JP2769279B2 - Genetically engineered D-amino acid oxidase producing bacteria - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a gene recombination method characterized by using Trigonopsis variabilis as a host, and to a D-protein which can be used for the production of an antibiotic cephalosporin C derivative and the like created using the method. It relates to an amino acid oxidase producing bacterium.

[0002]

2. Description of the Related Art D-amino acid oxidase is an enzyme that catalyzes the oxidative deamination of D-amino acids. This enzyme can be used to separate L-amino acids from racemic mixtures of DL-amino acids, to prepare keto acids from D-amino acids, or to prepare D-amino acids.
It is known that it is applied to amino acid analysis and has industrially useful properties. Yeast trigonopsis
D-amino acid oxidase produced by S. variabilis is
Not only does it oxidize D-amino acids, but also oxidizes cephalosporin C, a kind of antibiotic, to form 7-β- (5-carboxy-5-oxopentanamide) cephalosporanic acid. I have. It is also known that this compound reacts with H ₂ O ₂ produced at the same time to produce 7-β- (4-carboxybutanamide) cephalosporanic acid. These compounds provide intermediates for the synthesis of cephalosporin antibiotics which are important as pharmaceuticals (for example, Japanese Patent Publication No. 50-7158). These compounds further include H ₂ O ₂ and certain cephalosporins. A method for producing 7-aminocephalosporanic acid, which is a more useful intermediate material, by reacting an acylase has been invented (for example, Japanese Patent Publication No. 54-17032).

[0003] Regarding Trigonopsis variabilis, reports on conditions for high production of D-amino acid oxidase [Biotechnology letter].
s 14 (3) 195-200 (1992)], a report using Trigonopsis variabilis activated by freeze-thawing or treatment with an organic solvent or a surfactant (for example, Japanese Patent Publication No. 55-35118), -A report using the same enzyme extracted from the cells (for example, PCT W
O 90/12110).

On the other hand, examples of the use of recombinant DNA technology for the production of D-amino acid oxidase include those expressing the same enzyme gene of Trigonopsis variabilis in Escherichia coli (JP-A-63-74488) and Fusarium solani. The enzyme gene is expressed using Escherichia coli and Acremonium chrysogenum as hosts (Japanese Unexamined Patent Publication No. 2-200181) [Bio / Techno]
logic 9 188 (1991)]. However, when Escherichia coli or Acremonium chrysogenum is used as a host, these bacteria have modified enzymes such as cephalosporin C derivatives such as cephalosporinase and esterase, so that cephalosporin remains as it is. It could not be used for producing C derivatives and the like.

[0005]

The present inventors have already isolated the D-amino acid oxidase gene of Trigonopsis variabilis (JP-A-63-7118).
0). However, in the present invention, the D-amino acid oxidase
Focusing on the usefulness of Trigonopsis variabilis as a microorganism that produces
While establishing a transformation method using NA technology,
An object is to obtain Trigonopsis variabilis in which a large number of amino acid oxidase genes are stably inserted into a chromosome.

[0006]

Means for Solving the Problems The present inventors have found that, when Trigonopsis variabilis is used as a host, the gene is stably retained on the chromosome in multiple copies, and completed the present invention. That is, the present invention provides a Trigonopsis variabilis transformed with a D-amino acid oxidase gene, and a D-amino acid oxidase gene characterized in that a plurality of D-amino acid oxidase genes and a marker gene are retained on a chromosome.
D-amino acid oxidase activity compared to the parent strain
And high Esterase activity .

According to the present invention, the D-amino acid oxidase activity of Trigonopsis variabilis is increased, and the activity of the modifying enzyme esterase, which is problematic in the production of cephalosporin C derivatives and the like, is suppressed. It is possible to produce a cephalosporin C derivative or the like in high yield even when used as a cell. According to the genetic recombination method, a mutant strain can be obtained with higher reproducibility than the random mutation method. In addition, since a marker gene can be introduced into a mutant strain, not only is it easy to confirm gene retention, but also if a long-term culture is performed in industrial production or the like, selective pressure can be applied.

[0008] Trigonopsis variabilis is known to have various useful enzyme activities (for example, JP-A-2-117396) in addition to D-amino acid oxidase activity, and the transformation method of the present invention can be used. If this is the case, it becomes possible to give the bacterium the desired properties. Furthermore, when expressing a heterologous gene, Trigonopsis variabilis is useful as a host because not only can it stably maintain a large number of copies of the gene on the chromosome, but also it can be easily mass-cultured on an inexpensive medium.

As the D-amino acid oxidase gene in the present invention, a gene derived from Trigonopsis variabilis as it is, a gene obtained by converting the expression device to an efficient one, and other species such as Fusarium solani. A gene whose structure can be expressed in Trigonopsis variabilis can be used. However, those using a D-amino acid oxidase gene derived from Trigonopsis variabilis or an expression device are particularly preferred. Also,
Not only genomic DNA but also cDNA can be used.

[0010] As the host Trigonopsis variabilis, the CBS4095 strain (Centra
l Bureau voor Schimmelcul
strains) and its catalase-deficient strains (Biotechnology Institute of Technology FERM BP-4359), as well as various mutant strains of Trigonopsis variabilis. Among them, when producing a cephalosporin C derivative or the like, a catalase-deficient strain is preferable in order to react with H ₂ O ₂ .

[0011] As a DNA introduction method, a protoplast fusion method, a physical introduction method using electroporation or particle bombardment, a chemical introduction method using metal ions, DMSO or the like, which are usually used for yeast. According to the present invention, trigonopsis variabilis can be stably inserted into a chromosome in multiple copies by any known method. At the time of transformation, marker genes (marker genes) include various drug resistance genes, auxotrophic complement genes, and galactosidase genes.
A gene, such as ze, which can be subjected to a simple assay and whose structure can be expressed in Trigonopsis variabilis, can be used. Particularly, drug resistance genes such as a hygromycin B resistance gene and a G418 resistance gene are preferable. From the transformants into which the D-amino acid oxidase gene has been introduced together with such a marker gene, a strain having stable properties is selected and cultured in the same manner as a normal microorganism.

Further, Trigonopsis variabilis is
Cephalosporin C, 7-β- (5-carboxy-5-
Oxopentanamide) cephalosporanic acid, and 7
It is known that -β- (4-carboxybutanamide) cephalosporanic acid has an esterase activity of deacetylating the 3-position, and there is a problem that by-products of these deacetylated forms are formed in the reaction product. (EP
409521). The inventors of the present invention measured the esterase activity of a transformant having multiple copies of the D-amino acid oxidase gene and found, surprisingly, that the activity was significantly lower than that of the parent strain, thus completing the present invention. It is.

According to the present invention, it has been found that the target gene can be stably inserted into a chromosome with a high copy number by using Trigonopsis variabilis as a host. Therefore, it is possible to introduce another gene desired to be introduced into Trigonopsis variabilis. Even when used for transformation, it becomes possible to efficiently impart the desired properties to the present bacterium. Examples of such genes include useful enzyme genes originally produced by Trigonopsis variabilis such as an enantiomer-selective NADP-dependent oxidoreductase. In view of this, the present bacterium is very useful also as a host for expressing genes derived from heterologous organisms.

[0014]

The present invention will be specifically described below with reference to examples.

[0015]

Example 1 (Step 1) Cloning of D-amino acid oxidase gene Extraction of total DNA from Trigonopsis variabilis [Cryer et al.] [Reference: Method
In cells biology (Methods in C
Cell Biology, Volume 12, 39-44, Academic Press (Academic Press),
1975], according to Trigonopsis variabilis C.
Total DNA was extracted and purified from BS4095. This DNA
Take 40 μg, 4 units of MboI restriction enzyme and 37
The reaction was performed at 15 ° C. for 15 minutes. The cleaved DNA was extracted from the reaction solution with an equal amount of phenol / chloroform (1: 1 vol.), And the DNA was precipitated with ethanol.
Double concentration of TE buffer [10 mM Tris-HCl (pH
8.0) 1 mM EDTA]. D obtained
The entire amount of the NA solution was subjected to 0.7% agarose gel electrophoresis, a portion containing DNA corresponding to a size of 6 to 9 kb was recovered, and 30 μg of the vector pUC18 was replaced with BamH.
I, complete cleavage, mixing with the resulting DNA fragment,
4. Incubate the DNA ligase at 15 ° C. for 6 hours, and mix the vector with D. variabilis CBS 4095.
The NA fragment was ligated.

Next, a cephalosporinase-deficient mutant strain was constructed as an E. coli host. That is, Escherichia coli MC1061
(Malcom Casadavan, University of Chicago, USA
Obtained from Dr. Casadaban). The properties of this bacterium are described in Journal of Molecular Biology (JM
ol. Biol. ), 138 , 179-207, 198.
0) to N-methyl-N'-nitro-N
Colonies with increased sensitivity to cephalosporin C after treatment with nitrosoguanidine were selected. From among these, a strain with reduced cephalosporinase activity was selectively isolated, and one strain was named Escherichia coli MB65 and used as a host. The mutant Escherichia
Kori MB65 is the Institute of Biotechnology and Industrial Technology,
Deposited as FERM BP-4360. Next, the recombinant DNA obtained in the above step was introduced into Escherichia coli MB65 by transformation.
Colonies growing on an L-broth agar medium containing 0 μg / ml were collected and designated as a DNA library of Trigonopsis variabilis CBS4095.

2. Determination of amino acid sequence at amino terminal of D-amino acid oxidase 60 g of Trigonopsis variabilis CBS 4095
Wet cell is suspended in 60 ml of 0.1 M phosphate buffer (pH 7.5), sonicated for 5 minutes, and then centrifuged to obtain a supernatant. This 20 to 60% ammonium sulfate fraction is collected, 0.05M phosphate buffer (pH7.
5) Dialysis against DEAE-Sepharose
CL-6B (Pharmacia column scale 200ml, NaCl 0 → 0.5M gradient)
An active fraction is obtained by column chromatography. After dialysis against 0.05 M phosphate buffer (pH 7.5), HPL
CDEAE-5PW (21.5 mm x 1 manufactured by TOSO)
An active fraction is obtained by column chromatography (50 mm, NaCl 2 O → 0.5 M gradient). This was concentrated with Centriprep (manufactured by Amicon), and HPLC PROT
EIN CULUMN 300 (Waters)
7.8 mm x 300 mm Elution solvent: 0.1 M KPB
By applying the solution (pH 7.0) and an aqueous solution of 0.2 M NaCl), SDS-PAGE gave purified D-amino acid oxidase which produces a single band.

The amino acid sequence at the N-terminus of the amino group of the purified enzyme protein was determined using a Hunkapiller.
[Science 219, 650-659]
(1983)], and the amino acid sequence from the amino group end to the 41st position was determined. The obtained N-terminal amino acid sequence is shown in FIG. 3. Synthesis of DNA Probes Two sequences shown underlined in FIG. 1 are selected, and possible DNAs on the gene deduced from these amino acid sequences are selected.
An oligonucleotide mixture containing the entire base sequence was synthesized as shown in FIG. That is, oligonucleotides that can be obtained for each amino acid sequence are divided into two groups and synthesized to form a mixture, which is then mixed with DNA probes DAO-1 and DO-1.
They were named AO-2 and DAO-3 and DAO-4. All oligonucleotide synthesis was performed by Applied Biosystem's DN.
A Synthesizer Model 3
Performed using 80-A.

4. Selection and Separation of Candidate D-Amino Acid Axidase Clones from DNA Library Each of the DNA probes obtained in the above step 3 was purified by the method of Inglia et al. [Reference: Nucleic Acids Re.
s. ), 9 , 1627-1642, 1982] and T4 polynucleotide kinase and γ- ³² P-ATP.
Labeled with. Next, Escherichia coli obtained in the above step 1, which is a DNA library, was treated with 50 μg of ampicillin /
of L-broth agar medium containing L. ml, and this was subjected to a replica method using Whatman (Wha).
tman) 541 filter paper, lysozyme lysed, denatured DNA with alkali, neutralized with hydrochloric acid, and hybridized with DAO-1 and DAO-2. Hybridization was performed at 6 times the concentration of S
SC (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.5% Nonidet P-40
(Sigma, USA), DAO-1 or DAO-2
One and a half hours at 44 ° C. using about 2 × 10 ⁵ cpm / ml, and then twice at room temperature using 6-fold concentration SSC.
Subsequently, the filter paper was washed once at 44 ° C. using a 6-fold concentration of SSC. The back paper was dried and subjected to autoradiography (sensitivity conditions: -80 ° C, 3 hours). As a result, a large number of colonies positive for hybridization to DAO-1 or DAO-2 were found. Therefore, 40 strains were picked from the positive colonies, cultured in a liquid, and then subjected to the method of Birnboim et al. [Reference: Nucleic Acid Research (Nucleic Acid)]
sRes. ), 7 , 1513-1523, 1979]. Then these DN
After denaturing A by a conventional method, it was spotted on a nitrocellulose filter and hybridized with a DNA probe. Hybridization was performed at a 6-fold concentration of SSC and at a 10-fold concentration of Denhardt.
Solution (0.02% ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin) and labeled DNA probe at 44 ° C. (for DAO-1 and DAO-2) or 40 ° C. (DAO -3 and D
AO-4) for 1 hour, followed by a 6-fold concentration of S
Washed once at room temperature with SC, then once at 44 ° C. (for DAO-1 and DAO-2) or 40 ° C. (for DAO-3 and DAO-4). Thereafter, the filter was subjected to autoradiography (sensitivity conditions: -80 ° C, 3
Time), the probe shows a positive hybridization for DAO-1 or DAO-2, and
3 or 6 strains showing a positive effect on DAO-4 were also found. After cutting the plasmid DNAs from these six strains with various restriction enzymes and performing agarose gel electrophoresis,
Southern hybridization with a labeled DNA probe [Refer to the literature: Southern (Souther)
n) (1975) Journal of Molecular Biology (J. Mol. Biol.), 98 , 503-51.
7) was performed. As a result, a plasmid DNA in which DAO-2 and DAO-3 or DAO-4 were strongly hybridized positively in a DNA fragment of about 0.6 kb generated by EcoRI digestion was found. This plasmid is called p
DAOC2-12 was designated as a candidate for a D-amino acid oxidase clone.

[5] Identification of D-amino acid oxidase clone and determination of DNA nucleotide sequence The DNA nucleotide sequence of a 0.6 kb DNA fragment generated by cutting EcoRI from plasmid pDAOC2-12 was determined according to the method of Sanger et al. As a result, as shown in FIG. 3, the amino group N of the D-amino acid oxidase obtained in the above step was separated by the intron-like sequence existing in the eukaryotic gene.
A nucleotide sequence encoding an amino acid sequence completely identical to the terminal amino acid sequence was found, and it was found that this fragment contained a part of the D-amino acid oxidase gene. For the plasmid pDAOC2-12, a restriction enzyme cleavage map shown in FIG. 4 was created based on the results of restriction enzyme cleavage. Based on the above results, the DNA base sequence was determined for the downstream region in the gene reading direction from the base sequence already determined.
It was found that there was a nucleotide sequence encoding a protein consisting of 355 amino acids shown in FIG.
The intron-like sequence indicated by is deleted and displayed). Thus, DNA from Trigonopsis variabilis CBS 4095 in plasmid pDAOC2-12
It is assumed that the fragment completely contains the structural gene for D-amino acid oxidase.

6. Modification of D-amino acid oxidase gene 40 μg of plasmid pDAOC2-12 was ligated with EcoRI.
And HindIII, and the resulting fragment of about 0.45 kb was purified. 0.8 μg of the fragment was added to dATP, dGT.
P, dCTP, and TTP were added at a final concentration of 0.33 mM each,
Add 5 units of polymerase Klenow fragment, add 10 mM Tris-HCl (pH 7.5), 10 mM
MgCl ₂ , 1 mM dithiothreitol, 50 mM N
The reaction was carried out at 30 ° C. for 20 minutes in 30 μl of a reaction solution of aCl, and a DNA fragment having both ends blunt-ended was purified. About 0.2 μg of the BamHI linker (0.0175
O. D. ) And 1 unit of T4 DNA ligase, add 50
mM Tris-HCl (pH 7.5), 10 mM MgCl
_2. Reaction was carried out at 15 ° C. overnight in 20 μl of a reaction mixture containing 0.5 mM ATP and 5 mM dithiothreitol. After reaction D
The NA fragment was purified, cut at both ends with EcoRI and BamHI, and recovered as an EcoRI-BamHI fragment. On the other hand, the double-stranded DNA of phage M13mp18 was
And BamHI, followed by the EcoRI-
The BamHI fragment was added and ligated with T4 DNA ligase to construct phage M13M2-12-7 in which a part of the D-amino acid oxidase gene was incorporated.

Introduction of a BanI site in the intron-like sequence
Before and after the intron-like sequence as shown in FIG.
Of oligonucleotides complementary to sequences directly linked to
And named DAOE1. DAOE1 25pmol
e and 10 pmol of M13 primer Ml (Takara)
e is phosphorylated by T4 polynucleotide kinase and
Method of Messing [Literature: Method in
Enzymology (Methods Enzymo
l. ),101, 20-78, 1983].
Prepared single-stranded DNA of phage M13M2-12-7
Add about 0.5 pmole, heat at 95 ° C for 5 minutes, then room temperature
It was left until it became. Then add dATP, dGT
P, dCTP, TTP (0.4 mM each), ATP (0.
4 mM), DNA polymerase Klenow (Kleno)
w) Fragment (5 units), T4 DNA ligase (2 units)
In addition, each of 7 mM Tris-HCl (pH 7.5), MgC
l_Two, NaCl and 14 mM dithiothreitol
The reaction was performed at 37 ° C. for 30 minutes in 50 μl of the reaction solution. 0.
After stopping the reaction by adding 5 μl of 5M EDTA, meshing
(Messing) method (supra)
Sherichia Kori (Escherichia col
i) Transfection treatment of JM105 with reaction solution
Then, the phage-introduced bacteria were detected as plaques. Appear
Phage plaques from plaque-hybridization
[Reference: Benton et al. (197)
7) Science,196, 180-
182]³²DAOE1 and hive labeled with P
Rediscover and find positive plaques
Was. The phage in the positive plaque was re-equipped with Escherichia coli.
Infect JM105 and DAOE in the same manner as above.
Purify plaques that show hybridization with 1.
After separation, the phage present in the plaques
To conduct liquid culture to separate single-stranded DNA. 1 obtained
Analysis of the base sequence of the single-stranded DNA revealed that the
Of a nucleotide sequence having a BanI site lacking a tron-like sequence
From Trigonopsis variabilis CBS 4095
This phage was found to have a DNA fragment.
It was named M13ME1.

(Step 2) Preparation of Transformation Marker Gene Deletion of pHY300PLK BanI Site In constructing the plasmid after the construction, pHY300PLK was first selected from commercially available plasmids in order to obtain a plasmid without a BanI site that would hinder it. pHY300PLK
Was cut with a restriction enzyme BanI, blunt-ended and self-cyclized using a DNA blunting kit (manufactured by Takara), introduced into E. coli MB65, and placed on an L-broth agar medium containing 10 ug / ml of tetracycline. The colonies that had grown were selected, plasmid DNA was prepared by the method of Barnboim et al., A plasmid DNA having no BanI site was selected, and named pHY301.

2. Preparation of pTDAO1 Trigonopsis variabilis D without intron
-Plasmid pTDAO1 in which the amino acid oxidase gene was carried on pHY301 was prepared in the following manner. First, a 1700 bp fragment obtained by cleaving pDAOC2-12 with HindIII, 200 ng recovered after agarose electrophoresis, mixed with 1 ug of pUC18 cut with HindIII and treated with alkaline phosphatase, ligated with T4 DNA ligase, pDA
OC2-12V4 was obtained.

After pDAOC2-12V4 was cut with SalI, the plasmid was pDAOC12V4 which had been self-cyclized using T4 DNA ligase to remove the palindrome structure.
5 was produced. This pDAOC2-12V5 is connected to BamH
12 obtained by cutting with I and EcoRI
200 bp fragment collected after agarose gel electrophoresis, 1 ug of pHY301 digested with BglII, treated with alkaline phosphatase, and M13ME
1 was digested with BamHI and EcoRI, and a 470 bp fragment obtained by agarose electrophoresis
0ng was collected and the three were mixed and ligated with T4 DNA ligase to obtain pTDAO1.

3. Preparation of pTDAO2 A plasmid pTDAO2 having a promoter and a terminator portion of the D-amino acid oxidase gene of Trigonopsis variabilis was prepared in the following manner.
4. pTDAO1 was cut with SacI and BanI
1 μg of the 5 Kb fragment was recovered after agarose gel electrophoresis,
After blunt-ending using a DNA blunting kit (Takara) and treating with alkaline phosphatase,
g of BglII linker (manufactured by Takara, 8 bases), ligated with T4 DNA ligase, cut with BglII, and again subjected to agarose gel electrophoresis to recover a 5.5 Kb fragment. This was self-cyclized using T4 DNA ligase to obtain pTDAO2.

4. Preparation of pTHY83 The hygromycin B kinase gene was transferred to pTDAO2
The plasmid pTHY83 to be expressed using the promoter and terminator was prepared as follows. pTD
1 μg of AO2 was digested with BglII, treated with alkaline phosphatase, and pACTHY83 (EP A
0450758) was cut with BamHI, mixed with the 1.5 Kb fragment recovered after agarose gel electrophoresis,
Ligation with T4 DNA ligase yielded pTHY83.

(Step 3) Preparation of Transformant of Trigonopsis variabilis Preparation of protoplast YEPD medium (yeast extract 10 g / l, peptone 20 g)
/ L, glucose 20 g / l) 1.5 m of Trigonopsis variabilis CBS 4095 strain grown for 30 hours
1) and collected in a reducing buffer (0.05% mercaptoethanol, 10 mM Tris-HCl PH7.5).
After suspending in 1.5 ml, the mixture was incubated at 30 ° C. for 15 minutes, collected again, and collected with A-buffer (0.6 M sucrose, 2%) containing 4 mg / ml of Novozyme (manufactured by NOVO).
MgSO ₄ , 50 mM Tris-HCl PH 8.0)
Suspend in 2 ml and incubate at 30 degrees for 3 hours. The reaction solution is collected, washed once with A-buffer, and collected again.

2. DNA introduction by protoplast fusion The above protoplasts were suspended in 80 μl of B-buffer (0.75 M sucrose, 50 mM CaCl ₂ , 10 mM Tris-HCl PH 8.2) and 20 μl
A DNA solution (10 μg of pTHY83) was added, and 30
The temperature was kept for 3 minutes.

Thereafter, 900 μl of C-buffer (0.6 M sucrose, 50 mM CaCl ₂ , 3
8% polyethylene glycol, 10 mM Tris-H
ClPH8.2) is added, mixed, incubated at 30 ° C. for 30 minutes, and then collected, washed once with A-buffer, and collected again. (Step 4). Selection of Transformant The above protoplasts were suspended in 1.5 ml of A-buffer, and 300 μl each of 12.5 ml of a regeneration medium (yeast extract 10 g / l, peptone 20 g / l, glucose 20 g / l, sucrose 0.6 M, DL-methionine 3 g / l, agar 25 g / l) spread on 5 plates. After incubating at 30 ° C. for 12 hours, 7.5 ml of a regeneration medium containing 4 mg of hygromycin B is overlaid, and further cultured at 30 ° C. for 1-2 weeks, and colonies formed are selected. As a result, 13 hygromycin B resistant strains were obtained. On the other hand, instead of pTHY83, pHY300PLK
No hygromycin B resistant strain was obtained by the same procedure using Thus, it was confirmed that a transformation system of Trigonopsis variabilis could be established.

[0031]

Example 2 CB as Trigonopsis variabilis
In place of the S4095 strain, the catalase-deficient strain K
Using the C103 strain (FERM BP-4359) as a host, transformation was performed using pTHY83 according to the method of Example 1 to obtain 10 hygromycin B-resistant strains. On the other hand, a hygromycin B-resistant strain was not obtained by performing the same operation using pHY300PLK instead of pTHY83.

[0032]

Example 3 (Step 1) Preparation of pADH1 A plasmid pADH1 for introducing a D-amino acid oxidase gene simultaneously with a marker gene was prepared in the following manner. A 440 bp fragment obtained by digesting pDAOC2-12V5 with BamHI and SacI was recovered by agarose electrophoresis and collected at 100 ng.
1 μg of THY83 was digested with BamHI, treated with alkaline phosphatase, and pDAOC2-12
Was digested with BamHI and SacI, and a 2.4 Kb fragment obtained by agarose gel electrophoresis was used for 200 ng fragment.
g were collected, and the three were mixed and ligated with T4 DNA ligase to obtain pADH1.

(Step 2) Transformation using pADH1 Transformation was carried out using pADH1 as a Trigonopsis variabilis using the CBS4095 strain as a host according to the method of Example 1 to obtain eight hygromycin B resistant strains. . (Step 3) Measurement of D-amino acid oxidase activity of transformant using pADH1 The obtained hygromycin B-resistant strain was transformed into 3 ml of YEPD liquid medium containing 50 μg / ml of hygromycin B.
Was inoculated with one platinum loop, and cultured at 28 ° C. for 38 hours. 6 ml of 30% glycerol was added thereto, mixed, and stored at -70 degrees. 1 ml of this stock solution is added to a fermentation medium (glucose
2.5%, DL-methionine 0.3%, MgSO4
0.1%, KH2PO4 0.4%, C.I. S. L. 6
%, Shin-Etsu Silicone KM-72 0.5%, PH6.
5) Inoculate into a 500 ml flask containing 50 ml,
Cultivation at a temperature of 40 hours, adding 2 ml of 50% glucose,
The culture was further performed for 20 hours. 500 μl of this culture solution is 2 m
1 tube (Eppendorf) and 15 μl
Of toluene and stirred at room temperature for 2 hours using a micro tube mixer (speed 10 manufactured by Tommy),
After collecting the cells, the cells are washed with 1 ml of water and suspended again in 500 μl of water to obtain a toluene-activated cell suspension. This 10μ
of the reaction buffer (cephalosporin C)
11 mg / ml, 110 mM Tris-HCl PH
7.5) in a test tube and stirred at 25 ° C. for 10 minutes on a shaker (240 rpm), and then mixed with an equal amount of a reaction stop solution (NaOH 17 mM, acetic acid 13.3%),
Stop the reaction. The reaction solution was analyzed by HPLC (inert
Tosyl ODS-2 column, 5% acetonitrile-3% sodium acetate solvent, flow rate 1 ml per minute, detection at a wavelength of 254 nm), and formed 7-β- (5-carboxy-5-oxopentanamide) cephalosporanic acid and 7-β
-(4-Carboxybutanamide) cephalosporanic acid is quantified, and the D-amino acid oxidase activity that produces 1 μmol of both substances per minute is defined as 1 unit.

As a result, as shown in Table 1 (indicated by the activity value per 1 ml of the culture solution), compared to the parent strain CBS4095,
Many strains with significantly increased D-amino acid oxidase activity were obtained.

[0035]

Embodiment 4 CB as Trigonopsis variabilis
In place of the S4095 strain, the catalase-deficient strain K
Using the C103 strain (FERM BP-4359) as a host, transformation was performed using pADH1 according to the method of Example 1 to obtain 9 hygromycin B-resistant strains.

The D-amino acid oxidase activity of these strains was measured by the method of Example 3. As shown in Table 2, many strains having significantly increased D-amino acid oxidase activity were obtained as compared to the parent strain KC103. Was done. Moreover, on behalf of various transformants, total DNA was extracted and purified from the cells cultured for 60 hours under non-selective pressure according to the method of Cryer et al. 5 μg of this DNA is
What was cut with indIII, pDAOC2-12V
800b cut out from 4 with EcoRI and SacI
The p fragment was used as a probe for Southern hybridization. 2. naturally present on the chromosome of KC103
In addition to the 2 Kb band, the transformed strain had a 4.0 kb deep band derived from pADH1 and a faint band indicating random insertion. Bio Image Analyzer-BSA
2000K (Fuji Photo Film Co., Ltd.) and 3.2K
When the ratio of the radiation intensity of the 4.0 Kb band to the band b was measured, the results shown in Table 3 were obtained. From this,
By using this transformation method, the gene can be stably transformed into chromosome,
And that it can be inserted at a high copy number.

[0037]

Example 5 Esterase activity of various transformants using 7-β- (4-carboxybutanamide) cephalosporanic acid as a substrate was measured in the following manner. Toluene-treated activated cell suspensions are obtained from various transformants by the method of Step 3 of Example 3. 100 μl of this was mixed with 400 μl of esterase reaction buffer [2.5 mg / ml 7-β- (4
-Carboxybutanamide) cephalosporanic acid, 25
0 mM Tris-HCl pH 7.5]
After incubating for 30 minutes at room temperature, the reaction is stopped by mixing with 500 μl of methanol. The reaction solution was analyzed by HPLC (Zovac BP-NH ₂ column, 12% acetonitrile-8%
Acetic acid-4% methanol solvent, flow rate 1.8 ml per minute, detection at a wavelength of 254 nm) to produce deacetyl-7-β-
(4-Carboxybutanamide) Cephalosporanic acid is quantified, and the esterase activity that produces 1 μmol of the substance per minute is defined as 1 unit.

As a result, as shown in Table 4 (indicated by the activity value per 1 ml of the culture solution), CBS4095 and KC103 transformed by the D-amino acid oxidase gene (pADH1) and a part thereof (pTHY83) were:
The esterase activity was significantly lower than that of the parent strain.

[0039]

[Table 1]

[0040]

[Table 2]

[0041]

[Table 3]

[0042]

[Table 4]

[0043]

According to the present invention, Trigonopsis variabilis transformed by a genetic engineering technique has a higher D than that of the parent strain.
-Shows amino acid oxidase activity and low esterase activity, and is very useful for producing derivatives of cephalosporin C and the like. In addition, the use of a transformation system using Trigonopsis variabilis as a host enables efficient expression of various industrially useful genes.

[Brief description of the drawings]

FIG. 1. N-terminal amino acid sequence of D-amino acid oxidase

FIG. 2 is a DNA probe for cloning a D-amino acid oxidase gene.

FIG. 3 shows the nucleotide sequence of an EcoR fragment containing the N-terminal amino acid code of the D-amino acid oxidase gene.

FIG. 4 pDAOC2-12 restriction enzyme map

FIG. 5 shows the base sequence of the entire region of the D-amino acid oxidase gene code from which the intron portion has been deleted.

FIG. 6 shows the base sequence of the entire region of the D-amino acid oxidase gene code from which the intron portion has been deleted.

FIG. 7 is a schematic diagram showing the construction of plasmid 13ME1 having a part of the D-amino acid oxidase gene excluding introns.

FIG. 8 is a schematic diagram showing the construction of a plasmid pTDO1 having a D-amino acid oxidase gene excluding introns.

FIG. 9 is a schematic diagram showing the construction of a plasmid pTHY83 having a hygromycin B resistant marker.

FIG. 10 is a schematic diagram showing the construction of a plasmid pADH1 for introducing a D-amino acid oxidase gene. In the figure, the bold line is the DNA portion derived from Trigonopsis variabilis containing the D-amino acid oxidase gene. In the figure, only the restriction enzyme cleavage sites involved in the process are shown.

──────────────────────────────────────────────────続 き Continued on front page (51) Int.Cl. ⁶ identification code FI (C12N 9/06 C12R 1: 645) (58) Investigated field (Int.Cl. ⁶ , DB name) C12N 1/14-1 / 19 C12N 15/00-15/90 C12N 9/06 BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A plurality of D-amino acid oxidase genes
And D- characterized in that the marker gene is retained on the chromosome.
Parent transformed with the amino acid oxidase gene
D-amino acid oxidase activity is higher than that of
Trigonopsis variabilis with low sterase activity . 2. The Trigonopsis variabilis according to claim 1, wherein a plurality of D-amino acid oxidase genes and a marker gene are retained on a chromosome. 3. A method for genetic recombination using Trigonopsis variabilis as a host.