JPH062067B2 - Yeast NADPH-cytochrome P450 reductase producing strain - Google Patents

Description

TECHNICAL FIELD The present invention uses the yeast NADPH-cytochrome P450 reductase gene, an expression plasmid for large-scale production of the enzyme, a yeast strain carrying the plasmid, and the yeast. And a method for producing the enzyme. Further, the present invention relates to a method for producing NADPH-cytochrome P450 reductase in a large amount to increase the oxygen level in yeast cells and improve the activity of adding one-atom enzyme dependent on cytochrome P450 derived from mammals. The yeast strain obtained by the present invention produces cytochrome P450 and also produces large amounts of NADPH-cytochrome P450 reductase, and has high monoatomic oxygenation activity. Therefore, this strain can be used as a bioreactor for the synthesis of acetaminophen and steroids useful as pharmaceuticals.

Prior art and problems In the microsomal membrane of eukaryotic cells such as mammals and yeast,
There is an electron transport system composed of cytochrome P450 and NADPH-cytochrome P450 reductase. Cytochrome P450 is a heme protein located at the end of this electron transport system. Cytochrome P450 is an enzyme group consisting of many molecular species. Each molecular species has a wide range of substrate specificities, and since the substrate specificities overlap, a single atom can be used for a wide range of lipophilic compounds. It can catalyze oxygenation reactions. On the other hand, NADPH-cytochrome P450 reductase is a flavin enzyme containing one molecule of flavin adenine mononucleotide and one molecule of flavin mononucleotide in the molecule, and plays a role of transferring electrons from NADPH to cytochrome P450. Despite the molecular diversity of cytochrome P450, NADPH-
Cytochrome P450 reductase can supply electrons from NADPH to any cytochrome P450 molecular species. Moreover, the electron transfer to the cytochrome P450 by this NADPH-cytochrome P450 reductase exceeds the species of organisms,
For example, rat liver reductase is rabbit liver cytochrome P450.
The yeast reductase is rabbit or rat liver cytochrome
Electrons can be transferred to P450.

The present inventors have already isolated the rat liver cytochrome P450 gene and the rat liver NADPH-cytochrome P450 reductase gene, respectively (JP-A 61-56072 and JP-A 62-1908).
5) We succeeded in expressing these genes in yeast and producing functional enzyme proteins. Rat liver cytochrome P450 and reductase expressed in yeast were localized in the yeast microsomal membrane and exerted monoatomic oxygenation activity dependent on rat liver cytochrome P450. Furthermore, the present inventors produced a yeast strain that produces both cytochrome P450 and reductase, and showed that it exhibited cytochrome P450-dependent oxidative activity and could be used as a bioreactor for the oxidation reaction process of various compounds ( JP 62-10458
2). In addition, the inventors have used a genetic engineering and protein engineering technique to identify a chimeric cytochrome P450 between various cytochrome P450 molecular species and a novel monooxygenase having a reductase function in one molecule. Successful production (Japanese Patent Application No. 62-104583, Japanese Patent Application No. 61-7663)
3, Japanese Patent Application No. 61-187713). Furthermore, the inventors also isolated a gene of bovine adrenal cortex microsomal cytochrome P450 molecular species and expressed it in yeast to produce a yeast strain capable of oxidizing a steroid compound. (Japanese Patent Application No. 62-204101) On the other hand, NADPH-cytochrome P450
Regarding reductase, in addition to rat liver reductase gene, we succeeded in obtaining rabbit liver reductase gene (Japanese Patent Application No. 61-43443) and yeast reductase gene (Japanese Patent Application No. 62-325527). Thus, NADPH-cytochrome P450 reductase can be used as a bioreactor for oxidation reaction when used together with cytochrome P450. Yeast reductase was added to rat liver cytochrome P450, bovine adrenal cytochrome P450, NA
It can supply electrons from DPH, and the yeast reductase can supply electrons to cytochrome P450 very efficiently. Therefore, the production of a large amount of yeast reductase can be expected to improve the performance of the bioreactor for oxidation reaction.

The present inventors have succeeded in constructing an expression plasmid for expressing this in yeast using the gene for yeast NADPH-cytochrome P450 reductase. As a result, a yeast strain that produces a large amount of yeast reductase could be created. This facilitates isolation and purification of yeast reductase, that is, purification of yeast reductase has hitherto been carried out by solubilizing yeast microsomes with a surfactant and fractionating the solubilized microsomal protein with ammonium sulfate. It was possible by performing apatite, (diethylaminoethyl) DEAE-cellulose column chromatography (Y. Aoyama et al., Arch, Bioc.
hem. Biophys., 185, 362 (1985)), it is considered that the purification can be easily carried out because the reductase content of the yeast microsome was significantly increased. Further, the present inventors constructed an expression plasmid that simultaneously expresses yeast reductase and a mammalian-derived cytochrome P450 molecular species. Yeast transformed with the constructed expression plasmid showed cytochrome P4
In addition to producing 50, yeast reductase was produced in large quantities, and cytochrome P450-dependent oxidative activity was markedly increased. As a result, it is considered that the produced yeast strain has improved performance as a bioreactor for oxidation reaction.

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

The cDNA encoding the yeast NADPH-cytochrome P450 reductase used in the present invention is already known and can be isolated by a usual operation method. The expression plasmid expressing the fusion enzyme of the present invention can be constructed by inserting the yeast NADPH-cytochrome P450 reductase gene into an appropriate expression plasmid by a conventional method. As the expression plasmid, a known expression vector can be used. For example, a yeast expression vector pAAH5 (Me containing a promoter and terminator of yeast alcohol dehydrogenase (ADH1) gene
thods in Enzymology, 101, partC, p192-201), but any expression vector having a PGK promoter, G3PDH promoter, GAL10 promoter, etc. may be used as long as it has a promoter and terminator that function efficiently in the host, and particularly, , But not limited to. Moreover, the structure of the expression plasmid is not limited, and any structure may be used as long as it is stably retained in yeast. As a host, yeast such as Saccharomyces cerevisiae AH22 strain, Saccharomyces cerevisiae SHY3 strain and Saccharomyces cerevisiae NA87-11A strain can be conveniently used. Transformation with an expression plasmid containing the host yeast NADPH-cytochrome P450 reductase gene was performed using alkali metal (L
It can be carried out by a known method such as a method using iCl) or a protoplast method. By culturing the transformed yeast cells thus obtained, the yeast NADPH-cytochrome P450
A reductase can be produced. Cultivation of the transformed yeast obtained by the present invention can be carried out by an ordinary culture method.

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 pAAR1 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. Manufacturers that market these enzymes using the reaction volume of (for example, Takara Shuzo Co., Ltd.)
Was carried out under the reaction conditions attached to the product.

Figure 1 shows the construction of the expression plasmid pAAR1.

Λgt11 containing the yeast NADPH-cytochrome P450 reductase gene
The phage pgGYK (see Japanese Patent Application No. 62-325527) was digested with the restriction enzyme Kp.
Co-digested with mI and SacI, KpnI fragment of about 1.9Kb and about 0.9K
The KpnI-SacI fragment of b was recovered by low melting point agarose gel electrophoresis, and a commercially available cloning vector
A ligase reaction with pUC19 was performed to transform Escherichia coli JM109 strain. From each of the transformants, according to the method of Birnboim-Doly (Nucleic Acids Ref., 7,1513 (1979)), the plasmid DNA was prepared, and the plasmids into which the desired DNA fragment was inserted were respectively pUKN3 and pUKS31. And p
UKN3 was digested with Sau3AI and a DNA fragment of about 1.0 Kb was digested with pUC1.
Plasmid pUHB was obtained by inserting it into the BamHI site of 9. Similarly, pUKS31 was digested with Sau3AI and D350 of about 350 bp was digested.
Plasmid pUBH was obtained by inserting the NA fragment into the BamHI site of pUC19. Each of pUHB and pUBH has a structure having a reductase gene start codon and a stop codon on the HindIII side of the multi-cloning site of pUC19.
Then, DNA fragments prepared by co-digesting pUHB and pUBH with HindIII and BamHI, respectively, were prepared in advance.
BamHI site-deleted pUC19 (which can be adjusted by digesting pUC19 with HincII and SmaI and recombining) was ligase-reacted with HindIII-digested and alkaline phosphatase treated vector DNA fragments, and then E. coli JM109
The strain was transformed. A plasmid DNA was prepared from the transformant, and a plasmid containing one pUHB-derived DNA fragment and one pUBH-derived DNA fragment was designated as pUHBH.

On the other hand, the above-mentioned plasmid pUKN3 was digested with KpnI to give about 1.9 Kb.
Was prepared. Insert this into the KpnI site of pUKS31 and insert a plasmid with the correct reductase gene.
It was set to pUSKK6. Digest pUSKK6 with BamHI to obtain approximately 720 bp BamH
The I fragment was prepared and inserted into BamHI of plasmid pUHBH.
The plasmid in which the structural gene portion of the reductase was correctly regenerated was designated as pUAR. pUAR contains the structural gene for reductase,
The structural gene portion can be easily prepared by digesting with HindIII. pUAR was digested with HindIII and DN of about 2.1 Kb
A fragment was prepared, digested with HindIII, and treated with an alkaline phosphatase enzyme expression vector pAAH5 (Meth
ods in Enzymology, vol 101, P192), and then Escherichia coli JM109 strain was transformed. Plasmid DNA was prepared from the transformant, the DNA structure was confirmed, and the plasmid in which the reductase gene was inserted in the forward direction with respect to the ADH promoter and terminator was designated as pAAR1.

Example 2 Construction of Expression Plasmid pARR3 FIG. 2 shows the construction of the expression plasmid pARR3.

The plasmid pUSKK6 constructed in Example 1 contained about 350 bp of 5'upstream untranslated region and about 5'of 3'downstream untranslated region of the reductase gene.
Including 00bp. In these untranslated regions, nucleotide sequences specific to the promoter and terminator of the yeast gene are found, and it is considered that these function in yeast and can synthesize a reductase protein. Therefore, we constructed a plasmid expressing reductase under the control of the promoter and terminator of the reductase gene.

Plasmid pUSKK6 was co-digested with SphI, ScaI and SacI,
A 1.2 Kb SphI-ScaI fragment and an approximately 1.7 Kb ScaI-SacI fragment were prepared. On the other hand, the EcoRI site of the cloning vector pUC19 was made blunt by the fill-in reaction and then HindII.
I-linker (commercially available) was added. This vector is Sph
I, SacI co-digested, SphI-ScaI fragment prepared previously, S
Ligase reaction was performed with caI-SacI fragment and E. coli J
The M109 strain was transformed. Thus, a plasmid pUPR having HindIII sites introduced on both sides of the reductase gene was obtained.

The yeast expression vector pAAH5 was digested with BamHI, the ADH promoter and terminator regions were removed, the BamHI site was filled in, and then a HindIII linker was added. A ligase reaction was carried out between this vector and the HindIII and ScaI co-digested product of the above plasmid pUPR to transform E. coli. Plasmid DNA was prepared from the transformant and
By confirming the NA structure, a target expression plasmid pARR3 was obtained.

Example 3 Construction of expression plasmid pAMR1 Rat p450c (P-450MC) expression plasmid pAMC1 (Japanese Patent Application Laid-Open No. 61-
88878, described in JP-A-61-56072) is partially digested with BamHI,
The DNA fragment cut at only one site was recovered by low melting point agarose gel electrophoresis and treated with alkaline phosphatase. On the other hand, the yeast reductase expression plasmid pAAR1 constructed in Example 1 was partially digested with BamHI, and an about 4 Kb reductase expression unit consisting of the ADH promoter, reductase gene and ADH terminator was recovered by low melting point agarose gel electrophoresis. . Both prepared DNA fragments were ligated by ligase reaction, and then Escherichia coli JM109 strain was transformed. Plasmid DNA was prepared from the transformant and the DNA structure was analyzed. A plasmid containing one p450c expression unit and one reductase expression unit was designated as pAMR1
I named it.

Example 4 Construction of expression plasmid pAMR2 Rat P450c expression plasmid pAMC1 was digested with BamHI and
3.7Kb P450c expression unit (ADH promoter / P450
ccDNA.ADH terminator) was recovered by low melting point agarose gel electrophoresis. On the other hand, the yeast reductase expression plasmid pARR3 constructed in Example 2 was partially digested with BamHI to prepare a DNA fragment cleaved at only one site and treated with alkaline phosphatase. A BamHI fragment consisting of the P450c expression unit was inserted into this. For pARR3,
Although there are four BamHI sites, a plasmid in which a p450c expression unit was inserted into one of the BamHI sites at both ends of the reductase gene was named pAMR2.

Example 5 Construction of Expression Plasmid pARα Yeast expression vector pAAH5 can excise an expression unit consisting of an ADH promoter terminator by digestion with BamHI. Similarly, when a heterologous gene is inserted into the HindIII site of pAAH5, the expression unit can be excised by BamHI digestion. For example, when the P450c expression plasmid pAMC1 is cut with BamHI, the ADH promoter P450
A P450c expression unit consisting of ccDNA-ADH terminator can be easily prepared. However, it is not so easy when a BamHI site is present in the inserted heterologous gene. For example, in order to prepare the reductase expression unit described in Example 3, it was necessary to partially digest pAAR1 with BamHI and cut out a target DNA fragment of about 4 Kb. Therefore, an expression vector pAAH5N was constructed by replacing the BamHI site of pAAH5 with the NotI site. Since BamHI is a 6-base recognition enzyme, NotI is an 8-base recognition enzyme, so the frequency of NotI in the gene is considered to be significantly lower than that of BamHI.

Figure 3 shows the construction of the expression plasmid pARα.

The current vector pAAH5 was partially digested with BamHI and then treated with alkaline phosphatase. This and No shown below
A ligase reaction with tI linker (prepared using a DNA synthesizer) was performed to transform Escherichia coli DH1 strain.

Not1 linker: GATCGCGGCCGC CGCCGGCGCTGAC A plasmid DNA was prepared from the transformant to obtain a plasmid cleaved with NotI. This is similar to the above, BamH
I after digestion and alkaline phosphatase treatment, N
A ligase reaction with the otI linker was performed to transform Escherichia coli DH1 strain. In this way, the two BamHI sites of pAAH5 are
An expression vector pAAH5N substituted with tI was obtained.

Bovine adrenal P450 ₁₇ α expression plasmid PAα2 was transformed into HindIII and Eco
Co-digested with RI to prepare HindIII-EcoRI fragments of about 0.3 Kb and 1.5 Kb. This DNA fragment was digested with HindIII in advance, and ligase reaction was performed with pAAH5N treated with alkaline phosphatase to transform Escherichia coli DH1 strain.
A plasmid DNA was prepared from the transformant, and HindIII, E
The DNA structure was analyzed by digestion with coRI, and P450 ₁₇ α CDNA was identified for the ADH promoter terminator.
The plasmid in which the gene was inserted in the forward direction was designated as pANα.

On the other hand, yeast reductase expression plasmid pARR3 was partially digested with BamHI, and the NotI linker was inserted in the same manner as described above,
A plasmid pARRN in which the BamHI site on the 3'side of the reductase gene was replaced with NotI was obtained. After digesting pARRN with NotI,
The NotI fragment of about 1.8 Kb prepared from pANα was inserted. As a result of analyzing the plasmid DNA structure of the obtained transformant, a plasmid having one P450 ₁₇ α expression unit inserted was found.
It was pARα.

Example 6 Construction of Expression Plasmid pARγ FIG. 4 shows the construction of the expression plasmid pARγ.

Bovine adrenal P450 _C21 expression plasmid pUC21C2
No. 10093) was digested with HindIII to prepare a HindIII fragment of about 1.6 Kb. This DNA fragment was prepared in the same manner as in Example 5.
By inserting into the HindIII site of pAHH5N, a plasmid pANγ in which P450 _C21C DNA was inserted in the forward direction with respect to the ADH promoter and terminator was obtained. By digesting pANγ with NotI, a NotI fragment of about 3.6 Kb consisting of a P450 _C21 expression unit was prepared, and this was inserted into the NotI site of pARRN in the same manner as in Example 5 to give the desired expression plasmid pA.
Rγ was constructed.

Example 7 Transformation of Yeast with Expression Plasmid Saccharomyces cerevisiae AH22 strain (ATCC 38626) was transformed into 5
ml YPD medium (1% yeast extract, 2% polypeptone, 2%
After culturing in (% glucose) at 30 ° C. for 18 hours, 1 ml of the yeast culture solution was centrifuged to collect the cells. 0.2M LiC
The solution was washed with 1 ml of solution and then suspended in 20 μl of 1M LiCl solution.

30 μl of 70% polyethylene glycol 4000 solution,
10 μl (about 1 μg) of the expression plasmid solution was added, mixed well, and then incubated at 30 ° C. for 1 hour.
Then, after adding 140 μl of water and stirring well, this solution was added to an SD synthetic medium plate (2% glucose, 0.67%
Yeast nitrogen source amino acid-free, 20 μg / ml histidine, 2%
The transformant carrying the plasmid was obtained by plating on agar and incubating at 30 ° C. for 3 days. Yeast transformed with the plasmids pAAR1, pARR3, pAMR1, pAMR2, pARα and pARγ were respectively transformed into AH22 (pAAR1) strain and AM22 (pAAR1) strain.
RR3) strain, AM22 (pAMRI) strain, AH22 (pAMR2) strain, AH22 (pARα)
The strain was AH22 (pARγ) strain.

Example 8 Production of yeast NADPH-cytochrome P450 reductase The yeast AH22 (pAARI) strain, AH22 (pARR3) strain obtained in Example 7 and yeast A transformed with expression vector pAAH5 as a control
Each of the H22 (pAAH5) strains was cultured in SD synthetic medium (2% glucose, 0.67% yeast nitrogen source amino acid-free, 20 μg / ml histidine) to about 2 × 10 ⁷ cells / ml. To 0.9 ml of yeast culture solution, 0.1 ml of 2NHaOH and 8% 2-mercaptoethanol was added, and the mixture was incubated on ice for 10 minutes.
0.2 ml of 0% trichloroacetic acid was added, and the mixture was further incubated in ice for 10 minutes. The precipitate was collected by centrifugation at 12,000 rpm for 2 minutes, washed with ice-cold acetone, and then dried.

The prepared yeast total protein was sample buffer (4% sodium dodecyl sulfate, 0.16M Tris-hydrochloric acid (pH 6.8), 0.38M 2-mercaptoethanol, heated to 100 ° C for 5 minutes.
20% glycerol, 0.01% promphenol glue) 50
μl was added and dissolved by incubating at 100 ° C. This was subjected to 7.5% SDS-polyacrylamide gel and subjected to electrophoresis according to the method of Laemmli (Nature, 227, 680). After the electrophoresis, overlay the acrylamide gel and the nitrocellulose filter, and use the blotting buffer (25 mM Tris-hydrochloric acid (pH 8.8), 192 mM glycine,
A voltage of 30 V was applied in 20% methanol) to transfer the electrophoresed protein from the acrylamide gel to a nitrocellulose filter. Then, apply a nitrocellulose filter to a TBS buffer (50 mM Tris-HCl (pH 7.5), 200 mM).
3% gelatin, 0.05% zin2 after soaking in mM NaCl)
Incubated in TBS buffer containing 0 for 40 minutes at 37 ° C. Next, 100 μg of anti-yeast reductase antibody (Y. Aoya
ma et al., JBC, 259, 1661 (1984)), TBS buffer containing% gelatin, 0.05% Tween 20 at 37 ° C.
Incubated for 2 hours. After reaction with the antibody, in TBS buffer containing 0.05% Tween 20 at 37 ° C for 30 minutes 4
After repeated washings, the plate was incubated in TBS buffer containing 3% gelatin and 0.05% Tween 20 at 37 ° C. for 20 minutes. Subsequently, this nitrocellulose filter was treated with 2 μCi of [ ¹²⁵ I] Protein A (purchased from Amersham), TBS containing 1% gelatin and 0.05% Tween 20.
It was immersed in a buffer and incubated at 37 ° C. for 1 hour. 0.
30% at 37 ℃ in TBS buffer containing 05% Tween 20
After washing 4 times for each minute, the filter was air-dried and autoradiography was performed. AH22 (pAARI) strain, AH22 (pAARI
Both the RR3) strain and the AH22 (pAAH5) strain show a band of a protein that reacts with an anti-reductase antibody, and the migration position is the yeast reductase standard (Y. Aoyama et al., Arch. Biochem. Biophys.
185,362 (1978)). Simultaneous electrophoresis of a known amount of yeast reductase preparation, cut out the band part that reacts with anti-reductase antibody from the filter,
A calibration curve between the amount of reductase and the radioactivity was prepared by measuring the radioactivity with a γ-counter. It can react with the anti-reductase antibody of each yeast strain. The radioactivity of the band portion was measured, and the reductase production amount in yeast cells was measured from the prepared calibration curve. As a result, AH22 (pAA
RI) strain 9.4 × 10 ⁵ molecules / cell, AH22 (pARR3) strain 7.5 × 10 ⁵
It was revealed that the reductase of molecules / cells was produced.
On the other hand, the reductase content of the control AH22 (pAAH5) strain was 4 × 10 ⁴ molecules / cell or less. Therefore, A
It was revealed that the H22 (pAARI) strain and the AH22 (pARR3) strain produced about 20 times more reductase than the AH22 (pAAH5) strain.

On the other hand, the AH22 (pAARI) strain, the AH22 (pARR3) strain and the AH22 (pAAH5) strain were each cultured up to about 2 × 10 ⁷ cells / ml. About 2 x 10 ⁸
The bacterial cells were collected, suspended in a zymolyase solution (1.2 M sorbitol, 50 mM potassium phosphate (pH 7.2), 14 mM 2-mercaptoethanol, 0.4 mg / ml zymolyase 60,000), incubated at 30 ° C for 1 hour, and then suspended. Ferroplasts were prepared. Glass beads were added to this, and the cells were crushed by stirring for 3 minutes with a vortex mixer. Unbroken cells, nuclei, mitochondria, etc. were removed by centrifugation at 10,000 rpm for 20 minutes, and the supernatant was used as a crude extract to measure cytochrome C reducing activity.

Cytochrome C reducing activity is 200 mM potassium phosphate (pH 7.
7), 60 μM, EDTA, 1 mM KCN, 0.1 mM NADPH, 40 μM cytochrome C to the reaction mixture, the crude extract was added to bring the total volume to 3 ml, and the change in absorbance at 550 nm was measured at 30 ° C. Taking the oxidation-reduction molar absorption coefficient of cytochrome C as 21.2,
The reduced cytochrome C production rate was calculated. As shown in Table 1, the AH22 (pAAR1) strain and the AH22 (pARR3) strain were AH22 (pAAH5)
The cytochrome C reducing activity was about 20 times higher than that of the strain. From the above results, the reductase produced in yeast is
It was revealed that the molecule contains coenzymes FAD and FMN and has cytochrome C reducing activity.

Example 9 Simultaneous Expression of Cytochrome P450c and Yeast Reductase The yeast AH22 (pAMRI) and AH22 (pAMR2) strains obtained in Example 7 and the rat liver cytochrome P450c expressing yeast AH22 (pAMC1) strain were each about 2 × in SD synthesis medium. After culturing to 10 ⁷ cells / ml, add 1.5M acetanilide (methanol solution) to a final concentration of 25
It was added to be mM. Then, while continuing the shaking culture, a certain amount was taken after a certain period of time, the bacterial cells were removed by centrifugation, and the culture supernatant was subjected to HPCL (High Performance Liquid Chromatography) to determine the amount of acetaminophen produced. It was quantified. HPLC uses μBondapak C18 (4 x 30
(0 mm) and eluted with methanol: water: acetic acid (15: 84: 1%) and monitored the absorbance at 245 nm. Table 2 shows the acetaminophen-producing activity 6 hours after the reaction. AH22 (pAMR
1) and AH22 (pAMR2) showed about 15 times and about 11 times higher activity than the AH22 (pAMC1) strain, respectively. Rat liver cytochrome P450c and rat liver reductase co-expressing yeast AH22 (pARM
1) In the case of strains (described in Japanese Patent Application No. 62-104582), P450c expressing strains
The activity was increased only about 2-fold as compared with the AH22 (pAMC1) strain. Therefore, the results of this experiment show that the AH22 (pAMR1) strain and AH22 (pAMR2) are bioreactors for oxidation reaction.
It is shown that the strain is more useful than the AM22 (pARM1) strain.

Example 10 Co-expression of cytochrome P450 ₁₇ α and yeast reductase The yeast AH22 (pARα) strain obtained in Example 7 and bovine adrenal cytochrome P450 ₁₇ α-expressing yeast AH22 (PAα ₁ ) strain were each mixed in 5 ml of SD synthetic medium, After culturing up to about 10 8 × ⁶ cells / ml,
50 μl of 1 mM progesterone (ethanol solution) containing 10 μCi of [ ³ H] progesterone was added, and shaking culture was continued. After 1,2,6 hours, 1.0 ml was taken out, and the cells were removed by centrifugation to 0.8 ml of the culture supernatant.
Was added and stirred vigorously. After centrifugation, 1.0 ml of the dichloromethane layer was dried, the residue was dissolved in 20 μl of an equal volume of ethanol and ethyl acetate, and 10 μl was applied to a silica gel thin layer plate. Chloroform-ethyl acetate (3:
1), v / v) as a solvent, the plate was developed at room temperature for about 50 minutes, and then autoradiography was performed. The thin gel layer corresponding to the spots on the film was scraped off and the radioactivity was measured by a liquid scintillation counter. Was measured.

As a result, AH22 (pARα) strain showed 47,7 hours after 1,2,6 hours.
It was found that 8,95% of progesterone was converted to 17-hydroxyprogesterone. On the other hand, AH22 (p
The conversion rate of Aα1) strain from progesterone to 17-hydroxyprogesterone was 10, 20, 80% after 1, 2, 6 hours. Therefore, the AH22 (pARα) strain exhibited a conversion activity about 5 times higher than that of the AH22 (PAα1) strain in terms of the conversion rate at 1 hour, and was found to be useful as a steroid-oxidizing strain. .

Example 11 Simultaneous expression of cytochrome P450c ₂₁ and yeast reductase The yeast AH22 (pARγ) strain obtained in Example 7 and the bovine adrenal cytochrome P450c ₂₁ expressing yeast AH22 (pUC21C2) strain were respectively SD
After culturing in 5 ml of synthetic medium to about 8 × 10 ⁶ cells / ml, 50 μl of 1 mM progesterone (ethanol solvent) containing 10 μCi of [ ³ H] -progesterone, or 10 μCi
Containing [ ³ H] 17-hydroxyprogesterone 1 mM 17-
Hydroxylprogesterone (ethanol solution) (50 μl) was added, and shaking culture was continued 2, 5, 23 hours later, respectively.
2.0 ml of dichloromethane was added to 0.8 ml of the culture supernatant from which cells were removed by centrifugation, and the mixture was vigorously stirred. Then, the same operation as in Example 10 was performed to measure the radioactivity of the converted product.

When progesterone was used as a substrate, the conversion rate to 21-hydroxyprogesterone after 2, 5, and 23 hours was AH22 (p
ARγ) strain is 0.7, 2.3, 13 and AH22 (pUC21C2) strain is 0.2,
It was 1.2,11%. Therefore, the AH22 (pARγ) strain had slightly higher conversion activity than the AH22 (pUC21C2) strain. On the other hand, when using 17-hydroxyprogesterone as a substrate, the conversion rate to 11-deoxycortisol after 2, 5, 23 hours is 0.6, 4.4, 42% for AH22 (pARγ) strain,
AH22 (pUC21C2) strain was 0.4, 2.0, 30.6%. Therefore, it was revealed that the AH22 (pAPγ) strain showed about 1.5 to 2 times higher conversion activity than the AH22 (pUC21C2) strain.

Reference Example 1 Construction of Expression Plasmid pAα2 FIG. 5 shows an outline of the construction of the plasmid pAα2. 450 ₁₇
A plasmid pαNR (H) containing the amino terminal portion of α _c DNA (Japanese Patent Application No. 62-204101) was cleaved with restriction enzymes HphI and EcoRI at the same time. The reaction mixture was subjected to low-melting point agarose electrophoresis, and P4
50 ₁₇ About 250 corresponding to α-amino terminal coding region
The Kb fragment was recovered.

This fragment and a synthetic linker: 5′-AGCTTAAAAAAATGTGGCTGCTCCTGGCTGTC ATTTTTTTACACCGACGAGGACCGACA-5 ′ (having Hind III and HphI on the left and right ends, respectively, hereinafter, all synthetic DNAs were synthesized using Applied Biosystems 380A synthesizer). It was inserted into the HindIII-EcoRI site of plasmid pUC19 to obtain the desired plasmid pαN (H) 2. The plasmid pαN (H) 2 thus obtained and the plasmid pαC (H) containing the carboxyl-terminal coding region of P450 ₁₇ α (Japanese Patent Application No. 62-204101)
And were simultaneously digested with the restriction enzymes Hind III and EcoR I. The reaction mixture was subjected to low-melting point agarose electrophoresis, and fragments of about 285 bp and 1400 bp corresponding to the P450 ₁₇ α amino terminal and carboxyl terminal coding regions were recovered. Both of these fragments were inserted into the HindIII site of the yeast expression vector pAAH to obtain the P450 ₁₇ α single expression plasmid pAα2.

Effect of the Invention The yeast NADPH-cytochrome P450 reductase-producing yeast strain provided by the present invention produces about 20 times more reductase than the wild yeast strain used as a control. The reductase produced in yeast contains coenzymes necessary for activity,
It has the same function as yeast's original reductase. Therefore, it is considered that by containing the transformed yeast strain, a yeast having a high reductase content can be obtained, and the isolation and purification of the reductase can be facilitated.

Also, the yeast NADPH-cytochrome P4 provided by the present invention
The reductase-producing yeast strain is useful as a host for expressing a mammalian cytochrome P450 gene. When mammalian cytochrome P450 is produced in yeast and the bacterial cells are used as a bioreactor, in order to maximize the P450-dependent one-atom oxygenation activity, efficient interaction with reductase in yeast is required. Action is needed. Therefore, increasing the reductase content in yeast has the effect of increasing the P450-dependent activity. For example, when rat liver cytochrome P450c was produced using a reductase-producing yeast as a host, the P450c-dependent acetaminophen-producing activity was increased 1 to 16 times. In addition, bovine adrenal cytochrome P450 ₁₇ α and P450c ₂₁
Also in this case, the steroid-oxidizing activity of these P450-dependent yeast strains was increased 2- to 5-fold.

[Brief description of drawings]

FIG. 1 shows the steps for constructing the expression plasmid pAAR1 of the present invention. FIG. 2 shows the steps for constructing the expression plasmid pAAR3 of the present invention. FIG. 3 shows the steps for constructing the expression plasmid pARα of the present invention. FIG. 4 shows the steps for constructing the expression plasmid pARγ of the present invention. The restriction enzyme cleavage sites are B: BamHI, E: EcoRI, H: Hind III, Hc:
Hinc II, K: KpnI, N: NotI, S: Sau3AI, Sa: SacI, Sc: ScaI, Sm:
SmaI, Sp: SphI are shown. P and T are ADH promoter and terminator, respectively. Indicates the translation area.

Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12R 1: 865) (C12N 9/02 C12R 1: 865) (72) Inventor Hideo Okawa Takashizuka Takashi, Hyogo Prefecture 4 2-2-1 Sumitomo Kagaku Kogyo Co., Ltd., Examiner Kazuo Hirata (56) References JP-A-62-19085 JP-A-62-104582 Journal of Biochem history 103 [6] (1988) P: 1004-1010

Claims (16)

[Claims] 1. A plasmid containing DNA encoding rat liver cytochrome P450 and DNA encoding yeast NADPH-cytochrome P450 reductase, wherein both enzymes are expressed in yeast. 2. The plasmid according to claim 1, designated as pAMR1. 3. The plasmid according to claim 1, which is designated as pAMR2. 4. DNA encoding bovine adrenal cortex cytochrome P450 ₁₇ α and yeast NADPH-cytochrome P45.
A plasmid containing DNA encoding 0-reductase, wherein both enzymes are expressed in yeast. 5. The plasmid according to claim 4, which is designated as pARα. 6. A DNA encoding bovine adrenal cortex cytochrome P450 _C21 and yeast NADPH-cytochrome P45.
A plasmid containing DNA encoding 0-reductase, wherein both enzymes are expressed in yeast. 7. The plasmid according to claim 6, which is named pARγ. 8. A DNA encoding rat liver cytochrome P450, a DNA encoding bovine adrenal cortex cytochrome P450 ₁₇ α, or a bovine adrenal cortical cytochrome P45.
A yeast strain containing a DNA encoding 0 _C21 and a DNA encoding a yeast NADPH-cytochrome P450 reductase, and having a plasmid in which both of the above enzymes are expressed in yeast. 9. The yeast strain according to claim 8, which carries the plasmid pAMR2. 10. Saccharomyces cerevisiae AH22 / pAMR2
The yeast strain according to claim 9, which has been named. 11. The yeast strain according to claim 8, which carries the plasmid pAMR1. 12. Saccharomyces cerevisiae AH22 / pAMR1
The yeast strain according to claim 11, which is named. 13. The yeast strain according to claim 8, which carries the plasmid pARα. 14. Saccharomyces cerevisiae AH22 / pARα
14. The yeast strain according to claim 13, designated as. 15. The yeast strain according to claim 8, which carries the plasmid pARγ. 16. Saccharomyces cerevisiae AH22 / pARγ
16. The yeast strain according to claim 15, designated as.