JPH06181776A - Dna encoding serine hydroxylmethyltransferase - Google Patents

Abstract

PURPOSE:To obtain a new DNA encoding serine hydroxylmethyltransferase suitable for producing L-serine useful as medicine, food, etc. CONSTITUTION:A DNA encoding serine hydroxylmethyltransferase containing 154-1,458 base sequence among a base sequence of the formula. A bacterium such as Hyphomicrobium methylovorum (FERM BP-4,039), etc., capable of producing SHMT, is cultured, cells are collected and a chromosome DNA is prepared to give a mixture of a DNA fragment containing a DNA encoding SHMT. The mixture of the fragment is linked to a vector DNA and transduced into EcoL; JM109, etc., to give a gene bank of DNA encoding SHMT. A strain containing a recombinant vector containing DNA encoding SHMT is searched from the gene bank by hybridization method, a base sequence of DNA encoding SHMT is determined and purified to give the objective DNA.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a DNA encoding serine hydroxymethyltransferase (hereinafter abbreviated as SHMT), a recombinant vector incorporating the DNA and a microorganism containing the vector. S
HMT can be used to efficiently produce L-serine useful for medical treatment, foods and the like.

[0002]

SHMT has been found in various microorganisms in addition to rabbit liver, and the nucleotide sequences of DNAs encoding these SHMTs have also been reported [Plamann et al., Nu.
cleicAcids Research , 11 , 2065 (1983), JP-A-2-586
1, Chan et al., Gene , 101 , 51 (1990), Rossbach et al., Molecul
ar Microbiology , 5 , 39 (1991), Martin et al., Journal of B.
ilogical Chemistry, 262 , 5499 (1987), Steiert et al.,
DNA Sequence 1 , 107 (1990)]. Hyphomicrium (Hyphomicrium) used for efficient production of L-serine
As an SHMT produced by a microorganism belonging to the genus obium,
Hyphomicrobium m
SHMT produced by ethyloborum) GM2 is known [Eur. J. Biochem. , 162 , 533 (1987)]. However,
SHM produced by a microorganism belonging to the genus Hyphomicrobium
The nucleotide sequence of DNA encoding T has not been reported.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a DNA encoding SHMT which can be used for efficient production of L-serine, which is useful in medicine, foods and the like.

[0004]

The present invention relates to a DNA encoding SHMT, a recombinant vector incorporating the DNA and a microorganism containing the vector. SH
MT is an enzyme located in the middle of the pathway from glycine to acetyl-CoA through serine and pyruvate in the metabolic degradation process of threonine and glycine, and catalyzes the reaction of producing L-serine from glycine.

SHMT encoded by the DNA of the present invention
Is 1 to 4 of the amino acid sequence shown in SEQ ID NO: 2.
Contains the 35th amino acid sequence. Below, the DN of the present invention
The manufacturing method of A is shown. (A) Preparation of DNA Fragment Having DNA Encoding SHMT and Recombinant Vector The DNA fragment having DNA encoding SHMT can be obtained from any microorganism that produces SHMT encoded by the DNA of the present invention. It can be prepared.
For example, microorganisms belonging to the genus Hyphomicrobium, particularly Hyphomicrobium methyloboram GM2, are preferably used. Hyphomicrobium methyloboram GM2 has been deposited as FERM BP-4039 at the Institute of Microbial Technology, Institute of Industrial Science, as of October 20, 1992.

The microorganism is cultivated, the cells are collected by centrifugation, etc., and chromosomal DNA is prepared from the cells. This chromosomal DNA is partially or completely decomposed using restriction enzymes such as BamHI and HindIII, and a mixture of DNA fragments containing the DNA encoding SHMT is obtained by concentrating means such as ethanol precipitation. On the other hand, the vector DNA used in the present invention may be either a plasmid vector or a phage vector, so long as it can incorporate the DNA of the present invention and can be expressed in a host cell. For example, pBR322, pUC19 (Takara Shuzo), pBlueskrip
tSK (+) (Stratagene), pKK322-3 (Pharma
cia) and λZAP (Stratagene).

Next, a mixture of DNA fragments containing DNA encoding SHMT is ligated to the above vector DNA.
For the ligation, for example, Escherichia coli DNA ligase and T4 DNA ligase (manufactured by Takara Shuzo) are used. This recombinant plasmid
E. coli JM109, ME5427, DH5α, XL1-blue and HB101 strains are transformed or transduced to obtain a recombinant plasmid-containing strain, which is used as a gene bank of DNA encoding SHMT. Transformation and transduction can be performed, for example, by molecular cloning second
Edition; Maniatis et al., Cold Spring Harbor Laboratory, 1982)
It can be performed by the described method.

(B) Selection of Recombinant Vector-Containing Strain Containing SHMT-Encoding DNA A method for searching a strain containing a recombinant vector containing SHMT-encoding DNA from the above gene bank is as follows:
Known hybridization method (Molecular Genetics Experimental Method, p. 85, 1983, Kyoritsu Shuppan) and SHMT activity are deficient due to mutation of SHMT gene, and the requirement for glycine (hereinafter abbreviated as Gly) is required. It can be carried out by a method such as a complementation test using the fact that SHMT activity is expressed by introducing a recombinant vector containing a DNA encoding SHMT into the mutant strain shown, and the requirement for Gly disappears. As the probe DNA fragment used in the hybridization method, a DNA having the sequence is selected by comparing the nucleotide sequences of known SHMT-encoding DNAs and selecting a conserved nucleotide sequence commonly found in high frequency in all strains. SH derived from microorganisms based on fragments
PCR method (Polymerase) using DNA encoding MT
Amplify and separate by chain reaction method).

Examples of the plasmid containing the DNA encoding SHMT of the present invention include pAB2. Escherichia coli ME5, which is an E. coli containing pAB2
427 / pAB2 has been deposited as FERM BP-4040 at the Institute of Microbial Science and Technology of the Agency of Industrial Science as of October 20, 1992.

(C) Determination of nucleotide sequence of DNA encoding SHMT DNA encoding SHMT from the above recombinant plasmid
A known method for determining the nucleotide sequence of, for example, Sanger
Dideoxy method [Pro.Natl.Acad.Sci.USA , 74 ,
5463 (1977)].

The SHMT of the present invention thus obtained
Examples of the DNA encoding the DNA include a DNA containing the 154-1458th base sequence in the base sequence shown in SEQ ID NO: 1.

(D) Purification of SHMT Purification of SHMT encoded by the DNA of the present invention is carried out by a known purification method of SHMT [Miyazaki et al., Eur. J. Biochem., 16
2 , 533 (1987)] can be used. For example, nucleic acid removed from cell-free extract by protamine sulfate, ammonium sulfate fraction,
Chromatography such as DEAE-Sepharose, Phenyl Sepharose column, Matrex-gel-red A (Amicon
(Manufactured by K.K.) and affinity chromatography, gel filtration and the like.

(E) Measurement of SHMT activity The enzyme activity of SHMT is measured by a method of measuring L-serine produced using glycine as a substrate or a method of measuring formaldehyde remaining in the reaction solution [Miyazaki et al., Eur. J. Bioche.
m. , 162 , 533 (1987)].

General genetic engineering techniques such as plasmid preparation, extraction, purification, cleavage and ligation are carried out by molecular cloning (Molecular cloning 2nd edition, Mani).
atis et al., Cold Spring Harbor Laboratory, 1989)
The method described in 1. was used. Examples of the present invention will be shown below.

[0015]

【Example】

Example 1 (1) Chromosome DN of Hyphomicrobium methyloboram
Preparation of A Hyphomicrobium methyloboram GM2 (FERM BP-403
9) MII medium (ammonium hydrogen phosphate 3 g, dipotassium phosphate 2 g, sodium chloride 1 g, magnesium sulfate heptahydrate)
0.2 g, ferrous sulfate heptahydrate 10 mg; manganese sulphate pentahydrate 5 m
g, thiamine hydrochloride 10 μg, riboflavin 20 μg, calcium pantothenate 20 μg, pyridoxine hydrochloride 20 μg, biotin 1 μg, paraaminobenzoic acid 10 μg, nicotinic acid 1
Dissolve 0 μg in 1 liter of water, heat sterilize at 121 ° C for 20 minutes, cool to room temperature, and put in a test tube (diameter 16 mm, length 165 mm) containing 5 ml of 1% methanol-containing medium). , MII agar medium [2% agar (DIFCO
(MII medium containing the product) was cultured at 28 ° C. for 2 days to inoculate about 1 platinum loop of the cells, followed by shaking culture at 28 ° C. for 2 days. Subsequently, 5 ml of the above culture solution was inoculated into a 2 liter Erlenmeyer flask containing 500 ml of MII medium, and cultured again at 28 ° C. for 2 days with shaking.

The culture was centrifuged under cooling to collect the cells, and the cells were suspended in a 0.15 M aqueous sodium chloride solution and centrifuged to wash the cells. 2m of this fungus body
20 mM containing M ethylenediaminetetraacetic acid (EDTA)
It was suspended in 60 ml of Tris buffer (pH 7.5). A lysozyme solution [20 mg / ml of lysozyme was added to this suspension.
Buffer containing 2 mM EDTA (pH)
Dissolved in 7.5)] 7.5 ml was added, and the mixture was allowed to stand at 30 ° C. for 1 hour. To this, 7.5 ml of 20% sodium lauryl sulfate solution was added, and the mixture was slowly stirred. Next, 75 ml of phenol saturated with 10 mM Tris buffer (pH 7.5) containing 1 mM EDTA was added to this solution, and the mixture was sufficiently stirred. The solution is centrifuged and the aqueous layer 70
ml was collected. This phenol extraction operation was repeated 3 times, and to the resulting aqueous layer (75 ml) was added 2.5 M sodium acetate solution (7.5 ml), followed by ethanol (150 ml).
Was slowly added with stirring, and the precipitated chromosomal DNA was wound with a glass rod. 1m after drying this chromosomal DNA
10 mM Tris buffer containing M EDTA (pH 7.
5) Dissolve in 10 ml, add Ribonuclease A (manufactured by Sigma) to 50 μg / ml, and add 37
It was left still at 30 ° C. for 30 minutes. After the same phenol extraction as above, 1 ml of 2.5 M potassium acetate and 20 ml of ethanol were added, and the mixture was allowed to stand at 20 ° C. for 16 hours. Further, the pellet obtained by centrifugation was washed with 70% ethanol and dried to obtain a chromosomal DNA. This is a 10 mM Tris buffer containing 1 mM EDTA (pH
It was dissolved in 7.5) to obtain a chromosomal DNA solution. Save-
It was carried out at 20 ° C.

(2) Preparation of Probe for Hybridization (2-1) Amplification of DNA Fragment for Probe Known Escherichia coli [Plamann
Nuc. Acid. Res., 11 , 2065 (1983)], Salmonella typhimurium [Steiert et al.
DNA Sequence 1 , 107 (1990)], Bacillus stearothermophilus [Miyamoto
Et al., JP-A-2-5861], Bradyrhizobium japonicum
(Bradyrhizobium japonicum) [Rossbach et al., Molecular
Microbiology , 5 , 39 (1991)], the nucleotide sequences of the SHMT-encoding DNAs of microorganisms belonging to Microbiology , 5 , 39 (1991)] were compared, and a sequence commonly found in DNAs encoding SHMTs derived from any of the microorganisms was searched. A combination having a distance of about 500 base pairs from them was selected, and a DNA fragment complementary to the base sequence was used as a primer used for DNA amplification by the PCR method. That is, as the primers, two kinds of single-stranded DNA fragments having the base sequences shown in SEQ ID NOS: 3 and 4 were synthesized. The synthesis is carried out by the solid phase method by the phosphoramidite method [Beaucage et al., Tetrahedron Letter, 22 , 1
859 (1981)], an automatic DNA synthesizer 380A (manufactured by Applied Biosystems) was used.

The DNAs having the nucleotide sequences of SEQ ID NOs: 3 and 4 were used as 5′-side and 3′-side primers, respectively, by the PCR method, from the chromosomal DNA of Hyphomicrobium metylovoram prepared in (1) to produce SHMT. A partial fragment of the DNA coding for was amplified and separated as follows. Chromosome DN of Hyphomicrobium methyloboram prepared in (1) using Gene AmpDNA Amplification Reagent Kit (Takara Shuzo)
Using 1 ng / 100 μl of A as a template, 1. each of synthetic DNAs having the nucleotide sequences of SEQ ID NOS: 3 and 4 above.
DATP, dCTP, dG using 0 μM as a primer
TP and dTTP were each set to 200 μM, Taq DNA polymerase was set to 2.5 U / 100 μl, and the reaction was performed at 94 ° C. for 1.5 minutes, 40 ° C. for 2 minutes, and 72 ° C. for 2 minutes.
(Manufactured by K.K.), repeated 30 times, DN of about 500 base pairs
A fragment was obtained.

(2-2) Cloning of probe DNA fragment DNA which is a PCR reaction product obtained as described above
EcoRI and PstI were added to 20 μl of a solution containing 1 μg of the fragment for digestion and cleavage. Separately vector plasmid pU
20 μl of a solution containing 1 μg of C19 (manufactured by Takara Shuzo) was similarly digested and cleaved. The cleaved DNA fragment and plasmid DNA were purified by phenol extraction and ethanol precipitation. This purified DNA fragment 100
ng and 20 ng of purified plasmid DNA
The cells were suspended in a solution containing mM Tris buffer (pH 7.6), 66 mM magnesium chloride, 10 mM dithiothreitol (DTT) and 0.1 mM ATP. T4 to this
14 units after adding 10 units of DNA ligase (Takara Shuzo)
A recombinant plasmid was prepared by reacting at 16 ° C. for 16 hours and ligating both DNAs. Next, E. coli DH5 α
Strain (Takara Shuzo Co., Ltd.) with LB liquid medium (Bact-peptone 1%,
Bact-yeast extract 0.5%, sodium chloride 1%, pH
7.0) Inoculated into a 200 ml Erlenmeyer flask containing 50 ml, and cultured by shaking at 37 ° C. for 4 hours. Next, the bacterial cells were collected by centrifugation and suspended in 20 ml of a 50 mM calcium chloride solution cooled on ice. After still standing on ice for 20 minutes, the cells were collected by centrifugation under cooling and suspended in 40 ml of a cooled 50 mM calcium chloride solution to prepare a competent cell suspension used for transformation with a plasmid.

After adding 10 μl of the above recombinant plasmid solution to 100 μl of this competent cell suspension,
And allowed to stand for 10 minutes. Then, after heat treatment at 42 ° C. for 90 seconds, the cell suspension was treated with LB containing 100 μg / ml ampicillin and 20 μg / ml 5-bromo-4-chloroindolyl-β-galactoside (X-gal).
It was applied to an agar medium. This plate medium was cultured at 37 ° C. for 24 hours.

White colonies among the appearing colonies were cultured overnight in an LB liquid medium containing 100 μg / ml of ampicillin, and the cells were collected and plasmid DNA was prepared by a conventional method.
Was extracted. This plasmid is D prepared by PCR
It was confirmed by agarose gel electrophoresis that it contained a fragment having the same molecular weight as that of the EcoRI and PstI digestion product of the NA fragment, and it was used as a probe DNA fragment.

(3) Cloning of DNA encoding SHMT (3-1) Preparation of gene bank 10 μg of the chromosomal DNA of Hyphomicrobium methylobolum prepared in (1) was digested with HindIII and then subjected to agarose gel electrophoresis. Fragments less than 2 kb in length were removed. Next, pBluescript SK (+) (Stra
It was digested with HindIII using Tagene) and purified by phenol treatment and ethanol precipitation.

About 100 chromosomal DNAs digested with HindIII
ng and 20 ng of the vector plasmid, (2
-2) was ligated in the same manner as in 2) to prepare a recombinant plasmid.

(3-2) Colony hybridization 1 μg of the DNA fragment for probe prepared in (2-2) was used as a non-radio system DNA labeling and detection kit (DIG).
-ELISA method, catalog No. 1093657, manufactured by Boehringer Mannheim) and labeled with SH
It was used as a probe for detecting DNA encoding MT.

Next, the recombinant plasmid for gene bank prepared in (3-1) was treated with E. coli in the same manner as in (2-2).
It was transformed into DH5α and spread on LB agar medium containing 100 μg / ml of ampicillin. After the colonies were formed, a membrane filter NY13N (manufactured by Schleicher & Scheull) such as a nitrone membrane was placed on the plate agar, and the plate was brought into contact with the colonies on the plate agar, and then immediately peeled off. Turn it over and place it on another fresh LB agar medium containing 100 μg / ml ampicillin and leave at 37 ° C. for about 12 hours.
Incubated for hours. The membrane filter with colonies formed on the upper surface was peeled off from the agar medium and placed on a filter paper containing 0.5N sodium hydroxide for 10 minutes, and on a filter paper containing 0.5M Tris buffer (pH 7.3) for 5 minutes. 0.5M Tris buffer containing 5M sodium chloride (pH 7.
5) on filter paper containing 3) for 2 minutes 2 × SSC (0.3M sodium chloride, 0.03M sodium citrate, pH 7.
The mixture was allowed to stand for 5 minutes on a filter paper containing 0), and then heated at 80 ° C. for about 3 hours. This membrane filter was prepared as described above according to the method of non-radio system DNA labeling and detection kit (Boehringer Mannheim).
A colony having the target DNA was selected as a clone hybridized with the probe using a DNA detection probe encoding MT. 5 from about 18,000 colonies
A positive clone of the strain was obtained.

(4) Determination of nucleotide sequence of DNA encoding SHMT One strain was selected from the positive clones obtained in (3-2) and a plasmid was prepared by a conventional method. This plasmid was named pSA1 and its structure was confirmed by restriction enzyme digestion. A restriction enzyme map of a DNA fragment containing a DNA encoding SHMT of pSA1 is shown in FIG.

Next, a HindIII fragment of pSA1 (about 11 kb)
In the part that seems to code SHMT, (3-
It was identified by hybridization using the probe fragment used in 2), and the base sequence of the DNA encoding SHMT was determined. That is, Taq dye primer cycle s
equencing kit (Applied biosystem) or
Using the kit of Sequenase (United States Biochemical), did using the double-stranded DNA of pBlueskript as a template
The DNA base sequence was determined by an automatic DNA sequencer model 373A (manufactured by Applied Biosystem) by the eoxy chain termination method. The DNA sequence is shown in SEQ ID NO: 1. The amino acid sequence encoded by this DNA base sequence is shown in SEQ ID NO: 2. The molecular weight was calculated to be 45993, and the estimated molecular weight of SHMT produced by the known Hyphomicrobium methyloboram GM2 was 48,000 [Eur.J.Biochem. , 16
2 , 533 (1987)].

(5) Complementation test for mutation by DNA fragment encoding SHMT The cloned SHMT is encoded in the mutant strain E. coli ME5427, which has defective SHMT activity due to mutation of SHMT gene and shows Gly requirement. A plasmid containing DNA was introduced to examine whether the mutated SHMT gene was complementary.

First, as a subcloning of the SHMT-encoding DNA obtained as a 11 kb HindIII fragment, amplification of only the SHMT coding region was performed by the PCR method. First, a primer was designed so as to cover the entire DNA sequence of the coding region, and two types of DNA fragments having the nucleotide sequences of SEQ ID NOs: 5 and 6 were prepared (2-1).
Was synthesized in the same manner as.

The cloned SHMT plasmid (p
SA1) 10 ng as a template, 100 pmole each of synthetic primers having the nucleotide sequences of SEQ ID NOs: 5 and 6
The region encoding SHMT was amplified by using the same PCR method as in (2-1), and the DNA fragment was purified by agarose gel electrophoresis. Separately, a vector plasmid pKK223-3 (manufactured by Pharmacia) having a Taq promoter was cleaved with EcoRI, and a vector plasmid having a blunted cleaved end was prepared with a DNA Blunting Kit (manufactured by Takara Shuzo). The purified PCR product was added to T4
Ligation was performed by ligase reaction to prepare a recombinant plasmid, and this plasmid was named pAB2.

The mutant strain E. coli ME5427 was cultured in the same manner as in (2-2) to prepare competent cells for transformation. When this E. coli ME5427 was transformed with the above recombinant plasmid in the same manner as in (2-2), the resulting transformed clone lost the requirement for Gly, and pAB
It was confirmed that the DNA encoding SHMT on 2 was complementary to the mutation in E. coli.

(6) Measurement of SHMT activity Transformed clone E. coli ME5427 / pAB2 containing plasmid pAB2 obtained in (5) and E. coli ME5427 / pKK223-3 transformed with vector plasmid alone were treated with 100 mg of ampicillin. After culturing overnight in 500 ml of LB liquid medium containing 1 / l, the cells were collected by centrifugation and washed with a 0.15 M sodium chloride aqueous solution. The cells were suspended in 20 ml of 50 mM potassium phosphate buffer (pH 7.5), the suspension was treated with an ultrasonic disintegrator (Kaijo Denki) at 10 ° C or lower at 19 kHz for 15 minutes, and the cells were centrifuged. The body residue was removed and a cell-free extract was prepared.

SHMT activity of this cell-free extract was measured by Miyaza
According to the method of ki et al. [Eur. J. Biochem. , 162 , 533 (1987)], it was measured as follows. 0.5 M potassium phosphate buffer (pH 7.5), 0.4 mM dl-tetrahydrofolic acid, 0.3 mM pyridoxal phosphate, 25 mM
A reaction solution containing formaldehyde, 1.0 M Gly and cell-free extraction so that the total amount was 1.0 ml was reacted at 37 ° C. for 15 minutes, and the reaction was stopped by adding 0.3 ml of 15% trichloroacetic acid. As a control, a reaction solution obtained by removing Gly from the reaction solution was used.

After completion of the reaction, the mixture was centrifuged, and the amount of L-serine in the supernatant was measured by an amino acid analyzer (Amino Acid Analysis System manufactured by JASCO Corporation) using high performance liquid chromatography to quantify the reaction product. . L-serine 1μ per hour
The activity of mole generation was defined as 1 unit (U). The results are shown in Table 1.

[0035]

[Table 1]

As shown in Table 1, the E. coli ME5427 strain containing pAB2 produced SHMT, but the E. coli ME5427 strain containing only the vector did not produce SHMT at all.

[0037]

INDUSTRIAL APPLICABILITY The present invention provides a DNA encoding SHMT produced by Hyphomicrobium methyloboram used for efficient production of L-serine, a vector incorporating the DNA, and a microorganism containing the vector. .

[0038]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1489 Sequence type: Nucleic acid Sequence type: Genomic DNA Sequence characteristics Origin Biological name: Hyphomicobium methyloboram (Hyphomicr
obium methyloborum) Strain name: GM2 Sequence: CGCAACGGCC GTTCCCGCGC CCTTGTTACA CGGGGCTCGG GGTCGGTTGA GTGCTGGTGT 60 CCAGCCTCGT ACTATGGTTC AAAGCTCGTT GGCCGGACTT GACTCCGT GCTTC ATCla GCTCA GCTCA TCT ACACTCT CACTA CCA TCA TCACCA TCACCA TCACCA TCACCA TCACCA TCACCA TCACCA TCACCA TCACCA TCACACCA TCACCA TCACCA TCACACCA TCACCA TCACCA TCACACACCA TCACCA TCACCA TCACCA TCACCA TCACCA TCACCA TCACCA TCACCA TCACCA TCACCA TCACCA TCACCA TCACACCA TCACCA TCACCA TCACCA ACA CGT TTT TTC AAG TCG CAC GTG TCG GAG ACG 222 Gly Thr Ala Ser Thr Ser Arg Phe Phe Lys Ser His Val Ser Glu Thr 10 15 20 GAT CCC GAT ATT TTC AGC GCC ATC CAA AAG GAA TTT GGC CGC CAG CAG 270 Asp Pro Asp Ile Phe Ser Ala Ile Gln Lys Glu Phe Gly Arg Gln Gln 25 30 35 CAC GAG ATC GAG CTG ATC GCG TCT GAG AAC ATC GTT TCG CAG GCC GTT 318 His Glu Ile Glu Leu Ile Ala Ser Glu Asn Ile Val Ser Gln Ala Val 40 45 50 55 CTC GAT GCA GCT GGT TCG GTG CTG ACC AAC AAG TAT GCC GAG GGC TAT 366 Leu Asp Ala Ala Gly Ser Val Leu Thr Asn Lys Tyr Ala Glu Gly Tyr 60 65 70 CCG GGC AAG CGC TAC TAC GGC GGC TGC CAG TAC GTC GAC ATC GTT GAA 414 Pro Gly Lys Arg Tyr Tyr Gly Gly Cys Gln Tyr Val Asp Ile Val Glu 75 80 85 GAC ATC GCG ATC GAC CGC GCA AAG AAG CTC TTC AAC TGC GAA TTC GCG 462 Asp Ile Ala Ile Asp Arg Ala Lys Lys Leu Phe Asn Cys Glu Phe Ala 90 95 100 AAC GTG CAG CCG AAC TCC GGC AGC CAG GCG AAC CAG GGC GTG TTC AAT 510 Asn Val Gln Pro Asn Ser Gly Ser Gln Ala Asn Gln Gly Val Phe Asn 105 110 115 GCG CTC GCG CAG CCG GGC GAC ACC ATC CTC GGT CTC TCG CTC GCT GCC 558 Ala Leu Ala Gln Pro Gly Asp Thr Ile Leu Gly Leu Ser Leu Ala Ala 120 125 130 135 GGT GGT CAC TTG ACC CAC GGC GCG CCG GTG AAC CAG TCC GGC AAG TGG 606 Gly Gly His Leu Thr His Gly Ala Pro Val Asn Gln Ser Gly Lys Trp 140 145 150 TTC AAG GCC GTG CAC TAC ATG GTC AAG CCC GAC TCG CAC CTC ATC GAC 654 Phe Lys Ala Val His Tyr Met Val Lys Pro Asp Ser His Leu Ile Asp 155 160 165 ATG GAC GAA GTG CGC AAG CTG GCC CAG GAG CAC AAG CCG CGC ATC ATC 702 Met Asp Glu Val Arg Lys Leu Ala Gln Glu His Lys Pro Arg Ile Ile 170 175 180 ATC GCT GGT GGT TCG GCC TAT CCG CGC AAG ATC GAT TTC GCT GCA TTT 750 Ile Ala Gly Gly Ser Ala Tyr Pro Arg Lys Ile Asp Phe Ala Ala Phe 185 1 90 195 CGC GCG ATT GCG GAT GAG GTT GGC GCG ATC TTC CTC GTC GAT ATG GCG 798 Arg Ala Ile Ala Asp Glu Val Gly Ala Ile Phe Leu Val Asp Met Ala 200 205 210 215 CAC TTC GCC GGT CTT GTT GCG GCC GGT CTC ATT CCG AGC CCG TTC CCG 846 His Phe Ala Gly Leu Val Ala Ala Gly Leu Ile Pro Ser Pro Phe Pro 220 225 230 CAT GCC CAC GTC GTA ACG ACG ACG ACG CAC AAG ACG CTG CGC GGA CCC 894 His Ala His Val Val Thr Thr Thr Thr Thr His Lys Thr Leu Arg Gly Pro 235 240 245 CGT GGC GGC ATG ATT TTA ACG AAC GAC GCG GAC ATC GCG AAG AAG ATC 942 Arg Gly Gly Met Ile Leu Thr Asn Asp Ala Asp Ile Ala Lys Lys Ile 250 255 260 AAC TCG GCG ATC TTC CCT GGC ATT CAG GGC GGC CCG CTC ATG CAC GTT 990 Asn Ser Ala Ile Phe Pro Gly Ile Gln Gly Gly Pro Leu Met His Val 265 270 275 ATC GCC GGT AAG GCC GTC GCA TTC GGC GAG GCT CTG CGT CCG GAC TTC 1038 Ile Ala Gly Lys Ala Val Ala Phe Gly Glu Ala Leu Arg Pro Asp Phe 280 285 290 295 AAG GTC TAC ATC AAG CAG GTG ATG GAC AAC GCC CGC GCG CTC GGT GAA 1086 Lys Val Tyr Ile Lys Gln Val Met Asp Asn Ala Arg Ala Leu Gly Glu 300 305 310 GTG CTT GTG CAG AAC GGC TTC GCG CTC GTT TCT GGC GGC ACC GAC ACG 1134 Val Leu Val Gln Asn Gly Phe Ala Leu Val Ser Gly Gly Thr Asp Thr 315 320 325 CAC CTC GTT CTC GTC GAT CTG CGG CCG AAG AAG CTG ACC GGT ACG AAG 1182 His Leu Val Leu Val Asp Leu Arg Pro Lys Lys Leu Thr Gly Thr Lys 330 335 340 GCT GAG AAG GCG CTT GGC CGT GCC AAC ATC ACC TGC AAC AAG AAC GGC 1230 Ala Glu Lys Ala Leu Gly Arg Ala Asn Ile Thr Cys Asn Lys Asn Gly 345 350 355 ATT CCG TTC GAC CCC GAG AAG CCG ATG GTG ACG TCG GGC ATT CGT TTG 1278 Ile Pro Phe Asp Pro Glu Lys Pro Met Val Thr Ser Gly Ile Arg Leu 360 365 370 375 GGT TCG CCT GCA GGC ACG ACG CGC GGC TTC GGC GTT GCC GAA TTC CAG 1326 Gly Ser Pro Ala Gly Thr Thr Arg Gly Phe Gly Val Ala Glu Phe Gln 380 385 390 GAA ATC GGC CGC CTG ATC TCG GAA GTT CTG GAT GGC GTT GCG AAG AAC 1374 Glu Ile Gly Arg Leu Ile Ser Glu Val Leu Asp Gly Val Ala Lys Asn 395 400 405 GGC GAA GAT GGC AAC GGC GCC GTC GAA GCG GCC GTC AAG GCA AAG GCT 1422 Gly Glu Asp Gly Asn Gly Ala Val Glu Ala Ala Val Lys Ala Lys Ala 410 415 420 ATC GCT CTT TGC GAT CGC TTC CCG ATT TAC GCA TAACGCTGTT 1465 Ile Ala Leu Cys Asp Arg Phe Pro Ile Tyr Ala 425 430 434 TACGCACATT TGAAGATATG CTTG 1489

SEQ ID NO: 2 Sequence length: 434 Sequence type: Amino acid Sequence type: Protein Origin organism name: Hyphomicrium methyloboram
obium methyloborum) Strain name: GM2 Sequence: ATG TCG TCC GCA CCG GCT GCA GGT ACT GCT TCT ACG TCC CGT TTT TTC 48 Met Ser Ser Ala Pro Ala Ala Gly Thr Ala Ser Thr Ser Arg Phe Phe 1 5 10 15 AAG TCG CAC GTG TCG GAG ACG GAT CCC GAT ATT TTC AGC GCC ATC CAA 96 Lys Ser His Val Ser Glu Thr Asp Pro Asp Ile Phe Ser Ala Ile Gln 20 25 30 AAG GAA TTT GGC CGC CAG CAG CAC GAG ATC GAG CTG ATC GCG TCT GAG 144 Lys Glu Phe Gly Arg Gln Gln His Glu Ile Glu Leu Ile Ala Ser Glu 35 40 45 AAC ATC GTT TCG CAG GCC GTT CTC GAT GCA GCT GGT TCG GTG CTG ACC 192 Asn Ile Val Ser Gln Ala Val Leu Asp Ala Ala Gly Ser Val Leu Thr 50 55 60 AAC AAG TAT GCC GAG GGC TAT CCG GGC AAG CGC TAC TAC GGC GGC TGC 240 Asn Lys Tyr Ala Glu Gly Tyr Pro Gly Lys Arg Tyr Tyr Gly Gly Cys 65 70 75 80 CAG TAC GTC GAC ATC GTT GAA GAC ATC GCG ATC GAC CGC GCA AAG AAG 288 Gln Tyr Val Asp Ile Val Glu Asp Ile Ala Ile Asp Arg Ala Lys Lys 85 90 95 CTC TTC AAC TGC GAA TTC GCG AAC GTG CAG CCG AAC TCC GGC AGC CAG 336 Leu Phe Asn Cys Glu Phe Ala Asn Val Gln Pro Asn Ser Gly Ser Gln 100 105 110 GCG AAC CAG GGC GTG TTC AAT GCG CTC GCG CAG CCG GGC GAC ACC ATC 384 Ala Asn Gln Gly Val Phe Asn Ala Leu Ala Gln Pro Gly Asp Thr Ile 115 120 125 CTC GGT CTC TCG CTC GCT GCC GGT GGT CAC TTG ACC CAC GGC GCG CCG 432 Leu Gly Leu Ser Leu Ala Ala Gly Gly His Leu Thr His Gly Ala Pro 130 135 140 GTG AAC CAG TCC GGC AAG TGG TTC AAG GCC GTG CAC TAC ATG GTC AAG 480 Val Asn Gln Ser Gly Lys Trp Phe Lys Ala Val His Tyr Met Val Lys 145 150 155 160 CCC GAC TCG CAC CTC ATC GAC ATG GAC GAA GTG CGC AAG CTG GCC CAG 528 Pro Asp Ser His Leu Ile Asp Met Asp Glu Val Arg Lys Leu Ala Gln 165 170 175 GAG CAC AAG CCG CGC ATC ATC ATC GCT GGT GGT TCG GCC TAT CCG CGC 576 Glu His Lys Pro Arg Ile Ile Ile Ala Gly Gly Ser Ala Tyr Pro Arg 180 185 190 AAG ATC GAT TTC GCT GCA TTT CGC GCG ATT GCG GAT GAG GTT GGC GCG 624 Lys Ile Asp Phe Ala Ala Phe Arg Ala Ile Ala Asp Glu Val Gly Ala 195 200 205 ATC TTC CTC GTC GAT ATG GCG CAC TTC GCC GGT CTT GTT GCG GCC GGT 672 Ile Phe Leu Val Asp Met Ala His Phe Ala Gly Leu Val Ala Ala Gly 210 215 220 CTC ATT CCG AGC CCG TTC CCG CAT GCC CAC GTC GTA ACG ACG ACG ACG 720 Leu Ile Pro Ser Pro Phe Pro His Ala His Val Val Thr Thr Thr Thr 225 230 235 240 240 CAC AAG ACG CTG CGC GGA CCC CGT GGC GGC ATG ATT TTA ACG AAC GAC 768 His Lys Thr Leu Arg Gly Pro Arg Gly Gly Met Ile Leu Thr Asn Asp 245 250 255 GCG GAC ATC GCG AAG AAG ATC AAC TCG GCG ATC TTC CCT GGC ATT CAG 816 Ala Asp Ile Ala Lys Lys Ile Asn Ser Ala Ile Phe Pro Gly Ile Gln 260 265 270 GGC GGC CCG CTC ATG CAC GTT ATC GCC GGT AAG GCC GTC GCA TTC GGC 864 Gly Gly Pro Leu Met His Val Ile Ala Gly Lys Ala Val Ala Phe Gly 275 280 285 GAG GCT CTG CGT CCG GAC TTC AAG GTC TAC ATC AAG CAG GTG ATG GAC 912 Glu Ala Leu Arg Pro Asp Phe Lys Val Tyr Ile Lys Gln Val Met Asp 290 295 300 AAC GCC CGC GCG CTC GGT GAA GTG CTT GTG CAG AAC GGC TTC GCG CTC 960 Asn Ala Arg Ala Leu Gly Glu Val Leu Val Gln Asn Gly Phe Ala Leu 305 310 315 320 GTT TCT GGC GGC ACC GAC ACG CAC CTC GTT CTC GTC GAT CTG CGG CCG 1008 Val Se r Gly Gly Thr Asp Thr His Leu Val Leu Val Asp Leu Arg Pro 325 330 335 AAG AAG CTG ACC GGT ACG AAG GCT GAG AAG GCG CTT GGC CGT GCC AAC 1056 Lys Lys Leu Thr Gly Thr Lys Ala Glu Lys Ala Leu Gly Arg Ala Asn 340 345 350 ATC ACC TGC AAC AAG AAC GGC ATT CCG TTC GAC CCC GAG AAG CCG ATG 1104 Ile Thr Cys Asn Lys Asn Gly Ile Pro Phe Asp Pro Glu Lys Pro Met 355 360 365 GTG ACG TCG GGC ATT CGT TTG GGT TCG CCT GCA GGC ACG ACG CGC GGC 1152 Val Thr Ser Gly Ile Arg Leu Gly Ser Pro Ala Gly Thr Thr Arg Gly 370 375 380 TTC GGC GTT GCC GAA TTC CAG GAA ATC GGC CGC CTG ATC TCG GAA GTT 1200 Phe Gly Val Ala Glu Phe Gln Glu Ile Gly Arg Leu Ile Ser Glu Val 385 390 395 400 CTG GAT GGC GTT GCG AAG AAC GGC GAA GAT GGC AAC GGC GCC GTC GAA 1248 Leu Asp Gly Val Ala Lys Asn Gly Glu Asp Gly Asn Gly Ala Val Glu 405 410 415 GCG GCC GTC AAG GCA AAG GCT ATC GCT CTT TGC GAT CGC TTC CCG ATT 1296 Ala Ala Val Lys Ala Lys Ala Ile Ala Leu Cys Asp Arg Phe Pro Ile 420 425 430 TAC GCA 1302 Tyr Ala 434

SEQ ID NO: 3 Sequence length: 24 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Sequence: GCTGACCAAC AAATATGCTG AAGG 24

SEQ ID NO: 4 Sequence length: 29 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Sequence: ACCGGGTTCG GGTAGACGCC AGCAGCAAC 29

SEQ ID NO: 5 Sequence length: 18 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Sequence: ACATGTCGTC CGCACCGG 18

SEQ ID NO: 6 Sequence length: 20 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Sequence: GCGTAAACAG CGTTATGCGT 20

[Brief description of drawings]

FIG. 1 shows a restriction map of the HindIII fragment of recombinant plasmid pSA1.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1:19)

Claims (6)

[Claims] 1. 15 of the nucleotide sequences of SEQ ID NO: 1
A DNA encoding a serine hydroxymethyl transferase containing the nucleotide sequence of the 4th to 1458th positions. 2. Hyphomicrobium methyloboram GM
2. The DNA according to claim 1, which is produced by 2 (FERM BP-4039). 3. A recombinant vector incorporating the DNA according to claim 1. 4. A microorganism containing the recombinant vector according to claim 3. 5. A microorganism belonging to Escherichia coli containing the recombinant vector according to claim 3. 6. Escherichia coli ME5427 / pAB2 (FERM BP-4040) containing the recombinant vector according to claim 3.