JPH0813270B2 - Method for producing chimeric cytochrome P-450 - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to a chimeric cytochrome P-
45 0 process for the preparation of.

[0002]

2. Description of the Related Art Cytochrome P-450 (hereinafter P-450
Is a heme protein that exists widely in the living world from microorganisms to mammals, and there are many molecular species. Although each P-450 molecular species has different substrate specificity, they have a monoatomic oxygen addition function in common.
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 P-450c in large amounts in yeast (JP 61-88878). Cytochrome P-450c is a yeast NAD in yeast microsomes.
An electron transfer system is formed in cooperation with PH-cytochrome P-450 reductase, and a monoatomic oxygen addition reaction is shown. The present inventors have also invented a method for producing acetaminophen that hydroxylates the para position of acetanilide using a P-450c-expressing yeast strain (Japanese Patent Laid-Open No. 60-139128). Others, P-450c
P-expressing yeast or P-obtained from yeast
It is possible for 450c itself to react in the oxidation reaction step and the removal of organic compounds in industrial wastewater.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have found that P-4
By making a 50c variant, higher P-45
An attempt was made to create a strain showing 0 activity.

[0004]

SUMMARY OF THE INVENTION The present inventors have proposed a conventional P
Chimera P-45 with unique properties different from -450
0 gene is constructed from two or more P-450 genes, and an expression plasmid for expressing this chimeric P-450 gene in yeast cells and a yeast which holds the plasmid in the cells and produces the chimeric P-450. Succeeded in manufacturing. Then, by utilizing these, a method for producing the above chimeric cytochrome P-450 was established and the present invention was completed. That is, the present invention provides the following amino acid sequence
Coat rat liver cytochrome P450c represented by (I)
The following amino acids in the gene
Includes a heme binding region (HR2 region) consisting of sequence (II)
The C-terminal region of the rat liver cytochrome P450d gene
A heme binding region consisting of the following amino acid sequence (III)
Chimera with a C-terminal region containing (HR2 region)
A yeast expression plasmid containing the tochrome P-450 gene
Chimera characterized by culturing a yeast strain that retains
The present invention relates to a method for producing Tochrome P-450. Is provided.

[0005]

According to the present invention , it has an excellent oxidative activity,
Use as a bioreactor for various oxidation reactions
A chimeric cytochrome P-450 capable of producing easily
I can do it now.

The present invention will be described in more detail below. The chimeric cytochrome P-450 gene used in the present invention has, for example, an HR2 region (J. Biochem., 93.
p807-817, 1983), that is , the following amino acid
Acid sequence (II) The C-terminal region containing the region encoding
It can be produced by converting in the same region of 450 genes. As an example, rat liver cytochrome P-
Chimera P encoding the following amino acid sequence produced by replacing the C-terminal region containing the HR2 region of the 450c gene with the same region of rat liver cytochrome P-450d.
The -450 gene (referred to as P-450ccd gene) is preferred.

[Chemical Formula 3] SEQ ID NO: 1

Further, the gene represented by the following base sequence is preferable.

Embedded image SEQ ID NO: 2 Chimeric cytochrome P-450ccd used in the present invention
The gene includes, for example, plasmids pTF1, pTF2, pAMC1 and the like containing the cytochrome P-450c gene (JP-A-61-52284) and a plasmid pTZ330 [J. Bioche
m. , 96, 793-804, (1984)] and the HR2 region of the cytochrome P-450c gene is isolated.
It can be produced by replacing the C-terminal region with the same region of cytochrome P-450d. The plasmid pACCD1 according to the invention is preferred. Used in the present invention
From a yeast carrying a chimeric cytochrome P-450 gene
Genpu plasmid, for example, yeast <br/> onset Genbe compactors plasmid pAAH5 which holds the yeast alcohol dehydrogenase I promoter and the terminator (Washingt
on Research Foundation, Methods in Enzymology, 10
1 part C p192-201, Ammerer
It can be manufactured by these methods. In addition, the promoter of the yeast ADH1 gene is the Washington Rese
US Patent Application No. 29 of arch Foundation
It is included in 9,733 (9/31/81), and requires permission from the right holder when used for industrial or commercial purposes in the United States. ) And yeast expression vector pJD
B219 [Nature, 275 , 104 (197)
9)] or the like yeast expression vector can be produced by incorporating the chimeric cytochrome P-450 gene produced as described above. 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. Yeast gene bearing chimeric cytochrome P-450ccd gene
Genpu plasmid cells per in yeast strains transformed by pACCD1 or para-position hydroxylating activity of P-450 acetanilide per molecule, about 3 of yeast strains that have been transformed by conventional plasmid pAMC1
It can be seen that the efficiency is high and the utility as a bioreactor is high.

The yeast cells of the present invention can be obtained by transforming yeast belonging to the genus Saccharomyces with the plasmid containing the chimeric cytochrome P-450 gene by the alkali metal method or the protoplast method. Saccharomyces cerevisiae strain AH22 can be used, but is not limited to this strain.

[0009]

EXAMPLES Examples will be described below. 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. Example 1. Construction of plasmid pACCD1 Plasmid pACCD1 was constructed by the following method. About 5μ
g plasmid pTF1 (produced by the method described in JP-A-61-88878) was mixed with 20 units of each of restriction enzymes StuI and HindIII and reacted in 10 mM Tris-hydrochloric acid (pH 7.5), 50 mM NaCl at 37 ° C. for 1 hour. Then, 0.6% low melting point agarose gel electrophoresis was performed, a gel containing a DNA fragment of about 3.9 kb was cut out, and this was heated at about 65 ° C. for 5 minutes. 2 volumes of TE buffer [10 mM Tris-HCl, 0.5 mM
EDTA (pH 8.0)] was added, and then an equal amount of phenol saturated with TE buffer was added and stirred. 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. 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
After reacting for 1 hour at 60 ° C. in 8.0), about 100 ng of synthetic linker DNA having the following sequence and 5 units of T
4DNA ligase was mixed, and 66 mM Tris-HCl (pH 7.
6), 16 in 6.6 mM MgCl ₂ , 10 mM DTT, 1 mM ATP
The reaction was carried out at ℃ for 14 hours.

[SEQ ID NO: 3] E. coli DH1 (F ⁻ , recAl, endAl,
gyrA96, thi-l, hsdR17, supE44, λ -) were transformed, and plasmid DNA isolated from appearing colonies spread on plates containing ampicillin 100 [mu] g / ml, Hi
It was confirmed by cutting with ndIII or StuI that the cleavage sites for both of these restriction enzymes were newly generated, and the resulting plasmid was named pTFcc. Next, a known plasmid pTZ330 [J. Biochem., 9 containing a region encoding P-450d] was used.
6, p793-804 (1984)] was cut with StuI to obtain about 42
About 100 ng of 0 bp DNA fragment and about 1 ng of DNA treated with alkaline phosphatase after fragmenting pTFcc with StuI.
A ligase reaction was carried out by mixing 00 ng. Escherichia coli strain DH1 was transformed with the reaction mixture, plasmid DNA was prepared from the obtained colonies, and then 2 units of PvuII was added to 500 ng of plasmid DNA in 10 mM Tris-hydrochloric acid (pH 7.5), 50 mM NaCl. After reacting at 37 ° C. for 1 hour, 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 named pTFccd. next,
Approximately 100ng of pTFccd plus 2 units of SalI, 10mM
M Tris-hydrochloric acid (pH 7.5), 150 mM NaCl in 37 ℃ 1
After reacting for 5 hours, 5 units of DNA polymerase I
Klenow enzyme added, 67 mM calcium phosphate (pH 7.4),
6.7 mM MgCl ₂ , 1 mM _2- mercaptoethanol, 25 μ
MdATP, 25 μMdTTP, 25 μMdGTP, 2
The reaction was carried out in 5 μM dCTP at 25 ° C. for 1 hour and further treated with alkaline phosphatase. About 500ng
HindIII linker was added to carry out ligase reaction. Escherichia coli DH1 was transformed with the reaction mixture to prepare a plasmid DNA from the obtained colony, which was cleaved with HindIII and electrophoresed on 1% agarose gel to give a DNA of about 1.6 kb.
The presence of fragments was confirmed. The obtained plasmid was designated as pTFccd
I named it (H). About 1.6 after cutting pTFccd (H) with HindIII
A kb DNA fragment was recovered. Next, the yeast expression vector pA
About 100 ng of AH5 was cleaved with HindIII and treated with alkaline phosphatase, then about 200 ng of 1.6 kb DNA fragment
E. coli DH1 was transformed with the reaction mixture, and plasmid DNA was prepared from the obtained colonies. About 500ng of plasmid DNA
2 units of BamHI and StuI were added to 10 mM Tris-hydrochloric acid (pH 7.5) and 100 mM NaCl, and the mixture was reacted at 37 ° C for 1 hour, and then electrophoresed on 1% agarose gel. The orientation was confirmed, and the plasmid inserted in the forward direction was named pACCD1.

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, and the resulting pellet was mixed with 20 μl of 1 M LiCl solution, 30 μl of 70% polyethylene glycol 4000 solution, and about 1 μg of pACCD1.
μl of solution was added. Mix well and mix at 30 ° C for 1
After incubating for an hour, 140 μl of sterilized water was added and the mixture was stirred. Add this solution to the SD synthetic medium plate (2%
Glucose, 0.67% nitrogen source amino acid-free, 20 μg / ml
Transformed strain AH22 (pA) carrying pACCD1 by sprinkling it onto histidine, 2% agar and incubating at 30 ° C for 3 days.
Got CCD1).

Example 3 Quantification of chimeric cytochrome P-450 protein (abbreviated as P-450ccd) Saccharomyces cerevisiae AH22 (pAMC1) (JP-A 61-
56072) and the Saccharomyces cerevisiae AH22 (pACCD1) strain obtained in Example 2 in SD synthetic medium (2% glucose,
0.67% nitrogen source amino acid-free, 20 μg / ml histidine)
After culturing up to 1.5 x 10 ⁷ cells / ml each,
Suspend in zymolyce solution [1.2 M sorbitol, 50 mM potassium phosphate (pH 7.2), 14 mM 2-mercaptoethanol, 0.4 mg / ml zymolyce 60,000],
Incubated at 0 ° C for 30 minutes. Buffer solution (1% SD) heated to 100 ° C was added to the spheroplasts collected by centrifugation.
S, 50 mM Tris-HCl (pH 6.8), 10% mercaptoethanol, 40% glycerol, 0.02% bromophenol blue, 1 mM phenylmethylsulfonyl fluoride) was added for solubilization. Supernatant obtained by centrifugation (approximately 3X1
0 ⁶ cell fraction) was electrophoresed using 10% polyacrylamide gels. In addition, the protein in the gel is 25 mM
Electrophoretically blotted onto nitrocellulose filters in Tris-HCl (pH 8.3), 192 mM glycine-20% methanol. Next, the filter is put in TBS buffer [50 mM
After dipping in Tris-HCl (pH 7.5), 200 mM NaCl], 3
% In gelatin, 0.05% Tween20 in TBS buffer 3
After incubating at 7 ° C for 40 minutes, 30 µg of purified anti-P-450cIgG, 1% gelatin, 0.05% Tween20 was added.
Incubated for 2 hours at 37 ° C. in TBS buffer containing Then, in a buffer containing 0.05% Tween 20 at 37 ° C for 3
After repeating the operation of incubating for 0 minutes 4 times, 3%
37 ℃ in TBS buffer containing gelatin and 0.05% Tween20
And incubated for 20 minutes. Next, 2 μCi of [ ¹²⁵ I] -Protein A, 1% gelatin, 0.05% Tween2
After incubating in TBS buffer containing 0 at 37 ° C. for 70 minutes, in buffer containing 0.05% Tween 20 at 37 ° C.
The operation of incubating for 0 minutes was repeated 4 times. When the filter was air-dried and subjected to autoradiography, it was estimated that the AH22 (pACCD1) strain produced ^{5 to} 8 × 10 ⁵ molecules of P-450ccd per bacterium, based on the band density. P-450c in the pAMCl) strain
It was found to be about the same as the production volume of.

Example 4. P-450ccd containing heme
Quantitative AH22 (pACCD1) strain culture solution (SD synthetic medium, cell concentration about 1.5 × 10 ⁷ cells / ml) 100 ml was collected and
After suspending in 100 ml of 100 mM potassium phosphate buffer (pH 7.0), the mixture was centrifuged. Add 2 ml of the obtained pellet to 1
The cells were suspended in 00 mM potassium phosphate buffer (pH 7.0) and dispensed into two cuvettes in an amount of 1 ml each. After blowing carbon monoxide into the cuvette on the sample side, 5 to 10 mg of dithionite was added to both cuvettes, stirred, and then allowed to stand for 20 minutes. Then, stir the liquid in the cuvette to 400-5
The difference spectrum at 00 nm was measured, and ΔE447-490 = 91 mM ^-1
Based on Omura and Sato's value of cm ^-1 , P-450ccd
The concentration was calculated. As a result, it was found that AH22 (pACCD1) produced 3 to 4 × 10 ⁵ molecules of heme protein per cell, which was about the same as the production of P-450c in the AH22 (pAMCl) strain.

Example 5 Measurement of acetaminophen production by para-hydroxylation of acetanilide of bacterial cells Cultured up to about 1.4 × 10 ⁷ cells / ml in SD synthesis medium.
AH22 (pAMCl) strain and AH22 (pACCD1) strain, 1.5 M acetanilide (methanol solution) was added to each culture solution to make a final concentration of 25 mM. After that, shake culture (120 cycle / min) at 30 ° C, and take small aliquots every 1 hour,
The supernatant obtained by centrifugation was analyzed by HPLC to quantify the produced acetaminophen. The HPLC conditions are shown below. Column: μBondapak C18 (Φ 4 × 300 mm) Solvent: Methanol: Water: Acetic acid = 15: 84: 1 Detection; A245nm Flow rate; 2.0 ml / min Temperature; Room temperature (20-25 ° C.) Acetanilide per cell of AH22 (pACCD1) strain The para-hydroxylation activity was about 3 times higher than that of the AH22 (pAMCl) strain.

[Sequence Listing] SEQ ID NO: 1 Sequence length: 519 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Pro Ser Val Tyr Gly Phe Pro Ala Phe Thr Ser Ala Thr Glu 5 10 15 Leu Leu Leu Ala Val Thr Thr Phe Cys
Leu Gly Phe Trp Val Val 20 25 30 Arg Val Thr Thr Arg Thr Trp Val Pro Lys
Gly Leu Lys Ser Pro Pro 35 40 45 Gly Pro Trp Gly Leu Pro Phe Met Gly
His Val Leu Thr Leu Gly 50 55 60 Lys Asn Pro His Leu Ser Leu Thr Lys
Leu Ser Gln Gln Tyr Gly 65 70 75 Asp Val Leu Gln Ile Arg Ile Gly Ser
Thr Pro Val Val Val Leu 80 85 90 Ser Gly Leu Asn Thr Ile Lys Gln Ala
Leu Val Lys Gln Gly Asp 95 100 105 Asp Phe Lys Gly Arg Pro Asp Leu Tyr
Ser Phe Thr Leu Ile Ala 110 115 120 Asn Gly Gln Ser Met Thr Phe Asn Pro
Asp Ser Gly Pro Leu Trp 125 130 135 Ala Ala Arg Arg Arg Leu Ala Gln Asn Ala Leu Lys Ser Phe Ser 140 145 150 Ile Ala Ser Asp Pro Thr Leu Ala Ser
Ser Cys Tyr Leu Glu Glu 155 160 165 His Val Ser Lys Glu Ala Glu Tyr Leu
Ile Ser Lys Phe Gln Lys 170 175 180 Leu Met Ala Glu Val Gly His Phe Asp
Pro Phe Lys Tyr Leu Val 185 190 195 Val Ser Val Ala Asn Val Ile Cys Ala
Ile Cys Phe Gly Arg Arg 200 205 210 Tyr Asp His Asp Asp Gln Glu Leu Leu
Ser Ile Val Asn Leu Ser 215 220 225 Asn Glu Phe Gly Glu Val Thr Thr Gly Ser
Gly Tyr Pro Ala Asp Phe 230 235 240 Ile Pro Ile Leu Arg Tyr Leu Pro Asn
Ser Ser Leu Asp Ala Phe 245 250 255 Lys Asp Leu Asn Lys Lys Phe Tyr Ser
Phe Met Lys Lys Leu Ile 260 265 270 Lys Glu His Tyr Arg Thr Phe Glu Lys
Gly His Ile Arg Asp Ile 275 280 285 Thr Asp Ser Leu Ile Glu His Cys Gln
Asp Arg Arg Leu Asp Glu 290 295 300 Asn Ala Asn Val Gln Leu Ser Asp Asp
Lys Val Ile Thr Ile Val 305 310 315 Phe Asp Leu Phe Gly Ala Gly Phe Asp
Thr Ile Thr Thr Ala Ile 320 325 330 Ser Trp Ser Leu Met Tyr Leu Val Thr
Asn Pro Arg Ile Gln Arg 335 340 345 Lys Ile Gln Glu Glu Leu Asp Thr Val
Ile Gly Arg Asp Arg Gln 350 355 360 Pro Arg Leu Ser Asp Arg Pro Gln Leu
Pro Tyr Leu Glu Ala Phe 365 370 375 Ile Leu Glu Ile Tyr Arg Tyr Thr Ser
Phe Val Pro Phe Thr Ile 380 385 390 Pro His Ser Thr Thr Arg Asp Thr Ser
Leu Asn Gly Phe His Ile 395 400 405 Pro Lys Glu Cys Cys Ile Phe Ile Asn
Gln Trp Gln Val Asn His 410 410 415 420 Asp Glu Lys Gln Trp Lys Asp Pro Phe
Val Phe Arg Pro Glu Arg 425 430 435 Phe Leu Thr Asn Asp Asn Thr Ala Ile
Asp Lys Thr Leu Ser Glu 440 445 450 Lys Val Met Leu Phe Gly Leu Gly Lys
Arg Arg Cys Ile Gly Glu 455 460 465 Ile Pro Ala Lys Trp Glu Val Phe Leu
Phe Leu Ala Ile Leu Leu 470 475 480 His Gln Leu Glu Phe Thr Val Pro Pro Pro
Gly Val Lys Val Asp Leu 485 490 495 Thr Pro Ser Tyr Gly Leu Thr Met Lys
Pro Arg Thr Cys Glu His 500 505 510 Val Gln Ala Trp Pro Arg Phe Ser Lys 515 519 SEQ ID NO: 2 Sequence Length: 1560 Sequence Type: Nucleic Acid Number of Strands: Duplex Topology: Linear Sequence Type : Other nucleic acids Synthetic DNA Sequence ATGCCTTCTG TGTATGGATT CCCAGCCTTC ACATCAGCCA CAGAGCTGCT CTTGGCCGTC 60 ACCACATCTCT GCCTTGGATT CTGGGTGGTT AG
AGTCACAA GAACCTGGGT TCCCAAAGGT 120 CTGAAGAGTC CACCCGGACC CTGGGGCTTG CC
CTTCATGG GGCACGTGCT GACCCTGGGG 180 AAGAACCCCAC ACCTGTCACT GACAAAACTG AG
TCAGCAGT ATGGGGACGT GCTGCAGATC 240 CGTATTGGCT CCACACCCCGT GGTGGGTCTG AG
CGGCCCTGA ACACCCATCAA GCAGGCCCTG 300 GTGAAACAGG GGGATGACTT CAAAGGCCGG CC
AGACCTCT ACAGCTTCAC ACTATTCGCT 360 AATGGCCAGA GCATGACTTT CAACCCAGAC TC
TGGACCGC TGTGGGCTGC CCGCCGGCGC 420 CTGGCCCAGA ATGCCGCTGAA GAGTTTCTCC AT
AGCCTCAG ACCCAACACT GGCATCCCTCT 480 TGCTACTTGG AAGAGCACGT GAGCAAAGAG GC
TGAATACT TAATCAGCAA GTTCCAGAAG 540 CTGATGGCAG AGGTTGGCCA CTTCGACCCT TT
CAAGTTATT TGGTGGTGTC AGTGGCCAAT 600 GTCATCTGTG CCATATGCTT TGGCAGACGT TA
TGACCACG ATGACCAAGA GCTGCTCAGC 660 ATAGTCAATC TAAGCAATGA GTTTTGGGGAG GT
TACTGGTT CTGGATACCC AGCTGAACTTC 720 ATTCCTATCC TCCGTTACCT CCCTACTACTCT TC
CCTGGATG CCTTCAAGGA CTTGAATAAG 780 AAGTTCTACA GTTTTCATGAA GAAGCTAATC AA
AGAGCACT ACAGGACATT TGAGAAGGGC 840 CACATCCGGG ACATCACAGA CAGCCTCATT GA
GCATTGTC AGGACAGGAG GCTGGACGAG 900 AATGCCAATG TCCAGCTCTC AGATGATAAG GT
CATTACGA TTGTTTTTTGA CCTCTTTTGGA 960 GCTGGGTTTG ACACAATCAC AACTGCTACTC TC
TTGGAGCC TCATGTACCT GGTAACCAAC 1020 CCTAGGATAC AGAGAAAGAT CCAGGAGGAG TT
AGACACAG TGATTGGCAG GGATCGGCAG 1080 CCCCGGCTTT CTGACAGACC TCAGCTGCCC TA
TCTGGAGG CCTTCATCCT GGAGACTAC 1140 CGATACACAT CCTTTGTCCC CTCTACCATC CC
CCACAGCA CAACGAGGGA CACCTCACTG 1200 AATGGCTTCC ACATTCCCAA GGAGTGCTGCAT
CTTCATAA ACCAGTGGGCA GGTCAACCAT 1260 GATGAGAAGC AGTGGAAAGA CCCCTTTGTG TT
CCGCCCAG AGCGGTTTCT TACCAATGAC 1320 AACACGGCCA TCGACAAGAC CCTGAGTGAG AA
GGTGATGC TCTTCGGGCTT GGGAAAGCGC 1380 CGGTGCATTG GGGAGATCCC GGCCAAGTGG GA
AGTCTTCC TCTTCTTAGC CATCCTCTG 1440 CATCAGCTGGG AGTCTACGT GCCACCGGGC GT
GAAGGTGG ACCTGACACC CAGCTATGGG 1500 CTGACCATGA AGCCCAGAAC CTGTGAACAC GT
CCAGGCCT GGCCACGCTT CTCCAAGTGA 1560 SEQ ID NO: 3 Sequence Length: Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence CCTGGCCACG CTTCTCCAAG TGA GGACCGGTGC GAAGAGGTTC ACTTCGA '

Claims (6)

[Claims] 1. A rat represented by the following amino acid sequence (1):
To the gene encoding liver cytochrome P450c
The following amino acid sequence (II) in the gene
The C-terminal region containing the heme binding region (HR2 region)
The amino acid sequence of the liver cytochrome P450d gene
Includes a heme binding region (HR2 region) consisting of row (III)
Chimeric cytochrome P-450 with the C-terminal region replaced
A yeast strain carrying a yeast expression plasmid containing the gene
Chimeric cytochrome P-450 characterized by culturing
Manufacturing method 2. A chimeric cytochrome P-450 according to claim 1, which comprises culturing a yeast strain having a yeast expression plasmid containing the chimeric cytochrome P-450 gene encoding the amino acid sequence of SEQ ID NO: 1. Production method: SEQ ID NO: 1 3. A chimeric cytochrome P-450 according to claim 1, which comprises culturing a yeast strain carrying a yeast expression plasmid containing the chimeric cytochrome P-450 gene represented by the nucleotide sequence of SEQ ID NO: 2. Manufacturing method of ## STR2 ## SEQ ID NO: 2 4. The method for producing chimeric cytochrome P-450 according to claim 1, wherein the yeast strain is Saccharomyces cerevisiae AH22 strain. 5. The method for producing chimeric cytochrome P-450 according to claim 2, wherein the yeast strain is Saccharomyces cerevisiae AH22 strain. 6. The method for producing chimeric cytochrome P-450 according to claim 3, wherein the yeast strain is Saccharomyces cerevisiae AH22 strain.