下流に転写ターミネーターを連結することが好ましい。
本発明においては、適当なDNA、例えばベクターDNAに順
次IPP合成経路関連酵素遺伝子、転写プロモーター及び
転写ターミネーターを組み込んでもよく、転写方向が考
慮されていれば順不同で組み込んでもよい。また、遺伝
子発現を増強するエンハンサー配列、遺伝子発現を調節
する各種シスエレメントを転写ユニット周辺に組込んで
も良い。2. Preparation of recombinant DNA for expression or DNA fragment for genome integration In the present invention, in addition to culturing the above EUG strain alone, IPP
A recombinant DNA for expression or a DNA fragment for genomic integration containing a synthetic pathway-related enzyme gene was prepared and
It may be introduced into a strain and cultured. The recombinant DNA for expression for further transforming the EUG strain includes an IPP synthesis pathway-related enzyme gene, a transcription promoter and a transcription terminator DNA.
Can be obtained by connecting or inserting. Alternatively, a gene expression cassette (transcription unit) in which a transcription promoter and a transcription terminator are linked to the IPP synthesis pathway-related enzyme gene in advance may be prepared and then incorporated into a vector. The order of ligation and insertion is arbitrary, but it is preferable to ligate a transcription promoter upstream of the IPP synthesis pathway-related enzyme gene and ligate a transcription terminator downstream of the IPP synthesis pathway-related enzyme gene.
In the present invention, an IPP synthesis pathway-related enzyme gene, a transcription promoter and a transcription terminator may be sequentially incorporated into an appropriate DNA, for example, vector DNA, or in any order if the transcription direction is taken into consideration. In addition, an enhancer sequence that enhances gene expression and various cis elements that regulate gene expression may be incorporated around the transcription unit.

【００４３】IPP合成経路関連酵素遺伝子としては、以
下のものを例示することができる。 (a) ファルネシル二リン酸合成酵素遺伝子（FPP合成酵
素遺伝子） (b) ゲラニルゲラニル二リン酸合成酵素遺伝子（GGPP合
成酵素遺伝子） (c) ヒドロキシメチルグルタリルCoA還元酵素遺伝子（H
MG-CoA還元酵素遺伝子） (d) イソペンテニル二リン酸Δ-イソメラーゼ遺伝子（I
PPΔ-イソメラーゼ遺伝子） (e) メバロン酸キナーゼ遺伝子 (f) アセチルCoAアセチルトランスフェラーゼ遺伝子 (g) ヒドロキシメチルグルタリル-CoA合成酵素遺伝子 (h) メバロン酸リン酸キナーゼ遺伝子 (i) メバロン酸二リン酸脱炭酸酵素遺伝子Examples of the IPP synthetic pathway-related enzyme gene include the following. (a) Farnesyl diphosphate synthase gene (FPP synthase gene) (b) Geranylgeranyl diphosphate synthase gene (GGPP synthase gene) (c) Hydroxymethylglutaryl CoA reductase gene (H
MG-CoA reductase gene) (d) Isopentenyl diphosphate Δ-isomerase gene (I
PPΔ-isomerase gene) (e) Mevalonate kinase gene (f) Acetyl CoA acetyltransferase gene (g) Hydroxymethylglutaryl-CoA synthase gene (h) Mevalonate phosphate kinase gene (i) Mevalonate diphosphate dephosphate Carbonic acid enzyme gene

【００４４】FPP合成酵素遺伝子としては、例えばサッ
カロマイセス・セレビシアエERG20（配列番号１）、エ
シェリシア・コーライispA (配列番号３)、バチルス・
ステアロサーモフィラス(Bacillus stearothermophilu
s)由来のFPP合成酵素遺伝子（特開平5-219961号公報、
米国特許5,786,192号）、シネココッカス・エロンガタ
ス（Synechococcus elongatus）由来のFPP合成酵素遺伝
子（C. Ohto et al., (1999) Plant Mol. Biol., 40, 3
07-321）などが挙げられる。GGPP合成酵素遺伝子として
は、例えばサッカロマイセス・セレビシアエBTS1（配列
番号５）、スルフォロバス・アシドカルダリウス(Sulfo
lobus acidocaldarius)crtE（C. Ohto et al., (1998)
Biosci. Biotechnol. Biochem., 62, 1243-1246、特開
平7-308913号公報、米国特許5,773,273号）、サーマス
・サーモフィラス(Thermus thermophilus)Tth GGPP合成
酵素遺伝子（C. Ohto et al., (1999) Biosci. Biotech
nol. Biochem., 63, 261-270、特開平9-107974号公報、
米国特許6,107,072号）、シネココッカス・エロンガタ
ス（Synechococcus elongatus）由来のGGPP合成酵素遺
伝子（C. Ohto et al., (1999) Plant Mol. Biol., 40,
307-321）などが挙げられる。HMG-CoA還元酵素遺伝子
としては、サッカロマイセス・セレビシアエHMG1（配列
番号７）、HMG2、ストレプトマイセス(Streptomyces s
p.)CL190由来のHMG-CoA還元酵素遺伝子（S Takahashi e
t al., (1999) J. Bacteriol., 181, 1256-1263、）な
どが挙げられる。IPPΔ-イソメラーゼ遺伝子としては、
エシェリシア・コーライ（Escherichia coli）由来のid
i（配列番号25）などが挙げられる。その他のIPP合成経
路関連酵素遺伝子としては、以下の塩基配列を有するも
のを例示することができる。Examples of the FPP synthase gene include Saccharomyces cerevisiae ERG20 (SEQ ID NO: 1), Escherichia coli ispA (SEQ ID NO: 3), Bacillus
Bacillus stearothermophilu
s ) derived FPP synthase gene (JP-A-5-219961 JP,
U.S. Pat. No. 5,786,192), FPP synthase gene (C. Ohto et al. , (1999) Plant Mol. Biol., 40, 3) derived from Synechococcus elongatus .
07-321) and the like. Examples of the GGPP synthase gene include Saccharomyces cerevisiae BTS1 (SEQ ID NO: 5), Sulfolobus acidocaldarius ( Sulfo
lobus acidocaldarius ) crtE (C. Ohto et al. , (1998)
Biosci. Biotechnol. Biochem., 62, 1243-1246, JP-A-7-308913, US Pat. No. 5,773,273), Thermus thermophilus Tth GGPP synthase gene (C. Ohto et al. , (1999) Biosci. Biotech
nol. Biochem., 63, 261-270, JP 9-107974 A,
U.S. Patent No. 6,107,072), GGPP synthase gene (C. Ohto et al. , (1999) Plant Mol. Biol., 40, from Synechococcus elongatus ).
307-321) and the like. Examples of the HMG-CoA reductase gene, Saccharomyces cerevisiae HMG1 (SEQ ID NO: 7), HMG2, Streptomyces (Streptomyces s
p.) CL190-derived HMG-CoA reductase gene (S Takahashi e
T al. , (1999) J. Bacteriol., 181, 1256-1263,). As the IPPΔ-isomerase gene,
Id from Escherichia coli
i (SEQ ID NO: 25) and the like. Examples of other IPP synthesis pathway-related enzyme genes include those having the following base sequences.

【００４５】メバロン酸キナーゼ遺伝子ERG12（配列番
号26） アセチルCoAアセチルトランスフェラーゼ遺伝子ERG10
（配列番号27） HMG-CoA合成酵素遺伝子HMGS (ERG13)（配列番号28） メバロン酸リン酸キナーゼ遺伝子ERG8（配列番号29） メバロン酸二リン酸脱炭酸酵素遺伝子ERG19（配列番号
7）Mevalonate kinase geneERG12(Sequence number
No. 26) Acetyl CoA acetyl transferase geneERG10
(SEQ ID NO: 27) HMG-CoA synthase geneHMGS (ERG13)(SEQ ID NO: 28) Mevalonate phosphate kinase geneERG8(SEQ ID NO: 29) Mevalonate diphosphate decarboxylase geneERG19(Sequence number
7)

【００４６】また、宿主によっては非メバロン酸経路が
IPP合成の主要経路になっているので、IPP合成経路関連
遺伝子として非メバロン酸経路関連酵素遺伝子、たとえ
ばDXP合成酵素遺伝子、DXP還元異性化酵素遺伝子、MEP
シチジル転移酵素遺伝子、CDP-MEキナーゼ遺伝子、MECD
P合成酵素遺伝子など(K. Kaneda et al., (2001) PNAS,
98, 932-937、T. Kuzuyama et al., (2000) J. Bacter
iol., 182, 891-897、T. Kuzuyama et al., (2000) J.
Biol. Chem., 275, 19928-19932、S. Takahashi et a
l., (1998) PNAS, 95, 9879-9884)も利用することがで
きる。In addition, depending on the host, the non-mevalonate pathway may be
Since it is a major pathway of IPP synthesis, non-mevalonate pathway-related enzyme genes such as DXP synthase gene, DXP reductase gene, MEP are used as IPP synthesis pathway-related genes.
Cytidyl transferase gene, CDP-ME kinase gene, MECD
P synthase gene, etc. (K. Kaneda et al. , (2001) PNAS,
98, 932-937, T. Kuzuyama et al. , (2000) J. Bacter
iol., 182, 891-897, T. Kuzuyama et al. , (2000) J.
Biol. Chem., 275, 19928-19932, S. Takahashi et a
l. , (1998) PNAS, 95, 9879-9884) can also be used.

【００４７】これらの遺伝子は、公知の遺伝子単離方法
により、又は市販のキットを用いて当業者であれば容易
に得ることができる。また、本発明においては、上記IP
P合成経路関連酵素遺伝子の変異体（変異型遺伝子）を
使用することも可能である。上記IPP合成経路関連酵素
遺伝子の変異型は、その一部の領域（例えばHMG-CoA還
元酵素遺伝子では最大で2217塩基）が欠損した欠失型
や、野生型遺伝子又はこれら欠失型遺伝子の塩基配列の
うち1個又は数個-10数個の塩基が欠失、付加、置換又は
挿入した変異型遺伝子でもよい。1000個程度の塩基を付
加、置換又は挿入させることもできる。従って、変異型
遺伝子によりコードされるアミノ酸配列においても、IP
P合成経路関連酵素のアミノ酸配列（例えばFPP合成酵
素,配列番号２；GGPP合成酵素,配列番号６；野生型HMG-
CoA還元酵素,配列番号８）のうち1個又は数個（例えば1
個-10個、好ましくは1個-3個）、さらには300個以上の
アミノ酸に欠失、付加、置換又は挿入等の変異が生じて
もよく、野生型HMG-CoA還元酵素のアミノ酸配列（配列
番号８）のうち最大で739個のアミノ酸が欠失した欠失
型や、これら欠失型酵素から1個又は数個（例えば1個-1
0個、好ましくは1個-3個）のアミノ酸に欠失、置換又は
付加等の変異が生じてもよい。野生型FPP合成酵素に4ア
ミノ酸を付加した変異型酵素FHDEL、野生型FPP合成酵素
C末端側に300以上のアミノ酸を付加した変異型酵素であ
るFGGとFGGHDEL、野生型FPP合成酵素N末端側に300以上
のアミノ酸を付加した変異型酵素であるGGF、野生型FPP
合成酵素N末端側とC末端側にそれぞれ300以上と4つのア
ミノ酸を付加したGGFHDEL、野生型GGPP合成酵素C末端側
に300以上のアミノ酸を付加した変異型酵素であるGGFと
GGFHDEL、野生型GGPP合成酵素N末端側に300以上のアミ
ノ酸を付加した変異型酵素であるFGG、野生型GGPP合成
酵素N末端側に300以上のアミノ酸を付加しさらにC末端
側の10個のアミノ酸を欠失させたFGGHDELが変異型酵素
の例として挙げられる。These genes can be easily obtained by those skilled in the art by known gene isolation methods or by using commercially available kits. Further, in the present invention, the above IP
It is also possible to use a mutant of the P synthesis pathway-related enzyme gene (mutant gene). The mutant type of the above-mentioned IPP synthesis pathway-related enzyme gene has a deletion type in which a partial region (for example, 2217 bases at the maximum in the HMG-CoA reductase gene) is deleted, a wild type gene or the bases of these deletion type genes. It may be a mutant gene in which one or several to several tens of bases in the sequence are deleted, added, substituted or inserted. About 1000 bases can be added, substituted or inserted. Therefore, even in the amino acid sequence encoded by the mutant gene, IP
Amino acid sequences of P-synthesis pathway-related enzymes (eg FPP synthase, SEQ ID NO: 2; GGPP synthase, SEQ ID NO: 6; wild-type HMG-
CoA reductase, SEQ ID NO: 8) 1 or several (eg 1
Mutations such as deletion, addition, substitution or insertion may occur in 300 or more amino acids, and the amino acid sequence of the wild-type HMG-CoA reductase (10-10, preferably 1-3) SEQ ID NO: 8), which is a deletion type in which a maximum of 739 amino acids has been deleted, or one or several (eg, 1-1
Mutations such as deletion, substitution or addition may occur in 0, preferably 1 to 3) amino acids. Mutant enzyme FHDEL which added 4 amino acids to wild-type FPP synthase, wild-type FPP synthase
FGG and FGGHDEL, which are mutant enzymes with 300 or more amino acids added to the C-terminal side, wild-type FPP synthase GGF, which is a mutant enzyme with 300 or more amino acids added to the N-terminal side, and wild-type FPP
Synthetic enzyme GGFHDEL with N-terminal side and C-terminal side with 300 or more amino acids and four amino acids respectively, and wild-type GGPP synthase GGF which is a mutant enzyme with 300 or more amino acids added to C-terminal side.
GGFHDEL, a wild-type GGPP synthase FGG, which is a mutant enzyme with more than 300 amino acids added to the N-terminal side, and a wild-type GGPP synthase with more than 300 amino acids added to the N-terminal side and further 10 C-terminal amino acids An example of a mutant enzyme is FGGHDEL in which is deleted.

【００４８】また、本発明において使用することができ
る変異型遺伝子としては、ERG20の3’側に12塩基を付加
したpRS435FHDEL中の変異型ERG20、ERG20の3’側にBTS1
とGly Alaをコードする塩基配列約1000塩基を付加したp
RS435FGG中の変異型ERG20遺伝子、これにさらに3’側の
30塩基を欠失させたpRS435FGGHDEL中の変異型ERG20遺伝
子、ERG20の5’側にBTS1とGly Alaをコードする塩基配
列約1000塩基を付加したpRS435GGF中の変異型ERG20遺伝
子、ERG20の5’側にBTS1とGly Alaをコードする塩基配
列約1000塩基を付加しさらに3’側に12塩基を付加したp
RS435GGFHDEL中の変異型ERG20遺伝子、BTS1の3’側の30
塩基を欠失させたpRS435GGHDEL中の変異型BTS1、BTS1の
3’側にERG20とGly Alaをコードする塩基配列約1000塩
基を付加したpRS435GGF中の変異型BTS1遺伝子、これに
さらに12塩基を付加したpRS435GGFHDEL中の変異型BTS1
遺伝子、BTS1の5’側にERG20とGly Alaをコードする塩
基配列約1000塩基を付加したpRS435FGG中の変異型BTS1
遺伝子、BTS1の5’側にERG20とGly Alaをコードする塩
基配列約1000塩基を付加しさらに3’側の30塩基を欠失
させたpRS435FGGHDEL中の変異型BTS1遺伝子が挙げられ
る。The mutant genes that can be used in the present invention include mutant ERG20 in pRS435FHDEL in which 12 bases are added to the 3'side of ERG20 , and BTS1 on the 3'side of ERG20 .
And the base sequence encoding Gly Ala with about 1000 bases added p
Mutant ERG20 gene in RS435FGG
Mutant ERG20 gene in pRS435FGGHDEL with 30 bases deleted, mutant ERG20 gene in pRS435GGF with a nucleotide sequence encoding BTS1 and Gly Ala added to the 5'side of ERG20 , 5'side of ERG20 A nucleotide sequence encoding BTS1 and Gly Ala was added with about 1000 bases and 12 bases on the 3'side.
Mutant ERG20 gene in RS435GGFHDEL , 30 on the 3'side of BTS1
Mutated BTS1 and BTS1 in pRS435GGHDEL with deleted base
3 'side mutant BTS1 gene in pRS435GGF obtained by adding a nucleotide sequence of about 1000 bases encoding ERG20 and Gly Ala, variants in pRS435GGFHDEL added with further 12 bases to BTS1
Mutant BTS1 in pRS435FGG with about 1000 bases encoding ERG20 and Gly Ala added to the 5'side of the gene, BTS1
The mutant BTS1 gene in pRS435FGGHDEL, in which about 1000 bases encoding ERG20 and Gly Ala are added to the 5'side of the gene, BTS1 and 30 bases at the 3'side are deleted, can be mentioned.

【００４９】さらに、IPP合成経路関連酵素遺伝子をPCR
で増幅させる場合において、Taq DNAポリメラーゼ等の
忠実性（fidelity）の低いDNAポリメラーゼを用いたPCR
(polymerase chain reaction)で野生型DNAを増幅させた
DNA断片に生じる塩基の置換変異を「PCRエラー」とい
い、例えば、野生型HMG-CoA還元酵素遺伝子（配列番号
７）を鋳型に用いたときのPCRエラーによる塩基の置換
変異に起因する塩基配列を有するHMG-CoA還元酵素遺伝
子（「HMG1'」とする）（コードされるポリペプチドに
置換変異を生じ得る）も、本発明において使用すること
ができる。野生型HMG-CoA還元酵素遺伝子（配列番号
７）を鋳型にしたときの、PCRエラーによる塩基の置換
の態様を図4Aに示す。HMG1'は配列番号11に示す塩基配
列を有しており、これによってコードされるアミノ酸配
列を配列番号12に示す。図4Aにおいて、ヌクレオチドの
変異は、置換前の塩基（1文字表記）、HMG-CoA還元酵素
遺伝子の開始コドンの第1塩基を1としたときの塩基番
号、置換後の塩基（1文字表記）の順で表示してある。
アミノ酸の変異は、PCRエラー型HMG-CoA還元酵素のアミ
ノ酸配列において、置換前のアミノ酸残基（1文字表
記）、HMG-CoA還元酵素のアミノ酸番号、置換後のアミ
ノ酸残基（1文字表記）の順で表示してある。さらに、
上記PCRエラー型の塩基配列を部位特異的変異誘発法等
により一部修正することもでき、このような修正型のHM
G-CoA還元酵素（配列番号14）をコードする遺伝子（配
列番号13）も、本発明において使用することができる。Furthermore, PCR is carried out for the enzyme gene associated with the IPP synthesis pathway.
PCR using a low fidelity DNA polymerase such as Taq DNA polymerase for amplification
Amplified wild-type DNA by (polymerase chain reaction)
The substitution mutation of the base generated in the DNA fragment is called "PCR error", and for example, the nucleotide sequence caused by the substitution mutation of the base due to the PCR error when the wild-type HMG-CoA reductase gene (SEQ ID NO: 7) is used as a template A HMG-CoA reductase gene having the following ( designated as " HMG1 '") (which may cause a substitution mutation in the encoded polypeptide) can also be used in the present invention. FIG. 4A shows the mode of base substitution due to PCR error when the wild-type HMG-CoA reductase gene (SEQ ID NO: 7) was used as a template. HMG1 'has the base sequence shown in SEQ ID NO: 11, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 12. In FIG. 4A, the nucleotide mutation is the base before substitution (one-letter notation), the base number when the first base of the start codon of the HMG-CoA reductase gene is 1, and the base after substitution (one-letter notation). Are displayed in this order.
Amino acid mutation is the amino acid sequence of PCR error type HMG-CoA reductase before substitution (one letter notation), HMG-CoA reductase amino acid number, after substitution (one letter notation) Are displayed in this order. further,
It is also possible to partially correct the PCR error type nucleotide sequence by a site-directed mutagenesis method, etc.
The gene (SEQ ID NO: 13) encoding G-CoA reductase (SEQ ID NO: 14) can also be used in the present invention.

【００５０】また、HMG-CoA還元酵素の膜貫通ドメイン
と予想されている領域を欠損させた欠失体をコードする
HMG-CoA還元酵素遺伝子（PCRエラー型を含む）として、
例えば、PCRエラー型HMG-CoA還元酵素遺伝子HMG1'の欠
失型遺伝子HMG1Δの例を示す（図4B）。最上段は欠失の
ない遺伝子HMG1'である。細線（−）で表した部分は欠
失した領域である。それぞれの欠失型遺伝子において、
HMG1'遺伝子（配列番号11）のうちどの領域を欠失させ
たのかを表1に示す。HMG1'欠失型遺伝子は、欠失のパタ
ーンに従ってHMG1Δxxyで表す。xxは欠失のパターン
を、yは作業番号（任意の数字）を表す。図4Bには、HMG
1Δ02yの例として「Δ026」を表示した（他の欠失パタ
ーンのものも同様に表示した）。欠失は最大で1317塩
基。It also encodes a deletion product in which a region predicted to be a transmembrane domain of HMG-CoA reductase is deleted.
As HMG-CoA reductase gene (including PCR error type),
For example, an example of the deletion type gene HMG1Δ of the PCR error type HMG-CoA reductase gene HMG1 ′ is shown (FIG. 4B). At the top is the HMG1 'gene without deletion. The part indicated by a thin line (-) is the deleted region. In each deletion type gene,
Table 1 shows which region of the HMG1 'gene (SEQ ID NO: 11) was deleted. The HMG1 'deletion gene is represented by HMG1 Δxxy according to the pattern of deletion. xx represents a deletion pattern, and y represents a work number (arbitrary number). Figure 4B shows the HMG
1 As an example of Δ02y, “Δ026” is displayed (the other deletion patterns are also similarly displayed). The maximum deletion is 1317 bases.

【００５１】[0051]

【表1】 【table 1】

【００５２】また、本発明においては、IPP合成経路関
連酵素遺伝子から選ばれる一つの遺伝子又はその変異型
と、他の遺伝子又はその変異型とを連結し、生産される
ポリペプチドが融合タンパク質になるようにすることも
できる。本発明において、このような2種類以上の遺伝
子を結合させ、発現産物が融合タンパク質になるように
構築した遺伝子を「融合遺伝子」という。融合遺伝子の
塩基配列は、一方の野生型遺伝子の塩基配列を基準にす
ると1000塩基程度の付加変異型遺伝子と考えることもで
き、それにコードされるポリペプチドは300アミノ酸残
基以上を付加した付加変異型ポリペプチドと捉えること
もできる。融合遺伝子を作製するには、一方のDNAを適
当な制限酵素で切断し、これに、同じ制限酵素で切断し
ておいた他方のDNAを、該DNAによりコードされるタンパ
ク質のアミノ酸配列の読み枠がずれないように連結する
方法などが採用される。融合遺伝子化に用いる複数の遺
伝子結合領域に、それぞれの遺伝子翻訳産物が適切に機
能するコンフォメーションがとれるように人工的なヌク
レオチド配列を自由に挿入することもできる。たとえば
Gly Serをコードする5’ GGGTCC 3’（図29の「GS」）
などである。Further, in the present invention, one gene selected from the IPP synthesis pathway-related enzyme genes or its mutant form is linked to another gene or its mutant form, and the produced polypeptide becomes a fusion protein. You can also do so. In the present invention, a gene constructed by combining two or more kinds of such genes so that the expression product becomes a fusion protein is referred to as a "fusion gene". The base sequence of the fusion gene can be considered to be an addition mutation type gene of about 1000 bases based on the base sequence of one wild type gene, and the polypeptide encoded by it has an addition mutation of 300 amino acid residues or more added. It can be regarded as a type polypeptide. To prepare a fusion gene, one DNA is cleaved with an appropriate restriction enzyme, and the other DNA cleaved with the same restriction enzyme is added to the reading frame of the amino acid sequence of the protein encoded by the DNA. A method of connecting them so that they do not shift is adopted. An artificial nucleotide sequence may be freely inserted into a plurality of gene binding regions used for fusion gene conversion so that each gene translation product has a conformation in which it functions properly. For example
5'GGGTCC 3'coding Gly Ser ("GS" in Figure 29)
And so on.

【００５３】さらに、本発明においては、IPP合成経路
関連酵素遺伝子若しくはその変異型遺伝子又は前記融合
遺伝子の発現により生産されるタンパク質が小胞体に移
行するように改変したポリペプチドをコードする遺伝子
を作製し利用することもできる。小胞体移行型ポリペプ
チドとしては、C末端の小胞体保留シグナル（ER retent
ion signal）としてサッカロマイセス・セレビシアエで
はHis Asp Glu Leu（配列番号30））又はAsp Asp Glu L
eu（配列番号31）、一般的な真核生物のC末端シグナル
としてはLys Asp Glu Leu配列（配列番号32）が知ら
れ、機能は全く同等である小胞体保留シグナル（ER ret
ension signal）として働くことがわかっている（B.Lew
in “Genes V” (1994), Oxford University Press, Ne
w York, U.S.A., pp.279-318; B. Alberts et al. “Mo
lecular Biology of The Cell, third edtion” (199
4), Garland Publishing. Inc., New York, U.S.A., §
12−§13）。またこれらC末端4アミノ酸残基による小胞
体保留シグナル以外のシステムによる小胞体移行蛋白質
の一部分のドメイン、例えば小胞体への輸送シグナルと
して機能するN末端側のシグナルペプチド +H3N Met Met
Ser Phe Val Ser LeuLeu Leu Val Gly Ile Leu Phe Tr
p Ala Thr Glu Ala Glu Gln Leu Thr Lys CysGlu Val P
he Gln（配列番号33）などを用いることもできる（B.Le
win “GenesV” (1994), Oxford University Press, Ne
w York, U.S.A., pp.279-318; B. Alberts et al. “Mo
lecular Biology of The Cell, third edtion” (199
4), Garland Publishing. Inc., New York, U.S.A., §
12−§13）。小胞体シグナルを付加、置換又は挿入させ
たポリペプチドは、野生型ポリペプチドにさらに4アミ
ノ酸残基程度を付加、置換又は挿入した変異型ポリペプ
チドと見ることもでき、ポリペプチドをコードする遺伝
子は、野生型の遺伝子にさらに12塩基程度のヌクレオチ
ド配列を付加させた付加又は置換変異型遺伝子と見るこ
ともできる。Furthermore, in the present invention, a gene encoding a polypeptide modified so that the protein produced by the expression of the IPP synthesis pathway-related enzyme gene or its mutant gene or the fusion gene is transferred to the endoplasmic reticulum is prepared. You can also use it. As an endoplasmic reticulum-translocation-type polypeptide, the C-terminal endoplasmic reticulum retention signal (ER retent
(Is signal as His Asp Glu Leu (SEQ ID NO: 30)) or Asp Asp Glu L in Saccharomyces cerevisiae
eu (SEQ ID NO: 31), Lys Asp Glu Leu sequence (SEQ ID NO: 32) is known as a general eukaryotic C-terminal signal, and has the same function as the endoplasmic reticulum retention signal (ER ret).
It is known to work as a tension signal (B.Lew
in “Genes V” (1994), Oxford University Press, Ne
w York, USA, pp.279-318; B. Alberts et al. “Mo
lecular Biology of The Cell, third edtion ”(199
4), Garland Publishing. Inc., New York, USA, §
12-§13). In addition, a part of the endoplasmic reticulum translocation protein by a system other than the endoplasmic reticulum retention signal by these C-terminal 4 amino acid residues, for example, the N-terminal signal peptide that functions as a transport signal to the endoplasmic reticulum + H 3 N Met Met
Ser Phe Val Ser LeuLeu Leu Val Gly Ile Leu Phe Tr
p Ala Thr Glu Ala Glu Gln Leu Thr Lys CysGlu Val P
he Gln (SEQ ID NO: 33) can also be used (B.Le.
win “GenesV” (1994), Oxford University Press, Ne
w York, USA, pp.279-318; B. Alberts et al. “Mo
lecular Biology of The Cell, third edtion ”(199
4), Garland Publishing. Inc., New York, USA, §
12-§13). A polypeptide having an endoplasmic reticulum signal added, substituted or inserted can also be seen as a mutant polypeptide in which about 4 amino acid residues are further added, substituted or inserted into the wild-type polypeptide, and the gene encoding the polypeptide is It can also be regarded as an addition or substitution mutant type gene in which a nucleotide sequence of about 12 bases is further added to the wild type gene.

【００５４】遺伝子組換えのために使用されるDNAは、
宿主細胞に遺伝的に保持される可能性のあるものであれ
ば特に限定されず、例えば、プラスミド DNA、バクテリ
オファージ、レトロトランスポゾンDNA、酵母人工染色
体DNA（YAC: yeast artificial chromosome）等が挙げ
られる。また、ゲノムインテグレーションによる導入の
ための発現用組換えDNA断片としては、複製機能は必要
ではなく、PCR法又は化学合成により作製されたものを
使用することも可能である。The DNA used for gene recombination is
There is no particular limitation as long as it can be genetically retained in the host cell, and examples thereof include plasmid DNA, bacteriophage, retrotransposon DNA, yeast artificial chromosome (YAC), and the like. Moreover, the recombinant DNA fragment for expression for introduction by genome integration need not have a replication function, and it is also possible to use one produced by the PCR method or chemical synthesis.

【００５５】プラスミドDNAとしては、例えばpRS413、p
RS414、pRS415、pRS416、YCp50、pAUR112又はpAUR123な
どのYCp型大腸菌-酵母シャトルベクター、pYES2又はYEp
13などのYEp型大腸菌-酵母シャトルベクター、pRS403、
pRS404、pRS405、pRS406、pAUR101又はpAUR135などのYI
p型大腸菌-酵母シャトルベクター、大腸菌由来のプラス
ミド（pBR322、pBR325、pUC18、pUC19、pUC118、pUC11
9、pTV118N、pTV119N、pBluescript、pHSG298、pHSG396
又はpTrc99AなどのColE系プラスミド、pACYC177又はpAC
YC184などのp15A系プラスミド、pMW118、pMW119、pMW21
8又はpMW219などのpSC101系プラスミド等）、枯草菌由
来のプラスミド（例えばpUB110、pTP5等)などが挙げら
れ、ファージDNAとしてはλファージ（Charon4A、Charo
n21A、EMBL3、EMBL4、λgt10、λgt11、λZAP）、φX17
4、M13mp18又はM13mp19等が挙げられる。レトロトラン
スポゾンとしては、Ty因子などが挙げられる。YAC用ベ
クターとしてはpYACC2などが挙げられる。As the plasmid DNA, for example, pRS413, pRS
YCp type E. coli-yeast shuttle vector such as RS414, pRS415, pRS416, YCp50, pAUR112 or pAUR123, pYES2 or YEp
YEp-type E. coli-yeast shuttle vector such as 13, pRS403,
YI such as pRS404, pRS405, pRS406, pAUR101 or pAUR135
p-type E. coli-yeast shuttle vector, plasmid derived from E. coli (pBR322, pBR325, pUC18, pUC19, pUC118, pUC11
9, pTV118N, pTV119N, pBluescript, pHSG298, pHSG396
Or ColE plasmid such as pTrc99A, pACYC177 or pAC
P15A-based plasmids such as YC184, pMW118, pMW119, pMW21
8 or pMW101 and other pSC101-based plasmids), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, etc.), and the like.
n21A, EMBL3, EMBL4, λgt10, λgt11, λZAP), φX17
4, M13mp18, M13mp19 and the like. Examples of the retrotransposon include Ty factor. Examples of YAC vectors include pYACC2.

【００５６】宿主への組換えDNAの導入には、選択マー
カー遺伝子を用いる場合が多いが、マーカー遺伝子に起
因しない組換え体選抜の何らかのアッセイ法がある場合
は、必ずしもマーカー遺伝子は必要ではない。IPP合成
経路関連酵素遺伝子、その変異型酵素遺伝子、これらの
融合遺伝子、又はそれらがコードするポリペプチドに小
胞体シグナルを付加、置換又は挿入したポリペプチドを
コードする遺伝子を転写発現させるための転写プロモー
ターは、恒常発現型プロモーター又は誘導発現型プロモ
ーターを用いることができる。恒常発現型プロモーター
とは、主要代謝経路に関わる遺伝子の転写プロモーター
を意味し、どの生育条件でも転写活性を有するプロモー
ターである。誘導発現型プロモーターとは、特定の生育
条件で転写が誘導されるプロモーターである。A selectable marker gene is often used to introduce a recombinant DNA into a host, but a marker gene is not always necessary if there is any assay method for recombinant selection that does not result from the marker gene. Transcriptional promoter for transcribing a gene encoding a polypeptide in which an endoplasmic reticulum signal is added, substituted or inserted into an IPP synthetic pathway-related enzyme gene, a mutant enzyme gene thereof, a fusion gene thereof, or a polypeptide encoded by them A constitutive expression type promoter or an inducible expression type promoter can be used. The constitutive expression promoter means a transcription promoter of a gene involved in a major metabolic pathway, and has a transcription activity under any growth condition. The inducible expression type promoter is a promoter in which transcription is induced under specific growth conditions.

【００５７】転写プロモーターは、酵母等の宿主中で活
性を持つものであればいずれを用いてもよい。例えば酵
母での発現用として、GAL1プロモーター、GAL10プロモ
ーター、TDH3(GAP)プロモーター、ADH1プロモーター、T
EF2プロモーター等を用いることができる。また、大腸
菌での発現用として、trp、lac、trc、tacなどのプロモ
ーターを用いることができる。さらに、所望によりエン
ハンサーなどのシスエレメント、スプライシングシグナ
ル、ポリA付加シグナル、選択マーカーなどを連結する
ことができる。なお、選択マーカーとしては、URA3、LE
U2、TRP1、HIS3、ADE2、LYS2などの栄養非要求性の表現
型を指標とするマーカー遺伝子や、Ampr、Tetr、Cmr、K
mr、AUR1-C、can1等の抗生物質耐性遺伝子が挙げられ
る。Any transcription promoter may be used as long as it has activity in a host such as yeast. For example, for expression in yeast, GAL1 promoter, GAL10 promoter, TDH3 ( GAP ) promoter, ADH1 promoter, T
EF2 promoter etc. can be used. Further, for expression in E. coli, promoters such as trp , lac , trc , tac can be used. Furthermore, if desired, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker and the like can be linked. As a selection marker, URA3 , LE
U2 , TRP1 , HIS3 , ADE2 , LYS2 and other marker genes that use auxotrophic phenotypes as indicators, Amp r , Tet r , Cm r , K
Examples include antibiotic resistance genes such as m r , AUR1-C , and can1 .

【００５８】転写ターミネーターは、酵母等の宿主中で
活性を持つものであればいずれの遺伝子に由来するター
ミネーターを用いてもよい。例えば酵母での発現用とし
て、ADH1ターミネーター、CYC1ターミネーター等を用い
ることができる。本発明において遺伝子導入用組換えDN
Aとして作製された発現ベクターは、プラスミド名の次
に遺伝子名を表示することで命名及び識別することがで
きる。プラスミドとしてpRS434GAP又はpRS435GAPを使用
したときの発現ベクター名とその構成との関係を表2に
示す。例えば、プラスミドpRS434GAPにHMG1遺伝子を連
結した場合は「pRS434GAP-HMG1」のように、プラスミド
名の次に遺伝子名を表示することとする。この記載は、
pRS434、pRS444、pRS435GAP 及びpRS445プラスミドのい
ずれの場合も、上記プロモーターとの組み合わせによ
り、同じ要領で記載することができる。The transcription terminator may be a terminator derived from any gene as long as it has activity in a host such as yeast. For example, for expression in yeast, ADH1 terminator, CYC1 terminator and the like can be used. Recombinant DN for gene transfer in the present invention
The expression vector prepared as A can be named and identified by displaying the gene name next to the plasmid name. Table 2 shows the relationship between the expression vector name and its constitution when pRS434GAP or pRS435GAP was used as the plasmid. For example, when the HMG1 gene is ligated to the plasmid pRS434GAP, the gene name is displayed next to the plasmid name, such as “pRS434GAP-HMG1”. This description is
In the case of any of the pRS434, pRS444, pRS435GAP and pRS445 plasmids, the same procedure can be used in combination with the above promoter.

【００５９】[0059]

【表２】 [Table 2]

【００６０】3．組換え体の作製 本発明の組換え体は、本発明の組換えDNAを、各種IPP合
成経路関連酵素遺伝子（変異型遺伝子、融合遺伝子、及
び小胞体シグナルを付加、置換又は挿入したポリペプチ
ドをコードする遺伝子を含む）が発現し得るように前記
変異株、例えばEUG株に導入することにより得ることが
できる。3. Preparation of Recombinant The recombinant of the present invention is a recombinant DNA of the present invention, various IPP synthesis pathway-related enzyme gene (mutant gene, fusion gene, and a polypeptide having an endoplasmic reticulum signal added, substituted or inserted. (Including the encoding gene) so that it can be expressed and introduced into the mutant strain, for example, EUG strain.

【００６１】遺伝子が宿主細胞に導入されたか否かの確
認は、PCR(polymerase chain reaction)法、サザンハイ
ブリダイゼーション法等により行うことができる。例え
ば、組換え体からDNAを調製し、導入DNA特異的プライマ
ーを設計してPCRを行う。その後は、PCR増幅産物につい
てアガロースゲル電気泳動、ポリアクリルアミドゲル電
気泳動又はキャピラリー電気泳動等を行い、臭化エチジ
ウム、SYBR Green液等により染色するか、あるいはUV検
出器でDNAを検出し、増幅産物を1本のバンド又はピーク
として検出することにより、導入DNAを確認する。ま
た、予め蛍光色素等により標識したプライマーを用いて
PCRを行い、増幅産物を検出することもできる。Whether or not the gene has been introduced into the host cell can be confirmed by PCR (polymerase chain reaction) method, Southern hybridization method, or the like. For example, DNA is prepared from a recombinant, a primer specific to the introduced DNA is designed, and PCR is performed. After that, the PCR amplification product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis, etc. and stained with ethidium bromide, SYBR Green solution, etc., or the DNA is detected with a UV detector and the amplification product The introduced DNA is confirmed by detecting as a single band or peak. Also, use a primer that has been labeled with a fluorescent dye in advance.
Amplification products can also be detected by performing PCR.

【００６２】4．プレニルアルコールの生産 本発明において、プレニルアルコールは、上記変異株、
例えばEUG株を培養し、その培養物から採取することに
より得ることができる。また上記変異株、例えばEUG株
にIPP合成経路関連酵素遺伝子（変異型遺伝子及び融合
遺伝子を含む）を導入して組換え体を得た後、この組換
え体を転写抑制条件下で培養し、その培養物から採取す
ることにより得ることができる。「培養物」とは、培養
上清のほか、培養細胞若しくは培養菌体自体又は細胞若
しくは菌体の破砕物のいずれをも意味するものである。
本発明の変異株、例えばEUG株又はその組換え体を培養
する方法は、宿主の培養に用いられる通常の方法に従っ
て行われる。プレニルアルコールとしては、例えばFO
H、NOH又はGGOHが挙げられる。上記プレニルアルコール
は、それぞれ単独又は混合物として上記培養物中に蓄積
される。4. Production of prenyl alcohol In the present invention, prenyl alcohol is the above mutant strain,
For example, it can be obtained by culturing the EUG strain and collecting it from the culture. In addition, after the IPP synthesis pathway-related enzyme gene (including a mutant gene and a fusion gene) is introduced into the mutant strain, for example, EUG strain, to obtain a recombinant, the recombinant is cultured under a transcription repressing condition, It can be obtained by collecting from the culture. The term “culture” means not only the culture supernatant, but also the cultured cells or cultured cells themselves or the disrupted cells or cells.
The method for culturing the mutant strain of the present invention, for example, the EUG strain or its recombinant is carried out according to the usual method used for culturing a host. Examples of prenyl alcohol include FO
H, NOH or GGOH. The prenyl alcohols are accumulated in the culture individually or as a mixture.

【００６３】微生物を宿主として得られた組換え体を培
養する培地は、微生物が資化し得る炭素源、窒素源、無
機塩類等を含有し、組換え体の培養を効率的に行うこと
ができる培地であれば、複合天然培地、合成培地のいず
れを用いてもよい。炭素源としては例えば糖源を用いる
ことができ、通常はグルコースを含む。糖源は0.1-20%
(w/v)、好ましくは1-7% (w/v)であり、そのうちグルコ
ースの含有量は10-100%(w/w)、好ましくは50-100% (w/
w)であり、培養条件（使用宿主の種類、導入された組換
えベクターの種類、培養日数等）により適宜設定でき
る。但し、グルコースと併用してガラクトース、フラク
トース、スクロース、ラフィノース、ラクトース、デン
プン等の炭水化物、酢酸、プロピオン酸等の有機酸、エ
タノール、プロパノール等のアルコール類を炭素源とし
て使用してもよい。この場合、グルコース含有培地で培
養する前にガラクトース含有培地で前培養することも可
能である。前培養するときのガラクトースの含有量は、
混在する炭素源によって様々な値を取りうるが、通常糖
源あたり50-100% (w/w)、好ましくは糖源あたり100%(w/
w)である。EUG株の場合はガラクトース含有かつグルコ
ース非含有培地での培養がスクアレン合成酵素遺伝子ER
G9非転写抑制条件であり、グルコース含有培地での培養
がスクアレン合成酵素遺伝子ERG9転写抑制条件であり、
グルコース含培地での培養で、翻訳活性のあるスクアレ
ン合成酵素転写産物量が減少する。The medium for culturing the recombinant obtained by using the microorganism as a host contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the microorganism, and can efficiently cultivate the recombinant. As the medium, either a complex natural medium or a synthetic medium may be used. As the carbon source, for example, a sugar source can be used and usually contains glucose. 0.1-20% sugar source
(w / v), preferably 1-7% (w / v), of which the glucose content is 10-100% (w / w), preferably 50-100% (w / v).
w), which can be appropriately set depending on the culture conditions (type of host used, type of recombinant vector introduced, number of culture days, etc.). However, in combination with glucose, carbohydrates such as galactose, fructose, sucrose, raffinose, lactose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol may be used as a carbon source. In this case, it is also possible to pre-culture in a galactose-containing medium before culturing in a glucose-containing medium. The content of galactose when pre-cultured,
It can take various values depending on the mixed carbon source, but usually 50-100% (w / w) per sugar source, preferably 100% (w / w) per sugar source.
w). In the case of the EUG strain, squalene synthase gene ER can be cultured in a medium containing galactose and not glucose.
G9 non-transcriptional repressing condition, culture in glucose-containing medium is squalene synthase gene ERG9 transcriptional repressing condition,
Culturing in a glucose-containing medium reduces the amount of squalene synthase transcript having translation activity.

【００６４】窒素源としては、アンモニア、塩化アンモ
ニウム、硫酸アンモニウム、酢酸アンモニウム、リン酸
アンモニウム等の無機酸若しくは有機酸のアンモニウム
塩又はその他の含窒素化合物が挙げられ、その他ペプト
ン、肉エキス、コーンスティープリカー、各種アミノ酸
等が挙げられる。無機物としては、リン酸第一カリウ
ム、リン酸第二カリウム、リン酸マグネシウム、硫酸マ
グネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マンガ
ン、硫酸銅、炭酸カルシウム等が挙げられる。培養は、
通常、振盪培養又は通気攪拌培養などの好気的条件下、
26℃-42℃、好ましくは30℃で2-7日行う。pHの調整は、
無機又は有機酸、アルカリ溶液等を用いて行う。Examples of the nitrogen source include ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, and other nitrogen-containing compounds. Others include peptone, meat extract and corn steep liquor. , Various amino acids and the like. Examples of the inorganic substance include potassium primary phosphate, potassium secondary phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like. The culture is
Usually, aerobic conditions such as shaking culture or aeration stirring culture,
It is carried out at 26 ° C-42 ° C, preferably 30 ° C for 2-7 days. To adjust the pH,
It is performed using an inorganic or organic acid, an alkaline solution, or the like.

【００６５】転写プロモーターとして誘導型転写プロモ
ーターを用いた発現ベクターを用いて遺伝子導入した組
換え体を培養する場合は、必要に応じて転写誘導物質
（インデューサー）を培地に添加してもよい。例えばGA
L1プロモーターを用いた場合、インデューサーとしてガ
ラクトースを使用することができる。上記培養条件で培
養すると、高収率でプレニルアルコールを生産すること
ができる。また、プレニルアルコールを大量培養するに
は、ジャーファーメンター培養装置等を用いることもで
きる。In the case of culturing a recombinant introduced with an expression vector using an inducible transcription promoter as a transcription promoter, a transcription inducer (inducer) may be added to the medium, if necessary. GA for example
When using the L1 promoter, galactose can be used as an inducer. By culturing under the above culturing conditions, prenyl alcohol can be produced in high yield. In addition, a jar fermenter culture device or the like can be used for large-scale culture of prenyl alcohol.

【００６６】本発明において作製したプレニルアルコー
ル生産型変異細胞は、スクアレン合成酵素遺伝子欠損株
（erg9株；ATCC64031）におけるエルゴステロールのよ
うに特別な必須培地添加成分が無くても、翻訳活性のあ
るスクアレン合成酵素遺伝子転写産物量を調節すること
により生育かつプレニルアルコール高生産が可能である
が、通常培地に、さらにテルペノイド、油脂、界面活性
剤等を添加したり、窒素源や炭素源濃度を高くしてプレ
ニルアルコールの生産効率をさらに高めることもでき
る。これらの添加剤としては以下のものを例示できる。The prenyl alcohol-producing mutant cells produced in the present invention have a squalene having a translation activity even if there is no special essential medium addition component such as ergosterol in the squalene synthase gene-deficient strain ( erg9 strain; ATCC64031). Growth and high production of prenyl alcohol can be achieved by controlling the amount of synthase gene transcripts.However, terpenoids, oils and fats, surfactants, etc. can be added to the normal medium and the concentration of nitrogen and carbon sources can be increased. The production efficiency of prenyl alcohol can be further increased. The following can be illustrated as these additives.

【００６７】 テルペノイド： スクアレン、トコフェロール、IPP、DMAPP 油脂： 大豆油、魚油、アーモンド油、オリーブ油 界面活性剤： タージトール、トリトンX-305、スパン85、アデカノール L G-109 (旭電化製)、アデカノールLG-294(旭電化製)、アデカノールLG-295S (旭 電化製)、アデカノールLG-297 (旭電化製)、アデカノールB-3009A (旭電化製)、 アデカプロニックL-61 (旭電化製)[0067]   Terpenoids: squalene, tocopherol, IPP, DMAPP   Fats and oils: soybean oil, fish oil, almond oil, olive oil   Surfactants: Taditol, Triton X-305, Span 85, Adecanol L G-109 (Asahi Denka), ADEKA NOL LG-294 (Asahi Denka), ADEKA NOL LG-295S (Asahi Denka) Denka), Adekanol LG-297 (Asahi Denka), Adekanol B-3009A (Asahi Denka), ADEKA PRONIC L-61 (Made by Asahi Denka)

【００６８】油脂濃度は0.01%(w/v)以上、好ましくは1-
3%(w/v)であり、界面活性剤濃度は0.005-1%(w/v)、好ま
しくは0.05-0.5%(w/v)であり、テルペノイド濃度は0.01
%(w/v)以上、好ましくは1-3%(w/v)である。培養後、プ
レニルアルコールが菌体内又は細胞内に生産される場合
には、ホモジナイザー処理などを施して菌体又は細胞を
破砕することにより目的のプレニルアルコールを採取す
る。また、細胞を破砕せずに有機溶媒等で直接抽出して
もよい。あるいは、本発明のプレニルアルコールが菌体
外又は細胞外に生産される場合には、培養液をそのまま
使用するか、遠心分離等により菌体又は細胞を除去す
る。その後、有機溶媒による抽出等により前記培養物中
からプレニルアルコールを採取し、必要に応じてさらに
各種クロマトグラフィー等を用いて単離精製することが
できる。本発明において、各EUG株のプレニルアルコー
ル生産量、並びにプレニルアルコール生産に関するEUG
株とベクターとの好ましい組み合わせを例示すると表3
の通りである。The fat and oil concentration is 0.01% (w / v) or more, preferably 1-
3% (w / v), surfactant concentration 0.005-1% (w / v), preferably 0.05-0.5% (w / v), terpenoid concentration 0.01.
% (w / v) or more, preferably 1-3% (w / v). After the culture, when prenyl alcohol is produced in the cells or cells, the desired prenyl alcohol is collected by subjecting the cells or cells to homogenization to disrupt the cells or cells. Alternatively, the cells may be directly extracted with an organic solvent or the like without crushing. Alternatively, when the prenyl alcohol of the present invention is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. After that, prenyl alcohol can be collected from the culture by extraction with an organic solvent and the like, and if necessary, further isolated and purified by various chromatography and the like. In the present invention, the amount of prenyl alcohol produced by each EUG strain, and EUG relating to prenyl alcohol production
Examples of preferable combinations of strains and vectors are shown in Table 3
Is the street.

【００６９】[0069]

【表３】 [Table 3]

【００７０】(i) A451株由来のEUG5又はEUG8をグルコ
ース培地であるYM7培地で培養すると8.5-9.1mg/lのFOH
を生産し、糖源や窒素源を高濃度にし油を加えたYPDO7r
ich培地で培養すると57.5mg/lのFOHを生産し、0.43mg/l
のNOH、1.65mg/lのGGOHを生産した。YPH499株由来のEUG
12をYM7培地で培養すると7.4-40.6mg/lのFOHを生産す
る。YPH500株由来のEUG24又はEUG27をYM7培地で培養す
ると4.9-18.0mg/lのFOHを生産し、YPDO7rich培地で培養
すると42.2mg/lのFOHを生産し、1.75mg/lのNOH、1.26mg
/lのGGOHを生産した。W303-1A由来のEUG36、W303-1B由
来のEUG64を同様にYPDO7rich培地で培養すると、FOHを
それぞれ43mg/l、101.7m/l生産した。各EUG株はガラク
トース培地で前培養した後転写抑制条件であるグルコー
ス含有培地で本培養した。(I) When EUG5 or EUG8 derived from the A451 strain was cultured in YM7 medium which is a glucose medium, 8.5-9.1 mg / l FOH was obtained.
YPDO7r produced by producing high concentration sugar and nitrogen sources and adding oil
When cultivated in ich medium, it produces 57.5 mg / l FOH, 0.43 mg / l
Produced NOH, 1.65 mg / l GGOH. EUG derived from YPH499 strain
Cultivation of 12 in YM7 medium produces 7.4-40.6 mg / l FOH. YUG500 strain-derived EUG24 or EUG27 produced 4.9-18.0 mg / l FOH when cultured in YM7 medium, 42.2 mg / l FOH produced when cultured in YPDO7rich medium, 1.75 mg / l NOH, 1.26 mg.
Produced / l GGOH. When EUG36 derived from W303-1A and EUG64 derived from W303-1B were similarly cultured in YPDO7rich medium, FOH was produced at 43 mg / l and 101.7 m / l, respectively. Each EUG strain was pre-cultured in a galactose medium and then main-cultured in a glucose-containing medium which was a transcription repressing condition.

【００７１】(ii) TDH3（GAP）プロモーター下流にHMG
-CoA還元酵素遺伝子HMG1を組み込んでプラスミドを作製
し、これを宿主であるEUG株に導入すると、FOH蓄積量が
さらに高くなり、株によってはGGOH蓄積量も増加した。
EUG5で67.9mg/lのFOH、2.68mg/lのGGOHを生産した。GGO
H蓄積量の増加はEUG12、EUG27でも見られどちらも最大
で2.05mg/lのGGOHを生産した。 (iii) HMG1置換変異型遺伝子HMG1’の欠失型遺伝子HMG
ΔxyyをEUG5に導入するとYM7培地培養条件で、HMGΔxyy
導入前の8.5mg/lより高いFOH生産性を示し、最大でHMG
Δ122の12.1mg/l FOHであった。(Ii) HMG downstream of the TDH3 ( GAP ) promoter
-When a plasmid was prepared by incorporating the CoA reductase gene HMG1 and introduced into a host EUG strain, the FOH accumulation amount became higher and the GGOH accumulation amount also increased depending on the strain.
EUG5 produced 67.9 mg / l FOH and 2.68 mg / l GGOH. GGO
Increased amount of H accumulation was also seen in EUG12 and EUG27, both of which produced 2.05 mg / l of GGOH at the maximum. (iii) HMG1 substitution mutation type gene HMG1 'deletion type gene HMG
Introducing Δxyy into EUG5, HMG Δxyy
Higher FOH productivity than 8.5 mg / l before introduction, maximum HMG
It was 12.1 mg / l FOH with Δ122.

【００７２】(iv) TDH3（GAP）プロモーター下流にGGP
P合成酵素遺伝子BTS1を組み込んでプラスミドを作製
し、これを宿主である各EUG株に導入すると、FOH、GGOH
が生産される。EUG5へのBTS1導入によりBTS1導入前のYM
7培地培養による8.5mg/lより高いFOH生産性（11.0mg/
l）を示した。EUG12株を宿主にするとYM7培地で1.6mg/l
のGGOHを生産し、EUG27株を宿主にするとYM7培地で1.5m
g /lのGGOHを生産する。YPDO7rich培地で培養すると、E
UG5で19.8mg/l GGOH、EUG36で26.9mg/l GGOH、EUG64で6
2.7mg/l GGOHの生産性を示した。小胞体シグナルHDELを
C末端に持つ変異型GGPP合成酵素をコードする変異型BTS
1遺伝子を導入すると野生型BTS1と比べFOH生産性がEUG1
2で上昇（2.6mg/l→9.7mg/l）した。(Iv) GGP downstream of the TDH3 ( GAP ) promoter
When a plasmid was prepared by incorporating the P synthase gene BTS1 and introduced into each host EUG strain, FOH, GGOH
Is produced. YM before introducing BTS1 by introducing BTS1 to EUG5
FOH productivity higher than 8.5 mg / l with 7 medium culture (11.0 mg / l
l) is shown. 1.6 mg / l in YM7 medium with EUG12 strain as host
GGOH produced by using EUG27 strain as a host
Produces g / l GGOH. When cultured in YPDO7rich medium, E
19.8 mg / l GGOH for UG5, 26.9 mg / l GGOH for EUG36, 6 for EUG64
The productivity of 2.7 mg / l GGOH was shown. ER signal HDEL
Mutant BTS encoding mutant GGPP synthase with C-terminal
When one gene is introduced, FOH productivity is EUG1 compared to wild type BTS1.
It increased in 2 (2.6 mg / l → 9.7 mg / l).

【００７３】(v) YPH499株にERG20を導入してもプレニ
ルアルコールの生産はほとんどないのに対し（0.05mg/l
以下）、EUG12を宿主にすると4.5mg/lのFOHを生産し、G
GOH生産にも効果があり6.6mg/l GGOHを生産した。HDEL
を付加した変異型ERG20導入するとNOH生産に効果がある
場合があり、EUG12で0.62mg/lのNOHを生産した。 (vi) BTS1-ERG20融合遺伝子、あるいは、融合遺伝子の
末端がHDEL配列をコードする様にした遺伝子をA451に導
入しても0.26mg/l程度のGGOH生産であるのに対し、EUG5
に導入すると、6.5-6.6mg/l GGOHを生産する（YM7培
地）。(V) Introducing ERG20 into the YPH499 strain produced almost no prenyl alcohol (0.05 mg / l
Below), when EUG12 is used as a host, 4.5 mg / l FOH is produced, and G
It also had an effect on GOH production and produced 6.6 mg / l GGOH. HDEL
Introducing a mutant ERG20 with added may have an effect on NOH production. EUG12 produced 0.62 mg / l NOH. (vi) A BTS1-ERG20 fusion gene or a gene in which the end of the fusion gene encodes the HDEL sequence was introduced into A451, but GGOH production was about 0.26 mg / l, whereas EUG5
When introduced into, it produces 6.5-6.6 mg / l GGOH (YM7 medium).

【００７４】(vii) ERG20-BTS1融合遺伝子、あるい
は、融合遺伝子の末端がHDEL配列をコードする様にした
遺伝子をEUG5に導入すると(vi)と同様に0.79-2.43mg/l
のGGOHを生産する。 (viii) ERG19を各EUG株に導入すると、どの株もそれほ
どFOH生産は向上していないように見えるが、培養液OD
600値あたりのFOH生産量を比較すると、YPH株由来のEUG
12、EUG24で2倍から数倍FOH生産効率がよくなってい
た。(Vii) When the ERG20-BTS1 fusion gene or a gene in which the end of the fusion gene encodes the HDEL sequence was introduced into EUG5, 0.79-2.43 mg / l was obtained as in (vi).
To produce GGOH. (viii) When ERG19 was introduced into each EUG strain, FOH production did not seem to improve so much in all strains, but culture medium OD
Comparing FOH production per 600 values, EUG derived from YPH strain
12, EUG24 was 2 to several times more efficient in FOH production.

【００７５】[0075]

【実施例】以下、実施例により本発明をさらに具体的に
説明する。但し、本発明はこれら実施例にその技術的範
囲が限定されるものではない。本実施例は、以下の内容
を包含する。 (1) ゲノムDNA中のスクアレン合成酵素遺伝子転写プロ
モーターを転写抑制型プロモーターGAL1転写プロモータ
ーで置換して変異株(EUG株)を作製し、プレニルアルコ
ール生産菌とする。EUG株を転写抑制条件(グルコース含
有培地)で培養し、プレニルアルコールの生産量を測定
する。〔実施例1-2〕 (2) EUG株への遺伝子導入のため、Stratagene社のpRS
系各ベクター、Invitrogen社のpYES、又はS. cerevisia
e由来のゲノムDNAをもとにして、発現ベクターpRS435GA
Pなどを作製する。〔実施例3〕EXAMPLES The present invention will be described in more detail below with reference to examples. However, the technical scope of the present invention is not limited to these examples. This example includes the following contents. (1) A mutant strain (EUG strain) is prepared by replacing the squalene synthase gene transcription promoter in genomic DNA with a transcriptional repressible promoter GAL1 transcription promoter, and used as a prenyl alcohol-producing bacterium. The EUG strain is cultured under a transcription repressing condition (glucose-containing medium), and the amount of prenyl alcohol produced is measured. [Example 1-2] (2) pRS of Stratagene for gene introduction into EUG strain
System vector, pYES from Invitrogen, or S. cerevisia
Expression vector pRS435GA based on e- derived genomic DNA
Create P etc. Example 3

【００７６】(3) IPP合成経路関連酵素遺伝子のクロー
ニング 上記EUG株への導入用遺伝子として、HMG-CoA還元酵素遺
伝子、メバロン酸二リン酸脱炭酸酵素遺伝子、FPP合成
酵素遺伝子、GGPP合成酵素遺伝子、又はこれらの変異型
遺伝子をクローニングし、発現用プラスミドを作製す
る。〔実施例4-6,8〕 (4) (3)の各種遺伝子を導入して得た組換え体を転写抑
制条件(グルコース培地)で培養し、プレニルアルコール
の生産量を測定する。〔実施例7-8〕 (5) グルコース濃度を変化させた培地、あるいは、糖
源・窒素源や油添加物を加えた培地でEUG株を培養し、
プレニルアルコール生産に好適な条件を検討する。〔実
施例9-10〕 〔実施例1〕 各EUG株の作製 SGDから、スクアレン合成酵素遺伝子ERG9付近の遺伝子
地図を引き出し、ERG9転写プロモーター (ERG9p) 置換
用DNA断片増幅のためのPCRプライマーDNAを設計した
（図2）。形質転換体選択マーカー遺伝子URA3と転写プ
ロモーターGAL1pを含む1.8 kbp DNA断片（図2A(2)）
は、pYES2（図2C）をNaeIとNheIで切断後Klenow酵素で
平滑末端化し、セルフライゲーションにより2μ ori部
分を削除したpYES2Δ（図2B）を鋳型にしてPCR増幅によ
り調製した。PCRで使用したプライマーは以下の通りで
ある。(3) Cloning of IPP synthesis pathway-related enzyme gene As a gene for introduction into the above EUG strain, HMG-CoA reductase gene, mevalonate diphosphate decarboxylase gene, FPP synthase gene, GGPP synthase gene Alternatively, these mutant genes are cloned to prepare an expression plasmid. [Examples 4-6, 8] (4) The recombinants obtained by introducing the various genes of (3) are cultured under the transcription repressing condition (glucose medium), and the production amount of prenyl alcohol is measured. [Example 7-8] (5) a medium having a changed glucose concentration, or culturing the EUG strain in a medium containing a sugar source / nitrogen source or an oil additive,
Consider suitable conditions for prenyl alcohol production. [Example 9-10] [Example 1] Preparation of each EUG strain From SGD, a gene map in the vicinity of the squalene synthase gene ERG9 was extracted, and PCR primer DNA for amplification of ERG9 transcription promoter ( ERG9 p) substitution DNA fragment Was designed (Fig. 2). 1.8 kbp DNA fragment containing transformant selectable marker gene URA3 and transcription promoter GAL1 p (Fig. 2A (2))
Was prepared by PCR amplification using pYES2Δ (FIG. 2B), in which pYES2Δ (FIG. 2C) was cleaved with Nae I and Nhe I, blunt-ended with Klenow enzyme, and the 2 μ ori portion was deleted by self-ligation, as a template. The primers used in PCR are as follows.

【００７７】E-MCSf：5'- GCC GTT GAC AGA GGG TCC GA
G CTC GGT ACC AAG -3'（配列番号34） E-URA3r：5'- CAT ACT GAC CCA TTG TCA ATG GGT AAT A
AC TGA T -3'（配列番号35）E-MCSf: 5'- GCC GTT GAC AGA GGG TC C GA
G CTC GGT ACC AAG -3 '(SEQ ID NO: 34) E-URA3r: 5'- CAT ACT GAC CCA TTG TC A ATG GGT AAT A
AC TGA T -3 '(SEQ ID NO: 35)

【００７８】上記プライマーには、S.cerevisiae ゲノ
ム中のオープンリーディングフレームYHR189Wの下流部
分を含む0.7 kbpDNA断片と、ERG9の上流部分を含む 0.9
kbpDNA断片とでT/AライゲーションできるようにEam110
5I認識部位（配列中、下線部分）を加えてある。YHR189
W断片は、PCRプライマーYHR189WfとYHR189Wrを用いてYP
H499ゲノムDNAを鋳型にPCRにより調製し、ERG9断片は、
PCRプライマーERG9fとERG9rを用いてYPH499ゲノムDNAを
鋳型にPCRにより調製した。YPH499ゲノムDNAは、酵母ゲ
ノムDNA調製用キット「Genとるくん」（宝酒造）を用い
て調製した。The above-mentioned primers contained a 0.7 kbp DNA fragment containing the downstream part of the open reading frame YHR189W in the S. cerevisiae genome and 0.9 part containing the upstream part of ERG9.
Eam 110 to allow T / A ligation between kbpDNA fragment
A 5I recognition site (underlined in the sequence) is added. YHR189
The W fragment was isolated using the PCR primers YHR189Wf and YHR189Wr.
The H499 Genomic DNA was prepared by PCR as a template, ERG9 fragments,
Using the PCR primers ERG9f and ERG9r, YPH499 genomic DNA was prepared as a template by PCR. YPH499 genomic DNA was prepared using a yeast genomic DNA preparation kit "Gen Torukun" (Takara Shuzo).

【００７９】YHR189Wf：5'-TGT CCG GTA AAT GGA GAC-
3'(配列番号36) YHR189Wr：5'-TGT TCT CGC TGC TCG TTT-3'(配列番号3
7) ERG9f：5'-ATG GGA AAG CTA TTA CAA T-3'(配列番号38) ERG9r：5'-CAA GGT TGC AAT GGC CAT-3'(配列番号39)YHR189Wf: 5'-TGT CCG GTA AAT GGA GAC-
3 '(SEQ ID NO: 36) YHR189Wr: 5'-TGT TCT CGC TGC TCG TTT-3' (SEQ ID NO: 3
7) ERG9f: 5'-ATG GGA AAG CTA TTA CAA T-3 '(SEQ ID NO: 38) ERG9r: 5'-CAA GGT TGC AAT GGC CAT-3' (SEQ ID NO: 39)

【００８０】1.8 kbp DNA断片をEam1105Iで消化後、0.7
kbpDNA断片とライゲーションし、これを鋳型にしてプ
ライマーYHR189WfとE-MCSfを用いて2nd PCRを行った。
増幅した2.5 kbp DNA断片（図2A(5)）をEam1105Iで消化
後、0.9 kbp DNA断片とライゲーションし、これを鋳型
にしてプライマーYHR189W-3fとERG9-2rを用いて3rd PCR
を行った。増幅した3.4 kbp DNA断片を形質転換用DNA断
片とした。After digesting the 1.8 kbp DNA fragment with Eam 1105I,
It was ligated with a kbp DNA fragment, and using this as a template, 2nd PCR was carried out using the primers YHR189Wf and E-MCSf.
The amplified 2.5 kbp DNA fragment (Fig. 2A (5)) was digested with Eam 1105I, ligated with the 0.9 kbp DNA fragment, and this was used as a template for 3rd PCR using primers YHR189W-3f and ERG9-2r.
I went. The amplified 3.4 kbp DNA fragment was used as a transforming DNA fragment.

【００８１】YHR189W-3f：5'-CAA TGT AGG GCT ATA TAT
G-3'(配列番号40) ERG9-2r：5'-AAC TTG GGG AAT GGC ACA-3'(配列番号41) Zymo Research (Orange, CA) より購入したFrozen EZ y
east transformationII kitを用いて酵母に形質転換用D
NA断片の導入を行った。形質転換用DNA断片導入用宿主
として、A451、YPH499、YPH500、W303-1A及びW303-1Bを
用いた。YHR189W-3f: 5'-CAA TGT AGG GCT ATA TAT
G-3 '(SEQ ID NO: 40) ERG9-2r: 5'-AAC TTG GGG AAT GGC ACA-3' (SEQ ID NO: 41) Frozen EZ y purchased from Zymo Research (Orange, CA)
Use the east transformationII kit to transform yeast into yeast D
The NA fragment was introduced. A451, YPH499, YPH500, W303-1A and W303-1B were used as hosts for introducing the DNA fragment for transformation.

【００８２】組換え体は、SGR培地（SD(synthetic dext
rose)培地のグルコース成分をガラクトースとラフィノ
ースで置き換えたもの）をベースにし、CSM-URA (BIO 1
01 (Vista, CA) より購入) と、アデニン硫酸塩（終濃
度40 mg/l）とを加えた寒天培地（SGR-U培地）上で30℃
で培養し、生育してきた菌体をもう一度同じ培地に広
げ、培養しシングルコロニーアイソレーションを行っ
た。取得した組換え体は、EUG (ERG9p::URA3-GAL1p) 株
と名付け、A451由来のクローンをEUG1-10、YPH499由来
のクローンをEUG11-20、YPH500由来のクローンをEUG21-
30、W303-1A由来のクローンをEUG31-50、W303-1B由来の
クローンをEUG51-70とした。Recombinants were transformed into SGR medium (SD (synthetic dext
rose) medium with glucose components replaced with galactose and raffinose), and CSM-URA (BIO 1
01 (purchased from Vista, CA) and adenine sulfate (final concentration 40 mg / l) on agar medium (SGR-U medium) at 30 ℃
After culturing in, the grown bacterial cells were once again spread on the same medium and cultured to carry out single colony isolation. The obtained recombinants were named EUG ( ERG9 p :: URA3 - GAL1 p) strain, the A451-derived clone was EUG1-10, the YPH499-derived clone was EUG11-20, and the YPH500-derived clone was EUG21-.
The clones derived from 30, W303-1A were designated as EUG31-50 and the clones derived from W303-1B were designated as EUG51-70.

【００８３】SD培地でグルコースリプレッションにより
翻訳活性のあるスクアレン合成酵素遺伝子転写産物量が
減少し、増殖速度が低下したクローンを選抜し、A451か
らEUG8が、YPH499からEUG12が、YPH500からEUG27が得ら
れた。SD-U寒天培地とSGR-U寒天培地上での生育状態の
代表例を図5に示す。SGR-Uプレート（図5右パネル）で
は正常に生育するがグルコースリプレッションによりGA
L1プロモーター制御下のスクアレン合成酵素遺伝子転写
抑制された結果、翻訳活性のあるスクアレン合成酵素遺
伝子転写産物量が減少し、SD-Uプレート上（図5左パネ
ルSDで示した）では増殖速度が低下した株を得ることが
できた（図5ではEUG1-6のうちEUG5）。A clone in which the amount of squalene synthase gene transcript having translation activity was decreased by glucose repression in SD medium and the growth rate was decreased was selected, and A451 to EUG8, YPH499 to EUG12, and YPH500 to EUG27 were obtained. Was given. Figure 5 shows a representative example of the growth state on SD-U agar and SGR-U agar. It grows normally on the SGR-U plate (Fig. 5, right panel), but GA by glucose repression.
As a result of suppression of squalene synthase gene transcription under the control of the L1 promoter, the amount of squalene synthase gene transcript with translation activity decreased, and the growth rate decreased on the SD-U plate (shown in SD in Fig. 5, left panel). Was obtained (EUG5 of EUG1-6 in FIG. 5).

【００８４】翻訳活性のあるスクアレン合成酵素遺伝子
転写産物量が減少し、増殖速度が低下した組換え体のう
ち、EUG8、EUG12、EUG27から「Genとるくん」を用いて
ゲノムDNAを調製し、これを鋳型にし、YHR189WfとERG9-
2 (5'- TCA CGC TCT GTG TAAAGT GTA TA-3'(配列番号4
2)) をプライマーに用いてPCRを行い、増幅したDNA断片
の大きさをアガロースゲル電気泳動で調べた結果、URA3
とGAL1pを含む1.8 kbpのPCR断片がゲノムのスクアレン
合成酵素遺伝子座にインテグレートされていることが確
認できた。Among recombinants in which the amount of squalene synthase gene transcript having translation activity was decreased and the growth rate was decreased, genomic DNA was prepared from EUG8, EUG12, EUG27 using "Gen Torukun". With YHR189Wf and ERG9-
2 (5'- TCA CGC TCT GTG TAAAGT GTA TA-3 '(SEQ ID NO: 4
2)) was subjected to PCR using the primers, results of the size of the amplified DNA fragment was examined by agarose gel electrophoresis, URA3
It was confirmed that a 1.8-kbp PCR fragment containing GAL1 p and GAL1 p was integrated into the genomic squalene synthase locus.

【００８５】〔実施例2〕 各EUG株によるプレニルアル
コール生産 (1) プレニルアルコール生産量測定-1 (1-1) 培養 EUG8、EUG12、EUG27をSGR-U培地で前培養し、0.3 mlの
前培養液を30 mlのYM7培地、YME培地又はYM7(Gal)培地
にそれぞれ加え、300 ml 三角フラスコで30℃ 130 r.p.
m.の往復振盪培養で培養した。YM7培地とは、YM培地をN
aOHでpH7に調製した培地である。YME培地とは、YM7培地
に50 mg/lのエルゴステロールを加えた培地であってエ
タノール-タージトール溶液（50 mg/mlのストック液）
を1000分の一添加したものである。YM7(Gal)培地とは、
YM7培地のグルコースをガラクトースで置き換えたもの
である。Example 2 Production of prenyl alcohol by each EUG strain (1) Measurement of production amount of prenyl alcohol-1 (1-1) Culture EUG8, EUG12 and EUG27 were pre-cultured in SGR-U medium to give 0.3 ml of the medium. Add the culture broth to 30 ml of YM7 medium, YME medium or YM7 (Gal) medium, and in a 300 ml Erlenmeyer flask at 30 ℃ 130 rp.
The cells were cultured in a reciprocal shaking culture of m. YM7 medium is YM medium
It is a medium adjusted to pH 7 with aOH. YME medium is a medium in which 50 mg / l ergosterol is added to YM7 medium and is an ethanol-terditol solution (50 mg / ml stock solution).
Is added to 1/1000. What is YM7 (Gal) medium?
The glucose in YM7 medium was replaced with galactose.

【００８６】培養開始後、1日、2日、3日、4日、7日、8
日、9日目に2.5 mlの培養液を取り出しプレニルアルコ
ール生産量を定量した。すなわち、培養液にメタノール
を2.5 ml 加えて混合し、ペンタンを約5 ml 加えて激し
く攪拌後静置した。ペンタン層を新しいガラス試験管に
とり、ドラフト中でペンタンを気化させ溶質成分を濃縮
後、ガスクロマトグラフィー/質量分析（GC/MS）でプレ
ニルアルコールを同定、定量した。また、その際、菌体
増殖の度合いを調べるため、培養液50μlを水で30倍に
希釈し、600 nmの吸光度を測定した。After the start of culturing, 1 day, 2 days, 3 days, 4 days, 7 days, 8 days
On the 9th and 9th days, 2.5 ml of the culture solution was taken out and the amount of prenyl alcohol produced was quantified. That is, 2.5 ml of methanol was added to and mixed with the culture solution, about 5 ml of pentane was added, and the mixture was vigorously stirred and allowed to stand. The pentane layer was placed in a new glass test tube, pentane was vaporized in a draft, the solute components were concentrated, and then prenyl alcohol was identified and quantified by gas chromatography / mass spectrometry (GC / MS). At that time, in order to examine the degree of bacterial cell growth, 50 μl of the culture solution was diluted 30 times with water and the absorbance at 600 nm was measured.

【００８７】(1-2) GC/MS解析 ペンタン抽出画分をHP6890/5973 GC/MSシステム (Hewle
tt-Packard, Wilmington, DE) を用いて分離、同定、定
量した。使用カラムはHP-5MS (0.25 mm x 30 m、フィル
ム厚0.25 μm)であり、分析条件は以下の通りである。
本明細書中でのGC/MS分析条件は、全て同様である。(1-2) GC / MS analysis The pentane extraction fraction was analyzed using the HP 6890/5973 GC / MS system (Hewle
(tt-Packard, Wilmington, DE) was used for separation, identification and quantification. The column used is HP-5MS (0.25 mm x 30 m, film thickness 0.25 μm), and the analysis conditions are as follows.
All the GC / MS analysis conditions used herein are the same.

【００８８】インレット温度 250℃ 検出器温度 260℃ [MS ゾーン温度] MS Quad 150℃ MS Source 230℃ マススキャン範囲35-200 [インジェクションパラメーター] 自動インジェクションモード サンプル容量 2 μl 3回のメタノール洗浄及び2回のヘキサン洗浄 スプリット比 1/20 キャリヤーガス ヘリウム 1.0 ml/分 溶媒遅延 2 分 [オーブン加熱条件] 115℃90 秒 70℃/分で250℃まで加熱し、2分間維持 70℃/分で300℃まで加熱し、7分間維持 内部標準 エタノール中0.01 μl 1-ウンデカノール 信頼標準 (all-E)-ネロリドール (エーザイ) (all-E)-ファルネソール (Sigma) (all-E)-ゲラニルゲラニオール (エーザイ) スクアレン (東京化成工業)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 parameters] Automatic injection mode Sample volume 2 μl 3 times methanol wash and 2 times Hexane wash split ratio 1/20 Carrier gas Helium 1.0 ml / min Solvent delay 2 min [Oven heating conditions] 115 ° C 90 seconds 70 ° C / min heated to 250 ° C, maintained for 2 min 70 ° C / min up to 300 ° C Heat and hold for 7 minutes Internal standard 0.01 μl in ethanol 1-Undecanol Confidence standard ( all-E ) -nerolidol (Eisai) ( all-E ) -farnesol (Sigma) ( all-E ) -geranylgeraniol (Eisai) squalene (Tokyo Chemical Industry)

【００８９】(2) プレニルアルコール生産量測定-2 SD培地で増殖速度が低下したEUG株を1-5 mlのYM7培地中
で1-7日間、30℃ 130r.p.m.の往復振盪培養で培養し
た。培養開始後、1日、2日、3日、4日、7日に0.8-2.5 m
lの培養液を取り出し、前記(1)と同様にしてプレニルア
ルコール量を定量した。(2) Measurement of production amount of prenyl alcohol-2 EUG strain having a reduced growth rate in SD medium was cultivated in 1-5 ml of YM7 medium for 1-7 days by reciprocal shaking culture at 30 ° C. 130 rpm. . 0.8-2.5 m on the 1st, 2nd, 3rd, 4th and 7th days after the start of culture
The culture broth of 1 was taken out, and the amount of prenyl alcohol was quantified in the same manner as in (1) above.

【００９０】(3) 糖組成によるプレニルアルコール生産
量変化 YM7培地のグルコース (Glc) 成分を表4に示すように変
え、まず2日間30℃ 130r.p.m.の往復振盪培養で培養し
たのち、表5に示すように、YMGlc培地としては5%(w/v)G
lcを、YMGal培地としては5%(w/v)ガラクトース (Gal)を
加えさらに培養を7日目まで継続した。YMrich培地では
培養開始からYM7の全糖濃度を 6%(w/v)とし、その成分
は表4に示すようにしてある。結局、培地成分と培養時
間との関係は表5に示すようになる。培養開始後、2日、
4日、7日に2.5 mlの培養液を取り出し前記(1)と同様に
してプレニルアルコール生産量を定量した。(3) Changes in the amount of prenyl alcohol produced depending on the sugar composition The glucose (Glc) component of the YM7 medium was changed as shown in Table 4, and the cells were first cultivated for 2 days at 30 ° C. and 130 rpm in reciprocal shaking culture, and then Table 5 5% (w / v) G for YMGlc medium
lc and 5% (w / v) galactose (Gal) as a YMGal medium were added, and the culture was further continued until the 7th day. In YMrich medium, the total sugar concentration of YM7 was set to 6% (w / v) from the start of culture, and its components are shown in Table 4. Eventually, the relationship between the medium components and the culture time is as shown in Table 5. 2 days after the start of culture,
On the 4th and 7th days, 2.5 ml of the culture solution was taken out and the amount of prenyl alcohol produced was quantified in the same manner as in the above (1).

【００９１】[0091]

【表４】 [Table 4]

【表５】 [Table 5]

【００９２】(4) 結果と考察 (4-1) EUG株によるプレニルアルコール生産 A451、YPH499、YPH500由来でGlc増殖速度低下がみら
れ、ゲノムへのインテグレーションも完全に行われたEU
G株（それぞれEUG8、EUG12、EU27）を選び、プレニルア
ルコール生産量を測定した。結果を図6に示す。Glcを炭
素源としたYM7、YME培地で培養するとFOHを生産する系
が得られた。エルゴステロールを含むYME培地で培養し
た場合、EUG12とEUG27でFOH濃度が上昇する傾向が見ら
れ、それぞれ最大で40.6mg/l、18.0mg/lのFOHを蓄積し
た。A451株由来のEUG8と、YPH499株由来のEUG12及びYPH
500株由来のEUG27との間では生産プロファイルが異な
り、YPH系の株の方がより生産に適していると考えられ
る。そして、従来EUG9欠損株(erg9株： ATCC64031)で生
育に必須だったエルゴステロール(Erg)を添加せずに一
般的なYM7培地で4日培養するだけで30 mg/l程度のFOHを
生産する酵母が開発できた（図6のEUG12）。(4) Results and Discussion (4-1) Production of prenyl alcohol by EUG strain A451, YPH499, YPH500-derived Glc growth rate decrease was observed, and the integration into the genome was also completely performed in the EU.
G strains (EUG8, EUG12, EU27, respectively) were selected and the amount of prenyl alcohol produced was measured. The results are shown in Fig. 6. A system producing FOH was obtained by culturing in YM7 and YME medium using Glc as a carbon source. When cultured in YME medium containing ergosterol, the FOH concentration tended to increase in EUG12 and EUG27, and the maximum FOH concentrations were 40.6 mg / l and 18.0 mg / l, respectively. EUG8 from A451 strain and EUG12 and YPH from YPH499 strain
The production profile is different from that of EUG27 derived from 500 strains, and it is considered that the YPH strain is more suitable for production. Then, without adding ergosterol (Erg), which was essential for growth in the conventional EUG9- deficient strain ( erg9 strain: ATCC64031), it is cultivated in a general YM7 medium for 4 days to produce about 30 mg / l of FOH. Yeast was developed (EUG12 in Figure 6).

【００９３】次にEUG1-70のうちSD培地で増殖速度低下
のみられたクローンを培養し、プレニルアルコール生産
量を比較した（図7-11）。FOH生産量は、A451株由来の
もの（図7）ではEUG5（8.5 mg/l）、YPH499由来のもの
（図8）ではEUG12（17.8 mg/l）、YPH500由来のもの
（図9）ではEUG24（17.9 mg/l）及びEUG27（10.5 mg/
l）が、他のクローンと比較して高かった。また、W303-
1A由来のもの（図10）及びW303-1B由来のもの（図11）
では、それぞれの図に示す株が他のクローンと比較して
高かった。Next, among EUG1-70, clones showing a reduced growth rate in SD medium were cultured, and the amount of prenyl alcohol produced was compared (FIG. 7-11). The amount of FOH produced was from strain A451 (Fig. 7) EUG5 (8.5 mg / l), from YPH499 (Fig. 8) EUG12 (17.8 mg / l), and from YPH500 (Fig. 9) EUG24. (17.9 mg / l) and EUG27 (10.5 mg / l
l) was higher than other clones. Also, W303-
Derived from 1A (Fig. 10) and derived from W303-1B (Fig. 11)
Then, the strains shown in each figure were higher than the other clones.

【００９４】W303-1A由来：EUG31（16mg/l）、EUG33（4
1mg/l）、EUG35（38mg/l）、EUG36（43mg/l）、EUG38
（17mg/l）、EUG39（24mg/l）、EUG42（24mg/l）、EUG4
4（21mg/l）、EUG49（21mg/l） W303-1B由来：EUG51（28mg/l）、EUG56（16mg/l）、EUG
59（37mg/l）、EUG61（27mg/l）、EUG63（16mg/l）、EU
G64（38mg/l）Derived from W303-1A: EUG31 (16 mg / l), EUG33 (4
1mg / l), EUG35 (38mg / l), EUG36 (43mg / l), EUG38
(17mg / l), EUG39 (24mg / l), EUG42 (24mg / l), EUG4
4 (21 mg / l), EUG49 (21 mg / l) W303-1B origin: EUG51 (28 mg / l), EUG56 (16 mg / l), EUG
59 (37mg / l), EUG61 (27mg / l), EUG63 (16mg / l), EU
G64 (38mg / l)

【００９５】(4-2) グルコース濃度変化による生産性の
違い GlcとGalの糖濃度を変えてEUG8、EUG12、EUG27を培養し
た時のプレニルアルコール生産量を図12-14に示す。生
産物質はほとんどFOHのみであるが、A451由来株のEUG8
（図12）と、YPH由来株のEUG12（図13）及びEUG27（図1
4）との間では生産プロファイルが異なることがわか
る。EUG8ではいずれの条件でも初期培地条件の糖成分が
100% Glcのときに生産量が最大であったが、EUG12及びE
UG27では初期培地条件の糖成分が75%Glcの時に生産量が
よく、また、初期培地条件の糖成分が75% Glc (1%(w/v)
sugar)の後、2日目に終濃度5%(w/v)になるようにさら
にGlcを添加した条件でFOHをよく生産した。初期培地条
件の糖成分が75%Glc条件下で生育に最低限のERG9転写を
行わせることによって細胞増殖とFOHのバランスがちょ
うどよい条件になったと考えられる。YMGal培養条件に
おいて4日目以降にFOH生産が減少するのは、Gal添加でE
RG9の転写が促進され、Ergが細胞内で合成されるに従っ
て培地中のFOHを資化するためであることが予想され
る。FOH生産についてのみまとめた結果（図15）をみる
と、上記A451由来株EUG8と、YPH由来株EUG12及びEUG27
との生産プロファイルの違いがわかりやすい。また、
「初期条件の糖成分が75% Glc (1%(w/v) sugar)のの
ち、2日目に終濃度5%(w/v)になるようにさらにGlcを添
加した条件」ではOD600値がほとんど変わらないのに対
し、2日目から4日目、7日目とFOH生産量が増加している
ことから、このときの細胞は細胞***なしにFOHを生産
する生理的な状態であったと予想できる。このような菌
体を固定化できればFOH生産の効率よい系が開発できる
可能性が示された。(4-2) Difference in productivity due to changes in glucose concentration Fig. 12-14 shows the amount of prenyl alcohol produced when EUG8, EUG12, and EUG27 were cultured by changing the sugar concentrations of Glc and Gal. Almost all FOH is produced, but the A451-derived strain EUG8
(Fig. 12) and YUG-derived strains EUG12 (Fig. 13) and EUG27 (Fig. 1).
It can be seen that the production profile is different between 4). With EUG8, the sugar component of the initial medium condition was
Production was highest at 100% Glc, but EUG12 and E
In UG27, when the sugar component in the initial medium condition was 75% Glc, the production was good, and the sugar component in the initial medium condition was 75% Glc (1% (w / v).
FOH was well produced under the condition that Glc was further added to the final concentration of 5% (w / v) on the second day after (sugar). It is considered that the sugar component in the initial medium condition brought about the minimum ERG9 transcription for the growth under the condition of 75% Glc, and thus the condition in which the cell growth and FOH were balanced was just right. In YMGal culture conditions, the FOH production decreases after the 4th day because the addition of Gal causes E
It is expected that this is because transcription of RG9 is promoted, and Erg assimilates FOH in the medium as it is synthesized intracellularly. Looking at the results (Fig. 15) that summarizes only FOH production, the above A451-derived strain EUG8 and YPH-derived strains EUG12 and EUG27 are shown.
It is easy to understand the difference in the production profile between. Also,
The OD 600 was `` 600 % Glc (1% (w / v) sugar) in the initial condition, and then Glc added to the final concentration of 5% (w / v) on the second day ''. The FOH production increases from the 2nd day to the 4th day and the 7th day, while the value remains almost unchanged.Therefore, the cells at this time are in the physiological state of producing FOH without cell division. It can be expected that there was. It has been shown that if such cells can be immobilized, an efficient system for FOH production can be developed.

【００９６】本実施例で示したEUG株の開発により、翻
訳活性のあるスクアレン合成酵素遺伝子の転写産物量を
減少させることができる。そして、EUG株は、スクアレ
ン合成酵素コード領域に置換、挿入または欠失の変異を
導入して作製したスクアレン合成酵素遺伝子欠損株、た
とえばATCC64031などと異なり、ステロール取りこみ能
付加やErg添加などのスクアレン合成酵素欠損による致
死性を相補する特別なメカニズムを導入することなしに
YM7などの一般的な培地で培養すれば活性型プレニルア
ルコールであるFOHを発酵生産する系を作製できること
がわかった。また、EUG株は通常遺伝子組換え宿主株を
元にしているので、ATCC64031などと異なり、さらに遺
伝子工学的な改変を行うことが簡便にできさらに生産性
の高い株の開発に応用できる。By developing the EUG strain shown in this Example, the amount of the transcription product of the squalene synthase gene having translation activity can be reduced. Then, the EUG strain is different from the squalene synthase gene-deficient strain prepared by introducing a mutation of substitution, insertion or deletion in the squalene synthase coding region, for example, ATCC64031 and the like, and squalene synthesis such as sterol uptake ability addition and Erg addition Without introducing a special mechanism to complement lethality due to enzyme deficiency
It was found that a system for fermentative production of FOH, which is an active prenyl alcohol, can be prepared by culturing in a general medium such as YM7. In addition, since the EUG strain is usually based on a gene recombinant host strain, unlike the ATCC64031, it can be easily modified by genetic engineering and can be applied to the development of strains with high productivity.

【００９７】〔実施例3〕 発現ベクターの構築 (1) E. coli-S. cerevisiaeシャトルベクター Stratagene社よりプラスミドpRS404、pRS405を購入し
た。Invitrogen社 (Carlsbad, CA) からpYES2（図3）を
購入した。 (2) ゲノムDNAS . cerevisiaeゲノムDNAは、「Genとるくん」を購入
し、添付のプロトコルに従ってS. cerevisiae YPH499
からゲノムDNAを調製した。E. coli からのプラスミドD
NAは、Promega (Madison, WI) のWizard PureFection P
lasmid DNA Purification System を用いて調製した。[0097] Example 3 Construction of expression vector (1) E coli -. Bought S cerevisiae shuttle vector from Stratagene plasmids pRS404, pRS405.. PYES2 (Figure 3) was purchased from Invitrogen (Carlsbad, CA). (2) Genomic DNA For S. cerevisiae genomic DNA, purchase "Gen Torukun" and follow the protocol attached to S. cerevisiae YPH499.
Genomic DNA was prepared from Plasmid D from E. coli
NA is Wizard PureFection P from Promega (Madison, WI)
Prepared using the lasmid DNA Purification System.

【００９８】(3) pRSベクターへCYC1t断片の挿入CYC1 転写ターミネーターCYC1t断片はPCRで調製した。以
下のPCR用オリゴDNA：XhoI-Tcyc1FW とApaI-Tcyc1RVと
の組合せをPCR用プライマーDNAに用い、鋳型としてpYES
2を用いた。 XhoI-Tcyc1FW：5'- TGC ATC TCG AGG GCC GCA TCA TGT
AAT TAG -3'（配列番号43） ApaI-Tcyc1RV：5'- CAT TAG GGC CCG GCC GCA AAT TAA
AGC CTT CG -3'（配列番号44）[0098] (3) insertion of pRS vector into CYC1 t fragments CYC1 transcriptional terminator CYC1 t fragments were prepared by PCR. The following PCR oligo DNA: A combination of XhoI-Tcyc1FW and ApaI-Tcyc1RV was used as a PCR primer DNA, and pYES was used as a template.
2 was used. XhoI-Tcyc1FW: 5'- TGC ATC TCG AGG GCC GCA TCA TGT
AAT TAG -3 '(SEQ ID NO: 43) ApaI-Tcyc1RV: 5'- CAT TAG GGC CCG GCC GCA AAT TAA
AGC CTT CG -3 '(SEQ ID NO: 44)

【００９９】反応は、0.1 μg pYES2, 50 pmol プライ
マー DNA, MgSO4 含有1x Pfu バッファー(Promega, Mad
ison, WI), 10 nmol dNTP, 1.5 u Pfu DNA ポリメラー
ゼ (Promega)及び 1 μl Perfect Match ポリメラーゼ
エンハンサー (Stratagene)を含む50 μlの反応液を調
製し、95℃ 2分、(95℃ 45秒、60℃ 30秒、72℃ 1分)×
30サイクル、72℃ 5分、4℃ストックの反応条件で行っ
た。増幅した2種のDNAをそれぞれXhoIとApaIで切断し、
アガロースゲル電気泳動で260 bpのDNA断片を精製しCYC
1t-XAとした。pRS404、pRS405のXhoI-ApaI部位にCYC1t-
XAを挿入し、それぞれ、pRS404Tcyc、pRS405Tcycとし
た。The reaction was carried out using 1 x Pfu buffer containing 0.1 μg pYES2, 50 pmol primer DNA and MgSO 4 (Promega, Mad
ison, WI), 10 nmol dNTP, 1.5 u Pfu DNA Polymerase (Promega) and 1 μl Perfect Match Polymerase Enhancer (Stratagene) in 50 μl of reaction mixture was prepared at 95 ° C for 2 minutes (95 ° C for 45 seconds, 60 seconds). ℃ 30 seconds, 72 ℃ 1 minute) ×
It was carried out under the reaction conditions of 30 cycles, 72 ° C. for 5 minutes, and 4 ° C. stock. Cut the amplified two kinds of DNA with Xho I and Apa I,
A 260 bp DNA fragment was purified by agarose gel electrophoresis and CYC
It was 1t-XA. CYC1t- at the Xho I- Apa I site of pRS404 and pRS405
XA was inserted into pRS404Tcyc and pRS405Tcyc, respectively.

【０１００】(4) 転写プロモーターの調製 酵母ゲノムDNAを鋳型にしてPCRにより転写プロモーター
を含むDNA断片を調製した。使用したDNAプライマーは以
下の通りである。 SacI-Ptdh3FW：5'-CAC GGA GCT CCA GTT CGA GTT TAT C
AT TAT CAA-3'（配列番号45） SacII-Ptdh3RV：5'-CTC TCC GCG GTT TGT TTG TTT ATG
TGT GTT TAT TC -3'（配列番号46）(4) Preparation of transcription promoter A DNA fragment containing the transcription promoter was prepared by PCR using yeast genomic DNA as a template. The DNA primers used are as follows. SacI-Ptdh3FW: 5'-CAC GGA GCT CCA GTT CGA GTT TAT C
AT TAT CAA-3 '(SEQ ID NO: 45) SacII-Ptdh3RV: 5'-CTC TCC GCG GTT TGT TTG TTT ATG
TGT GTT TAT TC -3 '(SEQ ID NO: 46)

【０１０１】反応溶液は、0.46 μg 酵母ゲノムDNA, 10
0 pmol プライマー DNA, 1x ExTaqバッファー(宝酒造),
20 nmol dNTP, 0.5 u ExTaq DNAポリメラーゼ(宝酒造)
及び1 μl Perfect Match ポリメラーゼエンハンサーを
含む100 μl溶液を調製し、95℃ 2分、(95℃ 45秒、60
℃ 1分、72℃ 2分)×30サイクル、72℃ 4分、4℃ストッ
クの反応条件で行った。増幅したDNAをSacIとSacIIで切
断し、アガロースゲル電気泳動で0.7kbpのDNA断片を精
製し、TDH3pとした。The reaction solution was 0.46 μg yeast genomic DNA, 10
0 pmol primer DNA, 1x ExTaq buffer (Takara Shuzo),
20 nmol dNTP, 0.5 u ExTaq DNA polymerase (Takara Shuzo)
And 100 μl solution containing 1 μl Perfect Match Polymerase Enhancer were prepared at 95 ° C for 2 minutes, (95 ° C for 45 seconds, 60 ° C).
℃ 1 minute, 72 ℃ 2 minutes) × 30 cycles, 72 ℃ 4 minutes, 4 ℃ stock reaction conditions. The amplified DNA was cleaved with Sac I and Sac II, and a 0.7 kbp DNA fragment was purified by agarose gel electrophoresis to obtain TDH3 p.

【０１０２】(5) 2μ DNA複製開始領域の調製 YEpベクターであるpYES2をSspIとNheIで切断後、2μ DN
A複製開始点（2 μ ori）を含む1.5 kbp断片をアガロー
スゲル電気泳動により精製し、Klenow酵素で平滑末端化
し、このDNA断片を2μOriSNとした。 (6) YEp型発現ベクターの作製 pRS404Tcyc、pRS405TcycをBAP（bacterial alkaline ph
osphatase, 宝酒造）処理したNaeI部位に2μOriSNを挿
入し、E. coli SURE2に形質転換した後、プラスミドDNA
を調製した。これを、DraIII及びEcoRI、HpaI、又は、P
stI及びPvuIIにより切断後、アガロースゲル電気泳動
し、2μ oriの挿入とその向きをチェックした。作製し
たpRS404Tcyc、pRS405TcycにpYES2と同じ向きで2 μ or
iが挿入されたプラスミドをそれぞれpRS434Tcyc2μOr
i、pRS435Tcyc2μOriとし、pRS404Tcyc、pRS405Tcycにp
YES2と反対向きで2 μ oriが挿入されたプラスミドをそ
れぞれpRS444Tcyc2μOri、pRS445Tcyc2μOriとした。(5) Preparation of 2μ DNA replication initiation region After cutting YEp vector pYES2 with Ssp I and Nhe I, 2μ DN
A 1.5 kbp fragment containing the A replication origin (2 μ ori) was purified by agarose gel electrophoresis, blunt-ended with Klenow enzyme, and this DNA fragment was designated as 2 μ OriSN. (6) Preparation of YEp-type expression vector pRS404Tcyc and pRS405Tcyc were transformed into BAP (bacterial alkaline ph
osphatase, Takara Shuzo) 2 μOriSN was inserted into the treated Nae I site and transformed into E. coli SURE2.
Was prepared. Dra III and Eco RI, Hpa I, or P
After cutting with st I and Pvu II, agarose gel electrophoresis was performed to check the insertion of 2 μ ori and its orientation. 2 μ or in the same direction as pYES2 on the prepared pRS404Tcyc, pRS405Tcyc
pRS434Tcyc2μOr for each plasmid with i inserted
i, pRS435Tcyc 2 μOri, pRS404Tcyc, pRS405Tcyc
The plasmids in which 2 μ ori had been inserted in the opposite direction to YES2 were designated as pRS444Tcyc2 μOri and pRS445Tcyc2 μOri, respectively.

【０１０３】pRS434Tcyc2μOri、pRS435Tcyc2μOri、pR
S444Tcyc2μOri及びpRS445Tcyc2μOriの4種のプラスミ
ドのSacI-SacII部位に、転写プロモーターを含む断片TD
H3p(GAPp)を挿入しDNAをクローン化した。その結果、
(i) pRS434Tcyc2μOriからpRS434GAPが、 (ii) pRS435T
cyc2μOriからpRS435GAPが、(iii) pRS444Tcyc2μOri
からpRS444GAPが、 (iv) pRS445Tcyc2μOriからpRS445G
APが、それぞれ得られた（図16A-16D）。PRS434Tcyc2μOri, pRS435Tcyc2μOri, pR
S444Tcyc2μOri and pRS445Tcyc2μOri Four types of plasmid Sac I- Sac II site containing a transcription promoter fragment TD
H3 p ( GAP p) was inserted and the DNA was cloned. as a result,
(i) pRS434Tcyc2μOri to pRS434GAP, (ii) pRS435T
From cyc2μOri to pRS435GAP, (iii) pRS444Tcyc2μOri
From pRS444GAP, (iv) pRS445Tcyc2μOri to pRS445G
APs were obtained respectively (FIGS. 16A-16D).

【０１０４】(7) YEp型発現ベクターの酵母への導入 各YEp型発現ベクター約40 ngをFrozen-EZ Yeast Transf
ormation II (Zymo Research, Orange, CA) を用いた方
法でYPH499へ導入し（手順はキット同封の説明書に従っ
た）、SD-W寒天プレート(DOB+CSM-Trp, BIO101, Vista,
CA)上30℃で1000以上のTrp非要求性コロニーが観察さ
れたので、作製したYEp型発現ベクターのDNA複製領域が
機能し組換え体内でプラスミドとして正常に保持されて
いる事が確認された。(7) Introduction of YEp-type expression vector into yeast About 40 ng of each YEp-type expression vector was added to Frozen-EZ Yeast Transf
Ormation II (Zymo Research, Orange, CA) was used to introduce into YPH499 (procedure follows the instructions included in the kit), and SD-W agar plate (DOB + CSM-Trp, BIO101, Vista,
Since 1000 or more Trp non-auxotrophic colonies were observed on (CA) at 30 ° C, it was confirmed that the DNA replication region of the produced YEp type expression vector was functional and was normally retained as a plasmid in the recombinant body. .

【０１０５】〔実施例4〕 IPP合成経路関連酵素遺伝子
のクローニング 酵母cDNAからの遺伝子クローニングの際には、Clontech
社 (Palo Alto, CA)より購入したS. cerevisiae DBY746
由来のcDNA ライブラリ“Quick-Clone cDNA”を用い
た。 (1) ファルネシル二リン酸合成酵素遺伝子のクローニン
グS. cerevisiae由来のFPP合成酵素遺伝子ERG20：PCR(pol
ymerase chain reaction)法でS. cerevisiae FPP合成酵
素遺伝子ERG20約0.9 kbp断片（配列番号１）を、cDNAを
鋳型にして増幅した。PCRプライマーは以下の通りであ
る。Example 4 Cloning of IPP Synthetic Pathway-Related Enzyme Genes When cloning genes from yeast cDNA, Clontech was used.
S. cerevisiae DBY746 purchased from Palo Alto, CA
The derived cDNA library "Quick-Clone cDNA" was used. (1) Cloning of farnesyl diphosphate synthase gene FPP synthase gene ERG20 from S. cerevisiae : PCR (pol
S. cerevisiae FPP synthase gene ERG20 about 0.9 kbp fragment (SEQ ID NO: 1) was amplified by the ymerase chain reaction method using cDNA as a template. The PCR primers are as follows.

【０１０６】Primer 1 (SCFPS1):5'-ATG GCT TCA GAA A
AA GAA ATT AG-3'（配列番号47） Primer 2 (SCFPS 2):5'-CTA TTT GCT TCT CTT GTA AAC
TT-3'（配列番号48） Primer 1 (SCFPS1): 5'-ATG GCT TCA GAA A
AA GAA ATT AG-3 '(SEQ ID NO: 47) Primer 2 (SCFPS 2): 5'-CTA TTT GCT TCT CTT GTA AAC
TT-3 '(SEQ ID NO: 48)

【０１０７】上記反応溶液を用い、94℃ 45秒、55℃ 1
分、72℃ 2分を30サイクル行った。なお、後述のPCR
は、特別な記載がない限り上記と同様の条件で行った。
PCR断片をアガロースゲル電気泳動で精製後、pT7Blue-T
(Novagen, Madison,WI)へT/Aライゲーションによりク
ローニングした。ERG20はpT7Blue-T内のlacZと同じ向き
に挿入されていた。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、1-300塩基と610-
1059塩基の部分でのPCRエラーは無かった。なお、ERG20
によりコードされるアミノ酸配列を配列番号２に示す。
作成したプラスミドDNAをpT7ERG20とした。Using the above reaction solution, 94 ° C. for 45 seconds, 55 ° C. 1
30 minutes at 72 ° C for 2 minutes. The PCR described below
Was performed under the same conditions as above unless otherwise specified.
After the PCR fragment was purified by agarose gel electrophoresis, pT7Blue-T
(Novagen, Madison, WI) was cloned by T / A ligation. ERG20 was inserted in the same orientation as lacZ in pT7Blue-T. The nucleotide sequence of the cloned fragment was determined and compared with the SGD sequence.
There was no PCR error in the 1059 base portion. ERG20
The amino acid sequence encoded by is shown in SEQ ID NO: 2.
The prepared plasmid DNA was designated as pT7ERG20.

【０１０８】(2) ゲラニルゲラニル二リン酸合成酵素遺
伝子のクローニングS . cerevisiaeのGGPP合成酵素遺伝子BTS1（配列番号
３）のクローニングは、以下のように行った。GenBank
(http://www.ncbi.nlm.nih.gov/Genbank/index.html/)
にあるS. cerevisiae由来GGPP合成酵素遺伝子(アクセッ
ション番号（A.N.）： U31632)(Y. Jiang, et al., J.
Biol. Chem. 270 (37), 21793-21799 (1995))の情報を
もとに、該遺伝子によりコードされるタンパク質のN末
端、C末端にマッチするプライマーを作製し、これを用
いて酵母のcDNAライブラリー（Clontech No.CL7220-1）
を鋳型としたPCRを行った。(2) Cloning of geranylgeranyl diphosphate synthase gene S. cerevisiae GGPP synthase gene BTS1 (SEQ ID NO: 3) was cloned as follows. GenBank
(http://www.ncbi.nlm.nih.gov/Genbank/index.html/)
S. cerevisiae- derived GGPP synthase gene (accession number (AN): U31632) (Y. Jiang, et al ., J.
Based on the information of Biol. Chem. 270 (37), 21793-21799 (1995)), a primer matching the N-terminal and C-terminal of the protein encoded by the gene was prepared, and used to cDNA library (Clontech No.CL7220-1)
Was used as a template for PCR.

【０１０９】N末端側プライマー：5'-ATG GAG GCC AAG
ATA GAT GAG CT-3' (配列番号49) C末端側プライマー：5'-TCA CAA TTC GGA TAA GTG GTC
TA-3' (配列番号50) PCRは、Perfect Matchポリメラーゼエンハンサーを使用
し、94℃ 45秒の変性、55℃ 1分のアニーリング及び72
℃ 2分の伸長を1サイクルとしてこれを30サイクル行っ
た。N-terminal side primer: 5′-ATG GAG GCC AAG
ATA GAT GAG CT-3 '(SEQ ID NO: 49) C-terminal side primer: 5'-TCA CAA TTC GGA TAA GTG GTC
TA-3 '(SEQ ID NO: 50) PCR was performed using Perfect Match polymerase enhancer, denaturation at 94 ° C for 45 seconds, annealing at 55 ° C for 1 minute and 72 ° C.
This was repeated for 30 cycles, with extension of 2 minutes at ℃ as one cycle.

【０１１０】目的の断片（約1.0kbp）が確認されたの
で、BTS1断片をTAクローニング可能なpT7Blue Tベクタ
ーにクローニングし、BTS1の全領域の塩基配列を決定し
た。その結果、GenBankの配列と完全に一致し、S. cere
visiae由来の遺伝子であることを確認した。BTS1により
コードされるアミノ酸配列を配列番号４に示す。Since the target fragment (about 1.0 kbp) was confirmed, the BTS1 fragment was cloned into the pT7Blue T vector capable of TA cloning, and the nucleotide sequence of the entire region of BTS1 was determined. As a result, the sequence of GenBank was completely matched, and S. cere
It was confirmed that the gene was derived from visiae . The amino acid sequence encoded by BTS1 is shown in SEQ ID NO: 4.

【０１１１】(3) HMG-CoA還元酵素遺伝子のクローニン
グS . cerevisiae HMG-CoA還元酵素遺伝子HMG1のクローニ
ングは、以下のように行った。GenBankにあるS. cerevi
siae由来HMG-CoA還元酵素遺伝子HMG1 (A.N.：M22002)
(M. E. Basson, et al., Mol. Cell. Biol. 8, 3797-38
08 (1988):配列番号５)の情報をもとにN末端、C末端に
マッチするプライマーを作製し、これを用いて酵母のcD
NAライブラリー（Clontech）を鋳型としたPCRを行っ
た。(3) Cloning of HMG-CoA reductase gene S. cerevisiae HMG-CoA reductase gene HMG1 was cloned as follows. S. cerevi in GenBank
siae- derived HMG-CoA reductase gene HMG1 (AN: M22002)
(ME Basson, et al ., Mol. Cell. Biol. 8, 3797-38
08 (1988): A primer matching the N-terminal and C-terminal was prepared based on the information of SEQ ID NO: 5) and used to prepare yeast cD.
PCR was performed using the NA library (Clontech) as a template.

【０１１２】N末端側プライマー：5'-ATG CCG CCG CTA
TTC AAG GGA CT-3' (配列番号51) C末端側プライマー：5'-TTA GGA TTT AAT GCA GGT GAC
GG-3' (配列番号52) PCRは、Perfect Matchポリメラーゼエンハンサーを使用
し、94℃ 45秒の変性、55℃ 1分のアニーリング及び72
℃ 2分の伸長を1サイクルとしてこれを30サイクル行っ
た。N-terminal side primer: 5′-ATG CCG CCG CTA
TTC AAG GGA CT-3 '(SEQ ID NO: 51) C-terminal side primer: 5'-TTA GGA TTT AAT GCA GGT GAC
GG-3 '(SEQ ID NO: 52) PCR was performed using the Perfect Match polymerase enhancer, denaturing at 94 ° C for 45 seconds, annealing at 55 ° C for 1 minute, and 72 ° C.
This was repeated for 30 cycles, with extension of 2 minutes at ℃ as one cycle.

【０１１３】目的の断片(3.2kbp)が確認されたので、HM
G1をTAクローニング可能なpT7BlueTベクターにクローニ
ングし(これをpT7HMG1とした)、HMG1の塩基配列を決定
した。その結果、配列番号５の塩基配列及び配列番号６
のアミノ酸配列を確認した。決定された塩基配列は、Ge
nBankの配列に示した塩基配列と一部異なっており、PCR
エラーを起こしていた（図4A）。このPCRエラーを含ん
だ変異型HMG-CoA還元酵素遺伝子をHMG1'とする。Since a target fragment (3.2 kbp) was confirmed, HM
G1 was cloned into pT7BlueT vector capable of TA cloning (this was designated as pT7HMG1), and the nucleotide sequence of HMG1 was determined. As a result, the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6
Was confirmed. The determined nucleotide sequence is Ge
The nucleotide sequence is partly different from that shown in the nBank sequence, and PCR
It was making an error (Figure 4A). The mutant HMG-CoA reductase gene containing this PCR error is designated as HMG1 '.

【０１１４】(4) HMG-CoA還元酵素遺伝子のPCRエラーの
修正 PCRエラーは、pT7HMG1からHMG1遺伝子断片をサブクロー
ニングし、HMG1部分のPCRエラーによるアミノ酸置換変
異部分を修正した。HMG-CoA還元酵素遺伝子HMG1のPCRエ
ラー型DNAであるHMG1'を有するpT7HMG1からHMG1'遺伝子
断片をサブクローニングし、HMG1部分のPCRエラーによ
るアミノ酸置換変異部分を、部位特異的変異誘発法によ
り修正し、pALHMG106を作製した。作製方法の詳細は以
下の通りである。(4) Correction of PCR error of HMG-CoA reductase gene For PCR error, the HMG1 gene fragment from pT7HMG1 was subcloned, and the amino acid substitution mutation part of the HMG1 part due to the PCR error was corrected. Subcloning the HMG1 'gene fragment from pT7HMG1 having HMG1 ' which is a PCR error type DNA of HMG-CoA reductase gene HMG1 and correcting the amino acid substitution mutation part due to the PCR error of the HMG1 part by site-directed mutagenesis, pALHMG106 was created. The details of the manufacturing method are as follows.

【０１１５】プラスミドpT7HMG1をクローン化HMG1とし
て用いた。また、部位特異的変異導入用ベクターとして
pALTER-1を購入した(Promega)。部位特異的変異(site-d
irected mutagenesis)は、Promega発行の"Protocols an
d application guide, third edition, 1996 Promega,
ISBN 1-882274-57-1"に記載の方法で行った。変異導入
用オリゴは、次の3種類を化学合成した。The plasmid pT7HMG1 was used as cloned HMG1 . Also, as a vector for site-directed mutagenesis
We purchased pALTER-1 (Promega). Site-specific mutation (site-d
irected mutagenesis) is "Protocols an
d application guide, third edition, 1996 Promega,
ISBN 1-882274-57-1 ". The following three types of mutagenesis oligos were chemically synthesized.

【０１１６】HMG1(190-216) 5'-CCAAATAAAGACTCCAACACT
CTATTT-3'（配列番号53） HMG1(1807-1833) 5'-GAATTAGAAGCATTATTAAGTAGTGGA-3'
（配列番号54） HMG1(2713-2739) 5'-GGATTTAACGCACATGCAGCTAATTTA-3'
（配列番号55）HMG1 (190-216) 5'-CCAAATAAAGACTCCAACACT
CTATTT-3 '(SEQ ID NO: 53) HMG1 (1807-1833) 5'-GAATTAGAAGCATTATTAAGTAGTGGA-3'
(SEQ ID NO: 54) HMG1 (2713-2739) 5'-GGATTTAACGCACATGCAGCTAATTTA-3 '
(SEQ ID NO: 55)

【０１１７】部位特異的変異導入は、pT7HMG1をSmaI、A
paLI、SalIで切断し、3.2kbpのHMG1断片をアガロースゲ
ル電気泳動で調製した。これをpALTER-1のSmaI-SalI部
位に挿入し、pALHMG1を作製した。pALHMG1をアルカリ変
性後、上記変異導入オリゴ、リペアオリゴとしてAmp re
pair oligo(Promega)、及びノックアウトオリゴとしてT
et knockout oligo(Promega)をアニーリングさせ、E. c
oli ES1301(Promega)に導入後、125μg/mlアンピシリン
で部位特異的変異が導入されたプラスミドを保持する形
質転換体を集積培養し、プラスミドDNAを調製した。以
下の配列を有するプライマーを用いて塩基配列をチェッ
クしたところ、HMG1(190-216)、 HMG1(1807-1833)、 HM
G1(2713-2739)に相当する配列は全てこれらオリゴヌク
レオチドの配列に修正されていた（配列番号13）。な
お、修正された配列によりコードされるアミノ酸配列
（配列番号14）は、HMG1'によりコードされるアミノ酸
配列（配列番号12）と一致していた（サイレント変
異）。 HMG1(558-532) 5'-GTCTGCTTGGGTTACATTTTCTGAAAA-3'
（配列番号56） HMG1(1573-1599) 5'-CATACCAGTTATACTGCAGACCAATTG-3'
（配列番号57） HMG1(2458-2484) 5'-GAATACTCATTAAAGCAAATGGTAGAA-3'
（配列番号58） このHMG1内の配列が修正されたプラスミドをpALHMG106
とした（図17）。For site-directed mutagenesis, pT7HMG1 was cloned into Sma I, A
After cutting with pa LI and Sal I, a 3.2 kbp HMG1 fragment was prepared by agarose gel electrophoresis. This was inserted into the Sma I- Sal I site of pALTER-1 to prepare pALHMG1. After denaturing pALHMG1 with alkali, use Ampre as the above-mentioned mutation-introducing oligo and repair oligo.
pair oligo (Promega) and T as a knockout oligo
et knockout oligo (Promega) is allowed to anneal, E. c
After introduction into oli ES1301 (Promega), transformants carrying the plasmid in which the site-directed mutation was introduced with 125 μg / ml ampicillin were integrated and cultured to prepare plasmid DNA. When the nucleotide sequence was checked using a primer having the following sequence, HMG1 (190-216), HMG1 (1807-1833), HM
All the sequences corresponding to G1 (2713-2739) were corrected to the sequences of these oligonucleotides (SEQ ID NO: 13). The amino acid sequence encoded by the modified sequence (SEQ ID NO: 14) matched the amino acid sequence encoded by HMG1 '(SEQ ID NO: 12) (silent mutation). HMG1 (558-532) 5'-GTCTGCTTGGGTTACATTTTCTGAAAA-3 '
(SEQ ID NO: 56) HMG1 (1573-1599) 5'-CATACCAGTTATACTGCAGACCAATTG-3 '
(SEQ ID NO: 57) HMG1 (2458-2484) 5'-GAATACTCATTAAAGCAAATGGTAGAA-3 '
(SEQ ID NO: 58) The plasmid in which the sequence in HMG1 was corrected was designated as pALHMG106.
(Fig. 17).

【０１１８】(5) メバロン酸二リン酸脱炭酸酵素遺伝子
のクローニング PCR法でS. cerevisiaeメバロン酸二リン酸脱炭酸酵素遺
伝子ERG19(MVD1)約1.2kbp断片(配列番号７)をcDNAを鋳
型に増幅した。PCRプライマーは以下の通りである。 Primer 1 (SCU-1):5'-AAC TGC AGA TGA CCG TTT ACA CA
G CAT CCG T-3'（配列番号59） Primer 2 (SCU-2):5'-CGG AAT TCT TAT TCC TTT GGT AG
A CCA GTC T-3'（配列番号60） (下線部分は制限酵素認識部位)(5) Cloning of mevalonate diphosphate decarboxylase gene By PCR, S. cerevisiae mevalonate diphosphate decarboxylase gene ERG19 ( MVD1 ) about 1.2 kbp fragment (SEQ ID NO: 7) was used as a template. Amplified. The PCR primers are as follows. Primer 1 (SCU-1): 5'-AA C TGC AG A TGA CCG TTT ACA CA
G CAT CCG T-3 '(SEQ ID NO: 59) Primer 2 (SCU-2): 5'-CG G AAT TC T TAT TCC TTT GGT AG
A CCA GTC T-3 '(SEQ ID NO: 60) (restriction enzyme recognition site is underlined)

【０１１９】PCR断片をPstIとEcoRIで切断後アガロース
ゲル電気泳動で精製し、pT7BlueのPstI-EcoRI部位にク
ローニングした。これで、ERG19(MVD1)はpT7BlueのlacZ
と反対向きに挿入されることになる。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。なおERG19によりコードされ
るアミノ酸配列を配列番号８に示す。作成したプラスミ
ドDNAをpT7ERG19とした。The PCR fragment was cleaved with Pst I and Eco RI, purified by agarose gel electrophoresis, and cloned into the Pst I- Eco RI site of pT7Blue. Now the ERG19 ( MVD1 ) is lacZ on pT7Blue.
It will be inserted in the opposite direction. When the nucleotide sequence of the cloned fragment was determined and compared with the SGD sequence, there was no PCR error. The amino acid sequence encoded by ERG19 is shown in SEQ ID NO: 8. The prepared plasmid DNA was designated as pT7ERG19.

【０１２０】〔実施例5〕 変異型遺伝子のクローニン
グ 欠失型HMG-CoA還元酵素遺伝子のクローニング 実施例4(3)で作製したpT7HMG1をBamHI、SalI、ScaI処理
してPCRエラー配列を有するHMG1'遺伝子を取り出し、こ
れをpYES2(Invitrogen, Carlsbad, CA)のBamHI-XhoI部
位に導入した。得られた組換えベクターをpYES-HMG1と
した。ベクター内の塩基配列を確認したところ、配列番
号９の塩基配列であることを確認した。なお、pYES2
は、複製起点として酵母2μmDNAのori、及びガラクトー
スで誘導可能なGAL1プロモーターをもつ酵母発現用シャ
トルベクターである（図3）。Example 5 Cloning of Mutant Gene Cloning of Deletion HMG-CoA Reductase Gene pT7HMG1 prepared in Example 4 (3) was treated with Bam HI, Sal I and Sca I to obtain a PCR error sequence. The HMG1 'gene possessed was extracted and introduced into the Bam HI- Xho I site of pYES2 (Invitrogen, Carlsbad, CA). The obtained recombinant vector was designated as pYES-HMG1. When the base sequence in the vector was confirmed, it was confirmed to be the base sequence of SEQ ID NO: 9. Note that pYES2
Is a yeast expression shuttle vector having an ori of yeast 2 μm DNA as an origin of replication and a galactose-inducible GAL1 promoter (FIG. 3).

【０１２１】HMG-CoA還元酵素の膜貫通ドメインに対応
する領域を欠損させた欠失型HMG-CoA還元酵素遺伝子を
作製するため、前記の通り作製したpYES-HMG1を鋳型と
して、PCR法でベクター部分とともにHMG1コード領域の
一部分を欠失させた断片を調製した。得られた断片をKl
enow酵素で平滑末端にした後、セルフライゲーションに
より再び環化し、E. coli JM109へ形質転換させ、プラ
スミドDNAを調製した。プライマーとして使用した合成D
NA配列とその組合せを前記表1に示した。[0121] In order to prepare a deleted HMG-CoA reductase gene in which a region corresponding to the transmembrane domain of HMG-CoA reductase was deleted, a vector was prepared by PCR using pYES-HMG1 prepared as described above as a template. A fragment was prepared in which a part of the HMG1 coding region was deleted together with the part. The resulting fragment is Kl
After making the ends blunt with enow enzyme, they were circularized again by self ligation and transformed into E. coli JM109 to prepare plasmid DNA. Synthetic D used as a primer
The NA sequences and their combinations are shown in Table 1 above.

【０１２２】得られたプラスミドDNA内のHMG1上流、下
流のアミノ酸の読み枠がずれていないことと、その結合
部位近辺にPCRエラーによるアミノ酸置換が起きていな
いことを373A DNA sequencer (Perkin Elmer, Foster C
ity, CA) で確認した。その結果、結合部位近辺にPCRエ
ラーによるアミノ酸置換がなく、読み枠がずれずに遺伝
子を欠失する事のできた以下のプラスミドを得た。欠失
型HMG1遺伝子は、欠失のパターンに従ってΔ02y(yは任
意の作業番号を表す。)のように記載し、Δ02yを含むpY
ES2ベクターを例えばpYHMG026と記すこととする（他の
欠失体も同様）。373A DNA sequencer (Perkin Elmer, Foster) was confirmed that the reading frames of amino acids upstream and downstream of HMG1 in the obtained plasmid DNA were not displaced and that amino acid substitution due to PCR error did not occur near the binding site. C
ity, CA). As a result, the following plasmid was obtained in which there was no amino acid substitution near the binding site due to PCR error and the gene could be deleted without shifting the reading frame. The deletion type HMG1 gene is described as Δ02y (y represents an arbitrary work number) according to the deletion pattern, and pY containing Δ02y is described.
The ES2 vector will be referred to as, for example, pYHMG026 (same for other deletions).

【０１２３】HMG1Δ026：配列番号15HMG1 Δ044：配列番号16HMG1 Δ056：配列番号17HMG1 Δ062：配列番号18HMG1 Δ076：配列番号19HMG1 Δ081：配列番号20HMG1 Δ100：配列番号21HMG1 Δ112：配列番号22HMG1 Δ122：配列番号23HMG1 Δ133：配列番号24 プラスミド：pYHMG026, pYHMG027, pYHMG044, pYHMG04
5, pYHMG059, pYHMG062, pYHMG063, pYHMG065, pYHMG07
6, pYHMG081, pYHMG083, pYHMG085, pYHMG094,pYHMG10
0, pYHMG106, pYHMG107, pYHMG108, pYHMG109, pYHMG11
2, pYHMG122, pYHMG123, pYHMG125, pYHMG133, pYHMG13
4 〔実施例6〕 遺伝子のサブクローニング 今回使用した恒常発現型転写プロモーターをもつE. col
i- S. cerevisiae YEp型シャトルベクターであるpRSベ
クターは、実施例3において作製したものを使用した。 (1) FPP合成酵素遺伝子のサブクローニングS . cereviae由来のFPP合成酵素遺伝子ERG20：実施例4
(1)に記載のpT7ERG20 をXbaIとBamHIで切断し、アガロ
ースゲル電気泳動により1.1 kbpのERG20遺伝子断片を精
製した。これを、pRS435GAPとpRS445GAPのXbaI-BamHI部
位に挿入し、それぞれpRS435GAP-ERG20、pRS445GAP-ERG
20とした。 HMG1 Δ026: SEQ ID NO: 15 HMG1 Δ044: SEQ ID NO: 16 HMG1 Δ056: SEQ ID NO: 17 HMG1 Δ062: SEQ ID NO: 18 HMG1 Δ076: SEQ ID NO: 19 HMG1 Δ081: SEQ ID NO: 20 HMG1 Δ100: SEQ ID NO: 21 HMG1 Δ112: SEQ ID NO: 22 HMG1 Δ122: SEQ ID NO: 23 HMG1 Δ133: SEQ ID NO: 24 Plasmid: pYHMG026, pYHMG027, pYHMG044, pYHMG04
5, pYHMG059, pYHMG062, pYHMG063, pYHMG065, pYHMG07
6, pYHMG081, pYHMG083, pYHMG085, pYHMG094, pYHMG10
0, pYHMG106, pYHMG107, pYHMG108, pYHMG109, pYHMG11
2, pYHMG122, pYHMG123, pYHMG125, pYHMG133, pYHMG13
4 [Example 6] Gene subcloning E. col having a constitutive expression type transcription promoter used this time
As the pRS vector, which is an i - S . cerevisiae YEp type shuttle vector, the one prepared in Example 3 was used. . (1) FPP synthase gene Subcloning S FPP synthase gene from cereviae ERG20: Example 4
The pT7ERG20 described in (1) was cleaved with Xba I and Bam HI, and a 1.1 kbp ERG20 gene fragment was purified by agarose gel electrophoresis. This was inserted into the Xba I- Bam HI site of pRS435GAP and pRS445GAP, and pRS435GAP-ERG20 and pRS445GAP-ERG, respectively.
It was 20.

【０１２４】(2) GGPP合成酵素遺伝子又はその変異型遺
伝子のサブクローニングS . cereviae由来のGGPP合成酵素遺伝子BTS1：実施例4
(2)に記載のpT7Blue TベクターをBamHI、SalI処理してB
TS1断片を取り出し、これを、pYES2(Invitrogen社)のBa
mHI、XhoIサイトに導入した。得られた組換えベクター
をpYESGGPSとした。pYESGGPSをBamHIとMluIで切断し、
アガロースゲル電気泳動により1.3 kbp断片を精製し
た。これを、pRS435GAPとpRS445GAPのBamHI-MluI部位に
挿入し、それぞれpRS435GAP-BTS1、pRS445GAP-BTS1とし
た。(2) Subcloning of GGPP synthase gene or its mutant gene S. cereviae- derived GGPP synthase gene BTS1 : Example 4
Bam HI and pT7Blue T vector according to (2), Sal I treated to B
TS1 fragment was taken out, this, Ba of pYES2 (Invitrogen Corp.)
Introduced to m HI and Xho I sites. The obtained recombinant vector was designated as pYESGGPS. Disconnect pYESGGPS with Bam HI and Mlu I,
The 1.3 kbp fragment was purified by agarose gel electrophoresis. This was inserted into the Bam HI-Mlu I site of pRS435GAP and pRS445GAP to obtain pRS435GAP-BTS1 and pRS445GAP-BTS1, respectively.

【０１２５】(3) HMG-CoA還元酵素遺伝子又はその変異
型遺伝子のサブクローニング 実施例4(4)に記載のpALHMG106 （図17）をSmaIとSalIで
切断後、アガロースゲル電気泳動で3.2 kbpのPCRエラー
修正後のHMG1遺伝子断片を精製した。これをpRS434GA
P、pRS444GAPのSmaI-SalI部位へ挿入した。HMG1をサブ
クローン化したプラスミドは、XhoI、SpeI、NaeI及びSp
hIの各制限酵素マッピングと、挿入された3.2 kbp HMG1
遺伝子断片のボーダー領域の塩基配列確認とにより物理
地図をチェックし、計画通りに作製できたプラスミドを
選抜した。選抜したプラスミドはそれぞれ、pRS434GAP-
HMG1、pRS444GAP-HMG1とした。HMG-CoA還元酵素遺伝子
の欠失変異型遺伝子は、pYES2由来の欠失変異型HMG1を
組み込んであるプラスミド（実施例5記載）から上記と
同様にしてpRS434GAPへクローニングした。(3) Subcloning of HMG-CoA reductase gene or mutant gene thereof pALHMG106 (FIG. 17) described in Example 4 (4) was digested with Sma I and Sal I, and then 3.2 kbp by agarose gel electrophoresis. The HMG1 gene fragment after PCR error correction was purified. This is pRS434GA
P, pRS444GAP was inserted into the Sma I- Sal I site. The HMG1 subcloned plasmids consisted of Xho I, Spe I, Nae I and Sp
Each restriction enzyme mapping of h I and inserted 3.2 kbp HMG1
The physical map was checked by confirming the base sequence of the border region of the gene fragment, and the plasmids that were produced as planned were selected. The selected plasmids are pRS434GAP-
HMG1 and pRS444GAP-HMG1. The deletion mutant type gene of the HMG-CoA reductase gene was cloned into pRS434GAP in the same manner as above from the plasmid (described in Example 5) incorporating the deletion mutant type HMG1 derived from pYES2.

【０１２６】(4) メバロン酸二リン酸脱炭酸酵素遺伝子
のサブクローニング 実施例4(5)に記載のpT7ERG19をBamHI、SalIで切断後、
アガロースゲル電気泳動によりBamHI-SalI 1.5 kbpのER
G19遺伝子断片を精製し、pRS435GAPとpRS445GAPのBamHI
-SalI部位に挿入した。ERG19をサブクローン化したプラ
スミドはXbaI認識部位マッピングにより計画通りに作製
できたプラスミドを選抜した。選抜したプラスミドは、
それぞれpRS435GAP-ERG19、pRS445GAP-ERG19とした。(4) Subcloning of mevalonate diphosphate decarboxylase gene pT7ERG19 described in Example 4 (5) was cleaved with Bam HI and Sal I.
ER of Bam HI- Sal I 1.5 kbp by agarose gel electrophoresis
The G19 gene fragment was purified and pRS435GAP and pRS445GAP Bam HI
-Inserted at Sal I site. As the plasmid subcloning ERG19 , a plasmid that could be produced as planned by Xba I recognition site mapping was selected. The selected plasmid is
These were designated as pRS435GAP-ERG19 and pRS445GAP-ERG19, respectively.

【０１２７】〔実施例7〕 遺伝子導入EUG株によるプレ
ニルアルコール生産 (1) 使用した菌株 EUG株としてEUG5、EUG8、EUG12、EUG24、EUG27、EUG3
6、EUG64を用いた。 (2) 発現プラスミド メバロン酸二リン酸脱炭酸酵素遺伝子（ERG19）発現プ
ラスミドとしてpRS445GAP-ERG19を用いた。 HMG-CoA還元酵素遺伝子（HMG1）発現プラスミドとしてp
RS434GAP-HMG1、pRS444GAP-HMG1を用い、欠失型HMG1発
現プラスミドとしてはpRS434GAP-HMG026、pRS434GAP-HM
G044、pRS434GAP-HMG056、pRS434GAP-HMG062、pRS434GA
P-HMG076、pRS434GAP-HMG081、pRS434GAP-HMG100、pRS4
34GAP-HMG112、pRS434GAP-HMG122、pRS434GAP-HMG133を
用いた。上記ベクター名のHMG以下の番号は欠失パター
ンを示す (図4B)。欠失部分は 表1 に示したプライマー
DNAを用いPCRによって削除した。 FPP合成酵素遺伝子（ERG20）発現プラスミドとしてpRS4
35GAP-ERG20、pRS445GAP-ERG20を用いた。 GGPP合成酵素遺伝子（BTS1）発現プラスミドとしてpRS4
35GAP-BTS1、pRS445GAP-BTS1を用いた。 (3) 形質転換 Zymo Research (Orange, CA) より購入したFrozen EZ y
east transformationII kitを用いて酵母の形質転換を
行った。形質転換体は、SGR培地をベースにして適宜栄
養要求性を指標とした選択培地寒天プレート上で生育さ
せた。クローン化のため選択培地寒天プレート培養は2
回行った。[Example 7] Production of prenyl alcohol by transgenic EUG strain (1) EUG5, EUG8, EUG12, EUG24, EUG27, EUG3 as the strain EUG strain used
6, EUG64 was used. (2) Expression plasmid pRS445GAP-ERG19 was used as an expression plasmid for the mevalonate diphosphate decarboxylase gene ( ERG19 ). P as a HMG-CoA reductase gene ( HMG1 ) expression plasmid
Using RS434GAP-HMG1 and pRS444GAP-HMG1, pRS434GAP-HMG026 and pRS434GAP-HM are used as deletion type HMG1 expression plasmids.
G044, pRS434GAP-HMG056, pRS434GAP-HMG062, pRS434GA
P-HMG076, pRS434GAP-HMG081, pRS434GAP-HMG100, pRS4
34GAP-HMG112, pRS434GAP-HMG122 and pRS434GAP-HMG133 were used. The numbers below HMG in the vector name indicate the deletion pattern (FIG. 4B). The deletion part is the primer shown in Table 1.
It was deleted by PCR using DNA. PRS4 as an FPP synthase gene ( ERG20 ) expression plasmid
35GAP-ERG20 and pRS445GAP-ERG20 were used. PRS4 as GGPP synthase gene ( BTS1 ) expression plasmid
35GAP-BTS1 and pRS445GAP-BTS1 were used. (3) Transformation Frozen EZ y purchased from Zymo Research (Orange, CA)
The yeast was transformed using the east transformation II kit. The transformant was grown on a selective medium agar plate based on SGR medium and using auxotrophy as an index. 2 for selective medium agar plate culture for cloning
I went there.

【０１２８】(4) 培養 作製した形質転換体をSGR選択培地で前培養し、0.01-0.
05 mlの前培養液を1-5mlのYM7にそれぞれ加え、18mm径
の試験管で30℃ 130 r.p.m.の往復振盪培養で培養し
た。 (5) プレニルアルコール生産量測定 培養液にメタノールを等量加えて混合後、ペンタンを約
二倍量加えて激しく攪拌後静置した。ペンタン層を新し
いガラス試験管にとり、ドラフト中でペンタンを気化さ
せ溶質成分を濃縮後、GC/MSでプレニルアルコールを同
定、定量した。また、その際、菌体増殖の度合いを調べ
るため、培養液50μlを水で30倍に希釈し、600 nmの吸
光度を測定した。プレニルアルコール生産量は、実施例
２(1-2)と同様にHP6890/5973 GC/MSシステムを用いて測
定した。(4) Culture The prepared transformant was pre-cultured in an SGR selection medium to give 0.01-0.
05 ml of the preculture solution was added to each of 1-5 ml of YM7, and cultured in a 18 mm diameter test tube by reciprocal shaking culture at 30 ° C. and 130 rpm. (5) Measurement of prenyl alcohol production After adding an equal amount of methanol to the culture solution and mixing, about twice the amount of pentane was added, and the mixture was vigorously stirred and allowed to stand. The pentane layer was placed in a new glass test tube, pentane was vaporized in a fume hood, the solute component was concentrated, and then prenyl alcohol was identified and quantified by GC / MS. At that time, in order to examine the degree of bacterial cell growth, 50 μl of the culture solution was diluted 30 times with water and the absorbance at 600 nm was measured. The amount of prenyl alcohol produced was measured using the HP6890 / 5973 GC / MS system as in Example 2 (1-2).

【０１２９】(6) 結果と考察 (6-1) ERG19発現株のプレニルアルコール生産 まず初めに、メバロン酸二リン酸脱炭酸酵素遺伝子ERG1
9発現プラスミドをEUG株に導入してみた。pRS445GAP-ER
G19をA451由来のEUG5、YPH499由来のEUG12、YPH500由来
のEUG24株にそれぞれ導入し、プレニルアルコール生産
量を測定した (図18A)。どの株もそれほどFOH生産は向
上していないように見えるが、培養液OD6 00値あたりのF
OH生産量を比較すると、YPH株由来のEUG12、EUG24で2倍
から数倍FOH生産効率がよくなっていた (図18B)。従っ
て、本発明で作製した変異型細胞、すなわちスクアレン
合成酵素遺伝子を完全欠損又は部分欠損させることをせ
ずに、翻訳活性のあるスクアレン合成酵素遺伝子の転写
産物量を減少できる変異型細胞を宿主に用いれば、ERG1
9発現強化により菌体数が少ない環境でも効率よくFOHを
生産することができることがわかった。(6) Results and Discussion (6-1) Production of prenyl alcohol by ERG19- expressing strain First, the mevalonate diphosphate decarboxylase gene ERG1
The expression plasmid was introduced into the EUG strain. pRS445GAP-ER
G19 was introduced into each of A451-derived EUG5, YPH499-derived EUG12 and YPH500-derived EUG24 strains, and the amount of prenyl alcohol produced was measured (FIG. 18A). Which strains but does not seem to be that much FOH production improvement, culture OD 6 00 per value F
Comparing the OH production, EUG12 and EUG24 derived from the YPH strain had 2-fold to several-fold higher FOH production efficiency (Fig. 18B). Therefore, a mutant cell prepared in the present invention, that is, a mutant cell capable of reducing the amount of a transcription product of a squalene synthase gene having a translation activity as a host without completely or partially deleting the squalene synthase gene is used as a host. If used, ERG1
9 It was found that the enhanced expression enables efficient FOH production even in an environment where the number of bacterial cells is small.

【０１３０】(6-2) HMG1、HMG1D 発現株のプレニルアル
コール生産 まず、A451とA451由来のEUG8各5コロニーのプレニルア
ルコール生産量を図19に示す。宿主のEUG8は培養2日後
に平均2.5mg/l（最高3.3mg/l）のFOHを生産していた。p
RS434GAP-HMG1/EUG8（図20A）とpRS444GAP-HMG1/EUG8
（図20B）をそれぞれ5コロニーずつ培養し、プレニルア
ルコール生産量の平均と標準偏差をグラフ化したとこ
ろ、2日後にそれぞれ平均4.8mg/l（最高6.7mg/l）、平
均3.5mg/l（最高4.0mg/l）のFOH生産をしていた。EUG8
を宿主にした場合A451を宿主にした場合と比べてHMG1遺
伝子発現によるプレニルアルコール生産性向上効果が優
れていることがわかった。(6-2) Production of prenyl alcohol by strains expressing HMG1 and HMG1D First, FIG. 19 shows the amount of prenyl alcohol produced by 5 colonies of A451 and A451-derived EUG8. The host EUG8 produced an average of 2.5 mg / l (up to 3.3 mg / l) FOH after 2 days in culture. p
RS434GAP-HMG1 / EUG8 (Figure 20A) and pRS444GAP-HMG1 / EUG8
(Fig. 20B) were cultured in 5 colonies each, and the average and standard deviation of prenyl alcohol production were graphed. After 2 days, the average was 4.8 mg / l (maximum 6.7 mg / l) and the average was 3.5 mg / l ( The maximum FOH production was 4.0 mg / l). EUG8
It was found that the effect of improving the productivity of prenyl alcohol by the expression of HMG1 gene was superior when A451 was used as the host compared to when A451 was used as the host.

【０１３１】次に、HMG1遺伝子発現増強が、YPH499由来
のEUG12及びYPH500由来のEUG27に与える効果について調
べた。なお、HMG1をYPH499、YPH500に導入した株では、
すでに報告されているように(特開平5-192184号公報；
Donald et al. (1997) Appl.Environ. Microbiol. 63,
3341-3344)、SQの増加は観察されるがプレニルアルコー
ルはほとんど生産されないことを予備的実験により確認
してある。EUG12とEUG27の5コロニー由来の液体培養4日
目と7日目の生産量を測定した。EUG12、EUG27のプレニ
ルアルコール生産量を図21に示す。主要生産物はFOHで
あり、その他異性体のNOH がわずかに生産され、GGOHは
ほとんど生産されない。これにHMG1発現プラスミド(pRS
434GAP-HMG1, pRS444GAP-HMG1)を導入した結果を図22に
示す。HMG1遺伝子発現増強によりerg9の表現型がある程
度相補され、菌体量が増加しているのが顕著であった。
さらに、FOH生産量については、EUG12を用いた場合は宿
主に対して約2倍の効果があったが（図22A）、EUG27で
は同レベルであった（図22B）。一方、どちらの株でもG
GOH生産には効果がみられ最大でどちらも2.05mg/lのGGO
Hを蓄積した。GGOH生産については、pRS444GAP-HMG1導
入株の方が効果が高かった。従って、EUG12、EUG27を宿
主にした時、HMG1遺伝子がFOHだけでなくGGOH生産にも
効果があることが示された。ただし、7日培養で生産量
が低下する傾向はEUG株に特徴的なものである。HMG1の
各欠失型遺伝子をEUG5とEUG12で発現させた結果を図23-
24に示す。EUG5において、各欠失型HMG1遺伝子発現増強
により生産性の向上はそれほど顕著ではなかった。EUG1
2においては、HMG完全長遺伝子ほどではなかったがGGOH
生産性の向上に効果があった（図24B）。Next, the effect of enhanced HMG1 gene expression on EUG12 derived from YPH499 and EUG27 derived from YPH500 was examined. In addition, in the strain that introduced HMG1 into YPH499, YPH500,
As already reported (JP-A-5-192184;
Donald et al . (1997) Appl.Environ. Microbiol. 63,
3341-3344), it has been confirmed by preliminary experiments that an increase in SQ is observed but prenyl alcohol is hardly produced. The production amount on the 4th and 7th days of liquid culture derived from 5 colonies of EUG12 and EUG27 was measured. FIG. 21 shows the amount of prenyl alcohol produced by EUG12 and EUG27. The major product is FOH, the other isomer NOH is slightly produced, and GGOH is scarcely produced. The HMG1 expression plasmid (pRS
FIG. 22 shows the results of introducing 434GAP-HMG1 and pRS444GAP-HMG1). It was remarkable that the erg9 phenotype was complemented to some extent by the increased HMG1 gene expression and the bacterial cell amount was increased.
Furthermore, with respect to FOH production, when EUG12 was used, the effect was about twice as high as that on the host (Fig. 22A), but at the same level with EUG27 (Fig. 22B). On the other hand, G
Effective in GOH production, maximum 2.05 mg / l GGO in both cases
Accumulated H. Regarding GGOH production, the pRS444GAP-HMG1 introduced strain was more effective. Therefore, when EUG12 and EUG27 were used as hosts, it was shown that the HMG1 gene had an effect not only on FOH but also on GGOH production. However, the tendency that the production amount decreases after 7-day culture is characteristic of the EUG strain. Fig. 23- shows the result of expressing each deletion gene of HMG1 in EUG5 and EUG12.
Shown in 24. In EUG5, the productivity improvement due to the enhanced expression of each deletion type HMG1 gene was not so remarkable. EUG1
In 2, GGOH was less than that of HMG full-length gene
It was effective in improving productivity (Fig. 24B).

【０１３２】(6-3) ERG20発現株のプレニルアルコール
生産 pRS435GAP-ERG20/EUG8（図25A）とpRS445GAP-ERG20/EUG
8（図25B）をそれぞれ5コロニーずつ培養したときのFOH
生産量の平均は、それぞれ0.9 mg/l、0.5mg/lであった
（最大値は1.5mg/l）。次に、ERG20遺伝子発現増強がYP
H499、YPH500由来のEUG12、EUG27に与える効果について
調べた。pRS435GAP-ERG20とpRS445GAP-ERG20を導入した
株をそれぞれ5コロニーずつ培養し、プレニルアルコー
ル生産量の平均と標準偏差をグラフ化したところ、ERG2
0遺伝子発現のプレニルアルコール生産に対する効果
は、pRS435GAP-HMG1をEUG12、EUG27に導入した場合に、
7日培養でそれぞれ平均2.7 mg/l（最大値は3.6mg/l）、
1.1 mg/l（最大値は1.37mg/l）のGGOHを生産した（図26
A,B）。ERG20はFPP合成酵素をコードしているので、ERG
20発現増強によりGGOHが生産されるのは、実はGGPP合成
酵素はホモアリル性プライマー基質としてDMAPPでな
く、FPPを利用しERG20発現増強により細胞内FPP合成活
性があがった結果GGOH生産能が上昇したためであること
が示唆される。従って、ERG20はEUG株においてGGOH生産
に効果があることがわかった。(6-3) Prenyl alcohol production of ERG20 expressing strains pRS435GAP-ERG20 / EUG8 (FIG. 25A) and pRS445GAP-ERG20 / EUG
FOH when 5 colonies of 8 (Fig. 25B) were cultured.
The average production was 0.9 mg / l and 0.5 mg / l, respectively (maximum value was 1.5 mg / l). Next, the ERG20 gene expression enhancement is YP
The effects on EUG12 and EUG27 derived from H499 and YPH500 were examined. pRS435GAP-ERG20 and pRS445GAP-ERG20 strains introduced were cultured five colonies each, was graphed the mean and standard deviation of prenyl alcohol production, ERG2
The effect of 0 gene expression on prenyl alcohol production was as follows when pRS435GAP-HMG1 was introduced into EUG12 and EUG27.
Average of 2.7 mg / l in 7 days culture (maximum value is 3.6 mg / l),
It produced 1.1 mg / l (maximum 1.37 mg / l) GGOH (Fig. 26).
A, B). ERG20 encodes FPP synthase, so ERG
The reason why GGOH is produced by enhanced expression of 20 is that the GGPP synthase is actually not DMAPP as a homoallylic primer substrate, but because FPP is used to enhance ERG20 expression and intracellular FPP synthetic activity is increased, resulting in an increase in GGOH productivity. It is suggested that there is. Therefore, ERG20 was found to be effective in GGOH produced in EUG strain.

【０１３３】(6-4) BTS1発現株のプレニルアルコール生
産 pRS435GAP-BTS1/EUG8（図27A）とpRS445GAP-BTS1/EUG8
（図27B）をそれぞれ5コロニーずつ培養し、プレニルア
ルコール生産量の平均と標準偏差をグラフ化したとこ
ろ、BTS1遺伝子発現のプレニルアルコール生産に対する
効果は、pRS435GAP-BTS1を導入した場合に平均0.5 mg/l
程度（最大値は1.42mg/l）のGGOH生産性向上効果がみら
れた。A451株にpRS435GAP-BTS1を導入した場合でも0.1-
0.3 mg/l程度のGGOH生産能があることが予備実験でわか
っているので、EUG株を宿主にした場合でも同様にGGOH
生産に効果があることがわかった。(6-4) Prenyl alcohol production of BTS1 expressing strains pRS435GAP-BTS1 / EUG8 (FIG. 27A) and pRS445GAP-BTS1 / EUG8
(Fig. 27B) was cultured in 5 colonies each, and the average and standard deviation of the prenyl alcohol production was graphed.The effect of BTS1 gene expression on prenyl alcohol production was 0.5 mg / average when pRS435GAP-BTS1 was introduced. l
The effect of improving the GGOH productivity was observed at a level (maximum value 1.42 mg / l). Even if pRS435GAP-BTS1 was introduced into A451 strain, 0.1-
Preliminary experiments have shown that GGOH has an ability to produce GGOH of about 0.3 mg / l.
It turned out to be effective for production.

【０１３４】次に、BTS1遺伝子発現増強がYPH499、YPH5
00由来のEUG12、EUG27に与える効果について調べた。pR
S435GAP-BTS1とpRS445GAP-BTS1を導入した株をそれぞれ
5コロニーずつ培養し、プレニルアルコール生産量の平
均と標準偏差をグラフ化したところ、BTS1遺伝子発現の
プレニルアルコール生産に対する効果は、pRS435GAP-BT
S1を導入した場合に、7日培養で平均3.7-3.8 mg/l程度
（最大値はそれぞれ4.2mg/l、4.4mg/l）のGGOH生産性が
みられた（図28A,B）。YPH499、YPH500株にpRS435GAP-B
TS1を導入した場合でも0.1-0.2 mg/l程度のGGOH生産能
があることが予備実験でわかっているので、EUG株を宿
主にした場合通常の組換え体宿主よりGGOH生産に効果が
あることがわかった。Next, enhancement of BTS1 gene expression was observed in YPH499 and YPH5.
The effects on 00-derived EUG12 and EUG27 were examined. pR
S435GAP-BTS1 and pRS445GAP-BTS1 introduced strains respectively
When 5 colonies were cultured and the average and standard deviation of prenyl alcohol production were plotted, the effect of BTS1 gene expression on prenyl alcohol production was found to be pRS435GAP-BT.
When S1 was introduced, an average GGOH productivity of 3.7-3.8 mg / l (maximum values were 4.2 mg / l and 4.4 mg / l, respectively) was observed in 7-day culture (Fig. 28A, B). PRS435GAP-B added to YPH499 and YPH500 strains
Preliminary experiments have shown that even if TS1 is introduced, it has a GGOH-producing ability of about 0.1-0.2 mg / l. Therefore, when the EUG strain is used as a host, it is more effective than the normal recombinant host for GGOH production. I understood.

【０１３５】上記の結果から、本発明で作製した変異型
細胞、すなわちスクアレン合成酵素遺伝子を完全欠損又
は部分欠損させることをせずに、翻訳活性のあるスクア
レン合成酵素遺伝子転写産物量を減少できるようにした
変異型細胞（例えばEUG株）に、IPP合成経路関連酵素遺
伝子（例えばHMG1、ERG20又はBTS1）を含む発現用組換
えDNA又はゲノムインテグレート用DNAを導入して組換え
体を作製し、該組換え体を培養すると、翻訳活性を有す
るスクアレン合成酵素遺伝子の転写産物量が減少するこ
とによって、プレニルアルコールを製造できる系が構築
できたことになる。From the above results, it is possible to decrease the amount of squalene synthase gene transcript having translation activity, without completely or partially deleting the mutant cell prepared in the present invention, that is, the squalene synthase gene. Into the mutant cell (for example, EUG strain), the recombinant DNA for expression containing the IPP synthesis pathway-related enzyme gene (for example, HMG1 , ERG20 or BTS1) or the DNA for genome integration is introduced to prepare a recombinant, By culturing the recombinant, the amount of the transcription product of the squalene synthase gene having translation activity is reduced, which means that the system capable of producing prenyl alcohol was constructed.

【０１３６】〔実施例8〕 融合遺伝子導入EUG株による
プレニルアルコールの生産 実施例7でEUG株を宿主にしFPP合成酵素遺伝子を導入す
るとGGOH生産性が向上する予想外の結果が得られた。こ
れは、S. cerevisiae BTS1がコードしているGGPP合成酵
素は、プライマー基質としてDMAPPよりもFPPを好む事を
示唆している。よって、IPPからGGOH前駆体であるGGPP
への合成能をさらに強化するためには、FPP合成能を同
時に補強する必要があると考えられた。そこで、本実施
例では、BTS1とERG20の融合遺伝子を作製した。Example 8 Production of Prenyl Alcohol by Fusion Gene-Introduced EUG Strain In Example 7, when the EPP strain was used as a host and the FPP synthase gene was introduced, the unexpected result that the GGOH productivity was improved was obtained. This suggests that the GGPP synthase encoded by S. cerevisiae BTS1 prefers FPP over DMAPP as a primer substrate. Therefore, from IPP to GGOH precursor GGPP
It was considered necessary to reinforce the FPP synthesizing ability at the same time in order to further enhance the synthesizing ability to FPP. Therefore, in this example, a fusion gene of BTS1 and ERG20 was prepared.

【０１３７】本実施例では、融合遺伝子をS.cerevisiae
細胞内で発現させ、GGOH生産能が向上するかを確かめる
ことにした。また、あわせて小胞体シグナルをコードす
る塩基配列がBTS1、ERG20やその融合タンパク質遺伝子
の3’末端に位置するようにBTS1、ERG20、又はそれらの
融合遺伝子の変異型遺伝子を作製し、作製した遺伝子導
入によるプレニルアルコール生産に与える影響を調べる
ことにした。In this example, the fusion gene was cloned into S. cerevisiae .
It was confirmed that it could be expressed intracellularly to improve the GGOH productivity. In addition, BTS1 , ERG20 , or a mutant gene of these fusion genes is prepared so that the nucleotide sequence encoding the endoplasmic reticulum signal is also located at the 3'end of BTS1 , ERG20 or its fusion protein gene, and the prepared gene We decided to investigate the effect of introduction on prenyl alcohol production.

【０１３８】(1) 融合遺伝子導入用プラスミドDNAの作
製 GGPP合成酵素遺伝子BTS1をpYES2に組み込んだpYESGGPS
と、FPP合成酵素遺伝子ERG20をpT7に組み込んだpT7ERG2
0とを鋳型に用い、PCRを行った。使用したPCRプライマ
ーは以下の通りである。(1) Preparation of plasmid DNA for fusion gene introduction pYESGGPS in which GGPP synthase gene BTS1 was incorporated into pYES2
And pT7ERG2 in which the FPP synthase gene ERG20 was incorporated into pT7.
PCR was performed using 0 and as templates. The PCR primers used are as follows.

【０１３９】SacII-BTS1:5'-TCC CCG CGG ATG GAG GCC
AAG ATA GAT-3'（配列番号70） BTS1-XhoI:5'-CAA CTC GAG TCA CAA TTC GGA TAA GTG-
3'（配列番号71） ERG20HDEL-XbaI:5'-GCT CTA GAG TTC GTC GTG TTT GCT
TCT CTT GTA AAC TT-3'（配列番号72） BTS1HDEL-XhoI:5'-TAT CTC GAG TCA CAA TTC GTC ATG T
AA ATT GG-3'（配列番号73） BTSI-109I:5'-GCA GGG ACC CCA ATT CGG ATA AGT GGT C
-3'（配列番号74） 109I-BTS1:5'-GTA GGG TCC CTG GAG GCC AAG ATA GAT G
-3'（配列番号75） ERG20-109I:5'-GCA GGG ACC CTT TGC TTC TCT TGT AAA
CT-3'（配列番号76） 109I-ERG20:5'-GTA GGG TCC TCA GAA AAA GAA ATT AGG
AG-3'（配列番号77） -21:5'- TGT AAA ACG ACG GCC AGT-3'（配列番号78） T7:5'-TAA TAC GAC TCA CTA TAG GG-3'（配列番号79）SacII-BTS1: 5'-TCC CCG CGG ATG GAG GCC
AAG ATA GAT-3 '(SEQ ID NO: 70) BTS1-XhoI: 5'-CAA CTC GAG TCA CAA TTC GGA TAA GTG-
3 '(SEQ ID NO: 71) ERG20HDEL-XbaI: 5'-GC T CTA GA G TTC GTC GTG TTT GCT
TCT CTT GTA AAC TT-3 '(SEQ ID NO: 72) BTS1HDEL-XhoI: 5'-TAT CTC GAG TCA CAA TTC GTC ATG T
AA ATT GG-3 '(SEQ ID NO: 73) BTSI-109I: 5'-GCA GGG ACC C CA ATT CGG ATA AGT GGT C
-3 '(SEQ ID NO: 74) 109I-BTS1: 5'-GTA GGG TCC C TG GAG GCC AAG ATA GAT G
-3 '(SEQ ID NO: 75) ERG20-109I: 5'-GCA GGG ACC C TT TGC TTC TCT TGT AAA
CT-3 '(SEQ ID NO: 76) 109I-ERG20: 5'-GTA GGG TCC T CA GAA AAA GAA ATT AGG
AG-3 '(SEQ ID NO: 77) -21: 5'- TGT AAA ACG ACG GCC AGT-3' (SEQ ID NO: 78) T7: 5'-TAA TAC GAC TCA CTA TAG GG-3 '(SEQ ID NO: 79)

【０１４０】ERG20HDEL-XbaIの第3番目-第8番目の塩基
及びBTS1HDEL-XhoIの第4番目-第9番目の塩基（それぞ
れ、6塩基分を下線で施した部分）は、ベクター連結用
のSacII、XhoI又は XbaI認識部位を示す。また、BTSI-1
09I、109I-BTS1、ERG20-109I及び109I-ERG20の第4番目-
第10番目の塩基（それぞれ、7塩基分を下線で施した部
分）は融合遺伝子作製用のEcoO109I認識部位を示す。PC
Rは以下の反応液で行った。The 3rd to 8th bases of ERG20HDEL-XbaI and the 4th to 9th bases of BTS1HDEL-XhoI (each of which is an underlined portion of 6 bases) are Sac for vector ligation. II, Xho I or Xba I recognition site is shown. Also, BTSI-1
Fourth of 09I, 109I-BTS1, ERG20-109I and 109I-ERG20-
The 10th base (each underlined by 7 bases) represents an Eco O109I recognition site for producing a fusion gene. PC
For R, the following reaction solution was used.

【０１４１】 [0141]

【０１４２】KOD-Plusには1.6 μg/μlのKOD抗体が含ま
れている。反応条件は、94℃ で2分の反応後、94℃ 15
秒、55℃ 30秒及び68℃ 1分のサイクルを30サイクル行
い、その後68℃ 2分保温した。1st PCRは以下の鋳型、
プライマー（primer 1, primer 2）の組合せで行った。
(表6、図29)。PCR産物名も表6及び図29に示した。図29
において、最も左側の列に最終的なプラスミド名を記し
た。グレーの文字で示した配列はアミノ酸配列を意味
し、このうちGSは融合遺伝子の結合配列に導入し、HDEL
は小胞体シグナルとして、挿入又は欠失変異により遺伝
子3’末端に位置するようにした。くさび形矢印は、PCR
で使用したプライマーの位置と向きを示している。KOD-Plus contains 1.6 μg / μl KOD antibody. The reaction conditions are as follows: 94 ° C for 2 minutes, then 94 ° C 15
30 seconds, 55 ° C. for 30 seconds and 68 ° C. for 1 minute, followed by incubation at 68 ° C. for 2 minutes. 1st PCR is the following template,
A combination of primers (primer 1, primer 2) was used.
(Table 6, Figure 29). The PCR product names are also shown in Table 6 and FIG. Figure 29
In, the final plasmid name is shown in the leftmost column. The sequence shown in gray letters means the amino acid sequence, of which GS was introduced into the binding sequence of the fusion gene, HDEL
Was positioned as the endoplasmic reticulum signal at the 3'end of the gene by insertion or deletion mutation. Wedge-shaped PCR
The position and orientation of the primer used in Step 3 are shown.

【０１４３】[0143]

【表６】 [Table 6]

【０１４４】PCR産物#9、#10、#11、#12、#13、#14を制
限酵素EcoO109Iで消化後、#9と#11、#10と#12、#9と#1
3、#10と#14とをそれぞれライゲーションした。このラ
イゲーション溶液をPCRの鋳型とし、それぞれ、SacII-B
TS1と-21、T7とBTS1-XhoI、SacII-BTS1とERG20HDEL-Xba
I、T7とBTS1HDEL-XhoIを、それぞれprimer 1とprimer2
に用いて、2nd PCRを1st PCRと同じ条件で行い、2nd PC
R産物#9-#11、#10-#12、#9-#13、#10-#14を得た。After digesting the PCR products # 9, # 10, # 11, # 12, # 13 and # 14 with the restriction enzyme Eco O109I, # 9 and # 11, # 10 and # 12, # 9 and # 1.
3, # 10 and # 14 were ligated respectively. This ligation solution was used as a PCR template and SacII-B
TS1 and -21, T7 and BTS1-XhoI, SacII-BTS1 and ERG20HDEL-Xba
I, T7 and BTS1HDEL-XhoI, primer 1 and primer 2 respectively
2nd PCR under the same conditions as for 1st PCR,
R products # 9- # 11, # 10- # 12, # 9- # 13, # 10- # 14 were obtained.

【０１４５】#9-#11をSacIIとBamHIで切断後pRS435GAP
とpRS445GAPのSacII-BamHI部位に挿入し、それぞれpRS4
35GGFとpRS445GGFとした。#10-#12をXbaIとXhoIで切断
後pRS435GAPとpRS445GAPのXbaI-XhoI部位に挿入し、そ
れぞれpRS435FGGとpRS445FGGとした。#9-#13をSacIIとX
baIで切断後pRS435GAPのSacII-XbaI部位に挿入し、それ
ぞれpRS435GGFHDELとした。After cutting # 9- # 11 with Sac II and Bam HI, pRS435GAP
And pRS445GAP at the Sac II- Bam HI site, and pRS4
35GGF and pRS445GGF. After cutting # 10- # 12 with Xba I and Xho I, they were inserted into the Xba I- Xho I sites of pRS435GAP and pRS445GAP to obtain pRS435FGG and pRS445FGG, respectively. # 9- # 13 to Sac II and X
After cutting with ba I, they were inserted into Sac II- Xba I sites of pRS435GAP to obtain pRS435GGFHDEL.

【０１４６】#10-#14をXbaIとXhoIで切断後pRS435GAPと
pRS445GAPのXbaI-XhoI部位に挿入し、それぞれpRS435FG
GHDELとpRS445FGGHDELとした。#7をSacIIとXbaIで切断
後pRS435GAPとpRS445GAPのSacII-XbaI部位に挿入し、そ
れぞれpRS435FHDELとpRS445FHDELとした。#6をBamHIとX
hoIで切断後pRS435GAPとpRS445GAPのBamHI-XhoI部位に
挿入し、それぞれpRS435GGHDELとpRS445GGHDELとした。After cutting # 10- # 14 with Xba I and Xho I, pRS435GAP
Inserted at the Xba I- Xho I sites of pRS445GAP and pRS435FG
GHDEL and pRS445FGGHDEL. After cutting # 7 with Sac II and Xba I, it was inserted into the Sac II- Xba I sites of pRS435GAP and pRS445GAP to obtain pRS435FHDEL and pRS445FHDEL, respectively. # 6 to Bam HI and X
After cutting with ho I, they were inserted into Bam HI- Xho I sites of pRS435GAP and pRS445GAP to obtain pRS435GGHDEL and pRS445GGHDEL, respectively.

【０１４７】作製したプラスミドDNAは、DNAシークエン
シングにより設計通りの塩基配列を持っていることを確
認してある。BTS1-ERG20融合遺伝子をGGF、ERG20-BTS1
融合遺伝子をFGG、GGFの3’末端にHDELをコードする配
列が位置するように組換えた遺伝子をGGFHDEL、FGGの
3’末端にHDELをコードする配列が位置するように組換
えた遺伝子をFGGHDELとし、BTS1の3’末端にHDELをコー
ドする配列が位置するように組換えた遺伝子をGGHDELと
した。It has been confirmed by DNA sequencing that the prepared plasmid DNA has the designed nucleotide sequence. BTS1-ERG20 fusion gene to GGF, ERG20-BTS1
The fusion gene was FGG, and the gene that was recombined so that the sequence encoding HDEL was located at the 3'end of GGF was GGFHDEL or FGG.
The gene recombined so that the sequence encoding HDEL was located at the 3'end was designated as FGGHDEL, and the gene recombined so that the sequence encoding HDEL was located at the 3'end of BTS1 was designated as GGHDEL.

【０１４８】融合遺伝子でないBTS1、ERG20の発現プラ
スミドとして、pRS435GAP-BTS1（pRS435GGという）、pR
S445GAP-BTS1（pRS445GGという）、pRS435GAP-ERG20（p
RS435Fという）、pRS445GAP-ERG20（pRS445FGという）
を利用し、HMG1発現用に使用したプラスミドとしてpRS4
34TEF-HMG1とpRS434GAP-HMG1を利用した。As expression plasmids for BTS1 and ERG20 which are not fusion genes, pRS435GAP-BTS1 (referred to as pRS435GG), pR
S445GAP-BTS1 (referred to as pRS445GG), pRS435GAP-ERG20 (p
RS435F), pRS445GAP-ERG20 (called pRS445FG)
Using pRS4 as the plasmid used for HMG1 expression.
34TEF-HMG1 and pRS434GAP-HMG1 were used.

【０１４９】(2) 組換え体の作製 組換え体の作製は、Zymo Research (Orange, CA)のFroz
en EZ yeast transformation kitを用いて、上記の通り
作製したプラスミドを宿主に導入することにより行っ
た。宿主はEUG5、EUG12を使用し、対照としてA451、YPH
499、を使用した。(2) Recombinant Production Recombinant production was performed by Froz of Zymo Research (Orange, CA).
The plasmid prepared as described above was introduced into a host using the en EZ yeast transformation kit. EUG5 and EUG12 were used as hosts, and A451 and YPH were used as controls.
499 was used.

【０１５０】(3) プレニルアルコール生産量測定 A451とYPH499はSD培地に接種し、EUG株、及びEUG株を宿
主にしたさらなる組換え体をSGR選択培地に接種し、30
℃で培養し前培養液とした。培養した前培養液10又は25
μlを、1.0若しくは2.5 mlのYM7+ade (YM, pH7, 40 μ
g/ml アデニン硫酸塩) 培地又はYMO7 (YM7+ade, 1%(w/
v)大豆油, 0.1%(w/v) アデカノール LG-109 (Asahi Den
ka Kogyo, Tokyo, Japan)) 培地に加え、30℃で4日間又
は7日間130r.p.m.で往復振盪培養した。(3) Measurement of production amount of prenyl alcohol A451 and YPH499 were inoculated into SD medium, and EUG strain and a further recombinant hosting the EUG strain were inoculated into SGR selective medium.
Cultivation was carried out at ° C to obtain a preculture liquid. Pre-cultured medium 10 or 25
μl of 1.0 or 2.5 ml YM7 + ade (YM, pH7, 40 μl
g / ml adenine sulfate) medium or YMO7 (YM7 + ade, 1% (w /
v) Soybean oil, 0.1% (w / v) Adecanol LG-109 (Asahi Den
ka Kogyo, Tokyo, Japan)) The medium was added and cultured at 30 ° C. for 4 days or 7 days with reciprocal shaking at 130 rpm.

【０１５１】培養後、メタノールを等量加えて混合し、
ペンタンを約2倍量加えて激しく攪拌した。静置後、ペ
ンタン層を新しいガラス試験管にとり、ドラフト中でペ
ンタンを気化させ、溶質成分を濃縮後、GC/MSでプレニ
ルアルコールを同定・定量し、内部標準としてウンデカ
ノールを用いて定量した。また、その際、菌体増殖の度
合いを調べるため、培養液20 μlを水で30倍に希釈し、
600 nmの吸光度を測定した。プレニルアルコール生産量
は、実施例２(1-2)と同様にHP6890/5973 GC/MSシステム
を用いて測定した。After culturing, an equal amount of methanol was added and mixed,
About twice the amount of pentane was added, and the mixture was vigorously stirred. After standing, the pentane layer was placed in a new glass test tube, pentane was vaporized in a draft, solute components were concentrated, and then prenyl alcohol was identified and quantified by GC / MS, and quantified using undecanol as an internal standard. At that time, in order to examine the degree of bacterial growth, 20 μl of the culture solution was diluted 30 times with water,
The absorbance at 600 nm was measured. The amount of prenyl alcohol produced was measured using the HP6890 / 5973 GC / MS system as in Example 2 (1-2).

【０１５２】(4) 結果と考察 作製した各組換え体をYM7培地とYMO7培地で4日から7日
培養し、プレニルアルコール生産量を測定した。A451系
統の株を宿主としたときの結果を図30、31に示し、YPH4
99系統の株を宿主としたときの結果を図32、33に示し
た。図30、31において、GGFHDELはpRS435GGFHDELを示
す。「-1」は4日培養後の生産量、「-2」は7日培養後の
生産量を示す。YMO7培地では大豆油懸濁のため菌体量を
細胞数で示した。「10^3 cell/ul」は1マイクロリット
ルあたりの細胞数を1000で割った値を示す。(4) Results and Discussion Each recombinant thus prepared was cultured in YM7 medium and YMO7 medium for 4 to 7 days, and the amount of prenyl alcohol produced was measured. The results when the strain of A451 strain was used as a host are shown in Figs.
The results when 99 strains were used as hosts are shown in FIGS. 30 and 31, GGFHDEL represents pRS435GGFHDEL. "-1" indicates the production amount after 4 days of culture, and "-2" indicates the production amount after 7 days of culture. In YMO7 medium, the amount of cells was indicated by the number of cells because it was a soybean oil suspension. "10 ^ 3 cell / ul" indicates the value obtained by dividing the number of cells per microliter by 1000.

【０１５３】pRS435GGF/A451は、YM7培地で7日培養した
ときの生産量（図30、上パネルGGF/A451 -2）が平均0.2
6mg/l GGOH (最大0.28mg/l)であり、YMO7培地中では平
均0.98 mg/l (最大1.0mg/l)（図31、上パネルのGGF/A45
1-2）であった。一方、EUG5を宿主にした場合は、pRS43
5GGF導入により、YM7培地、7日間培養で平均6.6mg/l
（最大7.3mg/l）GGOHであり（図30、下パネルのGGF/EUG
5-2）、YMO7培地で培養すると平均9.6mg/l（最大10.1mg
/l）のGGOHを生産するようになった（図31、下パネルの
GGF/EUG5-2）。[0153] pRS435GGF / A451 produced 0.2 times on average in 7 days culture in YM7 medium (Fig. 30, upper panel GGF / A451 -2).
6 mg / l GGOH (up to 0.28 mg / l), average 0.98 mg / l (up to 1.0 mg / l) in YMO7 medium (Figure 31, upper panel GGF / A45).
It was 1-2). On the other hand, when EUG5 was used as a host, pRS43
By introducing 5GGF, YM7 medium, 6.6 mg / l on average for 7 days of culture
(Up to 7.3 mg / l) GGOH (Figure 30, lower panel GGF / EUG
5-2), when cultured in YMO7 medium, the average is 9.6 mg / l (maximum 10.1 mg
/ l) GGOH has come to be produced (Fig. 31, lower panel
GGF / EUG5-2).

【０１５４】pRS435GGFを導入したYPH499をYM7で7日間
培養したときに平均0.19mg/l（最大0.37mg/l）（図32、
上パネルのGGF/YPH499-2）、YMO7培地では平均2.5mg/l
（最大2.9mg/l）のGGOHを生産した（図33、上パネルのG
GF/YPH499-2）。一方、YPH499から作製したEUG12株を宿
主にしたときには、pRS435GGFやpRS435GGFHDEL導入株を
YMO培地で培養すると3.7-4.0mg/l（最大5.4-5.8mg/l）
のGGOHを生産し（図33、下パネルのGGF/EUG12-2, GGFHD
EL/EUG12-2）。したがって、YPH499やA451と言った通常
組換え宿主を用いてGGOH生産株を作製するよりも、本発
明で作製したスクアレン合成酵素遺伝子を完全欠損又は
部分欠損させることをせずに、翻訳活性のあるスクアレ
ン合成酵素遺伝子転写産物量を減少できるようにした変
異型細胞であるEUG5やEUG12を宿主にしたほうが、よりG
GOH高生産性の組換え体を作製できることが示された。When pPH435GGF-introduced YPH499 was cultured in YM7 for 7 days, the average was 0.19 mg / l (maximum 0.37 mg / l) (FIG. 32,
Upper panel GGF / YPH499-2), YMO7 medium average 2.5 mg / l
Produced (up to 2.9 mg / l) GGOH (Fig. 33, upper panel G
GF / YPH499-2). On the other hand, when the EUG12 strain prepared from YPH499 was used as a host, pRS435GGF or pRS435GGFHDEL-introduced strains were used.
3.7-4.0mg / l (maximum 5.4-5.8mg / l) when cultured in YMO medium
GGOH (Fig. 33, lower panel GGF / EUG12-2, GGFHD
EL / EUG12-2). Therefore, rather than producing a GGOH-producing strain using a normal recombinant host such as YPH499 or A451, the squalene synthase gene produced in the present invention does not have a complete deletion or partial deletion, and has translation activity. It is better to use EUG5 and EUG12, which are mutant cells that can reduce the amount of squalene synthase gene transcript, as a host.
It was shown that recombinants with high GOH productivity can be produced.

【０１５５】〔実施例9〕 各グルコース-ガラクトース
糖組成培地を用いたときの遺伝子導入EUG株によるプレ
ニルアルコール生産 (1) ベクターの宿主への導入及び培養 本実施例では、Glc-Gal糖組成変化により出芽酵母のプ
レニルアルコール生産がどう変わるかを確認する。併せ
て、BTS1-ERG20融合遺伝子発現によるプレニルアルコー
ル生産への効果も調べる。[Example 9] Production of prenyl alcohol by gene-transferred EUG strain when using each glucose-galactose sugar composition medium (1) Introduction of vector into host and culture In this example, Glc-Gal sugar composition changes To confirm the change in the production of prenyl alcohol in Saccharomyces cerevisiae. In addition, we will examine the effect of BTS1 - ERG20 fusion gene expression on prenyl alcohol production.

【０１５６】Zymo Research (Orange, CA) より購入し
たFrozen EZ yeast transformationII kitを用いて酵母
にベクターの導入を行った。ERG20、BTS1融合遺伝子発
現プラスミドとしてpRS435GGF、pRS435GGFHDELを用い
た。また、宿主としてEUG5、EUG12を用いた。対照の宿
主としてA451、YPH499を用いた。Vectors were introduced into yeast using the Frozen EZ yeast transformation II kit purchased from Zymo Research (Orange, CA). PRS435GGF and pRS435GGFHDEL were used as ERG20 and BTS1 fusion gene expression plasmids. In addition, EUG5 and EUG12 were used as hosts. A451 and YPH499 were used as control hosts.

【０１５７】各組換え体はSGR選択培地で前培養し、0.0
1-0.05mlの前培養液を1-5mlのYM7培地にそれぞれ加え、
18mm径の試験管で30℃、130r.p.m.の往復振盪培養条件
で培養した。YM7培地の糖成分（GlcとGalとの組成比）
として、0%、Glc-100%Gal、20%Glc-80%Gal、50%Glc-50%
Gal、75%Glc-25%Gal、100%Glc-0%Galの組成の各培地を
作製し、これらの培地でまず30℃、130r.p.m.の往復振
盪培養条件で培養した。2日培養後、終濃度5%(w/v)にな
るようにGlcをさらに加え、培養を7日目まで継続した。Each recombinant was pre-cultured in SGR selection medium to 0.0
Add 1-0.05 ml preculture to 1-5 ml YM7 medium,
It was cultivated in a test tube having a diameter of 18 mm under reciprocal shaking culture conditions of 30 ° C. and 130 rpm. Sugar component of YM7 medium (composition ratio of Glc and Gal)
As 0%, Glc-100% Gal, 20% Glc-80% Gal, 50% Glc-50%
Gal, 75% Glc-25% Gal, and 100% Glc-0% Gal compositions were prepared, and each medium was first cultured under reciprocal shaking culture conditions of 30 ° C. and 130 rpm. After 2 days of culture, Glc was further added so that the final concentration was 5% (w / v), and the culture was continued until the 7th day.

【０１５８】(2) 結果と考察 (2-1) EUG5のGGOH生産 EUG5にpRS435GGF、pRS435GGFHDELを導入したときのGGOH
生産の結果を図34に示す。pRS435GGF導入株（図34中、
「GGF/EUG5」と表示）及びpRS435GGFHDEL導入株（図34
中、「HDEL/EUG5」と表示）のいずれの場合も、2-4日の
培養、初期条件20-80%Glcが良好であった。(2) Results and Discussion (2-1) Production of GGOH of EUG5 GGOH when pRS435GGF and pRS435GGFHDEL were introduced into EUG5
The result of production is shown in FIG. pRS435GGF-introduced strain (in FIG. 34,
"GGF / EUG5") and pRS435GGFHDEL-introduced strain (Fig. 34)
In both cases, "HDEL / EUG5"), the initial condition of 20-80% Glc was good for 2-4 days of culture.

【０１５９】(2-2) EUG12のGGOH生産 EUG12にpRS435GGF、pRS435GGFHDELを導入したときのプ
レニルアルコール生産の結果を図35に示す。初期条件20
%Glcのときに、いずれの場合（宿主、発現プラスミド）
でも高いプレニルアルコールの生産性を示した。pRS435
GGF/EUG12（図35中、「GGF/EUG12」と表示）とpRS435GG
FHDEL/EUG12（図35中、「HDEL/EUG12」と表示）の初期
条件20%Glc、4日培養の場合は、それぞれ菌体がOD600＝
1.1、0.70に相当する量であっても、FOHは、それぞれ平
均7.6mg/l(最大10.5mg/l)、平均8.1mg/l(最大12.6mg/l)
（図面に表示せず）であった一方、GGOHは、それぞれ平
均5.4mg/l(最大7.0mg/l)、5.6mg/l（最大8.0mg/l）の生
産性があった。これは、菌体あたりの生産性としては非
常に効率が良いと考えられる。(2-2) Production of GGOH of EUG12 The results of prenyl alcohol production when pRS435GGF and pRS435GGFHDEL were introduced into EUG12 are shown in FIG. Initial condition 20
In case of% Glc, either case (host, expression plasmid)
However, it showed high productivity of prenyl alcohol. pRS435
GGF / EUG12 (displayed as "GGF / EUG12" in Figure 35) and pRS435GG
FHDEL / EUG12 (in FIG. 35, "HDEL / EUG12" and the display) in the case of initial conditions 20% Glc, 4 days culture, each cell OD 600 =
Even in amounts equivalent to 1.1 and 0.70, FOH has an average of 7.6 mg / l (maximum 10.5 mg / l) and an average of 8.1 mg / l (maximum 12.6 mg / l), respectively.
On the other hand, GGOH had an average productivity of 5.4 mg / l (maximum 7.0 mg / l) and 5.6 mg / l (maximum 8.0 mg / l), respectively (not shown in the drawing). This is considered to be very efficient in terms of productivity per cell.

【０１６０】〔実施例10〕 YPDO7rich培地を用いたと
きのプレニルアルコール生産量 (1) 培養とプレニルアルコール量測定 実施例1で作製したEUG5、EUG24、EUG36及びEUG64、並び
にそれらを宿主としてpRS434GAP-HMG1を導入した組換え
体、その作製した各組換え体にさらにpRS435GGを導入し
た組換え体をYPDO7rich培地（YPD, 1%(v/v) 大豆油, 0.
1%(v/v) アデカノール LG-109, 5%(w/v) Glc (終濃度 7
%(w/v) Glc), pH7）で30℃、130r.p.m.で往復振盪培養
し実施例2と同様にしてプレニルアルコール量を測定し
た。Example 10 Production of prenyl alcohol using YPDO7rich medium (1) Culture and measurement of prenyl alcohol EUG5, EUG24, EUG36 and EUG64 produced in Example 1 and pRS434GAP-HMG1 using them as hosts Introduced into the YPDO7rich medium (YPD, 1% (v / v) soybean oil, 0.
1% (v / v) Adecanol LG-109, 5% (w / v) Glc (final concentration 7
% (w / v) Glc), pH 7) was cultured at 30 ° C. and 130 rpm for reciprocal shaking, and the amount of prenyl alcohol was measured in the same manner as in Example 2.

【０１６１】(2) 結果と考察 培養液中のプレニルアルコール蓄積量と600nmの吸光度O
D600をグラフにまとめたものが図36である。YM培地より
窒素源と糖源を高濃度で含むYPDO7rich培地培養によ
り、より高濃度のプレニルアルコールを生産させること
ができた。EUG5をYPDO7rich培地で培養すると3日培養後
にFOHとGGOHをそれぞれ57.5mg/l、1.70mg/l生産した
（図36中「EUG5 in YPDO7rich」）。EUG24は培養3日後
にそれぞれ42.2mg/l、0.87mg/lを（図36中「EUG24 in Y
PDO7rich」）、EUG36は培養7日後にそれぞれ37.8mg/l、
0.98mg/lを（図36中「EUG36 in YPDO7rich」）、EUG64
は培養7日後にそれぞれ101.7mg/l、2.92mg・l（図36中
「EUG64 in YPDO7rich」）生産した。つまり、本発明で
作製したスクアレン合成酵素遺伝子を完全欠損又は部分
欠損させることをせずに、翻訳活性のあるスクアレン合
成酵素遺伝子転写産物量を減少できるようにした変異型
細胞、たとえばEUG64を宿主に用い、IPP合成経路関連酵
素遺伝子を含む発現用組換えDNA又はゲノムインテグレ
ート用DNAを導入して組換え体を作製し、組換え体を培
養した後、翻訳活性のあるスクアレン合成酵素遺伝子転
写産物量を減少させれば、単純なスクアレン合成酵素遺
伝子欠損株、たとえばS. cerevisiae ATCC64031とは異
なり、エルゴステロール要求性に起因する培地へのステ
ロール類の添加をしなくても培養液1リットルあたり0.1
gを超えるプレニルアルコール生産系を構築することが
できることがわかった。(2) Results and Discussion The amount of prenyl alcohol accumulated in the culture solution and the absorbance at 600 nm O
FIG. 36 shows a graph of D 600 . Higher concentration of prenyl alcohol could be produced by culturing YPDO7rich medium containing higher concentration of nitrogen source and sugar source than YM medium. When EUG5 was cultured in YPDO7rich medium, FOH and GGOH were produced at 57.5 mg / l and 1.70 mg / l, respectively, after 3 days of culture (“EUG5 in YPDO7rich” in FIG. 36). EUG24 was 42.2 mg / l and 0.87 mg / l after 3 days of culturing (see “EUG24 in Y in FIG. 36”).
PDO7rich "), EUG36 was 37.8 mg / l after 7 days of culture,
0.98mg / l ("EUG36 in YPDO7rich" in Figure 36), EUG64
Produced 101.7 mg / l and 2.92 mg · l (“EUG64 in YPDO7rich” in FIG. 36) after 7 days of culture, respectively. That is, a mutant cell capable of reducing the amount of squalene synthase gene transcript having translation activity without completely or partially deleting the squalene synthase gene prepared in the present invention, for example, EUG64 was used as a host. A recombinant squalene synthase gene transcript with translation activity is prepared by introducing a recombinant DNA for expression containing an IPP synthesis pathway-related enzyme gene or a DNA for genome integration into a recombinant, and culturing the recombinant. If, compared with a simple squalene synthase gene-deficient strain, such as S. cerevisiae ATCC64031, 0.1% per liter of culture medium is obtained without addition of sterols to the medium due to ergosterol requirement.
It has been found that it is possible to construct a prenyl alcohol production system in excess of g.

【０１６２】HMG1を導入した組換え体ではEUG5宿主の場
合3日培養後FOHを67.9mg/l生産した（図36中「HMG1/EUG
5 in YPDO7rich」）が、その他のEUG株では宿主よりFOH
生産性が向上しなかった。HMG1を導入した組換え体にさ
らにBTS1を導入したEUG5では、FOH生産を宿主並に確保
しつつGGOH高生産を示し、3日培養後に16.7mg/l、7日培
養後に19.8mg/l生産した（図36中「BTS1&HMG1/EUG5 in
YPDO7rich」）。特にEUG64を宿主とした場合7日培養後
に62.7mg/lのGGOHを生産した。つまり、本発明で作製し
た変異型細胞、すなわちスクアレン合成酵素遺伝子を完
全欠損又は部分欠損させることをせずに、翻訳活性のあ
るスクアレン合成酵素遺伝子転写産物量を減少できるよ
うにした変異型細胞（例えばEUG64）を宿主に用い、IPP
合成経路関連酵素遺伝子を含む発現用組換えDNA又はゲ
ノムインテグレート用DNAを導入して組換え体を作製
し、その組換え体を培養すると、翻訳活性のあるスクア
レン合成酵素遺伝子の転写産物量が減少することによっ
て、単純なスクアレン合成酵素遺伝子欠損株、例えばS.
cerevisiae ATCC64031株の培養液では検出されない種
類のプレニルアルコール（例えばGGOH）を、培養液1リ
ットルあたり0.06gを超えるレベルで生産させる系を構
築することができることがわかった。In the case of the EUG5 host, the recombinant into which HMG1 was introduced produced 67.9 mg / l of FOH after 3 days of culture (“HMG1 / EUG in FIG. 36”).
5 in YPDO7rich ”), but in other EUG strains,
The productivity did not improve. In EUG5 that further introduced BTS1 into the recombinant that introduced HMG1 , it showed high GGOH production while securing FOH production at the same level as the host, and produced 16.7 mg / l after 3 days of culture and 19.8 mg / l after 7 days of culture. (`` BTS1 & HMG1 / EUG5 in
YPDO7rich "). Especially when EUG64 was used as a host, 62.7 mg / l GGOH was produced after 7 days of culture. That is, the mutant cell produced in the present invention, that is, the mutant cell capable of reducing the amount of the squalene synthase gene transcript having translation activity without completely or partially deleting the squalene synthase gene ( For example, using EUG64) as a host,
When a recombinant is prepared by introducing a recombinant DNA for expression or a DNA for genome integration containing a synthetic pathway-related enzyme gene, and culturing the recombinant, the amount of the transcription product of the squalene synthase gene with translation activity decreases. By performing a simple squalene synthase gene-deficient strain, such as S.
It was found that it is possible to construct a system that produces a type of prenyl alcohol (eg, GGOH) that cannot be detected in the culture fluid of the cerevisiae ATCC64031 strain at a level exceeding 0.06 g per liter of the culture fluid.

【０１６３】[0163]

【発明の効果】本発明により、各種天然型の幾何異性を
有するプレニルアルコールの製造方法が提供される。本
発明によれば、各種天然型の幾何異性を有する活性型プ
レニルアルコールを大量に入手できるため、生体内にお
いて重要な膨大な種類のイソプレノイド・テルペノイド
化合物の工業生産に利用することができる点で、また、
活性型プレニルアルコールの新たな生理活性を見出すた
めの基本システムとして利用できる点で、本発明の方法
は有用である。INDUSTRIAL APPLICABILITY The present invention provides a method for producing prenyl alcohol having various natural geometric isomers. According to the present invention, since a large amount of active prenyl alcohol having various natural geometric isomers can be obtained, it can be used for industrial production of a huge variety of isoprenoid / terpenoid compounds that are important in vivo. Also,
The method of the present invention is useful in that it can be used as a basic system for finding a new physiological activity of active prenyl alcohol.

【０１６４】[0164]

【配列表】 SEQUENCE LISTING <110> TOYOTA JIDOSHA KABUSHIKI KAISHA <120> A METHOD OF PRODUCING PRENYLALCOHOL <130> P00-0911 <140> <141> <160> 79 <170> PatentIn Ver. 2.0 <210> 1 <211> 1059 <212> DNA <213> Saccharomyces cerevisiae <220> <221> CDS <222> (1)..(1056) <400> 1 atg gct tca gaa aaa gaa att agg aga gag aga ttc ttg aac gtt ttc 48 Met Ala Ser Glu Lys Glu Ile Arg Arg Glu Arg Phe Leu Asn Val Phe 1 5 10 15 cct aaa tta gta gag gaa ttg aac gca tcg ctt ttg gct tac ggt atg 96 Pro Lys Leu Val Glu Glu Leu Asn Ala Ser Leu Leu Ala Tyr Gly Met 20 25 30 cct aag gaa gca tgt gac tgg tat gcc cac tca ttg aac tac aac act 144 Pro Lys Glu Ala Cys Asp Trp Tyr Ala His Ser Leu Asn Tyr Asn Thr 35 40 45 cca ggc ggt aag cta aat aga ggt ttg tcc gtt gtg gac acg tat gct 192 Pro Gly Gly Lys Leu Asn Arg Gly Leu Ser Val Val Asp Thr Tyr Ala 50 55 60 att ctc tcc aac aag acc gtt gaa caa ttg ggg caa gaa gaa tac gaa 240 Ile Leu Ser Asn Lys Thr Val Glu Gln Leu Gly Gln Glu Glu Tyr Glu 65 70 75 80 aag gtt gcc att cta ggt tgg tgc att gag ttg ttg cag gct tac ttc 288 Lys Val Ala Ile Leu Gly Trp Cys Ile Glu Leu Leu Gln Ala Tyr Phe 85 90 95 ttg gtc gcc gat gat atg atg gac aag tcc att acc aga aga ggc caa 336 Leu Val Ala Asp Asp Met Met Asp Lys Ser Ile Thr Arg Arg Gly Gln 100 105 110 cca tgt tgg tac aag gtt cct gaa gtt ggg gaa att gcc atc aat gac 384 Pro Cys Trp Tyr Lys Val Pro Glu Val Gly Glu Ile Ala Ile Asn Asp 115 120 125 gca ttc atg tta gag gct gct atc tac aag ctt ttg aaa tct cac ttc 432 Ala Phe Met Leu Glu Ala Ala Ile Tyr Lys Leu Leu Lys Ser His Phe 130 135 140 aga aac gaa aaa tac tac ata gat atc acc gaa ttg ttc cat gag gtc 480 Arg Asn Glu Lys Tyr Tyr Ile Asp Ile Thr Glu Leu Phe His Glu Val 145 150 155 160 acc ttc caa acc gaa ttg ggc caa ttg atg gac tta atc act gca cct 528 Thr Phe Gln Thr Glu Leu Gly Gln Leu Met Asp Leu Ile Thr Ala Pro 165 170 175 gaa gac aaa gtc gac ttg agt aag ttc tcc cta aag aag cac tcc ttc 576 Glu Asp Lys Val Asp Leu Ser Lys Phe Ser Leu Lys Lys His Ser Phe 180 185 190 ata gtt act ttc aag act gct tac tat tct ttc tac ttg cct gtc gca 624 Ile Val Thr Phe Lys Thr Ala Tyr Tyr Ser Phe Tyr Leu Pro Val Ala 195 200 205 ttg gcc atg tac gtt gcc ggt atc acg gat gaa aag gat ttg aaa caa 672 Leu Ala Met Tyr Val Ala Gly Ile Thr Asp Glu Lys Asp Leu Lys Gln 210 215 220 gcc aga gat gtc ttg att cca ttg ggt gaa tac ttc caa att caa gat 720 Ala Arg Asp Val Leu Ile Pro Leu Gly Glu Tyr Phe Gln Ile Gln Asp 225 230 235 240 gac tac tta gac tgc ttc ggt acc cca gaa cag atc ggt aag atc ggt 768 Asp Tyr Leu Asp Cys Phe Gly Thr Pro Glu Gln Ile Gly Lys Ile Gly 245 250 255 aca gat atc caa gat aac aaa tgt tct tgg gta atc aac aag gca ttg 816 Thr Asp Ile Gln Asp Asn Lys Cys Ser Trp Val Ile Asn Lys Ala Leu 260 265 270 gaa ctt gct tcc gca gaa caa aga aag act tta gac gaa aat tac ggt 864 Glu Leu Ala Ser Ala Glu Gln Arg Lys Thr Leu Asp Glu Asn Tyr Gly 275 280 285 aag aag gac tca gtc gca gaa gcc aaa tgc aaa aag att ttc aat gac 912 Lys Lys Asp Ser Val Ala Glu Ala Lys Cys Lys Lys Ile Phe Asn Asp 290 295 300 ttg aaa att gaa cag cta tac cac gaa tat gaa gag tct att gcc aag 960 Leu Lys Ile Glu Gln Leu Tyr His Glu Tyr Glu Glu Ser Ile Ala Lys 305 310 315 320 gat ttg aag gcc aaa att tct cag gtc gat gag tct cgt ggc ttc aaa 1008 Asp Leu Lys Ala Lys Ile Ser Gln Val Asp Glu Ser Arg Gly Phe Lys 325 330 335 gct gat gtc tta act gcg ttc ttg aac aaa gtt tac aag aga agc aaa 1056 Ala Asp Val Leu Thr Ala Phe Leu Asn Lys Val Tyr Lys Arg Ser Lys 340 345 350 tag 1059 <210> 2 <211> 352 <212> PRT <213> Saccharomyces cerevisiae <400> 2 Met Ala Ser Glu Lys Glu Ile Arg Arg Glu Arg Phe Leu Asn Val Phe 1 5 10 15 Pro Lys Leu Val Glu Glu Leu Asn Ala Ser Leu Leu Ala Tyr Gly Met 20 25 30 Pro Lys Glu Ala Cys Asp Trp Tyr Ala His Ser Leu Asn Tyr Asn Thr 35 40 45 Pro Gly Gly Lys Leu Asn Arg Gly Leu Ser Val Val Asp Thr Tyr Ala 50 55 60 Ile Leu Ser Asn Lys Thr Val Glu Gln Leu Gly Gln Glu Glu Tyr Glu 65 70 75 80 Lys Val Ala Ile Leu Gly Trp Cys Ile Glu Leu Leu Gln Ala Tyr Phe 85 90 95 Leu Val Ala Asp Asp Met Met Asp Lys Ser Ile Thr Arg Arg Gly Gln 100 105 110 Pro Cys Trp Tyr Lys Val Pro Glu Val Gly Glu Ile Ala Ile Asn Asp 115 120 125 Ala Phe Met Leu Glu Ala Ala Ile Tyr Lys Leu Leu Lys Ser His Phe 130 135 140 Arg Asn Glu Lys Tyr Tyr Ile Asp Ile Thr Glu Leu Phe His Glu Val 145 150 155 160 Thr Phe Gln Thr Glu Leu Gly Gln Leu Met Asp Leu Ile Thr Ala Pro 165 170 175 Glu Asp Lys Val Asp Leu Ser Lys Phe Ser Leu Lys Lys His Ser Phe 180 185 190 Ile Val Thr Phe Lys Thr Ala Tyr Tyr Ser Phe Tyr Leu Pro Val Ala 195 200 205 Leu Ala Met Tyr Val Ala Gly Ile Thr Asp Glu Lys Asp Leu Lys Gln 210 215 220 Ala Arg Asp Val Leu Ile Pro Leu Gly Glu Tyr Phe Gln Ile Gln Asp 225 230 235 240 Asp Tyr Leu Asp Cys Phe Gly Thr Pro Glu Gln Ile Gly Lys Ile Gly 245 250 255 Thr Asp Ile Gln Asp Asn Lys Cys Ser Trp Val Ile Asn Lys Ala Leu 260 265 270 Glu Leu Ala Ser Ala Glu Gln Arg Lys Thr Leu Asp Glu Asn Tyr Gly 275 280 285 Lys Lys Asp Ser Val Ala Glu Ala Lys Cys Lys Lys Ile Phe Asn Asp 290 295 300 Leu Lys Ile Glu Gln Leu Tyr His Glu Tyr Glu Glu Ser Ile Ala Lys 305 310 315 320 Asp Leu Lys Ala Lys Ile Ser Gln Val Asp Glu Ser Arg Gly Phe Lys 325 330 335 Ala Asp Val Leu Thr Ala Phe Leu Asn Lys Val Tyr Lys Arg Ser Lys 340 345 350 <210> 3 <211> 900 <212> DNA <213> Escherichia coli <220> <221> CDS <222> (1)..(897) <400> 3 atg gac ttt ccg cag caa ctc gaa gcc tgc gtt aag cag gcc aac cag 48 Met Asp Phe Pro Gln Gln Leu Glu Ala Cys Val Lys Gln Ala Asn Gln 1 5 10 15 gcg ctg agc cgt ttt atc gcc cca ctg ccc ttt cag aac act ccc gtg 96 Ala Leu Ser Arg Phe Ile Ala Pro Leu Pro Phe Gln Asn Thr Pro Val 20 25 30 gtc gaa acc atg cag tat ggc gca tta tta ggt ggt aag cgc ctg cga 144 Val Glu Thr Met Gln Tyr Gly Ala Leu Leu Gly Gly Lys Arg Leu Arg 35 40 45 cct ttc ctg gtt tat gcc acc ggt cat atg ttc ggc gtt agc aca aac 192 Pro Phe Leu Val Tyr Ala Thr Gly His Met Phe Gly Val Ser Thr Asn 50 55 60 acg ctg gac gca ccc gct gcc gcc gtt gag tgt atc cac gct tac tca 240 Thr Leu Asp Ala Pro Ala Ala Ala Val Glu Cys Ile His Ala Tyr Ser 65 70 75 80 tta att cat gat gat tta ccg gca atg gat gat gac gat ctg cgt cgc 288 Leu Ile His Asp Asp Leu Pro Ala Met Asp Asp Asp Asp Leu Arg Arg 85 90 95 ggt ttg cca acc tgc cat gtg aag ttt ggc gaa gca aac gcg att ctc 336 Gly Leu Pro Thr Cys His Val Lys Phe Gly Glu Ala Asn Ala Ile Leu 100 105 110 gct ggc gac gct tta caa acg ctg gcg ttc tcg att tta agc gat gcc 384 Ala Gly Asp Ala Leu Gln Thr Leu Ala Phe Ser Ile Leu Ser Asp Ala 115 120 125 gat atg ccg gaa gtg tcg gac cgc gac aga att tcg atg att tct gaa 432 Asp Met Pro Glu Val Ser Asp Arg Asp Arg Ile Ser Met Ile Ser Glu 130 135 140 ctg gcg agc gcc agt ggt att gcc gga atg tgc ggt ggt cag gca tta 480 Leu Ala Ser Ala Ser Gly Ile Ala Gly Met Cys Gly Gly Gln Ala Leu 145 150 155 160 gat tta gac gcg gaa ggc aaa cac gta cct ctg gac gcg ctt gag cgt 528 Asp Leu Asp Ala Glu Gly Lys His Val Pro Leu Asp Ala Leu Glu Arg 165 170 175 att cat cgt cat aaa acc ggc gca ttg att cgc gcc gcc gtt cgc ctt 576 Ile His Arg His Lys Thr Gly Ala Leu Ile Arg Ala Ala Val Arg Leu 180 185 190 ggt gca tta agc gcc gga gat aaa gga cgt cgt gct ctg ccg gta ctc 624 Gly Ala Leu Ser Ala Gly Asp Lys Gly Arg Arg Ala Leu Pro Val Leu 195 200 205 gac aag tat gca gag agc atc ggc ctt gcc ttc cag gtt cag gat gac 672 Asp Lys Tyr Ala Glu Ser Ile Gly Leu Ala Phe Gln Val Gln Asp Asp 210 215 220 atc ctg gat gtg gtg gga gat act gca acg ttg gga aaa cgc cag ggt 720 Ile Leu Asp Val Val Gly Asp Thr Ala Thr Leu Gly Lys Arg Gln Gly 225 230 235 240 gcc gac cag caa ctt ggt aaa agt acc tac cct gca ctt ctg ggt ctt 768 Ala Asp Gln Gln Leu Gly Lys Ser Thr Tyr Pro Ala Leu Leu Gly Leu 245 250 255 gag caa gcc cgg aag aaa gcc cgg gat ctg atc gac gat gcc cgt cag 816 Glu Gln Ala Arg Lys Lys Ala Arg Asp Leu Ile Asp Asp Ala Arg Gln 260 265 270 tcg ctg aaa caa ctg gct gaa cag tca ctc gat acc tcg gca ctg gaa 864 Ser Leu Lys Gln Leu Ala Glu Gln Ser Leu Asp Thr Ser Ala Leu Glu 275 280 285 gcg cta gcg gac tac atc atc cag cgt aat aaa taa 900 Ala Leu Ala Asp Tyr Ile Ile Gln Arg Asn Lys 290 295 <210> 4 <211> 299 <212> PRT <213> Escherichia coli <400> 4 Met Asp Phe Pro Gln Gln Leu Glu Ala Cys Val Lys Gln Ala Asn Gln 1 5 10 15 Ala Leu Ser Arg Phe Ile Ala Pro Leu Pro Phe Gln Asn Thr Pro Val 20 25 30 Val Glu Thr Met Gln Tyr Gly Ala Leu Leu Gly Gly Lys Arg Leu Arg 35 40 45 Pro Phe Leu Val Tyr Ala Thr Gly His Met Phe Gly Val Ser Thr Asn 50 55 60 Thr Leu Asp Ala Pro Ala Ala Ala Val Glu Cys Ile His Ala Tyr Ser 65 70 75 80 Leu Ile His Asp Asp Leu Pro Ala Met Asp Asp Asp Asp Leu Arg Arg 85 90 95 Gly Leu Pro Thr Cys His Val Lys Phe Gly Glu Ala Asn Ala Ile Leu 100 105 110 Ala Gly Asp Ala Leu Gln Thr Leu Ala Phe Ser Ile Leu Ser Asp Ala 115 120 125 Asp Met Pro Glu Val Ser Asp Arg Asp Arg Ile Ser Met Ile Ser Glu 130 135 140 Leu Ala Ser Ala Ser Gly Ile Ala Gly Met Cys Gly Gly Gln Ala Leu 145 150 155 160 Asp Leu Asp Ala Glu Gly Lys His Val Pro Leu Asp Ala Leu Glu Arg 165 170 175 Ile His Arg His Lys Thr Gly Ala Leu Ile Arg Ala Ala Val Arg Leu 180 185 190 Gly Ala Leu Ser Ala Gly Asp Lys Gly Arg Arg Ala Leu Pro Val Leu 195 200 205 Asp Lys Tyr Ala Glu Ser Ile Gly Leu Ala Phe Gln Val Gln Asp Asp 210 215 220 Ile Leu Asp Val Val Gly Asp Thr Ala Thr Leu Gly Lys Arg Gln Gly 225 230 235 240 Ala Asp Gln Gln Leu Gly Lys Ser Thr Tyr Pro Ala Leu Leu Gly Leu 245 250 255 Glu Gln Ala Arg Lys Lys Ala Arg Asp Leu Ile Asp Asp Ala Arg Gln 260 265 270 Ser Leu Lys Gln Leu Ala Glu Gln Ser Leu Asp Thr Ser Ala Leu Glu 275 280 285 Ala Leu Ala Asp Tyr Ile Ile Gln Arg Asn Lys 290 295 <210> 5 <211> 1008 <212> DNA <213> Saccharomyces cerevisiae <220> <221> CDS <222> (1)..(1005) <400> 5 atg gag gcc aag ata gat gag ctg atc aat aat gat cct gtt tgg tcc 48 Met Glu Ala Lys Ile Asp Glu Leu Ile Asn Asn Asp Pro Val Trp Ser 1 5 10 15 agc caa aat gaa agc ttg att tca aaa cct tat aat cac atc ctt ttg 96 Ser Gln Asn Glu Ser Leu Ile Ser Lys Pro Tyr Asn His Ile Leu Leu 20 25 30 aaa cct ggc aag aac ttt aga cta aat tta ata gtt caa att aac aga 144 Lys Pro Gly Lys Asn Phe Arg Leu Asn Leu Ile Val Gln Ile Asn Arg 35 40 45 gtt atg aat ttg ccc aaa gac cag ctg gcc ata gtt tcg caa att gtt 192 Val Met Asn Leu Pro Lys Asp Gln Leu Ala Ile Val Ser Gln Ile Val 50 55 60 gag ctc ttg cat aat tcc agc ctt tta atc gac gat ata gaa gat aat 240 Glu Leu Leu His Asn Ser Ser Leu Leu Ile Asp Asp Ile Glu Asp Asn 65 70 75 80 gct ccc ttg aga agg gga cag acc act tct cac tta atc ttc ggt gta 288 Ala Pro Leu Arg Arg Gly Gln Thr Thr Ser His Leu Ile Phe Gly Val 85 90 95 ccc tcc act ata aac acc gca aat tat atg tat ttc aga gcc atg caa 336 Pro Ser Thr Ile Asn Thr Ala Asn Tyr Met Tyr Phe Arg Ala Met Gln 100 105 110 ctt gta tcg cag cta acc aca aaa gag cct ttg tat cat aat ttg att 384 Leu Val Ser Gln Leu Thr Thr Lys Glu Pro Leu Tyr His Asn Leu Ile 115 120 125 acg att ttc aac gaa gaa ttg atc aat cta cat agg gga caa ggc ttg 432 Thr Ile Phe Asn Glu Glu Leu Ile Asn Leu His Arg Gly Gln Gly Leu 130 135 140 gat ata tac tgg aga gac ttt ctg cct gaa atc ata cct act cag gag 480 Asp Ile Tyr Trp Arg Asp Phe Leu Pro Glu Ile Ile Pro Thr Gln Glu 145 150 155 160 atg tat ttg aat atg gtt atg aat aaa aca ggc ggc ctt ttc aga tta 528 Met Tyr Leu Asn Met Val Met Asn Lys Thr Gly Gly Leu Phe Arg Leu 165 170 175 acg ttg aga ctc atg gaa gcg ctg tct cct tcc tca cac cac ggc cat 576 Thr Leu Arg Leu Met Glu Ala Leu Ser Pro Ser Ser His His Gly His 180 185 190 tcg ttg gtt cct ttc ata aat ctt ctg ggt att att tat cag att aga 624 Ser Leu Val Pro Phe Ile Asn Leu Leu Gly Ile Ile Tyr Gln Ile Arg 195 200 205 gat gat tac ttg aat ttg aaa gat ttc caa atg tcc agc gaa aaa ggc 672 Asp Asp Tyr Leu Asn Leu Lys Asp Phe Gln Met Ser Ser Glu Lys Gly 210 215 220 ttt gct gag gac att aca gag ggg aag tta tct ttt ccc atc gtc cac 720 Phe Ala Glu Asp Ile Thr Glu Gly Lys Leu Ser Phe Pro Ile Val His 225 230 235 240 gcc ctt aac ttc act aaa acg aaa ggt caa act gag caa cac aat gaa 768 Ala Leu Asn Phe Thr Lys Thr Lys Gly Gln Thr Glu Gln His Asn Glu 245 250 255 att cta aga att ctc ctg ttg agg aca agt gat aaa gat ata aaa cta 816 Ile Leu Arg Ile Leu Leu Leu Arg Thr Ser Asp Lys Asp Ile Lys Leu 260 265 270 aag ctg att caa ata ctg gaa ttc gac acc aat tca ttg gcc tac acc 864 Lys Leu Ile Gln Ile Leu Glu Phe Asp Thr Asn Ser Leu Ala Tyr Thr 275 280 285 aaa aat ttt att aat caa tta gtg aat atg ata aaa aat gat aat gaa 912 Lys Asn Phe Ile Asn Gln Leu Val Asn Met Ile Lys Asn Asp Asn Glu 290 295 300 aat aag tat tta cct gat ttg gct tcg cat tcc gac acc gcc acc aat 960 Asn Lys Tyr Leu Pro Asp Leu Ala Ser His Ser Asp Thr Ala Thr Asn 305 310 315 320 tta cat gac gaa ttg tta tat ata ata gac cac tta tcc gaa ttg tga 1008 Leu His Asp Glu Leu Leu Tyr Ile Ile Asp His Leu Ser Glu Leu 325 330 335 <210> 6 <211> 335 <212> PRT <213> Saccharomyces cerevisiae <400> 6 Met Glu Ala Lys Ile Asp Glu Leu Ile Asn Asn Asp Pro Val Trp Ser 1 5 10 15 Ser Gln Asn Glu Ser Leu Ile Ser Lys Pro Tyr Asn His Ile Leu Leu 20 25 30 Lys Pro Gly Lys Asn Phe Arg Leu Asn Leu Ile Val Gln Ile Asn Arg 35 40 45 Val Met Asn Leu Pro Lys Asp Gln Leu Ala Ile Val Ser Gln Ile Val 50 55 60 Glu Leu Leu His Asn Ser Ser Leu Leu Ile Asp Asp Ile Glu Asp Asn 65 70 75 80 Ala Pro Leu Arg Arg Gly Gln Thr Thr Ser His Leu Ile Phe Gly Val 85 90 95 Pro Ser Thr Ile Asn Thr Ala Asn Tyr Met Tyr Phe Arg Ala Met Gln 100 105 110 Leu Val Ser Gln Leu Thr Thr Lys Glu Pro Leu Tyr His Asn Leu Ile 115 120 125 Thr Ile Phe Asn Glu Glu Leu Ile Asn Leu His Arg Gly Gln Gly Leu 130 135 140 Asp Ile Tyr Trp Arg Asp Phe Leu Pro Glu Ile Ile Pro Thr Gln Glu 145 150 155 160 Met Tyr Leu Asn Met Val Met Asn Lys Thr Gly Gly Leu Phe Arg Leu 165 170 175 Thr Leu Arg Leu Met Glu Ala Leu Ser Pro Ser Ser His His Gly His 180 185 190 Ser Leu Val Pro Phe Ile Asn Leu Leu Gly Ile Ile Tyr Gln Ile Arg 195 200 205 Asp Asp Tyr Leu Asn Leu Lys Asp Phe Gln Met Ser Ser Glu Lys Gly 210 215 220 Phe Ala Glu Asp Ile Thr Glu Gly Lys Leu Ser Phe Pro Ile Val His 225 230 235 240 Ala Leu Asn Phe Thr Lys Thr Lys Gly Gln Thr Glu Gln His Asn Glu 245 250 255 Ile Leu Arg Ile Leu Leu Leu Arg Thr Ser Asp Lys Asp Ile Lys Leu 260 265 270 Lys Leu Ile Gln Ile Leu Glu Phe Asp Thr Asn Ser Leu Ala Tyr Thr 275 280 285 Lys Asn Phe Ile Asn Gln Leu Val Asn Met Ile Lys Asn Asp Asn Glu 290 295 300 Asn Lys Tyr Leu Pro Asp Leu Ala Ser His Ser Asp Thr Ala Thr Asn 305 310 315 320 Leu His Asp Glu Leu Leu Tyr Ile Ile Asp His Leu Ser Glu Leu 325 330 335 <210> 7 <211> 3165 <212> DNA <213> Saccharomyces cerevisiae <220> <221> CDS <222> (1)..(3162) <400> 7 atg ccg ccg cta ttc aag gga ctg aaa cag atg gca aag cca att gcc 48 Met Pro Pro Leu Phe Lys Gly Leu Lys Gln Met Ala Lys Pro Ile Ala 1 5 10 15 tat gtt tca aga ttt tcg gcg aaa cga cca att cat ata ata ctt ttt 96 Tyr Val Ser Arg Phe Ser Ala Lys Arg Pro Ile His Ile Ile Leu Phe 20 25 30 tct cta atc ata tcc gca ttc gct tat cta tcc gtc att cag tat tac 144 Ser Leu Ile Ile Ser Ala Phe Ala Tyr Leu Ser Val Ile Gln Tyr Tyr 35 40 45 ttc aat ggt tgg caa cta gat tca aat agt gtt ttt gaa act gct cca 192 Phe Asn Gly Trp Gln Leu Asp Ser Asn Ser Val Phe Glu Thr Ala Pro 50 55 60 aat aaa gac tcc aac act cta ttt caa gaa tgt tcc cat tac tac aga 240 Asn Lys Asp Ser Asn Thr Leu Phe Gln Glu Cys Ser His Tyr Tyr Arg 65 70 75 80 gat tcc tct cta gat ggt tgg gta tca atc acc gcg cat gaa gct agt 288 Asp Ser Ser Leu Asp Gly Trp Val Ser Ile Thr Ala His Glu Ala Ser 85 90 95 gag tta cca gcc cca cac cat tac tat cta tta aac ctg aac ttc aat 336 Glu Leu Pro Ala Pro His His Tyr Tyr Leu Leu Asn Leu Asn Phe Asn 100 105 110 agt cct aat gaa act gac tcc att cca gaa cta gct aac acg gtt ttt 384 Ser Pro Asn Glu Thr Asp Ser Ile Pro Glu Leu Ala Asn Thr Val Phe 115 120 125 gag aaa gat aat aca aaa tat att ctg caa gaa gat ctc agt gtt tcc 432 Glu Lys Asp Asn Thr Lys Tyr Ile Leu Gln Glu Asp Leu Ser Val Ser 130 135 140 aaa gaa att tct tct act gat gga acg aaa tgg agg tta aga agt gac 480 Lys Glu Ile Ser Ser Thr Asp Gly Thr Lys Trp Arg Leu Arg Ser Asp 145 150 155 160 aga aaa agt ctt ttc gac gta aag acg tta gca tat tct ctc tac gat 528 Arg Lys Ser Leu Phe Asp Val Lys Thr Leu Ala Tyr Ser Leu Tyr Asp 165 170 175 gta ttt tca gaa aat gta acc caa gca gac ccg ttt gac gtc ctt att 576 Val Phe Ser Glu Asn Val Thr Gln Ala Asp Pro Phe Asp Val Leu Ile 180 185 190 atg gtt act gcc tac cta atg atg ttc tac acc ata ttc ggc ctc ttc 624 Met Val Thr Ala Tyr Leu Met Met Phe Tyr Thr Ile Phe Gly Leu Phe 195 200 205 aat gac atg agg aag acc ggg tca aat ttt tgg ttg agc gcc tct aca 672 Asn Asp Met Arg Lys Thr Gly Ser Asn Phe Trp Leu Ser Ala Ser Thr 210 215 220 gtg gtc aat tct gca tca tca ctt ttc tta gca ttg tat gtc acc caa 720 Val Val Asn Ser Ala Ser Ser Leu Phe Leu Ala Leu Tyr Val Thr Gln 225 230 235 240 tgt att cta ggc aaa gaa gtt tcc gca tta act ctt ttt gaa ggt ttg 768 Cys Ile Leu Gly Lys Glu Val Ser Ala Leu Thr Leu Phe Glu Gly Leu 245 250 255 cct ttc att gta gtt gtt gtt ggt ttc aag cac aaa atc aag att gcc 816 Pro Phe Ile Val Val Val Val Gly Phe Lys His Lys Ile Lys Ile Ala 260 265 270 cag tat gcc ctg gag aaa ttt gaa aga gtc ggt tta tct aaa agg att 864 Gln Tyr Ala Leu Glu Lys Phe Glu Arg Val Gly Leu Ser Lys Arg Ile 275 280 285 act acc gat gaa atc gtt ttt gaa tcc gtg agc gaa gag ggt ggt cgt 912 Thr Thr Asp Glu Ile Val Phe Glu Ser Val Ser Glu Glu Gly Gly Arg 290 295 300 ttg att caa gac cat ttg ctt tgt att ttt gcc ttt atc gga tgc tct 960 Leu Ile Gln Asp His Leu Leu Cys Ile Phe Ala Phe Ile Gly Cys Ser 305 310 315 320 atg tat gct cac caa ttg aag act ttg aca aac ttc tgc ata tta tca 1008 Met Tyr Ala His Gln Leu Lys Thr Leu Thr Asn Phe Cys Ile Leu Ser 325 330 335 gca ttt atc cta att ttt gaa ttg att tta act cct aca ttt tat tct 1056 Ala Phe Ile Leu Ile Phe Glu Leu Ile Leu Thr Pro Thr Phe Tyr Ser 340 345 350 gct atc tta gcg ctt aga ctg gaa atg aat gtt atc cac aga tct act 1104 Ala Ile Leu Ala Leu Arg Leu Glu Met Asn Val Ile His Arg Ser Thr 355 360 365 att atc aag caa aca tta gaa gaa gac ggt gtt gtt cca tct aca gca 1152 Ile Ile Lys Gln Thr Leu Glu Glu Asp Gly Val Val Pro Ser Thr Ala 370 375 380 aga atc att tct aaa gca gaa aag aaa tcc gta tct tct ttc tta aat 1200 Arg Ile Ile Ser Lys Ala Glu Lys Lys Ser Val Ser Ser Phe Leu Asn 385 390 395 400 ctc agt gtg gtt gtc att atc atg aaa ctc tct gtc ata ctg ttg ttt 1248 Leu Ser Val Val Val Ile Ile Met Lys Leu Ser Val Ile Leu Leu Phe 405 410 415 gtc ttc atc aac ttt tat aac ttt ggt gca aat tgg gtc aat gat gcc 1296 Val Phe Ile Asn Phe Tyr Asn Phe Gly Ala Asn Trp Val Asn Asp Ala 420 425 430 ttc aat tca ttg tac ttc gat aag gaa cgt gtt tct cta cca gat ttt 1344 Phe Asn Ser Leu Tyr Phe Asp Lys Glu Arg Val Ser Leu Pro Asp Phe 435 440 445 att acc tcg aat gcc tct gaa aac ttt aaa gag caa gct att gtt agt 1392 Ile Thr Ser Asn Ala Ser Glu Asn Phe Lys Glu Gln Ala Ile Val Ser 450 455 460 gtc acc cca tta tta tat tac aaa ccc att aag tcc tac caa cgc att 1440 Val Thr Pro Leu Leu Tyr Tyr Lys Pro Ile Lys Ser Tyr Gln Arg Ile 465 470 475 480 gag gat atg gtt ctt cta ttg ctt cgt aat gtc agt gtt gcc att cgt 1488 Glu Asp Met Val Leu Leu Leu Leu Arg Asn Val Ser Val Ala Ile Arg 485 490 495 gat agg ttc gtc agt aaa tta gtt ctt tcc gcc tta gta tgc agt gct 1536 Asp Arg Phe Val Ser Lys Leu Val Leu Ser Ala Leu Val Cys Ser Ala 500 505 510 gtc atc aat gtg tat tta ttg aat gct gct aga att cat acc agt tat 1584 Val Ile Asn Val Tyr Leu Leu Asn Ala Ala Arg Ile His Thr Ser Tyr 515 520 525 act gca gac caa ttg gtg aaa act gaa gtc acc aag aag tct ttt act 1632 Thr Ala Asp Gln Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe Thr 530 535 540 gct cct gta caa aag gct tct aca cca gtt tta acc aat aaa aca gtc 1680 Ala Pro Val Gln Lys Ala Ser Thr Pro Val Leu Thr Asn Lys Thr Val 545 550 555 560 att tct gga tcg aaa gtc aaa agt tta tca tct gcg caa tcg agc tca 1728 Ile Ser Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gln Ser Ser Ser 565 570 575 tca gga cct tca tca tct agt gag gaa gat gat tcc cgc gat att gaa 1776 Ser Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu 580 585 590 agc ttg gat aag aaa ata cgt cct tta gaa gaa tta gaa gca tta tta 1824 Ser Leu Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Leu Leu 595 600 605 agt agt gga aat aca aaa caa ttg aag aac aaa gag gtc gct gcc ttg 1872 Ser Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala Ala Leu 610 615 620 gtt att cac ggt aag tta cct ttg tac gct ttg gag aaa aaa tta ggt 1920 Val Ile His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu Gly 625 630 635 640 gat act acg aga gcg gtt gcg gta cgt agg aag gct ctt tca att ttg 1968 Asp Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser Ile Leu 645 650 655 gca gaa gct cct gta tta gca tct gat cgt tta cca tat aaa aat tat 2016 Ala Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys Asn Tyr 660 665 670 gac tac gac cgc gta ttt ggc gct tgt tgt gaa aat gtt ata ggt tac 2064 Asp Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr 675 680 685 atg cct ttg ccc gtt ggt gtt ata ggc ccc ttg gtt atc gat ggt aca 2112 Met Pro Leu Pro Val Gly Val Ile Gly Pro Leu Val Ile Asp Gly Thr 690 695 700 tct tat cat ata cca atg gca act aca gag ggt tgt ttg gta gct tct 2160 Ser Tyr His Ile Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser 705 710 715 720 gcc atg cgt ggc tgt aag gca atc aat gct ggc ggt ggt gca aca act 2208 Ala Met Arg Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr 725 730 735 gtt tta act aag gat ggt atg aca aga ggc cca gta gtc cgt ttc cca 2256 Val Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe Pro 740 745 750 act ttg aaa aga tct ggt gcc tgt aag ata tgg tta gac tca gaa gag 2304 Thr Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser Glu Glu 755 760 765 gga caa aac gca att aaa aaa gct ttt aac tct aca tca aga ttt gca 2352 Gly Gln Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala 770 775 780 cgt ctg caa cat att caa act tgt cta gca gga gat tta ctc ttc atg 2400 Arg Leu Gln His Ile Gln Thr Cys Leu Ala Gly Asp Leu Leu Phe Met 785 790 795 800 aga ttt aga aca act act ggt gac gca atg ggt atg aat atg att tct 2448 Arg Phe Arg Thr Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser 805 810 815 aaa ggt gtc gaa tac tca tta aag caa atg gta gaa gag tat ggc tgg 2496 Lys Gly Val Glu Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp 820 825 830 gaa gat atg gag gtt gtc tcc gtt tct ggt aac tac tgt acc gac aaa 2544 Glu Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys 835 840 845 aaa cca gct gcc atc aac tgg atc gaa ggt cgt ggt aag agt gtc gtc 2592 Lys Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val 850 855 860 gca gaa gct act att cct ggt gat gtt gtc aga aaa gtg tta aaa agt 2640 Ala Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu Lys Ser 865 870 875 880 gat gtt tcc gca ttg gtt gag ttg aac att gct aag aat ttg gtt gga 2688 Asp Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn Leu Val Gly 885 890 895 tct gca atg gct ggg tct gtt ggt gga ttt aac gca cat gca gct aat 2736 Ser Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala His Ala Ala Asn 900 905 910 tta gtg aca gct gtt ttc ttg gca tta gga caa gat cct gca caa aat 2784 Leu Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro Ala Gln Asn 915 920 925 gtt gaa agt tcc aac tgt ata aca ttg atg aaa gaa gtg gac ggt gat 2832 Val Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp 930 935 940 ttg aga att tcc gta tcc atg cca tcc atc gaa gta ggt acc atc ggt 2880 Leu Arg Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly 945 950 955 960 ggt ggt act gtt cta gaa cca caa ggt gcc atg ttg gac tta tta ggt 2928 Gly Gly Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Gly 965 970 975 gta aga ggc ccg cat gct acc gct cct ggt acc aac gca cgt caa tta 2976 Val Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gln Leu 980 985 990 gca aga ata gtt gcc tgt gcc gtc ttg gca ggt gaa tta tcc tta tgt 3024 Ala Arg Ile Val Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu Cys 995 1000 1005 gct gcc cta gca gcc ggc cat ttg gtt caa agt cat atg acc cac aac 3072 Ala Ala Leu Ala Ala Gly His Leu Val Gln Ser His Met Thr His Asn 1010 1015 1020 agg aaa cct gct gaa cca aca aaa cct aac aat ttg gac gcc act gat 3120 Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala Thr Asp 1025 1030 1035 1040 ata aat cgt ttg aaa gat ggg tcc gtc acc tgc att aaa tcc taa 3165 Ile Asn Arg Leu Lys Asp Gly Ser Val Thr Cys Ile Lys Ser 1045 1050 <210> 8 <211> 1054 <212> PRT <213> Saccharomyces cerevisiae <400> 8 Met Pro Pro Leu Phe Lys Gly Leu Lys Gln Met Ala Lys Pro Ile Ala 1 5 10 15 Tyr Val Ser Arg Phe Ser Ala Lys Arg Pro Ile His Ile Ile Leu Phe 20 25 30 Ser Leu Ile Ile Ser Ala Phe Ala Tyr Leu Ser Val Ile Gln Tyr Tyr 35 40 45 Phe Asn Gly Trp Gln Leu Asp Ser Asn Ser Val Phe Glu Thr Ala Pro 50 55 60 Asn Lys Asp Ser Asn Thr Leu Phe Gln Glu Cys Ser His Tyr Tyr Arg 65 70 75 80 Asp Ser Ser Leu Asp Gly Trp Val Ser Ile Thr Ala His Glu Ala Ser 85 90 95 Glu Leu Pro Ala Pro His His Tyr Tyr Leu Leu Asn Leu Asn Phe Asn 100 105 110 Ser Pro Asn Glu Thr Asp Ser Ile Pro Glu Leu Ala Asn Thr Val Phe 115 120 125 Glu Lys Asp Asn Thr Lys Tyr Ile Leu Gln Glu Asp Leu Ser Val Ser 130 135 140 Lys Glu Ile Ser Ser Thr Asp Gly Thr Lys Trp Arg Leu Arg Ser Asp 145 150 155 160 Arg Lys Ser Leu Phe Asp Val Lys Thr Leu Ala Tyr Ser Leu Tyr Asp 165 170 175 Val Phe Ser Glu Asn Val Thr Gln Ala Asp Pro Phe Asp Val Leu Ile 180 185 190 Met Val Thr Ala Tyr Leu Met Met Phe Tyr Thr Ile Phe Gly Leu Phe 195 200 205 Asn Asp Met Arg Lys Thr Gly Ser Asn Phe Trp Leu Ser Ala Ser Thr 210 215 220 Val Val Asn Ser Ala Ser Ser Leu Phe Leu Ala Leu Tyr Val Thr Gln 225 230 235 240 Cys Ile Leu Gly Lys Glu Val Ser Ala Leu Thr Leu Phe Glu Gly Leu 245 250 255 Pro Phe Ile Val Val Val Val Gly Phe Lys His Lys Ile Lys Ile Ala 260 265 270 Gln Tyr Ala Leu Glu Lys Phe Glu Arg Val Gly Leu Ser Lys Arg Ile 275 280 285 Thr Thr Asp Glu Ile Val Phe Glu Ser Val Ser Glu Glu Gly Gly Arg 290 295 300 Leu Ile Gln Asp His Leu Leu Cys Ile Phe Ala Phe Ile Gly Cys Ser 305 310 315 320 Met Tyr Ala His Gln Leu Lys Thr Leu Thr Asn Phe Cys Ile Leu Ser 325 330 335 Ala Phe Ile Leu Ile Phe Glu Leu Ile Leu Thr Pro Thr Phe Tyr Ser 340 345 350 Ala Ile Leu Ala Leu Arg Leu Glu Met Asn Val Ile His Arg Ser Thr 355 360 365 Ile Ile Lys Gln Thr Leu Glu Glu Asp Gly Val Val Pro Ser Thr Ala 370 375 380 Arg Ile Ile Ser Lys Ala Glu Lys Lys Ser Val Ser Ser Phe Leu Asn 385 390 395 400 Leu Ser Val Val Val Ile Ile Met Lys Leu Ser Val Ile Leu Leu Phe 405 410 415 Val Phe Ile Asn Phe Tyr Asn Phe Gly Ala Asn Trp Val Asn Asp Ala 420 425 430 Phe Asn Ser Leu Tyr Phe Asp Lys Glu Arg Val Ser Leu Pro Asp Phe 435 440 445 Ile Thr Ser Asn Ala Ser Glu Asn Phe Lys Glu Gln Ala Ile Val Ser 450 455 460 Val Thr Pro Leu Leu Tyr Tyr Lys Pro Ile Lys Ser Tyr Gln Arg Ile 465 470 475 480 Glu Asp Met Val Leu Leu Leu Leu Arg Asn Val Ser Val Ala Ile Arg 485 490 495 Asp Arg Phe Val Ser Lys Leu Val Leu Ser Ala Leu Val Cys Ser Ala 500 505 510 Val Ile Asn Val Tyr Leu Leu Asn Ala Ala Arg Ile His Thr Ser Tyr 515 520 525 Thr Ala Asp Gln Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe Thr 530 535 540 Ala Pro Val Gln Lys Ala Ser Thr Pro Val Leu Thr Asn Lys Thr Val 545 550 555 560 Ile Ser Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gln Ser Ser Ser 565 570 575 Ser Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu 580 585 590 Ser Leu Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Leu Leu 595 600 605 Ser Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala Ala Leu 610 615 620 Val Ile His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu Gly 625 630 635 640 Asp Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser Ile Leu 645 650 655 Ala Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys Asn Tyr 660 665 670 Asp Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr 675 680 685 Met Pro Leu Pro Val Gly Val Ile Gly Pro Leu Val Ile Asp Gly Thr 690 695 700 Ser Tyr His Ile Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser 705 710 715 720 Ala Met Arg Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr 725 730 735 Val Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe Pro 740 745 750 Thr Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser Glu Glu 755 760 765 Gly Gln Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala 770 775 780 Arg Leu Gln His Ile Gln Thr Cys Leu Ala Gly Asp Leu Leu Phe Met 785 790 795 800 Arg Phe Arg Thr Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser 805 810 815 Lys Gly Val Glu Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp 820 825 830 Glu Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys 835 840 845 Lys Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val 850 855 860 Ala Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu Lys Ser 865 870 875 880 Asp Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn Leu Val Gly 885 890 895 Ser Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala His Ala Ala Asn 900 905 910 Leu Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro Ala Gln Asn 915 920 925 Val Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp 930 935 940 Leu Arg Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly 945 950 955 960 Gly Gly Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Gly 965 970 975 Val Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gln Leu 980 985 990 Ala Arg Ile Val Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu Cys 995 1000 1005 Ala Ala Leu Ala Ala Gly His Leu Val Gln Ser His Met Thr His Asn 1010 1015 1020 Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala Thr Asp 1025 1030 1035 1040 Ile Asn Arg Leu Lys Asp Gly Ser Val Thr Cys Ile Lys Ser 1045 1050 <210> 9 <211> 1191 <212> DNA <213> Saccharomyces cerevisiae <220> <221> CDS <222> (1)..(1188) <400> 9 atg acc gtt tac aca gca tcc gtt acc gca ccc gtc aac atc gca acc 48 Met Thr Val Tyr Thr Ala Ser Val Thr Ala Pro Val Asn Ile Ala Thr 1 5 10 15 ctt aag tat tgg ggg aaa agg gac acg aag ttg aat ctg ccc acc aat 96 Leu Lys Tyr Trp Gly Lys Arg Asp Thr Lys Leu Asn Leu Pro Thr Asn 20 25 30 tcg tcc ata tca gtg act tta tcg caa gat gac ctc aga acg ttg acc 144 Ser Ser Ile Ser Val Thr Leu Ser Gln Asp Asp Leu Arg Thr Leu Thr 35 40 45 tct gcg gct act gca cct gag ttt gaa cgc gac act ttg tgg tta aat 192 Ser Ala Ala Thr Ala Pro Glu Phe Glu Arg Asp Thr Leu Trp Leu Asn 50 55 60 gga gaa cca cac agc atc gac aat gaa aga act caa aat tgt ctg cgc 240 Gly Glu Pro His Ser Ile Asp Asn Glu Arg Thr Gln Asn Cys Leu Arg 65 70 75 80 gac cta cgc caa tta aga aag gaa atg gaa tcg aag gac gcc tca ttg 288 Asp Leu Arg Gln Leu Arg Lys Glu Met Glu Ser Lys Asp Ala Ser Leu 85 90 95 ccc aca tta tct caa tgg aaa ctc cac att gtc tcc gaa aat aac ttt 336 Pro Thr Leu Ser Gln Trp Lys Leu His Ile Val Ser Glu Asn Asn Phe 100 105 110 cct aca gca gct ggt tta gct tcc tcc gct gct ggc ttt gct gca ttg 384 Pro Thr Ala Ala Gly Leu Ala Ser Ser Ala Ala Gly Phe Ala Ala Leu 115 120 125 gtc tct gca att gct aag tta tac caa tta cca cag tca act tca gaa 432 Val Ser Ala Ile Ala Lys Leu Tyr Gln Leu Pro Gln Ser Thr Ser Glu 130 135 140 ata tct aga ata gca aga aag ggg tct ggt tca gct tgt aga tcg ttg 480 Ile Ser Arg Ile Ala Arg Lys Gly Ser Gly Ser Ala Cys Arg Ser Leu 145 150 155 160 ttt ggc gga tac gtg gcc tgg gaa atg gga aaa gct gaa gat ggt cat 528 Phe Gly Gly Tyr Val Ala Trp Glu Met Gly Lys Ala Glu Asp Gly His 165 170 175 gat tcc atg gca gta caa atc gca gac agc tct gac tgg cct cag atg 576 Asp Ser Met Ala Val Gln Ile Ala Asp Ser Ser Asp Trp Pro Gln Met 180 185 190 aaa gct tgt gtc cta gtt gtc agc gat att aaa aag gat gtg agt tcc 624 Lys Ala Cys Val Leu Val Val Ser Asp Ile Lys Lys Asp Val Ser Ser 195 200 205 act cag ggt atg caa ttg acc gtg gca acc tcc gaa cta ttt aaa gaa 672 Thr Gln Gly Met Gln Leu Thr Val Ala Thr Ser Glu Leu Phe Lys Glu 210 215 220 aga att gaa cat gtc gta cca aag aga ttt gaa gtc atg cgt aaa gcc 720 Arg Ile Glu His Val Val Pro Lys Arg Phe Glu Val Met Arg Lys Ala 225 230 235 240 att gtt gaa aaa gat ttc gcc acc ttt gca aag gaa aca atg atg gat 768 Ile Val Glu Lys Asp Phe Ala Thr Phe Ala Lys Glu Thr Met Met Asp 245 250 255 tcc aac tct ttc cat gcc aca tgt ttg gac tct ttc cct cca ata ttc 816 Ser Asn Ser Phe His Ala Thr Cys Leu Asp Ser Phe Pro Pro Ile Phe 260 265 270 tac atg aat gac act tcc aag cgt atc atc agt tgg tgc cac acc att 864 Tyr Met Asn Asp Thr Ser Lys Arg Ile Ile Ser Trp Cys His Thr Ile 275 280 285 aat cag ttt tac gga gaa aca atc gtt gca tac acg ttt gat gca ggt 912 Asn Gln Phe Tyr Gly Glu Thr Ile Val Ala Tyr Thr Phe Asp Ala Gly 290 295 300 cca aat gct gtg ttg tac tac tta gct gaa aat gag tcg aaa ctc ttt 960 Pro Asn Ala Val Leu Tyr Tyr Leu Ala Glu Asn Glu Ser Lys Leu Phe 305 310 315 320 gca ttt atc tat aaa ttg ttt ggc tct gtt cct gga tgg gac aag aaa 1008 Ala Phe Ile Tyr Lys Leu Phe Gly Ser Val Pro Gly Trp Asp Lys Lys 325 330 335 ttt act act gag cag ctt gag gct ttc aac cat caa ttt gaa tca tct 1056 Phe Thr Thr Glu Gln Leu Glu Ala Phe Asn His Gln Phe Glu Ser Ser 340 345 350 aac ttt act gca cgt gaa ttg gat ctt gag ttg caa aag gat gtt gcc 1104 Asn Phe Thr Ala Arg Glu Leu Asp Leu Glu Leu Gln Lys Asp Val Ala 355 360 365 aga gtg att tta act caa gtc ggt tca ggc cca caa gaa aca aac gaa 1152 Arg Val Ile Leu Thr Gln Val Gly Ser Gly Pro Gln Glu Thr Asn Glu 370 375 380 tct ttg att gac gca aag act ggt cta cca aag gaa taa 1191 Ser Leu Ile Asp Ala Lys Thr Gly Leu Pro Lys Glu 385 390 395 <210> 10 <211> 396 <212> PRT <213> Saccharomyces cerevisiae <400> 10 Met Thr Val Tyr Thr Ala Ser Val Thr Ala Pro Val Asn Ile Ala Thr 1 5 10 15 Leu Lys Tyr Trp Gly Lys Arg Asp Thr Lys Leu Asn Leu Pro Thr Asn 20 25 30 Ser Ser Ile Ser Val Thr Leu Ser Gln Asp Asp Leu Arg Thr Leu Thr 35 40 45 Ser Ala Ala Thr Ala Pro Glu Phe Glu Arg Asp Thr Leu Trp Leu Asn 50 55 60 Gly Glu Pro His Ser Ile Asp Asn Glu Arg Thr Gln Asn Cys Leu Arg 65 70 75 80 Asp Leu Arg Gln Leu Arg Lys Glu Met Glu Ser Lys Asp Ala Ser Leu 85 90 95 Pro Thr Leu Ser Gln Trp Lys Leu His Ile Val Ser Glu Asn Asn Phe 100 105 110 Pro Thr Ala Ala Gly Leu Ala Ser Ser Ala Ala Gly Phe Ala Ala Leu 115 120 125 Val Ser Ala Ile Ala Lys Leu Tyr Gln Leu Pro Gln Ser Thr Ser Glu 130 135 140 Ile Ser Arg Ile Ala Arg Lys Gly Ser Gly Ser Ala Cys Arg Ser Leu 145 150 155 160 Phe Gly Gly Tyr Val Ala Trp Glu Met Gly Lys Ala Glu Asp Gly His 165 170 175 Asp Ser Met Ala Val Gln Ile Ala Asp Ser Ser Asp Trp Pro Gln Met 180 185 190 Lys Ala Cys Val Leu Val Val Ser Asp Ile Lys Lys Asp Val Ser Ser 195 200 205 Thr Gln Gly Met Gln Leu Thr Val Ala Thr Ser Glu Leu Phe Lys Glu 210 215 220 Arg Ile Glu His Val Val Pro Lys Arg Phe Glu Val Met Arg Lys Ala 225 230 235 240 Ile Val Glu Lys Asp Phe Ala Thr Phe Ala Lys Glu Thr Met Met Asp 245 250 255 Ser Asn Ser Phe His Ala Thr Cys Leu Asp Ser Phe Pro Pro Ile Phe 260 265 270 Tyr Met Asn Asp Thr Ser Lys Arg Ile Ile Ser Trp Cys His Thr Ile 275 280 285 Asn Gln Phe Tyr Gly Glu Thr Ile Val Ala Tyr Thr Phe Asp Ala Gly 290 295 300 Pro Asn Ala Val Leu Tyr Tyr Leu Ala Glu Asn Glu Ser Lys Leu Phe 305 310 315 320 Ala Phe Ile Tyr Lys Leu Phe Gly Ser Val Pro Gly Trp Asp Lys Lys 325 330 335 Phe Thr Thr Glu Gln Leu Glu Ala Phe Asn His Gln Phe Glu Ser Ser 340 345 350 Asn Phe Thr Ala Arg Glu Leu Asp Leu Glu Leu Gln Lys Asp Val Ala 355 360 365 Arg Val Ile Leu Thr Gln Val Gly Ser Gly Pro Gln Glu Thr Asn Glu 370 375 380 Ser Leu Ile Asp Ala Lys Thr Gly Leu Pro Lys Glu 385 390 395 <210> 11 <211> 3165 <212> DNA <213> Saccharomyces cerevisiae <220> <221> CDS <222> (1)..(3162) <400> 11 atg ccg ccg cta ttc aag gga ctg aaa cag atg gca aag cca att gcc 48 Met Pro Pro Leu Phe Lys Gly Leu Lys Gln Met Ala Lys Pro Ile Ala 1 5 10 15 tat gtt tca aga ttt tcg gcg aaa cga cca att cat ata ata ctt ttt 96 Tyr Val Ser Arg Phe Ser Ala Lys Arg Pro Ile His Ile Ile Leu Phe 20 25 30 tct cta atc ata tcc gca ttc gct tat cta tcc gtc att cag tat tac 144 Ser Leu Ile Ile Ser Ala Phe Ala Tyr Leu Ser Val Ile Gln Tyr Tyr 35 40 45 ttc aat ggt tgg caa cta gat tca aat agt gtt ttt gaa act gct cca 192 Phe Asn Gly Trp Gln Leu Asp Ser Asn Ser Val Phe Glu Thr Ala Pro 50 55 60 aat aaa gac ttc aac act cta ttt caa gaa tgt tcc cat tac tac aga 240 Asn Lys Asp Phe Asn Thr Leu Phe Gln Glu Cys Ser His Tyr Tyr Arg 65 70 75 80 gat tcc tct cta gat ggt tgg gta tca atc acc gcg cat gaa gct agt 288 Asp Ser Ser Leu Asp Gly Trp Val Ser Ile Thr Ala His Glu Ala Ser 85 90 95 gag tta cca gcc cca cac cat tac tat cta tta aac ctg aac ttc aat 336 Glu Leu Pro Ala Pro His His Tyr Tyr Leu Leu Asn Leu Asn Phe Asn 100 105 110 agt cct aat gaa act gac tcc att cca gaa cta gct aac acg gtt ttt 384 Ser Pro Asn Glu Thr Asp Ser Ile Pro Glu Leu Ala Asn Thr Val Phe 115 120 125 gag aaa gat aat aca aaa tat att ctg caa gaa gat ctc agc gtt tcc 432 Glu Lys Asp Asn Thr Lys Tyr Ile Leu Gln Glu Asp Leu Ser Val Ser 130 135 140 aaa gaa att tct tct act gat gga acg aaa tgg agg tta aga agt gac 480 Lys Glu Ile Ser Ser Thr Asp Gly Thr Lys Trp Arg Leu Arg Ser Asp 145 150 155 160 aga aaa agt ctt ttc gac gta aag acg tta gca tat tct ctc tac gat 528 Arg Lys Ser Leu Phe Asp Val Lys Thr Leu Ala Tyr Ser Leu Tyr Asp 165 170 175 gta ttt tca gaa aat gta acc caa gca gac ccg ttt gac gtc ctt att 576 Val Phe Ser Glu Asn Val Thr Gln Ala Asp Pro Phe Asp Val Leu Ile 180 185 190 atg gtt act gcc tac cta atg atg ttc tac acc ata ttc ggc ctc ttc 624 Met Val Thr Ala Tyr Leu Met Met Phe Tyr Thr Ile Phe Gly Leu Phe 195 200 205 aat gac atg agg aag acc ggg tca aat ttt tgg ttg agc gcc tct aca 672 Asn Asp Met Arg Lys Thr Gly Ser Asn Phe Trp Leu Ser Ala Ser Thr 210 215 220 gtg gtc aat tct gca tca tca ctt ttc tta gca ttg tat gtc acc caa 720 Val Val Asn Ser Ala Ser Ser Leu Phe Leu Ala Leu Tyr Val Thr Gln 225 230 235 240 tgt att cta ggc aaa gaa gtt tcc gca tta act ctt ttt gaa ggt ttg 768 Cys Ile Leu Gly Lys Glu Val Ser Ala Leu Thr Leu Phe Glu Gly Leu 245 250 255 cct ttc att gta gtt gtt gtt ggt ttc aag cac aaa atc aag att gcc 816 Pro Phe Ile Val Val Val Val Gly Phe Lys His Lys Ile Lys Ile Ala 260 265 270 cag tat gcc ctg gag aaa ttt gaa aga gtc ggt tta tct aaa agg att 864 Gln Tyr Ala Leu Glu Lys Phe Glu Arg Val Gly Leu Ser Lys Arg Ile 275 280 285 act acc gat gaa atc gtt ttt gaa tcc gtg agc gaa gag ggt ggt cgt 912 Thr Thr Asp Glu Ile Val Phe Glu Ser Val Ser Glu Glu Gly Gly Arg 290 295 300 ttg att caa gac cat ttg ctt tgt att ttt gcc ttt atc gga tgc tct 960 Leu Ile Gln Asp His Leu Leu Cys Ile Phe Ala Phe Ile Gly Cys Ser 305 310 315 320 atg tat gct cac caa ttg aag act ttg aca aac ttc tgc ata tta tca 1008 Met Tyr Ala His Gln Leu Lys Thr Leu Thr Asn Phe Cys Ile Leu Ser 325 330 335 gca ttt atc cta att ttc gaa ttg att tta act cct aca ttt tat tct 1056 Ala Phe Ile Leu Ile Phe Glu Leu Ile Leu Thr Pro Thr Phe Tyr Ser 340 345 350 gct atc tta gcg ctt aga ctg gaa atg aat gtt atc cac aga tct act 1104 Ala Ile Leu Ala Leu Arg Leu Glu Met Asn Val Ile His Arg Ser Thr 355 360 365 att atc aag caa aca tta gaa gaa gac ggt gtt gtt cca tct aca gca 1152 Ile Ile Lys Gln Thr Leu Glu Glu Asp Gly Val Val Pro Ser Thr Ala 370 375 380 aga atc att tct aag gca gaa aag aaa tcc gta tct tct ttc tta aat 1200 Arg Ile Ile Ser Lys Ala Glu Lys Lys Ser Val Ser Ser Phe Leu Asn 385 390 395 400 ctc agt gtg gtt gtc att atc atg aaa ctc tct gtc ata ctg ttg ttc 1248 Leu Ser Val Val Val Ile Ile Met Lys Leu Ser Val Ile Leu Leu Phe 405 410 415 gtc ttc atc aac ttt tat aac ttt ggt gca aat tgg gtc aat gat gcc 1296 Val Phe Ile Asn Phe Tyr Asn Phe Gly Ala Asn Trp Val Asn Asp Ala 420 425 430 ttc aat tca ttg tac ttc gat aag gaa cgt gtt tct cta cca gat ttt 1344 Phe Asn Ser Leu Tyr Phe Asp Lys Glu Arg Val Ser Leu Pro Asp Phe 435 440 445 att acc tcg aat gcc tct gaa aac ttt aaa gag caa gct att gtt agt 1392 Ile Thr Ser Asn Ala Ser Glu Asn Phe Lys Glu Gln Ala Ile Val Ser 450 455 460 gtc acc cca tta tta tat tac aaa ccc att aag tcc tac caa cgc att 1440 Val Thr Pro Leu Leu Tyr Tyr Lys Pro Ile Lys Ser Tyr Gln Arg Ile 465 470 475 480 gag gat atg gtt ctt cta ttg ctt cgt aat gtc agt gtt gcc att cgt 1488 Glu Asp Met Val Leu Leu Leu Leu Arg Asn Val Ser Val Ala Ile Arg 485 490 495 gat agg ttc gtc agt aaa tta gtt ctt tcc gcc tta gta tgc agt gct 1536 Asp Arg Phe Val Ser Lys Leu Val Leu Ser Ala Leu Val Cys Ser Ala 500 505 510 gtc atc aat gtg tat tta tta aat gct gct aga att cat acc agt tat 1584 Val Ile Asn Val Tyr Leu Leu Asn Ala Ala Arg Ile His Thr Ser Tyr 515 520 525 act gca gac caa ttg gtg aag act gaa gtc acc aag aag tct ttt act 1632 Thr Ala Asp Gln Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe Thr 530 535 540 gct cct gta caa aag gct tct aca cca gtt tta acc aat aaa aca gtc 1680 Ala Pro Val Gln Lys Ala Ser Thr Pro Val Leu Thr Asn Lys Thr Val 545 550 555 560 att tct gga tcg aaa gtc aaa agt tta tca tct gcg caa tcg agc tca 1728 Ile Ser Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gln Ser Ser Ser 565 570 575 tca gga cct tca tca tct agt gag gaa gat gat tcc cgc gat att gaa 1776 Ser Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu 580 585 590 agc ttg gat aag aaa ata cgt cct tta gaa gaa tta gaa gca tca tta 1824 Ser Leu Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Ser Leu 595 600 605 agt agt gga aat aca aaa caa ttg aag aac aaa gag gtc gct gcc ttg 1872 Ser Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala Ala Leu 610 615 620 gtt att cac ggt aag tta cct ttg tac gct ttg gag aaa aaa tta ggt 1920 Val Ile His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu Gly 625 630 635 640 gat act acg aga gcg gtt gcg gta cgt agg aag gct ctt tca att ttg 1968 Asp Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser Ile Leu 645 650 655 gca gaa gct cct gta tta gca tct gat cgt tta cca tat aaa aat tat 2016 Ala Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys Asn Tyr 660 665 670 gac tac gac cgc gta ttt ggc gct tgt tgt gaa aat gtt ata ggt tac 2064 Asp Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr 675 680 685 atg cct ttg ccc gtt ggt gtt ata ggc ccc ttg gtt atc gat ggt aca 2112 Met Pro Leu Pro Val Gly Val Ile Gly Pro Leu Val Ile Asp Gly Thr 690 695 700 tct tat cat ata cca atg gca act aca gag ggt tgt ttg gta gct tct 2160 Ser Tyr His Ile Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser 705 710 715 720 gcc atg cgt ggc tgt aag gca atc aat gct ggc ggt ggt gca aca act 2208 Ala Met Arg Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr 725 730 735 gtt tta act aag gat ggt atg aca aga ggc cca gta gtc cgt ttc cca 2256 Val Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe Pro 740 745 750 act ttg aaa aga tct ggt gcc tgt aag ata tgg tta gac tca gaa gag 2304 Thr Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser Glu Glu 755 760 765 gga caa aac gca att aaa aaa gct ttt aac tct aca tca aga ttt gca 2352 Gly Gln Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala 770 775 780 cgt ctg caa cat att caa act tgt cta gca gga gat tta ctc ttc atg 2400 Arg Leu Gln His Ile Gln Thr Cys Leu Ala Gly Asp Leu Leu Phe Met 785 790 795 800 aga ttt aga aca act act ggt gac gca atg ggt atg aat atg att tct 2448 Arg Phe Arg Thr Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser 805 810 815 aag ggt gtc gaa tac tca tta aag caa atg gta gaa gag tat ggc tgg 2496 Lys Gly Val Glu Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp 820 825 830 gaa gat atg gag gtt gtc tcc gtt tct ggt aac tac tgt acc gac aaa 2544 Glu Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys 835 840 845 aaa cca gct gcc atc aac tgg atc gaa ggt cgt ggt aag agt gtc gtc 2592 Lys Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val 850 855 860 gca gaa gct act att cct ggt gat gtt gtc aga aaa gtg tta aaa agt 2640 Ala Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu Lys Ser 865 870 875 880 gat gtt tcc gca ttg gtt gag ttg aac att gct aag aat ttg gtt gga 2688 Asp Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn Leu Val Gly 885 890 895 tct gca atg gct ggg tct gtt ggt gga ttt aac gca cgt gca gct aat 2736 Ser Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala Arg Ala Ala Asn 900 905 910 tta gtg aca gct gtt ttc ttg gca tta gga caa gat cct gca caa aat 2784 Leu Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro Ala Gln Asn 915 920 925 gtc gaa agt tcc aac tgt ata aca ttg atg aaa gaa gtg gac ggt gat 2832 Val Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp 930 935 940 ttg aga att tcc gta tcc atg cca tcc atc gaa gta ggt acc atc ggt 2880 Leu Arg Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly 945 950 955 960 ggt ggt act gtt cta gaa cca caa ggt gcc atg ttg gac tta tta ggt 2928 Gly Gly Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Gly 965 970 975 gta aga ggc cca cat gct acc gct cct ggt acc aac gca cgt caa tta 2976 Val Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gln Leu 980 985 990 gca aga ata gtt gcc tgt gcc gtc ttg gca ggt gaa tta tcc tta tgt 3024 Ala Arg Ile Val Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu Cys 995 1000 1005 gct gcc cta gca gcc ggc cat ttg gtt caa agt cat atg acc cac aac 3072 Ala Ala Leu Ala Ala Gly His Leu Val Gln Ser His Met Thr His Asn 1010 1015 1020 agg aaa cct gct gaa cca aca aaa cct aac aat ttg gac gcc act gat 3120 Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala Thr Asp 1025 1030 1035 1040 ata aat cgt ttg aaa gat ggg tcc gtc acc tgc att aaa tcc taa 3165 Ile Asn Arg Leu Lys Asp Gly Ser Val Thr Cys Ile Lys Ser 1045 1050 <210> 12 <211> 1054 <212> PRT <213> Saccharomyces cerevisiae <400> 12 Met Pro Pro Leu Phe Lys Gly Leu Lys Gln Met Ala Lys Pro Ile Ala 1 5 10 15 Tyr Val Ser Arg Phe Ser Ala Lys Arg Pro Ile His Ile Ile Leu Phe 20 25 30 Ser Leu Ile Ile Ser Ala Phe Ala Tyr Leu Ser Val Ile Gln Tyr Tyr 35 40 45 Phe Asn Gly Trp Gln Leu Asp Ser Asn Ser Val Phe Glu Thr Ala Pro 50 55 60 Asn Lys Asp Phe Asn Thr Leu Phe Gln Glu Cys Ser His Tyr Tyr Arg 65 70 75 80 Asp Ser Ser Leu Asp Gly Trp Val Ser Ile Thr Ala His Glu Ala Ser 85 90 95 Glu Leu Pro Ala Pro His His Tyr Tyr Leu Leu Asn Leu Asn Phe Asn 100 105 110 Ser Pro Asn Glu Thr Asp Ser Ile Pro Glu Leu Ala Asn Thr Val Phe 115 120 125 Glu Lys Asp Asn Thr Lys Tyr Ile Leu Gln Glu Asp Leu Ser Val Ser 130 135 140 Lys Glu Ile Ser Ser Thr Asp Gly Thr Lys Trp Arg Leu Arg Ser Asp 145 150 155 160 Arg Lys Ser Leu Phe Asp Val Lys Thr Leu Ala Tyr Ser Leu Tyr Asp 165 170 175 Val Phe Ser Glu Asn Val Thr Gln Ala Asp Pro Phe Asp Val Leu Ile 180 185 190 Met Val Thr Ala Tyr Leu Met Met Phe Tyr Thr Ile Phe Gly Leu Phe 195 200 205 Asn Asp Met Arg Lys Thr Gly Ser Asn Phe Trp Leu Ser Ala Ser Thr 210 215 220 Val Val Asn Ser Ala Ser Ser Leu Phe Leu Ala Leu Tyr Val Thr Gln 225 230 235 240 Cys Ile Leu Gly Lys Glu Val Ser Ala Leu Thr Leu Phe Glu Gly Leu 245 250 255 Pro Phe Ile Val Val Val Val Gly Phe Lys His Lys Ile Lys Ile Ala 260 265 270 Gln Tyr Ala Leu Glu Lys Phe Glu Arg Val Gly Leu Ser Lys Arg Ile 275 280 285 Thr Thr Asp Glu Ile Val Phe Glu Ser Val Ser Glu Glu Gly Gly Arg 290 295 300 Leu Ile Gln Asp His Leu Leu Cys Ile Phe Ala Phe Ile Gly Cys Ser 305 310 315 320 Met Tyr Ala His Gln Leu Lys Thr Leu Thr Asn Phe Cys Ile Leu Ser 325 330 335 Ala Phe Ile Leu Ile Phe Glu Leu Ile Leu Thr Pro Thr Phe Tyr Ser 340 345 350 Ala Ile Leu Ala Leu Arg Leu Glu Met Asn Val Ile His Arg Ser Thr 355 360 365 Ile Ile Lys Gln Thr Leu Glu Glu Asp Gly Val Val Pro Ser Thr Ala 370 375 380 Arg Ile Ile Ser Lys Ala Glu Lys Lys Ser Val Ser Ser Phe Leu Asn 385 390 395 400 Leu Ser Val Val Val Ile Ile Met Lys Leu Ser Val Ile Leu Leu Phe 405 410 415 Val Phe Ile Asn Phe Tyr Asn Phe Gly Ala Asn Trp Val Asn Asp Ala 420 425 430 Phe Asn Ser Leu Tyr Phe Asp Lys Glu Arg Val Ser Leu Pro Asp Phe 435 440 445 Ile Thr Ser Asn Ala Ser Glu Asn Phe Lys Glu Gln Ala Ile Val Ser 450 455 460 Val Thr Pro Leu Leu Tyr Tyr Lys Pro Ile Lys Ser Tyr Gln Arg Ile 465 470 475 480 Glu Asp Met Val Leu Leu Leu Leu Arg Asn Val Ser Val Ala Ile Arg 485 490 495 Asp Arg Phe Val Ser Lys Leu Val Leu Ser Ala Leu Val Cys Ser Ala 500 505 510 Val Ile Asn Val Tyr Leu Leu Asn Ala Ala Arg Ile His Thr Ser Tyr 515 520 525 Thr Ala Asp Gln Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe Thr 530 535 540 Ala Pro Val Gln Lys Ala Ser Thr Pro Val Leu Thr Asn Lys Thr Val 545 550 555 560 Ile Ser Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gln Ser Ser Ser 565 570 575 Ser Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu 580 585 590 Ser Leu Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Ser Leu 595 600 605 Ser Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala Ala Leu 610 615 620 Val Ile His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu Gly 625 630 635 640 Asp Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser Ile Leu 645 650 655 Ala Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys Asn Tyr 660 665 670 Asp Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr 675 680 685 Met Pro Leu Pro Val Gly Val Ile Gly Pro Leu Val Ile Asp Gly Thr 690 695 700 Ser Tyr His Ile Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser 705 710 715 720 Ala Met Arg Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr 725 730 735 Val Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe Pro 740 745 750 Thr Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser Glu Glu 755 760 765 Gly Gln Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala 770 775 780 Arg Leu Gln His Ile Gln Thr Cys Leu Ala Gly Asp Leu Leu Phe Met 785 790 795 800 Arg Phe Arg Thr Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser 805 810 815 Lys Gly Val Glu Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp 820 825 830 Glu Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys 835 840 845 Lys Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val 850 855 860 Ala Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu Lys Ser 865 870 875 880 Asp Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn Leu Val Gly 885 890 895 Ser Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala Arg Ala Ala Asn 900 905 910 Leu Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro Ala Gln Asn 915 920 925 Val Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp 930 935 940 Leu Arg Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly 945 950 955 960 Gly Gly Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Gly 965 970 975 Val Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gln Leu 980 985 990 Ala Arg Ile Val Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu Cys 995 1000 1005 Ala Ala Leu Ala Ala Gly His Leu Val Gln Ser His Met Thr His Asn 1010 1015 1020 Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala Thr Asp 1025 1030 1035 1040 Ile Asn Arg Leu Lys Asp Gly Ser Val Thr Cys Ile Lys Ser 1045 1050 <210> 13 <211> 3165 <212> DNA <213> Saccharomyces cerevisiae <220> <221> CDS <222> (1)..(3162) <400> 13 atg ccg ccg cta ttc aag gga ctg aaa cag atg gca aag cca att gcc 48 Met Pro Pro Leu Phe Lys Gly Leu Lys Gln Met Ala Lys Pro Ile Ala 1 5 10 15 tat gtt tca aga ttt tcg gcg aaa cga cca att cat ata ata ctt ttt 96 Tyr Val Ser Arg Phe Ser Ala Lys Arg Pro Ile His Ile Ile Leu Phe 20 25 30 tct cta atc ata tcc gca ttc gct tat cta tcc gtc att cag tat tac 144 Ser Leu Ile Ile Ser Ala Phe Ala Tyr Leu Ser Val Ile Gln Tyr Tyr 35 40 45 ttc aat ggt tgg caa cta gat tca aat agt gtt ttt gaa act gct cca 192 Phe Asn Gly Trp Gln Leu Asp Ser Asn Ser Val Phe Glu Thr Ala Pro 50 55 60 aat aaa gac tcc aac act cta ttt caa gaa tgt tcc cat tac tac aga 240 Asn Lys Asp Ser Asn Thr Leu Phe Gln Glu Cys Ser His Tyr Tyr Arg 65 70 75 80 gat tcc tct cta gat ggt tgg gta tca atc acc gcg cat gaa gct agt 288 Asp Ser Ser Leu Asp Gly Trp Val Ser Ile Thr Ala His Glu Ala Ser 85 90 95 gag tta cca gcc cca cac cat tac tat cta tta aac ctg aac ttc aat 336 Glu Leu Pro Ala Pro His His Tyr Tyr Leu Leu Asn Leu Asn Phe Asn 100 105 110 agt cct aat gaa act gac tcc att cca gaa cta gct aac acg gtt ttt 384 Ser Pro Asn Glu Thr Asp Ser Ile Pro Glu Leu Ala Asn Thr Val Phe 115 120 125 gag aaa gat aat aca aaa tat att ctg caa gaa gat ctc agc gtt tcc 432 Glu Lys Asp Asn Thr Lys Tyr Ile Leu Gln Glu Asp Leu Ser Val Ser 130 135 140 aaa gaa att tct tct act gat gga acg aaa tgg agg tta aga agt gac 480 Lys Glu Ile Ser Ser Thr Asp Gly Thr Lys Trp Arg Leu Arg Ser Asp 145 150 155 160 aga aaa agt ctt ttc gac gta aag acg tta gca tat tct ctc tac gat 528 Arg Lys Ser Leu Phe Asp Val Lys Thr Leu Ala Tyr Ser Leu Tyr Asp 165 170 175 gta ttt tca gaa aat gta acc caa gca gac ccg ttt gac gtc ctt att 576 Val Phe Ser Glu Asn Val Thr Gln Ala Asp Pro Phe Asp Val Leu Ile 180 185 190 atg gtt act gcc tac cta atg atg ttc tac acc ata ttc ggc ctc ttc 624 Met Val Thr Ala Tyr Leu Met Met Phe Tyr Thr Ile Phe Gly Leu Phe 195 200 205 aat gac atg agg aag acc ggg tca aat ttt tgg ttg agc gcc tct aca 672 Asn Asp Met Arg Lys Thr Gly Ser Asn Phe Trp Leu Ser Ala Ser Thr 210 215 220 gtg gtc aat tct gca tca tca ctt ttc tta gca ttg tat gtc acc caa 720 Val Val Asn Ser Ala Ser Ser Leu Phe Leu Ala Leu Tyr Val Thr Gln 225 230 235 240 tgt att cta ggc aaa gaa gtt tcc gca tta act ctt ttt gaa ggt ttg 768 Cys Ile Leu Gly Lys Glu Val Ser Ala Leu Thr Leu Phe Glu Gly Leu 245 250 255 cct ttc att gta gtt gtt gtt ggt ttc aag cac aaa atc aag att gcc 816 Pro Phe Ile Val Val Val Val Gly Phe Lys His Lys Ile Lys Ile Ala 260 265 270 cag tat gcc ctg gag aaa ttt gaa aga gtc ggt tta tct aaa agg att 864 Gln Tyr Ala Leu Glu Lys Phe Glu Arg Val Gly Leu Ser Lys Arg Ile 275 280 285 act acc gat gaa atc gtt ttt gaa tcc gtg agc gaa gag ggt ggt cgt 912 Thr Thr Asp Glu Ile Val Phe Glu Ser Val Ser Glu Glu Gly Gly Arg 290 295 300 ttg att caa gac cat ttg ctt tgt att ttt gcc ttt atc gga tgc tct 960 Leu Ile Gln Asp His Leu Leu Cys Ile Phe Ala Phe Ile Gly Cys Ser 305 310 315 320 atg tat gct cac caa ttg aag act ttg aca aac ttc tgc ata tta tca 1008 Met Tyr Ala His Gln Leu Lys Thr Leu Thr Asn Phe Cys Ile Leu Ser 325 330 335 gca ttt atc cta att ttc gaa ttg att tta act cct aca ttt tat tct 1056 Ala Phe Ile Leu Ile Phe Glu Leu Ile Leu Thr Pro Thr Phe Tyr Ser 340 345 350 gct atc tta gcg ctt aga ctg gaa atg aat gtt atc cac aga tct act 1104 Ala Ile Leu Ala Leu Arg Leu Glu Met Asn Val Ile His Arg Ser Thr 355 360 365 att atc aag caa aca tta gaa gaa gac ggt gtt gtt cca tct aca gca 1152 Ile Ile Lys Gln Thr Leu Glu Glu Asp Gly Val Val Pro Ser Thr Ala 370 375 380 aga atc att tct aag gca gaa aag aaa tcc gta tct tct ttc tta aat 1200 Arg Ile Ile Ser Lys Ala Glu Lys Lys Ser Val Ser Ser Phe Leu Asn 385 390 395 400 ctc agt gtg gtt gtc att atc atg aaa ctc tct gtc ata ctg ttg ttc 1248 Leu Ser Val Val Val Ile Ile Met Lys Leu Ser Val Ile Leu Leu Phe 405 410 415 gtc ttc atc aac ttt tat aac ttt ggt gca aat tgg gtc aat gat gcc 1296 Val Phe Ile Asn Phe Tyr Asn Phe Gly Ala Asn Trp Val Asn Asp Ala 420 425 430 ttc aat tca ttg tac ttc gat aag gaa cgt gtt tct cta cca gat ttt 1344 Phe Asn Ser Leu Tyr Phe Asp Lys Glu Arg Val Ser Leu Pro Asp Phe 435 440 445 att acc tcg aat gcc tct gaa aac ttt aaa gag caa gct att gtt agt 1392 Ile Thr Ser Asn Ala Ser Glu Asn Phe Lys Glu Gln Ala Ile Val Ser 450 455 460 gtc acc cca tta tta tat tac aaa ccc att aag tcc tac caa cgc att 1440 Val Thr Pro Leu Leu Tyr Tyr Lys Pro Ile Lys Ser Tyr Gln Arg Ile 465 470 475 480 gag gat atg gtt ctt cta ttg ctt cgt aat gtc agt gtt gcc att cgt 1488 Glu Asp Met Val Leu Leu Leu Leu Arg Asn Val Ser Val Ala Ile Arg 485 490 495 gat agg ttc gtc agt aaa tta gtt ctt tcc gcc tta gta tgc agt gct 1536 Asp Arg Phe Val Ser Lys Leu Val Leu Ser Ala Leu Val Cys Ser Ala 500 505 510 gtc atc aat gtg tat tta tta aat gct gct aga att cat acc agt tat 1584 Val Ile Asn Val Tyr Leu Leu Asn Ala Ala Arg Ile His Thr Ser Tyr 515 520 525 act gca gac caa ttg gtg aag act gaa gtc acc aag aag tct ttt act 1632 Thr Ala Asp Gln Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe Thr 530 535 540 gct cct gta caa aag gct tct aca cca gtt tta acc aat aaa aca gtc 1680 Ala Pro Val Gln Lys Ala Ser Thr Pro Val Leu Thr Asn Lys Thr Val 545 550 555 560 att tct gga tcg aaa gtc aaa agt tta tca tct gcg caa tcg agc tca 1728 Ile Ser Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gln Ser Ser Ser 565 570 575 tca gga cct tca tca tct agt gag gaa gat gat tcc cgc gat att gaa 1776 Ser Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu 580 585 590 agc ttg gat aag aaa ata cgt cct tta gaa gaa tta gaa gca tta tta 1824 Ser Leu Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Leu Leu 595 600 605 agt agt gga aat aca aaa caa ttg aag aac aaa gag gtc gct gcc ttg 1872 Ser Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala Ala Leu 610 615 620 gtt att cac ggt aag tta cct ttg tac gct ttg gag aaa aaa tta ggt 1920 Val Ile His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu Gly 625 630 635 640 gat act acg aga gcg gtt gcg gta cgt agg aag gct ctt tca att ttg 1968 Asp Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser Ile Leu 645 650 655 gca gaa gct cct gta tta gca tct gat cgt tta cca tat aaa aat tat 2016 Ala Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys Asn Tyr 660 665 670 gac tac gac cgc gta ttt ggc gct tgt tgt gaa aat gtt ata ggt tac 2064 Asp Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr 675 680 685 atg cct ttg ccc gtt ggt gtt ata ggc ccc ttg gtt atc gat ggt aca 2112 Met Pro Leu Pro Val Gly Val Ile Gly Pro Leu Val Ile Asp Gly Thr 690 695 700 tct tat cat ata cca atg gca act aca gag ggt tgt ttg gta gct tct 2160 Ser Tyr His Ile Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser 705 710 715 720 gcc atg cgt ggc tgt aag gca atc aat gct ggc ggt ggt gca aca act 2208 Ala Met Arg Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr 725 730 735 gtt tta act aag gat ggt atg aca aga ggc cca gta gtc cgt ttc cca 2256 Val Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe Pro 740 745 750 act ttg aaa aga tct ggt gcc tgt aag ata tgg tta gac tca gaa gag 2304 Thr Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser Glu Glu 755 760 765 gga caa aac gca att aaa aaa gct ttt aac tct aca tca aga ttt gca 2352 Gly Gln Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala 770 775 780 cgt ctg caa cat att caa act tgt cta gca gga gat tta ctc ttc atg 2400 Arg Leu Gln His Ile Gln Thr Cys Leu Ala Gly Asp Leu Leu Phe Met 785 790 795 800 aga ttt aga aca act act ggt gac gca atg ggt atg aat atg att tct 2448 Arg Phe Arg Thr Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser 805 810 815 aag ggt gtc gaa tac tca tta aag caa atg gta gaa gag tat ggc tgg 2496 Lys Gly Val Glu Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp 820 825 830 gaa gat atg gag gtt gtc tcc gtt tct ggt aac tac tgt acc gac aaa 2544 Glu Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys 835 840 845 aaa cca gct gcc atc aac tgg atc gaa ggt cgt ggt aag agt gtc gtc 2592 Lys Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val 850 855 860 gca gaa gct act att cct ggt gat gtt gtc aga aaa gtg tta aaa agt 2640 Ala Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu Lys Ser 865 870 875 880 gat gtt tcc gca ttg gtt gag ttg aac att gct aag aat ttg gtt gga 2688 Asp Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn Leu Val Gly 885 890 895 tct gca atg gct ggg tct gtt ggt gga ttt aac gca cat gca gct aat 2736 Ser Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala His Ala Ala Asn 900 905 910 tta gtg aca gct gtt ttc ttg gca tta gga caa gat cct gca caa aat 2784 Leu Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro Ala Gln Asn 915 920 925 gtc gaa agt tcc aac tgt ata aca ttg atg aaa gaa gtg gac ggt gat 2832 Val Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp 930 935 940 ttg aga att tcc gta tcc atg cca tcc atc gaa gta ggt acc atc ggt 2880 Leu Arg Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly 945 950 955 960 ggt ggt act gtt cta gaa cca caa ggt gcc atg ttg gac tta tta ggt 2928 Gly Gly Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Gly 965 970 975 gta aga ggc cca cat gct acc gct cct ggt acc aac gca cgt caa tta 2976 Val Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gln Leu 980 985 990 gca aga ata gtt gcc tgt gcc gtc ttg gca ggt gaa tta tcc tta tgt 3024 Ala Arg Ile Val Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu Cys 995 1000 1005 gct gcc cta gca gcc ggc cat ttg gtt caa agt cat atg acc cac aac 3072 Ala Ala Leu Ala Ala Gly His Leu Val Gln Ser His Met Thr His Asn 1010 1015 1020 agg aaa cct gct gaa cca aca aaa cct aac aat ttg gac gcc act gat 3120 Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala Thr Asp 1025 1030 1035 1040 ata aat cgt ttg aaa gat ggg tcc gtc acc tgc att aaa tcc taa 3165 Ile Asn Arg Leu Lys Asp Gly Ser Val Thr Cys Ile Lys Ser 1045 1050 <210> 14 <211> 1054 <212> PRT <213> Saccharomyces cerevisiae <400> 14 Met Pro Pro Leu Phe Lys Gly Leu Lys Gln Met Ala Lys Pro Ile Ala 1 5 10 15 Tyr Val Ser Arg Phe Ser Ala Lys Arg Pro Ile His Ile Ile Leu Phe 20 25 30 Ser Leu Ile Ile Ser Ala Phe Ala Tyr Leu Ser Val Ile Gln Tyr Tyr 35 40 45 Phe Asn Gly Trp Gln Leu Asp Ser Asn Ser Val Phe Glu Thr Ala Pro 50 55 60 Asn Lys Asp Ser Asn Thr Leu Phe Gln Glu Cys Ser His Tyr Tyr Arg 65 70 75 80 Asp Ser Ser Leu Asp Gly Trp Val Ser Ile Thr Ala His Glu Ala Ser 85 90 95 Glu Leu Pro Ala Pro His His Tyr Tyr Leu Leu Asn Leu Asn Phe Asn 100 105 110 Ser Pro Asn Glu Thr Asp Ser Ile Pro Glu Leu Ala Asn Thr Val Phe 115 120 125 Glu Lys Asp Asn Thr Lys Tyr Ile Leu Gln Glu Asp Leu Ser Val Ser 130 135 140 Lys Glu Ile Ser Ser Thr Asp Gly Thr Lys Trp Arg Leu Arg Ser Asp 145 150 155 160 Arg Lys Ser Leu Phe Asp Val Lys Thr Leu Ala Tyr Ser Leu Tyr Asp 165 170 175 Val Phe Ser Glu Asn Val Thr Gln Ala Asp Pro Phe Asp Val Leu Ile 180 185 190 Met Val Thr Ala Tyr Leu Met Met Phe Tyr Thr Ile Phe Gly Leu Phe 195 200 205 Asn Asp Met Arg Lys Thr Gly Ser Asn Phe Trp Leu Ser Ala Ser Thr 210 215 220 Val Val Asn Ser Ala Ser Ser Leu Phe Leu Ala Leu Tyr Val Thr Gln 225 230 235 240 Cys Ile Leu Gly Lys Glu Val Ser Ala Leu Thr Leu Phe Glu Gly Leu 245 250 255 Pro Phe Ile Val Val Val Val Gly Phe Lys His Lys Ile Lys Ile Ala 260 265 270 Gln Tyr Ala Leu Glu Lys Phe Glu Arg Val Gly Leu Ser Lys Arg Ile 275 280 285 Thr Thr Asp Glu Ile Val Phe Glu Ser Val Ser Glu Glu Gly Gly Arg 290 295 300 Leu Ile Gln Asp His Leu Leu Cys Ile Phe Ala Phe Ile Gly Cys Ser 305 310 315 320 Met Tyr Ala His Gln Leu Lys Thr Leu Thr Asn Phe Cys Ile Leu Ser 325 330 335 Ala Phe Ile Leu Ile Phe Glu Leu Ile Leu Thr Pro Thr Phe Tyr Ser 340 345 350 Ala Ile Leu Ala Leu Arg Leu Glu Met Asn Val Ile His Arg Ser Thr 355 360 365 Ile Ile Lys Gln Thr Leu Glu Glu Asp Gly Val Val Pro Ser Thr Ala 370 375 380 Arg Ile Ile Ser Lys Ala Glu Lys Lys Ser Val Ser Ser Phe Leu Asn 385 390 395 400 Leu Ser Val Val Val Ile Ile Met Lys Leu Ser Val Ile Leu Leu Phe 405 410 415 Val Phe Ile Asn Phe Tyr Asn Phe Gly Ala Asn Trp Val Asn Asp Ala 420 425 430 Phe Asn Ser Leu Tyr Phe Asp Lys Glu Arg Val Ser Leu Pro Asp Phe 435 440 445 Ile Thr Ser Asn Ala Ser Glu Asn Phe Lys Glu Gln Ala Ile Val Ser 450 455 460 Val Thr Pro Leu Leu Tyr Tyr Lys Pro Ile Lys Ser Tyr Gln Arg Ile 465 470 475 480 Glu Asp Met Val Leu Leu Leu Leu Arg Asn Val Ser Val Ala Ile Arg 485 490 495 Asp Arg Phe Val Ser Lys Leu Val Leu Ser Ala Leu Val Cys Ser Ala 500 505 510 Val Ile Asn Val Tyr Leu Leu Asn Ala Ala Arg Ile His Thr Ser Tyr 515 520 525 Thr Ala Asp Gln Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe Thr 530 535 540 Ala Pro Val Gln Lys Ala Ser Thr Pro Val Leu Thr Asn Lys Thr Val 545 550 555 560 Ile Ser Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gln Ser Ser Ser 565 570 575 Ser Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu 580 585 590 Ser Leu Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Leu Leu 595 600 605 Ser Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala Ala Leu 610 615 620 Val Ile His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu Gly 625 630 635 640 Asp Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser Ile Leu 645 650 655 Ala Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys Asn Tyr 660 665 670 Asp Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr 675 680 685 Met Pro Leu Pro Val Gly Val Ile Gly Pro Leu Val Ile Asp Gly Thr 690 695 700 Ser Tyr His Ile Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser 705 710 715 720 Ala Met Arg Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr 725 730 735 Val Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe Pro 740 745 750 Thr Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser Glu Glu 755 760 765 Gly Gln Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala 770 775 780 Arg Leu Gln His Ile Gln Thr Cys Leu Ala Gly Asp Leu Leu Phe Met 785 790 795 800 Arg Phe Arg Thr Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser 805 810 815 Lys Gly Val Glu Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp 820 825 830 Glu Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys 835 840 845 Lys Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val 850 855 860 Ala Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu Lys Ser 865 870 875 880 Asp Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn Leu Val Gly 885 890 895 Ser Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala His Ala Ala Asn 900 905 910 Leu Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro Ala Gln Asn 915 920 925 Val Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp 930 935 940 Leu Arg Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly 945 950 955 960 Gly Gly Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Gly 965 970 975 Val Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gln Leu 980 985 990 Ala Arg Ile Val Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu Cys 995 1000 1005 Ala Ala Leu Ala Ala Gly His Leu Val Gln Ser His Met Thr His Asn 1010 1015 1020 Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala Thr Asp 1025 1030 1035 1040 Ile Asn Arg Leu Lys Asp Gly Ser Val Thr Cys Ile Lys Ser 1045 1050 <210> 15 <211> 2925 <212> DNA <213> Saccharomyces cerevisiae <400> 15 atgccgccgc tattcaaggg actgaaacag atggcaaagc caattgccta tgtttcaaga 60 ttttcggcga aacgaccaat tcatataata cttttttctc taatcatatc cgcattcgct 120 tatctatccg tcattcagta ttacttcaat ggttggcaac tagattcaaa tagtgttttt 180 gaaactgctc caaataaaga cttcaacact ctatttcaag aatgttccca ttactacaga 240 gattcctctc tagatggttg ggtatcaatc accgcgcatg aagctagtga gttaccagcc 300 ccacaccatt actatctatt aaacctgaac ttcaatagtc ctaatgaaac tgactccatt 360 ccagaactag ctaacacggt ttttgagaaa gataatacaa aatatattct gcaagaagat 420 ctcagcgttt ccaaagaaat ttcttctact gatggaacga aatggaggtt aagaagtgac 480 agaaaaagtc ttttcgacgt aaagacgtta gcatattctc tctacgatgt attttcagaa 540 aatgtaaccc aagcagacca caaaatcaag attgcccagt atgccctgga gaaatttgaa 600 agagtcggtt tatctaaaag gattactacc gatgaaatcg tttttgaatc cgtgagcgaa 660 gagggtggtc gtttgattca agaccatttg ctttgtattt ttgcctttat cggatgctct 720 atgtatgctc accaattgaa gactttgaca aacttctgca tattatcagc atttatccta 780 attttcgaat tgattttaac tcctacattt tattctgcta tcttagcgct tagactggaa 840 atgaatgtta tccacagatc tactattatc aagcaaacat tagaagaaga cggtgttgtt 900 ccatctacag caagaatcat ttctaaggca gaaaagaaat ccgtatcttc tttcttaaat 960 ctcagtgtgg ttgtcattat catgaaactc tctgtcatac tgttgttcgt cttcatcaac 1020 ttttataact ttggtgcaaa ttgggtcaat gatgccttca attcattgta cttcgataag 1080 gaacgtgttt ctctaccaga ttttattacc tcgaatgcct ctgaaaactt taaagagcaa 1140 gctattgtta gtgtcacccc attattatat tacaaaccca ttaagtccta ccaacgcatt 1200 gaggatatgg ttcttctatt gcttcgtaat gtcagtgttg ccattcgtga taggttcgtc 1260 agtaaattag ttctttccgc cttagtatgc agtgctgtca tcaatgtgta tttattaaat 1320 gctgctagaa ttcataccag ttatactgca gaccaattgg tgaagactga agtcaccaag 1380 aagtctttta ctgctcctgt acaaaaggct tctacaccag ttttaaccaa taaaacagtc 1440 atttctggat cgaaagtcaa aagtttatca tctgcgcaat cgagctcatc aggaccttca 1500 tcatctagtg aggaagatga ttcccgcgat attgaaagct tggataagaa aatacgtcct 1560 ttagaagaat tagaagcatc attaagtagt ggaaatacaa aacaattgaa gaacaaagag 1620 gtcgctgcct tggttattca cggtaagtta cctttgtacg ctttggagaa aaaattaggt 1680 gatactacga gagcggttgc ggtacgtagg aaggctcttt caattttggc agaagctcct 1740 gtattagcat ctgatcgttt accatataaa aattatgact acgaccgcgt atttggcgct 1800 tgttgtgaaa atgttatagg ttacatgcct ttgcccgttg gtgttatagg ccccttggtt 1860 atcgatggta catcttatca tataccaatg gcaactacag agggttgttt ggtagcttct 1920 gccatgcgtg gctgtaaggc aatcaatgct ggcggtggtg caacaactgt tttaactaag 1980 gatggtatga caagaggccc agtagtccgt ttcccaactt tgaaaagatc tggtgcctgt 2040 aagatatggt tagactcaga agagggacaa aacgcaatta aaaaagcttt taactctaca 2100 tcaagatttg cacgtctgca acatattcaa acttgtctag caggagattt actcttcatg 2160 agatttagaa caactactgg tgacgcaatg ggtatgaata tgatttctaa gggtgtcgaa 2220 tactcattaa agcaaatggt agaagagtat ggctgggaag atatggaggt tgtctccgtt 2280 tctggtaact actgtaccga caaaaaacca gctgccatca actggatcga aggtcgtggt 2340 aagagtgtcg tcgcagaagc tactattcct ggtgatgttg tcagaaaagt gttaaaaagt 2400 gatgtttccg cattggttga gttgaacatt gctaagaatt tggttggatc tgcaatggct 2460 gggtctgttg gtggatttaa cgcacgtgca gctaatttag tgacagctgt tttcttggca 2520 ttaggacaag atcctgcaca aaatgtcgaa agttccaact gtataacatt gatgaaagaa 2580 gtggacggtg atttgagaat ttccgtatcc atgccatcca tcgaagtagg taccatcggt 2640 ggtggtactg ttctagaacc acaaggtgcc atgttggact tattaggtgt aagaggccca 2700 catgctaccg ctcctggtac caacgcacgt caattagcaa gaatagttgc ctgtgccgtc 2760 ttggcaggtg aattatcctt atgtgctgcc ctagcagccg gccatttggt tcaaagtcat 2820 atgacccaca acaggaaacc tgctgaacca acaaaaccta acaatttgga cgccactgat 2880 ataaatcgtt tgaaagatgg gtccgtcacc tgcattaaat cctaa 2925 <210> 16 <211> 3090 <212> DNA <213> Saccharomyces cerevisiae <400> 16 atgccgccgc tattcaaggg actgaaacag atggcaaagc caattgccta tgtttcaaga 60 ttttcggcga aacgaccaat tcatataata cttttttctc taatcatatc cgcattcgct 120 tatctatccg tcattcagta ttacttcaat ggttggcaac tagattcaaa tagtgttttt 180 gaaactgctc caaataaaga cttcaacact ctatttcaag aatgttccca ttactacaga 240 gattcctctc tagatggttg ggtatcaatc accgcgcatg aagctagtga gttaccagcc 300 ccacaccatt actatctatt aaacctgaac ttcaatagtc ctaatgaaac tgactccatt 360 ccagaactag ctaacacggt ttttgagaaa gataatacaa aatatattct gcaagaagat 420 ctcagcgttt ccaaagaaat ttcttctact gatggaacga aatggaggtt aagaagtgac 480 agaaaaagtc ttttcgacgt aaagacgtta gcatattctc tctacgatgt attttcagaa 540 aatgtaaccc aagcagaccc gtttgacgtc cttattatgg ttactgccta cctaatgatg 600 ttctacacca tattcggcct cttcaatgac atgaggaaga ccgggtcaaa tttttggttg 660 agcgcctcta cagtggtcaa ttctgcatca tcacttttct tagcattgta tgtcacccaa 720 tgtattctag gcaaagaagt ttccgcatta actctttttg aaggtttgcc tttcattgta 780 gttgttgttg gtttcaagca caaaatcaag attgcccagt atgccctgga gaaatttgaa 840 agagtcggtt tatctaaaag gattactacc gatgaaatcg tttttgaatc cgtgagcgaa 900 gagggtggtc gtttgattca agaccatttg ctttgtattt ttgcctttat cggatgctct 960 atgtatgctc accaattgaa gactttgaca aacttctgca tattatcagc atttatccta 1020 attttcgaat tgattttaac tcctacattt tattctgcta tcttagcgct tagactggaa 1080 atgaatgtta tccacagatc tactattatc aagcaaacat tagaagaaga cggtgttgtt 1140 ccatctacag caagaatcat ttctaaggca gaaaagaaat ccgtatcttc taactttggt 1200 gcaaattggg tcaatgatgc cttcaattca ttgtacttcg ataaggaacg tgtttctcta 1260 ccagatttta ttacctcgaa tgcctctgaa aactttaaag agcaagctat tgttagtgtc 1320 accccattat tatattacaa acccattaag tcctaccaac gcattgagga tatggttctt 1380 ctattgcttc gtaatgtcag tgttgccatt cgtgataggt tcgtcagtaa attagttctt 1440 tccgccttag tatgcagtgc tgtcatcaat gtgtatttat taaatgctgc tagaattcat 1500 accagttata ctgcagacca attggtgaag actgaagtca ccaagaagtc ttttactgct 1560 cctgtacaaa aggcttctac accagtttta accaataaaa cagtcatttc tggatcgaaa 1620 gtcaaaagtt tatcatctgc gcaatcgagc tcatcaggac cttcatcatc tagtgaggaa 1680 gatgattccc gcgatattga aagcttggat aagaaaatac gtcctttaga agaattagaa 1740 gcatcattaa gtagtggaaa tacaaaacaa ttgaagaaca aagaggtcgc tgccttggtt 1800 attcacggta agttaccttt gtacgctttg gagaaaaaat taggtgatac tacgagagcg 1860 gttgcggtac gtaggaaggc tctttcaatt ttggcagaag ctcctgtatt agcatctgat 1920 cgtttaccat ataaaaatta tgactacgac cgcgtatttg gcgcttgttg tgaaaatgtt 1980 ataggttaca tgcctttgcc cgttggtgtt ataggcccct tggttatcga tggtacatct 2040 tatcatatac caatggcaac tacagagggt tgtttggtag cttctgccat gcgtggctgt 2100 aaggcaatca atgctggcgg tggtgcaaca actgttttaa ctaaggatgg tatgacaaga 2160 ggcccagtag tccgtttccc aactttgaaa agatctggtg cctgtaagat atggttagac 2220 tcagaagagg gacaaaacgc aattaaaaaa gcttttaact ctacatcaag atttgcacgt 2280 ctgcaacata ttcaaacttg tctagcagga gatttactct tcatgagatt tagaacaact 2340 actggtgacg caatgggtat gaatatgatt tctaagggtg tcgaatactc attaaagcaa 2400 atggtagaag agtatggctg ggaagatatg gaggttgtct ccgtttctgg taactactgt 2460 accgacaaaa aaccagctgc catcaactgg atcgaaggtc gtggtaagag tgtcgtcgca 2520 gaagctacta ttcctggtga tgttgtcaga aaagtgttaa aaagtgatgt ttccgcattg 2580 gttgagttga acattgctaa gaatttggtt ggatctgcaa tggctgggtc tgttggtgga 2640 tttaacgcac gtgcagctaa tttagtgaca gctgttttct tggcattagg acaagatcct 2700 gcacaaaatg tcgaaagttc caactgtata acattgatga aagaagtgga cggtgatttg 2760 agaatttccg tatccatgcc atccatcgaa gtaggtacca tcggtggtgg tactgttcta 2820 gaaccacaag gtgccatgtt ggacttatta ggtgtaagag gcccacatgc taccgctcct 2880 ggtaccaacg cacgtcaatt agcaagaata gttgcctgtg ccgtcttggc aggtgaatta 2940 tccttatgtg ctgccctagc agccggccat ttggttcaaa gtcatatgac ccacaacagg 3000 aaacctgctg aaccaacaaa acctaacaat ttggacgcca ctgatataaa tcgtttgaaa 3060 gatgggtccg tcacctgcat taaatcctaa 3090 <210> 17 <211> 2973 <212> DNA <213> Saccharomyces cerevisiae <400> 17 atgccgccgc tattcaaggg actgaaacag atggcaaagc caattgccta tgtttcaaga 60 ttttcggcga aacgaccaat tcatataata cttttttctc taatcatatc cgcattcgct 120 tatctatccg tcattcagta ttacttcaat ggttggcaac tagattcaaa tagtgttttt 180 gaaactgctc caaataaaga cttcaacact ctatttcaag aatgttccca ttactacaga 240 gattcctctc tagatggttg ggtatcaatc accgcgcatg aagctagtga gttaccagcc 300 ccacaccatt actatctatt aaacctgaac ttcaatagtc ctaatgaaac tgactccatt 360 ccagaactag ctaacacggt ttttgagaaa gataatacaa aatatattct gcaagaagat 420 ctcagcgttt ccaaagaaat ttcttctact gatggaacga aatggaggtt aagaagtgac 480 agaaaaagtc ttttcgacgt aaagacgtta gcatattctc tctacgatgt attttcagaa 540 aatgtaaccc aagcagaccc gtttgacgtc cttattatgg ttactgccta cctaatgatg 600 ttctacacca tattcggcct cttcaatgac atgaggaaga ccgggtcaaa tttttggttg 660 agcgcctcta cagtggtcaa ttctgcatca tcacttttct tagcattgta tgtcacccaa 720 tgtattctag gcaaagaagt ttccgcatta actctttttg aaggtttgcc tttcattgta 780 gttgttgttg gtttcaagca caaaatcaag attgcccagt atgccctgga gaaatttgaa 840 agagtcggtt tatctaaaag gattactacc gatgaaatcg tttttgaatc cgtgagcgaa 900 gagggtggtc gtttgattca agaccatttg ctttgtattt ttgcctttat cggatgctct 960 atgtatgctc accaattgaa gactttgaca aacttctgca tattatcagc atttatccta 1020 attttcgaat tgattttaac tcctacattt tattctgcta tcttagcgct tagactggaa 1080 atgaatgtta tccacagatc tactattatc aagcaaacat tagaagaaga cggtgttgtt 1140 ccatctacag caagaatcat ttctaaggca gaaaagaaat ccgtatcttc tttcttaaat 1200 ctcagtgtgg ttgtcattat catgaaactc tctgtcatac tgttgttcgt cttcatcaac 1260 ttttataact ttggtgcaaa ttgggtcaat gatgccttca attcattgta cttcgataag 1320 gaacgtgttt ctctaccaga ttttattacc tcgaatgcct ctgaaaactt taaagagcaa 1380 cataccagtt atactgcaga ccaattggtg aagactgaag tcaccaagaa gtcttttact 1440 gctcctgtac aaaaggcttc tacaccagtt ttaaccaata aaacagtcat ttctggatcg 1500 aaagtcaaaa gtttatcatc tgcgcaatcg agctcatcag gaccttcatc atctagtgag 1560 gaagatgatt cccgcgatat tgaaagcttg gataagaaaa tacgtccttt agaagaatta 1620 gaagcatcat taagtagtgg aaatacaaaa caattgaaga acaaagaggt cgctgccttg 1680 gttattcacg gtaagttacc tttgtacgct ttggagaaaa aattaggtga tactacgaga 1740 gcggttgcgg tacgtaggaa ggctctttca attttggcag aagctcctgt attagcatct 1800 gatcgtttac catataaaaa ttatgactac gaccgcgtat ttggcgcttg ttgtgaaaat 1860 gttataggtt acatgccttt gcccgttggt gttataggcc ccttggttat cgatggtaca 1920 tcttatcata taccaatggc aactacagag ggttgtttgg tagcttctgc catgcgtggc 1980 tgtaaggcaa tcaatgctgg cggtggtgca acaactgttt taactaagga tggtatgaca 2040 agaggcccag tagtccgttt cccaactttg aaaagatctg gtgcctgtaa gatatggtta 2100 gactcagaag agggacaaaa cgcaattaaa aaagctttta actctacatc aagatttgca 2160 cgtctgcaac atattcaaac ttgtctagca ggagatttac tcttcatgag atttagaaca 2220 actactggtg acgcaatggg tatgaatatg atttctaagg gtgtcgaata ctcattaaag 2280 caaatggtag aagagtatgg ctgggaagat atggaggttg tctccgtttc tggtaactac 2340 tgtaccgaca aaaaaccagc tgccatcaac tggatcgaag gtcgtggtaa gagtgtcgtc 2400 gcagaagcta ctattcctgg tgatgttgtc agaaaagtgt taaaaagtga tgtttccgca 2460 ttggttgagt tgaacattgc taagaatttg gttggatctg caatggctgg gtctgttggt 2520 ggatttaacg cacgtgcagc taatttagtg acagctgttt tcttggcatt aggacaagat 2580 cctgcacaaa atgtcgaaag ttccaactgt ataacattga tgaaagaagt ggacggtgat 2640 ttgagaattt ccgtatccat gccatccatc gaagtaggta ccatcggtgg tggtactgtt 2700 ctagaaccac aaggtgccat gttggactta ttaggtgtaa gaggcccaca tgctaccgct 2760 cctggtacca acgcacgtca attagcaaga atagttgcct gtgccgtctt ggcaggtgaa 2820 ttatccttat gtgctgccct agcagccggc catttggttc aaagtcatat gacccacaac 2880 aggaaacctg ctgaaccaac aaaacctaac aatttggacg ccactgatat aaatcgtttg 2940 aaagatgggt ccgtcacctg cattaaatcc taa 2973 <210> 18 <211> 2457 <212> DNA <213> Saccharomyces cerevisiae <400> 18 atgccgccgc tattcaaggg actgaaacag atggcaaagc caattgccta tgtttcaaga 60 ttttcggcga aacgaccaat tcatataata cttttttctc taatcatatc cgcattcgct 120 tatctatccg tcattcagta ttacttcaat ggttggcaac tagattcaaa tagtgttttt 180 gaaactgctc caaataaaga cttcaacact ctatttcaag aatgttccca ttactacaga 240 gattcctctc tagatggttg ggtatcaatc accgcgcatg aagctagtga gttaccagcc 300 ccacaccatt actatctatt aaacctgaac ttcaatagtc ctaatgaaac tgactccatt 360 ccagaactag ctaacacggt ttttgagaaa gataatacaa aatatattct gcaagaagat 420 ctcagcgttt ccaaagaaat ttcttctact gatggaacga aatggaggtt aagaagtgac 480 agaaaaagtc ttttcgacgt aaagacgtta gcatattctc tctacgatgt attttcagaa 540 aatgtaaccc aagcagacaa ctttggtgca aattgggtca atgatgcctt caattcattg 600 tacttcgata aggaacgtgt ttctctacca gattttatta cctcgaatgc ctctgaaaac 660 tttaaagagc aagctattgt tagtgtcacc ccattattat attacaaacc cattaagtcc 720 taccaacgca ttgaggatat ggttcttcta ttgcttcgta atgtcagtgt tgccattcgt 780 gataggttcg tcagtaaatt agttctttcc gccttagtat gcagtgctgt catcaatgtg 840 tatttattaa atgctgctag aattcatacc agttatactg cagaccaatt ggtgaagact 900 gaagtcacca agaagtcttt tactgctcct gtacaaaagg cttctacacc agttttaacc 960 aataaaacag tcatttctgg atcgaaagtc aaaagtttat catctgcgca atcgagctca 1020 tcaggacctt catcatctag tgaggaagat gattcccgcg atattgaaag cttggataag 1080 aaaatacgtc ctttagaaga attagaagca tcattaagta gtggaaatac aaaacaattg 1140 aagaacaaag aggtcgctgc cttggttatt cacggtaagt tacctttgta cgctttggag 1200 aaaaaattag gtgatactac gagagcggtt gcggtacgta ggaaggctct ttcaattttg 1260 gcagaagctc ctgtattagc atctgatcgt ttaccatata aaaattatga ctacgaccgc 1320 gtatttggcg cttgttgtga aaatgttata ggttacatgc ctttgcccgt tggtgttata 1380 ggccccttgg ttatcgatgg tacatcttat catataccaa tggcaactac agagggttgt 1440 ttggtagctt ctgccatgcg tggctgtaag gcaatcaatg ctggcggtgg tgcaacaact 1500 gttttaacta aggatggtat gacaagaggc ccagtagtcc gtttcccaac tttgaaaaga 1560 tctggtgcct gtaagatatg gttagactca gaagagggac aaaacgcaat taaaaaagct 1620 tttaactcta catcaagatt tgcacgtctg caacatattc aaacttgtct agcaggagat 1680 ttactcttca tgagatttag aacaactact ggtgacgcaa tgggtatgaa tatgatttct 1740 aagggtgtcg aatactcatt aaagcaaatg gtagaagagt atggctggga agatatggag 1800 gttgtctccg tttctggtaa ctactgtacc gacaaaaaac cagctgccat caactggatc 1860 gaaggtcgtg gtaagagtgt cgtcgcagaa gctactattc ctggtgatgt tgtcagaaaa 1920 gtgttaaaaa gtgatgtttc cgcattggtt gagttgaaca ttgctaagaa tttggttgga 1980 tctgcaatgg ctgggtctgt tggtggattt aacgcacgtg cagctaattt agtgacagct 2040 gttttcttgg cattaggaca agatcctgca caaaatgtcg aaagttccaa ctgtataaca 2100 ttgatgaaag aagtggacgg tgatttgaga atttccgtat ccatgccatc catcgaagta 2160 ggtaccatcg gtggtggtac tgttctagaa ccacaaggtg ccatgttgga cttattaggt 2220 gtaagaggcc cacatgctac cgctcctggt accaacgcac gtcaattagc aagaatagtt 2280 gcctgtgccg tcttggcagg tgaattatcc ttatgtgctg ccctagcagc cggccatttg 2340 gttcaaagtc atatgaccca caacaggaaa cctgctgaac caacaaaacc taacaatttg 2400 gacgccactg atataaatcg tttgaaagat gggtccgtca cctgcattaa atcctaa 2457 <210> 19 <211> 2151 <212> DNA <213> Saccharomyces cerevisiae <400> 19 atgccgccgc tattcaaggg actgaaacag atggcaaagc caattgccta tgtttcaaga 60 ttttcggcga aacgaccaat tcatataata cttttttctc taatcatatc cgcattcgct 120 tatctatccg tcattcagta ttacttcaat ggttggcaac tagattcaaa tagtgttttt 180 gaaactgctc caaataaaga cttcaacact ctatttcaag aatgttccca ttactacaga 240 gattcctctc tagatggttg ggtatcaatc accgcgcatg aagctagtga gttaccagcc 300 ccacaccatt actatctatt aaacctgaac ttcaatagtc ctaatgaaac tgactccatt 360 ccagaactag ctaacacggt ttttgagaaa gataatacaa aatatattct gcaagaagat 420 ctcagcgttt ccaaagaaat ttcttctact gatggaacga aatggaggtt aagaagtgac 480 agaaaaagtc ttttcgacgt aaagacgtta gcatattctc tctacgatgt attttcagaa 540 aatgtaaccc aagcagacca taccagttat actgcagacc aattggtgaa gactgaagtc 600 accaagaagt cttttactgc tcctgtacaa aaggcttcta caccagtttt aaccaataaa 660 acagtcattt ctggatcgaa agtcaaaagt ttatcatctg cgcaatcgag ctcatcagga 720 ccttcatcat ctagtgagga agatgattcc cgcgatattg aaagcttgga taagaaaata 780 cgtcctttag aagaattaga agcatcatta agtagtggaa atacaaaaca attgaagaac 840 aaagaggtcg ctgccttggt tattcacggt aagttacctt tgtacgcttt ggagaaaaaa 900 ttaggtgata ctacgagagc ggttgcggta cgtaggaagg ctctttcaat tttggcagaa 960 gctcctgtat tagcatctga tcgtttacca tataaaaatt atgactacga ccgcgtattt 1020 ggcgcttgtt gtgaaaatgt tataggttac atgcctttgc ccgttggtgt tataggcccc 1080 ttggttatcg atggtacatc ttatcatata ccaatggcaa ctacagaggg ttgtttggta 1140 gcttctgcca tgcgtggctg taaggcaatc aatgctggcg gtggtgcaac aactgtttta 1200 actaaggatg gtatgacaag aggcccagta gtccgtttcc caactttgaa aagatctggt 1260 gcctgtaaga tatggttaga ctcagaagag ggacaaaacg caattaaaaa agcttttaac 1320 tctacatcaa gatttgcacg tctgcaacat attcaaactt gtctagcagg agatttactc 1380 ttcatgagat ttagaacaac tactggtgac gcaatgggta tgaatatgat ttctaagggt 1440 gtcgaatact cattaaagca aatggtagaa gagtatggct gggaagatat ggaggttgtc 1500 tccgtttctg gtaactactg taccgacaaa aaaccagctg ccatcaactg gatcgaaggt 1560 cgtggtaaga gtgtcgtcgc agaagctact attcctggtg atgttgtcag aaaagtgtta 1620 aaaagtgatg tttccgcatt ggttgagttg aacattgcta agaatttggt tggatctgca 1680 atggctgggt ctgttggtgg atttaacgca cgtgcagcta atttagtgac agctgttttc 1740 ttggcattag gacaagatcc tgcacaaaat gtcgaaagtt ccaactgtat aacattgatg 1800 aaagaagtgg acggtgattt gagaatttcc gtatccatgc catccatcga agtaggtacc 1860 atcggtggtg gtactgttct agaaccacaa ggtgccatgt tggacttatt aggtgtaaga 1920 ggcccacatg ctaccgctcc tggtaccaac gcacgtcaat tagcaagaat agttgcctgt 1980 gccgtcttgg caggtgaatt atccttatgt gctgccctag cagccggcca tttggttcaa 2040 agtcatatga cccacaacag gaaacctgct gaaccaacaa aacctaacaa tttggacgcc 2100 actgatataa atcgtttgaa agatgggtcc gtcacctgca ttaaatccta a 2151 <210> 20 <211> 1620 <212> DNA <213> Saccharomyces cerevisiae <400> 20 atgccgccgc tattcaaggg actgaaacat accagttata ctgcagacca attggtgaag 60 actgaagtca ccaagaagtc ttttactgct cctgtacaaa aggcttctac accagtttta 120 accaataaaa cagtcatttc tggatcgaaa gtcaaaagtt tatcatctgc gcaatcgagc 180 tcatcaggac cttcatcatc tagtgaggaa gatgattccc gcgatattga aagcttggat 240 aagaaaatac gtcctttaga agaattagaa gcatcattaa gtagtggaaa tacaaaacaa 300 ttgaagaaca aagaggtcgc tgccttggtt attcacggta agttaccttt gtacgctttg 360 gagaaaaaat taggtgatac tacgagagcg gttgcggtac gtaggaaggc tctttcaatt 420 ttggcagaag ctcctgtatt agcatctgat cgtttaccat ataaaaatta tgactacgac 480 cgcgtatttg gcgcttgttg tgaaaatgtt ataggttaca tgcctttgcc cgttggtgtt 540 ataggcccct tggttatcga tggtacatct tatcatatac caatggcaac tacagagggt 600 tgtttggtag cttctgccat gcgtggctgt aaggcaatca atgctggcgg tggtgcaaca 660 actgttttaa ctaaggatgg tatgacaaga ggcccagtag tccgtttccc aactttgaaa 720 agatctggtg cctgtaagat atggttagac tcagaagagg gacaaaacgc aattaaaaaa 780 gcttttaact ctacatcaag atttgcacgt ctgcaacata ttcaaacttg tctagcagga 840 gatttactct tcatgagatt tagaacaact actggtgacg caatgggtat gaatatgatt 900 tctaagggtg tcgaatactc attaaagcaa atggtagaag agtatggctg ggaagatatg 960 gaggttgtct ccgtttctgg taactactgt accgacaaaa aaccagctgc catcaactgg 1020 atcgaaggtc gtggtaagag tgtcgtcgca gaagctacta ttcctggtga tgttgtcaga 1080 aaagtgttaa aaagtgatgt ttccgcattg gttgagttga acattgctaa gaatttggtt 1140 ggatctgcaa tggctgggtc tgttggtgga tttaacgcac gtgcagctaa tttagtgaca 1200 gctgttttct tggcattagg acaagatcct gcacaaaatg tcgaaagttc caactgtata 1260 acattgatga aagaagtgga cggtgatttg agaatttccg tatccatgcc atccatcgaa 1320 gtaggtacca tcggtggtgg tactgttcta gaaccacaag gtgccatgtt ggacttatta 1380 ggtgtaagag gcccacatgc taccgctcct ggtaccaacg cacgtcaatt agcaagaata 1440 gttgcctgtg ccgtcttggc aggtgaatta tccttatgtg ctgccctagc agccggccat 1500 ttggttcaaa gtcatatgac ccacaacagg aaacctgctg aaccaacaaa acctaacaat 1560 ttggacgcca ctgatataaa tcgtttgaaa gatgggtccg tcacctgcat taaatcctaa 1620 <210> 21 <211> 1377 <212> DNA <213> Saccharomyces cerevisiae <400> 21 atgccgccgc tattcaaggg actgaaagca tcattaagta gtggaaatac aaaacaattg 60 aagaacaaag aggtcgctgc cttggttatt cacggtaagt tacctttgta cgctttggag 120 aaaaaattag gtgatactac gagagcggtt gcggtacgta ggaaggctct ttcaattttg 180 gcagaagctc ctgtattagc atctgatcgt ttaccatata aaaattatga ctacgaccgc 240 gtatttggcg cttgttgtga aaatgttata ggttacatgc ctttgcccgt tggtgttata 300 ggccccttgg ttatcgatgg tacatcttat catataccaa tggcaactac agagggttgt 360 ttggtagctt ctgccatgcg tggctgtaag gcaatcaatg ctggcggtgg tgcaacaact 420 gttttaacta aggatggtat gacaagaggc ccagtagtcc gtttcccaac tttgaaaaga 480 tctggtgcct gtaagatatg gttagactca gaagagggac aaaacgcaat taaaaaagct 540 tttaactcta catcaagatt tgcacgtctg caacatattc aaacttgtct agcaggagat 600 ttactcttca tgagatttag aacaactact ggtgacgcaa tgggtatgaa tatgatttct 660 aagggtgtcg aatactcatt aaagcaaatg gtagaagagt atggctggga agatatggag 720 gttgtctccg tttctggtaa ctactgtacc gacaaaaaac cagctgccat caactggatc 780 gaaggtcgtg gtaagagtgt cgtcgcagaa gctactattc ctggtgatgt tgtcagaaaa 840 gtgttaaaaa gtgatgtttc cgcattggtt gagttgaaca ttgctaagaa tttggttgga 900 tctgcaatgg ctgggtctgt tggtggattt aacgcacgtg cagctaattt agtgacagct 960 gttttcttgg cattaggaca agatcctgca caaaatgtcg aaagttccaa ctgtataaca 1020 ttgatgaaag aagtggacgg tgatttgaga atttccgtat ccatgccatc catcgaagta 1080 ggtaccatcg gtggtggtac tgttctagaa ccacaaggtg ccatgttgga cttattaggt 1140 gtaagaggcc cacatgctac cgctcctggt accaacgcac gtcaattagc aagaatagtt 1200 gcctgtgccg tcttggcagg tgaattatcc ttatgtgctg ccctagcagc cggccatttg 1260 gttcaaagtc atatgaccca caacaggaaa cctgctgaac caacaaaacc taacaatttg 1320 gacgccactg atataaatcg tttgaaagat gggtccgtca cctgcattaa atcctaa 1377 <210> 22 <211> 1302 <212> DNA <213> Saccharomyces cerevisiae <400> 22 atgccgccgc tattcaaggg actgaaacct ttgtacgctt tggagaaaaa attaggtgat 60 actacgagag cggttgcggt acgtaggaag gctctttcaa ttttggcaga agctcctgta 120 ttagcatctg atcgtttacc atataaaaat tatgactacg accgcgtatt tggcgcttgt 180 tgtgaaaatg ttataggtta catgcctttg cccgttggtg ttataggccc cttggttatc 240 gatggtacat cttatcatat accaatggca actacagagg gttgtttggt agcttctgcc 300 atgcgtggct gtaaggcaat caatgctggc ggtggtgcaa caactgtttt aactaaggat 360 ggtatgacaa gaggcccagt agtccgtttc ccaactttga aaagatctgg tgcctgtaag 420 atatggttag actcagaaga gggacaaaac gcaattaaaa aagcttttaa ctctacatca 480 agatttgcac gtctgcaaca tattcaaact tgtctagcag gagatttact cttcatgaga 540 tttagaacaa ctactggtga cgcaatgggt atgaatatga tttctaaggg tgtcgaatac 600 tcattaaagc aaatggtaga agagtatggc tgggaagata tggaggttgt ctccgtttct 660 ggtaactact gtaccgacaa aaaaccagct gccatcaact ggatcgaagg tcgtggtaag 720 agtgtcgtcg cagaagctac tattcctggt gatgttgtca gaaaagtgtt aaaaagtgat 780 gtttccgcat tggttgagtt gaacattgct aagaatttgg ttggatctgc aatggctggg 840 tctgttggtg gatttaacgc acgtgcagct aatttagtga cagctgtttt cttggcatta 900 ggacaagatc ctgcacaaaa tgtcgaaagt tccaactgta taacattgat gaaagaagtg 960 gacggtgatt tgagaatttc cgtatccatg ccatccatcg aagtaggtac catcggtggt 1020 ggtactgttc tagaaccaca aggtgccatg ttggacttat taggtgtaag aggcccacat 1080 gctaccgctc ctggtaccaa cgcacgtcaa ttagcaagaa tagttgcctg tgccgtcttg 1140 gcaggtgaat tatccttatg tgctgcccta gcagccggcc atttggttca aagtcatatg 1200 acccacaaca ggaaacctgc tgaaccaaca aaacctaaca atttggacgc cactgatata 1260 aatcgtttga aagatgggtc cgtcacctgc attaaatcct aa 1302 <210> 23 <211> 1203 <212> DNA <213> Saccharomyces cerevisiae <400> 23 atgccgccgc tattcaaggg actgaaatct gatcgtttac catataaaaa ttatgactac 60 gaccgcgtat ttggcgcttg ttgtgaaaat gttataggtt acatgccttt gcccgttggt 120 gttataggcc ccttggttat cgatggtaca tcttatcata taccaatggc aactacagag 180 ggttgtttgg tagcttctgc catgcgtggc tgtaaggcaa tcaatgctgg cggtggtgca 240 acaactgttt taactaagga tggtatgaca agaggcccag tagtccgttt cccaactttg 300 aaaagatctg gtgcctgtaa gatatggtta gactcagaag agggacaaaa cgcaattaaa 360 aaagctttta actctacatc aagatttgca cgtctgcaac atattcaaac ttgtctagca 420 ggagatttac tcttcatgag atttagaaca actactggtg acgcaatggg tatgaatatg 480 atttctaagg gtgtcgaata ctcattaaag caaatggtag aagagtatgg ctgggaagat 540 atggaggttg tctccgtttc tggtaactac tgtaccgaca aaaaaccagc tgccatcaac 600 tggatcgaag gtcgtggtaa gagtgtcgtc gcagaagcta ctattcctgg tgatgttgtc 660 agaaaagtgt taaaaagtga tgtttccgca ttggttgagt tgaacattgc taagaatttg 720 gttggatctg caatggctgg gtctgttggt ggatttaacg cacgtgcagc taatttagtg 780 acagctgttt tcttggcatt aggacaagat cctgcacaaa atgtcgaaag ttccaactgt 840 ataacattga tgaaagaagt ggacggtgat ttgagaattt ccgtatccat gccatccatc 900 gaagtaggta ccatcggtgg tggtactgtt ctagaaccac aaggtgccat gttggactta 960 ttaggtgtaa gaggcccaca tgctaccgct cctggtacca acgcacgtca attagcaaga 1020 atagttgcct gtgccgtctt ggcaggtgaa ttatccttat gtgctgccct agcagccggc 1080 catttggttc aaagtcatat gacccacaac aggaaacctg ctgaaccaac aaaacctaac 1140 aatttggacg ccactgatat aaatcgtttg aaagatgggt ccgtcacctg cattaaatcc 1200 taa 1203 <210> 24 <211> 975 <212> DNA <213> Saccharomyces cerevisiae <400> 24 atgccgccgc tattcaaggg actgaaaaag gatggtatga caagaggccc agtagtccgt 60 ttcccaactt tgaaaagatc tggtgcctgt aagatatggt tagactcaga agagggacaa 120 aacgcaatta aaaaagcttt taactctaca tcaagatttg cacgtctgca acatattcaa 180 acttgtctag caggagattt actcttcatg agatttagaa caactactgg tgacgcaatg 240 ggtatgaata tgatttctaa gggtgtcgaa tactcattaa agcaaatggt agaagagtat 300 ggctgggaag atatggaggt tgtctccgtt tctggtaact actgtaccga caaaaaacca 360 gctgccatca actggatcga aggtcgtggt aagagtgtcg tcgcagaagc tactattcct 420 ggtgatgttg tcagaaaagt gttaaaaagt gatgtttccg cattggttga gttgaacatt 480 gctaagaatt tggttggatc tgcaatggct gggtctgttg gtggatttaa cgcacgtgca 540 gctaatttag tgacagctgt tttcttggca ttaggacaag atcctgcaca aaatgtcgaa 600 agttccaact gtataacatt gatgaaagaa gtggacggtg atttgagaat ttccgtatcc 660 atgccatcca tcgaagtagg taccatcggt ggtggtactg ttctagaacc acaaggtgcc 720 atgttggact tattaggtgt aagaggccca catgctaccg ctcctggtac caacgcacgt 780 caattagcaa gaatagttgc ctgtgccgtc ttggcaggtg aattatcctt atgtgctgcc 840 ctagcagccg gccatttggt tcaaagtcat atgacccaca acaggaaacc tgctgaacca 900 acaaaaccta acaatttgga cgccactgat ataaatcgtt tgaaagatgg gtccgtcacc 960 tgcattaaat cctaa 975 <210> 25 <211> 549 <212> DNA <213> Escherichia coli <400> 25 atgcaaacgg aacacgtcat tttattgaat gcacagggag ttcccacggg tacgctggaa 60 aagtatgccg cacacacggc agacacccgc ttacatctcg cgttctccag ttggctgttt 120 aatgccaaag gacaattatt agttacccgc cgcgcactga gcaaaaaagc atggcctggc 180 gtgtggacta actcggtttg tgggcaccca caactgggag aaagcaacga agacgcagtg 240 atccgccgtt gccgttatga gcttggcgtg gaaattacgc ctcctgaatc tatctatcct 300 gactttcgct accgcgccac cgatccgagt ggcattgtgg aaaatgaagt gtgtccggta 360 tttgccgcac gcaccactag tgcgttacag atcaatgatg atgaagtgat ggattatcaa 420 tggtgtgatt tagcagatgt attacacggt attgatgcca cgccgtgggc gttcagtccg 480 tggatggtga tgcaggcgac aaatcgcgaa gccagaaaac gattatctgc atttacccag 540 cttaaataa 549 <210> 26 <211> 1332 <212> DNA <213> Saccharomyces cerevisiae <400> 26 atgtcattac cgttcttaac ttctgcaccg ggaaaggtta ttatttttgg tgaacactct 60 gctgtgtaca acaagcctgc cgtcgctgct agtgtgtctg cgttgagaac ctacctgcta 120 ataagcgagt catctgcacc agatactatt gaattggact tcccggacat tagctttaat 180 cataagtggt ccatcaatga tttcaatgcc atcaccgagg atcaagtaaa ctcccaaaaa 240 ttggccaagg ctcaacaagc caccgatggc ttgtctcagg aactcgttag tcttttggat 300 ccgttgttag ctcaactatc cgaatccttc cactaccatg cagcgttttg tttcctgtat 360 atgtttgttt gcctatgccc ccatgccaag aatattaagt tttctttaaa gtctacttta 420 cccatcggtg ctgggttggg ctcaagcgcc tctatttctg tatcactggc cttagctatg 480 gcctacttgg gggggttaat aggatctaat gacttggaaa agctgtcaga aaacgataag 540 catatagtga atcaatgggc cttcataggt gaaaagtgta ttcacggtac cccttcagga 600 atagataacg ctgtggccac ttatggtaat gccctgctat ttgaaaaaga ctcacataat 660 ggaacaataa acacaaacaa ttttaagttc ttagatgatt tcccagccat tccaatgatc 720 ctaacctata ctagaattcc aaggtctaca aaagatcttg ttgctcgcgt tcgtgtgttg 780 gtcaccgaga aatttcctga agttatgaag ccaattctag atgccatggg tgaatgtgcc 840 ctacaaggct tagagatcat gactaagtta agtaaatgta aaggcaccga tgacgaggct 900 gtagaaacta ataatgaact gtatgaacaa ctattggaat tgataagaat aaatcatgga 960 ctgcttgtct caatcggtgt ttctcatcct ggattagaac ttattaaaaa tctgagcgat 1020 gatttgagaa ttggctccac aaaacttacc ggtgctggtg gcggcggttg ctctttgact 1080 ttgttacgaa gagacattac tcaagagcaa attgacagct tcaaaaagaa attgcaagat 1140 gattttagtt acgagacatt tgaaacagac ttgggtggga ctggctgctg tttgttaagc 1200 gcaaaaaatt tgaataaaga tcttaaaatc aaatccctag tattccaatt atttgaaaat 1260 aaaactacca caaagcaaca aattgacgat ctattattgc caggaaacac gaatttacca 1320 tggacttcat aa 1332 <210> 27 <211> 1197 <212> DNA <213> Saccharomyces cerevisiae <400> 27 atgtctcaga acgtttacat tgtatcgact gccagaaccc caattggttc attccagggt 60 tctctatcct ccaagacagc agtggaattg ggtgctgttg ctttaaaagg cgccttggct 120 aaggttccag aattggatgc atccaaggat tttgacgaaa ttatttttgg taacgttctt 180 tctgccaatt tgggccaagc tccggccaga caagttgctt tggctgccgg tttgagtaat 240 catatcgttg caagcacagt taacaaggtc tgtgcatccg ctatgaaggc aatcattttg 300 ggtgctcaat ccatcaaatg tggtaatgct gatgttgtcg tagctggtgg ttgtgaatct 360 atgactaacg caccatacta catgccagca gcccgtgcgg gtgccaaatt tggccaaact 420 gttcttgttg atggtgtcga aagagatggg ttgaacgatg cgtacgatgg tctagccatg 480 ggtgtacacg cagaaaagtg tgcccgtgat tgggatatta ctagagaaca acaagacaat 540 tttgccatcg aatcctacca aaaatctcaa aaatctcaaa aggaaggtaa attcgacaat 600 gaaattgtac ctgttaccat taagggattt agaggtaagc ctgatactca agtcacgaag 660 gacgaggaac ctgctagatt acacgttgaa aaattgagat ctgcaaggac tgttttccaa 720 aaagaaaacg gtactgttac tgccgctaac gcttctccaa tcaacgatgg tgctgcagcc 780 gtcatcttgg tttccgaaaa agttttgaag gaaaagaatt tgaagccttt ggctattatc 840 aaaggttggg gtgaggccgc tcatcaacca gctgatttta catgggctcc atctcttgca 900 gttccaaagg ctttgaaaca tgctggcatc gaagacatca attctgttga ttactttgaa 960 ttcaatgaag ccttttcggt tgtcggtttg gtgaacacta agattttgaa gctagaccca 1020 tctaaggtta atgtatatgg tggtgctgtt gctctaggtc acccattggg ttgttctggt 1080 gctagagtgg ttgttacact gctatccatc ttacagcaag aaggaggtaa gatcggtgtt 1140 gccgccattt gtaatggtgg tggtggtgct tcctctattg tcattgaaaa gatatga 1197 <210> 28 <211> 1476 <212> DNA <213> Saccharomyces cerevisiae <400> 28 atgaaactct caactaaact ttgttggtgt ggtattaaag gaagacttag gccgcaaaag 60 caacaacaat tacacaatac aaacttgcaa atgactgaac taaaaaaaca aaagaccgct 120 gaacaaaaaa ccagacctca aaatgtcggt attaaaggta tccaaattta catcccaact 180 caatgtgtca accaatctga gctagagaaa tttgatggcg tttctcaagg taaatacaca 240 attggtctgg gccaaaccaa catgtctttt gtcaatgaca gagaagatat ctactcgatg 300 tccctaactg ttttgtctaa gttgatcaag agttacaaca tcgacaccaa caaaattggt 360 agattagaag tcggtactga aactctgatt gacaagtcca agtctgtcaa gtctgtcttg 420 atgcaattgt ttggtgaaaa cactgacgtc gaaggtattg acacgcttaa tgcctgttac 480 ggtggtacca acgcgttgtt caactctttg aactggattg aatctaacgc atgggatggt 540 agagacgcca ttgtagtttg cggtgatatt gccatctacg ataagggtgc cgcaagacca 600 accggtggtg ccggtactgt tgctatgtgg atcggtcctg atgctccaat tgtatttgac 660 tctgtaagag cttcttacat ggaacacgcc tacgattttt acaagccaga tttcaccagc 720 gaatatcctt acgtcgatgg tcatttttca ttaacttgtt acgtcaaggc tcttgatcaa 780 gtttacaaga gttattccaa gaaggctatt tctaaagggt tggttagcga tcccgctggt 840 tcggatgctt tgaacgtttt gaaatatttc gactacaacg ttttccatgt tccaacctgt 900 aaattggtca caaaatcata cggtagatta ctatataacg atttcagagc caatcctcaa 960 ttgttcccag aagttgacgc cgaattagct actcgcgatt atgacgaatc tttaaccgat 1020 aagaacattg aaaaaacttt tgttaatgtt gctaagccat tccacaaaga gagagttgcc 1080 caatctttga ttgttccaac aaacacaggt aacatgtaca ccgcatctgt ttatgccgcc 1140 tttgcatctc tattaaacta tgttggatct gacgacttac aaggcaagcg tgttggttta 1200 ttttcttacg gttccggttt agctgcatct ctatattctt gcaaaattgt tggtgacgtc 1260 caacatatta tcaaggaatt agatattact aacaaattag ccaagagaat caccgaaact 1320 ccaaaggatt acgaagctgc catcgaattg agagaaaatg cccatttgaa gaagaacttc 1380 aaacctcaag gttccattga gcatttgcaa agtggtgttt actacttgac caacatcgat 1440 gacaaattta gaagatctta cgatgttaaa aaataa 1476 <210> 29 <211> 1356 <212> DNA <213> Saccharomyces cerevisiae <400> 29 atgtcagagt tgagagcctt cagtgcccca gggaaagcgt tactagctgg tggatattta 60 gttttagata caaaatatga agcatttgta gtcggattat cggcaagaat gcatgctgta 120 gcccatcctt acggttcatt gcaagggtct gataagtttg aagtgcgtgt gaaaagtaaa 180 caatttaaag atggggagtg gctgtaccat ataagtccta aaagtggctt cattcctgtt 240 tcgataggcg gatctaagaa ccctttcatt gaaaaagtta tcgctaacgt atttagctac 300 tttaaaccta acatggacga ctactgcaat agaaacttgt tcgttattga tattttctct 360 gatgatgcct accattctca ggaggatagc gttaccgaac atcgtggcaa cagaagattg 420 agttttcatt cgcacagaat tgaagaagtt cccaaaacag ggctgggctc ctcggcaggt 480 ttagtcacag ttttaactac agctttggcc tccttttttg tatcggacct ggaaaataat 540 gtagacaaat atagagaagt tattcataat ttagcacaag ttgctcattg tcaagctcag 600 ggtaaaattg gaagcgggtt tgatgtagcg gcggcagcat atggatctat cagatataga 660 agattcccac ccgcattaat ctctaatttg ccagatattg gaagtgctac ttacggcagt 720 aaactggcgc atttggttga tgaagaagac tggaatatta cgattaaaag taaccattta 780 ccttcgggat taactttatg gatgggcgat attaagaatg gttcagaaac agtaaaactg 840 gtccagaagg taaaaaattg gtatgattcg catatgccag aaagcttgaa aatatataca 900 gaactcgatc atgcaaattc tagatttatg gatggactat ctaaactaga tcgcttacac 960 gagactcatg acgattacag cgatcagata tttgagtctc ttgagaggaa tgactgtacc 1020 tgtcaaaagt atcctgaaat cacagaagtt agagatgcag ttgccacaat tagacgttcc 1080 tttagaaaaa taactaaaga atctggtgcc gatatcgaac ctcccgtaca aactagctta 1140 ttggatgatt gccagacctt aaaaggagtt cttacttgct taatacctgg tgctggtggt 1200 tatgacgcca ttgcagtgat tactaagcaa gatgttgatc ttagggctca aaccgctaat 1260 gacaaaagat tttctaaggt tcaatggctg gatgtaactc aggctgactg gggtgttagg 1320 aaagaaaaag atccggaaac ttatcttgat aaataa 1356 <210> 30 <211> 4 <212> PRT <213> Saccharomyces cerevisiae <400> 30 His Asp Glu Leu 1 <210> 31 <211> 4 <212> PRT <213> Saccharomyces cerevisiae <400> 31 Asp Asp Glu Leu 1 <210> 32 <211> 4 <212> PRT <213> Saccharomyces cerevisiae <400> 32 Lys Asp Glu Leu 1 <210> 33 <211> 29 <212> PRT <213> Saccharomyces cerevisiae <400> 33 Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe Trp Ala 1 5 10 15 Thr Glu Ala Glu Gln Leu Thr Lys Cys Glu Val Phe Gln 20 25 <210> 34 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 34 gccgttgaca gagggtccga gctcggtacc aag 33 <210> 35 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 35 catactgacc cattgtcaat gggtaataac tgat 34 <210> 36 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 36 tgtccggtaa atggagac 18 <210> 37 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 37 tgttctcgct gctcgttt 18 <210> 38 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 38 atgggaaagc tattacaat 19 <210> 39 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 39 caaggttgca atggccat 18 <210> 40 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 40 caatgtaggg ctatatatg 19 <210> 41 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 41 aacttgggga atggcaca 18 <210> 42 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 42 tcacgctctg tgtaaagtgt ata 23 <210> 43 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 43 tgcatctcga gggccgcatc atgtaattag 30 <210> 44 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 44 cattagggcc cggccgcaaa ttaaagcctt cg 32 <210> 45 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 45 cacggagctc cagttcgagt ttatcattat caa 33 <210> 46 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 46 ctctccgcgg tttgtttgtt tatgtgtgtt tattc 35 <210> 47 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 47 atggcttcag aaaaagaaat tag 23 <210> 48 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 48 ctatttgctt ctcttgtaaa ctt 23 <210> 49 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 49 atggaggcca agatagatga gct 23 <210> 50 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 50 tcacaattcg gataagtggt cta 23 <210> 51 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 51 atgccgccgc tattcaaggg act 23 <210> 52 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 52 ttaggattta atgcaggtga cgg 23 <210> 53 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 53 ccaaataaag actccaacac tctattt 27 <210> 54 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 54 gaattagaag cattattaag tagtgga 27 <210> 55 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 55 ggatttaacg cacatgcagc taattta 27 <210> 56 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 56 gtctgcttgg gttacatttt ctgaaaa 27 <210> 57 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 57 cataccagtt atactgcaga ccaattg 27 <210> 58 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 58 gaatactcat taaagcaaat ggtagaa 27 <210> 59 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 59 aactgcagat gaccgtttac acagcatccg t 31 <210> 60 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 60 cggaattctt attcctttgg tagaccagtc t 31 <210> 61 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 61 tttcagtccc ttgaatagcg gcggcat 27 <210> 62 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 62 cacaaaatca agattgccca gtatgcc 27 <210> 63 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 63 agaagatacg gatttctttt ctgcttt 27 <210> 64 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 64 aactttggtg caaattgggt caatgat 27 <210> 65 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 65 ttgctcttta aagttttcag aggcatt 27 <210> 66 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 66 gcattattaa gtagtggaaa tacaaaa 27 <210> 67 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 67 cctttgtacg ctttggagaa aaaatta 27 <210> 68 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 68 tctgatcgtt taccatataa aaattat 27 <210> 69 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 69 aaggatggta tgacaagagg cccagta 27 <210> 70 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 70 tccccgcgga tggaggccaa gatagat 27 <210> 71 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 71 caactcgagt cacaattcgg ataagtg 27 <210> 72 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 72 gctctagagt tcgtcgtgtt tgcttctctt gtaaactt 38 <210> 73 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 73 tatctcgagt cacaattcgt catgtaaatt gg 32 <210> 74 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 74 gcagggaccc caattcggat aagtggtc 28 <210> 75 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 75 gtagggtccc tggaggccaa gatagatg 28 <210> 76 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 76 gcagggaccc tttgcttctc ttgtaaact 29 <210> 77 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 77 gtagggtcct cagaaaaaga aattaggag 29 <210> 78 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 78 tgtaaaacga cggccagt 18 <210> 79 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:synthetic DNA <400> 79 taatacgact cactataggg 20[Sequence list]                                SEQUENCE LISTING <110> TOYOTA JIDOSHA KABUSHIKI KAISHA <120> A METHOD OF PRODUCING PRENYLALCOHOL <130> P00-0911 <140> <141> <160> 79 <170> PatentIn Ver. 2.0 <210> 1 <211> 1059 <212> DNA <213> Saccharomyces cerevisiae <220> <221> CDS <222> (1) .. (1056) <400> 1 atg gct tca gaa aaa gaa att agg aga gag aga ttc ttg aac gtt ttc 48 Met Ala Ser Glu Lys Glu Ile Arg Arg Glu Arg Phe Leu Asn Val Phe   1 5 10 15 cct aaa tta gta gag gaa ttg aac gca tcg ctt ttg gct tac ggt atg 96 Pro Lys Leu Val Glu Glu Leu Asn Ala Ser Leu Leu Ala Tyr Gly Met              20 25 30 cct aag gaa gca tgt gac tgg tat gcc cac tca ttg aac tac aac act 144 Pro Lys Glu Ala Cys Asp Trp Tyr Ala His Ser Leu Asn Tyr Asn Thr          35 40 45 cca ggc ggt aag cta aat aga ggt ttg tcc gtt gtg gac acg tat gct 192 Pro Gly Gly Lys Leu Asn Arg Gly Leu Ser Val Val Asp Thr Tyr Ala      50 55 60 att ctc tcc aac aag acc gtt gaa caa ttg ggg caa gaa gaa tac gaa 240 Ile Leu Ser Asn Lys Thr Val Glu Gln Leu Gly Gln Glu Glu Tyr Glu  65 70 75 80 aag gtt gcc att cta ggt tgg tgc att gag ttg ttg cag gct tac ttc 288 Lys Val Ala Ile Leu Gly Trp Cys Ile Glu Leu Leu Gln Ala Tyr Phe                  85 90 95 ttg gtc gcc gat gat atg atg gac aag tcc att acc aga aga ggc caa 336 Leu Val Ala Asp Asp Met Met Asp Lys Ser Ile Thr Arg Arg Gly Gln             100 105 110 cca tgt tgg tac aag gtt cct gaa gtt ggg gaa att gcc atc aat gac 384 Pro Cys Trp Tyr Lys Val Pro Glu Val Gly Glu Ile Ala Ile Asn Asp         115 120 125 gca ttc atg tta gag gct gct atc tac aag ctt ttg aaa tct cac ttc 432 Ala Phe Met Leu Glu Ala Ala Ile Tyr Lys Leu Leu Lys Ser His Phe     130 135 140 aga aac gaa aaa tac tac ata gat atc acc gaa ttg ttc cat gag gtc 480 Arg Asn Glu Lys Tyr Tyr Ile Asp Ile Thr Glu Leu Phe His Glu Val 145 150 155 160 acc ttc caa acc gaa ttg ggc caa ttg atg gac tta atc act gca cct 528 Thr Phe Gln Thr Glu Leu Gly Gln Leu Met Asp Leu Ile Thr Ala Pro                 165 170 175 gaa gac aaa gtc gac ttg agt aag ttc tcc cta aag aag cac tcc ttc 576 Glu Asp Lys Val Asp Leu Ser Lys Phe Ser Leu Lys Lys His Ser Phe             180 185 190 ata gtt act ttc aag act gct tac tat tct ttc tac ttg cct gtc gca 624 Ile Val Thr Phe Lys Thr Ala Tyr Tyr Ser Phe Tyr Leu Pro Val Ala         195 200 205 ttg gcc atg tac gtt gcc ggt atc acg gat gaa aag gat ttg aaa caa 672 Leu Ala Met Tyr Val Ala Gly Ile Thr Asp Glu Lys Asp Leu Lys Gln     210 215 220 gcc aga gat gtc ttg att cca ttg ggt gaa tac ttc caa att caa gat 720 Ala Arg Asp Val Leu Ile Pro Leu Gly Glu Tyr Phe Gln Ile Gln Asp 225 230 235 240 gac tac tta gac tgc ttc ggt acc cca gaa cag atc ggt aag atc ggt 768 Asp Tyr Leu Asp Cys Phe Gly Thr Pro Glu Gln Ile Gly Lys Ile Gly                 245 250 255 aca gat atc caa gat aac aaa tgt tct tgg gta atc aac aag gca ttg 816 Thr Asp Ile Gln Asp Asn Lys Cys Ser Trp Val Ile Asn Lys Ala Leu             260 265 270 gaa ctt gct tcc gca gaa caa aga aag act tta gac gaa aat tac ggt 864 Glu Leu Ala Ser Ala Glu Gln Arg Lys Thr Leu Asp Glu Asn Tyr Gly         275 280 285 aag aag gac tca gtc gca gaa gcc aaa tgc aaa aag att ttc aat gac 912 Lys Lys Asp Ser Val Ala Glu Ala Lys Cys Lys Lys Ile Phe Asn Asp     290 295 300 ttg aaa att gaa cag cta tac cac gaa tat gaa gag tct att gcc aag 960 Leu Lys Ile Glu Gln Leu Tyr His Glu Tyr Glu Glu Ser Ile Ala Lys 305 310 315 320 gat ttg aag gcc aaa att tct cag gtc gat gag tct cgt ggc ttc aaa 1008 Asp Leu Lys Ala Lys Ile Ser Gln Val Asp Glu Ser Arg Gly Phe Lys                 325 330 335 gct gat gtc tta act gcg ttc ttg aac aaa gtt tac aag aga agc aaa 1056 Ala Asp Val Leu Thr Ala Phe Leu Asn Lys Val Tyr Lys Arg Ser Lys             340 345 350 tag 1059 <210> 2 <211> 352 <212> PRT <213> Saccharomyces cerevisiae <400> 2 Met Ala Ser Glu Lys Glu Ile Arg Arg Glu Arg Phe Leu Asn Val Phe   1 5 10 15 Pro Lys Leu Val Glu Glu Leu Asn Ala Ser Leu Leu Ala Tyr Gly Met              20 25 30 Pro Lys Glu Ala Cys Asp Trp Tyr Ala His Ser Leu Asn Tyr Asn Thr          35 40 45 Pro Gly Gly Lys Leu Asn Arg Gly Leu Ser Val Val Asp Thr Tyr Ala      50 55 60 Ile Leu Ser Asn Lys Thr Val Glu Gln Leu Gly Gln Glu Glu Tyr Glu  65 70 75 80 Lys Val Ala Ile Leu Gly Trp Cys Ile Glu Leu Leu Gln Ala Tyr Phe                  85 90 95 Leu Val Ala Asp Asp Met Met Asp Lys Ser Ile Thr Arg Arg Gly Gln             100 105 110 Pro Cys Trp Tyr Lys Val Pro Glu Val Gly Glu Ile Ala Ile Asn Asp         115 120 125 Ala Phe Met Leu Glu Ala Ala Ile Tyr Lys Leu Leu Lys Ser His Phe     130 135 140 Arg Asn Glu Lys Tyr Tyr Ile Asp Ile Thr Glu Leu Phe His Glu Val 145 150 155 160 Thr Phe Gln Thr Glu Leu Gly Gln Leu Met Asp Leu Ile Thr Ala Pro                 165 170 175 Glu Asp Lys Val Asp Leu Ser Lys Phe Ser Leu Lys Lys His Ser Phe             180 185 190 Ile Val Thr Phe Lys Thr Ala Tyr Tyr Ser Phe Tyr Leu Pro Val Ala         195 200 205 Leu Ala Met Tyr Val Ala Gly Ile Thr Asp Glu Lys Asp Leu Lys Gln     210 215 220 Ala Arg Asp Val Leu Ile Pro Leu Gly Glu Tyr Phe Gln Ile Gln Asp 225 230 235 240 Asp Tyr Leu Asp Cys Phe Gly Thr Pro Glu Gln Ile Gly Lys Ile Gly                 245 250 255 Thr Asp Ile Gln Asp Asn Lys Cys Ser Trp Val Ile Asn Lys Ala Leu             260 265 270 Glu Leu Ala Ser Ala Glu Gln Arg Lys Thr Leu Asp Glu Asn Tyr Gly         275 280 285 Lys Lys Asp Ser Val Ala Glu Ala Lys Cys Lys Lys Ile Phe Asn Asp     290 295 300 Leu Lys Ile Glu Gln Leu Tyr His Glu Tyr Glu Glu Ser Ile Ala Lys 305 310 315 320 Asp Leu Lys Ala Lys Ile Ser Gln Val Asp Glu Ser Arg Gly Phe Lys                 325 330 335 Ala Asp Val Leu Thr Ala Phe Leu Asn Lys Val Tyr Lys Arg Ser Lys             340 345 350 <210> 3 <211> 900 <212> DNA <213> Escherichia coli <220> <221> CDS <222> (1) .. (897) <400> 3 atg gac ttt ccg cag caa ctc gaa gcc tgc gtt aag cag gcc aac cag 48 Met Asp Phe Pro Gln Gln Leu Glu Ala Cys Val Lys Gln Ala Asn Gln   1 5 10 15 gcg ctg agc cgt ttt atc gcc cca ctg ccc ttt cag aac act ccc gtg 96 Ala Leu Ser Arg Phe Ile Ala Pro Leu Pro Phe Gln Asn Thr Pro Val              20 25 30 gtc gaa acc atg cag tat ggc gca tta tta ggt ggt aag cgc ctg cga 144 Val Glu Thr Met Gln Tyr Gly Ala Leu Leu Gly Gly Lys Arg Leu Arg          35 40 45 cct ttc ctg gtt tat gcc acc ggt cat atg ttc ggc gtt agc aca aac 192 Pro Phe Leu Val Tyr Ala Thr Gly His Met Phe Gly Val Ser Thr Asn      50 55 60 acg ctg gac gca ccc gct gcc gcc gtt gag tgt atc cac gct tac tca 240 Thr Leu Asp Ala Pro Ala Ala Ala Val Glu Cys Ile His Ala Tyr Ser  65 70 75 80 tta att cat gat gat tta ccg gca atg gat gat gac gat ctg cgt cgc 288 Leu Ile His Asp Asp Leu Pro Ala Met Asp Asp Asp Asp Leu Arg Arg                  85 90 95 ggt ttg cca acc tgc cat gtg aag ttt ggc gaa gca aac gcg att ctc 336 Gly Leu Pro Thr Cys His Val Lys Phe Gly Glu Ala Asn Ala Ile Leu             100 105 110 gct ggc gac gct tta caa acg ctg gcg ttc tcg att tta agc gat gcc 384 Ala Gly Asp Ala Leu Gln Thr Leu Ala Phe Ser Ile Leu Ser Asp Ala         115 120 125 gat atg ccg gaa gtg tcg gac cgc gac aga att tcg atg att tct gaa 432 Asp Met Pro Glu Val Ser Asp Arg Asp Arg Ile Ser Met Ile Ser Glu     130 135 140 ctg gcg agc gcc agt ggt att gcc gga atg tgc ggt ggt cag gca tta 480 Leu Ala Ser Ala Ser Gly Ile Ala Gly Met Cys Gly Gly Gln Ala Leu 145 150 155 160 gat tta gac gcg gaa ggc aaa cac gta cct ctg gac gcg ctt gag cgt 528 Asp Leu Asp Ala Glu Gly Lys His Val Pro Leu Asp Ala Leu Glu Arg                 165 170 175 att cat cgt cat aaa acc ggc gca ttg att cgc gcc gcc gtt cgc ctt 576 Ile His Arg His Lys Thr Gly Ala Leu Ile Arg Ala Ala Val Arg Leu             180 185 190 ggt gca tta agc gcc gga gat aaa gga cgt cgt gct ctg ccg gta ctc 624 Gly Ala Leu Ser Ala Gly Asp Lys Gly Arg Arg Ala Leu Pro Val Leu         195 200 205 gac aag tat gca gag agc atc ggc ctt gcc ttc cag gtt cag gat gac 672 Asp Lys Tyr Ala Glu Ser Ile Gly Leu Ala Phe Gln Val Gln Asp Asp     210 215 220 atc ctg gat gtg gtg gga gat act gca acg ttg gga aaa cgc cag ggt 720 Ile Leu Asp Val Val Gly Asp Thr Ala Thr Leu Gly Lys Arg Gln Gly 225 230 235 240 gcc gac cag caa ctt ggt aaa agt acc tac cct gca ctt ctg ggt ctt 768 Ala Asp Gln Gln Leu Gly Lys Ser Thr Tyr Pro Ala Leu Leu Gly Leu                 245 250 255 gag caa gcc cgg aag aaa gcc cgg gat ctg atc gac gat gcc cgt cag 816 Glu Gln Ala Arg Lys Lys Ala Arg Asp Leu Ile Asp Asp Ala Arg Gln             260 265 270 tcg ctg aaa caa ctg gct gaa cag tca ctc gat acc tcg gca ctg gaa 864 Ser Leu Lys Gln Leu Ala Glu Gln Ser Leu Asp Thr Ser Ala Leu Glu         275 280 285 gcg cta gcg gac tac atc atc cag cgt aat aaa taa 900 Ala Leu Ala Asp Tyr Ile Ile Gln Arg Asn Lys     290 295 <210> 4 <211> 299 <212> PRT <213> Escherichia coli <400> 4 Met Asp Phe Pro Gln Gln Leu Glu Ala Cys Val Lys Gln Ala Asn Gln   1 5 10 15 Ala Leu Ser Arg Phe Ile Ala Pro Leu Pro Phe Gln Asn Thr Pro Val              20 25 30 Val Glu Thr Met Gln Tyr Gly Ala Leu Leu Gly Gly Lys Arg Leu Arg          35 40 45 Pro Phe Leu Val Tyr Ala Thr Gly His Met Phe Gly Val Ser Thr Asn      50 55 60 Thr Leu Asp Ala Pro Ala Ala Ala Val Glu Cys Ile His Ala Tyr Ser  65 70 75 80 Leu Ile His Asp Asp Leu Pro Ala Met Asp Asp Asp Asp Leu Arg Arg                  85 90 95 Gly Leu Pro Thr Cys His Val Lys Phe Gly Glu Ala Asn Ala Ile Leu             100 105 110 Ala Gly Asp Ala Leu Gln Thr Leu Ala Phe Ser Ile Leu Ser Asp Ala         115 120 125 Asp Met Pro Glu Val Ser Asp Arg Asp Arg Ile Ser Met Ile Ser Glu     130 135 140 Leu Ala Ser Ala Ser Gly Ile Ala Gly Met Cys Gly Gly Gln Ala Leu 145 150 155 160 Asp Leu Asp Ala Glu Gly Lys His Val Pro Leu Asp Ala Leu Glu Arg                 165 170 175 Ile His Arg His Lys Thr Gly Ala Leu Ile Arg Ala Ala Val Arg Leu             180 185 190 Gly Ala Leu Ser Ala Gly Asp Lys Gly Arg Arg Ala Leu Pro Val Leu         195 200 205 Asp Lys Tyr Ala Glu Ser Ile Gly Leu Ala Phe Gln Val Gln Asp Asp     210 215 220 Ile Leu Asp Val Val Gly Asp Thr Ala Thr Leu Gly Lys Arg Gln Gly 225 230 235 240 Ala Asp Gln Gln Leu Gly Lys Ser Thr Tyr Pro Ala Leu Leu Gly Leu                 245 250 255 Glu Gln Ala Arg Lys Lys Ala Arg Asp Leu Ile Asp Asp Ala Arg Gln             260 265 270 Ser Leu Lys Gln Leu Ala Glu Gln Ser Leu Asp Thr Ser Ala Leu Glu         275 280 285 Ala Leu Ala Asp Tyr Ile Ile Gln Arg Asn Lys     290 295 <210> 5 <211> 1008 <212> DNA <213> Saccharomyces cerevisiae <220> <221> CDS <222> (1) .. (1005) <400> 5 atg gag gcc aag ata gat gag ctg atc aat aat gat cct gtt tgg tcc 48 Met Glu Ala Lys Ile Asp Glu Leu Ile Asn Asn Asp Pro Val Trp Ser   1 5 10 15 agc caa aat gaa agc ttg att tca aaa cct tat aat cac atc ctt ttg 96 Ser Gln Asn Glu Ser Leu Ile Ser Lys Pro Tyr Asn His Ile Leu Leu              20 25 30 aaa cct ggc aag aac ttt aga cta aat tta ata gtt caa att aac aga 144 Lys Pro Gly Lys Asn Phe Arg Leu Asn Leu Ile Val Gln Ile Asn Arg          35 40 45 gtt atg aat ttg ccc aaa gac cag ctg gcc ata gtt tcg caa att gtt 192 Val Met Asn Leu Pro Lys Asp Gln Leu Ala Ile Val Ser Gln Ile Val      50 55 60 gag ctc ttg cat aat tcc agc ctt tta atc gac gat ata gaa gat aat 240 Glu Leu Leu His Asn Ser Ser Leu Leu Ile Asp Asp Ile Glu Asp Asn  65 70 75 80 gct ccc ttg aga agg gga cag acc act tct cac tta atc ttc ggt gta 288 Ala Pro Leu Arg Arg Gly Gln Thr Thr Ser His Leu Ile Phe Gly Val                  85 90 95 ccc tcc act ata aac acc gca aat tat atg tat ttc aga gcc atg caa 336 Pro Ser Thr Ile Asn Thr Ala Asn Tyr Met Tyr Phe Arg Ala Met Gln             100 105 110 ctt gta tcg cag cta acc aca aaa gag cct ttg tat cat aat ttg att 384 Leu Val Ser Gln Leu Thr Thr Lys Glu Pro Leu Tyr His Asn Leu Ile         115 120 125 acg att ttc aac gaa gaa ttg atc aat cta cat agg gga caa ggc ttg 432 Thr Ile Phe Asn Glu Glu Leu Ile Asn Leu His Arg Gly Gln Gly Leu     130 135 140 gat ata tac tgg aga gac ttt ctg cct gaa atc ata cct act cag gag 480 Asp Ile Tyr Trp Arg Asp Phe Leu Pro Glu Ile Ile Pro Thr Gln Glu 145 150 155 160 atg tat ttg aat atg gtt atg aat aaa aca ggc ggc ctt ttc aga tta 528 Met Tyr Leu Asn Met Val Met Asn Lys Thr Gly Gly Leu Phe Arg Leu                 165 170 175 acg ttg aga ctc atg gaa gcg ctg tct cct tcc tca cac cac ggc cat 576 Thr Leu Arg Leu Met Glu Ala Leu Ser Pro Ser Ser His His Gly His             180 185 190 tcg ttg gtt cct ttc ata aat ctt ctg ggt att att tat cag att aga 624 Ser Leu Val Pro Phe Ile Asn Leu Leu Gly Ile Ile Tyr Gln Ile Arg         195 200 205 gat gat tac ttg aat ttg aaa gat ttc caa atg tcc agc gaa aaa ggc 672 Asp Asp Tyr Leu Asn Leu Lys Asp Phe Gln Met Ser Ser Glu Lys Gly     210 215 220 ttt gct gag gac att aca gag ggg aag tta tct ttt ccc atc gtc cac 720 Phe Ala Glu Asp Ile Thr Glu Gly Lys Leu Ser Phe Pro Ile Val His 225 230 235 240 gcc ctt aac ttc act aaa acg aaa ggt caa act gag caa cac aat gaa 768 Ala Leu Asn Phe Thr Lys Thr Lys Gly Gln Thr Glu Gln His Asn Glu                 245 250 255 att cta aga att ctc ctg ttg agg aca agt gat aaa gat ata aaa cta 816 Ile Leu Arg Ile Leu Leu Leu Arg Thr Ser Asp Lys Asp Ile Lys Leu             260 265 270 aag ctg att caa ata ctg gaa ttc gac acc aat tca ttg gcc tac acc 864 Lys Leu Ile Gln Ile Leu Glu Phe Asp Thr Asn Ser Leu Ala Tyr Thr         275 280 285 aaa aat ttt att aat caa tta gtg aat atg ata aaa aat gat aat gaa 912 Lys Asn Phe Ile Asn Gln Leu Val Asn Met Ile Lys Asn Asp Asn Glu     290 295 300 aat aag tat tta cct gat ttg gct tcg cat tcc gac acc gcc acc aat 960 Asn Lys Tyr Leu Pro Asp Leu Ala Ser His Ser Asp Thr Ala Thr Asn 305 310 315 320 tta cat gac gaa ttg tta tat ata ata gac cac tta tcc gaa ttg tga 1008 Leu His Asp Glu Leu Leu Tyr Ile Ile Asp His Leu Ser Glu Leu                 325 330 335 <210> 6 <211> 335 <212> PRT <213> Saccharomyces cerevisiae <400> 6 Met Glu Ala Lys Ile Asp Glu Leu Ile Asn Asn Asp Pro Val Trp Ser   1 5 10 15 Ser Gln Asn Glu Ser Leu Ile Ser Lys Pro Tyr Asn His Ile Leu Leu              20 25 30 Lys Pro Gly Lys Asn Phe Arg Leu Asn Leu Ile Val Gln Ile Asn Arg          35 40 45 Val Met Asn Leu Pro Lys Asp Gln Leu Ala Ile Val Ser Gln Ile Val      50 55 60 Glu Leu Leu His Asn Ser Ser Leu Leu Ile Asp Asp Ile Glu Asp Asn  65 70 75 80 Ala Pro Leu Arg Arg Gly Gln Thr Thr Ser His Leu Ile Phe Gly Val                  85 90 95 Pro Ser Thr Ile Asn Thr Ala Asn Tyr Met Tyr Phe Arg Ala Met Gln             100 105 110 Leu Val Ser Gln Leu Thr Thr Lys Glu Pro Leu Tyr His Asn Leu Ile         115 120 125 Thr Ile Phe Asn Glu Glu Leu Ile Asn Leu His Arg Gly Gln Gly Leu     130 135 140 Asp Ile Tyr Trp Arg Asp Phe Leu Pro Glu Ile Ile Pro Thr Gln Glu 145 150 155 160 Met Tyr Leu Asn Met Val Met Asn Lys Thr Gly Gly Leu Phe Arg Leu                 165 170 175 Thr Leu Arg Leu Met Glu Ala Leu Ser Pro Ser Ser His His Gly His             180 185 190 Ser Leu Val Pro Phe Ile Asn Leu Leu Gly Ile Ile Tyr Gln Ile Arg         195 200 205 Asp Asp Tyr Leu Asn Leu Lys Asp Phe Gln Met Ser Ser Glu Lys Gly     210 215 220 Phe Ala Glu Asp Ile Thr Glu Gly Lys Leu Ser Phe Pro Ile Val His 225 230 235 240 Ala Leu Asn Phe Thr Lys Thr Lys Gly Gln Thr Glu Gln His Asn Glu                 245 250 255 Ile Leu Arg Ile Leu Leu Leu Arg Thr Ser Asp Lys Asp Ile Lys Leu             260 265 270 Lys Leu Ile Gln Ile Leu Glu Phe Asp Thr Asn Ser Leu Ala Tyr Thr         275 280 285 Lys Asn Phe Ile Asn Gln Leu Val Asn Met Ile Lys Asn Asp Asn Glu     290 295 300 Asn Lys Tyr Leu Pro Asp Leu Ala Ser His Ser Asp Thr Ala Thr Asn 305 310 315 320 Leu His Asp Glu Leu Leu Tyr Ile Ile Asp His Leu Ser Glu Leu                 325 330 335 <210> 7 <211> 3165 <212> DNA <213> Saccharomyces cerevisiae <220> <221> CDS <222> (1) .. (3162) <400> 7 atg ccg ccg cta ttc aag gga ctg aaa cag atg gca aag cca att gcc 48 Met Pro Pro Leu Phe Lys Gly Leu Lys Gln Met Ala Lys Pro Ile Ala   1 5 10 15 tat gtt tca aga ttt tcg gcg aaa cga cca att cat ata ata ctt ttt 96 Tyr Val Ser Arg Phe Ser Ala Lys Arg Pro Ile His Ile Ile Leu Phe              20 25 30 tct cta atc ata tcc gca ttc gct tat cta tcc gtc att cag tat tac 144 Ser Leu Ile Ile Ser Ala Phe Ala Tyr Leu Ser Val Ile Gln Tyr Tyr          35 40 45 ttc aat ggt tgg caa cta gat tca aat agt gtt ttt gaa act gct cca 192 Phe Asn Gly Trp Gln Leu Asp Ser Asn Ser Val Phe Glu Thr Ala Pro      50 55 60 aat aaa gac tcc aac act cta ttt caa gaa tgt tcc cat tac tac aga 240 Asn Lys Asp Ser Asn Thr Leu Phe Gln Glu Cys Ser His Tyr Tyr Arg  65 70 75 80 gat tcc tct cta gat ggt tgg gta tca atc acc gcg cat gaa gct agt 288 Asp Ser Ser Leu Asp Gly Trp Val Ser Ile Thr Ala His Glu Ala Ser                  85 90 95 gag tta cca gcc cca cac cat tac tat cta tta aac ctg aac ttc aat 336 Glu Leu Pro Ala Pro His His Tyr Tyr Leu Leu Asn Leu Asn Phe Asn             100 105 110 agt cct aat gaa act gac tcc att cca gaa cta gct aac acg gtt ttt 384 Ser Pro Asn Glu Thr Asp Ser Ile Pro Glu Leu Ala Asn Thr Val Phe         115 120 125 gag aaa gat aat aca aaa tat att ctg caa gaa gat ctc agt gtt tcc 432 Glu Lys Asp Asn Thr Lys Tyr Ile Leu Gln Glu Asp Leu Ser Val Ser     130 135 140 aaa gaa att tct tct act gat gga acg aaa tgg agg tta aga agt gac 480 Lys Glu Ile Ser Ser Thr Asp Gly Thr Lys Trp Arg Leu Arg Ser Asp 145 150 155 160 aga aaa agt ctt ttc gac gta aag acg tta gca tat tct ctc tac gat 528 Arg Lys Ser Leu Phe Asp Val Lys Thr Leu Ala Tyr Ser Leu Tyr Asp                 165 170 175 gta ttt tca gaa aat gta acc caa gca gac ccg ttt gac gtc ctt att 576 Val Phe Ser Glu Asn Val Thr Gln Ala Asp Pro Phe Asp Val Leu Ile             180 185 190 atg gtt act gcc tac cta atg atg ttc tac acc ata ttc ggc ctc ttc 624 Met Val Thr Ala Tyr Leu Met Met Phe Tyr Thr Ile Phe Gly Leu Phe         195 200 205 aat gac atg agg aag acc ggg tca aat ttt tgg ttg agc gcc tct aca 672 Asn Asp Met Arg Lys Thr Gly Ser Asn Phe Trp Leu Ser Ala Ser Thr     210 215 220 gtg gtc aat tct gca tca tca ctt ttc tta gca ttg tat gtc acc caa 720 Val Val Asn Ser Ala Ser Ser Leu Phe Leu Ala Leu Tyr Val Thr Gln 225 230 235 240 tgt att cta ggc aaa gaa gtt tcc gca tta act ctt ttt gaa ggt ttg 768 Cys Ile Leu Gly Lys Glu Val Ser Ala Leu Thr Leu Phe Glu Gly Leu                 245 250 255 cct ttc att gta gtt gtt gtt ggt ttc aag cac aaa atc aag att gcc 816 Pro Phe Ile Val Val Val Val Gly Phe Lys His Lys Ile Lys Ile Ala             260 265 270 cag tat gcc ctg gag aaa ttt gaa aga gtc ggt tta tct aaa agg att 864 Gln Tyr Ala Leu Glu Lys Phe Glu Arg Val Gly Leu Ser Lys Arg Ile         275 280 285 act acc gat gaa atc gtt ttt gaa tcc gtg agc gaa gag ggt ggt cgt 912 Thr Thr Asp Glu Ile Val Phe Glu Ser Val Ser Glu Glu Gly Gly Arg     290 295 300 ttg att caa gac cat ttg ctt tgt att ttt gcc ttt atc gga tgc tct 960 Leu Ile Gln Asp His Leu Leu Cys Ile Phe Ala Phe Ile Gly Cys Ser 305 310 315 320 atg tat gct cac caa ttg aag act ttg aca aac ttc tgc ata tta tca 1008 Met Tyr Ala His Gln Leu Lys Thr Leu Thr Asn Phe Cys Ile Leu Ser                 325 330 335 gca ttt atc cta att ttt gaa ttg att tta act cct aca ttt tat tct 1056 Ala Phe Ile Leu Ile Phe Glu Leu Ile Leu Thr Pro Thr Phe Tyr Ser             340 345 350 gct atc tta gcg ctt aga ctg gaa atg aat gtt atc cac aga tct act 1104 Ala Ile Leu Ala Leu Arg Leu Glu Met Asn Val Ile His Arg Ser Thr         355 360 365 att atc aag caa aca tta gaa gaa gac ggt gtt gtt cca tct aca gca 1152 Ile Ile Lys Gln Thr Leu Glu Glu Asp Gly Val Val Pro Ser Thr Ala     370 375 380 aga atc att tct aaa gca gaa aag aaa tcc gta tct tct ttc tta aat 1200 Arg Ile Ile Ser Lys Ala Glu Lys Lys Ser Val Ser Ser Phe Leu Asn 385 390 395 400 ctc agt gtg gtt gtc att atc atg aaa ctc tct gtc ata ctg ttg ttt 1248 Leu Ser Val Val Val Ile Ile Met Lys Leu Ser Val Ile Leu Leu Phe                 405 410 415 gtc ttc atc aac ttt tat aac ttt ggt gca aat tgg gtc aat gat gcc 1296 Val Phe Ile Asn Phe Tyr Asn Phe Gly Ala Asn Trp Val Asn Asp Ala             420 425 430 ttc aat tca ttg tac ttc gat aag gaa cgt gtt tct cta cca gat ttt 1344 Phe Asn Ser Leu Tyr Phe Asp Lys Glu Arg Val Ser Leu Pro Asp Phe         435 440 445 att acc tcg aat gcc tct gaa aac ttt aaa gag caa gct att gtt agt 1392 Ile Thr Ser Asn Ala Ser Glu Asn Phe Lys Glu Gln Ala Ile Val Ser     450 455 460 gtc acc cca tta tta tat tac aaa ccc att aag tcc tac caa cgc att 1440 Val Thr Pro Leu Leu Tyr Tyr Lys Pro Ile Lys Ser Tyr Gln Arg Ile 465 470 475 480 gag gat atg gtt ctt cta ttg ctt cgt aat gtc agt gtt gcc att cgt 1488 Glu Asp Met Val Leu Leu Leu Leu Arg Asn Val Ser Val Ala Ile Arg                 485 490 495 gat agg ttc gtc agt aaa tta gtt ctt tcc gcc tta gta tgc agt gct 1536 Asp Arg Phe Val Ser Lys Leu Val Leu Ser Ala Leu Val Cys Ser Ala             500 505 510 gtc atc aat gtg tat tta ttg aat gct gct aga att cat acc agt tat 1584 Val Ile Asn Val Tyr Leu Leu Asn Ala Ala Arg Ile His Thr Ser Tyr         515 520 525 act gca gac caa ttg gtg aaa act gaa gtc acc aag aag tct ttt act 1632 Thr Ala Asp Gln Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe Thr     530 535 540 gct cct gta caa aag gct tct aca cca gtt tta acc aat aaa aca gtc 1680 Ala Pro Val Gln Lys Ala Ser Thr Pro Val Leu Thr Asn Lys Thr Val 545 550 555 560 att tct gga tcg aaa gtc aaa agt tta tca tct gcg caa tcg agc tca 1728 Ile Ser Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gln Ser Ser Ser                 565 570 575 tca gga cct tca tca tct agt gag gaa gat gat tcc cgc gat att gaa 1776 Ser Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu             580 585 590 agc ttg gat aag aaa ata cgt cct tta gaa gaa tta gaa gca tta tta 1824 Ser Leu Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Leu Leu         595 600 605 agt agt gga aat aca aaa caa ttg aag aac aaa gag gtc gct gcc ttg 1872 Ser Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala Ala Leu     610 615 620 gtt att cac ggt aag tta cct ttg tac gct ttg gag aaa aaa tta ggt 1920 Val Ile His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu Gly 625 630 635 640 gat act acg aga gcg gtt gcg gta cgt agg aag gct ctt tca att ttg 1968 Asp Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser Ile Leu                 645 650 655 gca gaa gct cct gta tta gca tct gat cgt tta cca tat aaa aat tat 2016 Ala Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys Asn Tyr             660 665 670 gac tac gac cgc gta ttt ggc gct tgt tgt gaa aat gtt ata ggt tac 2064 Asp Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr         675 680 685 atg cct ttg ccc gtt ggt gtt ata ggc ccc ttg gtt atc gat ggt aca 2112 Met Pro Leu Pro Val Gly Val Ile Gly Pro Leu Val Ile Asp Gly Thr     690 695 700 tct tat cat ata cca atg gca act aca gag ggt tgt ttg gta gct tct 2160 Ser Tyr His Ile Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser 705 710 715 720 gcc atg cgt ggc tgt aag gca atc aat gct ggc ggt ggt gca aca act 2208 Ala Met Arg Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr                 725 730 735 gtt tta act aag gat ggt atg aca aga ggc cca gta gtc cgt ttc cca 2256 Val Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe Pro             740 745 750 act ttg aaa aga tct ggt gcc tgt aag ata tgg tta gac tca gaa gag 2304 Thr Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser Glu Glu         755 760 765 gga caa aac gca att aaa aaa gct ttt aac tct aca tca aga ttt gca 2352 Gly Gln Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala     770 775 780 cgt ctg caa cat att caa act tgt cta gca gga gat tta ctc ttc atg 2400 Arg Leu Gln His Ile Gln Thr Cys Leu Ala Gly Asp Leu Leu Phe Met 785 790 795 800 aga ttt aga aca act act ggt gac gca atg ggt atg aat atg att tct 2448 Arg Phe Arg Thr Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser                 805 810 815 aaa ggt gtc gaa tac tca tta aag caa atg gta gaa gag tat ggc tgg 2496 Lys Gly Val Glu Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp             820 825 830 gaa gat atg gag gtt gtc tcc gtt tct ggt aac tac tgt acc gac aaa 2544 Glu Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys         835 840 845 aaa cca gct gcc atc aac tgg atc gaa ggt cgt ggt aag agt gtc gtc 2592 Lys Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val     850 855 860 gca gaa gct act att cct ggt gat gtt gtc aga aaa gtg tta aaa agt 2640 Ala Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu Lys Ser 865 870 875 880 gat gtt tcc gca ttg gtt gag ttg aac att gct aag aat ttg gtt gga 2688 Asp Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn Leu Val Gly                 885 890 895 tct gca atg gct ggg tct gtt ggt gga ttt aac gca cat gca gct aat 2736 Ser Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala His Ala Ala Asn             900 905 910 tta gtg aca gct gtt ttc ttg gca tta gga caa gat cct gca caa aat 2784 Leu Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro Ala Gln Asn         915 920 925 gtt gaa agt tcc aac tgt ata aca ttg atg aaa gaa gtg gac ggt gat 2832 Val Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp     930 935 940 ttg aga att tcc gta tcc atg cca tcc atc gaa gta ggt acc atc ggt 2880 Leu Arg Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly 945 950 955 960 ggt ggt act gtt cta gaa cca caa ggt gcc atg ttg gac tta tta ggt 2928 Gly Gly Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Gly                 965 970 975 gta aga ggc ccg cat gct acc gct cct ggt acc aac gca cgt caa tta 2976 Val Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gln Leu             980 985 990 gca aga ata gtt gcc tgt gcc gtc ttg gca ggt gaa tta tcc tta tgt 3024 Ala Arg Ile Val Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu Cys         995 1000 1005 gct gcc cta gca gcc ggc cat ttg gtt caa agt cat atg acc cac aac 3072 Ala Ala Leu Ala Ala Gly His Leu Val Gln Ser His Met Thr His Asn    1010 1015 1020 agg aaa cct gct gaa cca aca aaa cct aac aat ttg gac gcc act gat 3120 Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala Thr Asp 1025 1030 1035 1040 ata aat cgt ttg aaa gat ggg tcc gtc acc tgc att aaa tcc taa 3165 Ile Asn Arg Leu Lys Asp Gly Ser Val Thr Cys Ile Lys Ser                1045 1050 <210> 8 <211> 1054 <212> PRT <213> Saccharomyces cerevisiae <400> 8 Met Pro Pro Leu Phe Lys Gly Leu Lys Gln Met Ala Lys Pro Ile Ala   1 5 10 15 Tyr Val Ser Arg Phe Ser Ala Lys Arg Pro Ile His Ile Ile Leu Phe              20 25 30 Ser Leu Ile Ile Ser Ala Phe Ala Tyr Leu Ser Val Ile Gln Tyr Tyr          35 40 45 Phe Asn Gly Trp Gln Leu Asp Ser Asn Ser Val Phe Glu Thr Ala Pro      50 55 60 Asn Lys Asp Ser Asn Thr Leu Phe Gln Glu Cys Ser His Tyr Tyr Arg  65 70 75 80 Asp Ser Ser Leu Asp Gly Trp Val Ser Ile Thr Ala His Glu Ala Ser                  85 90 95 Glu Leu Pro Ala Pro His His Tyr Tyr Leu Leu Asn Leu Asn Phe Asn             100 105 110 Ser Pro Asn Glu Thr Asp Ser Ile Pro Glu Leu Ala Asn Thr Val Phe         115 120 125 Glu Lys Asp Asn Thr Lys Tyr Ile Leu Gln Glu Asp Leu Ser Val Ser     130 135 140 Lys Glu Ile Ser Ser Thr Asp Gly Thr Lys Trp Arg Leu Arg Ser Asp 145 150 155 160 Arg Lys Ser Leu Phe Asp Val Lys Thr Leu Ala Tyr Ser Leu Tyr Asp                 165 170 175 Val Phe Ser Glu Asn Val Thr Gln Ala Asp Pro Phe Asp Val Leu Ile             180 185 190 Met Val Thr Ala Tyr Leu Met Met Phe Tyr Thr Ile Phe Gly Leu Phe         195 200 205 Asn Asp Met Arg Lys Thr Gly Ser Asn Phe Trp Leu Ser Ala Ser Thr     210 215 220 Val Val Asn Ser Ala Ser Ser Leu Phe Leu Ala Leu Tyr Val Thr Gln 225 230 235 240 Cys Ile Leu Gly Lys Glu Val Ser Ala Leu Thr Leu Phe Glu Gly Leu                 245 250 255 Pro Phe Ile Val Val Val Val Gly Phe Lys His Lys Ile Lys Ile Ala             260 265 270 Gln Tyr Ala Leu Glu Lys Phe Glu Arg Val Gly Leu Ser Lys Arg Ile         275 280 285 Thr Thr Asp Glu Ile Val Phe Glu Ser Val Ser Glu Glu Gly Gly Arg     290 295 300 Leu Ile Gln Asp His Leu Leu Cys Ile Phe Ala Phe Ile Gly Cys Ser 305 310 315 320 Met Tyr Ala His Gln Leu Lys Thr Leu Thr Asn Phe Cys Ile Leu Ser                 325 330 335 Ala Phe Ile Leu Ile Phe Glu Leu Ile Leu Thr Pro Thr Phe Tyr Ser             340 345 350 Ala Ile Leu Ala Leu Arg Leu Glu Met Asn Val Ile His Arg Ser Thr         355 360 365 Ile Ile Lys Gln Thr Leu Glu Glu Asp Gly Val Val Pro Ser Thr Ala     370 375 380 Arg Ile Ile Ser Lys Ala Glu Lys Lys Ser Val Ser Ser Phe Leu Asn 385 390 395 400 Leu Ser Val Val Val Ile Ile Met Lys Leu Ser Val Ile Leu Leu Phe                 405 410 415 Val Phe Ile Asn Phe Tyr Asn Phe Gly Ala Asn Trp Val Asn Asp Ala             420 425 430 Phe Asn Ser Leu Tyr Phe Asp Lys Glu Arg Val Ser Leu Pro Asp Phe         435 440 445 Ile Thr Ser Asn Ala Ser Glu Asn Phe Lys Glu Gln Ala Ile Val Ser     450 455 460 Val Thr Pro Leu Leu Tyr Tyr Lys Pro Ile Lys Ser Tyr Gln Arg Ile 465 470 475 480 Glu Asp Met Val Leu Leu Leu Leu Arg Asn Val Ser Val Ala Ile Arg                 485 490 495 Asp Arg Phe Val Ser Lys Leu Val Leu Ser Ala Leu Val Cys Ser Ala             500 505 510 Val Ile Asn Val Tyr Leu Leu Asn Ala Ala Arg Ile His Thr Ser Tyr         515 520 525 Thr Ala Asp Gln Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe Thr     530 535 540 Ala Pro Val Gln Lys Ala Ser Thr Pro Val Leu Thr Asn Lys Thr Val 545 550 555 560 Ile Ser Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gln Ser Ser Ser                 565 570 575 Ser Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu             580 585 590 Ser Leu Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Leu Leu         595 600 605 Ser Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala Ala Leu     610 615 620 Val Ile His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu Gly 625 630 635 640 Asp Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser Ile Leu                 645 650 655 Ala Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys Asn Tyr             660 665 670 Asp Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr         675 680 685 Met Pro Leu Pro Val Gly Val Ile Gly Pro Leu Val Ile Asp Gly Thr     690 695 700 Ser Tyr His Ile Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser 705 710 715 720 Ala Met Arg Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr                 725 730 735 Val Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe Pro             740 745 750 Thr Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser Glu Glu         755 760 765 Gly Gln Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala     770 775 780 Arg Leu Gln His Ile Gln Thr Cys Leu Ala Gly Asp Leu Leu Phe Met 785 790 795 800 Arg Phe Arg Thr Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser                 805 810 815 Lys Gly Val Glu Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp             820 825 830 Glu Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys         835 840 845 Lys Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val     850 855 860 Ala Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu Lys Ser 865 870 875 880 Asp Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn Leu Val Gly                 885 890 895 Ser Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala His Ala Ala Asn             900 905 910 Leu Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro Ala Gln Asn         915 920 925 Val Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp     930 935 940 Leu Arg Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly 945 950 955 960 Gly Gly Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Gly                 965 970 975 Val Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gln Leu             980 985 990 Ala Arg Ile Val Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu Cys         995 1000 1005 Ala Ala Leu Ala Ala Gly His Leu Val Gln Ser His Met Thr His Asn    1010 1015 1020 Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala Thr Asp 1025 1030 1035 1040 Ile Asn Arg Leu Lys Asp Gly Ser Val Thr Cys Ile Lys Ser                1045 1050 <210> 9 <211> 1191 <212> DNA <213> Saccharomyces cerevisiae <220> <221> CDS <222> (1) .. (1188) <400> 9 atg acc gtt tac aca gca tcc gtt acc gca ccc gtc aac atc gca acc 48 Met Thr Val Tyr Thr Ala Ser Val Thr Ala Pro Val Asn Ile Ala Thr   1 5 10 15 ctt aag tat tgg ggg aaa agg gac acg aag ttg aat ctg ccc acc aat 96 Leu Lys Tyr Trp Gly Lys Arg Asp Thr Lys Leu Asn Leu Pro Thr Asn              20 25 30 tcg tcc ata tca gtg act tta tcg caa gat gac ctc aga acg ttg acc 144 Ser Ser Ile Ser Val Thr Leu Ser Gln Asp Asp Leu Arg Thr Leu Thr          35 40 45 tct gcg gct act gca cct gag ttt gaa cgc gac act ttg tgg tta aat 192 Ser Ala Ala Thr Ala Pro Glu Phe Glu Arg Asp Thr Leu Trp Leu Asn      50 55 60 gga gaa cca cac agc atc gac aat gaa aga act caa aat tgt ctg cgc 240 Gly Glu Pro His Ser Ile Asp Asn Glu Arg Thr Gln Asn Cys Leu Arg  65 70 75 80 gac cta cgc caa tta aga aag gaa atg gaa tcg aag gac gcc tca ttg 288 Asp Leu Arg Gln Leu Arg Lys Glu Met Glu Ser Lys Asp Ala Ser Leu                  85 90 95 ccc aca tta tct caa tgg aaa ctc cac att gtc tcc gaa aat aac ttt 336 Pro Thr Leu Ser Gln Trp Lys Leu His Ile Val Ser Glu Asn Asn Phe             100 105 110 cct aca gca gct ggt tta gct tcc tcc gct gct ggc ttt gct gca ttg 384 Pro Thr Ala Ala Gly Leu Ala Ser Ser Ala Ala Gly Phe Ala Ala Leu         115 120 125 gtc tct gca att gct aag tta tac caa tta cca cag tca act tca gaa 432 Val Ser Ala Ile Ala Lys Leu Tyr Gln Leu Pro Gln Ser Thr Ser Glu     130 135 140 ata tct aga ata gca aga aag ggg tct ggt tca gct tgt aga tcg ttg 480 Ile Ser Arg Ile Ala Arg Lys Gly Ser Gly Ser Ala Cys Arg Ser Leu 145 150 155 160 ttt ggc gga tac gtg gcc tgg gaa atg gga aaa gct gaa gat ggt cat 528 Phe Gly Gly Tyr Val Ala Trp Glu Met Gly Lys Ala Glu Asp Gly His                 165 170 175 gat tcc atg gca gta caa atc gca gac agc tct gac tgg cct cag atg 576 Asp Ser Met Ala Val Gln Ile Ala Asp Ser Ser Asp Trp Pro Gln Met             180 185 190 aaa gct tgt gtc cta gtt gtc agc gat att aaa aag gat gtg agt tcc 624 Lys Ala Cys Val Leu Val Val Ser Asp Ile Lys Lys Asp Val Ser Ser         195 200 205 act cag ggt atg caa ttg acc gtg gca acc tcc gaa cta ttt aaa gaa 672 Thr Gln Gly Met Gln Leu Thr Val Ala Thr Ser Glu Leu Phe Lys Glu     210 215 220 aga att gaa cat gtc gta cca aag aga ttt gaa gtc atg cgt aaa gcc 720 Arg Ile Glu His Val Val Pro Lys Arg Phe Glu Val Met Arg Lys Ala 225 230 235 240 att gtt gaa aaa gat ttc gcc acc ttt gca aag gaa aca atg atg gat 768 Ile Val Glu Lys Asp Phe Ala Thr Phe Ala Lys Glu Thr Met Met Asp                 245 250 255 tcc aac tct ttc cat gcc aca tgt ttg gac tct ttc cct cca ata ttc 816 Ser Asn Ser Phe His Ala Thr Cys Leu Asp Ser Phe Pro Pro Ile Phe             260 265 270 tac atg aat gac act tcc aag cgt atc atc agt tgg tgc cac acc att 864 Tyr Met Asn Asp Thr Ser Lys Arg Ile Ile Ser Trp Cys His Thr Ile         275 280 285 aat cag ttt tac gga gaa aca atc gtt gca tac acg ttt gat gca ggt 912 Asn Gln Phe Tyr Gly Glu Thr Ile Val Ala Tyr Thr Phe Asp Ala Gly     290 295 300 cca aat gct gtg ttg tac tac tta gct gaa aat gag tcg aaa ctc ttt 960 Pro Asn Ala Val Leu Tyr Tyr Leu Ala Glu Asn Glu Ser Lys Leu Phe 305 310 315 320 gca ttt atc tat aaa ttg ttt ggc tct gtt cct gga tgg gac aag aaa 1008 Ala Phe Ile Tyr Lys Leu Phe Gly Ser Val Pro Gly Trp Asp Lys Lys                 325 330 335 ttt act act gag cag ctt gag gct ttc aac cat caa ttt gaa tca tct 1056 Phe Thr Thr Glu Gln Leu Glu Ala Phe Asn His Gln Phe Glu Ser Ser             340 345 350 aac ttt act gca cgt gaa ttg gat ctt gag ttg caa aag gat gtt gcc 1104 Asn Phe Thr Ala Arg Glu Leu Asp Leu Glu Leu Gln Lys Asp Val Ala         355 360 365 aga gtg att tta act caa gtc ggt tca ggc cca caa gaa aca aac gaa 1152 Arg Val Ile Leu Thr Gln Val Gly Ser Gly Pro Gln Glu Thr Asn Glu     370 375 380 tct ttg att gac gca aag act ggt cta cca aag gaa taa 1191 Ser Leu Ile Asp Ala Lys Thr Gly Leu Pro Lys Glu 385 390 395 <210> 10 <211> 396 <212> PRT <213> Saccharomyces cerevisiae <400> 10 Met Thr Val Tyr Thr Ala Ser Val Thr Ala Pro Val Asn Ile Ala Thr   1 5 10 15 Leu Lys Tyr Trp Gly Lys Arg Asp Thr Lys Leu Asn Leu Pro Thr Asn              20 25 30 Ser Ser Ile Ser Val Thr Leu Ser Gln Asp Asp Leu Arg Thr Leu Thr          35 40 45 Ser Ala Ala Thr Ala Pro Glu Phe Glu Arg Asp Thr Leu Trp Leu Asn      50 55 60 Gly Glu Pro His Ser Ile Asp Asn Glu Arg Thr Gln Asn Cys Leu Arg  65 70 75 80 Asp Leu Arg Gln Leu Arg Lys Glu Met Glu Ser Lys Asp Ala Ser Leu                  85 90 95 Pro Thr Leu Ser Gln Trp Lys Leu His Ile Val Ser Glu Asn Asn Phe             100 105 110 Pro Thr Ala Ala Gly Leu Ala Ser Ser Ala Ala Gly Phe Ala Ala Leu         115 120 125 Val Ser Ala Ile Ala Lys Leu Tyr Gln Leu Pro Gln Ser Thr Ser Glu     130 135 140 Ile Ser Arg Ile Ala Arg Lys Gly Ser Gly Ser Ala Cys Arg Ser Leu 145 150 155 160 Phe Gly Gly Tyr Val Ala Trp Glu Met Gly Lys Ala Glu Asp Gly His                 165 170 175 Asp Ser Met Ala Val Gln Ile Ala Asp Ser Ser Asp Trp Pro Gln Met             180 185 190 Lys Ala Cys Val Leu Val Val Ser Asp Ile Lys Lys Asp Val Ser Ser         195 200 205 Thr Gln Gly Met Gln Leu Thr Val Ala Thr Ser Glu Leu Phe Lys Glu     210 215 220 Arg Ile Glu His Val Val Pro Lys Arg Phe Glu Val Met Arg Lys Ala 225 230 235 240 Ile Val Glu Lys Asp Phe Ala Thr Phe Ala Lys Glu Thr Met Met Asp                 245 250 255 Ser Asn Ser Phe His Ala Thr Cys Leu Asp Ser Phe Pro Pro Ile Phe             260 265 270 Tyr Met Asn Asp Thr Ser Lys Arg Ile Ile Ser Trp Cys His Thr Ile         275 280 285 Asn Gln Phe Tyr Gly Glu Thr Ile Val Ala Tyr Thr Phe Asp Ala Gly     290 295 300 Pro Asn Ala Val Leu Tyr Tyr Leu Ala Glu Asn Glu Ser Lys Leu Phe 305 310 315 320 Ala Phe Ile Tyr Lys Leu Phe Gly Ser Val Pro Gly Trp Asp Lys Lys                 325 330 335 Phe Thr Thr Glu Gln Leu Glu Ala Phe Asn His Gln Phe Glu Ser Ser             340 345 350 Asn Phe Thr Ala Arg Glu Leu Asp Leu Glu Leu Gln Lys Asp Val Ala         355 360 365 Arg Val Ile Leu Thr Gln Val Gly Ser Gly Pro Gln Glu Thr Asn Glu     370 375 380 Ser Leu Ile Asp Ala Lys Thr Gly Leu Pro Lys Glu 385 390 395 <210> 11 <211> 3165 <212> DNA <213> Saccharomyces cerevisiae <220> <221> CDS <222> (1) .. (3162) <400> 11 atg ccg ccg cta ttc aag gga ctg aaa cag atg gca aag cca att gcc 48 Met Pro Pro Leu Phe Lys Gly Leu Lys Gln Met Ala Lys Pro Ile Ala   1 5 10 15 tat gtt tca aga ttt tcg gcg aaa cga cca att cat ata ata ctt ttt 96 Tyr Val Ser Arg Phe Ser Ala Lys Arg Pro Ile His Ile Ile Leu Phe              20 25 30 tct cta atc ata tcc gca ttc gct tat cta tcc gtc att cag tat tac 144 Ser Leu Ile Ile Ser Ala Phe Ala Tyr Leu Ser Val Ile Gln Tyr Tyr          35 40 45 ttc aat ggt tgg caa cta gat tca aat agt gtt ttt gaa act gct cca 192 Phe Asn Gly Trp Gln Leu Asp Ser Asn Ser Val Phe Glu Thr Ala Pro      50 55 60 aat aaa gac ttc aac act cta ttt caa gaa tgt tcc cat tac tac aga 240 Asn Lys Asp Phe Asn Thr Leu Phe Gln Glu Cys Ser His Tyr Tyr Arg  65 70 75 80 gat tcc tct cta gat ggt tgg gta tca atc acc gcg cat gaa gct agt 288 Asp Ser Ser Leu Asp Gly Trp Val Ser Ile Thr Ala His Glu Ala Ser                  85 90 95 gag tta cca gcc cca cac cat tac tat cta tta aac ctg aac ttc aat 336 Glu Leu Pro Ala Pro His His Tyr Tyr Leu Leu Asn Leu Asn Phe Asn             100 105 110 agt cct aat gaa act gac tcc att cca gaa cta gct aac acg gtt ttt 384 Ser Pro Asn Glu Thr Asp Ser Ile Pro Glu Leu Ala Asn Thr Val Phe         115 120 125 gag aaa gat aat aca aaa tat att ctg caa gaa gat ctc agc gtt tcc 432 Glu Lys Asp Asn Thr Lys Tyr Ile Leu Gln Glu Asp Leu Ser Val Ser     130 135 140 aaa gaa att tct tct act gat gga acg aaa tgg agg tta aga agt gac 480 Lys Glu Ile Ser Ser Thr Asp Gly Thr Lys Trp Arg Leu Arg Ser Asp 145 150 155 160 aga aaa agt ctt ttc gac gta aag acg tta gca tat tct ctc tac gat 528 Arg Lys Ser Leu Phe Asp Val Lys Thr Leu Ala Tyr Ser Leu Tyr Asp                 165 170 175 gta ttt tca gaa aat gta acc caa gca gac ccg ttt gac gtc ctt att 576 Val Phe Ser Glu Asn Val Thr Gln Ala Asp Pro Phe Asp Val Leu Ile             180 185 190 atg gtt act gcc tac cta atg atg ttc tac acc ata ttc ggc ctc ttc 624 Met Val Thr Ala Tyr Leu Met Met Phe Tyr Thr Ile Phe Gly Leu Phe         195 200 205 aat gac atg agg aag acc ggg tca aat ttt tgg ttg agc gcc tct aca 672 Asn Asp Met Arg Lys Thr Gly Ser Asn Phe Trp Leu Ser Ala Ser Thr     210 215 220 gtg gtc aat tct gca tca tca ctt ttc tta gca ttg tat gtc acc caa 720 Val Val Asn Ser Ala Ser Ser Leu Phe Leu Ala Leu Tyr Val Thr Gln 225 230 235 240 tgt att cta ggc aaa gaa gtt tcc gca tta act ctt ttt gaa ggt ttg 768 Cys Ile Leu Gly Lys Glu Val Ser Ala Leu Thr Leu Phe Glu Gly Leu                 245 250 255 cct ttc att gta gtt gtt gtt ggt ttc aag cac aaa atc aag att gcc 816 Pro Phe Ile Val Val Val Val Gly Phe Lys His Lys Ile Lys Ile Ala             260 265 270 cag tat gcc ctg gag aaa ttt gaa aga gtc ggt tta tct aaa agg att 864 Gln Tyr Ala Leu Glu Lys Phe Glu Arg Val Gly Leu Ser Lys Arg Ile         275 280 285 act acc gat gaa atc gtt ttt gaa tcc gtg agc gaa gag ggt ggt cgt 912 Thr Thr Asp Glu Ile Val Phe Glu Ser Val Ser Glu Glu Gly Gly Arg     290 295 300 ttg att caa gac cat ttg ctt tgt att ttt gcc ttt atc gga tgc tct 960 Leu Ile Gln Asp His Leu Leu Cys Ile Phe Ala Phe Ile Gly Cys Ser 305 310 315 320 atg tat gct cac caa ttg aag act ttg aca aac ttc tgc ata tta tca 1008 Met Tyr Ala His Gln Leu Lys Thr Leu Thr Asn Phe Cys Ile Leu Ser                 325 330 335 gca ttt atc cta att ttc gaa ttg att tta act cct aca ttt tat tct 1056 Ala Phe Ile Leu Ile Phe Glu Leu Ile Leu Thr Pro Thr Phe Tyr Ser             340 345 350 gct atc tta gcg ctt aga ctg gaa atg aat gtt atc cac aga tct act 1104 Ala Ile Leu Ala Leu Arg Leu Glu Met Asn Val Ile His Arg Ser Thr         355 360 365 att atc aag caa aca tta gaa gaa gac ggt gtt gtt cca tct aca gca 1152 Ile Ile Lys Gln Thr Leu Glu Glu Asp Gly Val Val Pro Ser Thr Ala     370 375 380 aga atc att tct aag gca gaa aag aaa tcc gta tct tct ttc tta aat 1200 Arg Ile Ile Ser Lys Ala Glu Lys Lys Ser Val Ser Ser Phe Leu Asn 385 390 395 400 ctc agt gtg gtt gtc att atc atg aaa ctc tct gtc ata ctg ttg ttc 1248 Leu Ser Val Val Val Ile Ile Met Lys Leu Ser Val Ile Leu Leu Phe                 405 410 415 gtc ttc atc aac ttt tat aac ttt ggt gca aat tgg gtc aat gat gcc 1296 Val Phe Ile Asn Phe Tyr Asn Phe Gly Ala Asn Trp Val Asn Asp Ala             420 425 430 ttc aat tca ttg tac ttc gat aag gaa cgt gtt tct cta cca gat ttt 1344 Phe Asn Ser Leu Tyr Phe Asp Lys Glu Arg Val Ser Leu Pro Asp Phe         435 440 445 att acc tcg aat gcc tct gaa aac ttt aaa gag caa gct att gtt agt 1392 Ile Thr Ser Asn Ala Ser Glu Asn Phe Lys Glu Gln Ala Ile Val Ser     450 455 460 gtc acc cca tta tta tat tac aaa ccc att aag tcc tac caa cgc att 1440 Val Thr Pro Leu Leu Tyr Tyr Lys Pro Ile Lys Ser Tyr Gln Arg Ile 465 470 475 480 gag gat atg gtt ctt cta ttg ctt cgt aat gtc agt gtt gcc att cgt 1488 Glu Asp Met Val Leu Leu Leu Leu Arg Asn Val Ser Val Ala Ile Arg                 485 490 495 gat agg ttc gtc agt aaa tta gtt ctt tcc gcc tta gta tgc agt gct 1536 Asp Arg Phe Val Ser Lys Leu Val Leu Ser Ala Leu Val Cys Ser Ala             500 505 510 gtc atc aat gtg tat tta tta aat gct gct aga att cat acc agt tat 1584 Val Ile Asn Val Tyr Leu Leu Asn Ala Ala Arg Ile His Thr Ser Tyr         515 520 525 act gca gac caa ttg gtg aag act gaa gtc acc aag aag tct ttt act 1632 Thr Ala Asp Gln Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe Thr     530 535 540 gct cct gta caa aag gct tct aca cca gtt tta acc aat aaa aca gtc 1680 Ala Pro Val Gln Lys Ala Ser Thr Pro Val Leu Thr Asn Lys Thr Val 545 550 555 560 att tct gga tcg aaa gtc aaa agt tta tca tct gcg caa tcg agc tca 1728 Ile Ser Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gln Ser Ser Ser                 565 570 575 tca gga cct tca tca tct agt gag gaa gat gat tcc cgc gat att gaa 1776 Ser Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu             580 585 590 agc ttg gat aag aaa ata cgt cct tta gaa gaa tta gaa gca tca tta 1824 Ser Leu Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Ser Leu         595 600 605 agt agt gga aat aca aaa caa ttg aag aac aaa gag gtc gct gcc ttg 1872 Ser Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala Ala Leu     610 615 620 gtt att cac ggt aag tta cct ttg tac gct ttg gag aaa aaa tta ggt 1920 Val Ile His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu Gly 625 630 635 640 gat act acg aga gcg gtt gcg gta cgt agg aag gct ctt tca att ttg 1968 Asp Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser Ile Leu                 645 650 655 gca gaa gct cct gta tta gca tct gat cgt tta cca tat aaa aat tat 2016 Ala Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys Asn Tyr             660 665 670 gac tac gac cgc gta ttt ggc gct tgt tgt gaa aat gtt ata ggt tac 2064 Asp Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr         675 680 685 atg cct ttg ccc gtt ggt gtt ata ggc ccc ttg gtt atc gat ggt aca 2112 Met Pro Leu Pro Val Gly Val Ile Gly Pro Leu Val Ile Asp Gly Thr     690 695 700 tct tat cat ata cca atg gca act aca gag ggt tgt ttg gta gct tct 2160 Ser Tyr His Ile Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser 705 710 715 720 gcc atg cgt ggc tgt aag gca atc aat gct ggc ggt ggt gca aca act 2208 Ala Met Arg Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr                 725 730 735 gtt tta act aag gat ggt atg aca aga ggc cca gta gtc cgt ttc cca 2256 Val Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe Pro             740 745 750 act ttg aaa aga tct ggt gcc tgt aag ata tgg tta gac tca gaa gag 2304 Thr Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser Glu Glu         755 760 765 gga caa aac gca att aaa aaa gct ttt aac tct aca tca aga ttt gca 2352 Gly Gln Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala     770 775 780 cgt ctg caa cat att caa act tgt cta gca gga gat tta ctc ttc atg 2400 Arg Leu Gln His Ile Gln Thr Cys Leu Ala Gly Asp Leu Leu Phe Met 785 790 795 800 aga ttt aga aca act act ggt gac gca atg ggt atg aat atg att tct 2448 Arg Phe Arg Thr Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser                 805 810 815 aag ggt gtc gaa tac tca tta aag caa atg gta gaa gag tat ggc tgg 2496 Lys Gly Val Glu Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp             820 825 830 gaa gat atg gag gtt gtc tcc gtt tct ggt aac tac tgt acc gac aaa 2544 Glu Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys         835 840 845 aaa cca gct gcc atc aac tgg atc gaa ggt cgt ggt aag agt gtc gtc 2592 Lys Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val     850 855 860 gca gaa gct act att cct ggt gat gtt gtc aga aaa gtg tta aaa agt 2640 Ala Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu Lys Ser 865 870 875 880 gat gtt tcc gca ttg gtt gag ttg aac att gct aag aat ttg gtt gga 2688 Asp Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn Leu Val Gly                 885 890 895 tct gca atg gct ggg tct gtt ggt gga ttt aac gca cgt gca gct aat 2736 Ser Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala Arg Ala Ala Asn             900 905 910 tta gtg aca gct gtt ttc ttg gca tta gga caa gat cct gca caa aat 2784 Leu Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro Ala Gln Asn         915 920 925 gtc gaa agt tcc aac tgt ata aca ttg atg aaa gaa gtg gac ggt gat 2832 Val Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp     930 935 940 ttg aga att tcc gta tcc atg cca tcc atc gaa gta ggt acc atc ggt 2880 Leu Arg Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly 945 950 955 960 ggt ggt act gtt cta gaa cca caa ggt gcc atg ttg gac tta tta ggt 2928 Gly Gly Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Gly                 965 970 975 gta aga ggc cca cat gct acc gct cct ggt acc aac gca cgt caa tta 2976 Val Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gln Leu             980 985 990 gca aga ata gtt gcc tgt gcc gtc ttg gca ggt gaa tta tcc tta tgt 3024 Ala Arg Ile Val Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu Cys         995 1000 1005 gct gcc cta gca gcc ggc cat ttg gtt caa agt cat atg acc cac aac 3072 Ala Ala Leu Ala Ala Gly His Leu Val Gln Ser His Met Thr His Asn    1010 1015 1020 agg aaa cct gct gaa cca aca aaa cct aac aat ttg gac gcc act gat 3120 Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala Thr Asp 1025 1030 1035 1040 ata aat cgt ttg aaa gat ggg tcc gtc acc tgc att aaa tcc taa 3165 Ile Asn Arg Leu Lys Asp Gly Ser Val Thr Cys Ile Lys Ser                1045 1050 <210> 12 <211> 1054 <212> PRT <213> Saccharomyces cerevisiae <400> 12 Met Pro Pro Leu Phe Lys Gly Leu Lys Gln Met Ala Lys Pro Ile Ala   1 5 10 15 Tyr Val Ser Arg Phe Ser Ala Lys Arg Pro Ile His Ile Ile Leu Phe              20 25 30 Ser Leu Ile Ile Ser Ala Phe Ala Tyr Leu Ser Val Ile Gln Tyr Tyr          35 40 45 Phe Asn Gly Trp Gln Leu Asp Ser Asn Ser Val Phe Glu Thr Ala Pro      50 55 60 Asn Lys Asp Phe Asn Thr Leu Phe Gln Glu Cys Ser His Tyr Tyr Arg  65 70 75 80 Asp Ser Ser Leu Asp Gly Trp Val Ser Ile Thr Ala His Glu Ala Ser                  85 90 95 Glu Leu Pro Ala Pro His His Tyr Tyr Leu Leu Asn Leu Asn Phe Asn             100 105 110 Ser Pro Asn Glu Thr Asp Ser Ile Pro Glu Leu Ala Asn Thr Val Phe         115 120 125 Glu Lys Asp Asn Thr Lys Tyr Ile Leu Gln Glu Asp Leu Ser Val Ser     130 135 140 Lys Glu Ile Ser Ser Thr Asp Gly Thr Lys Trp Arg Leu Arg Ser Asp 145 150 155 160 Arg Lys Ser Leu Phe Asp Val Lys Thr Leu Ala Tyr Ser Leu Tyr Asp                 165 170 175 Val Phe Ser Glu Asn Val Thr Gln Ala Asp Pro Phe Asp Val Leu Ile             180 185 190 Met Val Thr Ala Tyr Leu Met Met Phe Tyr Thr Ile Phe Gly Leu Phe         195 200 205 Asn Asp Met Arg Lys Thr Gly Ser Asn Phe Trp Leu Ser Ala Ser Thr     210 215 220 Val Val Asn Ser Ala Ser Ser Leu Phe Leu Ala Leu Tyr Val Thr Gln 225 230 235 240 Cys Ile Leu Gly Lys Glu Val Ser Ala Leu Thr Leu Phe Glu Gly Leu                 245 250 255 Pro Phe Ile Val Val Val Val Gly Phe Lys His Lys Ile Lys Ile Ala             260 265 270 Gln Tyr Ala Leu Glu Lys Phe Glu Arg Val Gly Leu Ser Lys Arg Ile         275 280 285 Thr Thr Asp Glu Ile Val Phe Glu Ser Val Ser Glu Glu Gly Gly Arg     290 295 300 Leu Ile Gln Asp His Leu Leu Cys Ile Phe Ala Phe Ile Gly Cys Ser 305 310 315 320 Met Tyr Ala His Gln Leu Lys Thr Leu Thr Asn Phe Cys Ile Leu Ser                 325 330 335 Ala Phe Ile Leu Ile Phe Glu Leu Ile Leu Thr Pro Thr Phe Tyr Ser             340 345 350 Ala Ile Leu Ala Leu Arg Leu Glu Met Asn Val Ile His Arg Ser Thr         355 360 365 Ile Ile Lys Gln Thr Leu Glu Glu Asp Gly Val Val Pro Ser Thr Ala     370 375 380 Arg Ile Ile Ser Lys Ala Glu Lys Lys Ser Val Ser Ser Phe Leu Asn 385 390 395 400 Leu Ser Val Val Val Ile Ile Met Lys Leu Ser Val Ile Leu Leu Phe                 405 410 415 Val Phe Ile Asn Phe Tyr Asn Phe Gly Ala Asn Trp Val Asn Asp Ala             420 425 430 Phe Asn Ser Leu Tyr Phe Asp Lys Glu Arg Val Ser Leu Pro Asp Phe         435 440 445 Ile Thr Ser Asn Ala Ser Glu Asn Phe Lys Glu Gln Ala Ile Val Ser     450 455 460 Val Thr Pro Leu Leu Tyr Tyr Lys Pro Ile Lys Ser Tyr Gln Arg Ile 465 470 475 480 Glu Asp Met Val Leu Leu Leu Leu Arg Asn Val Ser Val Ala Ile Arg                 485 490 495 Asp Arg Phe Val Ser Lys Leu Val Leu Ser Ala Leu Val Cys Ser Ala             500 505 510 Val Ile Asn Val Tyr Leu Leu Asn Ala Ala Arg Ile His Thr Ser Tyr         515 520 525 Thr Ala Asp Gln Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe Thr     530 535 540 Ala Pro Val Gln Lys Ala Ser Thr Pro Val Leu Thr Asn Lys Thr Val 545 550 555 560 Ile Ser Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gln Ser Ser Ser                 565 570 575 Ser Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu             580 585 590 Ser Leu Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Ser Leu         595 600 605 Ser Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala Ala Leu     610 615 620 Val Ile His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu Gly 625 630 635 640 Asp Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser Ile Leu                 645 650 655 Ala Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys Asn Tyr             660 665 670 Asp Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr         675 680 685 Met Pro Leu Pro Val Gly Val Ile Gly Pro Leu Val Ile Asp Gly Thr     690 695 700 Ser Tyr His Ile Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser 705 710 715 720 Ala Met Arg Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr                 725 730 735 Val Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe Pro             740 745 750 Thr Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser Glu Glu         755 760 765 Gly Gln Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala     770 775 780 Arg Leu Gln His Ile Gln Thr Cys Leu Ala Gly Asp Leu Leu Phe Met 785 790 795 800 Arg Phe Arg Thr Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser                 805 810 815 Lys Gly Val Glu Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp             820 825 830 Glu Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys         835 840 845 Lys Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val     850 855 860 Ala Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu Lys Ser 865 870 875 880 Asp Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn Leu Val Gly                 885 890 895 Ser Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala Arg Ala Ala Asn             900 905 910 Leu Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro Ala Gln Asn         915 920 925 Val Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp     930 935 940 Leu Arg Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly 945 950 955 960 Gly Gly Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Gly                 965 970 975 Val Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gln Leu             980 985 990 Ala Arg Ile Val Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu Cys         995 1000 1005 Ala Ala Leu Ala Ala Gly His Leu Val Gln Ser His Met Thr His Asn    1010 1015 1020 Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala Thr Asp 1025 1030 1035 1040 Ile Asn Arg Leu Lys Asp Gly Ser Val Thr Cys Ile Lys Ser                1045 1050 <210> 13 <211> 3165 <212> DNA <213> Saccharomyces cerevisiae <220> <221> CDS <222> (1) .. (3162) <400> 13 atg ccg ccg cta ttc aag gga ctg aaa cag atg gca aag cca att gcc 48 Met Pro Pro Leu Phe Lys Gly Leu Lys Gln Met Ala Lys Pro Ile Ala   1 5 10 15 tat gtt tca aga ttt tcg gcg aaa cga cca att cat ata ata ctt ttt 96 Tyr Val Ser Arg Phe Ser Ala Lys Arg Pro Ile His Ile Ile Leu Phe              20 25 30 tct cta atc ata tcc gca ttc gct tat cta tcc gtc att cag tat tac 144 Ser Leu Ile Ile Ser Ala Phe Ala Tyr Leu Ser Val Ile Gln Tyr Tyr          35 40 45 ttc aat ggt tgg caa cta gat tca aat agt gtt ttt gaa act gct cca 192 Phe Asn Gly Trp Gln Leu Asp Ser Asn Ser Val Phe Glu Thr Ala Pro      50 55 60 aat aaa gac tcc aac act cta ttt caa gaa tgt tcc cat tac tac aga 240 Asn Lys Asp Ser Asn Thr Leu Phe Gln Glu Cys Ser His Tyr Tyr Arg  65 70 75 80 gat tcc tct cta gat ggt tgg gta tca atc acc gcg cat gaa gct agt 288 Asp Ser Ser Leu Asp Gly Trp Val Ser Ile Thr Ala His Glu Ala Ser                  85 90 95 gag tta cca gcc cca cac cat tac tat cta tta aac ctg aac ttc aat 336 Glu Leu Pro Ala Pro His His Tyr Tyr Leu Leu Asn Leu Asn Phe Asn             100 105 110 agt cct aat gaa act gac tcc att cca gaa cta gct aac acg gtt ttt 384 Ser Pro Asn Glu Thr Asp Ser Ile Pro Glu Leu Ala Asn Thr Val Phe         115 120 125 gag aaa gat aat aca aaa tat att ctg caa gaa gat ctc agc gtt tcc 432 Glu Lys Asp Asn Thr Lys Tyr Ile Leu Gln Glu Asp Leu Ser Val Ser     130 135 140 aaa gaa att tct tct act gat gga acg aaa tgg agg tta aga agt gac 480 Lys Glu Ile Ser Ser Thr Asp Gly Thr Lys Trp Arg Leu Arg Ser Asp 145 150 155 160 aga aaa agt ctt ttc gac gta aag acg tta gca tat tct ctc tac gat 528 Arg Lys Ser Leu Phe Asp Val Lys Thr Leu Ala Tyr Ser Leu Tyr Asp                 165 170 175 gta ttt tca gaa aat gta acc caa gca gac ccg ttt gac gtc ctt att 576 Val Phe Ser Glu Asn Val Thr Gln Ala Asp Pro Phe Asp Val Leu Ile             180 185 190 atg gtt act gcc tac cta atg atg ttc tac acc ata ttc ggc ctc ttc 624 Met Val Thr Ala Tyr Leu Met Met Phe Tyr Thr Ile Phe Gly Leu Phe         195 200 205 aat gac atg agg aag acc ggg tca aat ttt tgg ttg agc gcc tct aca 672 Asn Asp Met Arg Lys Thr Gly Ser Asn Phe Trp Leu Ser Ala Ser Thr     210 215 220 gtg gtc aat tct gca tca tca ctt ttc tta gca ttg tat gtc acc caa 720 Val Val Asn Ser Ala Ser Ser Leu Phe Leu Ala Leu Tyr Val Thr Gln 225 230 235 240 tgt att cta ggc aaa gaa gtt tcc gca tta act ctt ttt gaa ggt ttg 768 Cys Ile Leu Gly Lys Glu Val Ser Ala Leu Thr Leu Phe Glu Gly Leu                 245 250 255 cct ttc att gta gtt gtt gtt ggt ttc aag cac aaa atc aag att gcc 816 Pro Phe Ile Val Val Val Val Gly Phe Lys His Lys Ile Lys Ile Ala             260 265 270 cag tat gcc ctg gag aaa ttt gaa aga gtc ggt tta tct aaa agg att 864 Gln Tyr Ala Leu Glu Lys Phe Glu Arg Val Gly Leu Ser Lys Arg Ile         275 280 285 act acc gat gaa atc gtt ttt gaa tcc gtg agc gaa gag ggt ggt cgt 912 Thr Thr Asp Glu Ile Val Phe Glu Ser Val Ser Glu Glu Gly Gly Arg     290 295 300 ttg att caa gac cat ttg ctt tgt att ttt gcc ttt atc gga tgc tct 960 Leu Ile Gln Asp His Leu Leu Cys Ile Phe Ala Phe Ile Gly Cys Ser 305 310 315 320 atg tat gct cac caa ttg aag act ttg aca aac ttc tgc ata tta tca 1008 Met Tyr Ala His Gln Leu Lys Thr Leu Thr Asn Phe Cys Ile Leu Ser                 325 330 335 gca ttt atc cta att ttc gaa ttg att tta act cct aca ttt tat tct 1056 Ala Phe Ile Leu Ile Phe Glu Leu Ile Leu Thr Pro Thr Phe Tyr Ser             340 345 350 gct atc tta gcg ctt aga ctg gaa atg aat gtt atc cac aga tct act 1104 Ala Ile Leu Ala Leu Arg Leu Glu Met Asn Val Ile His Arg Ser Thr         355 360 365 att atc aag caa aca tta gaa gaa gac ggt gtt gtt cca tct aca gca 1152 Ile Ile Lys Gln Thr Leu Glu Glu Asp Gly Val Val Pro Ser Thr Ala     370 375 380 aga atc att tct aag gca gaa aag aaa tcc gta tct tct ttc tta aat 1200 Arg Ile Ile Ser Lys Ala Glu Lys Lys Ser Val Ser Ser Phe Leu Asn 385 390 395 400 ctc agt gtg gtt gtc att atc atg aaa ctc tct gtc ata ctg ttg ttc 1248 Leu Ser Val Val Val Ile Ile Met Lys Leu Ser Val Ile Leu Leu Phe                 405 410 415 gtc ttc atc aac ttt tat aac ttt ggt gca aat tgg gtc aat gat gcc 1296 Val Phe Ile Asn Phe Tyr Asn Phe Gly Ala Asn Trp Val Asn Asp Ala             420 425 430 ttc aat tca ttg tac ttc gat aag gaa cgt gtt tct cta cca gat ttt 1344 Phe Asn Ser Leu Tyr Phe Asp Lys Glu Arg Val Ser Leu Pro Asp Phe         435 440 445 att acc tcg aat gcc tct gaa aac ttt aaa gag caa gct att gtt agt 1392 Ile Thr Ser Asn Ala Ser Glu Asn Phe Lys Glu Gln Ala Ile Val Ser     450 455 460 gtc acc cca tta tta tat tac aaa ccc att aag tcc tac caa cgc att 1440 Val Thr Pro Leu Leu Tyr Tyr Lys Pro Ile Lys Ser Tyr Gln Arg Ile 465 470 475 480 gag gat atg gtt ctt cta ttg ctt cgt aat gtc agt gtt gcc att cgt 1488 Glu Asp Met Val Leu Leu Leu Leu Arg Asn Val Ser Val Ala Ile Arg                 485 490 495 gat agg ttc gtc agt aaa tta gtt ctt tcc gcc tta gta tgc agt gct 1536 Asp Arg Phe Val Ser Lys Leu Val Leu Ser Ala Leu Val Cys Ser Ala             500 505 510 gtc atc aat gtg tat tta tta aat gct gct aga att cat acc agt tat 1584 Val Ile Asn Val Tyr Leu Leu Asn Ala Ala Arg Ile His Thr Ser Tyr         515 520 525 act gca gac caa ttg gtg aag act gaa gtc acc aag aag tct ttt act 1632 Thr Ala Asp Gln Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe Thr     530 535 540 gct cct gta caa aag gct tct aca cca gtt tta acc aat aaa aca gtc 1680 Ala Pro Val Gln Lys Ala Ser Thr Pro Val Leu Thr Asn Lys Thr Val 545 550 555 560 att tct gga tcg aaa gtc aaa agt tta tca tct gcg caa tcg agc tca 1728 Ile Ser Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gln Ser Ser Ser                 565 570 575 tca gga cct tca tca tct agt gag gaa gat gat tcc cgc gat att gaa 1776 Ser Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu             580 585 590 agc ttg gat aag aaa ata cgt cct tta gaa gaa tta gaa gca tta tta 1824 Ser Leu Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Leu Leu         595 600 605 agt agt gga aat aca aaa caa ttg aag aac aaa gag gtc gct gcc ttg 1872 Ser Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala Ala Leu     610 615 620 gtt att cac ggt aag tta cct ttg tac gct ttg gag aaa aaa tta ggt 1920 Val Ile His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu Gly 625 630 635 640 gat act acg aga gcg gtt gcg gta cgt agg aag gct ctt tca att ttg 1968 Asp Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser Ile Leu                 645 650 655 gca gaa gct cct gta tta gca tct gat cgt tta cca tat aaa aat tat 2016 Ala Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys Asn Tyr             660 665 670 gac tac gac cgc gta ttt ggc gct tgt tgt gaa aat gtt ata ggt tac 2064 Asp Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr         675 680 685 atg cct ttg ccc gtt ggt gtt ata ggc ccc ttg gtt atc gat ggt aca 2112 Met Pro Leu Pro Val Gly Val Ile Gly Pro Leu Val Ile Asp Gly Thr     690 695 700 tct tat cat ata cca atg gca act aca gag ggt tgt ttg gta gct tct 2160 Ser Tyr His Ile Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser 705 710 715 720 gcc atg cgt ggc tgt aag gca atc aat gct ggc ggt ggt gca aca act 2208 Ala Met Arg Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr                 725 730 735 gtt tta act aag gat ggt atg aca aga ggc cca gta gtc cgt ttc cca 2256 Val Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe Pro             740 745 750 act ttg aaa aga tct ggt gcc tgt aag ata tgg tta gac tca gaa gag 2304 Thr Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser Glu Glu         755 760 765 gga caa aac gca att aaa aaa gct ttt aac tct aca tca aga ttt gca 2352 Gly Gln Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala     770 775 780 cgt ctg caa cat att caa act tgt cta gca gga gat tta ctc ttc atg 2400 Arg Leu Gln His Ile Gln Thr Cys Leu Ala Gly Asp Leu Leu Phe Met 785 790 795 800 aga ttt aga aca act act ggt gac gca atg ggt atg aat atg att tct 2448 Arg Phe Arg Thr Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser                 805 810 815 aag ggt gtc gaa tac tca tta aag caa atg gta gaa gag tat ggc tgg 2496 Lys Gly Val Glu Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp             820 825 830 gaa gat atg gag gtt gtc tcc gtt tct ggt aac tac tgt acc gac aaa 2544 Glu Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys         835 840 845 aaa cca gct gcc atc aac tgg atc gaa ggt cgt ggt aag agt gtc gtc 2592 Lys Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val     850 855 860 gca gaa gct act att cct ggt gat gtt gtc aga aaa gtg tta aaa agt 2640 Ala Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu Lys Ser 865 870 875 880 gat gtt tcc gca ttg gtt gag ttg aac att gct aag aat ttg gtt gga 2688 Asp Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn Leu Val Gly                 885 890 895 tct gca atg gct ggg tct gtt ggt gga ttt aac gca cat gca gct aat 2736 Ser Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala His Ala Ala Asn             900 905 910 tta gtg aca gct gtt ttc ttg gca tta gga caa gat cct gca caa aat 2784 Leu Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro Ala Gln Asn         915 920 925 gtc gaa agt tcc aac tgt ata aca ttg atg aaa gaa gtg gac ggt gat 2832 Val Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp     930 935 940 ttg aga att tcc gta tcc atg cca tcc atc gaa gta ggt acc atc ggt 2880 Leu Arg Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly 945 950 955 960 ggt ggt act gtt cta gaa cca caa ggt gcc atg ttg gac tta tta ggt 2928 Gly Gly Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Gly                 965 970 975 gta aga ggc cca cat gct acc gct cct ggt acc aac gca cgt caa tta 2976 Val Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gln Leu             980 985 990 gca aga ata gtt gcc tgt gcc gtc ttg gca ggt gaa tta tcc tta tgt 3024 Ala Arg Ile Val Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu Cys         995 1000 1005 gct gcc cta gca gcc ggc cat ttg gtt caa agt cat atg acc cac aac 3072 Ala Ala Leu Ala Ala Gly His Leu Val Gln Ser His Met Thr His Asn    1010 1015 1020 agg aaa cct gct gaa cca aca aaa cct aac aat ttg gac gcc act gat 3120 Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala Thr Asp 1025 1030 1035 1040 ata aat cgt ttg aaa gat ggg tcc gtc acc tgc att aaa tcc taa 3165 Ile Asn Arg Leu Lys Asp Gly Ser Val Thr Cys Ile Lys Ser                1045 1050 <210> 14 <211> 1054 <212> PRT <213> Saccharomyces cerevisiae <400> 14 Met Pro Pro Leu Phe Lys Gly Leu Lys Gln Met Ala Lys Pro Ile Ala   1 5 10 15 Tyr Val Ser Arg Phe Ser Ala Lys Arg Pro Ile His Ile Ile Leu Phe              20 25 30 Ser Leu Ile Ile Ser Ala Phe Ala Tyr Leu Ser Val Ile Gln Tyr Tyr          35 40 45 Phe Asn Gly Trp Gln Leu Asp Ser Asn Ser Val Phe Glu Thr Ala Pro      50 55 60 Asn Lys Asp Ser Asn Thr Leu Phe Gln Glu Cys Ser His Tyr Tyr Arg  65 70 75 80 Asp Ser Ser Leu Asp Gly Trp Val Ser Ile Thr Ala His Glu Ala Ser                  85 90 95 Glu Leu Pro Ala Pro His His Tyr Tyr Leu Leu Asn Leu Asn Phe Asn             100 105 110 Ser Pro Asn Glu Thr Asp Ser Ile Pro Glu Leu Ala Asn Thr Val Phe         115 120 125 Glu Lys Asp Asn Thr Lys Tyr Ile Leu Gln Glu Asp Leu Ser Val Ser     130 135 140 Lys Glu Ile Ser Ser Thr Asp Gly Thr Lys Trp Arg Leu Arg Ser Asp 145 150 155 160 Arg Lys Ser Leu Phe Asp Val Lys Thr Leu Ala Tyr Ser Leu Tyr Asp                 165 170 175 Val Phe Ser Glu Asn Val Thr Gln Ala Asp Pro Phe Asp Val Leu Ile             180 185 190 Met Val Thr Ala Tyr Leu Met Met Phe Tyr Thr Ile Phe Gly Leu Phe         195 200 205 Asn Asp Met Arg Lys Thr Gly Ser Asn Phe Trp Leu Ser Ala Ser Thr     210 215 220 Val Val Asn Ser Ala Ser Ser Leu Phe Leu Ala Leu Tyr Val Thr Gln 225 230 235 240 Cys Ile Leu Gly Lys Glu Val Ser Ala Leu Thr Leu Phe Glu Gly Leu                 245 250 255 Pro Phe Ile Val Val Val Val Gly Phe Lys His Lys Ile Lys Ile Ala             260 265 270 Gln Tyr Ala Leu Glu Lys Phe Glu Arg Val Gly Leu Ser Lys Arg Ile         275 280 285 Thr Thr Asp Glu Ile Val Phe Glu Ser Val Ser Glu Glu Gly Gly Arg     290 295 300 Leu Ile Gln Asp His Leu Leu Cys Ile Phe Ala Phe Ile Gly Cys Ser 305 310 315 320 Met Tyr Ala His Gln Leu Lys Thr Leu Thr Asn Phe Cys Ile Leu Ser                 325 330 335 Ala Phe Ile Leu Ile Phe Glu Leu Ile Leu Thr Pro Thr Phe Tyr Ser             340 345 350 Ala Ile Leu Ala Leu Arg Leu Glu Met Asn Val Ile His Arg Ser Thr         355 360 365 Ile Ile Lys Gln Thr Leu Glu Glu Asp Gly Val Val Pro Ser Thr Ala     370 375 380 Arg Ile Ile Ser Lys Ala Glu Lys Lys Ser Val Ser Ser Phe Leu Asn 385 390 395 400 Leu Ser Val Val Val Ile Ile Met Lys Leu Ser Val Ile Leu Leu Phe                 405 410 415 Val Phe Ile Asn Phe Tyr Asn Phe Gly Ala Asn Trp Val Asn Asp Ala             420 425 430 Phe Asn Ser Leu Tyr Phe Asp Lys Glu Arg Val Ser Leu Pro Asp Phe         435 440 445 Ile Thr Ser Asn Ala Ser Glu Asn Phe Lys Glu Gln Ala Ile Val Ser     450 455 460 Val Thr Pro Leu Leu Tyr Tyr Lys Pro Ile Lys Ser Tyr Gln Arg Ile 465 470 475 480 Glu Asp Met Val Leu Leu Leu Leu Arg Asn Val Ser Val Ala Ile Arg                 485 490 495 Asp Arg Phe Val Ser Lys Leu Val Leu Ser Ala Leu Val Cys Ser Ala             500 505 510 Val Ile Asn Val Tyr Leu Leu Asn Ala Ala Arg Ile His Thr Ser Tyr         515 520 525 Thr Ala Asp Gln Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe Thr     530 535 540 Ala Pro Val Gln Lys Ala Ser Thr Pro Val Leu Thr Asn Lys Thr Val 545 550 555 560 Ile Ser Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gln Ser Ser Ser                 565 570 575 Ser Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu             580 585 590 Ser Leu Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Leu Leu         595 600 605 Ser Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala Ala Leu     610 615 620 Val Ile His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu Gly 625 630 635 640 Asp Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser Ile Leu                 645 650 655 Ala Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys Asn Tyr             660 665 670 Asp Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr         675 680 685 Met Pro Leu Pro Val Gly Val Ile Gly Pro Leu Val Ile Asp Gly Thr     690 695 700 Ser Tyr His Ile Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser 705 710 715 720 Ala Met Arg Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr                 725 730 735 Val Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe Pro             740 745 750 Thr Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser Glu Glu         755 760 765 Gly Gln Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala     770 775 780 Arg Leu Gln His Ile Gln Thr Cys Leu Ala Gly Asp Leu Leu Phe Met 785 790 795 800 Arg Phe Arg Thr Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser                 805 810 815 Lys Gly Val Glu Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp             820 825 830 Glu Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys         835 840 845 Lys Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val     850 855 860 Ala Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu Lys Ser 865 870 875 880 Asp Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn Leu Val Gly                 885 890 895 Ser Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala His Ala Ala Asn             900 905 910 Leu Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro Ala Gln Asn         915 920 925 Val Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp     930 935 940 Leu Arg Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly 945 950 955 960 Gly Gly Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Gly                 965 970 975 Val Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gln Leu             980 985 990 Ala Arg Ile Val Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu Cys         995 1000 1005 Ala Ala Leu Ala Ala Gly His Leu Val Gln Ser His Met Thr His Asn    1010 1015 1020 Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala Thr Asp 1025 1030 1035 1040 Ile Asn Arg Leu Lys Asp Gly Ser Val Thr Cys Ile Lys Ser                1045 1050 <210> 15 <211> 2925 <212> DNA <213> Saccharomyces cerevisiae <400> 15 atgccgccgc tattcaaggg actgaaacag atggcaaagc caattgccta tgtttcaaga 60 ttttcggcga aacgaccaat tcatataata cttttttctc taatcatatc cgcattcgct 120 tatctatccg tcattcagta ttacttcaat ggttggcaac tagattcaaa tagtgttttt 180 gaaactgctc caaataaaga cttcaacact ctatttcaag aatgttccca ttactacaga 240 gattcctctc tagatggttg ggtatcaatc accgcgcatg aagctagtga gttaccagcc 300 ccacaccatt actatctatt aaacctgaac ttcaatagtc ctaatgaaac tgactccatt 360 ccagaactag ctaacacggt ttttgagaaa gataatacaa aatatattct gcaagaagat 420 ctcagcgttt ccaaagaaat ttcttctact gatggaacga aatggaggtt aagaagtgac 480 agaaaaagtc ttttcgacgt aaagacgtta gcatattctc tctacgatgt attttcagaa 540 aatgtaaccc aagcagacca caaaatcaag attgcccagt atgccctgga gaaatttgaa 600 agagtcggtt tatctaaaag gattactacc gatgaaatcg tttttgaatc cgtgagcgaa 660 gagggtggtc gtttgattca agaccatttg ctttgtattt ttgcctttat cggatgctct 720 atgtatgctc accaattgaa gactttgaca aacttctgca tattatcagc atttatccta 780 attttcgaat tgattttaac tcctacattt tattctgcta tcttagcgct tagactggaa 840 atgaatgtta tccacagatc tactattatc aagcaaacat tagaagaaga cggtgttgtt 900 ccatctacag caagaatcat ttctaaggca gaaaagaaat ccgtatcttc tttcttaaat 960 ctcagtgtgg ttgtcattat catgaaactc tctgtcatac tgttgttcgt cttcatcaac 1020 ttttataact ttggtgcaaa ttgggtcaat gatgccttca attcattgta cttcgataag 1080 gaacgtgttt ctctaccaga ttttattacc tcgaatgcct ctgaaaactt taaagagcaa 1140 gctattgtta gtgtcacccc attattatat tacaaaccca ttaagtccta ccaacgcatt 1200 gaggatatgg ttcttctatt gcttcgtaat gtcagtgttg ccattcgtga taggttcgtc 1260 agtaaattag ttctttccgc cttagtatgc agtgctgtca tcaatgtgta tttattaaat 1320 gctgctagaa ttcataccag ttatactgca gaccaattgg tgaagactga agtcaccaag 1380 aagtctttta ctgctcctgt acaaaaggct tctacaccag ttttaaccaa taaaacagtc 1440 atttctggat cgaaagtcaa aagtttatca tctgcgcaat cgagctcatc aggaccttca 1500 tcatctagtg aggaagatga ttcccgcgat attgaaagct tggataagaa aatacgtcct 1560 ttagaagaat tagaagcatc attaagtagt ggaaatacaa aacaattgaa gaacaaagag 1620 gtcgctgcct tggttattca cggtaagtta cctttgtacg ctttggagaa aaaattaggt 1680 gatactacga gagcggttgc ggtacgtagg aaggctcttt caattttggc agaagctcct 1740 gtattagcat ctgatcgttt accatataaa aattatgact acgaccgcgt atttggcgct 1800 tgttgtgaaa atgttatagg ttacatgcct ttgcccgttg gtgttatagg ccccttggtt 1860 atcgatggta catcttatca tataccaatg gcaactacag agggttgttt ggtagcttct 1920 gccatgcgtg gctgtaaggc aatcaatgct ggcggtggtg caacaactgt tttaactaag 1980 gatggtatga caagaggccc agtagtccgt ttcccaactt tgaaaagatc tggtgcctgt 2040 aagatatggt tagactcaga agagggacaa aacgcaatta aaaaagcttt taactctaca 2100 tcaagatttg cacgtctgca acatattcaa acttgtctag caggagattt actcttcatg 2160 agatttagaa caactactgg tgacgcaatg ggtatgaata tgatttctaa gggtgtcgaa 2220 tactcattaa agcaaatggt agaagagtat ggctgggaag atatggaggt tgtctccgtt 2280 tctggtaact actgtaccga caaaaaacca gctgccatca actggatcga aggtcgtggt 2340 aagagtgtcg tcgcagaagc tactattcct ggtgatgttg tcagaaaagt gttaaaaagt 2400 gatgtttccg cattggttga gttgaacatt gctaagaatt tggttggatc tgcaatggct 2460 gggtctgttg gtggatttaa cgcacgtgca gctaatttag tgacagctgt tttcttggca 2520 ttaggacaag atcctgcaca aaatgtcgaa agttccaact gtataacatt gatgaaagaa 2580 gtggacggtg atttgagaat ttccgtatcc atgccatcca tcgaagtagg taccatcggt 2640 ggtggtactg ttctagaacc acaaggtgcc atgttggact tattaggtgt aagaggccca 2700 catgctaccg ctcctggtac caacgcacgt caattagcaa gaatagttgc ctgtgccgtc 2760 ttggcaggtg aattatcctt atgtgctgcc ctagcagccg gccatttggt tcaaagtcat 2820 atgacccaca acaggaaacc tgctgaacca acaaaaccta acaatttgga cgccactgat 2880 ataaatcgtt tgaaagatgg gtccgtcacc tgcattaaat cctaa 2925 <210> 16 <211> 3090 <212> DNA <213> Saccharomyces cerevisiae <400> 16 atgccgccgc tattcaaggg actgaaacag atggcaaagc caattgccta tgtttcaaga 60 ttttcggcga aacgaccaat tcatataata cttttttctc taatcatatc cgcattcgct 120 tatctatccg tcattcagta ttacttcaat ggttggcaac tagattcaaa tagtgttttt 180 gaaactgctc caaataaaga cttcaacact ctatttcaag aatgttccca ttactacaga 240 gattcctctc tagatggttg ggtatcaatc accgcgcatg aagctagtga gttaccagcc 300 ccacaccatt actatctatt aaacctgaac ttcaatagtc ctaatgaaac tgactccatt 360 ccagaactag ctaacacggt ttttgagaaa gataatacaa aatatattct gcaagaagat 420 ctcagcgttt ccaaagaaat ttcttctact gatggaacga aatggaggtt aagaagtgac 480 agaaaaagtc ttttcgacgt aaagacgtta gcatattctc tctacgatgt attttcagaa 540 aatgtaaccc aagcagaccc gtttgacgtc cttattatgg ttactgccta cctaatgatg 600 ttctacacca tattcggcct cttcaatgac atgaggaaga ccgggtcaaa tttttggttg 660 agcgcctcta cagtggtcaa ttctgcatca tcacttttct tagcattgta tgtcacccaa 720 tgtattctag gcaaagaagt ttccgcatta actcttttt aaggtttgcc tttcattgta 780 gttgttgttg gtttcaagca caaaatcaag attgcccagt atgccctgga gaaatttgaa 840 agagtcggtt tatctaaaag gattactacc gatgaaatcg tttttgaatc cgtgagcgaa 900 gagggtggtc gtttgattca agaccatttg ctttgtattt ttgcctttat cggatgctct 960 atgtatgctc accaattgaa gactttgaca aacttctgca tattatcagc atttatccta 1020 attttcgaat tgattttaac tcctacattt tattctgcta tcttagcgct tagactggaa 1080 atgaatgtta tccacagatc tactattatc aagcaaacat tagaagaaga cggtgttgtt 1140 ccatctacag caagaatcat ttctaaggca gaaaagaaat ccgtatcttc taactttggt 1200 gcaaattggg tcaatgatgc cttcaattca ttgtacttcg ataaggaacg tgtttctcta 1260 ccagatttta ttacctcgaa tgcctctgaa aactttaaag agcaagctat tgttagtgtc 1320 accccattat tatattacaa acccattaag tcctaccaac gcattgagga tatggttctt 1380 ctattgcttc gtaatgtcag tgttgccatt cgtgataggt tcgtcagtaa attagttctt 1440 tccgccttag tatgcagtgc tgtcatcaat gtgtatttat taaatgctgc tagaattcat 1500 accagttata ctgcagacca attggtgaag actgaagtca ccaagaagtc ttttactgct 1560 cctgtacaaa aggcttctac accagtttta accaataaaa cagtcatttc tggatcgaaa 1620 gtcaaaagtt tatcatctgc gcaatcgagc tcatcaggac cttcatcatc tagtgaggaa 1680 gatgattccc gcgatattga aagcttggat aagaaaatac gtcctttaga agaattagaa 1740 gcatcattaa gtagtggaaa tacaaaacaa ttgaagaaca aagaggtcgc tgccttggtt 1800 attcacggta agttaccttt gtacgctttg gagaaaaaat taggtgatac tacgagagcg 1860 gttgcggtac gtaggaaggc tctttcaatt ttggcagaag ctcctgtatt agcatctgat 1920 cgtttaccat ataaaaatta tgactacgac cgcgtatttg gcgcttgttg tgaaaatgtt 1980 ataggttaca tgcctttgcc cgttggtgtt ataggcccct tggttatcga tggtacatct 2040 tatcatatac caatggcaac tacagagggt tgtttggtag cttctgccat gcgtggctgt 2100 aaggcaatca atgctggcgg tggtgcaaca actgttttaa ctaaggatgg tatgacaaga 2160 ggcccagtag tccgtttccc aactttgaaa agatctggtg cctgtaagat atggttagac 2220 tcagaagagg gacaaaacgc aattaaaaaa gcttttaact ctacatcaag atttgcacgt 2280 ctgcaacata ttcaaacttg tctagcagga gatttactct tcatgagatt tagaacaact 2340 actggtgacg caatgggtat gaatatgatt tctaagggtg tcgaatactc attaaagcaa 2400 atggtagaag agtatggctg ggaagatatg gaggttgtct ccgtttctgg taactactgt 2460 accgacaaaa aaccagctgc catcaactgg atcgaaggtc gtggtaagag tgtcgtcgca 2520 gaagctacta ttcctggtga tgttgtcaga aaagtgttaa aaagtgatgt ttccgcattg 2580 gttgagttga acattgctaa gaatttggtt ggatctgcaa tggctgggtc tgttggtgga 2640 tttaacgcac gtgcagctaa tttagtgaca gctgttttct tggcattagg acaagatcct 2700 gcacaaaatg tcgaaagttc caactgtata acattgatga aagaagtgga cggtgatttg 2760 agaatttccg tatccatgcc atccatcgaa gtaggtacca tcggtggtgg tactgttcta 2820 gaaccacaag gtgccatgtt ggacttatta ggtgtaagag gcccacatgc taccgctcct 2880 ggtaccaacg cacgtcaatt agcaagaata gttgcctgtg ccgtcttggc aggtgaatta 2940 tccttatgtg ctgccctagc agccggccat ttggttcaaa gtcatatgac ccacaacagg 3000 aaacctgctg aaccaacaaa acctaacaat ttggacgcca ctgatataaa tcgtttgaaa 3060 gatgggtccg tcacctgcat taaatcctaa 3090 <210> 17 <211> 2973 <212> DNA <213> Saccharomyces cerevisiae <400> 17 atgccgccgc tattcaaggg actgaaacag atggcaaagc caattgccta tgtttcaaga 60 ttttcggcga aacgaccaat tcatataata cttttttctc taatcatatc cgcattcgct 120 tatctatccg tcattcagta ttacttcaat ggttggcaac tagattcaaa tagtgttttt 180 gaaactgctc caaataaaga cttcaacact ctatttcaag aatgttccca ttactacaga 240 gattcctctc tagatggttg ggtatcaatc accgcgcatg aagctagtga gttaccagcc 300 ccacaccatt actatctatt aaacctgaac ttcaatagtc ctaatgaaac tgactccatt 360 ccagaactag ctaacacggt ttttgagaaa gataatacaa aatatattct gcaagaagat 420 ctcagcgttt ccaaagaaat ttcttctact gatggaacga aatggaggtt aagaagtgac 480 agaaaaagtc ttttcgacgt aaagacgtta gcatattctc tctacgatgt attttcagaa 540 aatgtaaccc aagcagaccc gtttgacgtc cttattatgg ttactgccta cctaatgatg 600 ttctacacca tattcggcct cttcaatgac atgaggaaga ccgggtcaaa tttttggttg 660 agcgcctcta cagtggtcaa ttctgcatca tcacttttct tagcattgta tgtcacccaa 720 tgtattctag gcaaagaagt ttccgcatta actcttttt aaggtttgcc tttcattgta 780 gttgttgttg gtttcaagca caaaatcaag attgcccagt atgccctgga gaaatttgaa 840 agagtcggtt tatctaaaag gattactacc gatgaaatcg tttttgaatc cgtgagcgaa 900 gagggtggtc gtttgattca agaccatttg ctttgtattt ttgcctttat cggatgctct 960 atgtatgctc accaattgaa gactttgaca aacttctgca tattatcagc atttatccta 1020 attttcgaat tgattttaac tcctacattt tattctgcta tcttagcgct tagactggaa 1080 atgaatgtta tccacagatc tactattatc aagcaaacat tagaagaaga cggtgttgtt 1140 ccatctacag caagaatcat ttctaaggca gaaaagaaat ccgtatcttc tttcttaaat 1200 ctcagtgtgg ttgtcattat catgaaactc tctgtcatac tgttgttcgt cttcatcaac 1260 ttttataact ttggtgcaaa ttgggtcaat gatgccttca attcattgta cttcgataag 1320 gaacgtgttt ctctaccaga ttttattacc tcgaatgcct ctgaaaactt taaagagcaa 1380 cataccagtt atactgcaga ccaattggtg aagactgaag tcaccaagaa gtcttttact 1440 gctcctgtac aaaaggcttc tacaccagtt ttaaccaata aaacagtcat ttctggatcg 1500 aaagtcaaaa gtttatcatc tgcgcaatcg agctcatcag gaccttcatc atctagtgag 1560 gaagatgatt cccgcgatat tgaaagcttg gataagaaaa tacgtccttt agaagaatta 1620 gaagcatcat taagtagtgg aaatacaaaa caattgaaga acaaagaggt cgctgccttg 1680 gttattcacg gtaagttacc tttgtacgct ttggagaaaa aattaggtga tactacgaga 1740 gcggttgcgg tacgtaggaa ggctctttca attttggcag aagctcctgt attagcatct 1800 gatcgtttac catataaaaa ttatgactac gaccgcgtat ttggcgcttg ttgtgaaaat 1860 gttataggtt acatgccttt gcccgttggt gttataggcc ccttggttat cgatggtaca 1920 tcttatcata taccaatggc aactacagag ggttgtttgg tagcttctgc catgcgtggc 1980 tgtaaggcaa tcaatgctgg cggtggtgca acaactgttt taactaagga tggtatgaca 2040 agaggcccag tagtccgttt cccaactttg aaaagatctg gtgcctgtaa gatatggtta 2100 gactcagaag agggacaaaa cgcaattaaa aaagctttta actctacatc aagatttgca 2160 cgtctgcaac atattcaaac ttgtctagca ggagatttac tcttcatgag atttagaaca 2220 actactggtg acgcaatggg tatgaatatg atttctaagg gtgtcgaata ctcattaaag 2280 caaatggtag aagagtatgg ctgggaagat atggaggttg tctccgtttc tggtaactac 2340 tgtaccgaca aaaaaccagc tgccatcaac tggatcgaag gtcgtggtaa gagtgtcgtc 2400 gcagaagcta ctattcctgg tgatgttgtc agaaaagtgt taaaaagtga tgtttccgca 2460 ttggttgagt tgaacattgc taagaatttg gttggatctg caatggctgg gtctgttggt 2520 ggatttaacg cacgtgcagc taatttagtg acagctgttt tcttggcatt aggacaagat 2580 cctgcacaaa atgtcgaaag ttccaactgt ataacattga tgaaagaagt ggacggtgat 2640 ttgagaattt ccgtatccat gccatccatc gaagtaggta ccatcggtgg tggtactgtt 2700 ctagaaccac aaggtgccat gttggactta ttaggtgtaa gaggcccaca tgctaccgct 2760 cctggtacca acgcacgtca attagcaaga atagttgcct gtgccgtctt ggcaggtgaa 2820 ttatccttat gtgctgccct agcagccggc catttggttc aaagtcatat gacccacaac 2880 aggaaacctg ctgaaccaac aaaacctaac aatttggacg ccactgatat aaatcgtttg 2940 aaagatgggt ccgtcacctg cattaaatcc taa 2973 <210> 18 <211> 2457 <212> DNA <213> Saccharomyces cerevisiae <400> 18 atgccgccgc tattcaaggg actgaaacag atggcaaagc caattgccta tgtttcaaga 60 ttttcggcga aacgaccaat tcatataata cttttttctc taatcatatc cgcattcgct 120 tatctatccg tcattcagta ttacttcaat ggttggcaac tagattcaaa tagtgttttt 180 gaaactgctc caaataaaga cttcaacact ctatttcaag aatgttccca ttactacaga 240 gattcctctc tagatggttg ggtatcaatc accgcgcatg aagctagtga gttaccagcc 300 ccacaccatt actatctatt aaacctgaac ttcaatagtc ctaatgaaac tgactccatt 360 ccagaactag ctaacacggt ttttgagaaa gataatacaa aatatattct gcaagaagat 420 ctcagcgttt ccaaagaaat ttcttctact gatggaacga aatggaggtt aagaagtgac 480 agaaaaagtc ttttcgacgt aaagacgtta gcatattctc tctacgatgt attttcagaa 540 aatgtaaccc aagcagacaa ctttggtgca aattgggtca atgatgcctt caattcattg 600 tacttcgata aggaacgtgt ttctctacca gattttatta cctcgaatgc ctctgaaaac 660 tttaaagagc aagctattgt tagtgtcacc ccattattat attacaaacc cattaagtcc 720 taccaacgca ttgaggatat ggttcttcta ttgcttcgta atgtcagtgt tgccattcgt 780 gataggttcg tcagtaaatt agttctttcc gccttagtat gcagtgctgt catcaatgtg 840 tatttattaa atgctgctag aattcatacc agttatactg cagaccaatt ggtgaagact 900 gaagtcacca agaagtcttt tactgctcct gtacaaaagg cttctacacc agttttaacc 960 aataaaacag tcatttctgg atcgaaagtc aaaagtttat catctgcgca atcgagctca 1020 tcaggacctt catcatctag tgaggaagat gattcccgcg atattgaaag cttggataag 1080 aaaatacgtc ctttagaaga attagaagca tcattaagta gtggaaatac aaaacaattg 1140 aagaacaaag aggtcgctgc cttggttatt cacggtaagt tacctttgta cgctttggag 1200 aaaaaattag gtgatactac gagagcggtt gcggtacgta ggaaggctct ttcaattttg 1260 gcagaagctc ctgtattagc atctgatcgt ttaccatata aaaattatga ctacgaccgc 1320 gtatttggcg cttgttgtga aaatgttata ggttacatgc ctttgcccgt tggtgttata 1380 ggccccttgg ttatcgatgg tacatcttat catataccaa tggcaactac agagggttgt 1440 ttggtagctt ctgccatgcg tggctgtaag gcaatcaatg ctggcggtgg tgcaacaact 1500 gttttaacta aggatggtat gacaagaggc ccagtagtcc gtttcccaac tttgaaaaga 1560 tctggtgcct gtaagatatg gttagactca gaagagggac aaaacgcaat taaaaaagct 1620 tttaactcta catcaagatt tgcacgtctg caacatattc aaacttgtct agcaggagat 1680 ttactcttca tgagatttag aacaactact ggtgacgcaa tgggtatgaa tatgatttct 1740 aagggtgtcg aatactcatt aaagcaaatg gtagaagagt atggctggga agatatggag 1800 gttgtctccg tttctggtaa ctactgtacc gacaaaaaac cagctgccat caactggatc 1860 gaaggtcgtg gtaagagtgt cgtcgcagaa gctactattc ctggtgatgt tgtcagaaaa 1920 gtgttaaaaa gtgatgtttc cgcattggtt gagttgaaca ttgctaagaa tttggttgga 1980 tctgcaatgg ctgggtctgt tggtggattt aacgcacgtg cagctaattt agtgacagct 2040 gttttcttgg cattaggaca agatcctgca caaaatgtcg aaagttccaa ctgtataaca 2100 ttgatgaaag aagtggacgg tgatttgaga atttccgtat ccatgccatc catcgaagta 2160 ggtaccatcg gtggtggtac tgttctagaa ccacaaggtg ccatgttgga cttattaggt 2220 gtaagaggcc cacatgctac cgctcctggt accaacgcac gtcaattagc aagaatagtt 2280 gcctgtgccg tcttggcagg tgaattatcc ttatgtgctg ccctagcagc cggccatttg 2340 gttcaaagtc atatgaccca caacaggaaa cctgctgaac caacaaaacc taacaatttg 2400 gacgccactg atataaatcg tttgaaagat gggtccgtca cctgcattaa atcctaa 2457 <210> 19 <211> 2151 <212> DNA <213> Saccharomyces cerevisiae <400> 19 atgccgccgc tattcaaggg actgaaacag atggcaaagc caattgccta tgtttcaaga 60 ttttcggcga aacgaccaat tcatataata cttttttctc taatcatatc cgcattcgct 120 tatctatccg tcattcagta ttacttcaat ggttggcaac tagattcaaa tagtgttttt 180 gaaactgctc caaataaaga cttcaacact ctatttcaag aatgttccca ttactacaga 240 gattcctctc tagatggttg ggtatcaatc accgcgcatg aagctagtga gttaccagcc 300 ccacaccatt actatctatt aaacctgaac ttcaatagtc ctaatgaaac tgactccatt 360 ccagaactag ctaacacggt ttttgagaaa gataatacaa aatatattct gcaagaagat 420 ctcagcgttt ccaaagaaat ttcttctact gatggaacga aatggaggtt aagaagtgac 480 agaaaaagtc ttttcgacgt aaagacgtta gcatattctc tctacgatgt attttcagaa 540 aatgtaaccc aagcagacca taccagttat actgcagacc aattggtgaa gactgaagtc 600 accaagaagt cttttactgc tcctgtacaa aaggcttcta caccagtttt aaccaataaa 660 acagtcattt ctggatcgaa agtcaaaagt ttatcatctg cgcaatcgag ctcatcagga 720 ccttcatcat ctagtgagga agatgattcc cgcgatattg aaagcttgga taagaaaata 780 cgtcctttag aagaattaga agcatcatta agtagtggaa atacaaaaca attgaagaac 840 aaagaggtcg ctgccttggt tattcacggt aagttacctt tgtacgcttt ggagaaaaaa 900 ttaggtgata ctacgagagc ggttgcggta cgtaggaagg ctctttcaat tttggcagaa 960 gctcctgtat tagcatctga tcgtttacca tataaaaatt atgactacga ccgcgtattt 1020 ggcgcttgtt gtgaaaatgt tataggttac atgcctttgc ccgttggtgt tataggcccc 1080 ttggttatcg atggtacatc ttatcatata ccaatggcaa ctacagaggg ttgtttggta 1140 gcttctgcca tgcgtggctg taaggcaatc aatgctggcg gtggtgcaac aactgtttta 1200 actaaggatg gtatgacaag aggcccagta gtccgtttcc caactttgaa aagatctggt 1260 gcctgtaaga tatggttaga ctcagaagag ggacaaaacg caattaaaaa agcttttaac 1320 tctacatcaa gatttgcacg tctgcaacat attcaaactt gtctagcagg agatttactc 1380 ttcatgagat ttagaacaac tactggtgac gcaatgggta tgaatatgat ttctaagggt 1440 gtcgaatact cattaaagca aatggtagaa gagtatggct gggaagatat ggaggttgtc 1500 tccgtttctg gtaactactg taccgacaaa aaaccagctg ccatcaactg gatcgaaggt 1560 cgtggtaaga gtgtcgtcgc agaagctact attcctggtg atgttgtcag aaaagtgtta 1620 aaaagtgatg tttccgcatt ggttgagttg aacattgcta agaatttggt tggatctgca 1680 atggctgggt ctgttggtgg atttaacgca cgtgcagcta atttagtgac agctgttttc 1740 ttggcattag gacaagatcc tgcacaaaat gtcgaaagtt ccaactgtat aacattgatg 1800 aaagaagtgg acggtgattt gagaatttcc gtatccatgc catccatcga agtaggtacc 1860 atcggtggtg gtactgttct agaaccacaa ggtgccatgt tggacttatt aggtgtaaga 1920 ggcccacatg ctaccgctcc tggtaccaac gcacgtcaat tagcaagaat agttgcctgt 1980 gccgtcttgg caggtgaatt atccttatgt gctgccctag cagccggcca tttggttcaa 2040 agtcatatga cccacaacag gaaacctgct gaaccaacaa aacctaacaa tttggacgcc 2100 actgatataa atcgtttgaa agatgggtcc gtcacctgca ttaaatccta a 2151 <210> 20 <211> 1620 <212> DNA <213> Saccharomyces cerevisiae <400> 20 atgccgccgc tattcaaggg actgaaacat accagttata ctgcagacca attggtgaag 60 actgaagtca ccaagaagtc ttttactgct cctgtacaaa aggcttctac accagtttta 120 accaataaaa cagtcatttc tggatcgaaa gtcaaaagtt tatcatctgc gcaatcgagc 180 tcatcaggac cttcatcatc tagtgaggaa gatgattccc gcgatattga aagcttggat 240 aagaaaatac gtcctttaga agaattagaa gcatcattaa gtagtggaaa tacaaaacaa 300 ttgaagaaca aagaggtcgc tgccttggtt attcacggta agttaccttt gtacgctttg 360 gagaaaaaat taggtgatac tacgagagcg gttgcggtac gtaggaaggc tctttcaatt 420 ttggcagaag ctcctgtatt agcatctgat cgtttaccat ataaaaatta tgactacgac 480 cgcgtatttg gcgcttgttg tgaaaatgtt ataggttaca tgcctttgcc cgttggtgtt 540 ataggcccct tggttatcga tggtacatct tatcatatac caatggcaac tacagagggt 600 tgtttggtag cttctgccat gcgtggctgt aaggcaatca atgctggcgg tggtgcaaca 660 actgttttaa ctaaggatgg tatgacaaga ggcccagtag tccgtttccc aactttgaaa 720 agatctggtg cctgtaagat atggttagac tcagaagagg gacaaaacgc aattaaaaaa 780 gcttttaact ctacatcaag atttgcacgt ctgcaacata ttcaaacttg tctagcagga 840 gatttactct tcatgagatt tagaacaact actggtgacg caatgggtat gaatatgatt 900 tctaagggtg tcgaatactc attaaagcaa atggtagaag agtatggctg ggaagatatg 960 gaggttgtct ccgtttctgg taactactgt accgacaaaa aaccagctgc catcaactgg 1020 atcgaaggtc gtggtaagag tgtcgtcgca gaagctacta ttcctggtga tgttgtcaga 1080 aaagtgttaa aaagtgatgt ttccgcattg gttgagttga acattgctaa gaatttggtt 1140 ggatctgcaa tggctgggtc tgttggtgga tttaacgcac gtgcagctaa tttagtgaca 1200 gctgttttct tggcattagg acaagatcct gcacaaaatg tcgaaagttc caactgtata 1260 acattgatga aagaagtgga cggtgatttg agaatttccg tatccatgcc atccatcgaa 1320 gtaggtacca tcggtggtgg tactgttcta gaaccacaag gtgccatgtt ggacttatta 1380 ggtgtaagag gcccacatgc taccgctcct ggtaccaacg cacgtcaatt agcaagaata 1440 gttgcctgtg ccgtcttggc aggtgaatta tccttatgtg ctgccctagc agccggccat 1500 ttggttcaaa gtcatatgac ccacaacagg aaacctgctg aaccaacaaa acctaacaat 1560 ttggacgcca ctgatataaa tcgtttgaaa gatgggtccg tcacctgcat taaatcctaa 1620 <210> 21 <211> 1377 <212> DNA <213> Saccharomyces cerevisiae <400> 21 atgccgccgc tattcaaggg actgaaagca tcattaagta gtggaaatac aaaacaattg 60 aagaacaaag aggtcgctgc cttggttatt cacggtaagt tacctttgta cgctttggag 120 aaaaaattag gtgatactac gagagcggtt gcggtacgta ggaaggctct ttcaattttg 180 gcagaagctc ctgtattagc atctgatcgt ttaccatata aaaattatga ctacgaccgc 240 gtatttggcg cttgttgtga aaatgttata ggttacatgc ctttgcccgt tggtgttata 300 ggccccttgg ttatcgatgg tacatcttat catataccaa tggcaactac agagggttgt 360 ttggtagctt ctgccatgcg tggctgtaag gcaatcaatg ctggcggtgg tgcaacaact 420 gttttaacta aggatggtat gacaagaggc ccagtagtcc gtttcccaac tttgaaaaga 480 tctggtgcct gtaagatatg gttagactca gaagagggac aaaacgcaat taaaaaagct 540 tttaactcta catcaagatt tgcacgtctg caacatattc aaacttgtct agcaggagat 600 ttactcttca tgagatttag aacaactact ggtgacgcaa tgggtatgaa tatgatttct 660 aagggtgtcg aatactcatt aaagcaaatg gtagaagagt atggctggga agatatggag 720 gttgtctccg tttctggtaa ctactgtacc gacaaaaaac cagctgccat caactggatc 780 gaaggtcgtg gtaagagtgt cgtcgcagaa gctactattc ctggtgatgt tgtcagaaaa 840 gtgttaaaaa gtgatgtttc cgcattggtt gagttgaaca ttgctaagaa tttggttgga 900 tctgcaatgg ctgggtctgt tggtggattt aacgcacgtg cagctaattt agtgacagct 960 gttttcttgg cattaggaca agatcctgca caaaatgtcg aaagttccaa ctgtataaca 1020 ttgatgaaag aagtggacgg tgatttgaga atttccgtat ccatgccatc catcgaagta 1080 ggtaccatcg gtggtggtac tgttctagaa ccacaaggtg ccatgttgga cttattaggt 1140 gtaagaggcc cacatgctac cgctcctggt accaacgcac gtcaattagc aagaatagtt 1200 gcctgtgccg tcttggcagg tgaattatcc ttatgtgctg ccctagcagc cggccatttg 1260 gttcaaagtc atatgaccca caacaggaaa cctgctgaac caacaaaacc taacaatttg 1320 gacgccactg atataaatcg tttgaaagat gggtccgtca cctgcattaa atcctaa 1377 <210> 22 <211> 1302 <212> DNA <213> Saccharomyces cerevisiae <400> 22 atgccgccgc tattcaaggg actgaaacct ttgtacgctt tggagaaaaa attaggtgat 60 actacgagag cggttgcggt acgtaggaag gctctttcaa ttttggcaga agctcctgta 120 ttagcatctg atcgtttacc atataaaaat tatgactacg accgcgtatt tggcgcttgt 180 tgtgaaaatg ttataggtta catgcctttg cccgttggtg ttataggccc cttggttatc 240 gatggtacat cttatcatat accaatggca actacagagg gttgtttggt agcttctgcc 300 atgcgtggct gtaaggcaat caatgctggc ggtggtgcaa caactgtttt aactaaggat 360 ggtatgacaa gaggcccagt agtccgtttc ccaactttga aaagatctgg tgcctgtaag 420 atatggttag actcagaaga gggacaaaac gcaattaaaa aagcttttaa ctctacatca 480 agatttgcac gtctgcaaca tattcaaact tgtctagcag gagatttact cttcatgaga 540 tttagaacaa ctactggtga cgcaatgggt atgaatatga tttctaaggg tgtcgaatac 600 tcattaaagc aaatggtaga agagtatggc tgggaagata tggaggttgt ctccgtttct 660 ggtaactact gtaccgacaa aaaaccagct gccatcaact ggatcgaagg tcgtggtaag 720 agtgtcgtcg cagaagctac tattcctggt gatgttgtca gaaaagtgtt aaaaagtgat 780 gtttccgcat tggttgagtt gaacattgct aagaatttgg ttggatctgc aatggctggg 840 tctgttggtg gatttaacgc acgtgcagct aatttagtga cagctgtttt cttggcatta 900 ggacaagatc ctgcacaaaa tgtcgaaagt tccaactgta taacattgat gaaagaagtg 960 gacggtgatt tgagaatttc cgtatccatg ccatccatcg aagtaggtac catcggtggt 1020 ggtactgttc tagaaccaca aggtgccatg ttggacttat taggtgtaag aggcccacat 1080 gctaccgctc ctggtaccaa cgcacgtcaa ttagcaagaa tagttgcctg tgccgtcttg 1140 gcaggtgaat tatccttatg tgctgcccta gcagccggcc atttggttca aagtcatatg 1200 acccacaaca ggaaacctgc tgaaccaaca aaacctaaca atttggacgc cactgatata 1260 aatcgtttga aagatgggtc cgtcacctgc attaaatcct aa 1302 <210> 23 <211> 1203 <212> DNA <213> Saccharomyces cerevisiae <400> 23 atgccgccgc tattcaaggg actgaaatct gatcgtttac catataaaaa ttatgactac 60 gaccgcgtat ttggcgcttg ttgtgaaaat gttataggtt acatgccttt gcccgttggt 120 gttataggcc ccttggttat cgatggtaca tcttatcata taccaatggc aactacagag 180 ggttgtttgg tagcttctgc catgcgtggc tgtaaggcaa tcaatgctgg cggtggtgca 240 acaactgttt taactaagga tggtatgaca agaggcccag tagtccgttt cccaactttg 300 aaaagatctg gtgcctgtaa gatatggtta gactcagaag agggacaaaa cgcaattaaa 360 aaagctttta actctacatc aagatttgca cgtctgcaac atattcaaac ttgtctagca 420 ggagatttac tcttcatgag atttagaaca actactggtg acgcaatggg tatgaatatg 480 atttctaagg gtgtcgaata ctcattaaag caaatggtag aagagtatgg ctgggaagat 540 atggaggttg tctccgtttc tggtaactac tgtaccgaca aaaaaccagc tgccatcaac 600 tggatcgaag gtcgtggtaa gagtgtcgtc gcagaagcta ctattcctgg tgatgttgtc 660 agaaaagtgt taaaaagtga tgtttccgca ttggttgagt tgaacattgc taagaatttg 720 gttggatctg caatggctgg gtctgttggt ggatttaacg cacgtgcagc taatttagtg 780 acagctgttt tcttggcatt aggacaagat cctgcacaaa atgtcgaaag ttccaactgt 840 ataacattga tgaaagaagt ggacggtgat ttgagaattt ccgtatccat gccatccatc 900 gaagtaggta ccatcggtgg tggtactgtt ctagaaccac aaggtgccat gttggactta 960 ttaggtgtaa gaggcccaca tgctaccgct cctggtacca acgcacgtca attagcaaga 1020 atagttgcct gtgccgtctt ggcaggtgaa ttatccttat gtgctgccct agcagccggc 1080 catttggttc aaagtcatat gacccacaac aggaaacctg ctgaaccaac aaaacctaac 1140 aatttggacg ccactgatat aaatcgtttg aaagatgggt ccgtcacctg cattaaatcc 1200 taa 1203 <210> 24 <211> 975 <212> DNA <213> Saccharomyces cerevisiae <400> 24 atgccgccgc tattcaaggg actgaaaaag gatggtatga caagaggccc agtagtccgt 60 ttcccaactt tgaaaagatc tggtgcctgt aagatatggt tagactcaga agagggacaa 120 aacgcaatta aaaaagcttt taactctaca tcaagatttg cacgtctgca acatattcaa 180 acttgtctag caggagattt actcttcatg agatttagaa caactactgg tgacgcaatg 240 ggtatgaata tgatttctaa gggtgtcgaa tactcattaa agcaaatggt agaagagtat 300 ggctgggaag atatggaggt tgtctccgtt tctggtaact actgtaccga caaaaaacca 360 gctgccatca actggatcga aggtcgtggt aagagtgtcg tcgcagaagc tactattcct 420 ggtgatgttg tcagaaaagt gttaaaaagt gatgtttccg cattggttga gttgaacatt 480 gctaagaatt tggttggatc tgcaatggct gggtctgttg gtggatttaa cgcacgtgca 540 gctaatttag tgacagctgt tttcttggca ttaggacaag atcctgcaca aaatgtcgaa 600 agttccaact gtataacatt gatgaaagaa gtggacggtg atttgagaat ttccgtatcc 660 atgccatcca tcgaagtagg taccatcggt ggtggtactg ttctagaacc acaaggtgcc 720 atgttggact tattaggtgt aagaggccca catgctaccg ctcctggtac caacgcacgt 780 caattagcaa gaatagttgc ctgtgccgtc ttggcaggtg aattatcctt atgtgctgcc 840 ctagcagccg gccatttggt tcaaagtcat atgacccaca acaggaaacc tgctgaacca 900 acaaaaccta acaatttgga cgccactgat ataaatcgtt tgaaagatgg gtccgtcacc 960 tgcattaaat cctaa 975 <210> 25 <211> 549 <212> DNA <213> Escherichia coli <400> 25 atgcaaacgg aacacgtcat tttattgaat gcacagggag ttcccacggg tacgctggaa 60 aagtatgccg cacacacggc agacacccgc ttacatctcg cgttctccag ttggctgttt 120 aatgccaaag gacaattatt agttacccgc cgcgcactga gcaaaaaagc atggcctggc 180 gtgtggacta actcggtttg tgggcaccca caactgggag aaagcaacga agacgcagtg 240 atccgccgtt gccgttatga gcttggcgtg gaaattacgc ctcctgaatc tatctatcct 300 gactttcgct accgcgccac cgatccgagt ggcattgtgg aaaatgaagt gtgtccggta 360 tttgccgcac gcaccactag tgcgttacag atcaatgatg atgaagtgat ggattatcaa 420 tggtgtgatt tagcagatgt attacacggt attgatgcca cgccgtgggc gttcagtccg 480 tggatggtga tgcaggcgac aaatcgcgaa gccagaaaac gattatctgc atttacccag 540 cttaaataa 549 <210> 26 <211> 1332 <212> DNA <213> Saccharomyces cerevisiae <400> 26 atgtcattac cgttcttaac ttctgcaccg ggaaaggtta ttatttttgg tgaacactct 60 gctgtgtaca acaagcctgc cgtcgctgct agtgtgtctg cgttgagaac ctacctgcta 120 ataagcgagt catctgcacc agatactatt gaattggact tcccggacat tagctttaat 180 cataagtggt ccatcaatga tttcaatgcc atcaccgagg atcaagtaaa ctcccaaaaa 240 ttggccaagg ctcaacaagc caccgatggc ttgtctcagg aactcgttag tcttttggat 300 ccgttgttag ctcaactatc cgaatccttc cactaccatg cagcgttttg tttcctgtat 360 atgtttgttt gcctatgccc ccatgccaag aatattaagt tttctttaaa gtctacttta 420 cccatcggtg ctgggttggg ctcaagcgcc tctatttctg tatcactggc cttagctatg 480 gcctacttgg gggggttaat aggatctaat gacttggaaa agctgtcaga aaacgataag 540 catatagtga atcaatgggc cttcataggt gaaaagtgta ttcacggtac cccttcagga 600 atagataacg ctgtggccac ttatggtaat gccctgctat ttgaaaaaga ctcacataat 660 ggaacaataa acacaaacaa ttttaagttc ttagatgatt tcccagccat tccaatgatc 720 ctaacctata ctagaattcc aaggtctaca aaagatcttg ttgctcgcgt tcgtgtgttg 780 gtcaccgaga aatttcctga agttatgaag ccaattctag atgccatggg tgaatgtgcc 840 ctacaaggct tagagatcat gactaagtta agtaaatgta aaggcaccga tgacgaggct 900 gtagaaacta ataatgaact gtatgaacaa ctattggaat tgataagaat aaatcatgga 960 ctgcttgtct caatcggtgt ttctcatcct ggattagaac ttattaaaaa tctgagcgat 1020 gatttgagaa ttggctccac aaaacttacc ggtgctggtg gcggcggttg ctctttgact 1080 ttgttacgaa gagacattac tcaagagcaa attgacagct tcaaaaagaa attgcaagat 1140 gattttagtt acgagacatt tgaaacagac ttgggtggga ctggctgctg tttgttaagc 1200 gcaaaaaatt tgaataaaga tcttaaaatc aaatccctag tattccaatt atttgaaaat 1260 aaaactacca caaagcaaca aattgacgat ctattattgc caggaaacac gaatttacca 1320 tggacttcat aa 1332 <210> 27 <211> 1197 <212> DNA <213> Saccharomyces cerevisiae <400> 27 atgtctcaga acgtttacat tgtatcgact gccagaaccc caattggttc attccagggt 60 tctctatcct ccaagacagc agtggaattg ggtgctgttg ctttaaaagg cgccttggct 120 aaggttccag aattggatgc atccaaggat tttgacgaaa ttatttttgg taacgttctt 180 tctgccaatt tgggccaagc tccggccaga caagttgctt tggctgccgg tttgagtaat 240 catatcgttg caagcacagt taacaaggtc tgtgcatccg ctatgaaggc aatcattttg 300 ggtgctcaat ccatcaaatg tggtaatgct gatgttgtcg tagctggtgg ttgtgaatct 360 atgactaacg caccatacta catgccagca gcccgtgcgg gtgccaaatt tggccaaact 420 gttcttgttg atggtgtcga aagagatggg ttgaacgatg cgtacgatgg tctagccatg 480 ggtgtacacg cagaaaagtg tgcccgtgat tgggatatta ctagagaaca acaagacaat 540 tttgccatcg aatcctacca aaaatctcaa aaatctcaaa aggaaggtaa attcgacaat 600 gaaattgtac ctgttaccat taagggattt agaggtaagc ctgatactca agtcacgaag 660 gacgaggaac ctgctagatt acacgttgaa aaattgagat ctgcaaggac tgttttccaa 720 aaagaaaacg gtactgttac tgccgctaac gcttctccaa tcaacgatgg tgctgcagcc 780 gtcatcttgg tttccgaaaa agttttgaag gaaaagaatt tgaagccttt ggctattatc 840 aaaggttggg gtgaggccgc tcatcaacca gctgatttta catgggctcc atctcttgca 900 gttccaaagg ctttgaaaca tgctggcatc gaagacatca attctgttga ttactttgaa 960 ttcaatgaag ccttttcggt tgtcggtttg gtgaacacta agattttgaa gctagaccca 1020 tctaaggtta atgtatatgg tggtgctgtt gctctaggtc acccattggg ttgttctggt 1080 gctagagtgg ttgttacact gctatccatc ttacagcaag aaggaggtaa gatcggtgtt 1140 gccgccattt gtaatggtgg tggtggtgct tcctctattg tcattgaaaa gatatga 1197 <210> 28 <211> 1476 <212> DNA <213> Saccharomyces cerevisiae <400> 28 atgaaactct caactaaact ttgttggtgt ggtattaaag gaagacttag gccgcaaaag 60 caacaacaat tacacaatac aaacttgcaa atgactgaac taaaaaaaca aaagaccgct 120 gaacaaaaaa ccagacctca aaatgtcggt attaaaggta tccaaattta catcccaact 180 caatgtgtca accaatctga gctagagaaa tttgatggcg tttctcaagg taaatacaca 240 attggtctgg gccaaaccaa catgtctttt gtcaatgaca gagaagatat ctactcgatg 300 tccctaactg ttttgtctaa gttgatcaag agttacaaca tcgacaccaa caaaattggt 360 agattagaag tcggtactga aactctgatt gacaagtcca agtctgtcaa gtctgtcttg 420 atgcaattgt ttggtgaaaa cactgacgtc gaaggtattg acacgcttaa tgcctgttac 480 ggtggtacca acgcgttgtt caactctttg aactggattg aatctaacgc atgggatggt 540 agagacgcca ttgtagtttg cggtgatatt gccatctacg ataagggtgc cgcaagacca 600 accggtggtg ccggtactgt tgctatgtgg atcggtcctg atgctccaat tgtatttgac 660 tctgtaagag cttcttacat ggaacacgcc tacgattttt acaagccaga tttcaccagc 720 gaatatcctt acgtcgatgg tcatttttca ttaacttgtt acgtcaaggc tcttgatcaa 780 gtttacaaga gttattccaa gaaggctatt tctaaagggt tggttagcga tcccgctggt 840 tcggatgctt tgaacgtttt gaaatatttc gactacaacg ttttccatgt tccaacctgt 900 aaattggtca caaaatcata cggtagatta ctatataacg atttcagagc caatcctcaa 960 ttgttcccag aagttgacgc cgaattagct actcgcgatt atgacgaatc tttaaccgat 1020 aagaacattg aaaaaacttt tgttaatgtt gctaagccat tccacaaaga gagagttgcc 1080 caatctttga ttgttccaac aaacacaggt aacatgtaca ccgcatctgt ttatgccgcc 1140 tttgcatctc tattaaacta tgttggatct gacgacttac aaggcaagcg tgttggttta 1200 ttttcttacg gttccggttt agctgcatct ctatattctt gcaaaattgt tggtgacgtc 1260 caacatatta tcaaggaatt agatattact aacaaattag ccaagagaat caccgaaact 1320 ccaaaggatt acgaagctgc catcgaattg agagaaaatg cccatttgaa gaagaacttc 1380 aaacctcaag gttccattga gcatttgcaa agtggtgttt actacttgac caacatcgat 1440 gacaaattta gaagatctta cgatgttaaa aaataa 1476 <210> 29 <211> 1356 <212> DNA <213> Saccharomyces cerevisiae <400> 29 atgtcagagt tgagagcctt cagtgcccca gggaaagcgt tactagctgg tggatattta 60 gttttagata caaaatatga agcatttgta gtcggattat cggcaagaat gcatgctgta 120 gcccatcctt acggttcatt gcaagggtct gataagtttg aagtgcgtgt gaaaagtaaa 180 caatttaaag atggggagtg gctgtaccat ataagtccta aaagtggctt cattcctgtt 240 tcgataggcg gatctaagaa ccctttcatt gaaaaagtta tcgctaacgt atttagctac 300 tttaaaccta acatggacga ctactgcaat agaaacttgt tcgttattga tattttctct 360 gatgatgcct accattctca ggaggatagc gttaccgaac atcgtggcaa cagaagattg 420 agttttcatt cgcacagaat tgaagaagtt cccaaaacag ggctgggctc ctcggcaggt 480 ttagtcacag ttttaactac agctttggcc tccttttttg tatcggacct ggaaaataat 540 gtagacaaat atagagaagt tattcataat ttagcacaag ttgctcattg tcaagctcag 600 ggtaaaattg gaagcgggtt tgatgtagcg gcggcagcat atggatctat cagatataga 660 agattcccac ccgcattaat ctctaatttg ccagatattg gaagtgctac ttacggcagt 720 aaactggcgc atttggttga tgaagaagac tggaatatta cgattaaaag taaccattta 780 ccttcgggat taactttatg gatgggcgat attaagaatg gttcagaaac agtaaaactg 840 gtccagaagg taaaaaattg gtatgattcg catatgccag aaagcttgaa aatatataca 900 gaactcgatc atgcaaattc tagatttatg gatggactat ctaaactaga tcgcttacac 960 gagactcatg acgattacag cgatcagata tttgagtctc ttgagaggaa tgactgtacc 1020 tgtcaaaagt atcctgaaat cacagaagtt agagatgcag ttgccacaat tagacgttcc 1080 tttagaaaaa taactaaaga atctggtgcc gatatcgaac ctcccgtaca aactagctta 1140 ttggatgatt gccagacctt aaaaggagtt cttacttgct taatacctgg tgctggtggt 1200 tatgacgcca ttgcagtgat tactaagcaa gatgttgatc ttagggctca aaccgctaat 1260 gacaaaagat tttctaaggt tcaatggctg gatgtaactc aggctgactg gggtgttagg 1320 aaagaaaaag atccggaaac ttatcttgat aaataa 1356 <210> 30 <211> 4 <212> PRT <213> Saccharomyces cerevisiae <400> 30 His Asp Glu Leu   1 <210> 31 <211> 4 <212> PRT <213> Saccharomyces cerevisiae <400> 31 Asp Asp Glu Leu   1 <210> 32 <211> 4 <212> PRT <213> Saccharomyces cerevisiae <400> 32 Lys Asp Glu Leu   1 <210> 33 <211> 29 <212> PRT <213> Saccharomyces cerevisiae <400> 33 Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe Trp Ala   1 5 10 15 Thr Glu Ala Glu Gln Leu Thr Lys Cys Glu Val Phe Gln              20 25 <210> 34 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 34 gccgttgaca gagggtccga gctcggtacc aag 33 <210> 35 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 35 catactgacc cattgtcaat gggtaataac tgat 34 <210> 36 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 36 tgtccggtaa atggagac 18 <210> 37 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 37 tgttctcgct gctcgttt 18 <210> 38 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 38 atgggaaagc tattacaat 19 <210> 39 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 39 caaggttgca atggccat 18 <210> 40 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 40 caatgtaggg ctatatatg 19 <210> 41 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 41 aacttgggga atggcaca 18 <210> 42 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 42 tcacgctctg tgtaaagtgt ata 23 <210> 43 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 43 tgcatctcga gggccgcatc atgtaattag 30 <210> 44 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 44 cattagggcc cggccgcaaa ttaaagcctt cg 32 <210> 45 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 45 cacggagctc cagttcgagt ttatcattat caa 33 <210> 46 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 46 ctctccgcgg tttgtttgtt tatgtgtgtt tattc 35 <210> 47 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 47 atggcttcag aaaaagaaat tag 23 <210> 48 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 48 ctatttgctt ctcttgtaaa ctt 23 <210> 49 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 49 atggaggcca agatagatga gct 23 <210> 50 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 50 tcacaattcg gataagtggt cta 23 <210> 51 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 51 atgccgccgc tattcaaggg act 23 <210> 52 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 52 ttaggattta atgcaggtga cgg 23 <210> 53 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 53 ccaaataaag actccaacac tctattt 27 <210> 54 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 54 gaattagaag cattattaag tagtgga 27 <210> 55 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 55 ggatttaacg cacatgcagc taattta 27 <210> 56 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 56 gtctgcttgg gttacatttt ctgaaaa 27 <210> 57 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 57 cataccagtt atactgcaga ccaattg 27 <210> 58 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 58 gaatactcat taaagcaaat ggtagaa 27 <210> 59 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 59 aactgcagat gaccgtttac acagcatccg t 31 <210> 60 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 60 cggaattctt attcctttgg tagaccagtc t 31 <210> 61 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 61 tttcagtccc ttgaatagcg gcggcat 27 <210> 62 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 62 cacaaaatca agattgccca gtatgcc 27 <210> 63 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 63 agaagatacg gatttctttt ctgcttt 27 <210> 64 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 64 aactttggtg caaattgggt caatgat 27 <210> 65 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 65 ttgctcttta aagttttcag aggcatt 27 <210> 66 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 66 gcattattaa gtagtggaaa tacaaaa 27 <210> 67 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 67 cctttgtacg ctttggagaa aaaatta 27 <210> 68 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 68 tctgatcgtt taccatataa aaattat 27 <210> 69 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 69 aaggatggta tgacaagagg cccagta 27 <210> 70 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 70 tccccgcgga tggaggccaa gatagat 27 <210> 71 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 71 caactcgagt cacaattcgg ataagtg 27 <210> 72 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 72 gctctagagt tcgtcgtgtt tgcttctctt gtaaactt 38 <210> 73 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 73 tatctcgagt cacaattcgt catgtaaatt gg 32 <210> 74 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 74 gcagggaccc caattcggat aagtggtc 28 <210> 75 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 75 gtagggtccc tggaggccaa gatagatg 28 <210> 76 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 76 gcagggaccc tttgcttctc ttgtaaact 29 <210> 77 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 77 gtagggtcct cagaaaaaga aattaggag 29 <210> 78 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 78 tgtaaaacga cggccagt 18 <210> 79 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 79 taatacgact cactataggg 20

【０１６５】[0165]

【配列表フリーテキスト】配列番号34-79: 合成DNA[Sequence list free text] SEQ ID NO: 34-79: Synthetic DNA

【図面の簡単な説明】[Brief description of drawings]

【図１】IPP合成経路関連酵素の代謝経路を示す図であ
る。FIG. 1 is a diagram showing metabolic pathways of IPP synthetic pathway-related enzymes.

【図２】ERG9遺伝子座周辺の物理地図と、ERG9プロモー
ター領域置換変異用DNA断片の構築図である。FIG. 2 shows a physical map around the ERG9 locus and a construction diagram of a DNA fragment for ERG9 promoter region substitution mutation.

【図３】発現シャトルベクターpYES2の物理地図。FIG. 3 is a physical map of the expression shuttle vector pYES2.

【図４】欠失型HMG1’の置換変異(A)及び構築図（B）を
示す図である。FIG. 4 is a diagram showing a substitution mutation (A) and a construction diagram (B) of deletion type HMG1 ′.

【図５】寒天プレート培地上でのEUG株のコロニー形成
を示した写真。FIG. 5 is a photograph showing colony formation of EUG strain on an agar plate medium.

【図６】EUG8、EUG12及びEUG27のプレニルアルコール生
産を示す図である。FIG. 6 is a diagram showing prenyl alcohol production of EUG8, EUG12 and EUG27.

【図７】A451由来のEUG株であるEUG5のプレニルアルコ
ール生産を示す図である。FIG. 7 is a diagram showing prenyl alcohol production of EUG5, which is an EUG strain derived from A451.

【図８】YPH499由来のEUG株であるEUG12のプレニルアル
コール生産を示す図である。FIG. 8 is a diagram showing the production of prenyl alcohol by EUG12, which is an EUG strain derived from YPH499.

【図９】YPH500由来のEUG株であるEUG24、EUG27のプレ
ニルアルコール生産を示す図である。FIG. 9 is a diagram showing the production of prenyl alcohol by YUG500-derived EUG strains EUG24 and EUG27.

【図１０】W303-1A由来の各EUG株のプレニルアルコール
生産を示す図である。FIG. 10 shows prenyl alcohol production of each EUG strain derived from W303-1A.

【図１１】W303-1B由来の各 EUG株のプレニルアルコー
ル生産を示す図である。FIG. 11 shows prenyl alcohol production of each EUG strain derived from W303-1B.

【図１２】培養液の初期糖濃度組成を変えた培地で培養
したときのEUG8によるプレニルアルコール生産を示す図
である。FIG. 12 is a diagram showing prenyl alcohol production by EUG8 when cultured in a medium in which the initial sugar concentration composition of the culture medium is changed.

【図１３】培養液の初期糖濃度組成を変えた培地で培養
したときのEUG12によるプレニルアルコール生産を示す
図である。FIG. 13 is a diagram showing the production of prenyl alcohol by EUG12 when cultured in a medium in which the initial sugar concentration composition of the culture medium was changed.

【図１４】培養液の初期糖濃度組成を変えた培地で培養
したときのEUG27によるプレニルアルコール生産を示す
図である。FIG. 14 is a diagram showing prenyl alcohol production by EUG27 when cultured in a medium in which the initial sugar concentration composition of the culture medium was changed.

【図１５】培養液の初期糖濃度組成を変えた培地で培養
したときのEUG8、EUG12及びEUG27によるFOH生産を示す
図である。FIG. 15 is a diagram showing FOH production by EUG8, EUG12, and EUG27 when cultured in a medium in which the initial sugar concentration composition of the culture medium was changed.

【図１６Ａ】プラスミドpRS434GAPを示す図である。FIG. 16A shows the plasmid pRS434GAP.

【図１６Ｂ】プラスミドpRS435GAPを示す図である。FIG. 16B shows the plasmid pRS435GAP.

【図１６Ｃ】プラスミドpRS444GAPを示す図である。FIG. 16C shows the plasmid pRS444GAP.

【図１６Ｄ】プラスミドpRS445GAPを示す図である。FIG. 16D shows the plasmid pRS445GAP.

【図１７】プラスミドpALHMG106の物理地図を示す図で
ある。FIG. 17 is a diagram showing a physical map of plasmid pALHMG106.

【図１８】ERG19発現プラスミドを導入したEUG株のプレ
ニルアルコール生産を示す図である。FIG. 18 is a diagram showing the production of prenyl alcohol by the EUG strain introduced with the ERG19 expression plasmid.

【図１９】A451又はEUG8によるプレニルアルコール生産
を示す図である。FIG. 19 shows prenyl alcohol production by A451 or EUG8.

【図２０】pRS434GAP-HMG1又はpRS444GAP-HMG1をEUG8に
導入したときのプレニルアルコール生産を示す図であ
る。FIG. 20 shows prenyl alcohol production when pRS434GAP-HMG1 or pRS444GAP-HMG1 was introduced into EUG8.

【図２１】EUG12又はEUG27によるプレニルアルコール生
産を示す図である。FIG. 21 shows prenyl alcohol production by EUG12 or EUG27.

【図２２】pRS434GAP-HMG1又はpRS444GAP-HMG1をEUG12
又はEUG27に導入したときのプレニルアルコール生産を
示す図である。FIG. 22: EUG12 of pRS434GAP-HMG1 or pRS444GAP-HMG1
FIG. 3 is a diagram showing the production of prenyl alcohol when introduced into EUG27.

【図２３】欠失型HMG1遺伝子発現プラスミドをEUG5へ導
入した株のプレニルアルコール生産量を示す図である。FIG. 23 is a diagram showing the amount of prenyl alcohol produced in a strain in which a deletion type HMG1 gene expression plasmid is introduced into EUG5.

【図２４】欠失型HMG1遺伝子発現プラスミドをEUG12へ
導入した株のプレニルアルコール生産量を示す図であ
る。FIG. 24 is a diagram showing the amount of prenyl alcohol produced in a strain in which a deletion type HMG1 gene expression plasmid was introduced into EUG12.

【図２５】ERG20遺伝子発現プラスミドをEUG8へ導入し
た株のプレニルアルコール生産量を示す図である。FIG. 25 is a diagram showing the amount of prenyl alcohol produced by a strain in which the ERG20 gene expression plasmid was introduced into EUG8.

【図２６】ERG20遺伝子発現プラスミドをEUG12又はEUG2
7へ導入した株のプレニルアルコール生産量を示す図で
ある。FIG. 26 shows that ERG20 gene expression plasmid was replaced with EUG12 or EUG2.
It is a figure which shows the amount of prenyl alcohol production of the strain introduce | transduced into 7.

【図２７】BTS1遺伝子発現プラスミドpRS435GAP-BTS1又
はpRS445GAP-BTS1をEUG8へ導入した株のプレニルアルコ
ール生産量を示す図である。FIG. 27 is a diagram showing the amount of prenyl alcohol produced in a strain in which the BTS1 gene expression plasmid pRS435GAP-BTS1 or pRS445GAP-BTS1 was introduced into EUG8.

【図２８】BTS1遺伝子発現プラスミドをEUG12又はEUG27
へ導入した株のプレニルアルコール生産量を示す図であ
る。FIG. 28: BTS1 gene expression plasmid was replaced with EUG12 or EUG27
It is a figure which shows the production amount of prenyl alcohol of the strain introduce | transduced into.

【図２９】BTS1とERG20の融合遺伝子及び小胞体シグナ
ルをC末端にもつポリペプチドをコードする遺伝子を作
製するために使用するプライマー並びにその位置及び方
向を示す図である。FIG. 29 is a diagram showing primers used for producing a gene encoding a fusion gene of BTS1 and ERG20 and a polypeptide having an endoplasmic reticulum signal at the C-terminus, and the position and direction thereof.

【図３０】A451、EUG5を宿主としたときのYM7培地中で
のGGOH生産量を測定した結果を示す図である。FIG. 30 is a diagram showing the results of measuring the amount of GGOH produced in a YM7 medium when A451 and EUG5 were used as hosts.

【図３１】A451、EUG5を宿主としたときのYMO7培地中で
のGGOH生産量を測定した結果を示す図である。FIG. 31 is a diagram showing the results of measuring the amount of GGOH produced in YMO7 medium when A451 and EUG5 were used as hosts.

【図３２】YPH499、EUG12を宿主としたときのYM7培地中
でのGGOH生産量を測定した結果を示す図である。FIG. 32 is a diagram showing the results of measuring the amount of GGOH produced in YM7 medium when YPH499 and EUG12 were used as hosts.

【図３３】YPH499、EUG12を宿主としたときのYMO7培地
中でのGGOH生産量を測定した結果を示す図である。FIG. 33 is a diagram showing the results of measuring the amount of GGOH produced in YMO7 medium when YPH499 and EUG12 were used as hosts.

【図３４】EUG5にpRS435GGFとpRS435GGFHDELを導入し、
所定の初期糖組成で培養したときのGGOHの生産量を示す
図である。FIG. 34: Introducing pRS435GGF and pRS435GGFHDEL into EUG5,
It is a figure which shows the production amount of GGOH when culture | cultivated by a predetermined initial sugar composition.

【図３５】EUG12にpRS435GGFとpRS435GGFHDELを導入
し、所定の初期糖組成で培養したときのGGOHの生産量を
示す図である。FIG. 35 is a diagram showing the production amount of GGOH when pRS435GGF and pRS435GGFHDEL were introduced into EUG12 and cultured with a predetermined initial sugar composition.

【図３６】EUG5、EUG24、EUG36及びEUG64並びにそれら
を宿主とした組換え体をYPDO7rich培地で培養したとき
のプレニルアルコール生産量とOD600値の時間変化。FIG. 36 shows time-dependent changes in prenyl alcohol production and OD 600 values when EUG5, EUG24, EUG36 and EUG64 and recombinants using them as a host were cultured in YPDO7rich medium.

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） Ｃ１２Ｒ 1:86） Ｃ１２Ｒ 1:865 (Ｃ１２Ｐ 7/02 Ｃ１２Ｎ 15/00 Ａ Ｃ１２Ｒ 1:865） Ｆターム(参考） 4B024 AA03 BA10 BA80 CA04 DA12 EA04 FA02 FA17 GA11 GA19 4B064 AC09 CA06 CA19 CC24 DA16 4B065 AA80X AB01 AC14 AC15 BA02 BB15 CA05 CA29 4H006 AA02 FC74 FE11 ─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. 7 Identification code FI theme code (reference) C12R 1:86) C12R 1: 865 (C12P 7/02 C12N 15/00 A C12R 1: 865) F term ( Reference) 4B024 AA03 BA10 BA80 CA04 DA12 EA04 FA02 FA17 GA11 GA19 4B064 AC09 CA06 CA19 CC24 DA16 4B065 AA80X AB01 AC14 AC15 BA02 BB15 CA05 CA29 4H006 AA02 FC74 FE11

Claims (21)

【特許請求の範囲】[Claims] 【請求項１】 翻訳活性のあるスクアレン合成酵素遺伝
子転写産物量を減少できるように変異させた変異型細胞
を培養し、得られる培養物からプレニルアルコールを採
取することを特徴とするプレニルアルコールの製造方
法。1. A method for producing prenyl alcohol, which comprises culturing a mutant cell mutated to reduce the amount of a squalene synthase gene transcript having a translation activity, and collecting prenyl alcohol from the resulting culture. Method. 【請求項２】 翻訳活性のあるスクアレン合成酵素遺伝
子転写産物量を減少できるように変異させた変異型細胞
に、IPP合成経路関連酵素遺伝子を含む発現用組換えDNA
又はゲノムインテグレート用DNAを導入して組換え体を
作製し、該組換え体を培養し、得られる培養物からプレ
ニルアルコールを採取することを特徴とするプレニルア
ルコールの製造方法。2. A recombinant cell for expression containing an IPP synthesis pathway-related enzyme gene in a mutant cell mutated to reduce the amount of squalene synthase gene transcript having translation activity.
Alternatively, a method for producing prenyl alcohol, comprising introducing a DNA for genome integration to prepare a recombinant, culturing the recombinant, and collecting prenyl alcohol from the resulting culture. 【請求項３】 スクアレン合成酵素遺伝子の転写プロモ
ーター領域を転写抑制型プロモーターで置換したプロモ
ーター領域を含む変異型細胞を、転写抑制条件下で培養
し、翻訳活性のあるスクアレン合成酵素遺伝子転写産物
量を減少させ、得られる培養物からプレニルアルコール
を採取することを特徴とするプレニルアルコールの製造
方法。3. A mutant cell containing a promoter region in which the transcription promoter region of the squalene synthase gene is replaced with a transcription repressing promoter is cultured under transcription repressing conditions to determine the amount of the squalene synthase gene transcript having translation activity. A method for producing prenyl alcohol, which comprises reducing prenyl alcohol from the resulting culture. 【請求項４】 スクアレン合成酵素遺伝子の転写プロモ
ーター領域を転写抑制型プロモーターで置換したしたプ
ロモーター領域を含み、翻訳活性のあるスクアレン合成
酵素遺伝子転写産物量を減少できるようにした変異型細
胞に、IPP合成経路関連酵素遺伝子を含む発現用組換えD
NA又はゲノムインテグレート用DNAを導入して組換え体
を作製し、該組換え体を転写抑制条件下で培養し、得ら
れる培養物からプレニルアルコールを採取することを特
徴とするプレニルアルコールの製造方法。4. A mutant cell comprising a promoter region in which the transcription promoter region of a squalene synthase gene is replaced with a transcription repressing promoter, and capable of reducing the amount of a transcription product of a squalene synthase gene having translation activity, to an IPP. Recombinant D for expression containing synthetic pathway-related enzyme genes
A method for producing prenyl alcohol, which comprises introducing a DNA for NA or genomic integration to prepare a recombinant, culturing the recombinant under a transcription repressing condition, and collecting prenyl alcohol from the resulting culture. . 【請求項５】 転写抑制型プロモーターがGAL1プロモー
ターである請求項３又は４記載の製造方法。5. The method according to claim 3, wherein the transcriptional repressor is GAL1 promoter. 【請求項６】 転写抑制条件がグルコース含有培地を用
いたものである請求項３又は４記載の製造方法。6. The production method according to claim 3, wherein the transcription repressing condition is one using a glucose-containing medium. 【請求項７】 IPP合成経路関連酵素遺伝子が、以下の
(a) - (l)の遺伝子からなる群から選ばれるいずれかの
遺伝子である請求項２又は４記載の製造方法。 (a) ファルネシル二リン酸合成酵素遺伝子 (b) ゲラニルゲラニル二リン酸合成酵素遺伝子 (c) ヒドロキシメチルグルタリルCoA還元酵素遺伝子 (d) イソペンテニル二リン酸Δ-イソメラーゼ遺伝子 (e) メバロン酸キナーゼ遺伝子 (f) アセチルCoAアセチルトランスフェラーゼ遺伝子 (g) ヒドロキシメチルグルタリル-CoA合成酵素遺伝子 (h) メバロン酸リン酸キナーゼ遺伝子 (i) メバロン酸二リン酸脱炭酸酵素遺伝子 (j) 上記(a) - (i)の遺伝子の変異型遺伝子 (k) 上記(a) - (i)の遺伝子からなる群から選ばれる遺
伝子又はその変異型遺伝子と、他の遺伝子又はその変異
型遺伝子との融合遺伝子 (l) 上記(a) - (k)の遺伝子に、コードするポリペプチ
ドが小胞体シグナルを有するように、付加、置換又は挿
入変異を施した遺伝子7. The IPP synthetic pathway-related enzyme gene has the following structure:
The method according to claim 2 or 4, wherein the gene is any gene selected from the group consisting of genes (a)-(l). (a) Farnesyl diphosphate synthase gene (b) Geranylgeranyl diphosphate synthase gene (c) Hydroxymethylglutaryl CoA reductase gene (d) Isopentenyl diphosphate Δ-isomerase gene (e) Mevalonate kinase gene (f) Acetyl CoA acetyltransferase gene (g) Hydroxymethylglutaryl-CoA synthase gene (h) Mevalonate phosphate kinase gene (i) Mevalonate diphosphate decarboxylase gene (j) Above (a)-( i) Mutant gene of the gene (k) (a)-a gene selected from the group consisting of the genes of (i) or a mutant gene thereof, and a fusion gene of another gene or a mutant gene thereof (l) Genes that have been subjected to addition, substitution or insertion mutation so that the encoded polypeptide has an endoplasmic reticulum signal in the above (a)-(k) gene 【請求項８】 小胞体シグナルが配列番号30、31又は32
に示されるものである請求項7記載の製造方法。8. The endoplasmic reticulum signal is SEQ ID NO: 30, 31 or 32.
8. The manufacturing method according to claim 7, which is shown in FIG. 【請求項９】 細胞が酵母である請求項1-8のいずれか
に記載の製造方法。9. The production method according to claim 1, wherein the cell is yeast. 【請求項１０】 酵母がサッカロマイセス・セレビシア
エである請求項９記載の製造方法。10. The method according to claim 9, wherein the yeast is Saccharomyces cerevisiae. 【請求項１１】 サッカロマイセス・セレビシアエが、
サッカロマイセス・セレビシアエA451株、YPH499株、YP
H500株、W303-1A株又はW303-1B株である請求項10記載の
製造方法。11. Saccharomyces cerevisiae
Saccharomyces cerevisiae A451 strain, YPH499 strain, YP
11. The production method according to claim 10, which is H500 strain, W303-1A strain or W303-1B strain. 【請求項１２】 プレニルアルコールが、ファルネソー
ル、ネロリドール及び/又はゲラニルゲラニオールであ
る請求項1-11のいずれかに記載の製造方法。12. The production method according to claim 1, wherein the prenyl alcohol is farnesol, nerolidol and / or geranylgeraniol. 【請求項１３】 転写抑制条件下で培養する前に、非転
写抑制条件下で培養することを特徴とする請求項3-12の
いずれかに記載の製造方法。13. The production method according to claim 3, wherein the culture is carried out under non-transcription-suppressing conditions before the culture is carried out under transcription-suppressing conditions. 【請求項１４】 転写抑制条件がグルコース含有培地を
用いたものであり、非転写抑制条件がガラクトース含有
培地を用いたものである請求項13記載の製造方法。14. The production method according to claim 13, wherein the transcription suppressing condition is a condition using a glucose-containing medium, and the non-transcription suppressing condition is a condition using a galactose-containing medium. 【請求項１５】 ファルネシル二リン酸合成酵素遺伝子
が、配列番号２又は４に示されるアミノ酸配列をコード
するものである請求項7記載の製造方法。15. The method according to claim 7, wherein the farnesyl diphosphate synthase gene encodes the amino acid sequence shown in SEQ ID NO: 2 or 4. 【請求項１６】 ゲラニルゲラニル二リン酸合成酵素遺
伝子が、配列番号６に示されるアミノ酸配列をコードす
るものである請求項7記載の製造方法。16. The method according to claim 7, wherein the geranylgeranyl diphosphate synthase gene encodes the amino acid sequence shown in SEQ ID NO: 6. 【請求項１７】 ヒドロキシメチルグルタリルCoA還元
酵素遺伝子が、配列番号８に示されるアミノ酸配列をコ
ードするものである請求項7記載の製造方法。17. The method according to claim 7, wherein the hydroxymethylglutaryl CoA reductase gene encodes the amino acid sequence shown in SEQ ID NO: 8. 【請求項１８】 ヒドロキシメチルグルタリルCoA還元
酵素遺伝子の変異型遺伝子が、配列番号11、13及び15-2
4に示されるいずれかの塩基配列を含むものである請求
項7記載の製造方法。18. The mutant genes of the hydroxymethylglutaryl CoA reductase gene have SEQ ID NOS: 11, 13 and 15-2.
8. The production method according to claim 7, which comprises any of the nucleotide sequences shown in 4. 【請求項１９】 メバロン酸二リン酸脱炭酸酵素遺伝子
が、配列番号10に示されるアミノ酸配列をコードするも
のである請求項7記載の製造方法。19. The method according to claim 7, wherein the mevalonate diphosphate decarboxylase gene encodes the amino acid sequence shown in SEQ ID NO: 10. 【請求項２０】 翻訳活性のあるスクアレン合成酵素遺
伝子転写産物量を減少できるように変異させた変異型細
胞。20. A mutant cell which has been mutated so as to reduce the amount of a transcription product of a squalene synthase gene having translation activity. 【請求項２１】 スクアレン合成酵素の転写プロモータ
ー領域を転写抑制型プロモーターで置換したプロモータ
ー領域を含み、翻訳活性のあるスクアレン合成酵素遺伝
子転写産物量を減少できるように変異させた変異型細
胞。21. A mutant cell comprising a promoter region in which the transcription promoter region of squalene synthase is replaced with a transcription repressing promoter, and mutated to reduce the amount of a transcription product of a squalene synthase gene having translation activity.
JP2001282978A 2000-12-28 2001-09-18 Method for producing prenyl alcohol Pending JP2003088368A (en)

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CA002433534A CA2433534A1 (en) 2000-12-28 2001-12-20 Process for producing prenyl alcohol
EP01272515A EP1354956A4 (en) 2000-12-28 2001-12-20 Process for producing prenyl alcohol
US10/450,941 US20040063182A1 (en) 2000-12-28 2001-12-20 Process for producing prenyl alcohol
PCT/JP2001/011215 WO2002053747A1 (en) 2000-12-28 2001-12-20 Process for producing prenyl alcohol
US12/060,434 US8759046B2 (en) 2000-12-28 2008-04-01 Process for producing prenyl alcohols

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006204215A (en) * 2005-01-28 2006-08-10 Toyota Motor Corp Method for producing ubiquinone
JP2008507974A (en) * 2004-07-27 2008-03-21 ザ レジェンツ オブ ザ ユニバーシティー オブ カリフォルニア Genetically modified host cells and methods of using the same for producing isoprenoid compounds
JP2009538601A (en) * 2006-05-26 2009-11-12 アミリス バイオテクノロジーズ， インコーポレイテッド Isoprenoid production

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000001650A1 (en) * 1998-07-06 2000-01-13 Dcv, Inc. Doing Business As Bio-Technical Resources Method of vitamin production

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000001650A1 (en) * 1998-07-06 2000-01-13 Dcv, Inc. Doing Business As Bio-Technical Resources Method of vitamin production

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008507974A (en) * 2004-07-27 2008-03-21 ザ レジェンツ オブ ザ ユニバーシティー オブ カリフォルニア Genetically modified host cells and methods of using the same for producing isoprenoid compounds
JP2006204215A (en) * 2005-01-28 2006-08-10 Toyota Motor Corp Method for producing ubiquinone
JP4655648B2 (en) * 2005-01-28 2011-03-23 トヨタ自動車株式会社 Method for producing ubiquinone
JP2009538601A (en) * 2006-05-26 2009-11-12 アミリス バイオテクノロジーズ， インコーポレイテッド Isoprenoid production

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