JPH02451A - Nadph-cytochrome p450 yeast-reducing enzyme gene - Google Patents

Abstract

PURPOSE:To enable production of a yeast-reducing enzyme by cloning a gene of NADPH-cytochrome P450 reducing enzyme from a yeast. CONSTITUTION:A gene composed of a specific amino acid sequence and coding NADPH-cytochrome P450 yeast-reducing enzyme is prepared. As a cell giving a source of gene coding the above-mentioned reducing enzyme, a yeast cell belonging to saccharomyces cerevisiae is preferably used. A cell capable of producing NADPH-cytochrome P450 yeast-reducing gene can be produced by integrating the acquired NADPH-cytochrome P450 yeast-reducing enzyme gene into a proper vector and introducing the vector into a cell of microorganism such as Escherichia coli or yeast or cell of animal. Especially, when a yeast cell is used, NADPH-cytochrome P450 yeast-reducing enzyme can be efficiently produced.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a gene encoding yeast NADP)I-cytochrome P450 reductase.

[Prior art and problems]

NADPH-cytochrome P450 reductase (NADPI
I; ferricytochrome oxidoreductase, EC
1, 6, 2, 4) are present in microsomes 11 of eukaryotic cells such as mammals and yeast, and are major enzymes constituting the electron transport system. This enzyme is a flavin enzyme containing one molecule of flavin adenine mononucleotide and one molecule of flavin mononucleotide in its molecule. This enzyme is NADPI
Electrons from I are transferred to cytochrome P450, the terminal enzyme of the microsomal electron transport chain. cytochrome l”4
50 is a group of enzymes consisting of many molecular species, each of which exhibits a wide range of substrate specificities, and because these substrate specificities overlap, they can exhibit oxidation reactions against a wide range of fat-soluble compounds. can.

Despite the molecular diversity of cytochrome P450, the same reductase is used to reduce NADP for all molecular species.
Electrons from H can be supplied. Electron transfer from NA[JpH to cytochrome P450 by this reductase occurs across biological species; for example, rat liver reductase transfers to rabbit liver cytochrome P450, and yeast reductase transfers to rabbit or rat liver cytochrome P450+IADPI.
can transfer electrons from

The present inventors have already isolated the runt liver cytochrome P450 gene and the runt liver NADPH-cytochrome P450 reductase gene (Japanese Patent Application Laid-Open No. 61-56072, Japanese Patent Application Publication No. 62-19085), and expressed these genes in yeast. We succeeded in producing an enzyme protein with oxidation activity. Rat liver cytochrome P expressed in yeast
450 and reductase were localized in yeast microsomal membranes and exerted oxidative activity dependent on rat liver cytochrome P450. Furthermore, the inventors produced a yeast strain that produces both cytochrome P450 and reductase, and demonstrated that this strain exhibits cytochrome P450-dependent oxidation activity and can be used as a bioreactor for oxidation reaction processes of various compounds ( JP-A-62-104582). In addition, the inventors succeeded in creating a new monooxygenase that can exert the functions of both cytochrome P450 and reductase enzymes in one molecule by linking the genes of both cytochrome P450 and reductase (Japanese Patent Application Laid-Open No. 61-187713 ).

In addition to the rat liver reductase gene, we also succeeded in obtaining the rabbit liver reductase gene (patent application 13443/1982).
.

In this way, reductase can be used as a bioreactor for oxidation reactions by using it together with cytochrome P450. However, when comparing the production of rat liver P450 and rat liver reductase in yeast, P
The production volume of 2S5 was higher.

One reason for this is thought to be that rat liver reductase has poor stability in yeast. Therefore, if a reductase from host yeast is used instead of rat reductase, yeast reductase is a protein of yeast itself, and it is thought that mass production will be possible.

Moreover, the supply of electrons from NADPH to rat P450 through the action of yeast reductase is extremely efficient.

Therefore, production of yeast reductase can be expected to improve the performance of bioreactors for oxidation reactions.

[Means of problem solving]

The present inventors have now succeeded in cloning the gene for NAI)P)I-cytochrome P450 reductase from yeast. This made it possible to produce yeast reductase.

That is, the first object of the present invention is to provide a gene encoding yeast reductase. For example, it is a gene containing the base sequence shown in FIG. 2, and furthermore, a gene containing the base sequence corresponding to the amino acid sequence shown in FIG.

As is well known, the yeast used in the examples of the present invention is Saccharomyces cerevisiae.

There are many other types of yeast (Bees cerevisiae), and the present invention also includes genes encoding yeast reductases derived from them.

Furthermore, according to genetic recombination technology, mutations can be made artificially in specific parts of the basic DNA without changing the basic characteristics of what is encoded by the DNA, or in such a way as to improve the characteristics. be able to. Similarly, the genes provided by the present invention can be artificially inserted, deleted, or replaced to have characteristics equivalent to or improved from those of natural genes. It contains a mutant gene.

Furthermore, for many amino acids, the DN that codes for them
There are multiple A base sequences. Therefore, the base sequence is not uniquely determined and may have many possibilities. In the case of the gene encoding the yeast reductase revealed by the present inventors, there are many possibilities for the DNA base sequence other than the base sequence of the natural gene, but the gene of the present invention It is not limited to only natural DNA base sequences, but includes other DNA base sequences that encode the amino acid sequences revealed by the present invention.

A second object of the present invention is to provide a recombinant DNA containing a base sequence encoding yeast reductase. Preferred DNA is DNA that is capable of self-replication within host microorganism cells (that is, includes a base sequence necessary for self-replication).
Particularly preferred is the one named pgGYR in the present invention.

The third object of the present invention is to obtain a DNA containing a base sequence encoding yeast reductase and capable of self-replication within a host microorganism cell.
The object of the present invention is to provide a transformed microorganism that retains the following. Particularly preferred microorganisms herein include E. lily Y1
090 (pgGYR).

The gene encoding yeast reductase does not have any intervening sequences except for some genes such as actin, so it can be expressed as is in host microorganism cells. By cloning the chromosomal DNA directly or from yeast cells by known methods (CARLSON, M. and BDTSTEI).
N, D, cell vol. 28, 145-154
p., 1982) and can be isolated by cloning the cDNA using an appropriate vector.

In the present invention, the cell serving as the source of the gene encoding the target reductase is a yeast cell, but preferably Saccharomyces cerevisiae.
refers to cells belonging to Saccharomyces cerevisiae, and more preferably Saccharomyces cerevisiae.
romyces cerevisiae) AlI22
strain (^TCC38626) etc. can be used.

As a method for cloning DNA, for example, chromosome D
In the case of NA, examples include a method in which a chromosomal DNA library is prepared using an appropriate vector such as Lambda Gill (Crunchyk) or Cosmindo Vector (Boehringer Mannheim) and then screened.

In the case of cDNA, for example, m prepared from yeast cells
Using RNA, reverse transcriptase and DNA polymerase ■ create two-terminal cDNA, and insert this into a vector such as pBR322 using a method such as the homopolymer method, or use the Okayama-Barb cloning vector (from Pharmacia). It is possible to use a method such as inserting the cDNA into another suitable vector such as a cDNA library (which can be done manually), creating a cDNA library using this, and selecting the desired cDNA clone from this library.

Regarding DNA selection, there are immunochemical methods using antibodies against yeast reductase, screening methods using positive hybridization translation assay, and synthetic DNA corresponding to the partial amino acid sequence of the protein.
Various methods can be used, such as a screening method by hybridization using a portion of DNA synthesized from the base sequence shown in FIG. 2 as a probe.

As the probe, in addition to the above, in the case of chromosomal DNA, cDNA obtained by the above-mentioned method can be used, and in the case of cDNA, chromosomal DNA obtained by the above-mentioned method can be used.

In addition, if only a portion of the yeast reductase DNA is obtained by the above method, this can be used as a probe to obtain the complete DNA.
NA can be obtained.

Yeast NADPH-Cytochrome P thus obtained
Cells that produce yeast NADPI+-cytochrome P450 reductase can be produced by incorporating the 450 reductase gene into a suitable vector and introducing it into microorganism cells such as Escherichia coli and yeast, or animal cells. In particular, when yeast cells are used, yeast NADPH-cytochrome P450 reductase can be efficiently produced as described above. Preferably, the expression vector contains genetic information that can be replicated in host cells, can propagate autonomously, is easy to isolate and purify from the host, and has a detectable marker. Various vectors are commercially available,
Restriction enzymes and the like used for cutting vectors are also commercially available.

Methods for using these are known. For example, for expression in Escherichia coli, expression vectors containing bromocous such as lac, tac, and ρ (these are commercially available as expression vectors or as promoter cartridges from Fa JL/7Sia PL) can be used.

For expression in yeast, an expression vector containing an alcohol dehydrogenase promoter, a phosphoglycerokinase promoter, a gluceroaldehyde 3-phosphate dehydrogenase promoter, etc. can be used. The cells that produce the produced yeast reductase can be used in the production of compounds useful as a bioreactor by combining them with cells that produce cytochrome P450.

Examples are given below to explain the present invention in more detail. The present invention is not limited only to the following examples,
Modifications common to the art of the invention may be made.

Example 1 Yeast - Isolation of enzyme cDNA clones (1) Preparation of yeast mRNA Saccharomyces cerevisiae
escerevisiae) At122 strain (ATCC
38626) in 100+d YPD medium (1% yeast extract, 2% polypeptone, 2% glucose) for 30
After shaking culture at ℃ for 18 hours, the bacterial cells were collected. After washing twice with water, the wet weight of the bacterial cells was measured, and 2Ir1 of disruption buffer (0.2M Tris-
Hydrochloric acid, pH 7, 5, 10+mM EDTA, 0.5M
NaCl), 2 g of glass beads, and 0.1% diethylokingate were added, and the mixture was placed in a portex mixer three times for 1 minute each. Next, extraction was performed twice with an equal volume of phenol/chloroform (1:lν/V) and once with an equal volume of chloroform, and then 2.5 times the volume of ethanol was added to the aqueous layer to precipitate the nucleic acids. Ta.

The obtained nucleic acid was passed through an oligo(dT) cellulose column to fractionate mRNA containing poly(A). Nucleic acids recovered by ethanol precipitation were added to equilibration buffer (lomM
Tris-HCl, pH 7.5, 1d EDTA.

30 A at 0.5M NaCl, 0.1% SO5)
It was dissolved so that zbo /Ial. This is 65
After heating at °C for 5 minutes, the column was applied to an oligo(dT) cellulose column equilibrated with an equilibration buffer and thoroughly washed with the equilibration buffer. After washing, elution was performed with sterile water, and the eluted poly(A) RNA fraction was subjected to ethanol precipitation by adding a 2% potassium acetate aqueous solution and 2 volumes of cold ethanol once, and the RNA was recovered. .

The RNA was washed with 90% alcohol and then dissolved in sterile water.

(2) Preparation of cDNA library cDNA synthesis from the prepared poly(A) RNA was performed using the cDNA synthesis system (commercially available from Amerjam) (
The test was carried out using the product code NO, llpH, 1256) according to the protocol attached to the product. That is,
After synthesizing cDNA complementary to mRNA using reverse transcriptase, inserting a second link and a gear knob into RNAjX using ribonuclease H, and repairing this using E. coli DNA polymerase I, the RNA strand is replaced with a DNA strand. did.

Finally, both ends of the DNA strand were equilibrated with T and DNA polymerase. A cDNA library was prepared using the ProtoClone Lambda gtll system commercially available from Promega Biotic. All methods followed the protocols attached to the products.

First, the Eco old site present in the double cDNA is converted into E
Methylated by coRI methylase, the subsequent εcoR
After protection from I digestion, EcoRI linkers were added to both ends. After cutting with EcoRI, the cDNA was ligated with the EcoRI arm of lambda gill, in vitro packaging was performed, and a cDNA library was prepared by infecting Escherichia coli strain Y1090 (attached to the product).

(3) Selection and production of yeast reductase cDNA clones
In order to select yeast reductase cDNA clones from the DNA library, immunochemical screening was performed using an antibody against yeast reductase. The anti-reductase rabbit antibody was prepared using purified yeast reductase (Aoyama,
Y et al., Arch, Biochea, Biophy
s, Vol. 185, No. 2, pp. 362-369,” 978
E. coli strain Y1090 (Biotic) was infected with the cDNA library, which can be easily prepared by administering the cDNA library (which can be purified by the method described in I spread it out to do so. onto the plate in advance] OmMIP
After soaking in TG (isopropyl β-D-thiogalactopyranoside), air-dried nitrocellulose filters were layered and incubated at 37°C for 3 hours and at 4″C for 1 hour. Peeled from the plate. filter,
Skim milk solution 5011M containing 0.5% skim milk (purchased from Difco) Lis-HCl, pl+8.
1505M NaCl) and incubated for 1 hour at room temperature. Next, 100 μ2 of the anti-reductase rabbit antibody was added to 50 μ2 of the neutralizing antibody (E. coli Y1090 strain cells were suspended in 100 μ2 of water and heat-treated at 95°C for 15 minutes, and incubated overnight at 4°C. skim milk solution 2 containing
0d and incubated overnight at room temperature.

The filter was washed three times for 10 minutes with 25 ml of T-skim milk solution (the above skim milk solution contains 0.05% Tween 20), and then washed with alkaline phosphatase-conjugated anti-heron IgG goat antibody (Promega Biotic Co., Ltd.). (purchased from) skim milk containing 10 μl? f7.20d and incubated for 1 hour at 37"C. Then 50mM Tris-HCl (pH 8)
After washing three times with 150 mM NaCl, 132 μl of nitro blue tetrazolium (dissolved in 70% diethylformamide to give 50 mg/-), 5011 g of 5-bromo-4-chloro-3-indolyl phosphate (dissolved in diethylformamide to give 50 mg/-) /mff1)6
11il/I! ALP slow-moving solution (100 Il Gatolisu hydrochloric acid (pH 9,5), 1100 II NaCl,
The color reaction was performed by immersing in 20 mM 5mM MgC1z) and incubating for 1 hour at room temperature. Finally, the filter was washed 2.3 times with water and then air-dried.

A total of 15,000 plaques were subjected to the above immunochemical screening, and two positive phage clones were finally obtained.

As described below, the two positive phage clones contained overlapping cDNA inserts and differed only in cDNA chain length.

The clone containing the longer cDNA insert of the two was named pgCYR.

Example 2 Fermentation reductase genomic DNA clone Raw nail bile As described below, the c DN obtained in Example 1-(3)
The A clone was not long enough to encode the full length of the yeast reductase protein.

Therefore, a yeast genome library was screened by plaque hybridization using this cDNA as a probe.

The yeast genome library was a yeast genome library commercially available from Krontschik (the DNA source was the yeast Saccharomyces cerevisiae strain X2180 (A T
CC26109), vector λgtll, product code number cyt, 101) was used. In this library, yeast genomic DNA was partially digested with the restriction enzyme EcoRI, and a DNA fragment corresponding to 1.5 to 6.0 kb was inserted into the EcoRr site of the vector λgtll.

The library was diluted and spread on plates, using the Quitar attached to the product as a guide, to give approximately 104 plaques per plate. DNA is removed by placing a nitrocellulose filter on the plate and leaving it for 1 minute.
was transferred to the filter. The filter is 01.5M N
aCl, 0.5!1 #aOH1■1.5M NaC
1,0,5M) Lis-hydrochloric acid (pH 8), ■3xss
The DNA was fixed on the filter by vacuum drying at 80°C.

Next, the filter ■3XSSC168°C11,5
time, ■3xSSC110xDenhardt (0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2% false serum albumin), 68"C51,5 hours, ■Hybridization buffer (5kM) Lis-HCl, pH 7,
5,loIIIM EDTA,,IM NaC1,1
0x Denhart, ttg/rrdl salmon testis DNA),
Treatments were performed at 68°C for 11 hours. After that, according to the instructions attached to the product, yeast reductase cDNA was converted to IEco by nick translation method using a nick translation kit (purchased from Amerjam).
The old RI-Bam fragment (the fragment with base numbers 474-1015 in the base sequence in Figure 2) was labeled with 32p,
The hybridization buffer containing this and the above-treated filter were packed in a plastic bag and kept at 6°C.
and incubated overnight. The filter that underwent hybridization was: ■2XSSC1 IO minutes at room temperature, ■1
X5SC10.1% SDS, 60°C 25 minutes, ■0°3
x ssc, 0.1% SO5, 60°C 125 minutes, ■0
．． After processing in the order of 1% 5SC10, 1% SO5, 60°C for 45 minutes, air drying and X-ray film (Kodank XAR
-5) and took an autoradiogram. In this way, one genomic DNA clone giving a positive signal was obtained and named pgGYR.

Yeast reductase c DN obtained in Example 1-(3) and 2
A, Restriction enzyme maps of insert DNA were prepared for each genomic DNA clone.

Prepare phage DNA and cut it with EcoRI to cut out the insert DNA and make pUC19.
was subcloned into the EcoR1 site of. However, in the case of genomic DNA clones, Ec on one side.

Since the R1 site was deleted, the DNA fragment was cut out by simultaneous digestion with EcoRI and 5acl, and pU
Subcloned between the EcoRI and Sac 1 sites of C19. Colon i1JM introduced with these plasmids
109 strains (Takara Shuzo Co., Ltd.) were cultured and cultured using Birnboim-Doly's method.
Plasmid DNA was prepared. Plasmid DNA is cut with various restriction enzymes, and the cut DNA fragments are reduced to 0.68%.
By comparing the chain lengths of DNA fragments cut with various restriction enzymes analyzed by agarose gel electrophoresis,
A restriction enzyme map shown in FIG. 1 was obtained.

The restriction maps of the two cDNA clones were negative, and they completely overlapped with the restriction maps of the genomic DNA clone.

The entire base sequence of the cDNA insert was determined using the pUc plasmid's forward and reverse primer DNA (purchased from 7 Almacia) and the 7-DEAZA Sequencing Kit (
(purchased from Takara Shuzo).

The nucleotide sequence around the EcoR1 site inside the insert was determined in the same manner after subcloning fragments cleaved with other restriction enzymes into puc19.

The cDNA clone pgCYR is located at base number 67 in Figure 2.
It was coded from the second to the last poly(A) till. This base sequence starts with tyrosine residue 669
Can code for amino acid residues, base numbers 2074-207
There was an open reading frame that ended at the 6th stop codon TAA. Two other reading frames in which the bases are shifted by one base are also possible as reading frames for proteins, but
In these cases, there are many stop codons in the middle of the base sequence, and it cannot be considered as the reading frame of a reductase.The result of converting the base sequence corresponding to the longest reading frame into amino acids is shown in Figure 2. This amino acid sequence shown in Fig. 1 is used for rat and rabbit NADPI+-
Sequence of cytochrome P450 reductase (DNA, Vol. 5, No. 1, pp. 1-1o, 1986 J, Biochem 100
5945-954 (described in 1986), a very well-positioned sequence was found. For example,
A sequence thought to be a region that binds FMN, a coenzyme of reductase (in rat and rabbit enzymes, NIIVFY
GSQTGTAEE), the sequence that was well guessed is base number 1.
It was seen from 78th to 222nd. In addition, a sequence containing a glycine residue characteristic of flavin enzymes (in rats, V
IFIVGPGTGIAPFMGFIQER) The corresponding sequence is base number 1606606, 665665.
It was the second time. Judging from these results, it has become clear that the amino acid sequence shown in Figure 2 is that of yeast reductase.

Next, the nucleotide sequence of the genomic DNA insert was determined by the same method. The determined base sequence and the result of converting it into an amino acid sequence are shown in Figure 2, Genome D.
The base sequence (base numbers 67-2076076) of the NA insert completely matched the base sequence of the cDNA insert.
The amino acid sequence of 50 reductase was found to be 22 amino acid residues longer than the sequence determined from cDNA. Therefore, yeast reductase was found to be a protein with a molecular weight of 76,756 and consisting of 691 amino acid residues.

[Brief explanation of the drawing]

Figure 1 shows yeast NADP I+-cytochrome P4
c DNA clone pgCY encoding 50 reductase
In Figure 0 showing the restriction enzyme map of R and the genomic DNA clone pgGYR, the blacked area indicates the DNA region encoding this enzyme. FIG. 2 shows the base sequences of insert DNA of cDNA clones and genomic DNA clones, and the amino acid sequence of yeast reductase deduced from the base sequences. The coding region of yeast reductase consists of base numbers 1 to 2.
It corresponds to number 073. However, A, C, D, E, F, G,)1. I, to,
L, M, N, P, Q, R, S, TV, w, y are alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, Represents arginine, serine, threonine, valine, tryptophan, and 1,000 rosine residues. The probe used for screening genomic DNA clones corresponds to base numbers 474 to 1015.

Claims (1)

[Scope of Claims] (1) Gene encoding yeast NADPH-cytochrome P450 reductase (2) Gene according to Claim 1 specified by the amino acid sequence shown in Figure 2 (3) Second Gene according to claim 1 specified by the base sequence shown in the figure (4) Recombinant DNA containing the base sequence encoding yeast NADPH-cytochrome P450 reductase (5) Self-propagation in microbial cells D as described in possible claim 4
NA (6) A microorganism that contains a base sequence encoding yeast NADPH-cytochrome P450 reductase and retains DNA capable of self-replication within microorganism cells. (7) The microorganism is Escherichia coli. microorganisms