JPH0581237B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel oxidase that has monoatomic oxygenation activity and the ability to supply reducing power from NADPH necessary for it in the same molecule, and a gene encoding the oxidase. , relates to an expression plasmid in yeast containing the gene and a yeast strain transformed with the expression plasmid. More specifically, the monoatomic oxygenation activity of cytochrome P450 (hereinafter referred to as P450) and
NADPH - an oxidizing enzyme that has the ability to supply reducing power from NADPHA possessed by cytochrome P450 reductase (hereinafter referred to as reductase) in the same molecule, a fused enzyme gene encoding the sensitizing enzyme, and a yeast expression plasmid containing the gene. The present invention also relates to a yeast strain transformed with the expression plasmid and a method for producing the oxidase, which comprises culturing the transformed yeast strain. BACKGROUND OF THE INVENTION P450 is a heme protein that exists widely in the living world, from microorganisms to mammals, and catalyzes monoatomic oxygenation reactions using a wide range of fat-soluble compounds as substrates. This broad substrate specificity exhibited by P450s is due to their molecular diversity. That is,
There are many molecular species of P450, each of which has a wide and overlapping substrate specificity, and can use a wide range of lipid-soluble compounds as substrates. However, the pathway that supplies electrons to many P450s is common, and in microsomes, reductases containing flavin adenine dinucleotide and flavin mononucleotide as coenzymes are mainly used.
Provides electrons from NADPH to P450 bound to substrate. Therefore, P450 exhibits a single-atom enzyme addition reaction only by binding to a substrate and conjugating with a reductase. The inventors have already discovered that
We isolated the genes for P450MC and rat reductase, expressed these genes using yeast as a host, and succeeded in producing an enzyme protein that exhibits a monoatomic oxygenation reaction (JP-A No. 61-88878, JP-A-61
-56702, patent application 62-019085, patent application 62-104582). In addition, we conducted further research with the aim of simultaneously expressing P450MC and reductase in yeast cells.
By creating a yeast strain that co-expresses P450MC and reductase (Japanese Patent Application Laid-open No. 63-104582), and by connecting both P450MC and reductase genes into a single gene, both electron transfer and substrate oxidation can be performed within a single molecule. Preparation of a yeast strain expressing a functional fusion enzyme (JP-A-63-
44888) was implemented. Glucocorticoids, which control carbohydrate metabolism and have the effect of depositing glycogen in the liver, are widely used as pharmaceuticals because they also have anti-inflammatory and anti-allergic effects. In the human body, it is biosynthesized through progesterone and 17-hydroxyprogesterone. Corticoids are often expensive as pharmaceuticals, and are currently produced by multi-step reactions using total synthesis or fermentation methods. The present inventors have already isolated the gene for P450 _17α present in the adrenal glands of bovines, expressed this gene using yeast as a host, and succeeded in producing 17-hydroxyprogesterone from progesterone (Patent Application No.
204101). P450 _17α originally exists in the adrenal glands and testicles of mammals, and is known to have hydroxylation activity at position 17 and side chain cleavage activity at positions _C17-20 . Produces stenedione (or dehydroepiandrostene from pregnenolone via 17-hydroxypregnenolone). However, the P450 _17α -expressing yeast strain produced by the present inventors showed a high hydroxylation activity at the 17-position, but no side chain cleavage activity at the C _17-20 positions, indicating that 17-hydroxyprogesterone (or 17-hydroxyprogesterone) pregnenolone)
only generated. This is thought to be due to the fact that environmental factors such as membrane components differ greatly between cells such as bovine adrenal glands and testicles and yeast, and the presence state of P450 ₁₇ α differs. This reactivity is very advantageous for producing glucocorticoids from progesterone via 17-hydroxyprogesterone. Structure of the invention This time, the inventors further developed their research and
Bovine P450 ₁₇ α gene and yeast reductase gene (patent application)
62-325527) into a single gene, which encodes an oxidase that has both the 1-atom oxygenation activity of P450 and the ability to supply reducing power from NADPH of NADPH-P450 reductase in the same molecule. A fusion enzyme gene was constructed and introduced into a yeast expression vector to construct an expression plasmid. The yeast strain into which the expression plasmid was introduced is P450
A fusion enzyme of yeast reductase and yeast reductase was produced and showed monoatomic oxygenation activity. Its oxidative activity is P450 ₁₇ α
Approximately 3~3% per bacterial cell compared to yeast strains expressing
5 times, and about 4 to 8 times per P450 molecule, indicating that it is highly useful for oxidation reaction processes. The regions involved in P450 _17α binding to microsomal membranes, substrate binding, and heme binding are, respectively,
It has already been predicted that the amino-terminal, central, and carboxyl-terminal HR2 regions. On the other hand, the regions involved in yeast reductase binding to microsomal membranes, flavin mononucleotide and flavin adenine dinucleotide binding, and NADPH binding range from positions 1 to 50 and positions 50 to 465, respectively, assuming the amino terminal methionine as number 1. It is also estimated that it is the 465th to 600th amino acid. The fusion enzyme gene of the present invention comprises a portion of the P450 ₁₇ α gene containing at least a region involved in substrate binding and a region involved in heme binding, and at least a portion of the yeast reductase gene (Patent Application No. 62-325527) containing flavin It can be constructed by connecting a region involved in mononucleotide or flavin adenine dinucleotide binding with a region including a region involved in NADPH binding with a linker. The cDNA corresponding to the coding region of P450 ₁₇ α and reductase used to construct the expression plasmid was
It can be produced by a conventional method used in the technical field of the present invention. For example, bovine adrenal P450 ₁₇
Regarding α, a plasmid containing this cDNA (J.BIOL.CHEM.2612475 (1986); Patent Application No. 62-
204101) by conventional methods. The same applies to the reductase gene, and the plasmid pgCYR (patent application 62
-305885). An expression plasmid that expresses the fusion enzyme of the present invention can be constructed by inserting the fusion gene constructed as described above into an appropriate expression plasmid using a conventional method. As the expression plasmid, a known expression vector can be used. For example, a yeast expression vector carrying the yeast alcohol dehydrogenase (ADHI) gene promoter and terminator
pAAH5 (available from Washington Research Foundation, Methods in Enzymology, 101 part
Cp.192-201, can be produced by the method of Ammerer et al. ), but there are no particular limitations as long as the expression vector has a promoter and terminator that function efficiently in the host, such as an expression vector having a PGK promoter, G3PDH promoter, or GAL10 promoter. Furthermore, the structure of the expression plasmid is not limited as long as it can be stably maintained in yeast. For expression of the fusion enzyme of the present invention, yeast such as S. cerevisiae strain AH22, S. cerevisiae SHY3 strain, and S. cerevisiae NA87-11A strain can be conveniently used as hosts. Transformation of these hosts with the expression plasmid containing the above-mentioned fusion gene of the present invention can be carried out by known methods such as a method using an alkali metal (LiCl) and a protoplast method. The fusion enzyme of the present invention can be produced by culturing the transformed yeast thus obtained. The transformed yeast obtained according to the present invention can be cultured by conventional culture methods. Hereinafter, the present invention will be explained in detail based on Examples, but the present invention is not limited to the Examples.
It is intended to include modifications to the extent that normally occur in the art. Example 1 Construction of expression plasmids Figure 1 shows plasmids pαLY1′, pαLY1, pαLY2,
The structures of pαLY3 and pαLY4 are shown. P450 _17α
The gene is shown as 〓〓, the reductase gene is shown as 〓〓, and the synthetic DNA linker is shown as 〓〓. The number on the top of each plasmid is the amino acid number of P450 ₁₇ α when the amino terminal Met is numbered 1, and the number on the bottom is the amino acid number of the reductase. Also▼
represents the site where the reductase is cleaved by papain. It is presumed that the amino terminal side of this site of the reductase is involved in binding to the microsomal membrane. For all plasmids, the P450 ₁₇ α portion contains its entire coding region. On the other hand, among the reductase parts, pαLY4 is the shortest and does not contain a papain cleavage site. A synthetic linker was inserted into the connection between the P450 ₁₇ α of pαLY4 and the reductase,
pαLY1′, pαLY1, pαLY2, and pαLY3 were constructed with the aim of facilitating efficient electron transfer by allowing both domains to easily collide. pαLY3 consists of 9 amino acids, pαLY1′ and
pαLY2 is 16 amino acids, pαLY1′ and pαLY1 are
27 amino acids longer than pαLY4. The production of each expression plasmid will be explained in detail below. Example 1-1 Construction of expression plasmid pαLY4 Figure 2 shows an outline of the construction of plasmid pαLY4. [Step 1] Construction of plasmid pUR(s) Plasmid pαNR(H) (patent application 62-204101) containing the amino terminal part of P450 ₁₇ αcDNA is
It was simultaneously cleaved with Hph and EcoR. The reaction mixture was subjected to low melting point agarose electrophoresis, and an approximately 250 kb fragment corresponding to the P450 ₁₇ α-amino terminal coding region was recovered. This fragment and a synthetic linker: 5'-AGCTTAAAAAAATGTGGCTGCTCCTGGCTGTC ATTTTTTTACACCGACGAGGACCGACA-5' (Hind and Hph are present at both left and right ends, respectively. All synthetic DNAs hereinafter were synthesized using Applied Biosystems Model 380A synthesizer.) were combined into a plasmid. Hind− of pUC19
Insert into EcoR site and target plasmid
pαN(H)2 was obtained. Plasmid pαN(H)2 thus obtained and plasmid pαC(H) (Japanese Patent Application No. 62-204101) containing the carboxyl terminal coding region of P450 _17α were simultaneously digested with restriction enzymes Hind and EcoR. The reaction mixture was subjected to low melting point agarose electrophoresis, and fragments of approximately 285 bp and 1400 bp corresponding to the P450 ₁₇ α-amino terminal and carboxyl terminal coding regions, respectively, were recovered. Both of these fragments were inserted into the Hind site of the yeast expression vector pAAH to obtain a plasmid pAα2 for expressing P450 _17α alone. The resulting plasmid pAα2 was digested with the restriction enzyme Hha
I cut it with. The reaction mixture was subjected to low melting point agarose electrophoresis, and a DNA fragment of about 240 bp corresponding to the P450 ₁₇ α carboxyl terminal coding region was recovered. This Hha−Hha fragment has 5′-
Synthetic linker SL1 with terminal phosphorylation and annealing: 5′-CCAGGCCTGGAAGGAAGCCCAGGCTGAGGGG GCGGTCCGGACCTTCCTTCGGGTCCGACTCCCCAGCT-5
′ (having Hha and Sal recognition sites at both left and right ends, respectively) was added, and a reaction was performed with T4 DNA ligase. Then, it was simultaneously digested with restriction enzymes Pst and Sal, and NaCl and ethanol were added to the reaction solution to perform ethanol precipitation. collected
P450 ₁₇ Plasmid Pst-Sal cleavage of approximately 140 bp corresponding to the α-carboxyl-terminal coding region
It was subcloned into the Pst and Sal sites of pUC18 to obtain plasmid pUα-PS. Next, plasmid pAα2 was simultaneously digested with restriction enzymes Hind and Pst, and the reaction mixture was subjected to low melting point agarose gel electrophoresis to recover an approximately 185 bp fragment corresponding to the P450 ₁₇ α amino-terminal coding region. On the other hand, the above plasmid pUα-PS was simultaneously digested with restriction enzymes Hind and Pst, and a fragment of approximately 2.8 kb was recovered in the same manner. A ligase reaction was performed between this fragment and the approximately 185 bp fragment corresponding to the P450 ₁₇ α-amino terminal coding region described above. Plasmid pUΔα was thus obtained. Furthermore, plasmid pAα2 was cut with the restriction enzyme Pst, and the reaction mixture was subjected to low melting point agarose gel electrophoresis, and a fragment of approximately 1200 bp corresponding to the central coding region of P450 _17α was recovered.
This fragment was inserted into the Pst site of the above plasmid pUΔα to obtain the target plasmid pUα(s). [Step 2] Construction of plasmid pUYR717 (B) Plasmid pgCYR (patent application 62-325527) containing yeast reductase cDNA insert was digested with restriction enzyme EcoR.
I cut it with. The reaction mixture was subjected to low-melting-point agarose gel electrophoresis, and fragments of approximately 410 bp and 1690 bp corresponding to the amino-terminal and carboxyl-terminal coding regions of the reductase were recovered, respectively. Each fragment of plasmid pUC19
Plasmid pUYR7, in which the amino-terminal fragment was subcloned into the EcoR site, and a plasmid in which the carboxyl-terminal fragment was inserted.
pUYR17 was obtained. Restriction enzyme plasmid pUYR17
It was partially digested with EcoR and a commercially available Hind linker was inserted to obtain plasmid pUYR17 (H). on the other hand,
The above plasmid pUYR7 was cleaved with the restriction enzyme EcoR, and the reaction mixture was subjected to low melting point agarose gel electrophoresis to recover an approximately 410 bp fragment corresponding to the amino-terminal coding region of the reductase. This fragment was inserted into the EcoR site of the above plasmid pUYR17 (H) to obtain plasmid pUYR717 (H). This plasmid pUYR717 (H) was combined with the restriction enzyme Pvu.
Co-digested with Hind. The reaction mixture was subjected to low melting point agarose gel electrophoresis, and approximately
A 2.0kb fragment was recovered. Approximately 2.0kb obtained in this way
Pvu-Hind fragment and synthetic linker SL2-1: 5'-GATCCCCCGTCGACCCCAG GGGGCAGCTGGGGTC-5' (has BamH and Pvu recognition sites at both left and right ends, and Sal recognition site inside) was inserted into the BamH-Hind site of plasmid pUC18. Insert it into
The target plasmid pUYR717 (B) was obtained. [Step 3] Construction of expression plasmid pαLY4 Plasmid pUYR717 obtained in Step 2
(B) was partially digested with restriction enzyme BstE, and further digested with restriction enzyme Pvu. This pUYR717 (B)
A commercially available Cla linker was inserted into the BstE-Pvu site to obtain plasmid pUYR4. Plasmid pUα(s) obtained in step 1 and the above plasmid
pUYR4 was simultaneously digested with restriction enzymes Hind and Sal. The reaction mixture was subjected to low melting point agarose gel electrophoresis, and fragments of approximately 1540 bp and 2020 bp corresponding to the P450 ₁₇ α or reductase coding region were recovered, respectively. Both obtained fragments were inserted into the Hind site of yeast expression vector pAAH5 to obtain the target plasmid pαLY4. Example 1-2 Construction of expression plasmid pαLY3 Figure 3 shows an outline of the expression plasmid pαLY3. The plasmid pUα(s) obtained in Step 2 of Example 1-1 and the plasmid pUYR717(B) obtained in Step 2 were simultaneously digested with restriction enzymes Hind and Sal. The reaction mixture was subjected to low-melting-point agarose gel electrophoresis to detect approximately 1540 bp and 1,540 bp, respectively, corresponding to P450 ₁₇ α or reductase coding region.
A 2050bp fragment was recovered. Both obtained fragments were inserted into the Hind site of yeast expression vector pAAH5 to obtain the target plasmid pαLY3. Example 1-3 Construction of expression plasmid pαLY2 Figure 4 shows an outline of the construction of plasmid pαLY2. Plasmid pUYR717 (H) obtained in Example 1-1, Step 2 was digested with restriction enzyme Pvu and Hind.
disconnected at the same time. The reaction mixture was subjected to low melting point agarose gel electrophoresis, and approximately 2.0kb of reductase cDNA was detected.
Fragments were recovered. The approximately 2.0kb obtained in this way
Pvu-Hind fragment and synthetic linker SL2-2: 5'-TCGACCCCATCCGATGACGGAGATATCACAG GGGGTAGGCTACTGCCTCTATAGTGTC-5' (having Sal and Pvu recognition sites at both left and right ends, respectively) was added to Sal-Hind of plasmid pUC18.
Insert into the desired plasmid
pUYR717(s) was obtained. The plasmid pUα(s) obtained in Example 1-1, Step 1, and the above plasmid pUYR717(s) were treated with restriction enzymes Hind and Sal.
disconnected at the same time. The reaction mixture was subjected to low melting point _agarose gel electrophoresis to detect approximately
Fragments of 1540bp and 2070bp were recovered. Both obtained fragments were inserted into the Hind site of yeast expression vector pAAH5 to obtain the target plasmid pαLY2. Example 1-4 Expression plasmid pαLY1′, pαLY1
Construction of plasmids pαLY1' and pαLY1 construction is shown in Figure 5. Synthetic linker SL3: 5'-CTGTGATATCTCCGTCATCGGACAGTAGCACGGCAAGCA GACACTATAGAGGCAGTAGCCTGTCATCGTGCCGTTCGT CTAGCCCCGCCAGGA GATCGGGGCGGTCCT-5' or synthetic linker SL4: 5'-TAAAGAGAAACTCCATCAAGGAACTG CTGATGTCCGAT ATTTCTCTTTGAGGTAGTTCCTTGACGACTACAGGCTA GACGGAGATATCACAG CTGCCTCTATAGTGTC−5′ Example 1-1, in which plasmid pUYR1' or pUYR1 was obtained by inserting the plasmid obtained in step 1
pUα(s) and the above plasmid pUYR1′ or
pUYR1 was co-digested with the expression enzymes Hind and Sal. The reaction mixture was subjected to low melting point agarose gel electrophoresis, and fragments of approximately 1540 bp, 2050 bp, or 2100 bp corresponding to the P450 ₁₇ α or reductase coding region were recovered. Both obtained fragments were inserted into the Hind site of the yeast expression vector pAAH5, and the target plasmid pαLY1′ or
pαLY1 was obtained. Example 2 Transformation of yeast using the constructed plasmid Saccharomyces cerevisiae strain AH22 [ATCC38626] was grown in 5 ml of YPD medium (1% yeast extract, 2% polypeptone, 2% glycose).
After culturing at 30°C for 18 hours, 1 ml of the yeast culture solution was centrifuged to collect the bacteria. The obtained bacterial cells were washed with 1 ml of 0.2M LiCl solution and then suspended in 20μ of 1M LiCl solution. Add 30μ of 70% polyethylene glycol 4000 solution, plasmids pαLY1′, pαLY1, pαLY2,
10μ of pαLY3 or pαLY4 solution (equivalent to approximately 1μg)
was added, thoroughly mixed, and then incubated at 30°C for 1 hour. Next, add 140μ of water,
After stirring well, this solution was spread on an SD synthetic medium plate (2% glucose, 0.67% amino acid-free yeast nitrogen source, 20 μg/ml histidine, 2% agar) and incubated at 30°C to generate plasmid pαLY1′. , pαLY1, pαLY2, pαLY3 and pαLY4 transformant AH22 (pαLY1′),
AH22 (pαLY1), AH22 (pαLY2), AH22
(pαLY3) and AH22 (pαLY4) were obtained, respectively. Example 3 Quantification of expression level of fusion protein of P450 ₁₇ α and reductase AH22 (pαLY1′), AH22 obtained in Example 2
(pαLY1), AH22 (pαLY2), AH22 (pαLY3) and AH22 (pαLY4) in SD synthetic medium (2% glucose, 0.67% amino acid-free yeast nitrogen source, 20 μg/ml)
Histidine) to about 2 x 10 ⁷ cells/ml, harvested the cells, washed with 100mM potassium phosphate PH7.0, and suspended in 2ml of 100mM potassium phosphate PH7.0. Dispense 1 ml of the bacterial suspension into two cuvettes, blow carbon monoxide into the sample-side cuvette, and add 5 to 50% dithionite to both cuvettes.
10 mg was added. After stirring well, add 400~
Measure the difference spectrum at 500nm, Δε=91mM ^-1 cm
Based on the value ^-1 , the heme-containing P450 amount was calculated. As a result, as shown in Table 1, AH22
(pαLY1′) strain, AH22 (pαLY1) strain, AH22
(pαLY2) strain, AH22 (pαLY3) strain and AH22
(pαLY4) strain is approximately 0.7×10 ⁵ per bacterial cell.
molecule, 0.4×10 ⁵ molecules, 0.9×10 ⁵ molecules, 0.7×10 ⁵ molecules, and 0.9×10 ⁵ molecules of heme-containing P450 protein. Example 4 Progesterone 17 of transformed yeast strain
Measurement of the amount of 17-hydroxyprogesterone produced by position hydroxylation Transformed yeast AH22 (pαLY1′) strain cultured in SD synthetic medium to approximately 0.7×10 ⁷ cells/ml, AH22
(pαLY1) strain, AH22 (pαLY2) strain, AH22
(pαLY3), AH22 (pαLY4) strain and AH22
(pAAH5) strain and AH22 that produced only P450 ₁₇ α
(pAα1) strain (patent application 62-204101) containing 1mM each containing 3μCi of [ ^3H ]-progesterone.
A progesterone-ethanol solution was added so that the final substrate concentration was 10 μM. Culture was carried out with shaking at 30°C, and after 0, 1, 2, and 6 hours, 1 ml aliquots were collected and centrifuged. To 0.8 ml of the supernatant obtained, 2 ml of dichloromethane was added, and the mixture was vigorously stirred. After centrifugation, 1 ml of the dichloromethane layer was dried, the residue was dissolved in 20μ of an equal volume solution of ethanol-ethyl acetate containing 0.5mM progesterone, 0.5mM 17-hydroxyprogesterone, and 0.5mM androstenedione, and 10μ was applied to a thin layer plate. . twenty five%
After developing with a chloroform solution containing ethyl acetate for 50 minutes at room temperature, irradiation with ultraviolet rays, scraping off the thin gel portion corresponding to the positions of progesterone, 17-hydroxyprogesterone, and androstenedione, and measuring the radioactivity using a liquid scintillation counter. was measured. As a result, the transformed yeast AH22 (pαLY1′) strain,
AH22 (pαLY1) strain, AH22 (pαLY2) strain, AH22
(pαLY3) strain and AH22 (pαLY4) strain after 6 hours, 88%, 86%, 89%, 90% and 89%, respectively.
It was found that % of progesterone was converted to 17-hydroxyprogesterone (Figure 6). on the other hand,
No activity was observed in the AH22 (pAAH5) strain used as a control. In addition, the production of androstenedione due to _C17-20 side chain cleavage activity was not observed in any of the strains. The amount of 17-hydroxyprogesterone produced per cell of each transformed strain was calculated based on the conversion rate to 17-hydroxyprogesterone one hour after addition of the substrate. AH22 producing only P450 ₁₇ α
(pAα1) strain (patent application 62204101) is set as 1, AH22 (pαLY1′) strain producing the fusion enzyme,
AH22 (pαLY1) strain, AH22 (pαLY2) strain, AH22
(pαLY3) strain and AH22 (pαLY4) strain, the increase was 4.7, 3.2, 5.0, 4.7, and 3.2, respectively. The bacterial cell used to compare the hydroxylation activity in the fusion enzyme system of the present invention and the hydroxylation activity in a conventional mixed enzyme system was the AH22 (pAα1) strain, which only produces P450 ₁₇ α. , endogenous yeast reductase is originally present in the yeast strain.
The amount is 0.3X10 ⁵ molecules/cell. That is,
The AH22 (pAα1) strain is a transformed yeast strain having a simple mixture of P450 ₁₇ α at a rate of 1.0×10 ⁵ molecules/cell and reductase at a rate of 0.3×10 ⁵ molecules/cell. From the above results, the enzyme fusion system using a fused enzyme of P450 ₁₇ α and reductase constitutes an electron transport system more efficiently than a simple enzyme mixed system of P450 ₁₇ α and reductase, and has a high uniatomic oxygen content. It has become clear that this reaction shows an addition reaction. Furthermore, the turnover rate (heme content listed in Table 1)
Regarding the hydroxylation activity at position 17 per P450 protein content, calculated based on the data in Table 1, AH22
(pαLY1′), AH22 (pαLY1), AH22 (pαLY2),
AH22 (pαLY3) is 6.7, 8.0, 5.6, 6.7, respectively,
The value is 1.0 for a simple enzyme mixture system for comparison.
It is clear that this is extremely high compared to . In addition, similar to the above test, the hydroxylation activity of the fusion enzyme system of the present invention and other mixed enzyme systems (in the unfused state, P450 ₁₇ α and exogenous yeast reductase are independent of each other) regarding the turnover rate. It also showed excellent effects in comparison with simple enzyme mixed systems (which exist in many countries). Therefore, it has become clear that these fusion enzymes have extremely high potency. Effects of the Invention The transformed yeast strain obtained by the present invention contains heme and has monoatomic oxygenation activity.
A fusion enzyme of P450 ₁₇ α and reductase was produced. The yeast strain of the present invention can be produced by adding progesterone to the culture solution and incubating it.
produced hydroxyprogesterone. On this occasion,
Six hours after substrate conversion, the conversion rate is very high, approximately 90%, and most of the product 17-hydroxyprogesterone is secreted into the medium. Therefore, 17-hydroxyprogesterone can be easily recovered by centrifugation, filtration, using an appropriate adsorbent, etc. On the other hand, yeast cells can be reused. Furthermore, the yeast strain of the present invention has a C _17-20
Since it does not have side chain cleavage activity, it is very convenient for producing glucocorticoids that are expensive as pharmaceuticals. Therefore, if the yeast strain of the present invention is used as a bioreactor for synthesizing the aforementioned steroids that are useful as pharmaceuticals, it will be possible to further simplify the current production by a multi-step reaction that combines fermentation and synthesis. It becomes possible. 【table】

[Brief explanation of the drawing]

Figure 1 shows the structure of the plasmid of the present invention and the number of amino acids of the fusion enzyme encoded by the plasmid. 〓〓 indicates the P450 ₁₇ α coding region, 〓〓 indicates the reductase coding region, 〓〓, 〓〓 indicate the synthetic linker. ▼ represents the site where the reductase is cleaved by papain. The number at the top of each plasmid is the amino acid number of P450 ₁₇ α when the amino terminal Met is set as number 1, and the number at the bottom is the amino acid number of the reductase. In addition, the number of amino acids of the fused enzyme is shown on the right side of the figure, divided into those derived from P450 _17α and those derived from the synthetic linker. Figure 2 shows the construction process of plasmid pαLY4 of the present invention. Figure 3 shows the construction process of plasmid pαLY3 of the present invention. Figure 4 shows the construction process of plasmid pαLY2 of the present invention. Figure-5
shows the construction steps of plasmids pαLY1′ and pαLY1 of the present invention. In all of Figures 2, 3, 4, and 5, Hd,
Hh, Ps, Hp, Sa, Ec, Pv, Bm, Bs, Cl are restriction enzymes Hind, Hha, Pst, Hph, respectively
, Sal, EcoR, Pvu, BamH, BstE
, shows the recognition site of Cla. 〓〓 indicates the P450 ₁₇ α coding region, 〓〓 indicates the reductase coding region, 〓〓, 〓〓 indicate the synthetic linker. Figure 6 shows the progesterone 17-position hydroxylation activity of various transformed strains. The vertical axis shows the conversion rate to 17-hydroxyprogesterone, and the horizontal axis shows the culture time. ×－×
AH22 (pAAH5) strain, ●−● AH22 (pαLY1′)
strain, ○−○ is AH22 (pαLY1) strain, □−□ is
AH22 (pαLY2) strain, ■−■ AH22 (pαLY3)
strain, △−△ is AH22 (pαLY4) strain, ▲−▲ is
AH22 (pAα1) is shown.

Claims (1)

[Scope of Claims] 1. A fusion enzyme gene in which a gene encoding bovine adrenal cytochrome P450 _17α and a gene encoding yeast NADPH-cytochrome P450 reductase are fused. 2. A yeast expression plasmid containing the gene according to claim 1 and expressing the oxidase. 3 Yeast expression plasmid pαLY1′, pαLY1,
The plasmid according to claim 2, specified as pαLY2, pαLY3 or pαLY4. 4. A transformed yeast strain carrying the yeast expression plasmid according to claim 2. 5 Satucharomyces cerevisiae-AH22
(pαLY1′) strain, AH22 (pαLY1) strain, AH22
(pαLY2) strain, AH22 (pαLY3) strain or AH22
The transformed yeast strain according to claim 4, which is identified as a (pαLY4) strain. 6 Bovine adrenal cytochrome P450 ₁₇ α and yeast NADPH
-Fusion enzyme with cytochrome P450 reductase. 7. A method for producing the oxidase, which comprises culturing the transformed yeast strain according to claim 4. 8. A method for producing 17-hydroxyprogesterone and 17-hydroxypregnenolone, which comprises hydroxylating progesterone and pregnenolone using the transformed yeast strain according to claim 4.