JP2001275689A - Phosphoserine phosphatase gene of coryneform bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a gene encoding a phosphoserine phosphatase of a coryneform bacterium and useful in a microbial industry for breeding an L-serine producing bacterium of a coryneform bacterium, or the like. SOLUTION: A DNA fraction complementary to the ser B deficit of Escherichia coli is isolated from a chromosomal DNA library of Brevibacterium flavum, and a DNA encoding a protein expressed by the following (A) or (B) as an open leading frame present in the DNA fraction is obtained. (A) A protein having a specific amino acid sequence derived from Brevibacterium flavum. (B) A protein consisting of an amino acid sequence including the substitution, loss, insertion, addition or inversion of one or more amino acids in the amino acid sequence of the above (A), and having phosphoserine phosphatase activity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a DNA encoding a phosphoserine phosphatase of a coryneform bacterium. The DNA can be used for microbial industry, such as breeding of L-serine-producing coryneform bacteria.

[0002]

2. Description of the Related Art As a conventional method for producing L-serine by fermentation, a strain capable of converting glycine and sugar to L-serine is used, and up to 14 g / L of L-serine in a medium containing 30 g / L of glycine. -There is a report that serine was produced. The conversion yield of glycine to L-serine in this method corresponds to 46% (Kubota K. Agr).
icultural Biological Chem
Istry, 49, 7-12, 1985). Also, using a strain capable of converting glycine and methanol to L-serine, 100 g / L glycine to 53 g / L L was used.
-Serine can be produced (T. Yoshida et a
l. Journal of Fermentatio
n and Bioengineering, Vol.
79, no. 2, 181-183. 1995). Also,
In the method using Nocardia bacteria, it is known that L-serine-producing ability is improved by breeding resistant bacteria such as serine hydroxamate and azaserine (Japanese Patent Publication No. 57-1235). However, in these methods, glycine which is a precursor of L-serine must be used, and the operation is complicated and disadvantageous in cost.

[0003] D-serine, α-methyl-serine, o-methylserine, isoserine, serine hydroxyl and the like are strains that can ferment L-serine directly from sugar and do not require addition of a precursor of L-serine to the medium. Corynebacterium glutamicum resistant to mate and 3-chloroalanine is known, but its L-serine accumulation is 0.8 g / L.
(Journal of the Japanese Society of Agricultural Chemistry, Vol. 48, No. 3, p201-208, 1974), and further improvement of the strain was desired for industrial direct fermentation of L-serine.

On the other hand, in a coryneform bacterium, a vector plasmid (see US Pat. No. 4,514,502) capable of autonomous growth in a cell and having a drug resistance marker gene, and a method for introducing the gene into the cell (Japanese Patent Laid-Open No. 2-2077)
No. 91) is disclosed, and L using these techniques is disclosed.
-Amino acid producing bacteria are being grown. Regarding L-serine, in coryneform bacteria having L-serine producing ability, a gene encoding D-3-phosphoglycerate dehydrogenase (serA gene) in which feedback inhibition by L-serine has been released (European Patent Application Publication No. 9
43,687) or a gene encoding phosphoserine phosphatase (serB) or a gene encoding phosphoserine transaminase (ser
C) It has been found that amplification of (EP-A-931,833) improves L-serine productivity. However, the serB gene is not compatible with Escherichia coli (GenBank accession X0304).
6, M30784), yeast (GenBank acc)
ession U36473), Helicobacter pylori (GenBank accession AF006)
039), but not known in coryneform bacteria.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made from the above viewpoint, and has as its object to provide a gene encoding phosphoserine phosphatase of coryneform bacteria.

[0006]

Means for Solving the Problems The inventors of the present invention have prepared a DNA encoding a SerB deletion of Escherichia coli from a chromosomal DNA library of Brevibacterium flavum.
The A fragment was obtained. Then, from the DNA fragment, a known s
subcloning an open reading frame having homology to the erB gene;
When introduced into a serB-deficient strain of E. coli, the serB deletion was not complemented. However, when the ORF was forcibly expressed using a lac promoter, it was found that the serB deletion was complemented, and it was confirmed that the ORF was a serB homologous gene of Brevibacterium flavum, and further, the operon was formed. This suggests that there is no promoter specific to the gene.

The present invention has been made as described above, and its gist is as follows. (1) A protein represented by the following (A) or (B): (A) a protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing; (B) the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acids, and having a phosphoserine phosphatase activity; protein. (2) DNA encoding a protein shown in the following (A) or (B). (A) a protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing; (B) the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acids, and having a phosphoserine phosphatase activity; protein. (3) a DNA that hybridizes with a DNA encoding a protein having the amino acid sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having phosphoserine phosphatase activity. (4) The DNA according to (3), wherein the stringent condition is that washing is performed at 60 ° C. at a salt concentration corresponding to 1 × SSC and 0.1% SDS. (5) DNA shown in the following (a) or (b) (2)
DNA. (A) DN containing a base sequence consisting of base numbers 210 to 1547 in the base sequence described in SEQ ID NO: 1 in the sequence listing
A. (B) Among the base sequences described in SEQ ID NO: 13 in the sequence listing,
A DNA that hybridizes with a probe having a base sequence consisting of base numbers 210 to 1547 or a part thereof under a stringent condition and encodes a protein having a phosphoserine phosphatase activity. (6) The DNA according to (5), wherein the stringent condition is that washing is performed at 60 ° C. at a salt concentration corresponding to 1 × SSC and 0.1% SDS. (7) A vector containing the DNA of any one of (1) to (6). (8) A bacterium in which the activity of phosphoserine phosphatase encoded by the DNA according to any one of (1) to (6) is increased. (9) A method for producing L-serine, comprising culturing the bacterium of (8) in a medium, producing and accumulating L-serine in the medium, and collecting L-serine from the medium.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The DNA of the present invention may be a Brevibacterium flavum, such as Brevibacterium flavum ATCC 14067.
From the chromosomal DNA of the strain, using the chromosomal DNA as a template, PCR (polymerase chain reaction) using a primer having the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing and a primer having the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing ). These primers were each provided with a restriction enzyme EcoR or Sal near the 5 'end.
The digestion of the amplification product with these restriction enzymes results in EcoRI and SalI
It can be inserted into a vector having a truncated end.

The nucleotide sequence of the above primer is determined by the Escherichia coli serB deletion strain ME8320 (thi, serB, z
ji-1 :: Tn10) (available from the National Institute of Genetics)
These primers are designed based on the nucleotide sequence of a DNA fragment that complements the erB deletion, and using these primers, the coding region and the adjacent region of the serB homologous gene (5 ′ untranslated region of about 200 bases and about 300 bases) 3 ′ untranslated region).

The DNA of the present invention obtained as described above
SEQ ID NO: 1 shows an example of the nucleotide sequence of SEQ. In addition, only the amino acid sequence is shown in SEQ ID NO: 2.

The serB homologous gene is ME8320.
Present in a DNA fragment that complements the serB deletion of the strain, and that is present in Escherichia coli and yeast (Saccharomyces
S. cerevisiae) and found as an open reading frame (ORF) having homology to the serB gene of S. cerevisiae. A DNA fragment containing a flanking region of sufficient length to contain this ORF and promoter is
PCR using the primers shown in SEQ ID NOs: 3 and 4
By Brevibacterium flavum ATCC 1406
Acquired from 7 strains and introduced into ME8320 strains.
The B deletion was not complemented. Therefore, initially, the O
RF was also not considered to be a serB homologous gene. However, when this ORF was ligated to the lacZ promoter and forcedly expressed, the serB deletion was complemented,
It was confirmed that RF was a serB homologous gene.

In addition, DN which complements the aforementioned serB deletion
In the A fragment, another ORF was found immediately upstream of the ORF of the serB homologous gene. These facts suggest that these ORFs form an operon and that there is no promoter region or the like immediately upstream of the serB homologous gene.

[0013] Initially, an attempt was made to obtain a serB homologous gene from Brevibacterium flavum using the sequence of a known serB gene. That is, the base sequence and amino acid sequence of the serB gene of another known species are compared, a region where the amino acid sequence is highly conserved among various organisms is searched, and PCR is performed based on the base sequence of the same region. Was prepared, and it was intended to amplify the serB homologous gene using the primer. But,
Such conserved regions were very small, and it was determined that it was difficult to obtain the target gene by PCR.
Therefore, a complementation test using a serB-deficient strain was performed.

[0014] The DNA of the present invention comprises
Cloning and PC by complementation test using defective strain
R, obtained by subcloning using the oligonucleotides prepared based on the nucleotide sequence of the DNA of the present invention, by hybridization with the chromosome D of Brevibacterium flavum.
It can also be obtained from the NA Library.

Preparation of genomic DNA library, hybridization, PCR, preparation of plasmid DNA,
Methods such as DNA cleavage and ligation and transformation are described in Sambroo
k, J., Fritsch, EF, Maniatis, T., Molecular Cloning, C
Old Spring Harbor Laboratory Press, 1.21 (1989).

The DNA of the present invention may comprise one or several amino acid substitutions at one or more positions, as long as the activity of the encoded phosphoserine phosphatase is not impaired.
It may encode a phosphoserine phosphatase containing deletions, insertions, additions, or inversions. here,
The term "several" varies depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, but is specifically 2 to 200, preferably 2 to 50, and more preferably 2 to 20. . Further, the DNA of the present invention differs from the amino acid sequence shown in SEQ ID NO: 2 by 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably as long as the activity of the encoded phosphoserine phosphatase is not impaired. 80% or more, particularly preferably 90%
It may encode an amino acid sequence having at least% homology.

DNA encoding a protein substantially the same as phosphoserine phosphatase as described above can be obtained by substituting, deleting, inserting, adding or reversing amino acid residues at a specific site by, for example, site-directed mutagenesis. It is obtained by modifying the nucleotide sequence to include the position. The modified DNA as described above can also be obtained by a conventionally known mutation treatment. Examples of the mutation treatment include a method of in vitro treating a DNA encoding phosphoserine phosphatase with hydroxylamine and the like, and a method of irradiating a microorganism having a DNA encoding phosphoserine phosphatase, for example, a bacterium belonging to the genus Escherichia with ultraviolet irradiation or N
-Methyl-N'-nitro-N-nitrosoguanidine (NTG)
Alternatively, a method of treating with a mutagen commonly used in mutagenesis treatment, such as nitrous acid, may be mentioned.

The substitution, deletion, insertion, addition, or inversion of a base as described above may occur naturally due to individual differences of microorganisms having phosphoserine phosphatase, differences in species or genera, and the like. The resulting mutation (mutant or variant) is also included.

The DNA having the above mutation is expressed in appropriate cells, and the expression product is examined for phosphoserine phosphatase activity, whereby DNA encoding a protein substantially identical to phosphoserine phosphatase can be obtained. In addition, from a DNA encoding a phosphoserine phosphatase having a mutation or a cell carrying the same, for example, a probe having a base sequence consisting of base numbers 210 to 1547 out of the base sequence shown in SEQ ID NO: 1 in the sequence listing is stringent. By isolating a DNA that hybridizes under the conditions and encodes a protein having phosphoserine phosphatase activity, a DNA that encodes a protein substantially identical to phosphoserine phosphatase can also be obtained. The term "stringent conditions" as used herein refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. It is difficult to quantify these conditions clearly, but as an example, DNAs with high homology
For example, DNAs having homology of 50% or more hybridize to each other and DNAs having lower homology do not hybridize to each other, or normal Southern hybridization washing conditions, for example, 60 ° C., 1 × SS
C, 0.1% SDS, preferably 0.1 × SSC,
Conditions for washing at a salt concentration corresponding to 0.1% SDS are exemplified. DNA encoding a protein having 60% or more, preferably 70% or more, more preferably 80% or more, particularly preferably 90% homology with the nucleotide sequence shown in SEQ ID NO: 1 and having phosphoserine phosphatase activity, 2 is a DNA of the present invention.

A part of the base sequence of SEQ ID NO: 1 can be used as a probe. Such a probe can be prepared by PCR using an oligonucleotide prepared based on the nucleotide sequence of SEQ ID NO: 1 as a primer and a DNA fragment containing the nucleotide sequence of SEQ ID NO: 1 as a template. When a DNA fragment having a length of about 300 bp is used as a probe, the washing conditions for hybridization are 50 ° C., 2 × SSC, 0.1% SDS.
Is mentioned.

Some of the genes that hybridize under the above-mentioned conditions have a stop codon in the middle,
Includes those that have lost activity due to mutations in the active center,
For them, the phosphoserine phosphatase activity can be linked to a commercially available active expression vector, for example, Lewis,
I. Pizer, J.M. B. C. 238 (1
2), can be easily removed by measuring according to the method of 3934-3944 (1963).

The DNA of the present invention is prepared by connecting a suitable vector, for example, a vector DNA capable of autonomous replication in cells of Escherichia coli and / or coryneform bacteria, to prepare a recombinant DNA, which is then transferred to Escherichia coli cells. If introduced, later operations will be easier. As a vector capable of autonomously replicating in Escherichia coli cells, a plasmid vector is preferable, and a vector capable of autonomous replicating in a host cell is preferable.
UC18, pBR322, pHSG299, pHSG3
99, pHSG398, RSF1010 and the like.

The preparation of the recombinant DNA is performed by using a transposon (WO02 / 02627, International Publication Pamphlet, WO9
3/18151 International Publication Pamphlet, European Patent Publication 0
445385, JP-A-6-46867, Verte
s, A. A. et al. , Mol. Mic
robiol. , 11, 739-746 (199
4), Bonami, C.I. , Et al. , Mo
l. Microbiol. , 14, 571-58
1 (1994), Vertes, A. et al. A. et
al. , Mol. Gen. Genet. , 2
45, 397-405 (1994); Jagar,
W. et al. , FEMS Microbio
logic Letters, 126, 1-6 (1
995), JP-A-7-107796, JP-A-7-32
7680), phage vector, chromosome recombination (Experiments in Molecular)
Genetics, Cold Spring Har
Bor Laboratory Press (197
2); Matsuyama, S .; and Mizus
Hima, S .; , J. et al. Bacteriol. , 162,
1196 (1985)) or the like.

When a DNA fragment capable of autonomously replicating a plasmid in a coryneform bacterium is inserted into these vectors, the vector is used as a so-called shuttle vector capable of autonomous replication in both Escherichia coli and coryneform bacteria. be able to.

The following are examples of such a shuttle vector. The microorganisms carrying each vector and the deposit numbers of the international depositary organizations are shown in parentheses. pHC4 Escherichia coli AJ12617
(FERM BP-3532) pAJ655 Escherichia coli AJ11882 (FERM BP-13
6), Corynebacterium glutamicum SR8201
(ATCC39135) pAJ1844 Escherichia coli AJ11883 (FERM BP-137), Corynebacterium glutamicum SR8202 (ATC
C39136) pAJ611 Escherichia coli AJ
11884 (FERM BP-138) pAJ3148
Corynebacterium glutamicum SR8203 (A
TCC39137) pAJ440 Bacillus subtilis AJ11901 (FERM BP-140)

These vectors can be obtained from the deposited microorganism as follows. The cells collected during the logarithmic growth phase were lysed using lysozyme and SDS, centrifuged at 30,000 × g, and polyethylene glycol was added to the supernatant obtained from the lysate. Cesium chloride-ethidium bromide equilibrium density gradient centrifugation Separation and purification by separation.

To transform a plasmid by introducing it into Escherichia coli A. The method of Morrison (Methods in Enzymology, 6
8,326, 1979) or by treating recipient cells with calcium chloride to increase DNA permeability (Man.
del, M .; and Higa, A .; , J. et al. Mo
l. , Biol. , 53, 159 (1970)).

In order to transform a coryneform bacterium by introducing a plasmid, an electric pulse method (Sugimoto et al., JP-A-2-20
No. 7791). Phosphoserine phosphatase can be produced by expressing the DNA of the present invention using an appropriate host-vector system.

As a host for expressing the DNA of the present invention, various prokaryotic cells including coryneform bacteria such as Brevibacterium flavum, Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae can be used. And various eukaryotic cells, animal cells, and plant cells. Among them, prokaryotic cells, in particular, coryneform bacteria, Escherichia
E. coli and Bacillus subtilis are preferred.

The DNA of the present invention does not have a promoter, for the expression upstream of DNA of the present invention, there lac acting in the host cells, trp, necessary to join promoters such as P _L. When a vector containing a promoter is used as the vector, ligation of the DNA of the present invention with the vector and the promoter can be performed at once. Such vectors include pMW219 containing the lacZ promoter (available from Nippon Gene).
And the like.

When the DNA of the present invention is highly expressed, a plasmid containing the DNA may be destabilized. In such a case, a low copy vector may be used. Transformation is
For example, as described for Escherichia coli K-12, recipient cells may be treated with calcium chloride and treated with D.
Methods to increase NA permeability (Mandel, M. and Higa, A., J. Mo
l., Biol., 53, 159 (1970)) and a method for preparing competent cells from cells in the growth stage and introducing DNA as described in Bacillus subtilis (Dunca).
n, CH, Wilson, GAand Young, FE, Gene, 1,153 (1977)
) Can be used. Alternatively, cells of a DNA recipient, such as are known for Bacillus subtilis, actinomycetes and yeast, are transformed into protoplasts or spheroplasts that readily incorporate the recombinant DNA and the recombinant DNA is introduced into the DNA recipient. Method (Chan
g, S.and Choen, SN, Molec.Gen., Genet., 168.111 (197
9); Bibb, MJ, Ward, JMand Hopwood, OA, Nature, 274,
398 (1978); Hinnen, A., Hicks, JBand Fink, GR, Proc. N
Atl. Acad. Sci. USA, 75 1929 (1978)) is also applicable. In addition, coryneform bacteria can be treated by the electric pulse method (Japanese Patent Laid-Open No. 2-20).
No. 7791) is also effective. These methods may be appropriately selected depending on the cells used as the host.

The cells into which the DNA of the present invention has been introduced as described above are cultured in a medium, and phosphoserine phosphatase is produced and accumulated in the culture, and the phosphoserine phosphatase is collected from the culture. Thereby, phosphoserine phosphatase can be produced. The medium used for the culture may be appropriately selected depending on the host used.

The phosphoserine phosphatase produced as described above may be used, if necessary, in the form of a conventional enzyme, such as ion exchange chromatography, gel filtration chromatography, adsorption chromatography, and solvent precipitation, from a cell extract or medium. It can be purified using a purification method.

The DNA of the present invention can be used for breeding L-serine producing bacteria such as coryneform bacteria. That is, phosphoserine phosphatase activity is imparted or enhanced by introducing the DNA of the present invention in a form in which it can be expressed, and as a result, L-serine producing ability is imparted or enhanced. In addition, the enhancement of phosphoserine phosphatase activity is not only attributable to the increase in the copy number of the serB homologous gene, but also to the increase of ser on the chromosome of Brevibacterium flavum.
It can also be performed by modifying the expression control sequence so as to enhance the expression of the B homologous gene. Chromosome DN
The modification of the expression control sequence on A is performed, for example, by replacing the expression control sequence such as the promoter of the operon containing the serB homologous gene with a strong one (see JP-A-1-215280).

Examples of coryneform bacteria that can be used for breeding L-serine producing bacteria include the following wild strains. Corynebacterium acetoside film ATCC13870 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium carnae ATCC15991 Corynebacterium glutamicum ATCC13032 (Brevibacterium divaricatum) ATCC14020 (Brevibacterium lactofermentum) ATCC13869・ Lilium) ATCC15990 Brevibacterium flavum ATCC14067 Corynebacterium meracecora ATCC17965 Brevibacterium saccharolyticum ATCC14066 Brevibacterium inmariofilm ATCC14068 Brevibacterium roseum ATCC13825 Brevibacterium Chiogenitarisu ATCC19240 micro Corynebacterium ammonia Fi lamb ATCC15354 Corynebacterium thermo-amino monocytogenes AJ12340 (FERM BP-1539)

A mutant strain having resistance to azaserine or β- (2-thienyl) -DL-alanine (EP-A-943,687) may also be used as a starting strain for breeding L-serine-producing bacteria. Can be.

The DNA of the present invention may be introduced into L-serine-producing bacteria together with other enzyme genes involved in L-serine biosynthesis. Such genes include D-
Gene encoding 3-phosphoglycerate dehydrogenase (serA) (EP-A-943,68)
No. 7), a gene encoding phosphoserine phosphatase (serB) and a gene encoding phosphoserine transaminase (serC) (EP-A-9).
31,833). For serA, L
D-3 in which feedback inhibition by serine is released
Mutated genes encoding phosphoglycerate dehydrogenase are preferred (EP-A-943,638)
No. 87).

A microorganism into which the DNA of the present invention has been introduced in a form capable of expression and which has the ability to produce L-serine is cultured in a medium, L-serine is accumulated in the medium, and the L-serine is removed from the medium. By recovering, L-serine can be produced directly from sugar. The microorganism into which the DNA of the present invention has been introduced can also be applied to a method for producing L-serine using a precursor of L-serine such as glycine as long as phosphoserine phosphatase is involved.

The medium used to produce L-serine using the microorganism into which the DNA of the present invention has been introduced includes a carbon source, a nitrogen source, inorganic salts, and, if necessary, amino acids,
An ordinary liquid medium containing an appropriate amount of organic trace nutrients such as vitamins is used. As the carbon source, glucose, sucrose, fructose, sugars such as galactose, starch saccharified liquid containing these sugars, sweet potato molasses, sugar beet molasses,
High test molasses, organic acids such as acetic acid, alcohols such as ethanol, glycerin and the like are also used. As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other organic nitrogen sources used supplementarily, such as oil cakes, soybean hydrolysate, casein hydrolyzate, other amino acids, corn steep liquor,
Peptides such as yeast or yeast extract and peptone are used. As the inorganic ions, phosphate ions, magnesium ions, calcium ions, iron ions, manganese ions, and the like are appropriately added. If the microorganism of the present invention has a required substance such as an amino acid, the required substance must be added.

Cultivation of microorganisms is usually carried out at a pH of 5 to 8 and at a temperature of 25.
It is carried out under aerobic conditions in the range of ４０40 ° C. Culture medium p
H is adjusted to a predetermined value within the above range by using an inorganic or organic acid, an alkaline substance, urea, calcium carbonate, ammonia gas or the like.

L-serine is collected from the fermentation broth by, for example, separating and removing the cells, treating with an ion exchange resin, or concentrating, cooling, crystallizing, membrane separation, or other known methods in combination. In order to remove impurities, purification may be performed using a conventional activated carbon adsorption method and a recrystallization method.

[0042]

The present invention will be described more specifically with reference to the following examples.

<1> Preparation of Brevibacterium flavum chromosome DNA library using high copy vector A chromosome is prepared from Brevibacterium flavum ATCC 14067, and the fragment becomes about 4 to 6 kb using restriction enzyme Sau3AI. Was partially decomposed as follows. The obtained fragment was ligated to a BamHI digestion of the coryneform bacterium and Escherichia coli shuttle vector pSAC4 with BamHI.

PSAC4 was prepared as follows. In order to enable the Escherichia coli vector pHSG399 (Takara Shuzo Co., Ltd.) to replicate autonomously in coryneform bacteria, a plasmid pHM1519 (Miwa, k. Et al., Agric) capable of autonomous replication in coryneform bacteria already obtained. .
Biol. Chem., 48 (1984) 2901-2903) was introduced (Japanese Unexamined Patent Publication No. 5-7491). Specifically, pH
M1519 was digested with restriction enzymes BamHI and KpnI to obtain a gene fragment containing an origin of replication, and the obtained fragment was blunt-ended using a Blunting kit manufactured by Takara Shuzo Co., Ltd., followed by SalI linker (Takara Shuzo Co., Ltd.). ) Using p
It was inserted into the SalI site of HSG399 to obtain pSAC4.

The ligation product was dissolved in TE buffer, and Escherichia coli DH5α was transformed by electroporation. An SOC medium (composition: bactotryptone 20 g / L, yeast extract 5 g / L, NaCl 0.5 g / L, glucose 10 g / L) was added to the transformant,
Incubate at 7 ° C. for 1 hour, and equilibrate 2 × LB medium (containing 40 mg / L of chloramphenicol. Composition of LB medium: 1% tryptone, 0.5% yeast extract, 0.1% NaCl)
%, Glucose 0.1%, pH 7) and cultured at 37 ° C. for 2 hours. 4 × L containing 40% glycerol in culture
After adding an equal amount of the B medium, the mixture was stored at -80 ° C.

The above culture was inoculated into an LB medium, and the plasmid was recovered from the obtained cells. The plasmid DNA was precipitated with ethanol and introduced into a serB-deficient strain ME8320 (thi, serB, zji-1 :: Tn10) of Escherichia coli (obtained from the National Institute of Genetics) by electroporation. The ME8320 strain contains 140 mg of vitamin B ₁
/ L cannot be grown on M9 medium, and L-serine 4
It was confirmed that the cells could be grown on the same medium containing 0 mg / L.

After the transformation, the cells are washed and vitamin B ₁
And chloramphenicol in an M9 agar medium containing 40 mg / L, and cultured at 37 ° C. for 3 to 4 days to form colonies. Plasmids were prepared from each colony, and the size was examined by electrophoresis. As a result, significant deletion was observed. It was considered that the high expression of the serB gene in the cells caused the instability of the plasmid, and the library was prepared again using a low-copy vector.

<2> Construction of Brevibacterium flavum chromosomal DNA library using low copy vector and isolation of SerB gene Chromosomes were prepared from Brevibacterium flavum ATCC 14067 and digested with Sau3AI. This reaction was adjusted so that the center of the distribution of the cut fragments was around 3 kbp or more. About 200 μg of the obtained digest was added to 10%
Separate by ４０40% sucrose density gradient centrifugation.
C LIQUID CHARG-ER (ADVANTEC) and MICRO TUBE PUMP
(EYELA) to fractionate 1 ml each. The sucrose density gradient centrifugation was performed using a Beckman SW28 rotor,
Performed at 0 ° C. and 260,000 rpm for 26 hours. A fraction having a DNA fragment whose distribution center was about 3 to 4 kbp or more was precipitated with ethanol, and then the DNA fragment was purified using Microcon-50 (Millipore).

The chromosomal DNA fragment obtained as described above was ligated to a low-copy vector pMW219 (Nippon Gene, BamHI-cut, dephosphorylated), and the ligation product was dissolved in TE buffer. Escherichia coli DH5α was transformed by electroporation. An SOC medium was added to the transformant, incubated at 37 ° C. for 1 hour, and an equal volume of 2 × LB medium (kanamycin 25
mg / L) and cultured at 37 ° C for 2 hours.
After adding an equal amount of 4 × LB medium containing 40% glycerol to the culture, the culture was stored at −80 ° C.

The above culture solution was inoculated into an LB medium, and the plasmid was recovered from the obtained cells. Plasmid DNA was precipitated with ethanol and introduced into ME8320 by electroporation.

After the transformation, the cells are washed, and vitamin B ₁
Then, the cells were spread on an M9 agar medium containing 25 mg / L of kanamycin and cultured at 37 ° C. for 3 to 4 days to form colonies. Each colony was again spread on the same medium and an LB medium containing 25 mg / L of kanamycin, a viable strain was selected, and a plasmid was prepared from the same strain.

In order to determine the nucleotide sequence of the inserted fragment of the obtained plasmid, the nucleotide sequence was first started from both ends of the multicloning site of the vector using universal primers. Then, about 300-400b
The reading was continued by p, and about 5 kbp each of both ends of the inserted fragment was determined. When an open reading frame (ORF) was searched for the determined base sequence,
One ORF was found that showed homology to a known phosphoserine phosphatase from another species (encoded by the serB gene). Although several regions having homology were found in the ORF, the homology with Escherichia coli was 43% in the amino acid sequence and 49.% in the base sequence.
The homology with yeast (Saccharomyces cerevisiae) is 36.6% in the amino acid sequence and 5 in the base sequence.
It was as low as 0.9%. The nucleotide sequence and the amino acid sequence were analyzed using the Genetyx-Mac computer program (software development, Tokyo). Homology analysis was performed by Lipman and Pe.
ason (Science, 227, 1435-1441, 1985).

The known ser determined as described above
The ORF having homology to the B gene and the nucleotide sequence of the adjacent region (SEQ ID NO: 1) and the amino acid sequence that can be encoded by the ORF (SEQ ID NO: 2) are shown in the sequence listing.

<3> Cloning of ORF having homology with serB In order to confirm whether the ORF homologous to the serB gene as described above is actually a serB homologous gene, the ORF and its upstream and downstream About 200-300b downstream
cloning of a chromosomal DNA fragment containing p
A complementation test was performed on the defective strain.

In order to obtain a target DNA fragment by PCR, primers having the nucleotide sequences shown in SEQ ID NOs: 3 and 4 were designed. Using these primers, PCR was performed using chromosomal DNA of Brevibacterium flavum ATCC 14067 as a template. The PCR reaction is
Using Pyrobest DNA polymerase (Takara Shuzo Co., Ltd.), 98 ° C. for 10 seconds, 55 ° C. for 30 seconds,
The reaction at 72 ° C. for 2 minutes was repeated 30 times.

Amplified DNA fragment and vector pMW
219 was digested with EcoRI and SalI and ligated to construct plasmid pMW219BSB. PCR
The absence of the error introduced by the reaction was confirmed by sequencing the amplified fragment. pMW219BS
In B, the ORF was inserted in the opposite direction to the lacZ promoter contained in the vector.

PMW219BSB was introduced into strain ME8320 in the same manner as in <2>, and kanamycin 25 mg / L was introduced.
Was spread on an LB medium containing. 10 formed colonies
Each strain was picked up, inoculated on M9 medium and cultured, but no growth was observed.

<4> Forced expression of ORF having homology with serB The orientation of the inserted fragment of pMW219BSB is changed, the ORF is oriented downstream in the lacZ promoter in the vector so that it is in the forward direction, and the ORF is forcibly expressed. I did it.
The obtained plasmid was introduced into ME8320 strain, and applied to an LB medium containing 25 mg / L of kanamycin. The formed colonies grew on the minimal medium.

From the above results, it was clarified that the ORF having homology with the above-mentioned serB gene has the ability to complement the serB deletion of Escherichia coli, and that the ORF is a serB homologous gene of Brevibacterium flavum. Was done.

In the cloned fragment obtained in the above <2>, another ORF was found immediately upstream of the ORF having homology to the serB gene. Therefore, these OR
F forms an operon, and since there is no promoter region or the like immediately upstream of the ORF having homology with the serB gene, it is considered that pMW219BSB did not complement the serB deletion.

[0061]

According to the present invention, DN encoding phosphoserine phosphatase of Brevibacterium flavum is provided.
A is provided. The DNA of the present invention is L-type coryneform bacterium.
-It can be used for breeding of serine-producing bacteria.

[0062]

[Sequence list] SEQUENCE LISTING <110> Ajinomoto Co., Inc. <120> Phosphoserine phosphatase gene of coryneform bacterium <130> P-8327 <140> <141> 2001-01-26 <150 > JP 2000-23341 <151> 2000-01-27 <160> 4 <170> PatentIn Ver. 2.0

<210> 1 <211> 1875 <212> DNA <213> Brevibacterium flavum <220> <221> CDS <222> (210) .. (1547) <400> 1 gaattcggta ccgcccagcc cttgcatctg actccagtcg ctaaaagcgt ctgatttaag 60 tcggtaccg actaaataag caccagcccc agcagagata atctgccggg gctggtgctt 120 ttcatattcc gacttggggc acccctgaat acatctcacc caatccccca aagctacaca 180 attgtccagc aacgactgat aaatctcca atg tcg tgt tcc gcg ctc aga cat 233 Met Ser Cys Ser Ala Leu Arg His 1 5 gag aca att gtt gcc gtg act gaa ctc atc cag aat gaa tcc caa gaa 281 Glu Thr Ile Val Ala Val Thr Glu Leu Ile Gln Asn Glu Ser Gln Glu 10 15 20 atc gct gag ctg gaa gcc gga cag cag gtt gca ttg cgt gaa ggt tat 329 Ile Ala Glu Leu Glu Ala Gly Gln Gln Val Ala Leu Arg Glu Gly Tyr 25 30 35 40 ctt cct gcg gtg atc aca gtg agc ggt aaa gac cgc cca ggt gtg act 377 Leu Pro Ala Val Ile Thr Val Ser Gly Lys Asp Arg Pro Gly Val Thr 45 50 55 gcc gcg ttc ttt agg gtc ttg tcc gct aat cag gtt cag gtc ttg gac 425 Ala Ala Phe Phe Arg Val Leu Ser Ala Asn Gln Val Gln Val Leu Asp 60 65 70 gtt gag cag tca atg ttc cgt ggc ttt ttg aac ttg gcg gcg ttt gtg 473 Val Glu Gln Ser Met Phe Arg Gly Phe Leu Asn Leu Ala Ala Phe Val 75 80 85 ggt atc gca cct gag cgt gtc gag acc gtc accca act gac 521 Gly Ile Ala Pro Glu Arg Val Glu Thr Val Thr Thr Gly Leu Thr Asp 90 95 100 acc ctc aag gtg cat gga cag tcc gtg gtg gtg gag ctg cag gaa act 569 Thr Leu Lys Val His Gly Gln Ser Val Val Val Glu Leu Gln Glu Thr 105 110 115 120 gtg cag tcg tcc cgt cct cgt tct tcc cat gtt gtt gtg gtg ttg ggg 617 Val Gln Ser Ser Arg Pro Arg Ser Ser His Val Val Val Val Leu Gly 125 130 135 gat ccg gtt gat gcg ttg gat att tcc cgc att ggt cag acc ctg gcg 665 Asp Pro Val Asp Ala Leu Asp Ile Ser Arg Ile Gly Gln Thr Leu Ala 140 145 150 gat tac gat gcc aac att gac acc att cgt ggt att tcg gat tac cct 713 Asp Tyr Asp Ala Asn Ile Asp Thr Ile Arg Gly Ile Ser Asp Tyr Pro 155 160 165 gtg acc ggc ctg gag ctg aag gtg act gtg ccg gat gtc agc cct ggt 761 Val Thr Gly Leu Glu Leu Lys Val Thr Val Pro Asp Val Ser Pro Gly 170 1 75 180 ggt ggt gaa gcg atg cgt aag gcg ctt gct gct ctt acc tct gag ctg 809 Gly Gly Glu Ala Met Arg Lys Ala Leu Ala Ala Lela Thr Ser Glu Leu 185 190 195 200 aat gtg gat att gcg att gag cgt tct ggt ctg cgt cgt tct aag 857 Asn Val Asp Ile Ala Ile Glu Arg Ser Gly Leu Leu Arg Arg Ser Lys 205 210 215 cgt ctg gtg tgc ttc gat tgt gat tcc acg ttg atc act ggt gag gtc 905 Arg Leu Val Cys Phe Asp Cys Ser Thr Leu Ile Thr Gly Glu Val 220 225 230 att gag atg ttg gcg gct cac gcg ggc aag gaa gct gaa gtt gcg gca 953 Ile Glu Met Leu Ala Ala His Ala Gly Lys Glu Ala Glu Val Ala Ala 235 240 245 gtt act cgt gcg atg cgc ggt gag ctc gat ttc gag gag tct ctg 1001 Val Thr Glu Arg Ala Met Arg Gly Glu Leu Asp Phe Glu Glu Ser Leu 250 255 260 cgt gag cgt gtg aag gcg ttg gct ggt ttg gat 10 Arg Glu Arg Val Lys Ala Leu Ala Gly Leu Asp Ala Ser Val Ile Asp 265 270 275 280 gag gtc gct gcc gct att gag ctg acc cct ggt gcg cgc acc acg atc 1097 Glu Val Ala Ala Ala Ile Glu Leu Thr Pro Gly Ala Ar g Thr Thr Ile 285 290 295 cgt acg ctg aac cgc atg ggt tac cag acc gct gtt gtt tcc ggt ggt 1145 Arg Thr Leu Asn Arg Met Gly Tyr Gln Thr Ala Val Val Ser Gly Gly 300 305 310 ttc atc cag gtg ttg gaa ggt ttg gct gag gag ttg gag ttg gat tat 1193 Phe Ile Gln Val Leu Glu Gly Leu Ala Glu Glu Leu Glu Leu Asp Tyr 315 320 325 gtc cgc gcc aac act ttg gaa atc gtt gat ggc aag ctg acc ggc aac 124 Thr Leu Glu Ile Val Asp Gly Lys Leu Thr Gly Asn 330 335 340 gtc acc ggc aag atc gtt gac cgc gct gcg aag gct gag ttc ctc cgt 1289 Val Thr Gly Lys Ile Val Asp Arg Ala Ala Lys Ala Glu Phe Leu Arg 345 350 355 360 gag ttc gct gcg gat tct ggc ctg aag atg tac cag act gtc gct gtc 1337 Glu Phe Ala Ala Asp Ser Gly Leu Lys Met Tyr Gln Thr Val Ala Val 365 370 375 375 ggt gat ggc gct aat gac atc gat atg ctc gcg ggt ctg ggt 1385 Gly Asp Gly Ala Asn Asp Ile Asp Met Leu Ser Ala Ala Gly Leu Gly 380 385 390 gtt gct ttc aac gcg aag cct gcg ctg aag gag att gcg gat act tcc 1433 Val Ala Phe Asn Ala Lys Leu Lys Glu Ile Ala Asp Thr Ser 395 400 405 gtg aac cac cca ttc ctc gac gag gtt ttg cac atc atg ggc att tcc 1481 Val Asn His Pro Phe Leu Asp Glu Val Leu His Ile Met Gly Ile Ser 410 415 420 cgc gac gag atc gat ctg gcg gat cag gaa gac ggc acc ttc cac cgc 1529 Arg Asp Glu Ile Asp Leu Ala Asp Gln Glu Asp Gly Thr Phe His Arg 425 430 430 435 440 gtt cca ttg acc aat gcc taaagattcg cttctcgacccc cc 445 cctcaaggcc cgggctagcg acgggccaca tagcgaggat ccttcggatc cttcgaccgt 1637 tcaggcaatg cagatcgcgt tgcacattcc gaaacagaat ccgccccggc ggacagatgt 1697 gttggaagcg gcggcgagga gtgtggtcaa gctttgcctc gacgaacgag tatccaccga 1757 tcctgatttt cgggcggcct tggaacgttg gtacggacac ttgattcgga aggtgtcacg 1817 tcgcgctcgt aatgcggcgt gggatcgggt gcaagattta cccggcgtga ctgtcgac 1875

<210> 2 <211> 446 <212> PRT <213> Brevibacterium flavum <400> 2 Met Ser Cys Ser Ala Leu Arg His Glu Thr Ile Val Ala Val Thr Glu 1 5 10 15 Leu Ile Gln Asn Glu Ser Gln Glu Ile Ala Glu Leu Glu Ala Gly Gln 20 25 30 Gln Val Ala Leu Arg Glu Gly Tyr Leu Pro Ala Val Ile Thr Val Ser 35 40 45 Gly Lys Asp Arg Pro Gly Val Thr Ala Ala Phe Phe Arg Val Leu Ser 50 55 60 Ala Asn Gln Val Gln Val Leu Asp Val Glu Gln Ser Met Phe Arg Gly 65 70 75 80 Phe Leu Asn Leu Ala Ala Phe Val Gly Ile Ala Pro Glu Arg Val Glu 85 90 95 Thr Val Thr Thr Gly Leu Thr Asp Thr Leu Lys Val His Gly Gln Ser 100 105 110 Val Val Val Glu Leu Gln Glu Thr Val Gln Ser Ser Arg Pro Arg Ser 115 120 125 Ser His Val Val Val Val Leu Gly Asp Pro Val Asp Ala Leu Asp Ile 130 135 140 Ser Arg Ile Gly Gln Thr Leu Ala Asp Tyr Asp Ala Asn Ile Asp Thr 145 150 155 160 Ile Arg Gly Ile Ser Asp Tyr Pro Val Thr Gly Leu Glu Leu Lys Val 165 170 175 Thr Val Pro Asp Val Ser Pro Gly Gly Gly Gly Glu Ala Met Arg Lys Ala 180 185 190 Leu Ala Ala Leu Thr Ser Glu Leu Asn Val Asp Ile Ala Ile Glu Arg 195 200 205 Ser Gly Leu Leu Arg Arg Ser Lys Arg Leu Val Cys Phe Asp Cys Asp 210 215 220 Ser Thr Leu Ile Thr Gly Glu Val Ile Glu Met Leu Ala Ala His Ala 225 230 235 240 Gly Lys Glu Ala Glu Val Ala Ala Val Thr Glu Arg Ala Met Arg Gly 245 250 255 Glu Leu Asp Phe Glu Glu Ser Leu Arg Glu Arg Val Lys Ala Leu Ala 260 265 270 270 Gly Leu Asp Ala Ser Val Ile Asp Glu Val Ala Ala Ala Ile Glu Leu 275 280 285 Thr Pro Gly Ala Arg Thr Thr Ile Arg Thr Leu Asn Arg Met Gly Tyr 290 295 300 Gln Thr Ala Val Val Ser Gly Gly Phe Ile Gln Val Leu Glu Gly Leu 305 310 315 320 Ala Glu Glu Leu Glu Leu Asp Tyr Val Arg Ala Asn Thr Leu Glu Ile 325 330 335 Val Asp Gly Lys Leu Thr Gly Asn Val Thr Gly Lys Ile Val Asp Arg 340 345 350 Ala Ala Lys Ala Glu Phe Leu Arg Glu Phe Ala Ala Asp Ser Gly Leu 355 360 365 Lys Met Tyr Gln Thr Val Ala Val Gly Asp Gly Ala Asn Asp Ile Asp 370 375 380 Met Leu Ser Ala Ala Gly Leu Gly Val Ala Phe Asn Ala Lys Pro Ala 385 390 395 400 Leu Lys Glu Ile Ala Asp Thr Ser Val Asn His Pro Phe Leu Asp Glu 405 410 415 Val Leu His Ile Met Gly Ile Ser Arg Asp Glu Ile Asp Leu Ala Asp 420 425 430 Gln Glu Asp Gly Thr Phe His Arg Val Pro Leu Thr Asn Ala 435 440 445

<210> 3 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 3 gctccagaat tcggtaccgc ccagcccttg catctg 36

<210> 4 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 4 catcgtcgac agtcacgccg ggtaaatctt gcaccc 36

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) C12R 1:19) (C12P 13/06 D (C12P 13/06 C12R 1:19) C12R 1:19) C12N 15/00 ZNAA (72) Inventor Masaichi Sugimoto 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Ajinomoto Co., Ltd. F-term in the laboratory (reference) 4B024 AA03 AA20 BA71 CA01 CA03 CA09 DA06 EA04 FA04 FA11 GA14 GA19 HA14 4B050 CC03 DD02 LL02 LL05 4B064 AE08 CA02 CA19 CC24 DA10 DA16 4B065 AA22Y AA26X AB01 BA03 CA17 CA41 CA60

Claims (9)

[Claims] 1. A protein represented by the following (A) or (B): (A) a protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing; (B) the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acids, and having a phosphoserine phosphatase activity; protein. 2. A DNA encoding a protein represented by the following (A) or (B): (A) a protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing; (B) the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acids, and having a phosphoserine phosphatase activity; protein. 3. A DNA that hybridizes with a DNA encoding a protein having the amino acid sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having a phosphoserine phosphatase activity.
NA. 4. The method according to claim 1, wherein the stringent condition is 1 × S
The DNA according to claim 3, wherein washing is performed at 60 ° C at a salt concentration corresponding to SC and 0.1% SDS. 5. The DNA according to claim 2, which is a DNA shown in (a) or (b) below. (A) DN containing a base sequence consisting of base numbers 210 to 1547 in the base sequence described in SEQ ID NO: 1 in the sequence listing
A. (B) Among the base sequences described in SEQ ID NO: 13 in the sequence listing,
A DNA that hybridizes with a probe having a base sequence consisting of base numbers 210 to 1547 or a part thereof under a stringent condition and encodes a protein having a phosphoserine phosphatase activity. 6. The method according to claim 1, wherein the stringent condition is 1 × S
The DNA according to claim 5, wherein washing is performed at 60 ° C at a salt concentration corresponding to SC and 0.1% SDS. 7. A vector comprising the DNA according to any one of claims 1 to 6. 8. D according to any one of claims 1 to 6,
A bacterium with increased activity of phosphoserine phosphatase encoded by NA. 9. A method for producing L-serine, comprising culturing the bacterium according to claim 8 in a medium, producing and accumulating L-serine in the medium, and collecting L-serine from the medium.