JPH02211880A - Yeast nadph-cytochrome p450 reductase-productive strain - Google Patents

Abstract

PURPOSE:To accomplish substantial enhancement in cytochrome-dependent oxidation activity by obtaining plasmid simultaneously manifesting yeast reductase and mammal-derived cytochrome P450 molecular species and by transforming yeast with this plasmid to effect mass production of both of them. CONSTITUTION:An established gene, cDNA coding yeast NAPH-cytochrome P450 reductase, is incorporated, by the conventional method, into a manifestation plasmid such as yeast manifestation vector PAAH5 holding both the promoter and terminator for yeast alcohol dehydrogenase gene. thus obtaining the objective plasmid.

Description

[Detailed description of the invention] Meaning! TECHNICAL FIELD The present invention relates to a yeast NADPH-cytochrome P450 reductase gene, an expression plasmid for mass-producing the enzyme, a yeast strain carrying the plasmid, and a method for producing the enzyme using the yeast. Furthermore, the present invention relates to a method for mass producing NADPH-cytochrome P450 reductase to increase the oxygen level within the yeast cells and improve monoatomic enzyme addition activity dependent on mammalian-derived cytochrome P450. The yeast strain obtained by the present invention produces cytochrome P450 and NADPH-cytochrome P450 reductase in large quantities, 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.

In the microsomal membrane of eukaryotic cells such as mammals and yeast, there are
Cytochrome P450 and NADPH-Cytochrome P450
There is an electron transport chain consisting of reductases.

Cytochrome P450 is a heme protein located at the end of this electron transport chain. Cytochrome P450 is an enzyme group consisting of many molecular species, and each molecular species exhibits a wide range of substrate specificities, and these substrate specificities overlap.
It can catalyze monoatomic oxygenation reactions for a wide range of fat-soluble compounds.

On the other hand, NADP)I-cytochrome P450 reductase is a flavoenzyme that contains one molecule of flavin adenine mononucleotide and one molecule of flavin mononucleotide in its molecule, and cytochrome P450, which plays the role of transferring electrons from NADPH to cytochrome P450, Despite exhibiting molecular diversity, NADPII-cytochrome P45
0 reductase can supply electrons from NAOP to any cytochrome P450 molecular species.

Moreover, the electron transfer to cytochrome P450 by NADPH-cytochrome P450 reductase transcends biological species; for example, rat liver reductase transfers to rabbit liver cytochrome P450, and yeast reductase transfers to rabbit or rat liver cytochrome P450. , electron transfer is possible.

The present inventors have already identified the rat liver cytochrome P450 gene and the rat liver NAI) pH-cytochrome P450 gene.
JP-A-61-56 discloses the isolation of reductase genes.
072, JP-A-62-19085), they succeeded in expressing these genes in yeast and producing a functional enzyme protein. Rat liver cytochrome P450 and reductase expressed in yeast were localized in yeast microsomal membranes and exhibited monoatomic oxygenation activity dependent on rat liver cytochrome P450. Furthermore, the inventors produced a yeast strain that produces both cytochrome P450 and reductase, and demonstrated that this strain exhibits cytochrome P450-dependent oxidation activity and can be used as a bioreactor for oxidation reaction processes of various compounds ( Japanese Patent Publication No. 62-10458
2) The inventors also used genetic engineering and fluorescent matter engineering methods to create chimeric cytochrome P450 and cytochrome P45 between various cytochrome P450 molecular species.
Succeeded in creating a new monooxygenase that has the functions of 0 and reductase in one molecule (Patent application 1045/1982)
83. Patent application 1986-76633. Special application Showa 6l-18
Furthermore, the inventors isolated the gene for the cytochrome P450 molecular species of bovine adrenal cortex microsomes and expressed it in yeast, thereby producing a yeast strain capable of oxidizing steroid compounds. (Japanese Patent Application No. 62-204101) On the other hand, regarding NADP) I-cytochrome P450 reductase, in addition to the rat liver reductase gene, the rabbit liver reductase gene (Japanese Patent Application No. 61-4
3443), yeast reductase gene (patent application 1986-32)
5527) was successfully acquired. In this way, N.A.D.
PI (-cytochrome P450 reductase is cytochrome P
By using it together with 450, it can be used as a bioreactor for oxidation reactions.

Yeast reductase can supply electrons from NADPII to rat liver cytochrome P450 and bovine adrenal cytochrome P450;
The electron supply to 0 is extremely efficient. Therefore, by producing yeast reductase in large quantities, it is expected that the performance of bioreactors for oxidation reactions will be improved.

The present inventors have successfully constructed an expression plasmid for expressing the yeast NADPH-cytochrome P450 reductase gene in yeast. As a result, they were able to create a yeast strain that produces yeast reductase in large quantities. This facilitates the isolation and purification of yeast reductase, i.e.
Conventionally, yeast reductase can be purified by solubilizing yeast microsomes with a surfactant, fractionating the solubilized microsomal proteins with ammonium sulfate, and then performing hydroxylapatite, (diethylaminoethyl) DEAE-cellulose column chromatography. There was (Y, ^ oya Rin a et al.

, ^rch, Biochem, Biophy
s,, 185. 362 (1985)), but it is thought that purification can be easily carried out because the reductase content of yeast microsomes has significantly increased. The present inventors also constructed an expression plasmid that simultaneously expresses yeast reductase and mammalian-derived cytochrome P450 molecular species. Yeast transformed with the constructed expression plasmid produced cytochrome P450 as well as yeast reductase in large quantities, and cytochrome P450-dependent oxidation activity was significantly increased. As a result, it is thought that the produced yeast strain had improved performance as a bioreactor for oxidation reactions.

The present invention will be explained in more detail below.

The cDN^ encoding the yeast NADPH-cytochrome P450 reductase used in the present invention is already known and can be isolated using normal procedures.The expression plasmid expressing the fusion enzyme of the present invention is yeast NADP The tocytochrome P450 reductase gene can be constructed by inserting it into an appropriate expression plasmid by a conventional method.01 As the expression plasmid, a known expression vector can be used.0 For example, yeast alcohol dehydrogenase (ADHI> gene promoter) and yeast expression vector pAAFI5 (Methods in
Enzy*ology+101. partc, pi92
-201), but promoter in PG, 63
Any expression vector having a promoter and terminator that functions efficiently in the host, such as an expression vector having a PDII promoter or a GALIO promoter, may be used, and there are no particular limitations on the structure of the expression plasmid. As a host, yeasts such as Saccharomyces cerevisiae A32 strain, Saccharomyces cerevisiae 5HYa strain, and Saccharomyces cerevisiae N may be used as long as they are stably retained in yeast.
A37-11A strain and the like can be conveniently used. Transformation with an expression plasmid containing the host yeast NADPH-cytochrome P450 reductase gene can be performed by a known method such as a method using an alkali metal (LiCI) or a protoplast method. By culturing the transformed yeast cells obtained in this way, yeast NA
The transformed yeast obtained according to the present invention, which can produce DPH cytochrome P450 reductase, can be cultured by a conventional culture method.

Hereinafter, the present invention will be described in detail based on examples. The present invention is not limited to the examples only, and ordinary changes in the technical field of the present invention can be made.

1 Construction of Plasmid R1 In the following examples, reactions such as DNA cleavage with restriction enzymes, DNA dephosphorylation with alkaline phosphatase, and DNA ligation with DNA ligase are usually performed in a reaction volume of 20 to 200 μi unless otherwise specified. Manufacturers who market these enzymes using
The reaction was carried out under the reaction conditions attached to the product.

Figure 1 shows the construction of expression plasmid pAAR1.

Yeast NADPI (-λgtll phage pgGYI containing the cytochrome P450 reductase gene?
= 325527), restriction enzymes Kpml and 5ac
Co-digested with KpnI fragment of approximately 1.9 Kb and approximately 0.1 Kb.
Each of the 9 Kb KpnI-3acl fragments was recovered by low melting point agarose gel electrophoresis, subjected to ligase reaction with commercially available cloning vector pUc19, and transformed into E. coli strain JM109.

From each transformant, Birnboim-Dol
y method (Nucleic Ac1ds Res,,
7.1513 (1979)), plasmid D
After adjusting the NA, the plasmids into which the desired DNA fragment had been inserted were added to pUl[N3. p[IKs31
And so. Plasmid 98HB was obtained by digesting pUKN3 with 5au3A I and inserting the approximately 1,0 b DNA fragment into the Ba-old site of pUc19. Similarly, pLIKs31 was digested with 5au3AI and
Plasmid pUBH was obtained by inserting a bp DNA fragment into the old Baa site of pUc19. pUHB
, pUBH each have a structure in which the start codon and stop codon of the reductase gene are located on the HindllI side of the ptlc19 multi-cloning site. Then, puon and pUBH were added to Hindllll, respectively.
The DNA fragment prepared by simultaneous digestion with p[1c1
9 (which can be prepared by digesting pUc19 with old ncll and 5eal and religating) was subjected to a ligase reaction with a vector DNA fragment that had been digested with old ndnT and treated with alkaline phosphatase, and then E. coli J? 1109 strain was transformed. Plasmid DNA was prepared from the transformant, and a plasmid containing one DNA fragment each derived from pUHB and pUBI+ was designated as pUHBH.

On the other hand, the aforementioned plasmid pUKN3 was digested with Kpn I to prepare an approximately 1.9 Kb Kpn r fragment. This was inserted into the pn1 site of plJKs31, and the plasmid in which the reductase gene was correctly inserted was designated pLlsKK6. pUsKK6 was digested with Bam old, approximately 720b
A Ba5al fragment of p was prepared and inserted into the Bam old of plasmid pUHBH. A plasmid in which the structural gene portion of the reductase was correctly reproduced was designated as pUAR. pUAR
contains the structural gene of the reductase, and the structural gene portion can be easily prepared by digesting with HindllI.

pUAR was digested with ) lindlI[, approx. IK
A yeast expression vector pAAR5 (Methods in Enzymolo
gy.

vol 101. After performing a glue gauze reaction with P192), E. coli strain JM 109 was transformed. Plasmid DNA was prepared from the transformant, and the DNA structure was
The plasmid in which the reductase gene is inserted in the forward direction against the ADH promoter and terminator is called pAA.
It was set as Rl.

2 Plasmid I? Construction of R3 Figure 2 shows the construction of the expression plasmid ρARR3.

The plasmid pUsKK6 constructed in [Example] contains about 350 bp of the 5" upstream untranslated region of the reductase gene and about 500 bp of the 3" downstream untranslated region. These untranslated regions contain the promoter and terminator of the yeast gene. Characteristic nucleotide sequences have been found in yeast, and these are thought to function in yeast to synthesize reductase proteins.
A plasmid expressing the reductase was constructed under the control of the promoter and terminator of the reductase gene.

Plasmid pusKK6 sph I, 5cal, 5
Co-digested with acI, approximately 1.2 Kb of 5phl-5ac
A 5cal-5acI fragment of approximately 1.7 Kb was prepared. On the other hand, cloning vector pUc19 Eco
After the R1 site was made blunt by a fill-in reaction, a former ndIII linker (commercially available) was added. This vector was co-digested with Sphr and 5acl, and a ligase reaction was performed with the previously prepared 5phr-5cal fragment and 5ealSacI fragment to transform Escherichia coli JM 109 strain. In this way, Hi on both sides of the reductase gene.
A plasmid pUPR into which the ndI[[ site was introduced was obtained.

The yeast expression vector pAA)15 was digested with BamHI,
Except for the ADH promoter and terminator region, Ba
After filling in the mHI site, the old ndll linker was added. This vector and the previous plasmid pUP
I? A glue gauze reaction was carried out with the Hindll+ and 5cal co-digested product of E. coli to transform E. coli. The desired expression plasmid pARR3 was obtained by preparing plasmid DNA from the transformant and confirming the DNA structure.

3 Plasmid A phylum R1 rat P450c (P-450MC) expression plasmid pAMc1 (JP-A-61-88878.JP-A-61-
56072) was partially digested with Ba11 old, and a DNA fragment that was cleaved at only one site was collected 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 Bam, and a reductase expression unit of about 4 and b consisting of the ADH promoter, reductase gene, and ADH terminator was transferred to a low melting point agarose gel. It was collected by electrophoresis. After ligating both of the prepared DNA fragments by ligase reaction, Escherichia coli JM 109 strain was transformed. Plasmid DNA was prepared from the transformed strain and the DNA structure was analyzed. The plasmid containing one P450c expression unit and one reductase expression unit was named pAMRl.

4 plasmids Rat P450c expression plasmid pAMc1 Ba11
The approximately 3.7 Kb P450c expression unit (
ADH promoter・P450cc DN A-A
DH 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 Ba+wHI to prepare a DNA fragment cut at only one site, and treated with alkaline phorphatase. In addition, P45
The 0c expression uni expression unit Ba■H1 fragment was inserted.

pARR3 has four old Bag sites, but one of the Ba*H1 sites at both ends of the reductase gene has a p
The plasmid into which the 450c expression unit has been inserted is pAM
It's named R2.

Example 5 Construction of expression plasmid ARα The expression unit consisting of the ADH promoter terminator can be excised from the yeast expression vector pA^5 by BamHI digestion. Hind of pAAH5
■When a foreign gene is inserted into the site, Ba1
1 The expression unit can be excised by old digestion. 0 For example, the P450c expression plasmid pAMc1 is
When cut with 1 old, ADH promoter P450ccD
A P450c expression unit consisting of the NA-ADH terminator can be easily prepared. However, if a Ba*HT site exists in the inserted heterologous gene, this is not so easy. For example, in order to prepare the reductase expression unit described in Example 3, pAARl must be
It was necessary to partially digest the DNA with 110ml and excise the desired DNA fragment of approximately 4 Kb. Therefore, Bam of pAA115
Expression vector pA with 111 site replaced with NotT site
Al15N was constructed. While Bag old is a 6-base recognition enzyme, NotT is an 8-base recognition enzyme, so it is thought that the frequency of Notl in the gene is significantly lower than that of Ba1l old.

Figure 3 shows the construction of the expression plasmid pARα.

After the expression vector pAAII5 was partially digested with Ba5a+, it was treated with alkaline phosphatase. This was subjected to a glue gauze reaction with the Notl linker shown below (prepared using a DNA synthesizer) to transform Escherichia coli strain DH1.

Notl linker: Plasmid DNA was prepared from the GATCGCGGCCGC transformant to obtain a plasmid that was cleaved with Notl. This is converted into Ba-
After digestion with III and alkaline phosphatase treatment, a glue gauze reaction with a Notl linker was performed to transform E. coli strain 0111. Thus, 2 of pAAH5
An expression vector pAAl15N was obtained in which one site of Bawl (1 site) was replaced with Notl.

Bovine adrenal P450 + , α expression plasmid PAα2 was co-digested with Bindl and EcoRI to prepare approximately 0.3 Kb and 1.5 Kb Hindll[-EcoRI fragments. This DNA fragment was previously digested with HindIII and treated with alkaline phosphatase to create pAA115.
A glue gauze reaction with N was performed to transform E. coli strain 081. Plasmid DNA was prepared from the transformed strain, and B
The DNA structure was analyzed by indl[[, EcoRI digestion, etc., and a plasmid in which P2S5 + qαC DNA was inserted in the forward direction relative to the ADH promoter terminator was named pANα.

On the other hand, yeast reductase expression plasmid pARR3 was transferred to Ba5
Partially digested with HI and as described above, Notl! J
A plasmid pARRN was obtained in which a linker was inserted and the old Bag site on the 3° side of the reductase gene was replaced with Notl.

After pARRN was digested with Notl, an approximately 1.8 Kb Notl fragment prepared from pANα was inserted. As a result of analyzing the plasmid DNA structure of the obtained transformed strain,
A plasmid into which one P2S5 r and α expression unit was inserted was designated as pARα.

6 Plasmid AR Figure 4 shows the construction of expression plasmid pAR7.

Bovine adrenal P450c*+ expression plasmid pUc21c2
(Feikoken Bibori No. 10093) was digested with old ndnl to prepare an approximately 1.6 Kb Hindllr fragment. In the same manner as in Example 5, this DNA fragment was transformed into ρAt(H5
Plasmid pAN 7 was obtained in which P450cmt cDNA was inserted in the forward direction with respect to the old ndI[1 site of N and AD) (promoter and terminator).
Digest pANγ with Notl to prepare an approximately 3.6 Kb Notl fragment consisting of the P450c expression unit, and insert this into the Not1 site of pARRN in the same manner as in Example 5 to obtain the desired expression plasmid. pA
I built R7.

7 Replacement of Saccharomyces cerevisiae AH2 strain (ATCC38
626), 5111 YPD medium (1% yeast extract,
2% polypeptone, 2% glucose) at 30°C for 1
After culturing for 8 hours, the laf yeast culture solution was centrifuged to collect bacteria. The bacterial cells were added to a 0.2M LiCl solution.
After washing with water, the cells were suspended in 2 μl of LM LiCl solution.

To this, 70% polyethylene glycol 4000ml solution 3
0μ! , expression plasmid solution 10μ! (approximately 1 μg) was added, thoroughly mixed, and then incubated at 30° C. for 1 hour. Next, 140〃j! After adding water and stirring thoroughly, this solution was added to an SD synthetic medium plate (2% glucose, 0.67% yeast nitrogen source amino acid free, 20M
g/di histidine, 2% agar) and incubated at 30°C for 3 hours.
A transformant retaining the plasmid was obtained by incubation for days. Plasmid pAAR1, pAR
Yeast transformed with R3, pAMRl, pAMR2, pARα, and pARγ were transformed with Al22 (pAARl), respectively.
) strain, Al22 (pARR3) strain, A)122 (PA
MRI) strain, Al22 (pAMR2) strain, Al22 (
pARα) strain and Al22(9ARγ) strain.

Yeast AH22 (pAARl) strain obtained in Example 7, A) 1
22 (pARR3) strain and as a control, yeast Al22 (p^^[5
) strain in SD synthetic medium (2% glucose, 0
．． The cells were cultured to approximately 2×10′ cells/id in 67% yeast (no nitrogen source amino acid, 20 Mg/d histidine). Yeast culture solution 0.9II11, 2N NaOH 18% 2-
After adding 0.1+d of mercaptoethanol and incubating in water for 10 minutes, 0.1+d of 30% trichloroacetic acid was added.
2d was added and further inked in water for 10 minutes. 12. The precipitate was collected by centrifugation at OOOrpm for 2 minutes, washed with water-cooled acetone, and then dried.

The prepared yeast total protein was mixed with sample buffer (4% sodium dodecyl sulfate, 0.1
6M) Lis-hydrochloric acid (pH 6,8), 0.38M 2
-Mercaptoethanol, 20% glycerol, 0°0
Add 50 μ2 of 1% bromophenol glue and add 100
T8 resolution was achieved by incubation at °C.

This was applied to a 7.5% 5DS-polyacrylamide gel using the method of Lae+1-1i (Nature, 227
．． Electrophoresis was performed according to 680). After electrophoresis, layer the acrylamide gel and nitrocellulose filter,
Plotting buffer (25mM) Lis-HCl (
Transfer the electrophoresed proteins from the acrylamide gel to a nitrocellulose filter by applying a voltage of 30 V in a solution (pH 8.8), 192 mM glycine, 20% methanol), and then transfer the nitrocellulose filter to a nitrocellulose filter using TBS.
Buffer (501 μM Tris-HCl (pH 7.5),
After soaking in 200mM NaCl), 3% gelatin,
in TBS buffer 1-3 containing 0.05% Twin 20
The ink was inked at 7°C for 40 minutes. Next, 100 μg of anti-yeast reductase antibody (Y, Aoyama et al.
+J, B, C, +259+1661 (1984))
The ink was incubated at 37°C for 2 hours in a TBS buffer containing 0.05% gelatin and 0.05% Twin 20. After the reaction with the antibody, washing was repeated four times at 37°C for 30 minutes each in TBS buffer containing 0.05% Twin 20, followed by washing in TBS buffer containing 3% gelatin and 0.05% Twin 20.
Ink evacuation was carried out in S buffer for 20 minutes at 37°C. Next, this nitrocellulose filter was
Ci [1tsI] T containing protein A (purchased from Amajam), 1% gelatin, 0.05% Twin 20
The ink was spread in BS buffer and inked at 37°C for 1 hour. After washing four times for 30 minutes each at 37°C in TBS buffer containing O,OS%Twin20, the filters were air-dried and autoradiography was performed. A
l22 (pAARI) strain, H22 (PARR3) strain,
All of the Al22 (PAAH5) strains show a protein band that reacts with the anti-reductase antibody, and the migration position is as follows.
Yeast reductase preparation (Y, Aoyama et al.,
Arch, Biochet Biophys, 185.3
62 (1978)). By simultaneously electrophoresing a known amount of yeast reductase preparation, cutting out the band that reacts with the anti-reductase antibody from the filter, and measuring the radioactivity with a T-counter, the relationship between the amount of reductase and the radioactivity can be determined. A calibration curve was created.

It can react with anti-reductase antibodies of each yeast strain.

The radioactivity of the band was measured, and the amount of reductase produced in the yeast cells was measured from the prepared calibration curve. As a result, it was found that the Al22 (pAARI) strain produced 9.4 x 10' molecules/cell of reductase, and the [22 (pARR3) strain produced 7.5 x 10 molecules/cell.

On the other hand, the reductase content of the control strain Al22 (pAAH5) was less than 4×10′ molecules/bacterial cell.

Therefore, AI(22(pAARl) strain, AI+22
(pAI'1R3) strain is A)122 (pAAI+5
) strain produced about 20 times more reductase.

On the other hand, A) 122 (PAARI) strain, ^H22 (pAR
R3) strain, 8) +22 (pAAH5) strain, about 2 each
The cells were cultured to X10' cells/d. Approximately 2 x 10 bacterial cells were collected and added to zymolyase solution (1.2M sorbitol,
5. OsM potassium phosphate (pH 7,2), 14mM
Spheroplasts were prepared by suspending the cells in 2-mercaptoethanol, 0.4 mg/d zymolyase (60,000) and incubating at 30°C for 1 hour. Glass peas were added to this, and the cells were crushed by stirring with a portex mixer for 3 minutes. 10.00Orp
Unbroken bacterial cells, nuclei, mitochondria, etc. were removed by centrifugation for 20 minutes, and the supernatant was used as a crude extract to measure cytochrome C reducing activity.

Cytochrome C reduction activity was determined by 20-axis potassium gallate (p
H7,7), 60μM, EDTA-1mMKC
N, 0.1mM NADP■, 40μM cytochrome C
Add the crude extract to the reaction mixture consisting of 3II1
1, and the change in absorbance at 550 n- was measured at 30°C.

The oxidized-reduced molar extinction coefficient of cytochrome C is 2162
The reduced cytochrome C production rate was calculated as Table 1
Eight F122 (PAARI) strains, Al1, as shown in
22 (pARR3) strain is AI (22 (pAAI+5
) strain, which showed a cytochrome C reducing activity approximately 20 times higher than that of the yeast strain, indicating that the reductase produced in yeast contains coenzymes FAD and PMN in the molecule and has cytochrome C reducing activity. It became clear.

Yeast AH22 (pAM old) obtained in Example 7, Al22
(pAMR2) strain and rat liver cytochrome P450c
After culturing the expression yeast AI (22 (pAMcl) strain) in SD synthetic medium to approximately 2 x 10 cells/d, 1.
5M acetanilide (methanol solution) at a final concentration of 25m
It was added so that it became M. After that, while continuing the shaking culture, a certain amount was collected after a certain period of time, the bacterial cells were removed by centrifugation, and the culture supernatant was subjected to HPLC (high performance liquid chromatography) to calculate the amount of acetaminophen produced. Quantitated. HPLC uses μBond as a column
Using apak Cl8 (4X30h*), elution was carried out with methanol:water:acetic acid (15:84:1%), and the absorbance at 245 nm was monitored. Table 2 shows the acetaminophen production activity after 6 hours of reaction. , AI22Al22(
pA, AI22Al22 (pA is AH22 (pAM
cl) strain, showed about 15 times and about 11 times higher activity, respectively. Co-expression of rat liver cytochrome P450c and rat liver reductase yeast AI2Al22 (pAR strain (
(described in patent application No. 62-104582), P450
Compared to the c expression strain AH22 (pAMcl) strain, the activity was
It only increased by about twice. Therefore, the results of this experiment demonstrate that AH22
This indicates that the (pAMl?l) strain and the AH22 (pAMti2) strain are more effective than the H22 (pARMI) strain.

The yeast AH22 (pARα) strain obtained in Example 7 and the yeast AH22 (PARα) expressing bovine adrenal cytochrome P450 + qα
Approximately 8
After culturing to 0" cells/d, IOμCi (Ql)
50 μl of 1 mM progesterone (ethanol solution) containing progesterone was added, and shaking culture was continued.
After 1 and 2.6 hours, 1.0 d was collected, and 2.0 ml of dichloromethane was added to 0.8 af of the culture supernatant from which bacterial cells were removed by centrifugation, and the mixture was vigorously stirred. After centrifugation, dichloromethane layer 1. Oad! Dry, dissolve the residual aroma in 20 μl of a solution with equal volumes of ethanol and ethyl acetate, and add 10 μl.
was applied to a silica gel thin layer plate. After developing the plate at room temperature for about 50 minutes using chloroform-ethyl acetate (3: L v/v) as a solvent, autoradiography was performed, and the thin gel portion corresponding to the spots formed on the film was peeled off. , radioactivity was measured using a liquid scintillation counter.

As a result, it was found that in the AH22 (pARα) strain, 47.78.95% of progesterone was converted to 17-hydroxyprogesterone after 1.2.6 hours. In contrast, the conversion rate of progesterone to 17-hydroxyprogesterone in the AH22 (pAα1) strain is 1,
After 2.6 hours, it was 10.20.80%. Therefore, the 8822 (pARα) strain exhibits approximately 5 times higher conversion activity than the 8H22 (PAα1) strain in terms of conversion rate at 1 hour, and is therefore useful as a steroid oxidation-reactive strain. found.

The yeast^)+22 (pARr) strain obtained in Example 7 and the bovine adrenal cytochrome P450Czr expressing yeast^t(22
(pUc21c2) strain, respectively, on SD synthetic medium 5Id.
After culturing to approximately 8 x 10" cells/d, 10 μC
i of 1mM progesterone (ethanol solvent) containing [3H]-progesterone 50. cl, louis, 10p
ci(7) ('H) 50 μl of 1mM 17-hydroxyprogesterone (ethanol solution) containing 17-hydroxyprogesterone was added and the shaking culture was continued for 2 and 5.23 hours, respectively. (2.0 m of dichloromethane was added to 0.8 d of the culture supernatant from which bacterial cells were removed by centrifugation and stirred vigorously.Then, the same operation as in Example 1o was carried out, and the radioactivity of the converted product was was measured.

When progesterone is used as a substrate, the conversion rate to 21-hydroxyprogesterone after 2.5.23 hours is 0.7.2.3.13 for the AI+22 (pARγ) strain and 0.0 for the AH22 (pUc21c2) strain. .2.1.2
．． It was 11%. Gatte, AH22 (pARr
) strain had slightly higher conversion activity than the AH22 (pUc21c2) strain. On the other hand, when 17-hydroxyprogesterone is used as a substrate, the conversion rate to 11-deoxycortisol after 2°5.23 hours is ^H22
(pARr) strain is 0.6.4.4.42%, A
1122 (pHc21c2) strain is 0.4.2.0.30
．． It was 6%. Therefore, A) 122(pAP r
) strain was found to exhibit approximately 1.5 to 2 times higher conversion activity than the AH22 (PUC21C2) strain.

1. Construction of plasmid Aα2 Figure 5 shows an outline of the construction of plasmid pAα2.
Plasmid ραNl containing the amino-terminal part of P450+yα cDNA? (Special @ 62-204101) was simultaneously digested with the restriction enzymes oph I and EcoRI, and the reaction mixture was subjected to low melting point agarose electrophoresis.
? Approximately 250, corresponding to the α-amino-terminal coding region
A fragment of Kb was recovered.

This fragment and synthetic linker: 5'-AGCTTAAAAAAAATGTGGCTGC
TCCTGGCTGTCATTDingTTTTACACCG
ACGAGGACCGACA-5' (contains old ndlll and Hph I at both left and right ends, respectively. All synthetic NAs hereinafter are 3-day 0A manufactured by Applied Biosystems.
Synthesized using a model synthesizer. ) into the old ndDI-EcoR1 site of plasmid pUc19,
The target plasmid pαN(H)2 was obtained. The thus obtained plasmid pcrN(H)2 and P45017
The plasmid pαC()I) (Japanese Patent Application No. 62-204101) containing the carboxyl-terminal coding region of α was simultaneously digested with the restriction enzymes old ndll and EcoRI.

The reaction mixture was subjected to low melting point agarose electrophoresis, and P2S5
．． approximately 285 bp, 1400 b corresponding to the 7α amino-terminal and carboxyl-terminal coding regions, respectively
A fragment of p. Both of these fragments were inserted into the H4ndm site of the yeast expression vector pAAl, and P4501? α
A single expression plasmid pAα2 was obtained.

Effects of the invention Yeast NADPH-cytochrome P provided by the present invention
The 450 reductase-producing yeast strain produces approximately 20 times more reductase than the control wild yeast strain.

The reductase produced in yeast contains coenzymes necessary for its activity and has the same function as the reductase native to yeast. Therefore, by culturing the transformed yeast strain, yeast with a high reductase content can be obtained, and it is considered that the isolation and purification of the reductase becomes easy.

Furthermore, the yeast NADPII-cytochrome P450 reductase-producing yeast strain provided by the present invention is useful as a host for expressing mammalian cytochrome P450 genes. When producing mammalian cytochrome P450 in yeast and using bacterial cells as a bioreactor, in order to maximize P2S5-dependent monoatomic oxygenation activity, efficient interaction with reductase in yeast is required. action is required. Therefore, the increase in reductase content in yeast is due to P2S
For example, when rat liver cytochrome P450c is produced using reductase Butsuza yeast as a host, the P450c-dependent acetaminophen production activity increases by 12 to 16 times. did. In addition, bovine adrenal cytochrome P450+tα and P450Cz
Also in the case of +, the steroid oxidation activity of these P2S5-dependent yeast strains increased 2 to 5 times.

Table 1 Reductase content of yeast NADPH-cytochrome P450 reductase producing strain and cytochrome c4 reduction activity strain (molecule/cell) (na+ol/win/s+g p
rotein')AH22(pAAf15) <
4.0X10' 26AH22
(pAARl) 9.4X10' 6
10AH22(pARR3) 7.4XIO'
570 Table 2 Acetaminophen production activity by transformed yeast strains Strains ^ [22 (pAMC5) 8022 (pAMRI) AH22 (pAMR2)

[Brief explanation of the drawing]

FIG. 1 shows the construction steps of the expression plasmid pAAR1 of the present invention. FIG. 2 shows the construction steps of the expression plasmid pAAR3 of the present invention. FIG. 3 shows the construction steps of the expression plasmid pARα of the present invention. FIG. 4 shows the construction steps of the expression plasmid pARγ of the present invention. FIG. 5 shows the construction process of plasmid p^α2 of Reference Example 1. Restriction enzyme cleavage sites are B: Ba+wH1, E: EcoR
I, H:H4nd111, Hc:H4ncI[+K:
Kpnl+N: Notl, S: 5au3Al, Sa
:5acl, Sc:5ca1. Ss:5acl, S
p: Indicates 5phl, also P. T indicates the ADH promoter and terminator, respectively, and in the figure, the symbol indicates the yeast reductase translation region, and = indicates P4.
501? = indicates the α translation region; = indicates the P450ct+ translation region. Figure UAR pAAR+ B

Claims (31)

[Claims] (1) Expression plasmid for mass production of yeast NADPH-cytochrome P450 reductase in yeast (2) The expression plasmid according to claim 1, which contains the promoter of the yeast NADPH-cytochrome P450 reductase gene. (3) The plasmid according to claim 2 named pARR3 (4) The expression plasmid according to claim 1, which contains the promoter of the yeast alcohol dehydrogenase gene. (5) The plasmid according to claim 4 named pAAR1 (6) Yeast strain that mass-produces yeast NADPH-cytochrome P450 reductase (7) The yeast strain according to claim 6, which carries an expression plasmid having a promoter of the yeast NADPH-cytochrome P450 reductase gene. (8) Yeast strain according to claim 7, which retains plasmid pARR3 (9) Saccharomyces cerevisiae AH22/pARR
The yeast strain of claim 8 named No. 3 (10) A yeast strain according to claim 6, which carries an expression plasmid having a promoter of a yeast alcohol dehydrogenase gene. (11) Yeast strain according to claim 10, which retains plasmid pAAR1 (12) Saccharomyces cerevisiae AH22/pAA
Yeast strain of claim 11 named R1 (13) A method for producing yeast NADPH-cytochrome P450 reductase, which comprises culturing a yeast strain that mass-produces yeast NADPH-cytochrome P450 reductase. (14) Expression plasmid that expresses mammalian cytochrome P450 in yeast and produces large amounts of yeast NADPH-cytochrome P450 reductase (15) Expression plasmid that expresses rat liver cytochrome P450 in yeast and produces large quantities of yeast NADPH-cytochrome P450 reductase (16) The plasmid according to claim 15 named pAMR1 (17) The plasmid according to claim 15 named pAMR2 (18) Cytochrome P450 of bovine adrenal cortex in yeast
_1_7α and yeast NADPH-cytochrome P4
Expression plasmid for mass production of 50 reductase (19) The plasmid according to claim 18 named pARα (20) Cytochrome P450 of bovine adrenal cortex in yeast
Expressing _c_2_1, yeast NADPH-cytochrome P
Expression plasmid for mass production of 450 reductase (21) The plasmid according to claim 20, named pARγ (22) producing mammalian-derived cytochrome P450,
Yeast strain that mass-produces yeast NADPH-cytochrome P450 reductase (23) A yeast strain carrying an expression plasmid that co-expresses a mammalian-derived cytochrome P450 gene and a yeast NADPH-cytochrome P450 reductase gene. (24) The yeast strain according to claim 23, which retains plasmid pAMR2. (25) Saccharomyces cerevisiae AH22/pAM
Yeast strain according to claim 24 named R2 (26) The yeast strain according to claim 23, which retains plasmid pAMR1 (27) Saccharomyces cerevisiae AH22/pAM
Yeast strain according to claim 26 named R1 (28) The yeast strain according to claim 23, which retains plasmid pARα (29) Saccharomyces cerevisiae AH22/pAR
Yeast strain according to claim 28 named α (30) Yeast strain according to claim 23, which retains plasmid pARγ (31) Saccharomyces cerevisiae AH22/pAR
Yeast strain according to claim 30 named γ