JPH0630584B2

JPH0630584B2 - Chimeric cytochrome P-450 gene constructed from a plurality of cytochrome P-450 genes, a plasmid for expression in yeast containing the same, a method for producing the same, and a yeast strain having these plasmids in cells

Info

Publication number: JPH0630584B2
Application number: JP24277385A
Authority: JP
Inventors: 利之榊; 義康薮崎; 秀郎大川
Original assignee: Agency of Industrial Science and Technology
Current assignee: National Institute of Advanced Industrial Science and Technology AIST
Priority date: 1985-10-31
Filing date: 1985-10-31
Publication date: 1994-04-27
Anticipated expiration: 2009-04-27
Also published as: JPS62104583A

Description

【発明の詳細な説明】本発明は、キメラチトクロムＰ−４５０遺伝子、それを
含む酵母発現プラスミド及びそれらのプラスミドは菌体
内に保持する酵母菌株並びにこれらの製造方法に関す
る。The present invention relates to a chimeric cytochrome P-450 gene, a yeast expression plasmid containing the same, a yeast strain that retains these plasmids in the cells, and a method for producing these.

チトクロムＰ−４５０（以下Ｐ−４５０と略称する）
は、微生物から哺乳動物にいたるまで広く生物界に存在
するヘムタンパク質であり、多くの分子種が存在する。
各々のＰ−４５０分子種は基質特異性を異にしている
が、共通して一原子酸素添加機能を有する。Cytochrome P-450 (hereinafter abbreviated as P-450)
Is a heme protein that exists widely in the living world from microorganisms to mammals, and many molecular species exist.
Although each P-450 molecular species has different substrate specificity, they have a monoatomic oxygen addition function in common.

近年、本発明者らは、酵母を宿主としてラット肝チトク
ロムＰ−４５０ｃ（Ｐ−４５０ｃ）を発現させる酵母発
現ベクターpAMC1を構築し酵母でＰ−４５０ｃを大量に
発現させることに成功した（特開昭61-88878)。In recent years, the present inventors have succeeded in constructing a yeast expression vector pAMC1 that expresses rat liver cytochrome P-450c (P-450c) using yeast as a host and expressing a large amount of P-450c in yeast (JP 61-88878).

チトクロムＰ−４５０ｃは酵母ミクロソームにおいて酵
母NADPH−チトクロムＰ−４５０還元酵素と連携して電
子伝達系を形成し、一原子酸素添加反応を示す。Cytochrome P-450c forms an electron transfer system in yeast microsomes in cooperation with yeast NADPH-cytochrome P-450 reductase and exhibits a monoatomic oxygenation reaction.

また、本発明者らは、Ｐ−４５０ｃ発現酵母菌株を用い
てアセトアニリドのパラ位を水酸化するアセトアミノフ
ェンの製法を発明した（特開昭60-139128)。その他、Ｐ
−４５０ｃを発現する酵母菌体あるいは酵母菌体から取
得したＰ−４５０ｃ自体を酸化反応工程や産業排水中の
有機化合物の除去に反応することが可能である。The present inventors have also invented a method for producing acetaminophen by hydroxylating the para position of acetanilide using a P-450c-expressing yeast strain (JP-A-60-139128). Other, P
Yeast cells expressing -450c or P-450c itself obtained from the yeast cells can be reacted in the oxidation reaction step or removal of organic compounds in industrial wastewater.

本発明者らは、Ｐ−４５０遺伝子について更に研究を重
ねた結果、従来のＰ−４５０とは異なる特異な性質を有
するキメラＰ−４５０遺伝子を２以上のＰ−４５０遺伝
子から構築することに成功し、更にこのキメラＰ−４５
０遺伝子を酵母菌体内で発現させる発現用プラスミド及
び当該プラスミドを菌体内に保持し該キメラＰ−４５０
を生産する酵母の製造に成功した。As a result of further research on the P-450 gene, the present inventors have succeeded in constructing a chimeric P-450 gene having two or more P-450 genes having a unique property different from that of conventional P-450. In addition, this chimera P-45
Expression plasmid for expressing 0 gene in yeast cells and the chimeric P-450 containing the plasmid in the cells
Succeeded in producing a yeast that produces

本発明により得られる酵母は、優れた酸化活性を有する
キメラＰ−４５０を生産し、この酵母菌体自身あるいは
キメラＰ−４５０を各種の酸化反応にバイオリアクター
として使用することが可能である。The yeast obtained by the present invention produces a chimeric P-450 having excellent oxidative activity, and the yeast cell itself or the chimeric P-450 can be used as a bioreactor for various oxidation reactions.

本発明のキメラＰ−４５０遺伝子は、例えば、ＨＲ２領
域(J.Biochem.,93p807-817,1983)を他の分子種のＰ−４
５０遺伝子の同領域で変換することにより製造すること
ができる。The chimeric P-450 gene of the present invention includes, for example, the HR2 region (J. Biochem., 93p807-817, 1983) of P-4 of other molecular species.
It can be produced by converting in the same region of 50 genes.

その一例として、ラット肝チトクロムＰ−４５０ｃ遺伝
子のＨＲ２領域を含むＣ末端領域をラット肝チトクロム
Ｐ−４５０ｄの同領域に置き換えることにより製造した
図１に記載した塩基配列およびアミノ酸配列で特定され
るキメラＰ−４５０遺伝子（Ｐ−４５０ccd遺伝子と呼
ぶ）を挙げることができる。本発明のキメラＰ−４５０
ccd遺伝子は、例えば、Ｐ−４５０ｃ遺伝子を含むプラ
スミドpTF1、pTF2,pAMClなど（特開昭61-52284)及びＰ−
４５０ｄをコードする領域を含むプラスミドpTZ330〔J.
Biochem.,96,793-804,(1984)に記載の方法で製造するこ
とができる〕からそれぞれ必要部分を分離しＰ−４５０
ｃ遺伝子のＨＲ２領域をＰ−４５０ｄの領域で置き換え
ることにより製造することができる。その具体的例とし
ては図２に本発明のキメラＰ−４５０遺伝子を含むプラ
スミドpACCD1の構築の概要を示した。As an example thereof, a chimera specified by the nucleotide sequence and amino acid sequence shown in FIG. 1 produced by replacing the C-terminal region containing the HR2 region of rat liver cytochrome P-450c gene with the same region of rat liver cytochrome P-450d. P-450 gene (referred to as P-450ccd gene) can be mentioned. Chimera P-450 of the present invention
Examples of the ccd gene include plasmids pTF1, pTF2, pAMCl containing the P-450c gene (JP-A-61-52284) and P-
A plasmid pTZ330 [J.
Biochem., 96, 793-804, (1984).]
It can be produced by replacing the HR2 region of the c gene with the region of P-450d. As a specific example, FIG. 2 shows an outline of the construction of the plasmid pACCD1 containing the chimeric P-450 gene of the present invention.

本発明のキメラＰ−４５０遺伝子を保持する酵母発現用
プラスミドは、例えば、酵母アルコールデヒドロゲナー
ゼＩプロモーターと同ターミネーターを保持する酵母発
現用ベクタープラスミドpAAH5(Washington Research Fo
undationから入手、Methods in Enzymology,101 part C
pl92-210,Ammererらの方法により製造できる。なお、
酵母ADH1遺伝子のプロモーターは、Washington Researc
h Foundationの米国特許第229,733に含まれており、米
国において工業的，商業的目的で使用する場合は権利者
からの権利許諾を必要とする。）や酵母発現ベクターpJ
DB219〔Nature,275,104(1979)〕等の酵母発現ベクター
に上述のように製造したキメラＰ−４５０遺伝子を組み
込むことにより製造することができる。この場合におい
て、酵母発現ベクター特に限定されるものではなく、ま
た、使用するプロモーターやターミネーターについて
も、酵母内で効率良く機能するプロモーター、ターミネ
ーターであればよく、これらに限定されるものでない。
また、プラスミド上のプロモーター、キメラチトクロム
Ｐ−４５０遺伝子、ターミネーター以外の構造も限定さ
れるものでなく、酵母内で安定に保持されるものであれ
ばよい。The yeast expression plasmid carrying the chimeric P-450 gene of the present invention is, for example, a yeast expression vector plasmid pAAH5 (Washington Research FoL) which holds the yeast alcohol dehydrogenase I promoter and the terminator.
Obtained from undation, Methods in Enzymology, 101 part C
It can be produced by the method of pl92-210, Ammerer et al. In addition,
The promoter of the yeast ADH1 gene is the Washington Researc
Included in US Foundation Patent No. 229,733 of the h Foundation and requires permission from the right holder for industrial or commercial use in the United States. ) Or yeast expression vector pJ
It can be produced by incorporating the chimeric P-450 gene produced as described above into a yeast expression vector such as DB219 [Nature, 275 , 104 (1979)]. In this case, the yeast expression vector is not particularly limited, and the promoter and terminator used may be any promoter and terminator that function efficiently in yeast, and are not limited thereto.
The structure other than the promoter on the plasmid, the chimeric cytochrome P-450 gene, and the terminator is not limited as long as it is stably retained in yeast.

キメラＰ−４５０ccd遺伝子を保持する酵母発現用プラ
スミドpACCD1により形質転換された酵母菌株における菌
体当たり、あるいはＰ−４５０一分子当たりのアセトア
ニリドのパラ位水酸化活性は、従来のプラスミドpAMC1
によって形質転換された酵母菌株の約３倍であり、バイ
オリアクターとして有用性が高いことがわかる。The para-hydroxylation activity of acetanilide per cell in a yeast strain transformed with the yeast expression plasmid pACCD1 carrying the chimeric P-450ccd gene or per P-450 molecule is determined by the conventional plasmid pAMC1.
It is about 3 times that of the yeast strain transformed by, and it can be seen that it is highly useful as a bioreactor.

また、本発明の酵母菌体は、キメラチトクロムＰ−４５
０遺伝子を含むプラスミドにより、アルカリ金属法、あ
るいはプロトプラスト法などでサッカロミセス属に属す
る酵母を形質転換することによって得られる。サッカロ
ミセス・セレビシェーAH22株を用いることができるが、
この株に限定されるものではない。Further, the yeast cell of the present invention is a chimeric cytochrome P-45.
It can be obtained by transforming a yeast belonging to the genus Saccharomyces by the alkali metal method, the protoplast method or the like with the plasmid containing 0 gene. Saccharomyces cerevisiae AH22 strain can be used,
It is not limited to this strain.

以下に実施例を挙げ本発明を更に詳細に説明する。本発
明は、以下の実施例のみに限定されるものではなく、本
発明の技術分野に於ける通常の変更をすることができ
る。Hereinafter, the present invention will be described in more detail with reference to examples. The present invention is not limited to the following examples, and can be modified in a usual manner in the technical field of the present invention.

実施例１プラスミドpACCD1の構築：プラスミドpACCD1を図２にその概要を示した方法により
構築した。Example 1 Construction of plasmid pACCD1: Plasmid pACCD1 was constructed by the method outlined in FIG.

約５μｇのプラスミドpTF1（特開昭61-88878に記載の方
法で製造）を各々２０ユニットの制限酵素StuI、Hind II
Iと混合し１０mMトリスー塩酸（pH7.5)、５０mM NaCl中
で３７℃１時間反応した後、0.6％低融点アガロースゲ
ル電気泳動を行い、約3.9kbのＤＮＡ断片を含むゲルを
切り出してこれを約６５℃で５分間加熱した。融解した
ゲルに２倍容のＴＥ緩衝液〔１０mMトリスー塩酸、0.5m
M EDTA(pH8.0)〕を加え、次にＴＥ緩衝液で飽和したフ
ェノールを等量加えて攪拌した。About 5 μg of plasmid pTF1 (manufactured by the method described in JP-A-61-88878) was added to each of 20 units of restriction enzymes StuI and HindII.
After mixing with I and reacting in 10 mM Tris-hydrochloric acid (pH 7.5), 50 mM NaCl for 1 hour at 37 ° C, 0.6% low melting point agarose gel electrophoresis is performed, and a gel containing a DNA fragment of about 3.9 kb is cut out and this is used. Heated at about 65 ° C. for 5 minutes. 2 volumes of TE buffer [10 mM Tris-HCl, 0.5 m
M EDTA (pH 8.0)] was added, and then an equal amount of phenol saturated with TE buffer was added and stirred.

遠心分離後、上層を分取し、２倍容のエタノールを加え
て沈澱したＤＮＡを回収した。以後のＤＮＡ断片の回収
はすべてこの方法で行った。After centrifugation, the upper layer was separated and 2 times volume of ethanol was added to collect the precipitated DNA. All subsequent recovery of DNA fragments was performed by this method.

約3.9kbのＤＮＡ断片約１００ngに１ユニットのアルカ
リホスファターゼを添加し５０mMトリスー塩酸（pH8.0)
中で６０℃１時間反応させた後、以下の配列を有する合
成リンカーＤＮＡ約１００ngおよび５ユニットのＴ４Ｄ
ＮＡリガーゼを混合し、６６mMトリス塩酸（pH7.6)、6.6
mM MgCl₂、１０mMＤＴＴ、１mMＡＴＰ中で１６℃、１４
時間反応させた。1 unit of alkaline phosphatase was added to about 100 ng of a DNA fragment of about 3.9 kb, and 50 mM Tris-HCl (pH 8.0) was added.
After reacting for 1 hour at 60 ° C. in a synthetic linker DNA having about 100 ng of the following sequence and 5 units of T4D
NA ligase was mixed, 66 mM Tris-HCl (pH 7.6), 6.6
mM MgCl ₂ , 10 mM DTT, 1 mM ATP at 16 ° C., 14
Reacted for hours.

CCTGGCCACGCTTCTCCAAGTGA GGACCGGTGCGAAGAGGTTCACTTCGA 反応後の混液により大腸菌ＤＨ１（Ｆ⁻，recAl,endAl,
gyrA96、thi-l、hsdR17、supE44、λ⁻）を形質転換し、１
００μｇ／mlのアンピリシンを含むプレートに広げ出現
するコロニーからプラスミドＤＮＡを単離し、Hind II
I、あるいはStuIで切断してこれら両制限酵素の切断部
位が新たに生成したことを確認し、得られたプラスミド
をpTFcccと名付けた。CCTGGCCACGCTTCTCCAAGTGA GGACCGGTGCGAAGAGGTTCACTTCGA E. coli DH1 (F ⁻ , recAl, endAl,
gyrA96, thi-l, hsdR17, supE44, λ ⁻ ) and transformed into 1
Plasmid DNA was isolated from the colonies that appeared by spreading on a plate containing 00 μg / ml of ampicillin, and Hind II
It was confirmed by cutting with I or StuI that new cleavage sites for these restriction enzymes were newly generated, and the resulting plasmid was named pTFccc.

次に、Ｐ−４５０ｄをコードする領域を含む公知のプラ
スミドpTZ330〔J.Biochem.,96,p793-804(1984)〕をStuI
で切断して得られた約４２０bpのＤＮＡ断片約１００ng
と、pTFccをStuIで断片した後、アルカリホスファター
ゼで処理をしたＤＮＡ約１００ngを混合してリガーゼ反
応を行った。反応混液により大腸菌ＤＨ１株を形質転換
し、得られたコロニーからプラスミドＤＮＡを調製し、
次に、５００ngのプラスミドＤＮＡに２ユニットのPvu
IIを加えて１０mMトリスー塩酸（（pH7.5)、５０mM NaC
l中で３７℃１時間反応させた後、１％アガロースゲル
電気泳動してそのパターンから、４２０bpのＤＮＡ断片
の挿入方向を確認し、正方向に挿入されたプラスミドを
pTFccdと名付けた。Next, a known plasmid pTZ330 [J. Biochem., 96, p793-804 (1984)] containing a region encoding P-450d was added to StuI.
Approximately 100 ng of a DNA fragment of approximately 420 bp obtained by digestion with
Then, pTFcc was fragmented with StuI, and about 100 ng of DNA treated with alkaline phosphatase was mixed to carry out a ligase reaction. Escherichia coli DH1 strain was transformed with the reaction mixture, and plasmid DNA was prepared from the obtained colonies.
Then add 2 units of Pvu to 500ng of plasmid DNA.
II was added to 10 mM Tris-hydrochloric acid ((pH7.5), 50 mM NaC
After reacting at 37 ° C for 1 hour in 1 liter, 1% agarose gel electrophoresis was performed to confirm the insertion direction of the 420 bp DNA fragment from the pattern, and the plasmid inserted in the forward direction was confirmed.
It was named pTFccd.

次に、約１００ngのpTFccdに２ユニットのSalIを加え、
１０mMＭトリスー塩酸（pH7.5)、１５０mM NaCl中で３
７℃１時間反応させた後、５ユニットのＤＮＡポリメラ
ーゼＩKlenow酵素を加え、６７mMリン酸カルシウム（pH
7.4）、６．７mM MgCl₂、1mM2−メルカプトエタノール、
２５μＭｄＡＴＰ、２５μＭｄＴＴＰ、２５μＭｄＧＴ
Ｐ、２５μＭｄＣＴＰ中で、２５℃、１時間反応させ、
更にアルカリホスファターゼ処理を施した。これに約５
００ngのHind IIIリンカーを加えてリガーゼ反応を行っ
た。反応混液により大腸菌ＤＨ１を形質転換し得られた
コロニーからプラスミドＤＮＡを調製しHind IIIで切断
した後、１％アガロースゲル電気泳動して約1.6kbのＤ
ＮＡ断片の存在を確認した。得られたプラスミドをpTFc
cd(H)と名付けた。Next, add 2 units of SalI to about 100ng of pTFccd,
3 in 10 mM Tris-HCl (pH 7.5), 150 mM NaCl
After reacting at 7 ° C for 1 hour, 5 units of DNA polymerase I Klenow enzyme was added, and 67 mM calcium phosphate (pH
7.4), 6.7 mM MgCl ₂ , 1 mM _2- mercaptoethanol,
25 μMdATP, 25 μMdTTP, 25 μMdGT
P, reacted in 25 μM dCTP at 25 ° C. for 1 hour,
Furthermore, alkaline phosphatase treatment was performed. About 5 to this
Ligase reaction was performed by adding 00 ng of Hind III linker. Escherichia coli DH1 was transformed with the reaction mixture to prepare a plasmid DNA from the obtained colony, which was cleaved with Hind III and electrophoresed on 1% agarose gel to give D of about 1.6 kb.
The presence of NA fragment was confirmed. The resulting plasmid was designated pTFc
I named it cd (H).

pTFccd(H)をHind IIIで切断し1.6kbのＤＮＡ断片を回収
した。pTFccd (H) was cleaved with HindIII to recover a 1.6 kb DNA fragment.

次に、酵母発現ベクターpAAH５約１００ngをHind IIIで
切断し、アルカリホスファターゼ処理を行った後、1.6k
bのＤＮＡ断片約２００ngと混合し、リガーゼ反応を行
った後、反応混液により大腸菌ＤＨ１を形質転換し、得
られたコロニーからプラスミドＤＮＡを調製した。約５
００ngのプラスミドＤＮＡに２ユニットのBamHI、StuI
を加え１０mMトリスー塩酸（pH7.5)、１００mM NaCl
中、３７℃で１時間反応させた後、１％アガロースゲル
電気泳動し、そのパターンから1.6kbのＤＮＡ断片の挿
入方向を確認し、正方向に挿入されたプラスミドをpACC
D1と名付けた。Next, about 100 ng of the yeast expression vector pAAH5 was cleaved with Hind III and treated with alkaline phosphatase, and then 1.6 k
After mixing with about 200 ng of the DNA fragment of b and carrying out a ligase reaction, Escherichia coli DH1 was transformed with the reaction mixture, and a plasmid DNA was prepared from the obtained colony. About 5
2 units of BamHI and StuI in 00ng of plasmid DNA
10 mM Tris-hydrochloric acid (pH 7.5), 100 mM NaCl
After reacting at 37 ° C for 1 hour in medium, 1% agarose gel electrophoresis was performed, the insertion direction of the 1.6 kb DNA fragment was confirmed from the pattern, and the plasmid inserted in the forward direction was pACC.
I named it D1.

実施例２：プラスミドpACCD1による酵母の形質転換ＹＰＤ培地（１％Yeast Extract、２％ポリペプトン、
２％グルコース）１mlにサッカロミセス・セレビシェAH
22株を植菌し、３０℃で１８時間振盪した後、遠心分離
により集菌した。得られた菌体を１mlの0.2ＭLiCl溶液
に懸濁した後、再び遠心分離し、得られたペレットに２
０μｌの１ＭLiCl溶液、３０μｌの７０％ポリエチレン
グリコール4000溶液、約１μｇのpACCD1を含む１０μｌ
の溶液を添加した。十分に混合した後、３０℃で１時間
インキュベートし、さらに１４０μｌの減菌水を加えて
攪拌した。この溶液をＳＤ合成培地プレート（２％グリ
コース、0.67％窒素源アミン酸不含、２０μｇ／mlヒス
チジン、２％寒天）の上にまき、３０℃で３日間インキ
ュベートし、pACCD1を保持する形質転換株AH22(pACCD1)
を得た。Example 2: Transformation of yeast with plasmid pACCD1 YPD medium (1% Yeast Extract, 2% polypeptone,
2% glucose) 1 ml to Saccharomyces cerevisiae AH
The 22 strains were inoculated, shaken at 30 ° C. for 18 hours, and then collected by centrifugation. The obtained bacterial cells were suspended in 1 ml of 0.2 M LiCl solution and then centrifuged again.
10 μl containing 0 μl 1M LiCl solution, 30 μl 70% polyethylene glycol 4000 solution, about 1 μg pACCD1
Solution was added. After mixing thoroughly, the mixture was incubated at 30 ° C. for 1 hour, 140 μl of sterilized water was added, and the mixture was stirred. This solution was spread on an SD synthetic medium plate (2% glucose, 0.67% nitrogen source amine acid-free, 20 μg / ml histidine, 2% agar), incubated at 30 ° C. for 3 days, and a transformant retaining pACCD1. AH22 (pACCD1)
Got

実施例３：キメラチトクロムＰ−４５０タンパク質（Ｐ
−４５０ccdと略記する）の定量サッカロミセス・セレビシェAH22(pAMCl)（特開昭61-56
072)と実施例２で得られたサッカロミセス・セレビシェ
AH22(pACCD1)株をＳＤ合成培地（２％グルコース、0.67
％窒素源アミン酸不含、２０μｇ／mlヒスチジン）で各
々1.5X10⁷cells/mlまで培養した後、集菌し、ザイモリ
エース溶液〔1.2Ｍソルビトール、５０mMリン酸カリウ
ム（pH7.2)、１４mM２−メルカプトエタノール、0.4mg/
mlザイモリエース60,000〕に懸濁し、３０℃で３０分イ
ンキュベートした。遠心分離により集めたスフェロプラ
ストに１００℃に熱した緩衝液（１％ＳＤＳ、５０mM T
ris-HCl(pH6.8)、１０％メルカプトエタノール、４０％
グリセロール、0.02％ブロモフェノールブルー、１mMフ
ェニルメチルスルホニルフロリド）を添加して可溶化し
た。遠心分離で得られた上清（約３Ｘ１０^６菌体分）を
１０％ポリアクリルアミドゲルを用いて電気泳動した。
さらに、ゲル中のタンパク質を２５mM Tris-HCl(pH8.
3)、１９２mMグリシン−２０％メタノール中で電気泳動
的にニトロセルロースフィルター上にブロットした。次
に、フィルターをＴＢＳ緩衝液〔５０mM Tris-HCl(pH7.
5)、２００mM NaCl〕に浸した後、３％ゼラチン、0.05
％Tween20を含むＴＢＳ緩衝液中、３７℃で４０分イン
キュベートし、さらに３０μｇの精製抗Ｐ−４５０ｃＩ
ｇＧ、１％ゼラチン、0.05％Tween20を含むＴＢＳ緩衝
液中３７℃で２時間インキュベートした。その後、0.05
％Tween20を含む緩衝液中３７℃で３０分インキュベー
トする操作を４回繰り返した後、３％ゼラチン、0.05％
Tween20を含むＴＢＳ緩衝液中３７℃で２０分インキュ
ベートした。次に、２μＣｉの〔¹²⁵I〕−プロテイン
Ａ、１％ゼラチン、0.05％Tween20を含むＴＢＳ緩衝液
中、３７℃で７０分インキュベートした後、0.05％Twee
n20を含む緩衝液中、３７℃で３０分インキュベートす
る操作を４回繰り返した。Example 3: Chimeric cytochrome P-450 protein (P
-450 ccd) Saccharomyces cerevisiae AH22 (pAMCl) (JP-A-61-56)
072) and Saccharomyces cerevisiae obtained in Example 2.
AH22 (pACCD1) strain was added to SD synthetic medium (2% glucose, 0.67
% Nitrogen source amine acid-free, 20 μg / ml histidine), each was cultivated to 1.5 × 10 ⁷ cells / ml, and then the cells were collected, and zymolyce solution [1.2 M sorbitol, 50 mM potassium phosphate (pH 7.2), 14 mM 2-mercapto was used. Ethanol, 0.4 mg /
ml Zymo Ace 60,000], and the mixture was incubated at 30 ° C. for 30 minutes. Buffer solution (1% SDS, 50 mM T) heated to 100 ° C was added to the spheroplasts collected by centrifugation.
ris-HCl (pH6.8), 10% mercaptoethanol, 40%
Glycerol, 0.02% bromophenol blue, 1 mM phenylmethylsulfonyl fluoride) was added to solubilize. The supernatant obtained by centrifugation (approximately 3 × 10 ⁶ bacterial cells) was electrophoresed on a 10% polyacrylamide gel.
Furthermore, the protein in the gel was added to 25 mM Tris-HCl (pH 8.
3), electrophoretically blotted onto nitrocellulose filters in 192 mM glycine-20% methanol. Next, the filter was washed with TBS buffer [50 mM Tris-HCl (pH 7.
5), 200 mM NaCl] and then 3% gelatin, 0.05
Incubated in TBS buffer containing% Tween 20 at 37 ° C. for 40 minutes, and further added 30 μg of purified anti-P-450cI.
Incubated in TBS buffer containing gG, 1% gelatin, 0.05% Tween 20 for 2 hours at 37 ° C. Then 0.05
After incubating for 30 minutes at 37 ° C in a buffer containing% Tween20 4 times, 3% gelatin, 0.05%
Incubated in TBS buffer containing Tween 20 for 20 minutes at 37 ° C. Next, after incubating at 37 ° C. for 70 minutes in a TBS buffer containing 2 μCi of [ ¹²⁵ I] -Protein A, 1% gelatin and 0.05% Tween 20, 0.05% Twee was used.
The operation of incubating at 37 ° C. for 30 minutes in a buffer containing n20 was repeated 4 times.

フィルターを風乾燥した後、オートラジオグラフィーを
行ったところ、バンドの濃さからAH22(pACCD1)株は菌体
当たり５〜８Ｘ１０^５分子のＰ−４５０ccdで生産して
いることが推定され、AH22(pAMCl)株におけるＰ−４５
０ｃの生産量とほぼ同程度であることがわかった。After air-drying the filter, autoradiography was performed, and it was estimated that the strain AH22 (pACCD1) was produced with 5-8 × 10 ⁵ molecules of P-450ccd per cell, based on the band density. p-45 in the pAMCl) strain
It was found that the production was almost the same as that of 0c.

実施例４：ヘムを含有するＰ−４５０ccdの定量 AH22(pACCD1)株の培養液（ＳＤ合成培地、菌体濃度約1.
5X10⁷cells/ml)100mlを集菌し、１０mlの１００mMリン
酸カリウム緩衝液（pH7.0)に懸濁した後、遠心分離し
た。得られたペレットを新たに２mlの１００mMリン酸カ
リウム緩衝液（pH7.0)に懸濁し２本のキュベットに１ml
ずつ分注した。サンプル側のキュベットに一酸化炭素を
吹き込んだ後、両キュベット内にジチオナイト５〜１０
mgを添加し、攪拌した後、２０分間放置した。その後、
キュベット内の液を攪拌して４００〜５００nmの差スペ
クトルを測定し、△Ｅ447-490＝91mM^-1cm^-1という大
村、佐藤らの値を元にしてＰ−４５０ccd濃度を算出し
た。Example 4: Quantification of P-450 ccd containing heme AH22 (pACCD1) strain culture medium (SD synthetic medium, cell concentration of about 1.
100 ml of 5 × 10 ⁷ cells / ml) was collected, suspended in 10 ml of 100 mM potassium phosphate buffer (pH 7.0), and then centrifuged. The obtained pellet was newly suspended in 2 ml of 100 mM potassium phosphate buffer solution (pH 7.0), and 1 ml was put in two cuvettes.
We dispensed each. After blowing carbon monoxide into the cuvette on the sample side, dithionites 5-10 were placed in both cuvettes.
After adding mg and stirring, it was left for 20 minutes. afterwards,
The liquid in the cuvette was stirred and the difference spectrum at 400 to 500 nm was measured, and the P-450 ccd concentration was calculated based on the value of ΔE447-490 = 91 mM ^-1 cm ^{-1 by} Omura and Sato.

その結果、AH22(pACCD1)は菌体あたり３〜４Ｘ１０^５分
子のヘムタンパク質を産生していることがわかり、AH22
(pAMCl)株におけるＰ−４５０ｃの生産とほぼ同程度で
あることがわかった。As a result, it was found that AH22 (pACCD1) produces 3 to 4 × 10 ⁵ molecules of heme protein per cell,
It was found that the production was almost the same as that of P-450c in the (pAMCl) strain.

実施例５：菌体のアセトアニリドのパラ位水酸化による
アセトアミノフェン生成量の測定ＳＤ合成培地中で約1.4X10⁷cells/mlまで培養したAH22
(pAMCl)株、AH22(pACCD1)株、それぞれの培養液中に1.5
Ｍアセトアニリド（メタノール溶液）を添加し、終濃度
２５mMとした。その後、３０℃で振盪培養（１２０cycl
e/min)し、１時間ごとに少量ずつ分取し、遠心分離で得
た上清をHPLCで分析し生成したアセトアミノフェンを定
量した。HPLCの条件を以下に示す。Example 5: Measurement of acetaminophen production by para-hydroxylation of bacterial acetanilide AH22 cultured to approximately 1.4 × 10 ⁷ cells / ml in SD synthesis medium.
(pAMCl) strain, AH22 (pACCD1) strain, 1.5 in each culture
M acetanilide (methanol solution) was added to give a final concentration of 25 mM. Then, shake culture (120 cycl
e / min), and aliquots were taken in small amounts every hour, and the supernatant obtained by centrifugation was analyzed by HPLC to quantify the acetaminophen produced. The HPLC conditions are shown below.

カラム：μBondapak C18（Φ４Ｘ３００mm）溶媒；メタノール：水：酢酸＝１５：８４：１検出；Ａ２４５ｎｍ流速；2.0ml/min 温度；室温（２０〜２５℃）図３に示したようにAH22(pACCD1)株の菌体あたりのアセ
トアニリドのパラ位水酸化活性はAH22(pAMCl)株の約３
倍であった。Column: μBondapak C18 (Φ4 × 300 mm) solvent; methanol: water: acetic acid = 15: 84: 1 detection; A245nm flow rate; 2.0 ml / min temperature; room temperature (20-25 ° C.) As shown in FIG. 3, AH22 (pACCD1) strain The para-hydroxylation activity of acetanilide per cell of Escherichia coli is about 3 that of AH22 (pAMCl) strain
It was double.

[Brief description of drawings]

図１（その１および２）は、本発明のキメラＰ−４５０
遺伝子Ｐ−４５０ccd ｃＤＮＡのコーディング領域の
塩基配列およびそれから推定されるアミノ酸配列を示す
図である。図２は、本発明のプラスミドpACCD1の構築の概要を表す
図である。図３は、AH22(pACCD1)株、AH22(pAMCl)株の培養液中の
生成アセトアミノフェン濃度（nmol/ml)及び菌体濃度
（Ｘ１０⁸cells/ml)の経時変化を示したものである。
●、○はそれぞれAH22(pACCD1)株のアセトアミノフェン
濃度及び菌体濃度を示す。 ▲、△はそれぞれAH22(pAMCl)株のアセトアミノフェン
濃度及び菌体濃度を示す。FIG. 1 (Nos. 1 and 2) shows the chimeric P-450 of the present invention.
It is a figure which shows the base sequence of the coding region of gene P-450ccd cDNA, and the amino acid sequence deduced from it. FIG. 2 is a diagram showing an outline of construction of the plasmid pACCD1 of the present invention. FIG. 3 shows the time-dependent changes in the concentration of acetaminophen (nmol / ml) produced and the concentration of cells (X10 ⁸ cells / ml) in the culture solution of AH22 (pACCD1) strain and AH22 (pAMCl) strain. .
● and ○ indicate acetaminophen concentration and bacterial cell concentration of AH22 (pACCD1) strain, respectively. ▲ and △ represent the acetaminophen concentration and the bacterial cell concentration of the AH22 (pAMCl) strain, respectively.

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.⁵ 識別記号庁内整理番号ＦＩ技術表示箇所Ｃ１２Ｒ 1:865） (Ｃ１２Ｎ 9/02 Ｃ１２Ｒ 1:865） (54)【発明の名称】複数のチトクロムＰ−450遺伝子から構築したキメラチトクロムＰ−450遺伝子、およびそれを含む酵母内発現用プラスミドとその製造法、ならびに菌体内にそれらのプラスミドを保有する酵母菌株─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12R 1: 865) (C12N 9/02 C12R 1: 865) (54) [Title of invention] Multiple Chimeric cytochrome P-450 gene constructed from cytochrome P-450 gene, plasmid for expression in yeast containing the same, method for producing the same, and yeast strain having the plasmid in bacterial cells

Claims

[Claims]

1. A gene encoding rat liver cytochrome P450c represented by the following amino acid sequence (I), which has a C-terminal containing a heme-binding region (HR2 region) consisting of the following amino acid sequence (II) in the gene: C containing a heme binding region (HR2 region) consisting of the following amino acid sequence (III) of rat liver cytochrome P450d gene
Chimeric cytochrome P-450 gene replaced in the terminal region a) Amino acid sequence (I) Met Pro Ser Val Tyr Gly Phe Pro Ala Phe Thr Ser Al
a Thr Glu Leu Leu Leu Ala Val Thr Thr Phe Cys Leu
Gly Phe Trp Val Val Arg Val Thr Arg Thr Trp Val Pr
o Lys Gly Leu Lys Ser Pro Pro Gly Pro Trp Gly Leu
Pro Phe Ile Gly His Val Leu Thr Leu Gly Lys Asn Pr
o His Leu Ser Leu Thr Lys Leu Ser Gln Gln Tyr Gly
Asp Val Leu Gln Ile Arg Ile Gly Ser Thr Pro Val Va
l Val Leu Ser Gly Leu Asn Thr Ile Lys Gln Ala Leu
Val Lys Gln Gly Asp Asp Phe Lys Gly Arg Pro Asp Le
u Tyr Ser Phe Thr Leu Ile Ala Asn Gly Gln Ser Met
Thr Phe Asn Pro Asp Ser Gly Pro Leu Trp Ala Ala Ar
g Arg Arg Leu Ala Gln Asn Ala Leu Lys Ser Phe Ser
Ile Ala Ser Asp Pro Thr Leu Ala Ser Ser Cys Tyr Le
u Glu Glu His Val Ser Lys Glu Ala Glu Tyr Leu Ile
Ser Lys Phe Gln Lys Leu Met Ala Glu Val Gly His Ph
e Asp Pro Phe Lys Tyr Leu Val Val Ser Val Ala Asn
Val Ile Cys Ala Ile Cys Phe Gly Arg Arg Tyr Asp Hi
s Asp Asp Gln Glu Leu Leu Ser Ile Val Asn Leu Ser
Asn Glu Phe Gly Glu Val Thr Gly Ser Gly Tyr Pro Al
a Asp Phe Ile Pro Ile Leu Arg Tyr Leu Pro Asn Ser
Ser Leu Asp Ala Phe Lys Asp Leu Asn Lys Lys Phe Ty
r Ser Phe Met Lys Lys Leu Ile Lys Gln His Tyr Arg
Thr Phe Glu Lys Gly His Ile Arg Asp Ile Thr Asp Se
r Leu Ile Glu His Cys Gln Asp Arg Arg Leu Asp Glu
Asn Ala Asn Val Gln Leu Ser Asp Asp Lys Val Ile Th
r Ile Val Phe Asp Leu Phe Gly Ala Gly Phe Asp Thr
Ile Thr Thr Ala Ile Ser Trp Ser Leu Met Tyr Leu Va
l Thr Asn Pro Arg Ile Gln Arg Lys Ile Gln Glu Glu
Leu Asp Thr Val Ile Gly Arg Asp Arg Gln Pro Arg Le
u Ser Asp Arg Pro Gln Leu Pro Tyr Leu Glu Ala Phe
Ile Leu Glu Thr Phe Arg His Ser Ser Phe Val Pro Ph
e Thr Ile Pro His Ser Thr Ile Arg Asp Thr Ser Leu
Asn Gly Phe Tyr Ile Pro Lys Gly His Cys Val Phe Va
l Asn Gln Trp Gln Val Asn His Asp Gln Glu Leu Trp
Gly Asp Pro Asn Glu Phe Arg Pro Glu Arg Phe Leu Th
r Ser Ser Gly Thr Leu Asp Lys His Leu Ser Glu Lys
Val Ile Leu Phe Gly Leu Gly Lys Arg Lys Cys Ile Gl
y Glu Thr Ile Gly Arg Leu Glu Val Phe Leu Phe Leu
Ala Ile Leu Leu Gln Gln Met Glu Phe Asn Val Ser Pr
o Gly Glu Lys Val Asp Met Thr Pro Ala Tyr Gly Leu
Thr Leu Lys His Ala Arg Cys Glu His Phe Gln Val Gl
n Met Arg Ser Ser Gly Pro Gln His Leu Gln Ala b) Amino acid sequence (II) Phe Gly Leu Gly Lys Arg Lys Cys Ile Gly Glu Thr Il
e Gly Arg Leu Glu Val Phe Leu Phe c) Amino acid sequence (III) Phe Gly Leu Gly Lys Arg Arg Cys Ile Gly Glu Ile Pr
o Ala Lys Trp Glu Val Phe Leu Phe

2. The chimeric cytochrome P-450 gene according to claim 1, which encodes the following amino acid sequence: Met Pro Ser Val Tyr Gly Phe Pro Ala Phe 10 Thr Ser Ala Thr Glu Leu Leu Leu Leu Ala Val 20 Thr Thr Phe Cys Leu Gly Phe Trp Val Val 30 Arg Val Thr Arg Thr Trp Val Pro Lys Gly 40 Leu Lys Ser Pro Pro Gly Pro Trp Gly Leu 50 Pro Phe Met Gly His Val Leu Thr Leu Gly 60 Lys Asn Pro His Leu Ser Leu Thr Lys Leu 70 Ser Gln Gln Tyr Gly Asp Val Leu Gln Ile 80 Arg Ile Gly Ser Thr Pro Val Val Val Leu 90 Ser Gly Leu Asn Thr Ile Lys Gln Ala Leu 100 Val Lys Gln Gly Asp Asp Phe Lys Gly Arg 110 Pro Asp Leu Tyr Ser Phe Thr Leu Ile Ala 120 Asn Gly Gln Ser Met Thr Phe Asn Pro Asp 130 Ser Gly Pro Leu Trp Ala Ala Arg Arg Arg 140 Leu Ala Gln Asn Ala Leu Lys Ser Phe Ser 150 Ile Ala Ser Asp Pro Thr Leu Ala Ser Ser 160 Cys Tyr Leu Glu Glu His Val Ser Lys Glu 170 Ala Glu Tyr Leu Ile Ser Lys Phe Gln Lys 180 Leu Met Ala Glu Val Gly His Phe Asp Pro 19 0 Phe Lys Tyr Leu Val Val Ser Val Ala Asn 200 Val Ile Cys Ala Ile Cys Phe Gly Arg Arg 210 Tyr Asp His Asp Asp Gln Glu Leu Leu Ser 220 Ile Val Asn Leu Ser Asn Glu Phe Gly Glu 230 Val Thr Gly Ser Gly Tyr Pro Ala Asp Phe 240 Ile Pro Ile Leu Arg Tyr Leu Pro Asn Ser 250 Ser Leu Asp Ala Phe Lys Asp Leu Asn Lys 260 Lys Phe Tyr Ser Phe Met Lys Lys Leu Ile 270 Lys Glu His Tyr Arg Thr Phe Glu Lys Gly 280 His Ile Arg Asp Ile Thr Asp Ser Leu Ile 290 Glu His Cys Gln Asp Arg Arg Leu Asp Glu 300 Asn Ala Asn Val Gln Leu Ser Asp Asp Lys 310 Val Ile Thr Ile Val Phe Asp Leu Phe Gly 320 Ala Gly Phe Asp Thr Ile Thr Thr Ala Ile 330 Ser Trp Ser Leu Met Tyr Leu Val Thr Asn 340 Pro Arg Ile Gln Arg Lys Ile Gln Glu Glu 350 Leu Asp Thr Val Ile Gly Arg Asp Arg Gln 360 Pro Arg Leu Ser Asp Arg Pro Gln Leu Pro 370 Tyr Leu Glu Ala Phe Ile Leu Glu Ile Tyr 380 Arg Tyr Thr Ser Phe Val Pro Phe Thr Ile 390 Pro His Ser Thr Thr Arg Asp Thr Ser Leu 400 Asn Gly Phe His Ile Pro Lys Glu Cys Cys 410 Ile Phe Ile Asn Gln Trp Gln Va l Asn His 420 Asp Glu Lys Gln Trp Lys Asp Pro Phe Val 430 Phe Arg Pro Glu Arg Phe Leu Thr Asn Asp 440 Asn Thr Ala Ile Asp Lys Thr Leu Ser Glu 450 Lys Val Met Leu Phe Gly Leu Gly Lys Arg 460 Arg Cys Ile Gly Glu Ile Pro Ala Lys Trp 470 Glu Val Phe Leu Phe Leu Ala Ile Leu Leu 480 His Gln Leu Glu Phe Thr Val Pro Pro Gly 490 Val Lys Val Asp Leu Thr Pro Ser Tyr Gly 500 Leu Thr Met Lys Pro Arg Thr Cys Glu His 510 Val Gln Ala Trp Pro Arg Phe Ser Lys 519

3. The chimeric cytochrome P according to claim 1, which is represented by the following base sequence.
-450 genes

4. A gene encoding rat liver cytochrome P450c represented by the following amino acid sequence (I), which has a C-terminal containing a heme-binding region (HR2 region) consisting of the following amino acid sequence (II) in the gene. The region is defined as the following amino acid sequence of rat liver cytochrome P450d gene.
(III) A yeast expression plasmid containing a chimeric cytochrome P-450 gene replaced with a C-terminal region containing a heme binding region (HR2 region) a) Amino acid sequence (I) Met Pro Ser Val Tyr Gly Phe Pro Ala Phe Thr Ser Al
a Thr Glu Leu Leu Leu Ala Val Thr Thr Phe Cys Leu
Gly Phe Trp Val Val Arg Val Thr Arg Thr Trp Val Pr
o Lys Gly Leu Lys Ser Pro Pro Gly Pro Trp Gly Leu
Pro Phe Ile Gly His Val Leu Thr Leu Gly Lys Asn Pr
o His Leu Ser Leu Thr Lys Leu Ser Gln Gln Tyr Gly
Asp Val Leu Gln Ile Arg Ile Gly Ser Thr Pro Val Va
l Val Leu Ser Gly Leu Asn Thr Ile Lys Gln Ala Leu
Val Lys Gln Gly Asp Asp Phe Lys Gly Arg Pro Asp Le
u Tyr Ser Phe Thr Leu Ile Ala Asn Gly Gln Ser Met
Thr Phe Asn Pro Asp Ser Gly Pro Leu Trp Ala Ala Ar
g Arg Arg Leu Ala Gln Asn Ala Leu Lys Ser Phe Ser
Ile Ala Ser Asp Pro Thr Leu Ala Ser Ser Cys Tyr Le
u Glu Glu His Val Ser Lys Glu Ala Glu Tyr Leu Ile
Ser Lys Phe Gln Lys Leu Met Ala Glu Val Gly His Ph
e Asp Pro Phe Lys Tyr Leu Val Val Ser Val Ala Asn
Val Ile Cys Ala Ile Cys Phe Gly Arg Arg Tyr Asp Hi
s Asp Asp Gln Glu Leu Leu Ser Ile Val Asn Leu Ser
Asn Glu Phe Gly Glu Val Thr Gly Ser Gly Tyr Pro Al
a Asp Phe Ile Pro Ile Leu Arg Tyr Leu Pro Asn Ser
Ser Leu Asp Ala Phe Lys Asp Leu Asn Lys Lys Phe Ty
r Ser Phe Met Lys Lys Leu Ile Lys Glu His Tyr Arg
Thr Phe Glu Lys Gly His Ile Arg Asp Ile Thr Asp Se
r Leu Ile Glu His Cys Gln Asp Arg Arg Leu Asp Glu
Asn Ala Asn Val Gln Leu Ser Asp Asp Lys Val Ile Th
r Ile Val Phe Asp Leu Phe Gly Ala Gly Phe Asp Thr
Ile Thr Thr Ala Ile Ser Trp Ser Leu Met Tyr Leu Va
l Thr Asn Pro Arg Ile Gln Arg Lys Ile Gln Glu Glu
Leu Asp Thr Val Ile Gly Arg Asp Arg Gln Pro Arg Le
u Ser Asp Arg Pro Gln Leu Pro Tyr Leu Glu Ala Phe
Ile Leu Glu Thr Phe Arg His Ser Ser Phe Val Pro Ph
e Thr Ile Pro His Ser Thr Ile Arg Asp Thr Ser Leu
Asn Gly Phe Tyr Ile Pro Lys Gly His Cys Val Phe Va
l Asn Gln Trp Gln Val Asn His Asp Gln Glu Leu Trp
Gly Asp Pro Asn Glu Phe Arg Pro Glu Arg Phe Leu Th
r Ser Ser Gly Thr Leu Asp Lys His Leu Ser Glu Lys
Val Ile Leu Phe Gly Leu Gly Lys Arg Lys Cys Ile Gl
y Glu Thr Ile Gly Arg Leu Glu Val Phe Leu Phe Leu
Ala Ile Leu Leu Gln Gln Met Glu Phe Asn Val Ser Pr
o Gly Glu Lys Val Asp Met Thr Pro Ala Tyr Gly Leu
Thr Leu Lys His Ala Arg Cys Glu His Phe Gln Val Gl
n Met Arg Ser Ser Gly Pro Gln His Leu Gln Ala b) Amino acid sequence (II) Phe Gly Leu Gly Lys Arg Lys Cys Ile Gly Glu Thr Il
e Gly Arg Leu Glu Val Phe Leu Phe c) Amino acid sequence (III) Phe Gly Leu Gly Lys Arg Arg Cys Ile Gly Glu Ile Pr
o Ala Lys Trp Glu Val Phe Leu Phe

5. A yeast expression plasmid Met Pro Ser Val Tyr Gly Phe Pro Ala Phe 10 Thr Ser containing a chimeric cytochrome P-450 gene encoding the following amino acid sequence. Ala Thr Glu Leu Leu Leu Ala Val 20 Thr Thr Phe Cys Leu Gly Phe Trp Val Val 30 Arg Val Thr Arg Thr Trp Val Pro Lys Gly 40 Leu Lys Ser Pro Pro Gly Pro Trp Gly Leu 50 Pro Phe Met Gly His Val Leu Thr Leu Gly 60 Lys Asn Pro His Leu Ser Leu Thr Lys Leu 70 Ser Gln Gln Tyr Gly Asp Val Leu Gln Ile 80 Arg Ile Gly Ser Thr Pro Val Val Val Leu 90 Ser Gly Leu Asn Thr Ile Lys Gln Ala Leu 100 Val Lys Gln Gly Asp Asp Phe Lys Gly Arg 110 Pro Asp Leu Tyr Ser Phe Thr Leu Ile Ala 120 Asn Gly Gln Ser Met Thr Phe Asn Pro Asp 130 Ser Gly Pro Leu Trp Ala Ala Arg Arg Arg 140 Leu Ala Gln Asn Ala Leu Lys Ser Phe Ser 150 Ile Ala Ser Asp Pro Thr Leu Ala Ser Ser 160 Cys Tyr Leu Glu Glu His Val Ser Lys Glu 170 Ala Glu Tyr Leu Ile Ser Lys Phe Gln Lys 180 Leu Me t Ala Glu Val Gly His Phe Asp Pro 190 Phe Lys Tyr Leu Val Val Ser Val Ala Asn 200 Val Ile Cys Ala Ile Cys Phe Gly Arg Arg 210 Tyr Asp His Asp Asp Gln Glu Leu Leu Ser 220 Ile Val Asn Leu Ser Asn Glu Phe Gly Glu 230 Val Thr Gly Ser Gly Tyr Pro Ala Asp Phe 240 Ile Pro Ile Leu Arg Tyr Leu Pro Asn Ser 250 Ser Leu Asp Ala Phe Lys Asp Leu Asn Lys 260 Lys Phe Tyr Ser Phe Met Lys Lys Leu Ile 270 Lys Glu His Tyr Arg Thr Phe Glu Lys Gly 280 His Ile Arg Asp Ile Thr Asp Ser Leu Ile 290 Glu His Cys Gln Asp Arg Arg Leu Asp Glu 300 Asn Ala Asn Val Gln Leu Ser Asp Asp Lys 310 Val Ile Thr Ile Val Phe Asp Leu Phe Gly 320 Ala Gly Phe Asp Thr Ile Thr Thr Ala Ile 330 Ser Trp Ser Leu Met Tyr Leu Val Thr Asn 340 Pro Arg Ile Gln Arg Lys Ile Gln Glu Glu 350 Leu Asp Thr Val Ile Gly Arg Asp Arg Gln 360 Pro Arg Leu Ser Asp Arg Pro Gln Leu Pro 370 Tyr Leu Glu Ala Phe Ile Leu Glu Ile Tyr 380 Arg Tyr Thr Ser Phe Val Pro Phe Thr Ile 390 Pro His Ser Thr Thr Arg Asp Thr Ser Leu 400 Asn Gly Phe His Ile Pro Lys Glu Cys Cy s 410 Ile Phe Ile Asn Gln Trp Gln Val Asn His 420 Asp Glu Lys Gln Trp Lys Asp Pro Phe Val 430 Phe Arg Pro Glu Arg Phe Leu Thr Asn Asp 440 Asn Thr Ala Ile Asp Lys Thr Leu Ser Glu 450 Lys Val Met Leu Phe Gly Leu Gly Lys Arg 460 Arg Cys Ile Gly Glu Ile Pro Ala Lys Trp 470 Glu Val Phe Leu Phe Leu Ala Ile Leu Leu 480 His Gln Leu Glu Phe Thr Val Pro Pro Gly 490 Val Lys Val Asp Leu Thr Pro Ser Tyr Gly 500 Leu Thr Met Lys Pro Arg Thr Cys Glu His 510 Val Gln Ala Trp Pro Arg Phe Ser Lys 519

6. The yeast expression plasmid according to claim 4, which contains a chimeric cytochrome P-450 gene represented by the following nucleotide sequence.

7. A gene encoding rat liver cytochrome P450c represented by the following amino acid sequence (I), which has a C-terminal containing a heme-binding region (HR2 region) consisting of the following amino acid sequence (II) in the gene: C containing a heme binding region (HR2 region) consisting of the following amino acid sequence (III) of rat liver cytochrome P450d gene
Yeast strain carrying a yeast expression plasmid containing the chimeric cytochrome P-450 gene replaced in the terminal region a) Amino acid sequence (I) Met Pro Ser Val Tyr Gly Phe Pro Ala Phe Thr Ser Al
a Thr Glu Leu Leu Leu Ala Val Thr Thr Phe Cys Leu
Gly Phe Trp Val Val Arg Val Thr Arg Thr Trp Val Pr
o Lys Gly Leu Lys Ser Pro Pro Gly Pro Trp Gly Leu
Pro Phe Ile Gly His Val Leu Thr Leu Gly Lys Asn Pr
o His Leu Ser Leu Thr Lys Leu Ser Gln Gln Tyr Gly
Asp Val Leu Gln Ile Arg Ile Gly Ser Thr Pro Val Va
l Val Leu Ser Gly Leu Asn Thr Ile Lys Gln Ala Leu
Val Lys Gln Gly Asp Asp Phe Lys Gly Arg Pro Asp Le
u Tyr Ser Phe Thr Leu Ile Ala Asn Gly Gln Ser Met
Thr Phe Asn Pro Asp Ser Gly Pro Leu Trp Ala Ala Ar
g Arg Arg Leu Ala Gln Asn Ala Leu Lys Ser Phe Ser
Ile Ala Ser Asp Pro Thr Leu Ala Ser Ser Cys Tyr Le
u Glu Glu His Val Ser Lys Glu Ala Glu Tyr Leu Ile
Ser Lys Phe Gln Lys Leu Met Ala Glu Val Gly His Ph
e Asp Pro Phe Lys Tyr Leu Val Val Ser Val Ala Asn
Val Ile Cys Ala Ile Cys Phe Gly Arg Arg Tyr Asp Hi
s Asp Asp Gln Glu Leu Leu Ser Ile Val Asn Leu Ser
Asn Glu Phe Gly Glu Val Thr Gly Ser Gly Tyr Pro Al
a Asp Phe Ile Pro Ile Leu Arg Tyr Leu Pro Asn Ser
Ser Leu Asp Ala Phe Lys Asp Leu Asn Lys Lys Phe Ty
r Ser Phe Met Lys Lys Leu Ile Lys Glu His Tyr Arg
Thr Phe Glu Lys Gly His Ile Arg Asp Ile Thr Asp Se
r Leu Ile Glu His Cys Gln Asp Arg Arg Leu Asp Glu
Asn Ala Asn Val Gln Leu Ser Asp Asp Lys Val Ile Th
r Ile Val Phe Asp Leu Phe Gly Ala Gly Phe Asp Thr
Ile Thr Thr Ala Ile Ser Trp Ser Leu Met Tyr Leu Va
l Thr Asn Pro Arg Ile Gln Arg Lys Ile Gln Glu Glu
Leu Asp Thr Val Ile Gly Arg Asp Arg Gln Pro Arg Le
u Ser Asp Arg Pro Gln Leu Pro Tyr Leu Glu Ala Phe
Ile Leu Glu Thr Phe Arg His Ser Ser Phe Val Pro Ph
e Thr Ile Pro His Ser Thr Ile Arg Asp Thr Ser Leu
Asn Gly Phe Tyr Ile Pro Lys Gly His Cys Val Phe Va
l Asn Gln Trp Gln Val Asn His Asp Gln Glu Leu Trp
Gly Asp Pro Asn Glu Phe Arg Pro Glu Arg Phe Leu Th
r Ser Ser Gly Thr Leu Asp Lys His Leu Ser Glu Lys
Val Ile Leu Phe Gly Leu Gly Lys Arg Lys Cys Ile Gl
y Glu Thr Ile Gly Arg Leu Glu Val Phe Leu Phe Leu
Ala Ile Leu Leu Gln Gln Met Glu Phe Asn Val Ser Pr
o Gly Glu Lys Val Asp Met Thr Pro Ala Tyr Gly Leu
Thr Leu Lys His Ala Arg Cys Glu His Phe Gln Val Gl
n Met Arg Ser Ser Gly Pro Gln His Leu Gln Ala b) Amino acid sequence (II) Phe Gly Leu Gly Lys Arg Lys Cys Ile Gly Glu Thr Il
e Gly Arg Leu Glu Val Phe Leu Phe c) Amino acid sequence (III) Phe Gly Leu Gly Lys Arg Arg Cys Ile Gly Glu Ile Pr
o Ala Lys Trp Glu Val Phe Leu Phe

8. The yeast strain Met Pro Ser Val Tyr Gly Phe Pro according to claim 7, which holds a yeast expression plasmid containing a chimeric cytochrome P-450 gene encoding the following amino acid sequence. Ala Phe 10 Thr Ser Ala Thr Glu Leu Leu Leu Ala Val 20 Thr Thr Phe Cys Leu Gly Phe Trp Val Val 30 Arg Val Thr Arg Thr Trp Val Pro Lys Gly 40 Leu Lys Ser Pro Pro Gly Pro Trp Gly Leu 50 Pro Phe Met Gly His Val Leu Thr Leu Gly 60 Lys Asn Pro His Leu Ser Leu Thr Lys Leu 70 Ser Gln Gln Tyr Gly Asp Val Leu Gln Ile 80 Arg Ile Gly Ser Thr Pro Val Val Val Leu 90 Ser Gly Leu Asn Thr Ile Lys Gln Ala Leu 100 Val Lys Gln Gly Asp Asp Phe Lys Gly Arg 110 Pro Asp Leu Tyr Ser Phe Thr Leu Ile Ala 120 Asn Gly Gln Ser Met Thr Phe Asn Pro Asp 130 Ser Gly Pro Leu Trp Ala Ala Arg Arg Arg 140 Leu Ala Gln Asn Ala Leu Lys Ser Phe Ser 150 Ile Ala Ser Asp Pro Thr Leu Ala Ser Ser 160 Cys Tyr Leu Glu Glu His Val Ser Lys Glu 170 Ala Glu Tyr Leu Ile Ser Lys Phe Gln Lys 180 Leu Met Ala Glu Val Gly His Phe Asp Pro 190 Phe Lys Tyr Leu Val Val Ser Val Ala Asn 200 Val Ile Cys Ala Ile Cys Phe Gly Arg Arg 210 Tyr Asp His Asp Asp Gln Glu Leu Leu Ser 220 Ile Val Asn Leu Ser Asn Glu Phe Gly Glu 230 Val Thr Gly Ser Gly Tyr Pro Ala Asp Phe 240 Ile Pro Ile Leu Arg Tyr Leu Pro Asn Ser 250 Ser Leu Asp Ala Phe Lys Asp Leu Asn Lys 260 Lys Phe Tyr Ser Phe Met Lys Lys Leu Ile 270 Lys Glu His Tyr Arg Thr Phe Glu Lys Gly 280 His Ile Arg Asp Ile Thr Asp Ser Leu Ile 290 Glu His Cys Gln Asp Arg Arg Leu Asp Glu 300 Asn Ala Asn Val Gln Leu Ser Asp Asp Lys 310 Val Ile Thr Ile Val Phe Asp Leu Phe Gly 320 Ala Gly Phe Asp Thr Ile Thr Thr Ala Ile 330 Ser Trp Ser Leu Met Tyr Leu Val Thr Asn 340 Pro Arg Ile Gln Arg Lys Ile Gln Glu Glu 350 Leu Asp Thr Val Ile Gly Arg Asp Arg Gln 360 Pro Arg Leu Ser Asp Arg Pro Gln Leu Pro 370 Tyr Leu Glu Ala Phe Ile Leu Glu Ile Tyr 380 Arg Tyr Thr Ser Phe Val Pro Phe Thr Ile 390 Pro His Ser Thr Thr Arg Asp Thr Ser Leu 400 Asn Gly Phe His Ile Pro Lys Glu Cys Cys 410 Ile Phe Ile Asn Gln Trp Gln Val Asn His 420 Asp Glu Lys Gln Trp Lys Asp Pro Phe Val 430 Phe Arg Pro Glu Arg Phe Leu Thr Asn Asp 440 Asn Thr Ala Ile Asp Lys Thr Leu Ser Glu 450 Lys Val Met Leu Phe Gly Leu Gly Lys Arg 460 Arg Cys Ile Gly Glu Ile Pro Ala Lys Trp 470 Glu Val Phe Leu Phe Leu Ala Ile Leu Leu 480 His Gln Leu Glu Phe Thr Val Pro Pro Gly 490 Val Lys Val Asp Leu Thr Pro Ser Tyr Gly 500 Leu Thr Met Lys Pro Arg Thr Cys Glu His 510 Val Gln Ala Trp Pro Arg Phe Ser Lys 519

9. The yeast strain according to claim 7, which retains a yeast expression plasmid containing a chimeric cytochlorom P-450 gene represented by the following nucleotide sequence.

10. The yeast strain according to claim 7, wherein the yeast strain is Saccharomyces cerevisiae AH22 strain.

11. The yeast strain according to claim 8, wherein the yeast strain is Saccharomyces cerevisiae AH22 strain.

12. The yeast strain according to claim 9, wherein the yeast strain is Saccharomyces cerevisiae AH22 strain.