JPH0832235B2 - Chimeric P450 expression plasmid and yeast strain carrying the plasmid - Google Patents

Description

TECHNICAL FIELD The present invention relates to a chimeric P450 expression plasmid and a yeast strain carrying the plasmid. The yeast strain obtained by the present invention has the ability to efficiently convert the mature form of rat liver P450 _C25 by completely cleaving the signal peptide of chimeric P450. Using the enzyme produced by the above yeast strain (mature rat liver P450 _C25 ) and ferredoxin and NADPH-ferredoxin reductase, steroids useful as pharmaceuticals and active vitamin
It can be synthesized like D _3.

<Prior Art> P450 is a heme protein that exists widely in the living world from microorganisms to mammals, and catalyzes a one-atom oxygenation reaction using various lipid-soluble compounds as substrates as terminal enzymes of the electron transfer system. The P450 molecular species that is the terminal enzyme is diverse,
Since each exhibits a different substrate specificity, a very wide range of lipophilic compounds can be hydroxylated. Mammalian
The P450-dependent electron transport system is divided into its microsomal type localized in microsomes and mitochondrial type localized in mitochondria. In the former, NADPH-P450 reductase (reductase), which contains flavin adenine dinucleotide and flavin mononucleotide as coenzymes in the molecule, supplies electrons from NADPH to P450, and in the latter, flavin adenine dinucleotide coenzyme. In the molecule, NADPH-ferredoxin reductase and ferredoxin, which contains non-heme iron as a coenzyme in the molecule, supply electrons from NADPH to P450, thereby hydroxylating various lipid-soluble substrates. To catalyze.

The present inventors have already succeeded in carrying out an engineeringly useful hydroxylation reaction by using a recombinant yeast strain that expresses several types of microsomal P450s in yeast. That is, rat liver P450 _MC gene, bovine adrenal P450 ₁₇ α gene and bovine adrenal P450 _C21 gene are isolated, and expression plasmids containing these genes are prepared, and yeast are transformed with these expression plasmids. , A yeast strain producing a microsomal P450 was obtained (JP-A-61)
-56072, JP-A-1-47380, JP-A-2-31680). These yeast strains showed monoatomic enzyme addition activity depending on the respective microsomal P450 molecular species. Moreover, the rat liver reductase gene and the yeast reductase gene were isolated, and the enzyme having P450 reducing ability was successfully expressed in yeast (JP-A-62-19085, JP-A-2-211880). Furthermore, the inventors have succeeded in producing a yeast strain (Japanese Patent Application Laid-Open No. 62-104582) that produces both microsomal P450 and reductase yeast in yeast, and a novel monooxygenase having the functions of both enzymes in one molecule. (JP-A-63-44888, JP-A-2-23870,
JP-A-2-249488). By the above-mentioned techniques, the present inventors have enabled the synthesis of acetaminophen and steroid hormone intermediates useful as pharmaceuticals by using these microsomal P450-producing yeast strains.

On the other hand, there is no known example of expressing a membrane protein, such as a mitochondrial P450, which has a complex structure containing a heme in the molecule and is localized in the inner mitochondrial membrane in yeast. .

<Problems to be Solved by the Invention> Biological reactions involving mitochondrial P450s have many synthetic reactions of physiologically important compounds, such as active vitamin D _3, and thus have high industrial applicability. Therefore, the creation of a yeast strain that produces mitochondrial P450 has been desired.

<Means for Solving the Problems> The inventors of the present invention efficiently determine the maturity (enzyme) of rat liver P450 _C25 , which is a membrane protein having a complex structure containing heme in the molecule, in yeast. As a result of sincerity efforts to produce it, in place of the original signal sequence that normally functions in the living body of the rat, an exogenous signal sequence possessed by a protein that has a completely different structure and function from the P450 molecular species described above By exchanging them, the mature rat liver P450 _C25 having a strong activity was successfully produced in yeast, and the present invention was completed.

That is, the present invention has a signal sequence consisting of N-terminal 29 amino acid residues of yeast cytochrome C oxidase subunit IV at the N-terminal and a mature rat liver P450 _C25 consisting of 501 amino acid residues at the C-terminal. The present invention provides a plasmid expressing a chimeric P450 and a yeast strain carrying the plasmid.

Mature rat liver P4 partially purified from the above yeast strains
50 _C25 is conjugated with bovine adrenal adrenodoxin (hereinafter abbreviated as ADX) and bovine adrenal NADPH-adrenodoxin reductase (hereinafter abbreviated as ADR) to produce THC or 1α-OH-vitamin D ₃ to TeHC. Or 1α, 25-
It was possible to produce (OH) ₂ vitamin D ₃ , respectively.

 Hereinafter, the present invention will be described in more detail.

The rat liver P450 _C25 used in the present invention is synthesized in the cytoplasm as a precursor consisting of 533 amino acids in the living body of rat, and then the translocation signal existing at the N-terminal thereof is recognized and incorporated into the inner mitochondrial membrane. (Localized in mitochondria), where processing is performed, and the mature form of 501 amino acids (molecular weight about 53KD)
become. In the present invention, the first object is to make the rat liver P450 _C25 precursor capable of _{exhibiting the} same action in yeast as described above.

Even when the rat liver P450 _C25 precursor, which has the original signal sequence that normally functions in the rat body, is expressed in yeast, it is taken up into the inner mitochondrial membrane in yeast as in the rat body, and then normal. However, it was not completely processed into a mature form consisting of 501 amino acids. Therefore, the enzyme activity per P450 molecule is low. Yeast strains obtained by the present invention, in place of the original signal sequence that normally functions in the living body of the rat, the foreign signal sequence that the protein has a completely different structure and function from the P450 molecular species described above. The exchanged rat liver P450 _C25 precursor is expressed in yeast. Coincidentally, the unique signal peptide of the rat liver P450 _c25 precursor was completely cleaved in yeast. As a result, in the yeast, the conversion to mature rat liver P450 _C25 was efficiently performed, and it became possible to achieve the above object.

CDNA encoding rat liver 450 _C25 used in the present invention
Is already known, and can be isolated by a usual operation method. For example, the rat liver P450 _C25 gene is expressed in plasmid pLMT25 (Usui et al., (1990) FEBS 262,135-
138).

The plasmid expressing the rat liver P450 _C25 precursor of the present invention is the N-terminal of the yeast cytochrome C oxidase subunit IV.
It has a signal sequence consisting of 29 amino acid residues at the N-terminus,
Mature rat liver P450 _C25 consisting of 501 amino acid residues
A yeast expression vector pAAH5 (Methods in En) containing a yeast alcohol dehydrogenase (ADH) gene promoter and terminator containing a chimeric P450 gene having a terminal
zymology, 101, partC, p192-201). Saccharomyces cerevisiae AH22 strain is preferable as a host. A plasmid expressing a chimeric P450 having a signal sequence consisting of 29 amino acid residues at the N-terminal of yeast cytochrome C oxidase subunit IV at the N-terminal and mature rat liver P450 _C25 consisting of 501 amino acid residues at the C-terminal The transformation of the host yeast by the method can be performed by a known method such as a method using an alkali metal (LiCl) or a protoplast method. The transformed yeast cells thus obtained express the chimeric P450 in yeast,
Next, by completely cleaving the specific signal sequence of the chimeric P450, mature rat liver P450
_It has the ability to efficiently convert to _C25 . By culturing the transformed yeast according to a usual culturing method, mature rat liver P450 _C25 can be produced.

<Examples> Hereinafter, the present invention will be described in detail based on Examples. The present invention is not limited to the examples, and it is possible to make ordinary modifications in the technical field of the present invention.

Example 1 Construction of Expression Plasmid pAC25 In the following examples, reactions such as digestion of DNA with a restriction enzyme, dephosphorylation of DNA with alkaline phosphatase, and binding of DNA with a DNA ligase were generally 20 to 200 μm unless otherwise specified. Was carried out under the reaction conditions attached to the product by the manufacturer (for example, Takara Shuzo Co., Ltd.) that commercially sells these enzymes. The rat liver P450 _C25 expression plasmid pAC25 was constructed as shown in FIG.

Plasmid pLMT25 (Usu containing the rat liver P450 _C25 gene
i et al., (1990) FEBS 262,135-138) with restriction enzyme EcoR
About 3 including the P450 _C25 N-terminal region co-digested with I and SacI
Approximately 158 including 20 bp EcoRI-SacI fragment and C-terminal region
The 0 bp SacI-EcoRI fragment was recovered by low melting agarose gel electrophoresis. These fragments were subcloned into the commercially available vector plasmid pUC19 at EcoRI and SacI sites to obtain pUC25N and pUC25C, respectively. pUC25N for EcoRI, Nco
A fragment of about 2880 bp obtained by co-digestion with I and the synthetic linker LC252: Perform a ligase reaction with (having EcoRI and NcoI recognition sites on the left and right ends, and Hind III recognition site on the inside).
Escherichia coli HB101 strain was transformed. From the obtained transformant, a plasmid DNA was prepared according to the method of Birnboim-Doly and analyzed by restriction enzyme digestion to obtain a target plasmid having a synthetic linker inserted therein. By further determining the nucleotide sequence, the sequence of the synthetic linker portion was confirmed, and the obtained plasmid was designated as pUC25NH.

On the other hand, pUC25C containing the region of rat liver P450 _C25 C-terminal was
The approximately 4 kb fragment obtained by simultaneous digestion with NcoI and EcoRI was ligated with a commercially available Hind III linker to give E. coli.
The HB101 strain was transformed. Transformant to plasmid DNA
Was prepared, and the plasmid in which the HindIII site was created in the 3'untranslated region of P450 _C25 was designated as pUC25CH.

PUC25NH and pUC25CH were respectively digested with Hind III and SacI to obtain fragments of about 270 bp and 1370 bp, respectively, and a commercially available vector plasmid pUC18 was subjected to Hind III digestion and alkaline phosphatase treatment, followed by ligase reaction. Escherichia coli HB101 strain was transformed. Plasmid DNA was prepared from the transformants, and pUC25NH and pUC25CH
The plasmid containing each one of the derived DNA fragments was designated as pUC25H. pUC25H was digested with Hind III to prepare a cDNA fragment of about 1640 bp encoding a rat liver P450 _C25 precursor, and this fragment and a yeast expression vector pAAH5N treated with alkaline phosphatase after Hind III digestion (JP-A-2-211880). After carrying out a ligase reaction with E. coli, the Escherichia coli HB101 strain was transformed. Plasmid DNA was prepared from the transformant, the DNA structure was confirmed, and the plasmid in which the rat liver P450 _C25 precursor gene was inserted in the forward direction with respect to the ADH promoter and terminator was designated as pAC25.

Example 2 Construction of Expression Plasmid pACC253 The present inventors have already shown that when a bovine adrenal mitochondrial protein is expressed in yeast, the signal peptide portion thereof is expressed in yeast cytochrome c oxidase subunit IV (hereinafter abbreviated as COX IV). It has been found that the expression level is remarkably increased by substituting it with the one described in JP-A-4-30793.
Therefore, mature rat liver P450 in yeast mitochondria
Plasmid pACC2 in which the signal sequence part was replaced with that of CX IV for the purpose of stably highly expressing _C25.
53 was constructed as shown in FIG.

Plasmid p containing the N-terminal gene of rat liver P450 _C25
UC25N was co-digested with PstI and SacI to recover a fragment of about 150 bp. This fragment and the synthetic linker LC253: (Hind III and PstI recognition sites are located on the left and right ends respectively)
Then, a ligase reaction was carried out with the HindIII-SacI fragment of the commercially available vector pU19 to transform Escherichia coli HB101 strain. Plasmid DNA was prepared from the transformant and analyzed by restriction enzyme digestion to obtain the desired plasmid into which the fragment and the synthetic linker were inserted. Furthermore, the sequence of the synthetic linker portion was confirmed by determining the base sequence, and the obtained plasmid was designated as pBSCC253. This plasmid and the above-mentioned plasmid pUC25CH were co-digested with Hind III and Sac I, and COX I
A gene fragment (approximately 260 bp) encoding the V signal peptide and rat liver P450 _C25 N-terminal portion and rat liver P450 _C25
A gene fragment (about 1370 bp) encoding the C-terminal part was recovered. E. coli HB101 strain was transformed by triple ligation of these fragments with a commercially available vector plasmid pUC18-HindIII fragment. Plasmid DNA was prepared from the transformant and analyzed by restriction enzyme digestion,
A plasmid pUCC253H having both fragments inserted was obtained. pUCC25
3H was digested with Hind III to prepare a chimeric P450 cDNA fragment of about 1630 bp, which was digested with Hind III and treated with alkaline phosphatase to obtain a yeast expression vector pAAH5N (JP-A-2-21).
1880) and then Escherichia coli HB101 strain was transformed. Plasmid DN prepared from transformants
The structure of A was confirmed by restriction enzyme digestion, and the plasmid in which the chimeric P450 gene was inserted in the forward direction with respect to the ADH promoter and terminator was designated as pACC253.

Example 3 Transformation of Yeast with Expression Plasmid Saccharomyces cerevisiae AH22 (ATCC38626) [ρ
0] strain (although mitochondrial-like organelles exist,
Strains that do not have mitochondrial DNA) and [ρ +] strains (which have normal mitochondria and their DNA)
After culturing in D medium (1% yeast extract, 2% polypeptone, 2% glucose) at 30 ° C. for 18 hours, 1 ml of yeast culture solution was centrifuged to collect the cells. 0.2M LiCl solution
After washing with 1 ml, it was suspended in 20 μ of a 1M LiCl solution.
To this, 70μ polyethylene glycol 4000 solution 30μ,
Add 10μ of expression plasmid solution (about 1μg DNA),
After mixing well, it was incubated at 30 ° C. for 1 hour. Next, add 140 μ of water and add SD synthetic medium plate (2% glucose, 0.67% yeast nitrogen source amino acid-free, 20%
A transformant carrying a plasmid was obtained by plating on a μg / ml histidine, 2% agar) and incubating at 30 ° C. for 3 days. The yeasts transformed with the plasmids pAC25 and pACC253 were used as AH22 [ρ0] (pAC25) strain and AH2 strain, respectively.
2 [ρ +] (pAC25) strain, AH22 [ρ0] (pACC253) strain, A
The strain was designated as H22 [ρ +] (pACC253) strain.

Example 4 Production of rat liver P450 _C25 AH22 [ρ0] (pAC25) strain obtained in Example 3, AH22 [ρ
+] (PAC25) strain, AH22 [ρ0] (pACC253) strain, AH22
[Ρ +] (pACC253) strain and control AH22 [ρ
0] (pAAH5) strain and AH22 [ρ +] (pAAH5) strain on SD synthetic medium (2% glucose, 0.67% amino acid-free yeast nitrogen source,
20μg / ml histidine) 300ml each about 2 × 10 ⁷ cells / m
After culturing to l, the cells were collected, washed with 100 mM potassium phosphate (pH 7.0), and then suspended in 2 ml of 100 mM potassium phosphate (pH 7.0). 1 ml of the bacterial suspension was dispensed into each of two cuvettes, carbon monoxide was blown into the sample-side cuvette, and then 5 to 10 mg of dithionite was added to both cuvettes.
After stirring well, measure the difference spectrum of 400-500 nm,
The heme-containing P450 amount was calculated based on Δε (450 nm-490 nm) = 91 mM ⁻¹ cm m ⁻¹ . As a result, AH22 [ρ0], [ρ
+] (PAC25) strain and AH22 [ρ0], [ρ +] (pACC2
53) All strains contain approximately 2 × 10 ⁴ molecules of heme-containing P per cell.
It was found to produce 450 proteins. In contrast, controls AH22 [ρ0] and [ρ +] (pAAH
5) Heme-containing P450 was not produced in the strain.

Example 5 Measurement of P450 _C25- Dependent Oxidative Activity in Reconstitution System In a reconstitution system with bovine adrenal ADX and bovine adrenal ADR, control AH22 [ρ +] (pAAH5) strain, AH22 [ρ
The P450 _C25- dependent hydroxylation activity was measured using the organelle fraction prepared from the +] (pAC25) strain and the AH22 [ρ +] (pACC253) strain.

The cell fraction from the transformed yeast cells was according to the following method. Approximately 4 x 10 ¹⁰ from transformant approximately x 10 ⁷ cells / ml culture
The bacterial cells were collected, suspended in a zymolyase solution (10 mM Tris-hydrochloric acid (pH 7.5), 2.0 M sorbitol, 0.1 mM dithiothreitol, 0.1 mM EDTA, 0.3 mg / ml zymolyase 10T), and then suspended at 30 ° C. for 1 hour. Spheroplasts were prepared by incubation for a period of time. Sonication buffer (10mM
The cells were suspended in Tris-hydrochloric acid (pH 7.5), 0.65 M sorbitol, 0.1 mM dithiothreitol, 0.1 mM EDTA, 1 μg / ml leupeptin, 1 μg / ml pepstatin) and homogenized with a Teflon homogenizer to disrupt the cells. The precipitate 1 was removed by centrifugation at 3000 xg for 5 minutes to obtain a supernatant 1 '. Precipitate 1 was suspended in sonication buffer and homogenized again, then 3000 × g, 5
After centrifugation for 1 minute, a precipitate 1 (unbroken cell / nuclear fraction) and a supernatant 1 ″ were obtained. The supernatants 1 ′ and 1 ″ were combined to obtain a supernatant 1 and centrifuged at 10,000 × g for 20 minutes, A precipitate 2 (mitochondrial fraction) and a supernatant 2 were obtained. 120,000 more supernatant 2
Xg, centrifuge for 70 minutes, precipitate 3 (microsome fraction)
And supernatant 3 (cytoplasmic fraction). When the mitochondrial fraction and the microsome fraction were used for the measurement of oxidative activity, 0.5% final concentration of cholic acid was added to each yeast cell organelle fraction to solubilize the protein from the mitochondria or microsomal membrane in advance.

THC 27-position hydroxylation activity was measured by the following method. 100 mM
Tris-hydrochloric acid (pH 7.8), 0.5 mM EDTA, 14 nmol [ ³ H] TH
C, 4 nmol bovine adrenal ADX, 0.1 U bovine adrenal ADR, 100 mM NADPH
Solubilized yeast cell organelle fraction (containing 0.34 mg protein) was added to the reaction mixture consisting of
Incubated for a period of time at 37 ° C. The reaction was stopped by adding 5 ml of ethyl acetate, the mixture was stirred with a vortex mixer for 1 minute and then centrifuged, and 4 ml of the ethyl acetate layer was dried. This was dissolved in a small amount of ethyl acetate and subjected to silica gel thin layer chromatography (TLC). TLC is ethyl acetate: acetone = 7.
3 was developed as a solvent. After development, the radioactivity of the substrate and product parts of the TLC plate was measured using a scanner. The THC27 hydroxylation activity in the reconstituted system was measured. As a result, when the mitochondrial fraction of AH22 [ρ +] (pAAH5) strain was used, no radioactivity peak could be detected at the TeHC position. In some cases, the TeHC peak could be detected. From the peak areas of substrate and product, AH22 [ρ
+] (PAC25) strain mitochondrial fraction, microsome fraction, AH22 [ρ +] (pACC253) strain mitochondrial fraction and microsome fraction were used, and the reaction time was about 27, 13, 26, 25, respectively. It was found that% THC was converted to TeHC.

On the other hand, the 1α-OH-vitamin D ₃ 25-position hydroxylation activity was measured as follows. 100 mM Tris-HCl (pH 7.8), 0.5 mM
EDTA, 100 nmol 1α-OH-vitamin D, 4 nmol bovine adrenal AD
Each solubilized yeast cell organelle fraction was added to a reaction mixture consisting of X, 0.1 U bovine adrenal ADR, and 100 mM NADPH to make the total volume 0.5 ml, and the mixture was incubated at 37 ° C for a certain period of time. The reaction was stopped by adding 5 ml of benzene, stirred for 1 minute with a vortex mixer, and then centrifuged to dry 4 ml of the benzene layer. This was dissolved in a small amount of isopropanol and analyzed by HPLC. The HPLC conditions are shown below.

1. Column; Fine Pack Sil 5 (manufactured by JASCO Corporation) 2. Solvent: hexane: methanol: isopropanol = 8
4: 8: 8 3. Flow rate; 1.2 ml / min 4. Column temperature; room temperature 5. Detection wavelength; 265 nm 1α-OH-vitamin D ₃ 25-position hydroxylation activity was measured and AH22 [ρ +] (pAAH5 ) Strain showed no activity, but the transformant strain had HP
A peak could be detected at the position of 1α, 25- (OH) ₂ vitamin D ₃ by LC analysis. From the peak height, the amount of 1α, 25- (OH) ₂ vitamin D ₃ produced by the mitochondrial fraction and microsomal fraction of the AH22 [ρ +] (pAC25) strain containing 0.34 mg protein was about 1 hour each at a reaction time of 1 hour. Calculated to be 23 pmol and 12 pmol. In addition, chimeric P450 producing AH22 [ρ
The mitochondrial fraction and the microsomal fraction of the +] (pACC253) strain also showed similar activity. Above, rat liver P4
From the results of 50 _C25- dependent hydroxylation activity measurement, mature rat liver P450 _C25 produced in yeast contains heme necessary for activity expression in the molecule, and in the reconstitution system, bovine adrenal ADX and It was found that by constructing the electron transfer system with bovine adrenal ADR, the oxidative activity dependent on mature rat liver P450 _C25 can be exerted.

<Effects of the Invention> With the transformed yeast cell of the present invention, the mature form (enzyme) of rat liver 450 _C25 , which is a membrane protein having a complex structure containing a heme in the molecule, is efficiently used in yeast for the first time. It has become possible to produce.

Furthermore, mature P450 _C25 crudely purified from the transformant
In the reconstitution system of bovine adrenal ADX and bovine adrenal ADR
TeHC and 1α, 25 (OH) ₂ vitamin D ₃ were produced from C and 1α-OH-vitamin D ₃ , respectively. That is, P45
0 _C25 showed both hydroxylation activity of THC27 position and l [alpha]-OH @ - vitamin D ₃ 25-position. Currently localized to mitochondria
As a P450 molecular species, expression of bovine adrenal P450 _SCC , P450 ₁₁ β, etc. in xenogeneic cells (monkey kidney-derived COSl cells) has been attempted, but both have low production and low activity.
In addition, rat liver P450 _C25 has been reported to be expressed in COS1 cells, but only activity against THC has been detected. This time, the present inventors were the first mitochondrial P450 to successfully express a function in yeast. The mature rat liver P450 _C25 produced in yeast was both hydroxylated at the THC27 position and at the 1α-OH vitamin D ₃ 25 position. It showed activity.

Several kinds of P450s are involved in the biosynthesis of bile acids from cholesterol in the liver, of which P450 _C25 is T
Catalyzes the conversion of HC to TeHC. Patients with cerebral tendon xanthomatosis have been congenitally deficient in this enzyme. This disease is characterized by xanthoma and exacerbated neuropathy, and xanthoma occurs in the patient's Achilles tendon, lungs, and brain, resulting in cataracts, impaired intelligence, and other serious symptoms. It is known that cholestanol in serum of patients with this disease is increased from 10 times to 100 times of the normal value, and at present, the diagnostic method using this and a THC labeled with a radioisotope are used for this enzyme. The method of measuring the activity of is used. By culturing the transformed yeast strain obtained by the present invention, large-scale production of mature rat liver P450 _C25 has become possible, so if an isolated and purified antibody to this enzyme is prepared,
Since it can be used to detect P450 _C25 with high sensitivity, it is considered to be effective for the diagnosis of cerebral tendon xanthomatosis.

On the other hand, in order for vitamin D ₃ to become active, hydroxylation at the 25th position and then hydroxylation at the 1α position are required. Vitamin D ₃ taken up from food or biosynthesized in the body becomes active 1α, 25- (OH) ₂ vitamin D ₃ in the body as a hormone responsible for regulating calcium metabolism, and also in cells. It plays an important role in promoting differentiation and regulating cellular immune function. Therefore, 1α, 25- (OH) ₂ vitamin D ₃ is effective for the treatment of osteoporosis, chronic renal failure, vitamin D resistant rickets, bone lesions of osteomalacia, hypoparathyroidism disease and the like. Currently, 1α, 25- (OH) ₂ Vitamin D
_{Since 3} can be produced in small quantities, it depends on a complicated chemical synthesis method. INDUSTRIAL APPLICABILITY According to the present invention, mature rat liver P450 _C25 produced in yeast and bovine adrenal glands ADX and ADR can be combined to be used as a bioreactor for active vitamin D ₃ production. Further, the present inventors have already succeeded in expressing ADX, which is one of the components of the mitochondrial electron transfer system, in yeast. ADX partially purified from this yeast cell is a bovine adrenal ADR purified preparation and bovine adrenal P450SC.
Pregnenolone could be produced from cholesterol together with the C-purified preparation (JP-A-4-30793). In addition, the bovine adrenal ADR gene is known and its expression is easy. Furthermore, we have P450 and NADPH-cytochrome P.
A technique for co-expressing 450 reductase in yeast has also been developed (JP-A-62-104582). Therefore, in the future, P450 _C25 and A
By creating a strain that simultaneously expresses DX and ADR, it is possible to further simplify the complicated chemical synthesis method that is currently performed by using it as a bioreactor for steroid compound oxidation reaction.

[Brief description of drawings]

FIG. 1 shows the steps for constructing the expression plasmid pAC25 of the present invention. FIG. 2 shows the steps for constructing the expression plasmid pACC253 of the present invention. Restriction enzyme sites, Ec: EcoI, Nc: NcoI, Sc: SacI,
Hd: Hind III, Nt: NotI and Ps: PstI are shown. Also in the figure Is the rat P450 _C25 translation region, Represents the translation region of the enzyme cytochrome c oxidase subunit IV, and P and T represent the ADH promoter and terminator, respectively.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1: 865) (C12N 9/02 C12R 1: 865) (72) Inventor Hideo Okawa Takarazuka, Hyogo Prefecture 4-2-1 Takashi Ichi, Sumitomo Kagaku Kogyo Co., Ltd. (56) References FEBS Lett. p135-138 (1990)

Claims (6)

[Claims] 1. Yeast cytochrome C oxidase subunit IV
A mature rat liver P45 consisting of 501 amino acid residues having a signal sequence consisting of N-terminal 29 amino acid residues of
A plasmid expressing a chimeric P450 having _C25 at the C terminus 2. Yeast cytochrome C oxidase subunit IV
A chimeric P450 consisting of a nucleotide sequence encoding a signal sequence consisting of 29 amino acid residues at the N-terminal and a nucleotide sequence encoding a mature rat liver P450 _C25 consisting of 501 amino acid residues
Plasmid containing gene 3. pACC253 represented by the following restriction map 4. A yeast strain carrying the plasmid according to claim 1. 5. A yeast strain carrying pACC253 according to claim 2. 6. A Saccharomyces cerevisiae AH22 carrying pACC253 according to claim 3.