JP2005151999A - Regulatory gene for nitrile-hydratase gene expression - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a regulatory gene DNA encoding a peptide having activation ability of a nitrile-hydratase gene promoter, a recombinant DNA including the regulatory gene DNA, and a transformant transformed by the recombinant DNA. <P>SOLUTION: The regulatory gene DNA encoding a polypeptide having activation ability of a nitrile-hydratase gene promoter is isolated. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a regulatory gene DNA derived from Rhodococcus bacteria and encoding a polypeptide having the ability to activate a nitrile hydratase gene promoter, a recombinant DNA containing this DNA, and a trait transformed with the recombinant DNA. Concerning the converter.

  The method for producing amides using nitrile hydratase, which is an enzyme that hydrates nitriles to produce amides, is industrially superior because it is a reaction at normal temperature and pressure, and a high conversion rate. It has been used as a method. In particular, Rhodococcus bacteria are known to accumulate significant amounts of enzymes in the cells, and have high catalytic activity (Japanese Patent Publication Nos. 4-4873, 62-91189, and 2). -470 and JP-A-2-84198).

  Nitrile hydration with the nitrile hydratase gene cloned by the genetic recombination method allows multiple copies of the same gene to be present in the cell, thus dramatically improving the catalytic ability of microorganisms compared to conventional methods. Can be expected to increase. In addition, improvement of the enzyme using gene recombination technology is facilitated by methods such as site-specific mutation and random mutation. The nitrile hydratase gene derived from the genus Rhodococcus has been obtained from the N774 strain (Japanese Patent Laid-Open No. 2-119778) or the J1 strain (Japanese Patent Laid-Open No. 4-21379). When the N774 nitrile hydratase gene is incorporated into a Rhodococcus-E. Coli composite plasmid vector (Japanese Patent Laid-Open No. 5-64589) and introduced into a Rhodococcus bacterium, high activity is obtained (Japanese Patent Laid-Open No. 5-68566). ). On the other hand, no nitrile hydratase gene derived from Rhodococcus rhodochrous J1 strain, which has extremely high performance as compared with N774 strain nitrile hydratase, has been reported so far.

  The inventors have found that when the gene derived from the J1 strain is introduced, the expression of the nitrile hydratase gene promoter is not functioning, and the regulatory gene required for the function of the promoter has not been found so far. I thought it was because it was not included in the DNA region that was acquired. Therefore, as a result of searching for a regulatory gene that should exist somewhere on the chromosomal DNA of the J1 strain, it was found upstream of the nitrile hydratase structural gene, and this was used to detect the presence of the nitrile J1 strain in the recombinant genus Rhodococcus. The present invention was completed by highly expressing hydratase activity.

  That is, the present invention is derived from a Rhodococcus bacterium, a regulatory gene DNA that encodes a polypeptide having the ability to activate a nitrile hydratase gene promoter, a recombinant DNA containing this DNA, and a recombinant DNA that is transformed with the recombinant DNA. The present invention relates to a transformant.

  By making the regulatory gene of the present invention coexist in the same genus Rhodococcus as the nitrile hydratase gene containing the promoter region, mass production of the nitrile hydratase is made possible. Further, by introducing a heterologous gene under the promoter, high expression of the heterologous protein becomes possible.

  The present invention is described in detail below. The present invention is carried out by the following steps.

(1) Chromosomal DNA preparation:
Chromosomal DNA is isolated and prepared from Rhodococcus rhodochrous strain J1.

(2) Preparation of DNA library:
From the plasmid having the nitrile hydratase gene of the J1 strain, the gene portion derived from the J1 strain is excised and labeled with a radioisotope as a probe.

  After cleaving the chromosomal DNA with a restriction enzyme, a DNA fragment fraction containing the target gene is detected by the Southern hybridization method (Southern E. M., J. Mol. Biol. 98 503 (1975)) using the above probe. This fraction is inserted into the plasmid vector pUC19 to form a library.

(3) Production of transformants and selection of recombinant DNA:
A transformant using the recombinant DNA library prepared in step (2) is prepared, and colony hybridization method (R. Bruce Wallace. Et al. Nuc. Acids Res. 9 879 (1981)), a transformant containing the desired recombinant DNA is selected. Furthermore, the target recombinant DNA is reconfirmed by Southern hybridization.

(4) Purification of recombinant plasmid and construction of restriction enzyme map:
The plasmid is purified from the recombinant obtained in step (3). The plasmid thus obtained is called pNHU10. This is analyzed by electrophoresis for those cleaved with various restriction enzymes, and a restriction enzyme map is prepared.

(5) Production of a recombinant plasmid comprising a region containing a regulatory gene and a nitrile hydratase gene and a vector plasmid capable of replicating in Rhodococcus:
Recombination consisting of the plasmid obtained in step (4), the plasmid pNHJ10H containing the nitrile hydratase gene J1 and the composite plasmid vector pK4, the region containing the regulatory gene and the nitrile hydratase gene and the vector plasmid replicable in the genus Rhodococcus The body plasmids pHK18 and pHK19 are prepared.

(6) Transformation of Rhodococcus bacteria and production of nitrile hydratase using transformants:
Rhodococcus bacteria are transformed with the plasmid obtained in step (5), and it is confirmed that nitrile hydratase derived from the J1 strain is expressed.

(7) Deletion plasmid and nitrile hydratase activity:
A plasmid obtained by removing various regions from the plasmid obtained in step (5) is prepared, and which region is essential for the expression of nitrile hydratase is examined. A region necessary for expression is specified in a portion other than the nitrile hydratase structural gene region.

(8) Determination of nucleotide sequence:
The base sequence is determined for the region specified in step (7).

  Rhodococcus rhodochrous J1 strain has been deposited with the Biotechnology Institute of Technology as FERM BP-1478. Moreover, the plasmid pNHJ10H containing the nitrile hydratase gene is a transformant TG1 / pNHJ10H (FERM BP-2777) containing this, and the complex plasmid vector pK4 is the transformant Rhodococcus rhodochrous ATCC12674 / pK4 (FERM BP -3731), plasmid pNHU10 has been deposited as a transformant JM109 / pNHU10 (FERM P-14568) containing it.

  Hereinafter, the present invention will be specifically described by way of examples. However, the technical scope of the present invention is not limited by these examples.

In the examples, the following abbreviations were used.
TE solution: 10 mM Tris-HCl (pH7.8) -1 mM EDTA (pH8.0)
TNE solution: 50 mM Tris-HCl (pH 8.0) -1 mM EDTA (pH 8.0) -50 mM NaCl
STE solution: 50 mM Tris-HCl (pH8.0) -1 mM EDTA (pH8.0) -35 mM sucrose
2xYT medium: 1.6% tryptone, 1% yeast extract, 0.5% NaCl
MY medium: 1% polypeptone, 0.3% yeast extract, 0.3% malt extract, 1% glucose

1) Preparation of chromosomal DNA Rhodococcus rhodochrous J1 strain (FERM BP-1478) in 100 ml medium (glucose, KH ₂ PO ₄ , K ₂ HPO ₄ , MgSO ₄ .7H ₂ O, yeast extract, peptone, CoCl ₂ , urea The cells were collected after culturing at 28 ° C. for 2 days in 1 L of water, pH 7.2). The obtained bacterial cells were washed with a TEN solution, suspended in 10 ml of TE solution, added with 4 ml of 0.25 M EDTA, 10-20 mg of lysozyme, 10-20 mg of achromoprotease and 10 ml of 10% SDS, and added at 37 ° C. After standing for a period of time, the supernatant was obtained by centrifuging. To 20 ml of the supernatant, 0.7 ml of a 2.5 M sodium acetate solution and diethyl ether were added and centrifuged, and the upper layer was discarded, and twice the ethanol was added to the lower layer, and the DNA was wound up with a glass rod. This is immersed in TE solution: ethanol (volume ratio) 2: 1, 1: 9, 0:10 for 5 minutes, then dissolved in 2-4 ml of TE solution, and 10 μl of ribonuclease A and ribonuclease T1 mixture. And treated at 37 ° C. Next, an equal amount of phenol saturated with a TE solution was added, and an equal amount or more of ether was added to the upper layer after centrifugation to obtain a lower layer. This was dialyzed overnight with 2 L of TE solution to which a small amount of chloroform was added, followed by a second dialysis for 3 to 4 hours to obtain 4 ml of a chromosomal DNA preparation.

2) Preparation of DNA library
Plasmid pNHJ10H (JP-A-4-21379, Biochim. Biophys. Acta 1129, 23-33 (1991)) in which a 6.0 kb DNA fragment containing the J1 strain H-type nitrile hydratase gene was incorporated into the pUC19 vector was transformed with restriction enzymes SacI and EcoRI. After cleavage, the 0.37 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. To 10 μl of pNHJ10H, 3 μl of a restriction enzyme buffer (10-fold concentration), 2 μl each of restriction enzymes SacI and EcoRI, and 13 μl of sterilized water were added and reacted at 37 ° C. for 2 hours.

  Agarose gel electrophoresis was performed on a sample obtained by cleaving J1 strain chromosomal DNA with EcoRI and EcoRV. A 0.37 kb SacI-EcoRI fragment was labeled with the radioactive isotope 32P, and a DNA fragment of about 4.3 kb including the upstream region was detected by Southern hybridization using this as a probe. A DNA fragment fraction containing a 4.3 kb fragment hybridized with the probe was excised from an agarose gel and inserted into a plasmid vector pUC19 cleaved with SmaI and EcoRI.

  pUC19 was cleaved as follows. To 10 μl of pUC19, 3 μl of restriction enzyme buffer (10-fold concentration), 2 μl each of restriction enzymes SmaI and EcoRI and 13 μl of sterilized water were added and reacted at 30 ° C. for 2 hours. An equal amount of TE solution saturated phenol was added to the reaction solution, and the mixture was stirred and then centrifuged to separate it into an upper layer (aqueous layer) and a lower layer. This operation was repeated twice for the upper layer, the same amount of chloroform was added, and the same extraction operation was repeated twice. 3 μl of 3M sodium acetate and 90 μl of ethanol were added to the upper layer, allowed to stand at −80 ° C. for 30 minutes, centrifuged, dried and dissolved in TE solution.

  Insertion into pUC19 by reacting 3 μl of the DNA fragment fraction containing 4.3 kb fragment with SmaI, EcoRI cleaved pUC19 μl prepared as described above and TAKARA ligation kit overnight at 4 ° C. It was.

3) Preparation of transformants and selection of recombinant DNA E. coli JM109 strain (obtained from Takara Shuzo Co., Ltd.) was cultured in 10 ml of 2xYT medium at 37 ° C for 12 hours, then inoculated with 1% of the same medium for 2 hours. Cultured. After collecting the cells by centrifugation, 5 ml of a cold 50 mM CaCl ₂ solution was added, allowed to stand at 0 ° C. for 40 minutes, centrifuged again, and suspended in 0.25 ml of a cold 50 mM CaCl ₂ solution. Add 60 μl of the solution (DNA library) containing the recombinant plasmid prepared in step (2), leave it at 0 ° C for 40 minutes, give a heat shock at 42 ° C for 2 minutes, and add 1 ml of 2xYT medium. The mixture was further cultured with shaking at 37 ° C for 60 minutes. 100 μl of this was plated on 2 × YT agar medium containing 50 μg / ml of ampicillin and cultured at 37 ° C. Colony hybridization was performed on the transformant colonies grown on the agar medium, and transformants containing the upstream region of the nitrile hydratase gene were selected. That is, colonies grown on an agar medium were transferred onto a nitrocellulose filter, the cells were dissolved and DNA was fixed, and this was treated with the probe (0.37 kb fragment) prepared in step (2). Colonies containing the desired recombinant DNA were selected by the graph method. Further, a recombinant plasmid was extracted from the selected colony, hybridized with the above-mentioned probe by Southern hybridization, and reconfirmed that the selected colony was a transformant containing the target gene.

4) Purification of recombinant plasmid and preparation of restriction enzyme map The transformant selected in step (3) is cultured overnight at 37 ° C in 100 ml of 2xYT medium, collected, washed with TNE solution, and washed with STE. 8 ml of the solution and 10 mg of lysozyme were added and left at 0 ° C. for 5 minutes. 4 ml of 0.25 M EDTA was added, and further 2 ml of 10% SDS and 5 M NaCl were added at room temperature, and the mixture was allowed to stand at 0 to 4 ° C. for 3 hours. This was ultracentrifuged, 1/2 equivalent of 30% PEG6000 was added to the supernatant, left overnight at 0-4 ° C., centrifuged, and dissolved in TNE to make 7.5 ml. After adding CsCl, protein removal was performed by centrifugation, and 300-500 mg / ml of ethidium bromide solution was added, transferred to a seal tube, heat sealed, and ultracentrifuged. cccDNA was recovered, and an equivalent amount of water-saturated isopropyl alcohol was added to remove ethidium bromide, and about 3 ml of recombinant plasmid purified by dialysis with TE was obtained. This was named pNHU10. This recombinant plasmid was cleaved with several kinds of restriction enzymes to prepare a restriction enzyme map shown in FIG.

5) Preparation of recombinant plasmid consisting of a region containing regulatory gene and nitrile hydratase gene and vector plasmid replicable in Rhodococcus The plasmid pNHU10 obtained in step (4) is included in pNHJ10H containing nitrile hydratase gene It overlaps with the DNA fragment derived from J1 strain by 0.37 kb, and further includes up to 3.9 kb upstream. A 1.4 kb FspI-EcoRI fragment and a 0.9 kb ScaI-EcoRI fragment were excised from pNHU10 and inserted into the Escherichia coli-Rhodococcus complex plasmid vector pK4 to prepare plasmids pHJK16 and pHJK17. 5.9 kb EcoRI fragment derived from pNHJ10H was inserted into these plasmids to prepare pHJK18 and pHJK19, respectively. These manufacturing processes are shown in FIGS. In addition, in pHJK18 of FIG. 1, the thick arrows indicate the positions and directions of the regulatory genes found in the present invention, and the thin arrows indicate the positions and directions of the two subunit genes of the nitrile hydratase gene. Further, in pHJK19 of FIG. 2, the thick arrows indicate the positions and directions of the regulatory genes found in the present invention, and the thin arrows indicate the positions and directions of the two subunit genes of the nitrile hydratase gene.

6) Transformation of Rhodococcus bacteria and production of nitrile hydratase using transformants Rhodococcus rhodochrous ATCC12674 cells in the logarithmic growth phase were collected by centrifugation and washed three times with ice-cold sterile water. And suspended in a 15% PEG6000 (polyethylene glycol 6000) solution (bacterial cell concentration of 10 ⁹ cells / ml or more). 1 μg of the plasmid pHK18 DNA or pHK19 DNA in step (5) and 100 μl of the cell suspension were mixed and ice-cooled. A mixture of DNA and bacterial cells was placed in a gene pulser chamber, cooled on 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 solution was allowed to stand for 10 minutes under ice-cooling, and after heat treatment at 37 ° C. for 5 minutes, 1 ml of MY medium was added and shaken at 25 ° C. for 3 hours. The mixture 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 an MY agar medium containing kanamycin that the colonies that appeared were clearly kanamycin resistant.

Rhodococcus rhodochrous recombinant thus produced, Rhodococcus rhodochrous ATCC12674 / pHJK18 or Rhodococcus rhodochrous ATCC12674 / pHJK19, medium (glycerol 10 g, polypeptone 5 g, yeast extract 3 g, malt extract 3 g, KH ₂ PO ₄ 1 G, K ₂ HPO ₄ 1 g, CoCl ₂ · 6H ₂ O 0.01 g, urea 0.75 g or 3.75 g (pH 7.0) / L) were cultured at 28 ° C for 2 days, and the activity of nitrile hydratase was measured. . The cells were collected from the culture by centrifugation, washed with 150 mM NaCl, and suspended in 0.1 M HEPES-KOH (pH 7.2) containing 0.35% n-butyric acid. Cells were disrupted by sonication, the cell membrane was removed by centrifugation to obtain a cell extract, and nitrile hydratase activity was measured. The activity measurement was performed as follows. Add 0.5 ml of 100 mM potassium phosphate buffer (pH 7.0) and 1 ml of 200 mM acrylonitrile to 0.5 ml of cell extract appropriately diluted with water, react at 20 ° C for 10 minutes, and then add 0.2 ml of 1N HCl. The reaction was stopped by adding. The acrylamide produced was quantified by HPLC.

  The values in Table 1 below were obtained for cell extracts of Rhodococcus rhodochrous ATCC12674 / pHJK18 and Rhodococcus rhodochrous ATCC12674 / pHJK19. In the table, urea is an inducer of nitrile hydratase enzyme.

7) Deletion plasmid and nitrile hydratase activity
In pHJK18 and pHJK19, it was thought that there were still many regions that were not necessary for nitrile hydratase expression.When a deletion plasmid was prepared and nitrile hydratase activity was examined, the regulatory gene region essential for expression of activity was It was revealed that it was between 1.9 kb from ScaI to AccIII.

8) Determination of base sequence When the DNA base sequence was determined for the region specified in step (7), the only long open reading frame having the amino acid sequence shown in SEQ ID NO: 1 was found. The base sequence was determined by the chain termination method (Sanger F. Science 214, 1205-1210 (1980)) using Tth DNA polymerase. The base sequence of this open reading frame is shown in SEQ ID NO: 2.

The figure which shows the preparation process of recombinant plasmid pHJK18. The figure which shows the preparation process of recombinant plasmid pHJK19.

Claims (2)

  A regulatory gene DNA encoding a polypeptide having the amino acid sequence of SEQ ID NO: 1 and having the ability to activate a nitrile hydratase gene promoter.   A regulatory gene DNA having the ability to activate the nitrile hydratase gene promoter having the base sequence of SEQ ID NO: 2.

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