JPH0673459B2 - Yeast cells containing expression plasmids PARM1 and PARM1 for expression of rat liver cytochrome P-450C and NADPH-cytochrome P-450 reductase in yeast at the same time - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to rat liver cytochrome P-450c and NADPH-
The present invention relates to an expression plasmid for simultaneously expressing a cytochrome P-450 reductase gene in yeast, and a yeast cell that produces rat liver cytochrome P-450c and NADPH-cytochrome P-450 reductase transformed with the plasmid.

The electron transport system existing in mammalian liver microsomes includes cytochrome P-450 (hereinafter abbreviated as P-450) and NADPH-.
It is a complex enzyme system composed of cytochrome P-450 reductase (hereinafter abbreviated as Fpt), and has a unique oxidative activity for various substrates.

The present inventors have already isolated the rat liver cytochrome P-450c gene (the nucleotide sequence of the gene is Nuclei
c Acids Res., 12 , 6, p2929-2938 (1984). ). Furthermore, we succeeded in expressing this gene in yeast. In addition, a yeast expression plasmid pAMC1 that expresses P-450c was constructed, and a large amount of P-450c was produced using the yeast cells transformed with the plasmid.
Succeeded in exhibiting high oxidative activity (Japanese Patent Laid-Open No. 61-
88878).

Further, by incorporating the rat liver Fpt gene into a yeast expression vector, constructing a yeast expression plasmid pARF1 and transforming with the plasmid, rat liver Fpt gene was obtained.
Was produced (Japanese Patent Laid-Open No. 62-19085).

The present inventors further proceeded with the research, and this time, from pTRF2 prepared by remodeling the P-450c expression plasmid pAMC1 and the Fp1 expression plasmid pARF1, the yeast alcohol dehydrogenase I (abbreviated as ADH1) gene was prepared. P-450c and Fpt sandwiched between promoter and terminator
BamHI with gene part as promoter and terminator
It was excised with the plasmid pAAH5 (Washigton Researc
Available from h Foundation, Methodstn Euzymology, 10
1, partc, p192 to 201, can be produced by the method of Amere et al.
The yeast ADH1 gene promoter is the Washington Resea
rch Foundation US Application No. 299,733,
When used for industrial or commercial purposes in the United States, a right permission from the right holder is required. The plasmid pARM1 was constructed by inserting it into the BamHI site of (1), and yeast was transformed with the plasmid to successfully produce yeast that simultaneously produces P-450c and Fpt.

The yeast cell obtained by the present invention has about twice as high oxidative activity as the yeast cell that produces only P-450c. Can be advantageously applied to the oxidative removal of Also,
The yeast transformant obtained by the present invention has the ability to specifically and efficiently hydroxylate only the p-position of acetanilide, and can be used for the production of acetaminophen useful as an antipyretic and analgesic agent. It has excellent properties.

The plasmid of the present invention can be prepared, for example, by the following method. That is, the plasmids ARF1 and p described in the patent application (Japanese Patent Application Laid-Open No. 61-88878) relating to the inventions of the present inventors
Plasmid pTRF2 containing RFCC5 to ADH1 gene promoter, Fpt-encoding gene and ADH1 terminator
Was created by the method outlined in Fig. 2. From this, 4.1kb
The DNA fragment of is cut out with the restriction enzyme BamHI. On the other hand, a 3.7 kb DNA containing an ADH1 gene promoter, a gene encoding P-450c and an ADH1 gene terminator from the plasmid pAMC1 described in the patent application (Japanese Patent Application Laid-Open No. 61-88878) relating to the present inventors. The fragment is cut out with the restriction enzyme BamHI. Next, these DNA fragments were digested with the enzyme expression plasmid p.
AAH5 (Methods in Enzymology 101, part C P192−201, A
can be manufactured by the method of mmerer et al., Wshington
(Available from the Research Foundation) and inserted into the BamHI site.

Furthermore, by using this plasmid to transform yeast, for example, Saccharomyces cerevisiae strain AH22 (ATCC38626), a transformed yeast that simultaneously produces rat liver cytochrome P-450c and Fpt can be produced.

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

Example 1 Construction of plasmid pTRF2 Step 1: Oligonucleotide adapters 5'AGCTTTAAACATGGGGG3 'and 5'AGTCCCCCATGTTT with HindIII and HinfI cleavage sites on both sides.
AA3 ′ was synthesized using Applied Biosystems 380A type DNA synthesizer.

The obtained oligonucleotide adapter was purified by high performance liquid chromatography and then phosphorylated at the 5'end.

Step 2: About 5 μg of plasmid pRFCC5 (which can be produced by the method described in JP-A-62-19085) is added to 250 μl of the restriction enzyme reaction solution M [1.
Dissolved in 0 mM Tris-hydrochloric acid (pH 7.5), 10 mM MgCl ₂ , 1 mM dithiothreitol, 50 mM NaCl], and 25 units of PvuII
And HindIII are added, and a cleavage reaction is performed at 37 ° C for 1 hour to prepare a DNA fragment encoding the amino group terminal side of Fpt.
Dissolve in 00 μl of restriction enzyme reaction solution M and add 15 units of Hinf
A HinfI-PvuII fragment of about 700 bp was prepared by cutting with I.

On the other hand, the plasmid pBR322 (about 2 μg) was digested with 10 units of PvuII, HindIII, and SalI in 50 μl of the restriction enzyme reaction solution M at 37 ° C. for 1 hour to give about
A 2.3 kb HindIII-PvuII fragment was prepared.

The resulting HinfI-PvuII fragment (about 0.5 μg) and HindIII-Pv
The ligase reaction solution [66 mM Tris-HCl (pH 7.6), 66 mM MgCl ₂ , 10 mM DTT, uII fragment (about 0.5 μg) together with about 1 μg of the oligonucleotide adapter obtained in step 1
1 mM ATP] and incubated at 15 ° C for 20 hours, and circularization was performed with 32 units of T4 DNA ligase.

Escherichia coli DH1 strain was transformed with the reaction mixture, and plasmid DNA was prepared from the obtained transformant. Of the plasmid DNAs, the one that gives 1800, 690, and 450 bp DNA fragments when cleaved by AhaIII, and that gives a 700 bp DNA fragment when cleaved by PvuII and HindIII was designated as plasmid pRFA1.

Step 3: 52 units of HindIII in plasmid pRFA1 (about 1 μg),
PvuII was added, and cut in 50 μl of restriction enzyme reaction solution M for 1 hour at 37 ° C., and then subjected to low melting point agarose gel electrophoresis for about 700 b.
A DNA fragment of p was prepared. On the other hand, the plasmid pRFCC5 was cut with HindIII and PvuII in the same manner as in step 2 to prepare a DNA fragment encoding the carboxy group terminal side of Fpt.

The surface DNA fragment was previously cleaved with HindIII and treated with alkaline phosphatase expression vector pAAH5 (0.5 μm
g) and mixed with 50 μl of ligase reaction mixture to obtain 32
Units of T4 DNA ligase were added and incubated at 15 ° C overnight.

The reaction mixture was used to transform E. coli DH1 strain.

The obtained transformant was used in L medium (containing 10 g polypeptone, 5 g yeast extract, 5 g NaCl, 1 g glucose per 1).
Incubate in the method of Birnboim et al. [Nucleic Acids Res.,
7, 1513-1523 (1979)] and prepared by cutting with BamHI and NcoI to obtain 2.1, 1.4 and 0.7 kb of DNA.
The plasmid that gave the gene was designated as pTRF2. This plasmid pTR
The restriction map of F2 is shown in FIG.

Example 2: Construction of plasmid pARM1 Step 1: Approximately 5 μg of plasmid pAMC1 in 15 μl of restriction enzyme reaction solution H [10 mM Tris-hydrochloric acid (pH 7.5), 10 mM MgCl ₂ , 1 mM dithiothreitol, 150 mM NaCl] 15 units. After digestion with the restriction enzyme BamHI (purchased from Takara Shuzo Co., Ltd.), a 3.7 Kb DNA fragment containing the ADH1 promoter, the gene encoding P-450c and the ADH1 terminator was recovered by low melting point agarose gel electrophoresis.

Step 2: Approximately 5 μg of plasmid pTRF2 was similarly treated with the restriction enzyme BamHI.
After cleaving with 4., a 4.1 Kb DNA fragment containing the ADH1 promoter, the gene encoding Fpt and the ADH1 terminator was recovered by low melting point agarose gel electrophoresis.

Step 3: Plasmid pAAH5 (about 5 μg) was similarly digested with the restriction enzyme Bam.
After cutting with HI, 2 units of Escherichia coli alkaline phosphatase (purchased from Takara Shuzo) was added and reacted at 60 ° C. for 1 hour.

After phenol-chloroform extraction, DNA was recovered by ethanol precipitation.

Step 4: The DNA fragments obtained in steps 1 to 3 were mixed in 50 μl of ligase reaction solution, 32 units of T4 DNA ligase (purchased from Takara Shuzo) was added, and the mixture was incubated at 15 ° C. for 20 hours.

Subsequently, the reaction mixture was used to transform E. coli DH1 strain.

The obtained transformant was cultured in L medium (containing 10 g polypeptone, 5 g yeast extract, 5 g NaCl, 1 g glucose per 1) and subjected to the method of Birnboim et al. (Nucleic Acids Re
s., 7 , 1513-1523).

The plasmid DNA was cleaved with the restriction enzyme BamHI, and a plasmid containing both the 3.7 kb DNA fragment and the 4.1 kb DNA fragment was designated as pARM1.

In addition, the size of the DNA fragment was determined by agarose gel electrophoresis after cutting plasmid pARM1 with restriction enzymes HindIII and NcoI. As a result, P-450c and Fpt genes were "head to tail" as shown in Fig. 1. Turned out to be connected.

The insertion direction of the BamHI fragment in the plasmid pARM1 has not been determined, but it is considered that the insertion direction of the ADH1 promoter is not related to the expression level, so the plasmid inserted in the reverse direction is essentially the same as the plasmid pARM1. .

Example 3: Transformation of yeast with plasmid pARM1 5 ml of YPD medium (1% yeast extract, 2% polypeptone, 2% glucose) was added to Saccharomyces cerevisiae (sacch).
aromyces cerevisiae) AH22 strain (ATCC38626)
It was cultured at 30 ° C for 16 to 18 hours. Centrifuge 1 ml of culture solution (100
The cells were collected at 00 rpm for 1 minute), and the obtained cells were washed with 0.2M LiCl. 20 μl of the obtained bacterial cells
Suspended in M LiCl, plasmid pARM1 DNA about 1 μg, 70%
Add 30 μl of polyethylene glycol 4000 and add 1 at 30 ℃
Incubated for hours. Then, 140 μl of sterilized water was added, 100 μl of each was spread on an SD plate (0.67% yeast nitrogen source-free amino acid, 2% glucose, 20 μg / ml histidine, 2% agar) and incubated at 30 ° C. to transform Saccharomyces Cerevisiae (saccharomyces
cerevisiae) AH22 (pARM1) strain was obtained.

Example 4: Measurement of P-450 content of AH22 (pARM1) strain The above-mentioned transformant AH22 (pARM1) strain and AH22 (pAMC1) strain.
100 ml of each culture medium (SD synthetic medium, cell concentration: about 2 × 10 ⁷ cells / ml) was collected, suspended in 20 ml of 100 mM potassium phosphate buffer (pH 7.0), and then centrifuged. The obtained pellet was suspended again in 2 ml of 100 mM potassium phosphate buffer (pH 7.0) and dispensed in 1 cuvette into 2 cuvettes. Carbon monoxide was blown into the cuvette on the sample side, and then into both cuvettes. Add 5-10 mg of dithionite and after stirring, add about 15
Let stand for a minute. The difference spectrum at 400 to 500 nm was measured, and P was calculated based on the value of ΔE447−490 = 91 mM ⁻¹ cm ^−1.
The -450 content was calculated. As a result, the P-450 content per cell of the AH22 (pARM1) strain was higher than that of the AH22 (pAMC1) strain,
Found to be almost the same Example 5: Measurement of acetaminophen production by acetanilide p-hydroxylation of AH22 (pARM1) strain AH22 (pA cultured in SD synthetic medium to about 1.5 × 10 ⁷ cells / ml)
RM1), AH22 (pAMC1) strain culture medium, 1.5M acetanilide (methanol solution) was added respectively, final concentration 25mM
And Then, while continuing the shaking culture, a fixed amount was taken every hour, and the culture supernatant obtained by centrifugation was subjected to HPLC to quantify the produced acetaminophen. HPCL is
Using μBondapak C18 (4x300mm), methanol: water:
Eluted with acetic acid (15: 84: 1 v / v) and detected at A 245 nm. As a result, the P-450c-requiring acetanilide p-hydroxylation activity of the AH22 (pARM1) strain was about twice that of the AH22 (pAMCl) strain, and the Fp coexpressed with P-450c in the AH22 (pARMCl) strain.
It was found that t increased the activity.

[Brief description of drawings]

FIG. 1 is a diagram showing an outline of the method for constructing plasmid pARM1 of the present invention and a restriction enzyme diagram of plasmid pARM1. In the figure, the part painted in black is P-450cc DNA, and the shaded part is Fptc.
Indicates DNA. FIG. 2 is a diagram showing an outline of a construction method of the plasmid pTRF2 and a restriction enzyme map of the plasmid pTRF2. In the figure, the part painted in black represents the Fpt cDNA, and the shaded part represents the synthetic adapter used for the construction. FIG. 3 (3-1 and 3-2) is a diagram showing the nucleotide sequence of the P-450c coding region and the amino acid sequence deduced therefrom. FIG. 4 (4-1 and 4-2) is a diagram showing the base sequence of the Fpt coding region and the amino acid sequence deduced therefrom.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number for FI Technical location (C12N 1/19 C12R 1: 865) (54) [Title of invention] Rat liver cytochrome P-450C and NADPH-cytochrome Yeast cells containing the expression plasmids PARM1 and PARM1 for the purpose of simultaneously expressing P-450 reductase in yeast

Claims (4)

[Claims] 1. An expression plasmid capable of simultaneously expressing a rat liver cytochrome P-450c gene and a mammalian NADPH-cytochrome P-450 reductase gene in yeast cells, which comprises mammalian NADPH-cytochrome P-. A plasmid having at least two 450 reductase genes and having at least one structure in which a rat liver cytochrome P-450c gene is inserted in a connecting region while the genes are present. 2. The plasmid according to claim 1, which is pARM1 consisting of about 22400 base pairs and represented by the following restriction enzyme map. 3. An expression plasmid capable of simultaneously expressing a rat liver cytochrome P-450c gene and a mammalian NADPH-cytochrome P-450 reductase gene in yeast cells, which comprises mammalian NADPH-cytochrome P-. A yeast strain having a plasmid having at least two 450 reductase genes and having at least one structure in which a rat liver cytochrome P-450c gene is inserted in a connecting region while the genes are present. 4. The yeast strain according to claim 3, which comprises about 22400 base pairs and retains pARM1 represented by the following restriction enzyme map.