JPH09264A - Genes that regulate nitrile hydratase gene expression - Google Patents

Abstract

(57) [Summary] [Structure] Gene D encoding a polypeptide substantially containing the amino acid sequence represented by SEQ ID NO: 1 and having a function of regulating a nitrile hydratase gene promoter.
Recombinant DN incorporating NA, the DNA and a nitrile hydratase gene containing a promoter region into a vector
A transformant obtained by transforming A and the recombinant DNA. The present invention provides a gene encoding a polypeptide having a function of regulating the nitrile hydratase gene promoter.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a gene DN, which is derived from a bacterium of the genus Rhodococcus and which encodes a polypeptide having a function of regulating the nitrile hydratase gene promoter.
A, a recombinant DNA containing this DNA, and a transformant transformed with the recombinant DNA.

[0002]

2. Description of the Related Art Microorganisms belonging to the genus Rhodococcus are known as industrially useful microbial catalysts because of their strong physical strength and ability to accumulate a large amount of enzymes in cells. It is used for production of amides or acids by hydration or hydrolysis (JP-A-2-470, JP-A-3-251192). Attempts have been made to improve the microorganisms belonging to the genus Rhodococcus into more useful ones by a method of gene recombination, for example, as a host-
Development of vector systems is underway (Japanese Patent Laid-Open No. 5-6458).
No. 9). On the other hand, there is still little information on the expression system of genes of microorganisms belonging to the genus Rhodococcus, and only the regulatory gene and promoter of the H-type nitrile hydratase of Rhodococcus rhodochrous J1 strain have been clarified ( Journal of Japan Society for Biotechnology).

When expressing heterologous genes in recombinants, the polypeptide produced can often be harmful to the host bacterium. Therefore, if the polypeptide is produced from the early stage of growth, the growth of the host bacterium may be inhibited, resulting in a decrease in productivity. Therefore, in a gene expression system of a microorganism belonging to the genus Rhodococcus, a negative control is performed in the early stage of growth of the host bacterium, and the negative control can be released by the addition of an inducer after the middle stage of growth. Was strongly desired.

[0004]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a gene that regulates nitrile hydratase gene expression and a recombinant in which the gene is incorporated.

[0005]

[Means for Solving the Problems] The inventors of the present invention conducted extensive research based on the above problems and searched for a gene that regulates the expression of the L-type nitrile hydratase gene of Rhodococcus rhodochrous J1 strain. Its presence was found upstream of the gene, and it was confirmed that the gene was a negative regulatory gene, leading to the completion of the present invention. That is, the present invention is a gene DNA encoding a polypeptide substantially containing the amino acid sequence represented by SEQ ID NO: 1 and having a function of regulating the nitrile hydratase gene promoter. Examples of such a gene include those having the base sequence represented by SEQ ID NO: 2.

Here, the expression "substantially contains an amino acid sequence" means that deletion, substitution, addition or the like of some of the amino acids is possible as long as this peptide has a function of regulating the nitrile hydratase gene promoter. It indicates that it is acceptable. Therefore, in the amino acid sequence represented by SEQ ID NO: 1, those in which the first amino acid, methionine (Met), is deleted are also included in the polypeptide due to this amino acid sequence change. Further, the DNA of the present invention which encodes a polypeptide includes not only the DNA having the nucleotide sequence represented by SEQ ID NO: 2 but also degenerate isomers encoding the same polypeptide which differ only in a degenerate codon. is there. Further, “regulating a promoter” means, for example, changing the expression level of a promoter depending on the presence or absence of an inducer.

Furthermore, the present invention is a recombinant DNA in which the gene DNA and a nitrile hydratase gene containing a promoter region are incorporated into a vector. further,
The present invention is a transformant obtained by transforming the recombinant DNA. Hereinafter, the present invention will be described in detail. The DNA of the present invention, a recombinant incorporating the DNA, and a transformant transformed with the recombinant are provided by the following steps.

(1) Preparation of chromosomal DNA: Rhodococcus
Chromosomal DNA is isolated and prepared from Rhodochrous J1 strain (hereinafter sometimes referred to as "J1 strain"). Chromosomal DNA
Any known method can be used for the separation and preparation of. For example, the method of Saito and Miura (Biochim.
Biophys. Acta, 72, 619 (1963)).

(2) Preparation of DNA library: J1 strain L
An appropriate restriction enzyme is allowed to act on a plasmid having the type nitrile hydratase gene to cut out the gene portion derived from the J1 strain, which is labeled with a radioisotope to be used as a probe. After cutting the chromosomal DNA prepared in step (1) with a restriction enzyme, Southern hybridization (Southern E.
M., J. Mol. Biol. 98, 503 (1975)), the DNA fragment fraction containing the target gene is detected using the above probe. This fraction is used as a plasmid vector, eg pUC1.
8. Insert into pUC19 etc. to make a DNA library.

(3) Preparation of transformant and recombinant DN
Selection of A: A transformant is prepared from the recombinant DNA library prepared in the step (2), and the transformant is selected from the transformants.
Colony hybridization using the probe prepared in (2) (R. Bruce Wallace. Et al. Nuc. Acids
Res. 9, 879 (1981)) selects a transformant containing the target recombinant DNA. Furthermore, the target recombinant DNA is reconfirmed by the Southern hybridization method.

(4) Purification of recombinant plasmid and construction of restriction enzyme map: A plasmid is prepared from the recombinant obtained in step (3). Preparation is by the method of Birnboim and Doly (Nu
cleic Acid Res., 7, 1513-1523 (1979)). The plasmid thus obtained is called pNLU10. The digested product with various restriction enzymes is analyzed by electrophoresis to prepare a restriction enzyme map.

(5) Construction of a recombinant plasmid comprising a region containing a regulatory gene and a nitrile hydratase gene and a vector plasmid replicable in Rhodococcus: plasmid pNLU10 obtained in step (4), J1 strain L-type nitrile Plasmid pNHJ containing the hydratase gene
From 20L and the composite plasmid vector pK4, a recombinant plasmid pLJK10 consisting of a region containing a regulatory gene and a nitrile hydratase gene and a vector plasmid replicable in Rhodococcus is prepared.

(6) Transformation of Rhodococcus and Production of Nitrile Hydratase Using the Transformant: Process
Rhodococcus bacteria are transformed with the plasmid pLJK10 obtained in (5), and it is confirmed that nitrile hydratase derived from J1 strain is expressed. Transformation and confirmation of gene expression can be performed by known methods. For example, the transformation is performed by the electroporation method, the protoplast method, or the like. For confirmation of gene expression, nitrile hydratase activity is measured. For example, nitrile hydratase may be allowed to act with benzonitrile as a substrate, and the resulting benzamide may be quantified by an appropriate chromatography method (eg, HPLC).

(7) Deletion plasmid and nitrile hydratase activity: Plasmids pLJK20 to pLJK60 were prepared by removing various regions from the plasmid pLJK10 obtained in step (5), and which region was used to control the expression of nitrile hydratase. Find out if you are involved. That is, regarding the recombinant having a plasmid excluding various regions, using benzonitrile as a substrate in the same manner as in step (6) and quantifying the benzamide produced, the region involved in the regulation of nitrile hydratase expression was determined. Identify.

(8) Determination of base sequence: The base sequence of the region specified in step (7) is determined. The base sequence can be determined by a known method, for example, the dideoxy method using a radiolabel or a fluorescent label, the Maxam-Gilbert method and the like. In addition, Rhodococcus rhodochrous J1
The strain has been deposited as Rhodococcus rhodochrous J-1 (FERM BP-1478) at the Institute of Biotechnology, Institute of Biotechnology, AIST. In addition, the plasmid pNHJ20L containing the nitrile hydratase gene is a transformant TG1 / pNHJ20L containing this plasmid.
(FERM BP-2778) as a composite plasmid vector pK4
Is a transformant containing this R. rhodochrous ATCC 1267
As a 4 / pK4 (FERM BP-3731) plasmid, the plasmid pNLU10 contained the transformant E. coli JM109 / pNLU10 (FERM P
-14993) have been deposited with the Institute of Biotechnology, Institute of Industrial Science and Technology.

[0016]

EXAMPLES The present invention will be described in more detail below with reference to examples. However, the present invention is not limited by these examples. In this example, the following abbreviations were used for buffer solutions and media. TE: 10 mM Tris-HCl (pH7.8) -1 mM EDTA (pH8.0) TNE: 50 mM Tris-HCl (pH8.0) -1 mM EDTA (pH8.0)-
50 mM NaCl STE: 50 mM Tris-HCl (pH8.0) -1 mM EDTA (pH8.0)-
35 mM sucrose 2xYT medium: 1.6% tryptone, 1% yeast extract, 0.1%.
5% NaCl MY medium: 1% polypeptone, 0.3% yeast extract, 0.3
% Malt extract, 1% glucose

[Example 1] (1) Preparation of chromosomal DNA Rhodococcus rhodochrous J1 strain was treated with 100 mL of a medium (glucose, KH ₂ PO ₄ , K ₂ HPO ₄ , MgSO ₄ / 7H ₂ O, yeast extract, peptone, CoCl 2). ₂ , after culturing with urea, water 1L, pH7.2),
The bacteria were collected. After washing with TEN, suspend in 10 mL TE and add 0.25
Add 4 mL of M EDTA, 10 to 20 mg of lysozyme, 10 to 20 mg of achromoprotease, and 10 mL of 10% SDS, and add 3 mL at 37 ° C.
After leaving for a while, it was centrifuged to obtain a supernatant. Add 0.7 mL of 2.5 M sodium acetate solution and diethyl ether, centrifuge, discard the upper layer, add 2 times dissolved ethanol to the lower layer, and add DNA with a glass rod.
Wound up. This is TE: ethanol (volume ratio) 2:
After soaking in each of the 1: 1, 9 and 0:10 solutions for 5 minutes, 2
It was dissolved in ˜4 mL of TE, 10 μL of a mixture of RNase A and T1 was added, and the mixture was treated at 37 ° C. Next, an equal amount of phenol saturated with TE was added, and an equal amount or more of ether was added to the upper layer after centrifugation to obtain a lower layer. A small amount of chloroform was added to this. 2
After dialysis against L TE overnight, the second dialysis was performed for 3 to 4 hours to obtain 4 ml of chromosomal DNA preparation.

(2) Preparation of DNA library 9.4 kb D containing J1 strain L-type nitrile hydratase gene
Plasmid pNH in which the NA fragment was integrated into pUC19 vector
J20L (Japanese Patent Laid-Open No. 4-111379, Biochim. Biophys. Acta
1129, 23-33 (1991)) was cleaved with restriction enzymes SacI and EcoRI, and a 1 kb SacI-EcoRI fragment was separated by 1% agarose gel electrophoresis and cut out from the gel and recovered. Cleavage with a restriction enzyme was performed as follows. For pNHJ20L 10 μL, 3 μL of restriction enzyme buffer (10 times concentration), restriction enzyme S
Add 2 μL each of acI and EcoRI, 13 μL of sterilized water,
The reaction was carried out at 37 ° C for 2 hours.

An agarose gel electrophoresis was carried out on a sample prepared by cutting the J1 strain chromosomal DNA with EcoRI. the above
The 1.35 kb SacI-EcoRI fragment was labeled with the radioactive isotope ³² P, and using this as a probe, a DNA fragment of about 7.5 kb including the upstream region was detected by the Southern hybridization method. A DNA fragment fraction containing a 7.5 kb fragment hybridized with the probe was cut out from an agarose gel,
It was inserted into the plasmid vector pUC18 that had been cut with EcoRI.

Cleavage of pUC18 was performed as follows. pUC18
For 10 μL, 3 μL of restriction enzyme buffer (10 times concentration),
Add 2 μL of each restriction enzyme EcoRI and 13 μL of sterilized water, and add 3
The reaction was performed at 0 ° C. for 2 hours. An equal amount of TE-saturated phenol was added to the reaction solution, and the mixture was stirred and then centrifuged to separate the upper layer (aqueous layer) and the lower layer. After this operation was repeated twice for the upper layer, the same amount of chloroform was added, and the same extraction operation was repeated twice. To the upper layer, 3 μL of 3M sodium acetate and 90 μL of ethanol were added, and the mixture was allowed to stand at −80 ° C. for 30 minutes, centrifuged, dried and dissolved in TE. 3 μL of the DNA fragment fraction containing the 7.5 kb EcoRI fragment was prepared as described above and EcoRI-cut pU
C18 1μL and TAKARA Ligation Kit at 4 ℃
Insert into pUC18 by reacting overnight at
This was the NA library.

(3) Preparation of transformant and recombinant DN
Selection of A After culturing the E. coli JM109 strain in 10 mL of 2xYT medium at 37 ° C for 12 hours, 1% was inoculated into the same medium and cultured for 2 hours. After collecting the cells by centrifugation, add 5 mL of cold 50 mM CaCl ₂ solution,
After leaving it at 0 ℃ for 40 minutes, centrifuge again, and cool 50 mM CaCl ₂ solution.
Suspended in 0.25 mL. Add a solution (DNA library) containing the recombinant plasmid prepared in step (2) to this.
Add 60 μL, leave at 0 ° C for 40 minutes, heat shock at 42 ° C for 2 minutes, add 1mL of 2xYT medium, and add 60mL at 37 ° C.
The cells were cultured with shaking for minutes. Add 100 μL of this to ampicillin 50
The cells were seeded on 2xYT agar medium containing µg / mL and cultured at 37 ° C.
The transformant colonies grown on the agar medium were subjected to colony hybridization to select transformants containing the upstream region of the nitrile hydratase gene. That is, the colonies grown on the agar medium were transferred to a nitrocellulose filter, the cells were dissolved to fix the DNA, and this was treated with the probe (1.35 kb fragment) prepared in step (2) and autoradioactive. A colony containing the target recombinant DNA was selected by the graph method. Furthermore, recombinant plasmids were extracted from the selected colonies and hybridized with the above-mentioned probe by Southern hybridization, and it was reconfirmed that the selected colonies were transformants containing the target gene.

(4) Purification of recombinant plasmid and construction of restriction enzyme map The transformant selected in step (3) was cultured in 100 mL of 2xYT medium at 37 ° C overnight, and the cells were collected and washed with TNE. Then S
TE (8 mL) and lysozyme (10 mg) were added, and the mixture was left at 0 ° C. for 5 minutes. Add 4 mL of 0.25 M EDTA and add 10% SD at room temperature.
S2 ml and 5 M NaCl 5 ml were added, and the mixture was allowed to stand at 0 to 4 ° C for 3 hours. This is ultracentrifuged and 1% of 30% PEG6000 is added to the supernatant.
Two equivalents were added, the mixture was left overnight at 0 to 4 ° C., centrifuged, and dissolved in TNE to make 7.5 mL. After adding CsCl, the protein was removed by centrifugation, 300-500 mg / mL of ethidium bromide solution was added, and the mixture was transferred to a seal tube, heat-sealed, and ultracentrifuged. c
The cDNA was recovered, water-saturated isopropyl alcohol was added in an equivalent amount or more to remove ethidium bromide, and dialyzed with TE.
About 3 mL of purified recombinant plasmid was obtained. This is pNLU
I named it 10. This recombinant plasmid was cleaved with several kinds of restriction enzymes to prepare the restriction enzyme map shown in FIG.

(5) Construction of Recombinant Plasmid Composed of Region Containing Regulatory Gene and NHase Gene and Vector Plasmid Replicable in Rhodococcus The plasmid pNLU10 obtained in step (4) is pNHJ20L containing nitrile hydratase gene. 1.35 kb overlaps with the DNA fragment derived from the J1 strain contained in, and further includes up to 6.15 kb upstream. 6.15 kb EcoRI-SacI from pNLU10
The fragment was cut out and inserted into the plasmid pBluescript SK + to prepare the plasmid pNLU20. Next, 9.4 from pNHJ20L
The kb SacI fragment was cut out and inserted into pNLU20 in the correct orientation to prepare plasmid pNLU30. Further pNLU
A 14.55 kb HindIII fragment was excised from 30 and inserted into a composite plasmid vector pK4 for Escherichia coli-Rhodococcus bacteria to prepare a plasmid pLJK10. The production process of pLJK10 is shown in FIG.

(6) Transformation of bacteria of the genus Rhodococcus and production of nitrile hydratase using the transformant Cells of the logarithmic growth phase of Rhodococcus rhodochrous ATCC 12674 strain were collected by centrifugation and sterilized with ice-cold sterile water. Three
Washed twice and 15% PEG6000 (polyethylene glycol 600
0) Suspended in the solution (cell concentration 10 ⁹ cells / mL or more). Plasmid DNA or vector DN prepared in step (5)
A 1 μg and a bacterial cell suspension 100 μL were mixed and cooled on ice. The mixture of DNA and bacterial cells was placed in a chamber for Gene Pulser, cooled with ice, and then subjected to electric pulse treatment with a capacitance of 25 μF, a resistance of 400 ohms, and an electric field strength of 20 kV / cm.

The electric pulse treatment liquid is allowed to stand for 10 minutes under ice cooling,
After heat treatment at 37 ℃ for 5 minutes, add 1 ml of MY medium and mix at 25 ℃ for 3 minutes.
Shake time. It was applied to MY agar medium containing 50 μg / ml kanamycin and cultured at 28 ° C. for 2 days. It was confirmed by applying to a separately prepared kanamycin-containing MY agar medium that the emerged colonies were clearly kanamycin resistant.

The thus-produced recombinant Rhodococcus bacterium was cultured in a medium containing crotonamide (2 g / L) as an inducer (glycerol 10 g, polypeptone 5 g, yeast extract 3 g, malt extract 3 g, KH ₂ PO ₄ 1 g, K _2). HPO ₄ 1g, Co
Cl ₂ · 6H ₂ O 0.01 g, (pH 7.0) / L) was cultured at 28 ° C. for 2 days, and nitrile hydratase activity was measured. The cells were collected from the culture solution by centrifugation, washed with 150 mM NaCl, and then 0.3
The cells were suspended in 0.1 M HEPES-KOH (pH 7.2) containing 5% n-butyric acid. The activity was measured as follows. To 0.25 mL of bacterial cell suspension diluted appropriately with water, 0.25 mL of 100 mM potassium phosphate buffer (pH 7.0) and 0.5 mL of 12 mM benzonitrile.
In addition, after reacting at 20 ° C. for 10 minutes, the reaction was stopped by adding 0.1 mL of 1N HCl. The benzamide produced was quantified by HPLC.

ATCC12674 / pLJK10 produced 0.016 benzamide when cultured in a medium containing no inducer.
While it was as low as mM, 1.1 mM benzamide was produced when crotonamide was added. ATCC1267
In 4 / pK4, benzamide formation was not observed in any case.

(7) Deletion plasmid and nitrile hydratase activity From the deletion plasmid pLJK10, deletion plasmids pLJK20-60 were prepared. As shown in FIG. 3, 8.3 kb from pNLU30 was used to construct plasmids pLJK20 to pLJK60.
KpnI fragment, 11.8 kb EcoRV-HindIII fragment, 5.45 kb P
The stI fragment and the 3.2 kb KpnI fragment were cut out and inserted into pK4. However, pLJK10, pLJK2
For 0 and pLJK30, the end of the cleaved fragment was
Ligation was performed after smoothing using the ing Kit. When the nitrile hydratase activity of the recombinants having these deletion plasmids was examined, the results shown in Table 1 were obtained.

[0029]

[Table 1]

From the results shown in Table 1, in the recombinants having the deletion plasmid containing the gene of the present invention (for example, pLJK20 and pLJK30), the addition of crotonamide increased the nitrile hydratase activity and increased the amount of benzamide produced. I found out that On the other hand, it was found that a recombinant having a deletion plasmid not containing the gene of the present invention (for example, pLJK50 and pLJK60) exhibits high nitrile hydratase activity regardless of addition or non-addition of crotonamide. . Therefore, it was revealed that the negative regulatory gene of the present invention exists upstream of the nitrile hydratase gene.

(8) Determination of base sequence As a result of determining the DNA base sequence of the region shown in FIG. 3 (region shown in the bottom column of FIG. 3), the base sequence shown in SEQ ID NO: 3 was obtained. The unique long open reading frame (presented at the 118th to 1191st positions in the nucleotide sequence shown in SEQ ID NO: 3) having the amino acid sequence shown in NO: 1 was found. The nucleotide sequence can be determined by Tth DNA polyme
Chain termination method using rase (Sanger F.
Science 214, 1205-1210 (1980)). The base sequence of this open reading frame is SEQ ID NO: 2.
It was shown to.

[0032]

INDUSTRIAL APPLICABILITY The present invention provides a gene encoding a polypeptide having a function of regulating the nitrile hydratase gene promoter. By allowing this regulatory gene and a heterologous gene introduced downstream of the nitrile hydratase gene promoter to coexist in the same cell, it becomes possible to regulate the expression of the heterologous protein by an inducer involved in the expression of nitrile hydratase.

[0033]

[Sequence list]

SEQ ID NO: 1 Sequence length: 357 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin organism name: Rhodococcus rh
Odochrous) strain name: J1 sequence: MetArgArgArgGlyPheProThrSerArgAsnAlaSerSerHis 15 ProArgLeuGlyLeuLeuThrSerLysSerGlyProGlySerLeu 30 PheAlaThrSerIleAspAsnAlaAlaMetLeuAlaLeuGluGlu 45 IleAsnAlaGluGlyGlyIleHisGlySerProValGluLeuIle 60 ValGlyAspAspAlaThrAspProGlnThrGlyValMetGluAla 75 ArgArgLeuValGlnAlaGlyCysArgThrIleLeuValAlaAla 90 ThrSerAlaThrTyrThrAlaValAlaGluAlaLeuSerAspAla 105 ProValLeuLeuValGlnProValMetAsnGluGlyGlyArgGly 120 AspArgLeuArgLeuGlnLeuGlyGluArgProAspAspGlnLeu 135 AlaAlaAlaValLysProLeuMetArgMetGluGlyValArgArg 150 TrpPheLeuAlaGlyAspAspTyrCysTrpProArgThrThrHis 165 GlyLeuAlaArgGlnLeuLeuProArgTyrGlyAlaGlyValVal 180 GlyGluGluPheAlaSerLeuGlyThrArgAspPheAlaProIle 195 IleGluSerIleGlnLysSerGlyAlaAspIleValLeuSerThr 210 PheValGlyAlaAspAlaValAlaPheGluArgGlnCysTyrAla 225 MetGlyLeuArgAspGlnCysIleThrLeuAlaProAlaLeuGlu 240 GluSerThrLeuThrTyrIleGlyAspGluAlaAlaArgGlyLeu 255 TyrSerValSerGlyTyrPheGlnGlyLeuGlnThrGluGlyAsn 270 AlaSerLeuLeuLysArgTyrArgAlaGlnPheGlyProTrpAla 285 ProProLeuSerThrLe uSerGluSerValPheGluAlaValHis 300 IleTrpTrpGlnAlaAlaArgLysAlaGluAspSerGluArgIle 315 AlaAlaAlaMlaAetMathArgHisGlyGluPheGlnLeuGlaLuProArgGlyArgGlyThr 330 ValThrLeuSerGluGrArg

SEQ ID NO: 2: Sequence length: 1074 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear Origin Biological name: Rhodococcus rhodococcus
odochrous) Strain name: J1 Sequence: ATG CGG CGT CGA GGG TTC CCT ACC TCT CGA AAC GCC TCT TCC CAT 45 CCC CGT CTG GGA TTG TTG ACG TCG AAA TCC GGA CCC GGC AGC CTC 90 TTC GCC ACC TCG ATT GAC AAC GCC GCC ATG CTG GCA CTG GAG GAA 135 ATC AAT GCC GAG GGC GGT ATC CAC GGC AGT CCA GTC GAG TTG ATC 180 GTG GGT GAT GAC GCC ACA GAC CCG CAG ACC GGC GTC ATG GAA GCG 225 CGA CGA CTC GTC CAG GCA GGC TGC CGG ACC ATT CTG GTC GCC GCC 270 ACA TCG GCT ACC TAC ACC GCC GTT GCC GAG GCG CTC AGT GAT GCA 315 CCC GTG CTG CTG GTA CAG CCG GTC ATG AAC GAA GGA GGG CGC GGC 360 GAT CGT CTC CGC CTC CAG CTC GGT GAG CGG CCG GAC GAC GAC TTG 405 GCA GCA GCC GTG AAA CCA CTC ATG CGG ATG GAG GGG GTG CGA CGA 450 TGG TTT CTG GCC GGC GAC GAT TAT TGT TGG CCC CGT ACG ACA CAC 495 GGC CTG GCA CGC CAA CTG TTG CCT CGG TAC GGC GCC GGA GTC GTC CG GGG GAA GAG TTC GCG TCT CTG GGA ACT CGT GAC TTC GCC CCG ATC 585 ATC GAA TCG ATC CAG AAA TCG GGT GCC GAC ATC GTA CTG TCG ACC 630 TTC GTC GGA GCT GAT GCT GTT GCG TTC GAG CGG CAA TGC TAT GCA 675 ATG GGA TTG CGG GAT CAG TGC ATC ACT CTG GCG CCC GCC TTG GAG 720 GAG TCC ACG CTG ACG TAC ATC GGC GAC GAA GCG GCC CGT GGC CTC 765 TAC TCC GTC TCG GGG TAC TTC CAA GGA TTA CAA ACG GAA GGT AAC C810 GCC TCG CTG AAG AGG TAC CGG GCG CAA TTC GGT CCT TGG GCA 855 CCG CCG CTG TCG ACG TTG AGC GAG TCT GTG TTC GAG GCA GTT CAC 900 ATA TGG TGG CAA GCG GCC CGG AAA GCC GAG GAT AGC GAG CGG ATC 945 GCC GCG GCA ATG CGT CAC GGC GAG TTT CAG CTA CCG CGG GGC ACC 990 GTC ACC TTG AGC GAG GAC CGA CTC AGG CAG CAG CTC CAT TTG ACG 1035 AAA GCG ACC GGC ACA GAA CTG TAT CCT GCC CTC GGA TGA 1074

SEQ ID NO: 3: Sequence length: 1911 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Origin Biologic name: Rhodococcus rh
Odochrous) strain name: J1 sequence: GATATCGTGC ACGACCAGCC CTGGCGAGGG CGTATCAAAC TCCGTTTCGA 50 AGCCGCAAAC GGTGGTACGA GGGTTCGGCT CATCGCCGAA CTCGACCAGC 100 CCGGATTGGA ATGGTTGATG CGGCGTCGAG GGTTCCCTAC CTCTCGAAAC 150 GCCTCTTCCC ATCCCCGTCT GGGATTGTTG ACGTCGAAAT CCGGACCCGG 200 CAGCCTCTTC GCCACCTCGA TTGACAACGC CGCCATGCTG GCACTGGAGG 250 AAATCAATGC CGAGGGCGGT ATCCACGGCA GTCCAGTCGA GTTGATCGTG 300 GGTGATGACG CCACAGACCC GCAGACCGGC GTCATGGAAG CGCGACGACT 350 CGTCCAGGCA GGCTGCCGGA CCATTCTGGT CGCCGCCACA TCGGCTACCT 400 ACACCGCCGT TGCCGAGGCG CTCAGTGATG CACCCGTGCT GCTGGTACAG 450 CCGGTCATGA ACGAAGGAGG GCGCGGCGAT CGTCTCCGCC TCCAGCTCGG 500 TGAGCGGCCG GACGACCAGT TGGCAGCAGC CGTGAAACCA CTCATGCGGA 550 TGGAGGGGGT GCGACGATGG TTTCTGGCCG GCGACGATTA TTGTTGGCCC 600 CGTACGACAC ACGGCCTGGC ACGCCAACTG TTGCCTCGGT ACGGCGCCGG 650 AGTCGTCGGG GAAGAGTTCG CGTCTCTGGG AACTCGTGAC TTCGCCCCGA 700 TCATCGAATC GATCCAGAAA TCGGGTGCCG ACATCGTACT GTCGACCTTC 750 GTCGGAGCTG ATGCTGTTGC GTTCGAGCGG CAATGCTATG CAATGGGATT 800 GCGGGATCAG TGCATCA CTC TGGCGCCCGC CTTGGAGGAG TCCACGCTGA 850 CGTACATCGG CGACGAAGCG GCCCGTGGCC TCTACTCCGT CTCGGGGTAC 900 TTCCAAGGAT TACAAACGGA AGGTAACGCC TCGCTTCTGA AGAGGTACCG 950 GGCGCAATTC GGTCCTTGGG CACCGCCGCT GTCGACGTTG AGCGAGTCTG 1000 TGTTCGAGGC AGTTCACATA TGGTGGCAAG CGGCCCGGAA AGCCGAGGAT 1050 AGCGAGCGGA TCGCCGCGGC AATGCGTCAC GGCGAGTTTC AGCTACCGCG 1100 GGGCACCGTC ACCTTGAGCG AGGACCGACT CAGGCAGCAG CTCCATTTGA 1150 CGAAAGCGAC CGGCACAGAA CTGTATCCTG CCCTCGGATG AGTTGAGACC 1200 TCGCTTGCTG ATACCGAAGA CCAGGGCATA TGCCTGGTCA TATCGGCGTG 1250 CTCGCTCCGT CTGCAGTTTC GTTCCCGGCC GGACGGATGC GCAACCACCG 1300 AGCGCAACTC GGGGTCGTCT GTGCATCAGC GTCGCCAGGA TTGCTTCCGG 1350 CTACGCTCGG GTCCCGCGTT ACCGCCATTC GACGCGATGT CGATCCCGGT 1400 CAAGGCGACA ATGACAGCTG CCAGATCCTC CTCTGTGGGA TCGGGACCGG 1450 TCGCGGAGGT CGAGGCTTTC TGCGAGATCT GCTTCCCTCT CGGGGGTCGT 1500 TCTGACTTCA TTCTTCGCCT TCCTTTGTGA ACGTCTCTCT GCGACACTTC 1550 CGGCCACAGT CAGCATCTTT TGAGATAGCT CCGAATGCAG ACGGTTCACG 1600 ATTCGTTCCG GGGGCTCGAC GAACGTCTTC CAGTCTCCGA AACAAACAGC 1650 A GTGTGGTCG AATTTGTGGC CGTCAGCGCG GTTGAGCCAC AGAGTGAATG 1700 CGCGGCAATC GAACTCAGAT TATCGGGCCA TTGCCCCACT GTCCACAGCG 1750 CGTGAGCT 750 CCGACAGGAGCTAGCTGACTAGCT

[Brief description of drawings]

FIG. 1 is a restriction map of recombinant plasmid pNLU10.

FIG. 2 is a process drawing of the recombinant plasmid pLJK10.

FIG. 3 shows a recombinant deletion plasmid.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12R 1:01) (C12N 1/21 C12R 1:01) (C12N 1/21 C12R 1:19) (C12N 9/88 C12R 1:01)

Claims (4)

[Claims] 1. A gene DNA encoding a polypeptide substantially containing the amino acid sequence represented by SEQ ID NO: 1 and having a function of regulating a nitrile hydratase gene promoter. 2. The gene DNA according to claim 1, wherein the gene encoding a polypeptide having the function of regulating the nitrile hydratase gene promoter has the nucleotide sequence represented by SEQ ID NO: 2. 3. A recombinant DNA in which the gene DNA according to claim 1 or 2 and a nitrile hydratase gene containing a promoter region are incorporated into a vector. 4. A transformant obtained by transforming with the recombinant DNA according to claim 3.