The expression of mitochondrial P450 in yeast is disclosed. The mitochondrial P450 includes a chimeric P450 in which a signal sequence present at the N-terminus of a mammalian mitochondrial P450 has been substituted by a signal of a yeast mitochondrial protein and a chimeric P450 in which the latter signal has been further substituted by a targeting signal sequence to microsome. An expression plasmid for producing the enzyme in a large scale, a recombinant yeast strain carrying the expression plasmid, a process for producing the enzyme by the use of the recombinant yeast, and a process for producing 5.beta.-cholestane-3.alpha., 7.alpha., 12.alpha., 27-tetrol, 25-hydroxyvitamin D.sub.3 and 1.alpha.,25-dihydroxyvitamin D.sub.3 are also disclosed.

BACKGROUND OF THE INVENTION 
The present invention relates to the expression of mitochondrial P450 in 
yeast. The mitochondrial P450 includes a chimeric P450 in which a signal 
sequence present at the N-terminus of a mammalian mitochondrial P450 has 
been substituted by a signal of a yeast mitochondrial protein and a 
chimeric P450 in which the latter signal has been further substituted by a 
targeting signal sequence to microsome. More specifically, the present 
invention relates to genes which encode a rat liver mitochondria 
P450.sub.c25 precursor, a chimeric P450 in which a matured P450.sub.c25 is 
connected with 29 amino acid residues which is believed to be the 
mitochondrial targeting signal of the yeast cytochrome c oxidase subunit 
IV (COXIV), and a chimeric P450 in which a signal sequence comprising 15 
amino acid residues at the N-terminus of bovine adrenal P450.sub.17 
.alpha. which is believed to be the microsomal targeting signal is 
followed by a matured P450.sub.c25, respectively. 
The present invention further relates to an expression plasmid for 
producing the enzyme in a large scale, a recombinant yeast strain carrying 
the expression plasmid, a process for producing the enzyme by the use of 
the recombinant yeast, and a process for producing 
58-cholestane-3.alpha.,7.alpha.,12.alpha.,27-tetrol (referred to 
hereinafter as TeHC), 25-hydroxy-vitamin D.sub.3 and 
1.alpha.,25-dihydroxyvitamin D.sub.3. 
The term chimeric P450 used in this specification means hereinafter P450 
comprising a foreign N-terminal targeting signal sequence and a matured 
mitochondrial P450 at C-terminus. 
P450 is a hemoprotein existing widely in biological fields from 
microorganisms to mammals and catalyzes as a terminal enzyme of electron 
transport chains monooxygenation toward a variety of lipophilic compounds 
as substrates. 
P450 as the terminal enzyme in electron transport chains has a variety of 
molecular forms, which exhibit different substrate specificities and thus 
can catalyze the hydroxylation of a wide variety of lipophilic compounds. 
P450-dependent electrontransport chains in mammals are classified into two 
groups; microsomal and mitochondrial types. 
In microsomes, NADPH-cytochrome P450 reductase (reductase) containing 
flavin adenine dinucleotide and flavin mononucleotide as cofactors in the 
molecule transfers electrons from NADPH to P450. In mitochondria, 
NADPH-ferredoxin reductase containing flavin adenine dinucleotide as a 
cofactor in the molecule and ferredoxin containing non-heme iron as a 
cofactor in the molecule transfer electrons from NADPH to P450. 
The present inventors have already succeeded in industrially useful 
hydroxylations by producing several microsomal P450s and reductases in 
yeast and using their recombinant yeast strains. 
That is, yeast strains producing the enzymes have been obtained by 
isolating genes for rat liver P450c, bovine adrenal P450.sub.17 .alpha. 
and P450.sub.c21, respectively, making expression plasmids containing 
these genes, respectively, and transforming yeast with the expression 
plasmids (Japanese Patent Kokai (Laid-Open) Nos. 56072/1986, 47380/1989 
and 31680/1990). 
These yeast strains exhibited monooxygenase activities depending on P450 
molecular forms produced therein, respectively. 
The present inventors have also succeeded in the expression of the enzyme 
having P450 reducing ability within yeast by isolating rat liver reductase 
gene or yeast reductase gene (Japanese Patent Kokai (Laid-Open) Nos. 
19085/1987 and 51525/1990). 
Moreover, the present inventors have succeeded in creating a yeast strain 
which produces both P450 and reductase (Japanese Patent Kokai (Laid-Open) 
No. 104582/1987) and novel monooxygenases having the functions of both 
enzymes (Japanese Patent Kokai (Laid-Open) Nos. 44888/1988 and 23870/1990, 
and Japanese patent Application No. 71250/1989). 
By these techniques, the present inventors have successfully produced 
acetaminophene or steroid hormone intermediates useful as medicines with 
these P450-producing yeast strains. 
On the other hand, the mitochondrial P450 participates in many biological 
reactions which synthesize physiologically important compounds such as 
active vitamin D.sub.3 and steroid hormones, and thus the creation of a 
yeast strain producing the mitochondrial P450 molecular form has a high 
industrial applicability. 
However, there has not hitherto been reported the expression of an active 
mitochondrial P450 in yeast. 
Furthermore, two enzymes, ferredoxin and ferredoxin reductase, are required 
in addition of P450 for the expression of mitochondorial P450-dependent 
monooxygenase activity. 
Thus, the present inventors have tried to create a bioreactor for the 
expression of the mitochondorial P450 in yeast and of the mitochondrial 
P450-dependent monooxygenase activity. 
Rat liver P450.sub.c25 which is a mitochondrial P450 is synthesized as a 
precursor comprising 533 amino acids in cytoplasm and then transported to 
mitochondria. During these processes, 32 amino acids at the amino terminus 
acting as a mitochondrial targeting signal are removed to yield a matured 
P450.sub.c25 comprising 501 amino acids (molecular weight: 57 KD) on the 
mitochondrial membrane. 
The present inventors have already found that when a bovine adrenal 
mitochondrial protein is expressed in yeast, the substitution of its 
signal peptide part by that of yeast cytochrome c oxidase subunit IV 
(referred to hereinafter as COXIV) increases the expression amount 
remarkably (Japanese Patent Application No. 136496/1990), 
SUMMARY OF THE INVENTION 
Rat liver P450.sub.c25 gene or a gene encoding the chimeric P450 (COX-C25) 
in which its own signal sequence had been substituted by the COXIV signal 
sequence was linked to the downstream of the promoter of a yeast alcohol 
dehydrogenase (ADH) gene for construction of expression plasmids. As a 
result, yeast strains transformed with these expression plasmids produced 
P450s containing heme. 
It was also found that the P450.sub.c25 and the chimetic P450 (COX-C25) 
thus produced were transported to the mitochondria in yeast and processed 
into the matured forms. Furthermore, the P450.sub.c25 and the chimeric 
P450 (cox-c25) partially purified from these transformed yeast strains 
successfully produced TeHC or 1.alpha., 25-dihydroxyvitamin D.sub.3 from 
THC or 1.alpha.-hydroxyvitamin D.sub.3 by coupling with bovine adrenal 
adrenodoxin (referred to hereinafter as ADX) and bovine adrenal 
NADPH-adrenodoxin reductase (referred to hereinafter as ADR). 
Furthermore, the present inventors have constructed a plasmid for 
expressing the chimeric P450 (17.alpha.-c25) in which the mitochondrial 
targeting signal substituted by the N-terminal 15 amino acids derived from 
bovine adrenal P450.sub.17.alpha., which is believed to be a microsomal 
targeting signal of P450.sub.17.alpha. order to enhance the production of 
the enzyme inherently existing in mitochondria by exchanging its 
localization into microsomes. 
The present inventors have also constructed a plasmid which express 
simultaneously the chimeric P450 (17.alpha.-c25), the matured ADX and the 
matured ADR for the purpose of constructing a novel electron transport 
chain with the chimeric P450 (17.alpha.-c25) on microsomal membrane and 
ADX and ADR in cytoplasm and exhibiting the monooxygenase activity. In 
this connection, these three enzymes were expressed with different 
promoters and terminators, because the use of the same promoter and the 
same terminator would cause recombination in the plasmid during the 
cultivation of the recombinant yeast. 
It was found that all of the P450.sub.c25, the chimeric P450 (COX-c25) and 
the chimeric P450 (17.alpha.-c25) expressed in yeast in the present 
invention exhibited hydroxylation activities to THC, vitamin D.sub.3 and 
1.alpha.-hydroxyvitamin D.sub.3 and thus were active forms. Thus, the 
yeast expression system is useful for expressing the mitochondrial P450. 
Particularly, the chimeric P450 (17.alpha.-c25) produces a large amount of 
the enzyme and has a high activity. 
While P450.sub.c25 and the chimeric P450 (COX-c25) were expressed in 
mitochondria in yeast, any chimetic P450 (17.alpha.-c25) expressed existed 
in microsomal fraction. Thus, the N-terminal 15 amino acid residues 
derived from P450.sub.17 .alpha. played a role of a targeting signal of 
the chimeric P450 (17.alpha.-c25) to microsomal membrane in yeast, and the 
P450.sub.c25 which is inherently mitochondrial membrane protein was 
successfully expressed in yeast microsome. 
The production amount of the chimetic P450 (17.alpha.-c25) was about 
2.times.10.sup.5 molecules/cell, and thus the substitution of the signal 
successfully increased the production amount to about 4 times. Moreover, 
the microsomal fraction exhibited high hydroxylation activities to THC, 
vitamin D.sub.3 and 1.alpha.-hydroxyvitamin D.sub.3 by adding ADX and ADR 
to the fraction. 
Particularly, the hydroxylation-activity to 1.alpha.-hydroxyvitamin D.sub.3 
of the chimeric P450(17.alpha.-c25) in the microsomal fraction was by far 
higher than that of the original matured P450.sub.c25 comprising 501 amino 
acids in the mitochondrial fraction. 
The present inventors has also succeeded in construction of a novel 
electron transport chain from NADPH to the chimeric P450 (17.alpha.-c25) 
through ADX and ADR on yeast microsomal membrane, as the simultaneous 
expression strain of chimetic P450 (17.alpha.-c25), ADX and ADR exhibits 
monooxygenase activity. 
Therefore, the strain can be used as a simple bioreactor in which the 
hydroxylation of steroid compounds or an active vitamin D.sub.3 is 
performed in one step. 
Particularly, the strain can easily recover reaction products by adding a 
substrate to the culture of the microorganism and extracting with an 
organic solvent or the like after a certain period of time, so that it is 
useful as a bioreactor.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention will be explained in detail below. 
cDNA encoding the rat liver P450.sub.c25 used in the present invention is 
well known and can be isolated by the conventional procedures. The signal 
sequence portion to mitochondria can be substituted by a signal sequence 
of the other mitochondrial protein such as COXIV or the like. In this 
case, P450 is transported into mitochondria. On the other hand, if this 
signal sequence is substituted by a signal sequence to microsome, P450 is 
transported into microsome. As the transfer signal to microsome, not only 
bovine adrenal P450.sub.17.alpha. but also a transfer signal of the other 
microsmal P450 can be used. 
Furthermore, as the matured mitochondrial P450 existing at the C-terminus 
of the chimeric P450 of the present invention, not only the rat liver 
P450.sub.c25 but also the other mitochondrial P450 can be used. 
cDNA encoding COXIV and bovine adrenal P450.sub.17.alpha. used in the 
present invention is well known and can be isolated by the conventional 
procedures. 
For example, the expression plasmid for expressing the chimeric P450 
(17.alpha.-c25) can be constructed by synthesizing cDNA corresponding the 
N-terminal amino acid sequence of bovine adrenal P450.sub.17.alpha., 
linking to the cDNA region encoding the matured rat liver P450.sub.c25, 
and inserting the gene into the yeast expression vector pAAH5 which 
retains the promoter and the terminator of the yeast alcohol dehydrogenase 
I (referred to hereinafter as ADH) gene (Methods in Enzymology, 101, part 
C, 192-201). 
The plasmid for simultaneously expressing the three enzymes can be 
constructed by inserting the expression units of the matured ADR and the 
matured ADX into he expression plasmid for the chimeric P450 
(17.alpha.-c25). In this connection, the promoter and the terminator are 
not limited to ADH and may be the promoters and the terminators which 
function efficiently in yeast. The promoter and the terminator for 
expressing simultaneously the three enzymes are not the same and 
preferably different from each other, because recombination on plasmid 
will occur only with a low probability upon cultivation. The expression 
amount of the enzyme is scarcely affected by the position or the direction 
of the three enzymes to be inserted into the expression unit. 
Yeast is used as a host, and the yeast Saccharomyces cerevisiae strain AH22 
is preferred. 
Transformation of yeast as a host by the expression plasmid can be 
conducted by the well-known methods such as that with an alkali metal 
(LiCl) or protoplast. Transformed yeast obtained by the present invention 
can be cultivated in an ordinary culture medium containing glucose, 
nitrogen sources or the like by the conventional methods. P450.sub.c25 and 
the chimetic P450 can be produced by cultivating the transformed yeast 
strain thus obtained. 
The present invention is described in detail with reference to Examples. 
However, it goes without saying that the present invention is not limited 
to Examples. 
EXAMPLE 1 
Construction of Expression Plasmid pAC25 
In the following Examples, the reactions such as the cutting of DNA with 
restriction enzymes, the dephosphorylation of DNA with alkaline 
phosphatase, the ligation of DNAs with DNA ligase were conducted generally 
with a reaction volume of 20.gtoreq.200 .mu.l Under the reaction 
conditions described in the instructions appended in the products of the 
manufacturers (e.g. Takara Shuzo Co., Ltd.) of these enzymes, unless 
otherwise specified. Rat liver P450.sub.c25 expression plasmid was 
constructed in such a procedure as shown in FIG. 1. 
Plasmid pLMT25 containing rat liver P450.sub.c25 gene (Usui et al., (1990) 
FEBS Lett., 262, 135-138) was doubly digested with restriction enzymes 
EcoRI and SacI, and the ca. 320 bp EcoRI-SacI fragment encoding the region 
of the amino terminal side of P450.sub.c25 and the ca. 1580 bp SacI-EcoRI 
fragment encoding the region of the carboxy terminal side were recovered 
by the low melting agarose gel electrophoresis. These fragments were 
subcloned at the EcoRI and SacI sites of a commercially available vector 
plasmid pUC19 to give pUC25N and pUC25C, respectively. A fragment of about 
2880 bp obtained by the doubly digestion of pUC25N with EcoRI and NcoI, 
and a synthetic linker LC252: 
EQU (SEQ. ID NO.1) 5'AATTCAAGCTTAAAAAAATGGCTCTGTTGAGCCGCATGAGA 
EQU (SEQ. ID NO.2) 3'GTTCGAATTTTTTTACCGACACAACTCGGCGTACTCT 
EQU CTGAGATGGGCGCTTCTGGACACTCGTGTGATGGGC 3' 
EQU GACTCTACCCGCGAAGACCTGTGAGCACACTACCCGGTAC 5' 
which has EcoRI and NcoI recognition sites at right and left ends, 
respectively, and HindIII recognition site between the both ends, were 
ligated for transform E. coli strain HB101. 
Plasmid DNA was prepared from the transformant thus obtained according to 
the Birnboim-Doly's method and analyzed by the digestion with restriction 
enzymes to obtain the aimed plasmid in which the synthetic linker was 
inserted. Further, the sequence of the synthetic linker part was confirmed 
by the determination of the base sequence. Thus, the plasmid obtained was 
named pUC25NH. 
The plasmid pUC25C encoding the region of the carboxy terminal side of 
P450.sub.c25 was digested with NcoI and fill-in ligated with a 
commercially available HindIII linker to transform E. coli strain HB101. 
Plasmid DNA was prepared from the transformant, and the plasmid in which a 
HindIII site was created in the 3'-non-coding region of P450.sub.c25 was 
named pUC25CH. 
The fragments of about 270 bp and 1370 bp obtained by the doubly digestion 
of pUC25NH and pUC25CH with HindIII and SecI, respectively, and a 
commercially available vector plasmid pUC18 which had been digested with 
HindIII and treated with alkaline phosphatase were ligated to transform E. 
coli strain HB101. A plasmid DNA was prepared from the transformant, and 
the plasmid containing both DNA fragments derived from pU25NH and pUC25CH, 
respectively, was named pUC25H. pUC25H was digested with Hind III, and a 
cDNA fragment of ca. 1640 bp encoding the rat liver P450.sub.c25 precursor 
was prepared. After the fragment and the yeast expression vector pAAH5N 
which had been digested with HindIII and then treated with alkaline 
phosphatase (Japanese Patent Application No. 202785/1988) were ligated, E. 
coli strain HB101 was transformed. A plasmid DNA was prepared from the 
transformant, the DNA structure was confirmed by restriction enzyme 
digestion and the plasmid in which the rat liver P450.sub.c25 gene had 
been inserted in the right orientation between the ADH promoter and the 
terminator was named pAC25. 
EXAMPLE 2 
Construction of Expression Plasmid pACC253 
Reference is made to FIG. 3. 
Plasmid pUC25N containing the gene encoding the region of the amino 
terminal side of the rat liver P450.sub.c25 was doubly digested with PstI 
and SacI to recover a fragment of about 150 bp. The fragment, the 
synthetic linker LC253: 
##STR1## 
which has HindIII and PstI recognition sites at right and left ends, 
respectively, and the HindIII-SacI fragment of a commercially available 
vector pUC19 were ligated, and E. coli strain HB101 was transformed. 
Plasmid DNA was prepared from the transformant thus obtained and analyzed 
by the digestion with restriction enzymes to obtain the aimed plasmid in 
which the fragment and the synthetic linker were inserted. Further, the 
sequence of the synthetic linker part was confirmed by the determination 
of the base sequence. Thus, the plasmid obtained was named pBSCC253. The 
plasmid and the aforementioned plasmid pUC25CH were doubly digested with 
HindIII and SacI, and a DNA fragment (ca. 260 bp) coding for the signal 
peptide of COXIV and the amino terminal part of P450.sub.c25 and a DNA 
fragment (ca. 1370 bp) coding for the carboxy terminal part of 
P450.sub.c25 were recovered separately. The triple ligation of these 
fragments and a commercially available vector plasmid pUC18-HindIII 
fragment was conducted to transform E. coli strain HB101. A plasmid DNA 
was prepared from the transformant and analyzed by the digestion with 
restriction enzymes to obtain a plasmid pUCC253H in which the fragments 
had been inserted. pUCC253H was digested with HindIII to prepare a 
modified cDNA fragment (ca.1630 bp) for the rat liver P450.sub.c25, which 
was ligated with the yeast expression vector pAAH5N which had been 
digested with HindIII and then treated with alkaline phosphatase (Japanese 
Patent Application No. 202785/1988), and then E. coli strain HB 101 was 
transformed. The structure of the plasmid DNA prepared from the 
transformant was confirmed, and the plasmid in which the rat liver 
P450.sub.c25 modified gene had been inserted in the right orientation 
between the ADH promoter and the terminator was named pACC253. 
EXAMPLE 3 
Construction of Expression Plasmid pAMS25 and Simultaneous Expression 
Plasmid pRXMS25 
The chimeric P450 (17.alpha.-c25) expression plasmid pAMS25 was constructed 
according to FIG. 5. 
The plasmid pUC25N used in the rat liver P450.sub.c25 expression plasmid 
pAC25 illustrated in Example 1 was partially digested with PstI and then 
with EcoRI. Into the DNA fragment obtained was inserted a synthetic 
linker: sequence (SEQ ID NO:5) 
EQU AATTCAAGCT TAAAAAAATG TGGCTGCTCC TGGCTGTCTT TCTGCTCACC CTCGCCTATT60 GTTCGA 
ATTTITTTAC ACCGACGAGG ACGACAGAA AGACGAGTGG GAGCGGATAA TAGCGATCCC TGCA 74 
ATCGCTAGGG 
and a HindIII-SacI fragment was prepared from the plasmid thus obtained. 
Also, a SacI-HindIII fragment was prepared from pUC25CH and doubly 
inserted into the HindIII site of pUC19 for obtaining a plasmid pUMS25. 
Next, an expression plasmid pAMS25 was constructed by inserting the HindIII 
fragment obtained from pUMS25 into the Hind III site of a vector pAAH5N. 
An expression plasmid pGX was obtained by inserting a Hind III fragment 
coding for the matured ADX (described in Japanese Patent Application No. 
136496/1990) between the PGK promoter and the terminator of a vector pGAHN 
(containing the PGK promoter and the terminator in place of the ADH 
promoter and the terminator of a vector pAAH5N) (described in Japanese 
Patent Application No. 136496/1990). pPR was also prepared by inserting a 
Hind III fragment coding for the matured ADR (described in Japanese Patent 
Kokai No. 112986/1988) between the GAP promoter and the terminator of a 
vector pPAHN, which contains the GAP promoter and the terminator (Japanese 
Patent Kokai (Laid-Open) No. 112986/1988) in place of the ADH promoter and 
the terminator of the vector pAAH5N). Simultaneous expression plasmid pRX5 
for ADR and ADX was obtained by ligating the DNA fragment obtained by 
partially digesting pPR with NotI and the NotI fragment obtained from pGX. 
Then, pRX5 was partially digested with NotI, ligated with a NotI fragment 
(3.7 kb) obtained from pAMS25 to obtain the simultaneous expression 
plasmid pRXMS25 for the chimeric P450.sub.c25, ADX and ADR (See FIG. 6). 
EXAMPLE 4 
Transformation of Yeast with Expression Plasmid 
Saccharomyces cerevisiae AH22 (ATCC 38626) was cultivated in 5 ml of a YPD 
culture medium (1% yeast extract, 2% polypeptone, 2% glucose) at 
30.degree. C. for 18 hours, and the yeast cells were collected by 
centrifuging 1 ml of the culture. The yeast cells were washed with 1 ml of 
a 0.2M LiCl solution and then suspended into 20 .mu.l of a 1M LiCl 
solution. 
To this suspension were added 30 .mu.l of a 70% polyethylene glycol 4000 
solution and 10 .mu.l of respective expression plasmid solutions (about 1 
.mu.g DNA), and the mixture was stirred well and incubated at 30.degree. 
C. for 1 hour. Then, 140 .mu.l of water was added to the mixture, and the 
resulting mixture was inoculated on an SD synthetic culture medium plate 
(2% glucose, 0.67% yeast nitrogen base without no amino acids, 20 .mu.g/ml 
histidine, 2% agar) and incubated at 30.degree. C. for 3 days to give a 
transformant in which the plasmid was maintained. 
EXAMPLE 5 
Production of P450.sub.c25 and Chimera P450.sub.c25 
The strains AH22/pAC25, AH22/pACC253, AH22/pAMS25 and AH22/pRXMS25 obtained 
in Example 4 were cultivated in 300 ml of a synthetic culture medium (8% 
glucose, 5.4% yeast nitrogen base without amino acids, 160 .mu.g/ml 
histidine) to a density about 2.times.10.sup.7 cells/ml, and the 
recombinant yeast cells were collected, washed with 100 mM potassium 
phosphate (pH 7.0) and suspended into 2 ml of the same buffer solution. A 
1 ml portion of the suspension was dispensed into each of the two 
cuvettes, carbon monoxide was blown into the sample cuvette, and then 5-10 
mg of dithionite was added to both of the cuvettes. After sufficiently 
stirring the mixtures, the differential spectra were measured in the wave 
length of 400-500 nm to calculate the amount of the heme-containing P450 
based on .DELTA..epsilon. (450 nm-490 nm)=91 mM.sup.-1 
.multidot.cm.sup.-1. 
As the result, it was found that the strains AH22/pAC25, AH22/pACC253, 
AH22/pAMS25 and AH22/pRXMS25 produced a heme-containing P450 in an amount 
of 5.times.10.sup.4 molecules, 3.times.10.sup.4 molecules, 
2.times.10.sup.5 molecules and 1.times.10.sup.5 molecules per cell, 
respectively. On the other hand, no production of the heme-containing P450 
was observed in the control strain AH22/pAAH5. 
EXAMPLE 6 
Measurement of the Hydroxylation Activity at 25 Position of 
1.alpha.-hydroxy-vitamin D.sub.3 and the Hydroxylation Activity at 27 
Position of THC 
A mitochondrial fraction prepared from the strain AH22/pAC25 and a 
microsomal fraction prepared from the strain AH22/pAMS25 were used for the 
measurement of hydroxylation activities. Subcellular fractionation was 
prepared from a transformant yeast strain according to the following 
method. About 4.times.10.sup.10 cells were collected from a culture 
containing about 2.times.10.sup.7 cells/ml, suspended in Zymolyase 
solution [10 mM Tris-HCl (pH 7.5), 2.0M sorbitol, 0.1 mM dithiothreitol, 
0.1 mM EDTA, 0.3 mg/ml Zymolyase 100T] and incubated at 30.degree. C. for 
1 hour to prepare spheroplasts. The spheroplasts were suspended in a 
sonication buffer (10 mM Tris-HCl (pH 7.5), 0.65M sorbitol, 0.1 mM 
dithiothreitol, 0.1 mM EDTA, 1 .mu.g/ml leupeptin, 1 .mu.g/ml pepstatin) 
and homogenized with a Teflon homogenizer to disrupt the cells. 
Precipitate 1 was removed by centrifugation at 3000.times.g for 5 minutes 
to give supernatant 1'. Precipitate 1 was resuspended in a sonication 
buffer, homogenized again, centrifuged at 3000.times.g for 5 minutes to 
give precipitate 1 (undisrupted cells and nucleus fractions) and 
supernatant 1". Supernatant 1 comprising supernatant 1' and supernatant 
1"was further centrifuged at 10,000.times.g for 20 minutes to give 
precipitate 2 (mitochondrial fraction) and supernatant 2. Supernatant 2 
was further centrifuged at 120,000.times.g for 70 minutes to give 
precipitate 3 (microsomal fraction) and supernatant 3 (soluble fraction). 
Reaction systems (2 ml) shown in the following but NADPH were mixed and 
preincubated at 37.degree. C. for 5 minutes, and reaction was initiated by 
the addition of NADPH. After 5 and 10 minutes, 0.5 ml portions of reaction 
solutions were taken, respectively, and 5 ml of benzene was added to the 
removed samples. The benzene layer obtained by centrifugation was dried 
completely and analyzed by HPLC under the condition listed below. 
[Reaction 
______________________________________ 
1. Mitochondrial fraction: strain AH22/pAC25 
(containing 0.1 nmol P450.sub.c25), 
Microsomal fraction: strain AH22/pAMS25 
(containing 0.8 nmol chimeric P450 (17.alpha.-c25), 
2. Bovine adrenal adrenodoxin reductase 
0.8 nmol, 
3. Bovine adrenal adrenodoxin 
8.0 nmol, 
4. Substrate final concentration 
200 .mu.M, 
5. NADPH final concentration 
1.0-2.0 mM, 
6. Tris-HCl (pH 7.8) 100.0 mM, 
7. EDTA 0.5-1.0 mM. 
______________________________________ 
[HPLC Analysis Condition] 
Column: .mu.Bondapak C18 (.phi.4.times.300 mm), 
Detection: A265 (1.alpha.-hydroxyvitamin D3), Radioactive detector (.sup.3 
H-THC), 
Flow rate: 1.0 ml/min, 
Temperature: 50.degree. C., 
Elution condition 
0-5 min: 80% acetonitrile-20% water solution 
5-15 min: linear concentration gradient of 80% -100% acetonitrile, 
15-25 min: 100% acetonitrile 
When 1.alpha.-hydroxyvitamin D.sub.3 was used as a substrate, the 
25-hydroxylated product were observed in the mitochondrial fraction of the 
strain AH22/pAC25 and the microsomal fraction of the strain AH22/pAMS25. 
On the other hand, no 25-hydroxylated products were observed in the 
control strain AH22/pAAH5. Thus, it was suggested that the hydroxylation 
activities are dependent on P450.sub.c25 and the chimetic P450 
(17.alpha.-c25). 
In addition, no activity was detected in the absence of either NADPH, ADX 
or ADR, so that the hydroxylation activities were believed to be exhibited 
with the construction of the electron transfer chain, 
NADPH.fwdarw.ADR.fwdarw.P450.sub.c25 or chimeric P450 (17.alpha.-c25), 
which was formed on the addition of NADPH, ADX and ADR. 
In the microsomal fraction of the strain AH22/pAMS25, the calculated 
turnover number was 7.4 moI/mol P450/min, and thus it was found that the 
rate was about 50 times higher than the calculated turnover number 
obtained in the mitochondrial fraction of the strain AH22/pAC25 of 0.14 
mol/mol P450/min. When [.sup.3 H]-THC was used as a substrate, the 
transformation of THC to the 27-hydroxylated product (TeHC) was observed 
in the mitochondrial fraction of the strain AH22/pAC25 and the microsomal 
fraction of the strain AH22/PAMS25. No activity was observed in the 
control strain AH22/pAAH5. Thus, it was suggested that the activity was 
dependent on P450.sub.c25 and chimetic P450 (17.alpha.-c25). The turnover 
numbers calculated in the mitochondrial fraction of the strain AH22/pAC25 
and the microsomal fraction of the strain AH22/pAMS25 were 20 mol/mol 
P450/min and 23 mol/mol P450/min, respectively. 
EXAMPLE 7 
Measurement of the 27-hydroxylation Activity of THC in the Triple 
Simultaneous Expression Strain AH22/pRXMS25 
To the culture of the strain AH22/pRXMS25 (1.6.times.10.sup.7 cells/ml) was 
added [.sup.3 H]-THC so that the final concentration is 10 .mu.M, and the 
incubation was continued. A 1 ml portion of the culture after 14 hour 
cultivation was taken out and extracted with 2 ml of dichloromethane, and 
the dichloromethane layer was evaporated to dryness, dissolved in 80 .mu.l 
of acetonitrile, of which a 50 .mu.l portion was analyzed by HPLC under 
the same condition as described in Example 6. As a result, it was found 
that 19% of the substrate THC added had been transformed into TeHC. Such 
transformation was not observed in the control strain AH22/pAAH5, and thus 
it was found that the chimetic P450.sub.c25, ADX and ADR constructed an 
electron transport chain and exhibited a monooxygenase activity in the 
strain AH22/pRXMS25. 
The transformed yeast provided by the present invention produces 
P450.sub.c25 containing heme in the molecule and the chimeric 
P450.sub.c25. 
P450.sub.c25 and the chimeric P450.sub.c25 exhibited 25-hydroxylation 
activities to vitamin D.sub.3 and 1.alpha.-hydroxyvitamin D.sub.3 and 
27-hydroxylation activity to THC by the addition of bovine adrenal ADX and 
ADR. 
While P450.sub.c25 is a protein inherently present in mitochondrial inner 
membrane, conversion from mitochondria to microsome for its localization 
in yeast cells was succeeded by removing the mitochondrial targeting 
signal and adding the N-terminal 15 amino acid residues derived from the 
microsomal P450.sub.17.alpha., which functions as a targeting signal to 
microsomal membrane. Such a conversion of the localization of the membrane 
enzyme has not hitherto been reported in the literatures. While the 15 
amino acid residues at the N-terminus of P450.sub.17.alpha. are highly 
hydrophobic and is believed to play a role as a targeting signal to 
microsomal membrane, it has no specificity as a signal and can be replaced 
with a signal of the other microsomal membrane proteins. 
The production amount of P450.sub.c25 per yeast cell could be increased to 
about 4 times and the 25-hydroxylation activity of 1.alpha.-hydroxyvitamin 
D.sub.3 per P450 molecule could be also increased to about 50 times by 
changing mitochondria into microsome for the localization of membrane 
enzyme based on the substitution of the signal. Thus, the ability of the 
25-hydroxylation of 1.alpha.-hydroxyvitamin D.sub.3 per cell was assumed 
to be raised to about 200 times. It is possible to produce 1.alpha., 
25-dihydroxyvitamin D.sub.3 which plays a role for regulating calcium 
metabolism, stimulating the differentiation of cells and controlling 
cellular immune systems and is useful for the treatment of osteoporosis, 
chronic renal insufficiency, vitamin D resistant rickets or the like by 
using P450.sub.c25 or the chimeric P450.sub.c25 as a bioreactor. 
In addition, the use of the cells of the strain which expresses 
simultaneously chimeric P450.sub.c25, ADX and ADR as a bioreactor is a 
useful means for simplifying the currently used complex method for 
chemical synthesis of an active vitamin D.sub.3. 
Further, P450.sub.c25 produced in yeast can be easily purified, and an 
antibody against P450.sub.c25 can be used as a diagnostic agent of 
cerebrotendinous xanthomatosis. It has been definitely shown that the 
patients of cerebrotendinous xanthomatosis are congenitally defficient in 
the enzyme. The disease is characterized in xanthomatosis or the 
neuropathy of animus, and xanthomatosis is generated at Achilles tendon, 
lung and brain of the patient resulting in serious symptoms such as 
cataract, lowering of intelligence or the like. It is known that the 
cholestanol level in the serum of the patients is in a proportion of 
10-100 to the normal level, and diagnostic methods utilizing this 
phenomenon or methods of measuring the activity of the enzyme with THC 
labelled with a radioisotope are currently used. The production of 
P450.sub.c25 in a large scale has become possible by cultivating the 
strain of the transformed yeast obtained by the present invention, so that 
the preparation of an antibody against the isolated and purified enzyme 
makes it possible to detect P450.sub.c25 at a high sensitivity and is 
believed to be effective for the diagnosis of cerebrotendinous 
xanthomatosis. 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 5 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AATTCAAGCTTAAAAAAATGGCTCTGTTGAGCCGCATGAGA41 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CTGAGATGGGCGCTTCTGGACACTCGTGTGATGGGC36 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 61 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
AGCTTAAAAAAATGCTTTCACTACGTCAATCGATAAGATTTTTCAAGCCAGCCACAAGAAC61 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 49 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
TTTGTGTAGCTCTAGATATCTGCTTCAGCAAAAACCCGCGATCCCTGCA49 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 74 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
AATTCAAGCTTAAAAAAATGTGGCTGCTCCTGGCTGTCTTTCTGCTCACCCTCGCCTATT60 
TAGCGATCCCTGCA74 
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