JPH0636744B2 - Chimeric cytochrome P-450 gene - Google Patents

Description

TECHNICAL FIELD The present invention relates to a chimeric cytochrome P-450 gene, a yeast expression plasmid containing the same, a yeast strain that retains these plasmids in the cells, and a method for producing them.

Prior Art and Problems Cytochrome P-450 (hereinafter abbreviated as P-450)
Is a heme protein that exists widely in the living world from microorganisms to mammals, and many molecular species exist.
Although each P-450 molecular species has different substrate specificity, they commonly catalyze a monoatomic enzyme addition reaction for a lipophilic substrate.

In recent years, the present inventors have succeeded in constructing a yeast expression vector pAMC1 that expresses rat liver cytochrome P-450C (P-450C) using yeast as a host and producing a large amount of P-450C in the yeast (Japanese Patent Application Sho 59-1229
53).

In yeast, P-450C is present in microsomes, forms an electron transfer system in cooperation with yeast NADPH-cytochrome P-450 reductase, and is a monoatomic oxygenation reaction (oxidation activity).
Indicates.

As described in the inventors' patent application (Japanese Patent Application No. 60-139128), using a P-450C expressing yeast strain,
It is possible to produce acetaminophen by para-hydroxylating acetanilide (Japanese Patent Application No. 60-13912).
8).

Furthermore, the present inventors have succeeded in constructing a chimeric P-450 gene from two or more kinds of P-450 genes and expressing it in yeast cells (Japanese Patent Application No. 60-24277).
3). The substrate specificity of the chimeric P-450ccd described in this patent application is derived from P-450C,
The chimeric P-450 has high oxidative activity and P-450C substrate specificity.

Background of the Invention As a result of various studies, the present inventors have found that the region that determines the substrate specificity of P-450, that is, the substrate binding site, is rat liver P
At −450d, the 184th to 370th positions from the N-terminus
Second amino acid sequence, and P-
In the case of P-450MC, the region involved in the localization of 450C to yeast microsomes is a region of about several tens of amino acid residues (about 30) from its N terminus, and is produced by a gene containing this region. It was revealed that P-450, which localizes to microsomes, is stably maintained and exhibits oxidative activity.

Structure of the Invention The present invention is a chimera P constructed by ligating a corresponding C-terminal side portion of a gene encoding P-450 of another molecular species after a gene region encoding an amino acid terminal region portion of P-450C. A 450 gene, a yeast expression plasmid containing the gene and expressing it in yeast cells,
Provided are a transformant yeast created by transforming with the expression plasmid, and a chimeric P-450 of the expression product.

Means for Solving Problems The chimeric P-450 gene of the present invention encodes a region involved in the localization of P-450C to yeast microsomes, that is, a region containing several tens of amino acid residues from the N-terminus.
A fragment of the -450C gene is isolated from, for example, the plasmids pTF1, pTF2, pAMC1 and the like containing the P-450C gene (Japanese Patent Application No. 59-169447) and ligated to the C-terminal portion of P-450 of another molecular species. It can be manufactured.

More specifically, for example, a region encoding the N-terminal 186 amino acid residues of the P-450C gene is followed by a corresponding C-terminal portion of the P-450 gene of another molecular species, such as P-450d. (J. Biochem. 96,793-804, (198
4)) can be obtained by connecting the regions encoding the 184th to 513th amino acids (the chimeric P-450 constructed in this manner is the chimeric P-450c).
dd2).

The C-terminal portion of P-450d is a plasmid pTZ330 [J. Biochem., 96, containing the region encoding P-450d.
793-804, (1984) can be produced by the method described.)
Can be separated from. Chimeric plasmid pACD
A schematic of D2 construction is shown in FIG.

The thus-obtained chimeric P-450 of the present invention has the substrate specificity of various P-450 molecular species used on the C-terminal side, and according to the present invention, it has various substrate specificities. It is possible to produce a chimeric P-450 having.

The yeast expression plasmid carrying the chimeric P-450 gene of the present invention is, for example, the yeast expression vector plasmid pAAH5 (Wachingt) which holds the promoter and the terminator of the yeast alcohol dehydrogenase I gene.
on Research Foundation, Methods in Enz
ymology, 101 part C p192-201, Ammerer et al.) and yeast expression vector oJDB219 [Natu
re, 275 , 104 (1979)], etc., and the chimeric P-450 gene produced as described above is incorporated into the yeast expression vector. In this case, the yeast expression vector is not particularly limited, and the promoter and terminator used may be any promoter and terminator that function efficiently in yeast, and are not limited to these. Absent. In addition, a promoter on a plasmid, a chimeric P-450 gene,
The structure other than the terminator is not limited,
What is necessary is that it can be stably retained in yeast.

The acetanilide p-position hydroxylation activity per bacterial cell or per p-450 molecule in the yeast strain transformed with the yeast expression plasmid pACDD2 carrying the chimeric P-450cdd gene is the same as that of the conventional plasmid p.
It is about 5 to 6 times that of the yeast strain transformed with AMC1, and it can be seen that it is highly useful as a bioreactor. The yeast cell of the present invention can be obtained by transforming a yeast belonging to the genus Saccharomyces with the plasmid containing the chimeric P-450 gene by the alcal metal method or the prohyplast method. Saccharomyces cerevisiae AH22 strain can be used,
It is not limited to this strain.

Hereinafter, the present invention will be described in more detail with reference to examples. The present invention is not limited to the following examples, and can be modified in a usual manner in the technical field of the present invention.

Example 1 Construction of plasmid pACDD2 FIG. 2 shows an outline of the construction of plasmid pACDD2.

Patent application for the invention of the present inventors (Japanese Patent Application No. Sho 59-122)
593), the plasmid pTF1 described in
After partial digestion with I and further with StuI, low melting point agarose gel electrophoresis was performed to excise a gel containing a DNA fragment of about 4.2 kb, which was heated at 65 ° C. for 5 minutes. To the melted gel, 2 volumes of TE buffer [10 mM Tris-hydrochloric acid 0.5 mM EDTA (pH 8.0)] was added, and then an equal amount of phenol saturated with TE buffer was added and stirred. After centrifugation, the upper layer was separated, and double the volume of cold ethanol was added to precipitate the DNA for recovery. All subsequent recovery of DNA fragments was performed by this method. DNA of about 4.2 kb
After treating about 100 ng of the fragment with alkaline phosphatase,
Known plasmid pTZ330 (J. Biochem. 96 793-80
4, (1984) was mixed with about 50 ng of a DNA fragment obtained by cutting with BaI and StuI, and a ligase reaction was carried out. E. coli DH1 (F-recA1, endA1, gyrA96, thi
-1, hsdR17, supE44, λ-), 100 μg / m
The plasmid DNA was isolated from the colonies that spread and appeared on the plate containing ampicillin. Obtained DNA about 1
00ng was partially cleaved with BalI and then treated with alkaline phosphatase to obtain about 190 bp B obtained from pTF1.
A ligase reaction was carried out by mixing with about 50 ng of an alI-BalI DNA fragment. E. coli DH transformed with the reaction mixture
Plasmid DNA was prepared from 1 colony and pTFc
It was named dc (Fig. 2).

About 100 ng of a DNA fragment of about 3.9 kb obtained by cleaving pTFcdc with StuI and HindIII was treated with alkaline phosphatase, and then mixed with about 100 ng of a synthetic linker DNA having the sequence shown below to carry out a ligase reaction. It was Nucleotide sequence of synthetic linker: CCTGGCCACGCTTCTCCAAGTGA GGACCGGGTGCGAAGAGGTTCACTTC
Plasmid DNA was prepared from E. coli DH1 colonies transformed with the GA reaction mixture. This plasmid DN
About 4 A obtained by cleaving about 100 ng of A with StuI and treating with alkaline phosphatase, and cleaving pTZ330 with StuI.
Ligase was performed by mixing with about 100 ng of 20 bp DNA. A plasmid DNA was prepared from a colony of Escherichia coli DH1 transformed with the reaction mixture and named pTFcfd.

Then, about 100 ng of pTFcdd was cleaved with Sa1I, filled in with a DNA polymerase I Klenow enzyme, and further treated with alkaline phosphatase. About 500 ng of HindIII linker was added to this and ligase reaction was performed. Escherichia coli DH1 was transformed with the reaction mixture to prepare plasmid DNA from the resulting colonies,
The DNA structure was confirmed by cutting with HindIII, and the obtained plasmid was named pTFcdd (H). pTFc
Cleavage of dd (H) with HindIII and D of about 1.6 kb
The NA fragment was recovered.

Next, yeast expression vector pAAH5 (Washington Researc
Available from h Fundation, Method in Enzymology, 101
part C p192-201), approx. 100
ng was digested with HindIII, treated with alkaline phosphatase, mixed with about 200 ng of a 1.6 kb DNA fragment, subjected to ligase reaction, and transformed into Escherichia coli DH1 strain with the reaction mixture. A plasmid DNA was prepared from the colony, cut with BamHI and StuI to confirm the DNA structure, and the obtained plasmid was named pACDD2.

Example 2 Transformation of yeast with plasmid pACDD2 YPD medium (1% Yeast Extract, 2% polypeptone,
2% glucose) 1 m was inoculated with Saccharomyces cerevisiae AH22 strain and shaken at 30 ° C. for 18 hours,
The cells were collected by centrifugation.

The obtained cells were suspended in 1 m of 0.2 M LiC solution and then centrifuged again.
A 70 μl polyethylene glycol 4000 solution of 30 μl of the MLiC solution and a 10 μl solution containing about 1 μg of pACDD2 were added. After mixing thoroughly, the mixture was incubated at 30 ° C. for 1 hour, 140 μm of sterilized water was further added, and the mixture was stirred. This solution was spread on an SD synthetic medium plate (2% glucose, 0.67% nitrogen source amino acid-free, 20 μ1 / m1 histidine, 2% agar), incubated at 30 ° C. for 3 days, and transformed strain AH2 carrying pACDD2.
2 (pACDD2) was obtained.

Example 3 Chimeric P-450 protein (P-450c
Analysis of dd) The AH22 (pACDD2) strain obtained in Example 2 was treated with SD synthetic medium (2% glucose, 0.67% nitrogen source amino acid-free, 2
After culturing up to 1.5 × 10 ⁷ cells / ml with 0 μl / ml histidine), the cells were collected, and zymolyase solution (1.2 M sorbitol, 50 mM potassium phosphate (pH 7.2), 14 m) was collected.
It was suspended in M2-mercaptoethanol, 0.4 mg / m Zymolyase 60,000) and incubated at 30 ° C for 30 minutes. 100 to spheroplasts collected by centrifugation
Buffer heated to ℃ (1% SDS, 50 mM Tris-HCl (pH 6.
8), 10% mercaptoethanol, 40% glycerol, 0.02% bromophenol blue, 1 mM phenylmethylsulfonylfluoride) was added for solubilization. The supernatant obtained by centrifugation (approximately 3 × 10 ⁶ bacterial cells) was electrophoresed on a 10% polyacrylamide gel. In addition, the proteins in the gel were electrophoretically blotted onto nitrocellulose filters in 25 mM Tris-HCl (pH 8.3), 192 mM glycine-20% methanol. Next, the filter was immersed in a TBS buffer solution [50 mM Tris-HCl (pH 7.5) 200 mM NaCl], and then TBS containing 3% gelatin 0.05% Tween 20.
Incubate for 40 minutes at 37 ° C in buffer, then 3
0 μg of purified anti-P-450MC IgG, 1% gelatin, 0.
Incubated for 2 hours at 37 ° C. in TBS containing 05% Tween20. After that, the procedure of incubating in a buffer containing 0.05% Tween 20 for 30 minutes at 37 ° C. was repeated 4 times, and then TB containing 3% gelatin and 0.05% Tween 20 was repeated.
Incubated in S buffer for 20 minutes at 37 ° C. Next, 2 μCi of [ ¹²⁵ I] -Protein A, 1% gelatin
70% at 37 ° C in TBS buffer containing 0.05% Tween 20
TB containing 0.05% Tween 20 after a minute incubation
The operation of incubating in S at 37 ° C. for 30 minutes was repeated 4 times. When the filter was air-dried and then autoradiography was performed, a band of P-450cdd was observed at almost the same electrophoretic position as that of rat P-450C. From the band intensity, it was estimated that the AH22 (pACDD2) strain produced at least 2 × 10 ⁵ molecules of P-450cdd per cell.

Example 4 Quantification of P-450 cdd containing heme Saccharomyces cerevisia
e) AH22 (pACDD2) strain culture medium (SD synthesis medium, bacterial cell concentration of about 1.5 × 10 ⁷ cells / ml) was collected in 100 ml, and 10 ml of 100 mM potassium phosphate buffer (pH 7.0) was collected.
And then centrifuged. The obtained pellet was newly suspended in 2m of 100mM potassium phosphate buffer solution (pH 7.0) and dispensed into each of two cuvettes in an amount of 1ml. After blowing carbon monoxide into the cuvette on the sample side, 5 to 10 mg of dithionite was added to both cuvettes, stirred, and then allowed to stand for 20 minutes. Then, stir the liquid in the cuvette and measure the difference spectrum at 400 to 500 nm.
90 = 91mM ^-1 cm ^-1 P-45 based on the values of Omura and Sato et al.
The 0 concentration was calculated. As a result, Saccharomyces serevisiae AH22 (pACD
Strain D2) was found to produce about 1.5 × 10 ⁵ molecules of heme protein per cell.

Example 5 Measurement of acetanilide p-position hydroxylation activity of yeast cells Saccharomyces cerevisiae cultured in SD synthetic medium up to about 1.4 × 10 ⁷ cells / ml
AH22 (pAAH5), AH22 (pAMCl) strain and AH22 (pACD) cultured up to 1.2 × 10 ⁷ cells / ml.
1.5M acetanilide (methanol solution) was added to the culture solution of strain D2) to give a final concentration of 25 mM.

Then, shake culture (120 cycle / min) at 30 ° C., aliquots are taken in small portions every hour, and the supernatant obtained by centrifugation is subjected to HPLC.
The resulting acetaminophen was quantified. HP
The LC conditions are shown below.

Column: μBondapak C18 (φ4mm × 300mm) Solvent: Methanol: Water: Acetic acid = 15: 84: 1 (V / V) Detection: A245nm Flow rate: 2.0 ml / min Temperature: Room temperature (20-25 ° C) Culture 0,4, P-450 content per bacterium in 7 hours was AH2
2 (pAMCl) strain is about 5.5 × 10 ⁵ molecule / bacillus, AH22
The (pACDD2) strain had about 1.5 × 10 ⁵ molecules / bacillus, but the control AH22 (pAAH5) strain had P-4.
50 could not be detected. AH22 from the activity of each strain
The activity of the (pAAH5) strain was determined. By dividing the P-450-dependent activity by the P-450 content, AH22
It was found that the activity per molecule of P-450cdd applied to the (pACDD2) strain was about 5 to 6 times higher than the activity per molecule of P-450MC in the AH22 (pAMC1) strain.

Example 6 Measurement of ^7- Ethoxycoumarin O-Deethylation Activity of Yeast Cells In a culture solution of AH22 (pACDD2) and AH22 (pAMC1) strains cultured up to about 1.2 × 10 ⁷ cells / m in SD synthesis medium. 20 mM 7-ethoxycoumarin (50% aqueous methanol solution) was added to a final concentration of 0.5 mM. Then, shake culture was performed at 30 ° C., 0.25 m was collected every hour, and 0.2 ml of the supernatant obtained by centrifugation was mixed with 20% trichloroacetic acid 2%.
3.5 μchloroform (1.0 m) was added, and the mixture was vigorously stirred. After centrifugation, the chloroform layer 0.5m has 2.0m of 0.
After adding 01N NaOH-0.1M NaC aqueous solution, vigorously stirring and centrifuging, the water layer was excited at a wavelength of 366 nm and a fluorescence wavelength of 452 nm.
And the product 7-hydroxycoumarin was quantified.

As a result, P-450c in AH22 (pACDD2) strain
The activity per molecule of dd was found to be about 15% of the activity per molecule of P-450C applied to the AH22 (pACM1) strain. The acetanilide p-position hydroxylation activity described above is
It is known that P-450d is higher than P-450C, but
On the contrary, it is known that P-450C has higher ethoxycoumarin O-deethylation activity. From the results obtained here, it can be inferred that P-450cdd has the substrate specificity of P-450d.

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence and amino acid sequence of pACDD2. FIG. 2 shows a schematic of the construction of plasmid pACDD2. Sa, P, B, St, H are restriction enzymes SalI, PatI, BalI, StuI, H respectively
The cleavage site of indIII is shown. Figure 3 shows AH22 (pACDD2) and AH22 (pAM
C1) Concentration of produced acetamiphene in the culture solution (nmol
/ Ml) and bacterial cell concentration (× 10 ⁸ cells / ml) over time. {Circle around (1)} and □ indicate the acetaminophen concentration and the bacterial cell concentration of the AH22 (pACDD2) strain, respectively. The triangles and triangles show the acetaminophen concentration and the bacterial cell concentration of AH22 (pAMC1), respectively. FIG. 4 is a diagram showing changes over time in the concentration of produced 7-hydroxycoumarin and the concentration of cells in the culture solution of each strain, as in FIG.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location (C12N 1/19 C12R 1: 865)

Claims (6)

[Claims] 1. An amino acid terminal region containing a region that controls localization of rat liver cytochrome P-450c gene to yeast microsomes, and a region excluding the amino group terminal side of rat liver cytochrome P-450d gene below. Chimeric cytochrome P-450 gene represented by nucleotide sequence 2. The following region consisting of an amino acid terminal region containing a region controlling localization of rat liver cytochrome P-450c gene to yeast microsomes and a region excluding the amino group terminal side of rat liver cytochrome P-450d gene: A yeast expression plasmid containing a chimeric cytochrome P-450 gene represented by a nucleotide sequence and expressing the gene in yeast 3. The following region consisting of an amino acid terminal region containing a region responsible for localization of rat liver cytochrome P-450c gene to yeast microsomes and a region excluding the amino group terminal side of rat liver cytochrome P-450d gene: A yeast strain containing a chimeric cytochrome P-450 gene represented by a nucleotide sequence and transformed with a yeast expression plasmid that expresses the gene in yeast. 4. The expression plasmid pA according to claim 2, which is represented by the following restriction enzyme map.
CDD2 5. The yeast strain according to claim 3, which retains the expression plasmid pACDD2 represented by the following restriction enzyme map. 6. The method according to claim 5, wherein the yeast strain is Saccharomyces cerevisiae AH22 strain.
Strain according to item