JP3387525B2 - Fusion oxidase of cytochrome P450c and yeast NADPH-cytochrome P450 reductase, gene encoding the enzyme, and method for producing the enzyme - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel fusion oxidase which has in one molecule the one-atom oxygenation activity of cytochrome P450 (hereinafter referred to as P450) and the ability to supply reducing power from NADPH, which is necessary therefor. A gene encoding an enzyme, a yeast expression plasmid containing the gene,
It relates to a method for producing acetaminophen, which comprises using a yeast strain transformed with the expression plasmid and the fusion enzyme.

The yeast cells transformed with the plasmid obtained by the present invention produce a fusion enzyme composed of P450 and a reductase and have a monoatomic oxygenation activity derived from this, and Alternatively, the fusion enzyme obtained from bacterial cells can be used as a bioreactor for the synthesis of acetaminophen, which is useful as a drug.

[0003]

2. Description of the Related Art P450 is a heme protein widely existing in the living world from microorganisms to mammals, and catalyzes a one-atom oxygen addition reaction using a wide range of fat-soluble compounds as substrates. This broad substrate specificity of P450 is due to P
Due to 450 molecular diversity. Although there are many molecular species in P450, the pathways that supply electrons to each P450 are common, and in microsomes, mainly flavin adenine dinucleotide and flavin mononucleotide are contained as coenzymes in the reduction. The enzyme is NADPH
Donates electrons to the substrate-bound P450. Therefore,
P450 exhibits a one-atom oxygen addition reaction only by binding to a substrate and coupling with a reductase.

[0004]

The present inventors have succeeded in expressing a fusion enzyme of rat P450 and rat NADPH-P450 reductase in yeast, but it is possible to increase the activity per cell. Wanted

[0005]

Means for Solving the Problems The inventors of the present invention have conducted a sincere study for the purpose of obtaining a yeast having high activity. As a result, a yeast having a fusion enzyme having P450c at the N-terminal side and a yeast reductase at the C-terminal side was yeast. When expressed in Escherichia coli, the expression level of the fusion enzyme using yeast reductase in yeast increased about 3 times that in the case of using rat reductase, and the activity per fusion enzyme molecule was higher than that of rat reductase. It was also found that when yeast reductase was used, it was about 5 times higher. This fusion enzyme is considered to have a very high electron transfer efficiency within the fusion enzyme molecule. Due to the increase in the expression level and the activity per molecule, the activity per cell of the novel fusion enzyme-expressing strain of the present invention is about 15 times higher than that of the fusion enzyme-expressing strain using rat reductase. The fusion enzyme of the invention and the yeast strain expressing the enzyme are extremely useful as bioreactors.

The fusion enzyme gene of the present invention comprises a region involved in microsome membrane binding of a microsomal protein and P
DNA encoding at least a region involved in substrate binding and a region involved in heme binding of 450c, and a region involved in at least flavin mononucleotide or flavin adenine dinucleotide binding of yeast reductase and NADPH
It can be constructed by connecting DNAs encoding regions involved in binding.

For example, the fusion enzyme gene of the present invention can be produced by connecting a yeast reductase gene to the 3'end side of rat liver cytochrome P450c gene, and the fusion oxidase produced by the expression of this gene is , P450c having one atom oxygenation activity and NA
It is an oxygen oxide that also has the ability to supply reducing power from NADPH possessed by DPH-P450 reductase in the same molecule.

[0008] As the above-mentioned region involved in microsome binding, it is preferable to use those having a microsome binding ability such as those derived from rat liver P450c or yeast reductase, as well as N-terminal regions of other microsomal P450s and microsomal proteins. it can. The regions involved in the binding of P450c to the microsome membrane and the binding of the substrate and heme binding are homologous region-2 (HR2) (J. Bioche) at the N-terminal portion, the central portion and the C-terminal side, respectively.
m.93,807-817) region, and these regions or an essential part of its function can be used in the present invention.

Binding of yeast reductase to microsome membranes,
The regions involved in flavin mononucleotide, flavin adenine dinucleotide binding, and NADPH binding are 1 to 50th, 50 to 465th, and 465 to 6th, respectively, where the amino-terminal methionine is number 1.
It is said that the amino acids are up to the 00th position, and these regions or their essential regions can be used.

Since the P450c gene is expressed as a fusion enzyme in a single molecule, the P450c gene should not contain a site corresponding to a translation stop codon. It can also be constructed by connecting the above-mentioned regions of both genes directly or via a linker so that the frames of both genes are not displaced. The cDNA corresponding to the coding region of P450c and the reductase used in the construction of the expression plasmid can be produced by a conventional method used in the technical field of the present invention.
For example, in the case of P450c, the plasmid pAMP19 containing this cDNA (Japanese Patent Laid-Open No. 63-44888) and the reductase gene can be extracted from pgCYR (Japanese Patent Application No. 62-325527) in a conventional manner.

The yeast expression vector expressing the fusion enzyme of the present invention can be constructed by inserting the fusion enzyme gene constructed as described above into the cloning site of an appropriate expression vector by a conventional method. Examples of yeast expression vectors include yeast expression vector pAAH5 (available from Washington Research Fundation, Methods in Enzymology, 101 part C, p192) that holds the promoter and the terminator of the yeast alcohol dehydrogenase (ADH1) gene.
-201, can be produced by the method of Ammerer et al.), Etc., but any expression vector having a PGK promoter, G3PDH promoter, GAL10 promoter, etc. that has a promoter and terminator that function more efficiently in the host yeast can be used. It is not particularly limited. The structure of the expression vector is not particularly limited as long as it can be stably retained in yeast.

The yeast used as a host for expressing the fusion enzyme of the present invention is not particularly limited. For example, yeast belonging to the genus Saccharomyces, Schizosaccharomyces, Candida, etc. can be used. Cereviche AH22 strain, Saccharomyces cerevisiae SHY3 strain, Saccharomyces cerevisiae NA87-11A strain and the like can be conveniently used as hosts.

Transformation of these hosts with an expression plasmid containing the above fusion enzyme gene can be carried out by a known method such as a method using alkali metal (LiCl) or a protoplast method. In the case of using the alkali metal (LiCl), the host cells are cultured in YPD medium, the cells are collected, washed with Licl, and then suspended in a solution of Licl and polyethylene glycol 4000. Add the sample DNA solution to this suspension and add 1
It should be left for about an hour. By this operation, the transformed yeast can be obtained on an appropriate selective medium plate.

In the case of the protoplast method, host yeast cultured in YPD medium is collected and treated with a cell wall lysing enzyme such as thymolyase in an isocratic solution containing sorbitol and the like. As a result, protoplastized yeast is obtained. Transformation is possible by adding the sample DNA and polyethylene glycol solution to the protoplast solution and keeping it warm. The transformant can be obtained by overlaying a protoplast suspension on a selective medium plate and then culturing at 30 ° C for 5 to 7 hours.

The fusion enzyme of the present invention can be produced by culturing the transformed yeast thus obtained. Cultivation of the transformed yeast obtained by the present invention can be performed by an ordinary culture method. Any medium can be used as long as it has a composition capable of growing yeast, but it is necessary to apply selective pressure to prevent the plasmid from falling off. For example,
In mutation strain host cell requires a specific amino acid, if it contains a gene complementing the amino acid auxotrophy as the marker on the plasmid, by culturing the transformed yeast minimal medium without amino acid, plasmid The target gene can be expressed while preventing the loss of the target gene.

The yeast strain of the present invention expressing the fusion enzyme or the fusion enzyme gene of the present invention is used as a bioreactor for the synthesis of acetaminophenone. Acetanilide was added to the transformed yeast culture solution or the microsome fraction prepared from the transformed yeast, or the purified fusion oxidase at a final concentration of 25 mM, and the mixture was incubated at 30 ° C. for a certain period of time, and then the product was For example, by HPLC, by separating, the acetanilide p
It is possible to obtain acetaminophenone which is a hydroxylated compound. When using the microsome fraction, NAD is used as the reaction system.
It is necessary to add a PH or NADPH regeneration system.

[0017]

The activity per cell of the novel fusion enzyme-expressing strain of the present invention is about 15 times higher than that of the fusion enzyme-expressing strain using P450c and rat reductase. The enzyme-expressing strain has extremely high utility as a bioreactor, and is useful as a bioreactor when synthesizing acetaminophenone.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to the examples, and generally includes changes to the extent that they are carried out in the field of the present invention. Example 1 Construction of expression plasmid pAFCR1 pAMP19 containing cDNA of rat P450c (JP-A-63-448)
88) was co-digested with restriction enzymes HindIII and XhoI,
A 1.6 kb DNA fragment was obtained. On the other hand, yeast NADPH-P4
PBYR717 (H) containing 50 reductase (JP-A 2-451) was digested with PvuII, and XhoI linker of Chemical formula 1 was

Embedded image After inserting SEQ ID NO: 1 and changing the PvuII site into an XhoI site, XhoI and Hi
Co-digestion with ndIII gave a fragment of approximately 2.1 kb. Plasmid pAFCR1 was obtained by inserting both of these fragments into the HindIII site of vector pAAH5 at the same time.

Example 2 Transformation of Yeast with Plasmid pAFCR1 YPD medium (1% yeast extract, 2% polypeptone, 2% glucose) was inoculated with Saccharomyces cerevisiae AH22 strain and shaken at 30 ° C. for 5 × 10 ⁷ The cells were collected by centrifugation at the time when the cells became / ml. The cells obtained from 1 ml of the culture solution were suspended in 1.0 ml of 0.2M LiCl solution and then centrifuged again, and the pellets obtained were 20 μl of 1M LiCl solution, 30 μl of 70% polyethylene glycol 4000 solution,
10 μl of solution containing 1.0 μg of pAFCR1 was added. After mixing well, the mixture was incubated at 30 ° C. for 1 hour, and 140 μl of sterilized water was added and stirred. SD this solution
Spread on a synthetic medium plate (2.0% glucose, 0.67% nitrogen source amino acid-free, 20 μg / ml histidine, 2.0% agar), incubate at 30 ° C for 3 days, and plasmid pAF
A transformant AH22 / pAFCR1 carrying CR1 was obtained.

Example 3 Quantification of fusion enzyme in AH22 / pAFCR1 strain AH22 / pAFCR1 and control in a medium containing 8% glucose, 5.4% nitrogen source amino acid-free, 160 μg / ml histidine.
Each of the AH22 / pAAH5 strains was cultured to 2 × 10 ⁷ cells / ml.
0.1 ml of 2N NaOH-8% 2-mercaptoethanol aqueous solution was added to 0.9 ml of each culture and incubated at 0 ° C for 10 minutes. Furthermore, 0.2 ml of 30% trichloroacetic acid aqueous solution was added, and the mixture was incubated at 0 ° C. for 10 minutes and then centrifuged (10,000 × g, 5 minutes). The pellet obtained was washed with 1 ml of acetone and then dried, and the buffer solution [1% SDS, 50 mM Tris-
50 μl of HCl (pH 8), 10% 2-mercaptoethanol, 40% glycerol, 0.02% bromophenol blue] was added for solubilization. Further, this was electrophoresed using a 10% polyacrylamide gel, and the protein in the gel was electrophoretically blotted on a nitrocellulose filter in a buffer [25 mM Tris, 192 mM glycine, 20% methanol].

Next, the filter was put into a TBS buffer solution [50 mM T
ris-HCl (pH 7.5), 200 mM NaCl, and then 3% gelatin
4% at 37 ° C in TBS buffer containing 0.05% Tween 20
Incubate for 0 minutes and add 30 μg of anti-yeast reducing enzyme.
TBS buffer containing elementary antibody, 1% gelatin, 0.05% Tween20
Incubated in liquid at 37 ° C. for 2 hours. Then 0.
30 minutes at 37 ℃ in TBS buffer containing 05% Tween20
Incubate 4 times and then use 3% gelatin
20 minutes in TBS buffer containing 0.05% Tween20
Incubated. Then 10 μCi of [¹²⁵I] −Protein
A, in TBS buffer containing 1% gelatin and 0.05% Tween20
After incubating at 37 ℃ for 60 minutes, 0.05% Tween20
Incubate for 30 minutes at 37 ° C in TBS buffer containing
This operation was repeated 4 times. After air-drying the filter,
Autoradiography was performed. AH22 / pAAH5 strain
For samples containing 50 ng or 100 ng of yeast reductase
Concentration of yeast reductase band in AH22 / pAFCR
From the comparison of the blackening concentration of the fusion enzyme band in one strain, A
The expression level of the fusion enzyme in the H22 / pAFCR1 strain was about 2 x 10 ^Five
 Presumed to be molecules / cells. This is a rat P450c and
Amount of fusion enzyme expressed in a strain expressing the fusion enzyme
Equivalent to about 3 times.

Example 4 Quantification of the amount of fusion enzyme containing heme AH cultured in SD synthetic medium to about 2 × 10 ⁷ cells / ml
100 ml of 22 / pAFCR1 strain cells were collected and suspended in 2 ml of 100 mM potassium phosphate (pH 7.0). Dispense 1 ml of the bacterial suspension into each of two cuvettes, blow carbon monoxide into the sample-side cuvette, and then dithionite 5-10 in both cuvettes.
mg was added. After stirring well, the difference spectrum at 400-500 nm was measured, and Δε = from the difference in absorbance between 447 nm and 490 nm.
The amount of heme-containing fusion enzyme was calculated based on the value of 91 mM ^-1 cm ^-1 . As a result, it was found that the AH22 / pAFCR1 strain produces about 2 × 10 ⁵ molecules of heme-containing fusion enzyme per cell.

Example 5 Measurement of Acetaminophen Production by Transformed Yeast Strain by Acetanilide p Hydroxylation Transformed yeast AH22 / pAFCR1 and AH22 / pAMP19 strains cultured in SD synthesis medium to about 2 × 10 ⁷ cells / ml 1.5M acetanilide (methanol solution) was added to each of the culture broths to give a final concentration of 25 mM. Then, while shaking culture was continued, a fixed amount of the culture solution was taken every hour, and the culture solution supernatant obtained by removing the cells by centrifugation was subjected to HPLC (high performance liquid chromatography) to quantify the acetaminophen produced. did. HPLC was carried out using a μ Bondapak C18 column, eluting with methanol: water: acetic acid = 15: 84: 1, and was detected by absorbance at 245 nm. As a result, the activity per cell of the fusion enzyme producing strain AH22 / pAFCR1 containing yeast reductase was found to be higher than that of the fusion enzyme producing strain AH22 containing rat reductase.
It was found that the activity was about 15 times that of / pAMP19 and that the activity per fusion enzyme molecule was about 5 times.

[Sequence list]

SEQ ID NO: 1 Sequence length: 12 Sequence type: Nucleic acid Number of chains: double-stranded Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array CAGCTCGAGCTG GTCGAGCTCGAC

[Brief description of drawings]

FIG. 1 is a diagram showing a method for constructing plasmito pAFCR1.

[Explanation of symbols]

Cleavage site by H restriction enzyme Hind III Cleavage site by X restriction enzyme Xhol

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI C12R 1: 865) (C12P 13/00 C12R 1: 865) (72) Inventor Hideo Okawa 14-3- Asahigaoka-cho, Ashiya-shi, Hyogo 403 (56) Reference JP-A-63-44888 (JP, A) JP-A-2-451 (JP, A) DNA, 1987, Vol. 6, No. 3, p. 189-197 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 C12P 13/00 BIOSIS (DIALOG) BIOTECHNOLOGY ABST RACT (DIALOG) JISST file (JOIS) WPI (DIALOG) SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / Gene Seq

Claims (9)

(57) [Claims] 1. A DN consisting microsomal type protein microsomal membrane coupled to encoding regions involved sequences and regions and heme binds to encoding regions involved sequences involved in base quality binding sites of rat liver cytochrome P450c
Consists DNA encoding regions involved in area and NADPH binding involved in off La <br/> bottles mononucleotide or flavin adenine dinucleotide-binding yeast reductase gene linked to the 3 'end is A, rat liver cytochrome P4
One-atom oxygenation activity of 50c and yeast NADPH-
A fusion gene encoding a fusion oxidase having the ability to supply reducing power of cytochrome P450 reductase 2. A yeast expression plasmid containing the gene according to claim 1 and expressing the oxidase. 3. A yeast expression plasmid pAFCR1 4. A transformed yeast strain carrying the yeast expression plasmid according to claim 2 or 3. 5. A yeast strain according to claim 4, wherein belonging to the Saccharomyces genus 6. A Saccharomyces cerevisiae AH22 strain carrying the yeast expression plasmid pAFCR1. 7. A region involved in microsomal membrane binding of a microsomal protein and rat liver cytochrome P450.
The c gene has at least a region involved in substrate binding and a region involved in heme binding on the N-terminal side, and the C-terminal side is at least a region involved in flavin mononucleotide or flavin adenine dinucleotide binding of the yeast reductase gene and NADPH. A fusion oxidase consisting of a region involved in binding and having the one-atom oxygenation activity of rat liver cytochrome P450c and the ability to supply reducing power of yeast NADPH-cytochrome P450 reductase 8. A method for producing an oxidase, which comprises culturing the transformed yeast strain according to claim 4, 5, or 6. 9. The method of acetaminophen transformed yeast strains or yeast according to claim 4, 5 or 6, wherein characterized in that the hydroxylating p-position of the acetanilide with recombinant fusion oxidase produced