JPH0223870A - Fused oxidation enzyme of cytochrome p45017alpha and yeast nadph-cytochrome p450 reductase - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to the improvement of l-atom oxygenation activity and the NA required for it.
The present invention relates to a novel oxidase having the ability to supply reducing power from DPH in its molecule, a gene encoding the oxidase, a yeast expression plasmid containing the gene, and a yeast strain transformed with the expression plasmid.

More specifically, the l atom oxygenation activity and NADPI of cytochrome P450 (hereinafter referred to as P2S5)
An oxidase having within the same molecule the ability to supply reducing power from NADPH possessed by I cytochrome P450 reductase (hereinafter referred to as reductase), a fusion enzyme gene encoding the oxidase, a yeast expression plasmid containing the gene, and The present invention relates to a yeast strain transformed with the expression plasmid and a method for producing the oxidase, which comprises culturing the transformed yeast strain.

Honko Oku □□□ Background P2S5 is a heme protein that exists in a wide range of living organisms, from microorganisms to mammals, and catalyzes monoatomic oxygenation reactions using a wide range of fat-soluble compounds as substrates. P2S
This broad substrate specificity exhibited by 5 is due to the molecular diversity of P2S5. That is, there are many molecular species of P2S5, each of which has a wide range of substrate specificities and overlaps, and can use a wide range of lipid-soluble compounds as substrates. However, the thread that supplies electrons to many P2S5s is common, and in microsomes, reductases containing flavin adenine dinucleotide and flavin mononucleotide as coenzymes in their molecules convert electrons from NADPH into P2S5s that bind substrates. supply to Therefore, P2S5 exhibits a single-atom enzyme addition reaction only by binding to a substrate and conjugating with a reductase.

The present inventors have already isolated the genes for P2S5 anal and runt reductases present in the liver of rats, and succeeded in expressing these genes using yeast as a host to produce an enzyme protein that exhibits a monoatomic oxygenation reaction. I did (time opening 61-
88878, Hours 61-56702, Special 62-019
085, Special No. 62-104582), and further research to create a yeast strain that co-expresses P450MC and reductase with the aim of simultaneously expressing P450MC and reductase in yeast cells (Jikai No. 63-104582). Construction of a yeast strain that expresses a fused enzyme that has both the functions of electron transfer and substrate oxidation within a single molecule by linking both the P450MC and reductase genes into a single gene (Jikai 63- 44888) was carried out.

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 via progesterone and 17-hydroxyprogesterone. Corticoids are often expensive as pharmaceuticals, and are currently produced by a multi-step reaction using total synthesis or fermentation. We succeeded in producing 17-hydroxyprogesterone from progesterone by expressing this gene (Special VA62-204101).
), P450+, α are originally present in mammalian adrenal glands, Suimaru, etc., and have hydroxylation activity at position 17 and C+t-Z. It is known to have side chain cleavage activity, and produces androstenedione from progesterone via 17-hydroxyprogesterone (or dehydroepiandrostene from pregnenolone via 17-hydroxypregnenolone). However, P2 produced by the present inventors
S51? The α-expressing yeast strain showed high hydroxylation activity at position 17, but C+t-X. No side chain cleavage activity was shown at 17
- Only hydroxyprogesterone (or 17-hydroxypregnenolone) was produced. 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 raimaru cells and yeast, and the state of existence of P450+vα differs. This reactivity is very advantageous for producing glucocorticoids from progesterone via 17-hydroxyprogesterone.

Bottom of the Invention This time, the present inventors further developed their research and developed bovine P4.
50rvα gene and yeast reductase gene (Special 62-3
25527) to form a single gene, and the monoatomic oxygenation activity and NADP of P2S5
A fusion enzyme gene encoding an oxidase that has the ability of II-P450 reductase to supply reducing power from NADPII in the same molecule was constructed, and this was introduced into a yeast expression hector to construct an expression plasmid.

The yeast strain into which the expression plasmid was introduced produced a fusion enzyme of P2S5 and yeast reductase, and exhibited l-atom oxygenation activity. The oxidation activity was approximately 3 to 5 times higher per bacterial cell and approximately 4 to 8 times higher per 1450 molecules than a yeast strain expressing P450I and α alone, indicating that it is highly useful for oxidation reaction processes. .

The regions involved in P2S5 + qα binding to the microsomal membrane, substrate binding, and heme binding are the amino-terminal, central, and carboxyl-terminal HR regions, respectively.
It is already estimated that it will be in the 2GW region.

On the other hand, the regions involved in yeast reductase binding to the microsomal membrane, flavin mononucleotide and flavin adenine dinucleotide binding, and NADPH binding are from 1 to 50 and from 50 to 465, respectively, assuming the amino terminal methionine as number 1. It is also estimated that it is the amino acid number 465 to 600.

The fusion enzyme gene of the present invention combines a small portion of the P45017α gene (including a region involved in substrate binding and a region involved in heme binding) and a yeast reductase gene (particularly IHN
o, 62-325527), by connecting at least a region involved in flavin mononucleotide or flavin adenine dinucleotide binding with a region including a region involved in NADPH binding with a linker.

P4501 used for expression plasmid construction? Corresponds to the coding region of α and reductase. For example, for bovine adrenal P450 + qα, a plasmid (J, B I
OL, CHEM, 261.2475 (1986
); time 62-204101) by a conventional method. The same applies to the reductase gene, and the plasmid pgCYR (
62305885).

An expression plasmid that expresses the fusion enzyme of the present invention can be constructed by inserting the fusion gene constructed horizontally as described above into an appropriate expression plasmid using a conventional method.0 As the expression plasmid, a known expression vector can be used. For example, yeast expression vector pAAH5 (Washington Re5e) carrying the promoter and terminator of the yeast alcohol dehydrogenase (MDIll) gene.
Available from arch Foundation, Meth
ods in Enzya+ology+ 101p
It can be produced by the method of art Cp, 1192-201A+werer et al. ), but F'lJ promoter, C3PI)+1bomoy-2
There are no particular limitations on the expression vector, as long as it has a promoter and terminator that function efficiently in the host, such as an expression vector with the GALIO promoter.The structure of the expression plasmid is also not limited. Any material that can be stably maintained within the container may be used.

For expression of the fusion enzyme of the present invention, yeast such as S. cerevisiae^H22 strain, S. cerevisiae 5HYS strain, and S. cerevisiae NA37 are used.
-11A strain etc. can be conveniently used as a host. 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 (LiCI) 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 described in detail based on five examples, but the present invention is not limited to the examples and includes modifications to the extent normally practiced in the field of the present invention.

Example 1. - Construction of plasmids Figure 1 shows plasmids pαLY1', pαLY1. pαL
Y2. +1αLY3. ραLY4. The structure of is shown. The P2S5.7α gene is indicated by m, the reductase gene by loco, and the synthetic NA linker by ■. The number on the top of each plasmid is the amino acid number of P2S51ffα when the amine terminal Met is numbered 1, and the number on the bottom is the amino acid number of the reductase. In addition, ma 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 P45017α portion contains its entire coding head. On the other hand, among the reductase parts, pαLY4 is the shortest and does not contain a papain cleavage site. By inserting a synthetic linker into the connection between P450+yα of pαLY4 and the reductase, we aimed to make electron transfer more efficient by making it easier for both domains to collide.
αLYI”, pαLYI, pαLY2. pαLY3
．． was built. pαLY3 has 9 amino acids, pαLYI
° and pαLY2 are for 16 amino acids, ραLYI' and pα1. Yl is 27 amino acids longer than pcxLY4. The production of each expression plasmid will be explained in detail below.

Example 1-1. Construction diagram of plasmid αLY4 2
An outline of the construction of plasmid pαLY4 is shown.

[Step 1] Construction of plasmid pUR(s) P450
1? α, plasmid pα containing the amino-terminal part of DN^
The reaction mixture obtained by simultaneously cleaving NR(H) (Special I! 1162-204101) with restriction enzyme 11ph■ and EcoRI was subjected to low melting point agarose electrophoresis, and P45017α
An approximately 250 Kb fragment corresponding to the amino-terminal coding region was recovered.

This fragment and synthetic linker 5'-^GCTTAAAA^^^TGTGGCTGCT
CCTGGCTGTCATTTTTTTACACCGA
CGAGGACCGACA-5' (has Hindnl and Hphr at both left and right ends, respectively.Hereinafter, synthetic N
All A were synthesized using a 380A synthesizer manufactured by Applied Biosystems. ) and the plasmid pH
The target plasmid pαN(11)2 was obtained by inserting into the old nd[1-EcoR1 site of c19. The thus obtained plasmid pαN(11)2, P450+, α
The plasmid pαC(It) (Special training 62-204101) containing the carboxyl-terminal coding region of was simultaneously digested with the restriction enzymes old ndl[[ and EcoRI.

The reaction mixture was subjected to low melting point agarose electrophoresis, and P450
1, approximately 285 bp and 1400 b corresponding to the α-amino-terminal and carboxyl-terminal coding regions, respectively.
A fragment of p. Both of these fragments were inserted into the llindllll site of yeast-expressed Hector pAA+1, and P45
0+, cr single expression plasmid p^α2 was obtained. The resulting plasmid paα2 was digested with the restriction enzyme 11hal, and the reaction mixture was subjected to low melting point agarose electrophoresis.
An approximately 240 bp DNA fragment corresponding to the 5017α carboxyl terminal coding region was recovered. This Hhal
- Phosphorylate the 5′-end of the Hhal fragment in advance,
Annealed synthetic linker-5LI: 5'-CCAGGCCTGGAAGGAAGCCCA
GGCTGAGGGGGGCGGTCCGGACCTTC
CTTCGGGTCCGACTCCCCAGCT-5(
It has Hhal and 5all recognition sites at both left and right ends, respectively. ) was added, and a reaction was performed using T4 DNA ligase. Then, it was simultaneously digested with restriction enzymes Pstl and 5all, NaCl and ethanol were added to the reaction solution, and ethanol precipitation was performed.
Approximately 140 corresponding to the carboxyl-terminal coding region
Pstl-5a11 cleavage of bp into plasmid pUc18
PsLl, 5all site was subcloned to obtain plasmid pUα-Ps.
was simultaneously digested with the restriction enzymes old ndl[l and Pstl, and the reaction mixture was subjected to low melting point agarose gel electrophoresis.
Approximately 18 corresponding to the 01tα amino-terminal coding region
A 5 bp fragment was recovered. On the other hand, the above plasmid puα
-Ps was simultaneously digested with the restriction enzymes old ndDI and Pstl, and a fragment of approximately 2.8 kb was recovered in the same manner. This fragment was subjected to a glue gauze reaction with the aforementioned fragment of about 185 bp corresponding to the P45017α amino-terminal coding region. Plasmid ptlΔα was thus obtained. Furthermore, plasmid pAα2 was cut with restriction enzyme Pstl,
The reaction mixture was subjected to low melting point agarose gel electrophoresis, and P4
501? Approximately 120, corresponding to the central coding region of α
A 0 bp fragment was recovered. This fragment was added to the above plasmid p
It was inserted into the Pst1 site of uΔα to obtain the target plasmid pUα(s).

[Step 2] Plasmid pUYR717(B) (7
) Construct plasmid pgCYR (special training 62-325527) containing yeast reductase cDN^ insert with restriction enzyme E
Cut with coRI. 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 coding region and the carboxyl-terminal coding region of the reductase were recovered, respectively. Each fragment was added to EcoR1 of plasmid pUc19.
A plasmid pUYR7 in which the amino-terminal fragment was inserted and a plasmid pUYR17 in which the carboxyl-terminal fragment was inserted were obtained. Plasmid pUYR17 was partially cut with the restriction enzyme EcoRI, and a commercially available 11ind[[[linker was inserted and plasmid pUYR17
YR17(It) was obtained. On the other hand, the above plasmid pUY
R7 was cleaved with the restriction enzyme EcoRI, and the reaction mixture was subjected to low melting point agarose gel electrophoresis to recover an approximately 410 bp fragment corresponding to the reductase amino-terminal coding region.

This fragment was added to Eco of the above plasmid pUYR17(H).
It was inserted into the R1 site to obtain plasmid pUYR717(H). This plasmid pUYR7170f) was co-digested with the restriction enzymes Pvu II and old ndl[I, and the reaction mixture was subjected to low melting point agarose gel electrophoresis.
An approximately 2.0 kb fragment of NA was recovered. The approximately 2.0 kb Pvu II-Hindl[l fragment thus obtained and the synthetic linker -5L2-1:5'-GATCCCCCGT
CGACCCCAGGGGGCAGCTGGGGGTC-
5" (has Bag old and PνuIl recognition sites at both left and right ends, and Sal+ recognition site inside) into the BamHI-11indI [1 site of plasmid p11c18] to create the target plasmid pUYR717 (B)
I got it.

[Step 3] Construction of expression plasmid pαLY4 Plasmid pUYR717 (B) obtained in step 2 was partially digested with restriction enzyme BstEII, and further digested with restriction enzyme Pvu.
It was cut at l. Bs tE of this pHYR717(B)
Commercially available C1ar linker at II-Pvu 11 site
was inserted to obtain plasmid pHYR4. Plasmid ρUα(s) obtained in step 1 and the above plasmid pU
A reaction mixture in which Y114 was simultaneously digested with the restriction enzymes Hindl [I and 5all] was subjected to low melting point agarose gel electrophoresis to obtain approximately 1540 bρ and 2020 bp corresponding to P450+yα or reductase coding region, respectively.
fragments were recovered. Both obtained fragments were inserted into the Hindl[[ site of the yeast expression vector pAAH5 to obtain the target plasmid pαLY4.

1-2. Figure 3 of Plasmid αLY3 shows an outline of the expression plasmid pαLY3. Example 1-1,
The plasmid puα(s) obtained in Step 2 and the plasmid ρUYR717(B) obtained in Step 2 were simultaneously digested with the restriction enzymes Hindl[I and 5a11. The reaction mixture was subjected to low melting point agarose gel electrophoresis, and P4501
Fragments of approximately 1540 bp and 2050 bp corresponding to the 7α or reductase coding region, respectively, were recovered. Both of the obtained fragments were inserted into the yeast expression vector pAAH5.
Insert the target plasmid pαLY3 into the ndl1 site.
I got it.

Example 1-3 Collection of plasmid αLY2 In 4, an outline of the construction of plasmid pαLY2 was shown. Example 1-
1. Plasmid ptlYR717 obtained in step 2
The reaction mixture obtained by simultaneously cleaving (H) with restriction enzymes Pvu II and Hindnl was subjected to low melting point agarose gel electrophoresis, and an approximately 2.0 kb fragment of reductase cDNA was recovered. Approximately 2. Okb's Pvu■-Hln
dll [fragment and synthetic linker-3L2-2:5'-TC
GACCCCCATCCGATGACGGAGATATC
ACAGGGGGGTAGGCTACTGCCTCTAT
AGTGTC-5 (5all on both left and right ends, Pv
It has a uU recognition site. ) of plasmid pHc1B 5
all-1 (indl11 site) to obtain the target plasmid pHYR717(s). Example 1-1.
The plasmid pυα (S) obtained in step 1 and the above plasmid pUYR717 (s) were added using the restriction enzyme old ndl.
The 0 reaction mixture that was simultaneously cleaved with lr and 5all was subjected to low melting point agarose gel electrophoresis, and P4503. α or reductase coding region, each approximately 15
Fragments of 40 bp and 2070 bp were recovered.

Both of the obtained fragments were inserted into yeast expression vector pAAI (5's l1
Insert the target plasmid pαLY into the ind■ site.
I got 2.

Example 1-4, current plasmid αtyt', pαLY
Figure 5 shows an outline of the construction of plasmids pαLYI' and pαLYI. Example 1-1. Synthetic linker -5L3 in the pvu If site of plasmid pUYR717 (B) obtained in step 2: 5' -CTGTGATATCTCCGTCATCGG
ACAGTAGCACGGCAAGCAGACACTA
TAGAGGCAGTAGCCTGTCATCGTGC
CGTTCGTCTAGCCCCGCCAGGA GATCGGGGCGGTCCCT-5' or synthetic linker-3L4: 5' -TAAAGAGAAAACTCCATCAAGG
AACTGCTGATGTCCGATATTTCTCT
TTGAG[:TAGTTCCTTGACGACTAC
Example 1 in which plasmid pUYR1' or pHYR1 was obtained by inserting AGGCT^GACGGAGATATCACAG CTGCCTCTATAGTGTC-5'
-1. Plasmid pUα(s) obtained in step 1 and the above plasmid pUYR1° or pUYI? The 0 reaction mixture obtained by co-digesting 1 with the expressing enzymes H4ndTll and 5aXl was subjected to low melting point agarose gel electrophoresis, and P45017
Approximately 1540 bp, 2050 bp, or 2100 bp corresponding to α or reductase coding region, respectively.
A bp fragment was recovered. Both of the obtained fragments were inserted into the old ndl11 site of the yeast expression vector pAAH5 to obtain the target plasmid pαLYI' or pαLYI.

2. Transformation of yeast using the prepared plasmid Saccharomyces cerevisiae
cescerevisiae) Al22 strain (ATCC3
8626) to 5IIIl vp.

After culturing in medium (1% yeast extract, 2% polypeptone, 2% glycose) at 30°C for 18 hours, 1rsl
The yeast culture solution was centrifuged to collect the bacteria. The obtained bacterial cells were reduced to 0.
After washing with 2M LiCl solution 14, IM LiCl
It was suspended in 20 μl of solution. To this, add 30 μl of 70% polyethylene glycol 4000 solution to plasmid pαLYI.
10 μi (corresponding to about 1 μg) of p a LYI, pcrLY2, p α LY3 or p a LY4 solution was added, thoroughly mixed, and then incubated at 30° C. for 1 hour. Then, after adding 140 μl of water and stirring well, this solution was plated on an SD synthetic medium plate (2% glucose, 0.67% amino acid-incompatible yeast nitrogen source, 20 μg/
rdt histidine, 2% agar) and incubating at 30°C.
pαLY1, pαLY2, pαIJ3 and pαLY4
Transformant al122 (pαLYI'), which carries AI
(22(+)αLYI), At122(pαLY2),
Al22(pαLY3) and Al22(pαLY4)
were obtained respectively.

Al22 (pαIJI'), Al12 obtained in Example 2
2 (pαLYI), A1122 (pαLY2), A) 1
22 (pαLY3) and Al22 (pαLY4) were cultured in SD synthetic medium (2% glucose, 0.67% amino acid-incompatible yeast nitrogen source, 20 μg 7 dl histidine) to approximately 2 x 10' cells/d, and harvested to 100 mM.
After washing with potassium phosphate pH7.0, 100s' potassium phosphate pH7.0 was used. o, suspended in 2I11. The bacterial suspension was dispensed into two cuvettes each having a length of 1 m, and after blowing carbon monoxide into the sample-side cuvette, 5 to 1 g of dithionite was added to both cuvettes. After stirring well, measure the difference spectrum between 400 and 500 nm, and find that Δε=
Heme-containing P4501i was calculated based on the value of 91mM-'cm-'. As a result, as shown in Table 1, the strains Al22 (pαLYI'), AI+22 (ραLY
I) strain, Al122 (pαLY2) strain, Al22 (
pαLY3) strain and Al22 (pαLY4) strain
Approximately 0.7 XIO' molecules and 0.4 per bacterial cell, respectively.
XIO' molecule, 0.9 XIO' molecule, 0.7 XIO
'molecule, and 0.9 XIO' molecule of heme-containing P45
It was found that 0 protein was produced.

Measurement of production amount Transformed yeast strain AH22 (pαLYI'), Al2, cultured to approximately 0.7 x 10' cells/d in SD synthetic medium
2 (pαLYI) strain, A) 122 (pαLY2) strain,
Al22 (pαLY3), Al22 (pαLY4) and Al122 (pAAH5) strains and P45017α
8022 (pAα1) strain (Special 1!162) that produced only
-204101), 3 μCi of each [3
(2) A 1mM progesterone-ethanol solution containing progesterone was added so that the final substrate concentration was 10 μH. Culture with shaking at 30°C, and after 0, 1, and 2.6 hours, collect ljd and centrifuge to obtain supernatant 0.8d.
21d dichloromethane was added to the solution and stirred vigorously. After centrifugation, the dichloromethane layer 11d was dried, and the residue was dissolved in 20 μl of an equal volume solution of ethanol-ethyl acetate containing 0.5 PengH progesterone, 0.5-A17-hydroxyprogesterone, and 0.5 mM androstenedione. was applied to a thin layer plate. After developing for 50 minutes at room temperature with a chloroform solution containing 25% ethyl acetate, progesterone, 17
- The thin gel portion corresponding to the positions of hydroxyprogesterone and androstenedione was scraped off, and the radioactivity was measured using a liquid synchronization counter.

As a result, transformed yeast AH22 (pαLYI') strain,
Al22 (p αLY1) strain, Al122 (p αLY
2) strain, Al122 (pαLY3) strain and Al22
(pαLY4) strain after 6 hours, 88% and 86%, respectively.
%, 89%, 90% and 89% progesterone 1
It was found that it was converted to 7-hydroxyprogesterone (Figure 6). On the other hand, Al22 (
No activity was observed in the pA^ll5) strain. Also C
Production of androstenedione due to side chain cleavage activity at position 174 was not observed in any of the strains.

Based on the conversion rate to 17-hydroxyprogesterone 1 hour after substrate addition, 17-hydroxyprogesterone per bacterial cell of various transformed strains was calculated.
-Hydroxyprogesterone production was calculated. P45
01? When the Al122 (ρ^αl) strain (special 1162204101) that only produces α is set as 1, the AlI22 (pαLYI') strain that produces the fusion enzyme, the Al1
22 (+1αLY1) strain, Al22 (pαLY2) strain,
Al122(pαLY3) strain and Al122(ραL
Y4) strain, 4.7.3.2.5.0.4.
It increased to 7° and 3.2.

The above results revealed that the fused enzyme of P45017α and reductase constitutes an electron transport chain more efficiently than when P450+ and α are expressed alone, and exhibits a reaction with high l-atom oxygenation.

In addition, the transformed yeast strain obtained by the present invention contains heme, has l-atom oxygenation activity, and has produced a fusion enzyme of P2S5.1α and a reductase. 17-hydroxyprogesterone was produced by adding progesterone to the solution and inking it. At this time, the loss rate 6 hours after substrate conversion was approximately 90.
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, since the yeast strain of the present invention does not have any -2° side chain cleavage activity, it is very convenient for producing glucocorticoids, which are expensive as pharmaceuticals. Therefore, if the yeast strain of the present invention is used as a bioreactor for the synthesis of the aforementioned steroids, which are useful as pharmaceuticals, it will be possible to further simplify the current production process using a multi-step reaction that combines fermentation and synthesis. Become.

Al22(pαLY4) 0.9
3.24, Brief Explanation of Figures = 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.

The opening indicates the P450+, α coding region, the opening = the reductase coding region, and the symbols ■ and synthetic linker. Ma represents the site where the reductase is cleaved by papain. The numbers at the top of each plasmid are the amino acid numbers of P45ol and α when the amino terminal Met is numbered 1, and the numbers at the bottom are the amino acid numbers of the reductase. Furthermore, the number of amino acids of the fused enzyme is shown on the right end of the figure, divided into those derived from P2S5 + tα 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 plasmids pctLY1'' and pαL' of the present invention.
/1 construction process is shown. Figure 2. 3. 4. In all 5, 11d, llh, Ps, tip, Sa,
Ec, Pv, Bm, Bs, CI are restriction enzymes old ndl[l, Hhal, Pstl, Hphl,
Sa! I, EcoRIPvu■, Bam1l I
, BstE n , and C1a l recognition sites.

a is the P450+, α coding region, mouth=] is the reductase coding region, ■, is the synthetic linker.0 Figure 6 shows the progesterone 17-position hydroxylation activity of various transformed strains. The vertical axis is the 17- The conversion rate to hydroxyprogesterone is shown on the horizontal axis, and the culture time is shown on the horizontal axis. ×□×
is At122 (pAAII5) strain, ・□・ is Al22
(pαLYI°) strain, ○□O is AlI22 (pαLY
I) Strain, mouth □ mouth is Al22 (pαLY2) stock, ■□■
is AH22(pαLY3) strain, Δ□Δ is AH22(pαLY3) strain, and Δ□Δ is AH22(pαLY3) strain.
LY4) strain, Mumu indicates AH22 (ρ^αl) strain.

1EcoR1, HindI[1 l EcoRI, Hind m Figure 2 (Part 1) puα+S+ Figure 2 (Part 2) 1) U’n pU"l'R71L a.

Figure 2 (Part 4) c. c. c. p〆■R pUYR7+7[H] pUYR7171B+ Figure 2 (Part 3) plJlIts+ pUYR7171B+ No. figure ↓5a11. city m1 pUYR717 concave ↓S11. H+nd m LIals pLIYR717tBl ↓Pvu[1 pUYR+' (or ptJYRI)

Claims (8)

[Claims] (1) Monoatomic oxygenation activity of bovine adrenal cytochrome P450_1_7α and yeast NADPH-cytochrome P4
A fusion enzyme gene encoding an oxidase that has the ability to supply reducing power of 50 reductase (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) Saccharomyces cerevisiae AH22 (pαLY
1') strain, AH22 (pαLY1) strain, AH22 (pα
LY2) strain, AH22 (pαLY3) strain or AH2
2 (pαLY4) strain according to claim 4. (6) Monoatomic oxygenation activity of bovine liver cytochrome P450_1_7α and yeast NADPH-cytochrome P4
Oxidase that has the ability to supply reducing power of 50 reductase (7) A method for producing the oxidase, which comprises culturing the transformed yeast strain according to claim 4. (8) 17- characterized by progesterone and pregnenolone hydroxylation by the transformed yeast strain according to claim 4;
Method for producing hydroxyprogesterone and 17-hydroxypregnenolone