JPH0870871A - DNA fragment containing a gene encoding tryptophan synthase - Google Patents

Abstract

PURPOSE: To obtain the DNA fragment containing a gene coding a specific Trp B protein and a gene coding a specific Trp A protein, enabling to obtain a Conynebacterium bacterium having a high tryptophan synthetase-producing ability, and useful in the fields of foods, medicines, livestock, etc. CONSTITUTION: This DNA fragment contains a gene coding Trp B protein expressed by an amino acid sequence ranging from the No.1 amino acid to the No.417 amino acid in the sequence of the formula and a gene coding Trp A protein expressed by an amino acid sequence ranging from the No.418 amino acid to the No.697 amino acid.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to DN containing a gene encoding tryptophan synthase derived from coryneform bacteria.
A fragment. Tryptophan synthase is an enzyme that catalyzes the biosynthesis of L-tryptophan in vivo, but L-tryptophan is one of the essential amino acids and is industrially important because of its high demand in the fields of food, medicine and livestock. It is an amino acid.

[0002]

BACKGROUND OF THE INVENTION In prokaryotes, the gene encoding tryptophan synthase is Escherichia coli.
ia coli ) -derived gene [Nucl. Acids Res., Vol. 9, p.
6647 (1981)], Bacillus subtil
is ) -derived gene [Gene, Vol.34, p.169, (1985)],
Brevibacterium lactofermentum (Brevibac
t erium lactofermentum) derived from the gene [Nucl. Acids R
es., Vol.14, p.10113 (1986)] and the like have been isolated.
However, regarding the gene encoding tryptophan synthase derived from Brev ibacterium flavum , which is an industrially important bacterium,
As far as the inventors know, there are no reports.

[0003]

The present invention has been made for the purpose of providing a DNA fragment containing a gene encoding a tryptophan synthase derived from a coryneform bacterium, which can be expressed in the coryneform bacterium.

[0004]

As a result of intensive studies to solve the above problems, the present inventors have found that a gene encoding tryptophan synthase can be isolated from a certain coryneform bacterium, The present invention has been completed.

Thus, according to the present invention, the gene encoding the TrpB protein represented by the amino acid sequence from amino acid No. 1 to amino acid No. 417 and the amino acid No. 418 to amino acid No. 697 in the sequence of SEQ ID No. 1 in the sequence listing below. There is provided a DNA fragment containing a gene encoding the TrpA protein represented by the amino acid sequences above. Hereinafter, the present invention will be described in more detail.

The "gene encoding tryptophan synthase" of the present invention means TrpB protein and TrpA.
It is composed of two types of proteins, indole and L
-Tryptophan synthase (EC 4.2.), Which is an enzyme that catalyzes the reaction to produce L-tryptophan from serine.
Trp B gene and trp A encoding 1.20)
It means a gene, and hereinafter, this may be abbreviated as "tryptophan synthase gene" and a DNA fragment containing the gene may be abbreviated as " trp BA fragment".

Although the trpBA fragment of the present invention can be synthesized after its nucleotide sequence is determined, it is usually a coryneform bacterium, for example, Brevibacterium flavum MJ-233 ( F
ERM BP-1497) present on the chromosome of the strain
It can be separated and obtained from the digested fragments generated by digesting NA with an appropriate restriction enzyme by the method described below. First, the coryneform bacterium, such as Brevibacterium flavum MJ-233, is cultured according to a conventional method (see, for example, JP-A-51-130592), cells are collected from the culture, and chromosomal DNA is extracted from the cells. To do. Chromosomal DNA can be easily extracted from bacterial cells by the method described in paragraph (A) of Example 1 of JP-A-5-15378, for example.

This chromosomal DNA is cut out using an appropriate restriction enzyme, for example, BglII , and the resulting cut DNA fragment is cloned into an appropriate vector, for example pBluescriptII SK ⁺ (Toyobo Co., Ltd.), and tryptophan. A tryptophan-requiring Escherichia coli mutant lacking synthase is transformed. This transformant strain is cultured under an appropriate selective pressure, the bacterial cells are recovered from the culture, and the bacterial cells are collected from the bacterial cells by a conventional method, for example, alkali-
The plasmid is extracted by the SDS method. This plasmid is cleaved with an appropriate restriction enzyme, the cleaved fragment is separated and analyzed by agarose gel electrophoresis, and the inserted DNA fragment derived from Brevibacterium flavum MJ-233 chromosome is extracted from the gel to obtain the trp of the present invention. The BA fragment can be obtained.

The tryptophan-requiring Escherichia coli mutant strain is, for example, Escherichia coli ATCC23718.
(Defective gene: trp B) and Escherichia coli A
Examples thereof include TCC23717 (defective gene: trp A). The presence of the trp B gene and the trp A gene on the trp BA fragment of the present invention can be easily confirmed by a complementation test for the above-mentioned tryptophan-requiring Escherichia coli mutant strain.

As one of the trp BA fragments of the present invention thus obtained, the above Brevibacterium flavum MJ
It can be mentioned DNA fragment of size about 3.1kb obtained by cutting chromosomal DNA of -233 with restriction enzyme Bgl II. This size tr is about 3.1 kb
nucleotide sequence of p BA fragment dideoxynucleotide enzymatic method using plasmid pUC18 or pUC19 or the like (dideoxy chain termination method; Sanger, F et al,.
Proc. Nat. Acad. Sci., USA, 74 , 5463, 1977). The sequence thus determined in the DNA fragment of about 3.1 kb in size is shown in SEQ ID NO: 1 in the sequence listing below. From the open reading frame present in this sequence, the gene encoding the TrpB protein ( trp B gene) has amino acid number 1 in the sequence.
To the amino acid number 417, and the gene encoding the TrpA protein ( trp A gene) has the amino acid number 418 in the sequence.
Was found to encode the amino acid sequence from amino acid number 619 to amino acid number 619. The DNA fragment containing the gene encoding the tryptophan synthase of the present invention, which comprises the above trp B gene and trp A gene, is not limited to those isolated from natural bacterial chromosomal DNA, for example, coryneform bacterial chromosomal DNA, and A DNA synthesizer used, for example, Beckman system
It may be one synthesized using 1 Plus (System-1 Plus).

As described above, the DNA fragment of the present invention obtained from bacterial chromosomal DNA of Brevibacterium flavum MJ-233 or the like has a nucleotide sequence that does not substantially impair the function of encoding tryptophan synthase. Some of the bases may be replaced with other bases, may be deleted, new bases may be inserted, or even a part of the base sequence may be transposed Well, and may be a nucleotide sequence that hybridizes to those nucleotide sequences, and any of these derivatives is included in the DNA fragment containing the gene encoding the tryptophan synthase of the present invention. The present invention has been described above, but the present invention will be described more specifically with reference to the following examples. It should be understood, however, that the examples are to be considered as an aid in gaining a specific appreciation of the invention and are not to be construed as limiting the scope of the invention in any way.

[0012]

【Example】

Example 1 Brevibacterium flavum MJ-233-derived tryp
Cloning of DNA fragment containing tophan synthase gene
Of (A) Brevibacterium flavum MJ-233 of total DNA extracted Brevibacterium flavum MJ-233 (FERM BP-
1497) is a semi-synthetic medium A medium [composition: urea 2
g, (NH ₄ ) ₂ SO ₄ 7 g, K ₂ HPO ₄ 0.5 g, KH _{2
PO 4 0.5g, MgSO 4 0.5g,} FeSO 4 · 7H 2
O 6 mg, MnSO ₄ .4-6H ₂ O 6 mg, yeast extract 2.5 g, casamino acid 5 g, biotin 200 μg,
Thiamine hydrochloride (200 μg) and glucose (20 g) were dissolved in distilled water to make 1 liter.] After culturing in 1 liter until the late logarithmic growth phase, cells were recovered. A solution containing the obtained bacterial cells at a concentration of 10 mg / ml [composition:
10 mM NaCl, 20 mM Tris buffer (pH 8.
0) 1 mM EDTA · 2Na] was suspended in 15 ml.
Proteinase K was added to the suspension at a final concentration of 100 μg / ml and this was incubated at 37 ° C. for 1 hour.
Next, sodium dodecyl sulfate was added to a final concentration of 0.5%, and the mixture was incubated at 50 ° C for 6 hours to lyse the cells. After adding an equal amount of a phenol / chloroform solution to the resulting lysate and gently shaking at room temperature for 10 minutes, the total amount was 5,000 at 10 to 12 ° C for 5,000 minutes.
It was subjected to centrifugation at xg and the supernatant fraction was collected. Sodium acetate was added to the supernatant fraction to a concentration of 0.3 M, and then 2 volumes of ethanol were gently added. The DNA existing between the aqueous layer and the ethanol layer was picked up with a glass rod, washed with 70% ethanol and air dried. The obtained DNA was a solution [composition: 10 mM Tris buffer (pH 7.5), 1 mM EDTA · 2Na].
After adding 5 ml and leaving still at 4 degreeC overnight, it used for the experiment.

(B) Preparation of DNA Fragment Containing Tryptophan Synthase Gene 25 μg of chromosomal DNA of Brevibacterium flavum prepared in the above (A) was digested with restriction enzyme Bgl II 50
Cleavage by reacting with units for 1 hour at 30 ℃
A Bgl II degradation product solution of NA was prepared. To the solution of this degradation product, 1 μ of the plasmid pBluescriptII SK ⁺ (manufactured by Toyobo Co., Ltd.)
g was allowed to react with the restriction enzyme Bgl II at 37 ° C. for 1 hour and mixed with a solution of the decomposed product.
mM Tris buffer (pH 7.6), 10 mM dithiothreitol, 1 mM ATP, 10 mM MgCl ₂ , and T4 DNA ligase 1 unit were added to each unit and reacted at 16 ° C for 15 hours to decompose the DNA degradation product. Combined.

Using the obtained solution, the conventional method [M. Mandel,
A.Higa; J. Mol. Biol., 53 , 159 (1970)], an Escherichia coli mutant strain, Escherichia coli ATCC23718 (tryptophan synthase-deficient mutant strain, that is, a tryptophan-requiring strain) was transformed, and the obtained trait was obtained. A selective medium containing the transformant containing 50 μg / ml of ampicillin [composition; K ₂ HP
O ₄ 7 g, KH ₂ PO ₄ 2 g, (NH ₄ ) ₂ SO ₄ 1 g, M
0.1 g of gSO ₄ .7H ₂ O, ₂ g of glucose, and 16 g of agar were dissolved in distilled water to make 1 liter]. The strain grown on the selective medium was inoculated into L medium containing 50 μg / ml of ampicillin at the final concentration, and this was cultured at 37 ° C. for 7 hours. The culture solution was centrifuged at 4 ° C. for 10 minutes at 8,000 × g to recover the bacterial cells. Alkali-SDS method [T. Maniatis,
EF Fritsch, J. Sambrook, Molecular cloning, p. 90
~ 91 (1982)] was used to extract the plasmid. The plasmid was cleaved with the restriction enzyme Bgl II, and the cleaved fragment was subjected to agarose gel electrophoresis.
BluescriptII SK ⁺ size of about 3.1kb to Bgl II site of
The insertion of the DNA fragment was confirmed.

(C) E. coli mutation requiring tryptophan
Complementation test for strains Using the plasmid solution prepared in (B) above, tryptic
Tryptophan, which lacks each step of the tophan biosynthesis system, is required.
Escherichia coli ATCC23720, which is a mutant strain of Escherichia coli
(Defective gene:trpD), ATCC23719 (defective inheritance
Child:trpC), the same ATCC23718 (defective gene:trp
B), the same ATCC23717 (defective gene:trpTransformation of A)
After replacement, Escherichia coli ATCC23718 strain and
And about 10 in ATCC23717 strain^Five Cells / μg DNA
A transformant strain that grows on the selection medium was obtained. This
This gives the size of the plasmid prepared in (B) above.
About 3.1 kbBglII-BglII tr on the DNA fragment
Tryptophan synthase inheritance consisting of pB and trpA
It was confirmed that the child was included.

Example 2 Tryptophan synthase inheritance
Determination of nucleotide sequence of DNA fragment containing offspring DNA of about 3.1 kb which was confirmed to contain tryptophan synthase in (C) of Example 1 above
The nucleotide sequence of the fragment was determined by the following operation, and the site of the tryptophan synthase gene existing on this DNA fragment was identified. DNA fragment solution of about 3.1 kb
14 μl was reacted with the restriction enzyme Sau 3AI at 37 ° C. for 1 to 5 minutes to partially decompose the DNA fragment. In addition, cloning vector pUC118 (manufactured by Takara Shuzo) was used as restriction enzyme B.
After cutting with am HI, dephosphorylation treatment was performed. The obtained vector fragment and the partially decomposed DNA fragment were mixed, and the final concentration of each mixture was 50 mM Tris buffer (p
H7.6), 10 mM dithiothreitol, 1 mM AT
P, 10 mM MgCl ₂ , and T4 DNA ligase
Each component was added so as to be 1 unit, and reacted at 16 ° C. for 10 hours for binding.

Similar to the above, DNA of about 3.1 kb in size
14 μl of the fragment solution was reacted with the restriction enzyme Taq I 50 units for 5 to 8 minutes to partially decompose the DNA fragment. The cloning vector pUC118 was reacted with a restriction enzyme Acc I, cleaved and then dephosphorylated, and this was ligated to the partially degraded DNA fragment in the same manner as above.

Using the thus obtained plasmid mixture, the calcium chloride method [Journal of Molecular Biology, 53 , 159 (1
970), and transforming Escherichia coli JM109 strain (manufactured by Takara Shuzo Co., Ltd.) with ampicillin 50 μg /
selective medium containing 10 ml [tryptone 10 g, yeast extract
5 g, 5 g of sodium chloride and 16 g of agar are dissolved in 1 liter of distilled water]. The strain grown in the above medium was liquid-cultured according to a conventional method, and the plasmid DNA was extracted from the obtained culture solution. Extracted plasmid DNA
Base sequence of plasmid pUC118 (Takara Shuzo)
Using the dideoxynucleotide enzymatic method [dideoxy chai
n termination method, Sanger, F. et al., Proc. Natl. Aca
d. Sci. USA, 74 , 5463 (1977)].

Specifically, the plasmid DNA extracted from the above-mentioned culture solution was used as Catalyst 80 manufactured by Perkin Elmer.
0 Molecular Biology Lab Station (CA
TALYST 800 Moleculer Biology Labostation ； Prkin-El
mer) according to the protocol and then the nucleotide sequence of the inserted DNA fragment of each plasmid was determined by a 373A DNA sequencer manufactured by Perkin Elmer. And the concatenation of these individual sequences is
Elmer sequence analysis software Inherit (I
(NHERIT) to determine the entire base sequence of a DNA fragment having a size of about 3.1 kb. The sequence is shown in SEQ ID NO: 1 in the Sequence Listing below. The presence of two open reading frames was confirmed in the determined nucleotide sequence, and by comparison of homology with the known trp B gene and trp A gene of Escherichia coli, they were identified as Brevibacterium flavum MJ-233. It was found to be the trp B gene (base numbers 889 to 2139) and the trp A gene (base numbers 2139 to 2978).

[0020]

EFFECTS OF THE INVENTION By breeding and improving a coryneform bacterium using a DNA fragment containing a gene encoding tryptophan synthase provided by the present invention, it becomes possible to obtain a coryneform bacterium having a high tryptophan synthase-producing ability. .

[0021]

[Sequence Listing] SEQ ID NO: the length of one sequence: 3118 Number of strand: double strand sequence type: nucleic acid Topology: linear sequence type: Genomic DNA Origin: Organism Name: Brevibacterium flavum (Brevibacteri
um flavum ) Strain name: MJ-233 Sequence features: Characteristic symbol: CDS Location: 889-2142 Method for determining characteristic: E Characteristic symbol: CDS Location: 2139-2981 Method for determining characteristic: E sequence AGATCTTTGG CGTCAACCAC CGCAACCTGC ATGATCTGTC CATTGATTTG GATCGTTCAC 60 GTCGCCTGTC CAAGCTCATT CCAGCAGATG CCGTGCTCGT GTCTGAGTCT GGCGTGCGCG 120 ATACCGAAAC CGTCCGCCAG CTAGGTGGGC ACTCCAATGC ATTCCTCGTT GGCTCCCAGC 180 TGACCAGCCA GGAAAACGTC GATCTGGCAG CCCGCGAATT GGTCTACGGC CCCAACAAAG 240 TCTGCGGACT CACCTCACCA AGTGCAGCAC AAACCGCTCG CGCAGCGGGT GCGGTCTACG 300 GCGGGCTCAT CTTCGAAGAG GCATCACCAC GCAATGTTTC ACGTGAAACA TCGCAAAAAA 360 TCATCGCCGC AGAGCCCAAC CTGCGCTACG TCGCGGTCAG CCGTCGCACC TCCGGGTACA 420 AGGATTTGCT TGTCGACGGC ATCTTCGCCG TACAAATCCA CGCCCCACTG CAGGGCAGCG 480 TCGAAGCAGA AAAGGCATTG ATCGCCGCCG TTCGTGAAGA GGTTGGACCG CAGGTCCAGG 540 TCTGGCGC GATCTCGATG TCCAGCCCCT TGGGGGCTGA AGTGGAGGA GTGCGGCGGAGA GCTAATT CTTGATGCCC ATGAAGGTGG CAGCGGGGAA GTATTCGACT 660 GGGCTACGGT GCCAGCCGCT GTGAAGGCAA AGTCTTTGCT CGCGGGAGGC ATCTCTCCGG 720 ACAACGCTGC GCAGGCACTC GCTGTGGGCT GCGCAGGTTT GGACATCAAC TCTGGCGTGG 780 AATACCCCGC CGGTGCAGGC ACGTGGGCTG GGGCGAAAGA CGCCGGCGCG CTGCTGAAAA 840 TTTTCGCAAC CATCTCCACA TTCCACTACT AAAGGTTTAA ATAGGATC ATG ACT GAA 897 Met Thr Glu 1 AAA GAA AAC TTG GGT GGC TCC ACG CTG CTG CCT GCA TAC TTC GGT GAA 945 Lys Glu Asn Leu Gly Gly Ser Thr Leu Leu Pro Ala Tyr Phe Gly Glu 5 10 15 TTC GgC GGC CAG TTC GTC GCG GAA TCC CTC CTG CCT GCT CTC GAC CAG 993 Phe Gly Gly Gln Phe Val Ala Glu Ser Leu Leu Pro Ala Leu Asp Gln 20 25 30 35 CTG gAG AAG GCC TTC GTT GAC GCG ACC AAC AGC CCA GAG TTC CGC GAA 1041 Leu Glu Lys Ala Phe Val Asp Ala Thr Asn Ser Pro Glu Phe Arg Glu 40 45 50 GAA CTc GGC GGC TAC CTC CGC gAT TAC CTC GGC CGC CCA ACC CCG CTG 1089 Glu Leu Gly Gly Tyr Leu Arg Asp Tyr Leu Gly Arg Pro Thr Pro Leu 55 60 65 ACC GAA TGC TCC AAC CTG CCA CTC TCA GGC GAA GGC AAA GGC TTT GCG 1137 Thr Glu Cys Se r Asn Leu Pro Leu Ser Gly Glu Gly Lys Gly Phe Ala 70 75 80 CGG aTC TTC CTC AAG CGC GAA GAC CTC GTC CAC GGC GGT GCA CAC AAA 1185 Arg Ile Phe Leu Lys Arg Glu Asp Leu Val His Gly Gly Ala His Lys 85 90 95 ACT AAC CAG GTG ATC GGC CAG GTG CTG CTT GCC AAG CGC ATG GGC AAA 1233 Thr Asn Gln Val Ile Gly Gln Val Leu Leu Ala Lys Arg Met Gly Lys 100 105 110 115 ACC CGC ATC ATC GCA GAG ACC GGC GcA GGC CAG CAC GGC ACC GCC ACC 1281 Thr Arg Ile Ile Ala Glu Thr Gly Ala Gly Gln His Gly Thr Ala Thr 120 125 130 GcT CTC GCA TGT GCG CTC ATG GGC CTC GAG TGC GTT GTC TAC ATG GGC 1329 Ala Leu Ala Cys Ala Leu Met Gly Leu Glu Cys Val Val Tyr Met Gly 135 140 145 GCC AAG GAC GTT GCC CGC CAG CAG CCC AAC GTC TAC CGC ATG CAG CTG 1377 Ala Lys Asp Val Ala Arg Gln Gln Pro Asn Val Tyr Arg Met Gln Leu 150 155 160 CAC GGC GCG AAG GTC ATC CCC GTG GAA TcT GGT TCC GGC ACC CTG AAG 1425 His Gly Ala Lys Val Ile Pro Val Glu Ser Gly Ser Gly Thr Leu Lys 165 170 175 GAC GCC GTG AAT GAA GCG CTG CGC GAT TGG ACC GCA ACC TTC CAC GAG 1473 A sp Ala Val Asn Glu Ala Leu Arg Asp Trp Thr Ala Thr Phe His Glu 180 185 190 195 TCC CAC TAC CTT CTC GGC ACC GCC GCC GGC CCG CAT CCA TTC CCA ACC 1521 Ser His Tyr Leu Leu Gly Thr Ala Ala Gly Pro His Pro Phe Pro Thr 200 205 210 ATc GTG CGT GAA TTC CAC AAG GTG ATC TCT GaG GAA GCC AAG GCA CAG 1569 Ile Val Arg Glu Phe His Lys Val Ile Ser Glu Glu Ala Lys Ala Gln 215 220 225 ATG CTA GAG CGC ACC GGC AAG CTT CCC GAC GTT GTG GTC GCC TGT GTC 1617 Met Leu Glu Arg Thr Gly Lys Leu Pro Asp Val Val Val Ala Cys Val 230 235 240 GGT GGT GGC TCC AAC GCC ATC GGC ATG TTC GCA GAC TTC ATT GAC GAT 1665 Gly Gly Gly Ser Asn Ala Ile Gly Met Phe Ala Asp Phe Ile Asp Asp 245 250 255 GAA GgC GTA GAG CTC GTC GGC GCT GAG CCA GCC GGT GAA GGC CTC GAC 1713 Glu Gly Val Glu Leu Val Gly Ala Glu Pro Ala Gly Glu Gly Leu Asp 260 265 270 275 TCC GGC AAG CAC GGC GCA ACC ATC ACC AAC GGT CAG ATC GGC ATC CTA 1761 Ser Gly Lys His Gly Ala Thr Ile Thr Asn Gly Gln Ile Gly Ile Leu 280 285 290 CAC GGC ACC CGT TCC TAC CTG ATG CGC AAC TCC GAC GGC CAA GTG GAA 1809 His Gly Thr Arg Ser Tyr Leu Met Arg Asn Ser Asp Gly Gln Val Glu 295 300 305 GAG TCC TAC TCC ATC TCC GCC GGA CTT GAT TAC CCA GGC GTC GGC CCA 1857 Glu Ser Tyr Ser Ile Ser Ala Gly Leu Asp Tyr Pro Gly Val Gly Pro 310 315 320 CAG CAC GCA CAC CTG CAC GCC ACC GGC CGC GCC ACC TAC GTT GGT ATC 1905 Gln His Ala His Leu His Ala Thr Gly Arg Ala Thr Tyr Val Gly Ile 325 330 335 ACC GAC GCC GAA GCC CTC CAA GCA TTC CAG TAT CTC GCC CGC TAC GAA 1953 Thr Asp Ala Glu Ala Leu Gln Ala Phe Gln Tyr Leu Ala Arg Tyr Glu 340 345 350 355 GGC ATC ATC CCC GCA CTG GAA TCC TCA CAC GCA TTC GCC TAC GCA CTC 2001 Gly Ile Ile Pro Ala Leu Glu Ser Ser His Ala Phe Ala Tyr Ala Leu 360 365 370 AAG CGC GCC AAG ACC GCC GAA GAG GAA GGC CAG AAC TTA ACC ATC CTC 2049 Lys Arg Ala Lys Thr Ala Glu Glu Glu Gly Gln Asn Leu Thr Ile Leu 375 380 385 GTC TCC CTA TCC GGC CGT GGC GAC AAG GAC GTT GAC CAC GTG CGC CGC 2097 Val Ser Leu Ser Gly Arg Gly Asp Lys Asp Val Asp His Val Arg Arg 390 395 400 ACC CTC GAA GAA AAT CCA GAA CTG ATC CTG AAG GAC AAC CGATG AGC CGT 2147 Thr Leu Glu Glu Asn Pro Glu Leu Ile Leu Lys Asp Asn Arg 405 410 415 Met Ser Arg 418 420 TAC GAC GAT CTT TTT GCA CGC cTC GAC ACG GCA GGG GAG GGC GCC TTT 2195 Tyr Asp Asp Leu Phe Ala Arg Leu Asp Thr Ala Gly Glu Gly Ala Phe 425 430 435 GTT CCC TTC ATC ATG CTG AGC GAC CCT TCA CCA GAG GAG GCT TTC CAG 2243 Val Pro Phe Ile Met Leu Ser Asp Pro Ser Pro Glu Glu Ala Phe Gln 440 445 450 ATC ATC TCC ACA GCA ATC GAA GCT GGC GcA GAT GCA CTG GAA CTT GGC 2291 Ile Ile Ser Thr Ala Ile Glu Ala Gly Ala Asp Ala Leu Glu Leu Gly 455 460 465 GTA CCT TTC TCC GAC CCA GTT GCC GAT GGC CCC ACC GTC GCG GAA TCC 2339 Val Pro Phe Ser Asp Pro Val Ala Asp Gly Pro Thr Val Ala Glu Ser 470 475 480 CAC CTC CGC GCA CTC GAC GGC GGC GCC ACC GTA GAC AGC GCA CTC GAG 2387 His Leu Arg Ala Leu Asp Gly Gly Ala Thr Val Asp Ser Ala Leu Glu 485 490 495 500 CAG ATC AAG CGC GTG CGC GCA GCC TAC CCA GAG GTT CCC ATC GGA ATG 2435 Gln Ile Lys Arg Val Arg Ala Ala Tyr Pro Glu Val Pro Ile Gly Met 505 510 515 CTC ATC TAC GGC AAC GTT CCT TTC ACC CGT GGC TTG GAT CGC TTC TAC 2483 Leu Ile Tyr Gly Asn Val Pro Phe Thr Arg Gly Leu Asp Arg Phe Tyr 520 525 530 CAA GAG TTC GCT GAA GCT GGC GCA GAC TCC ATC CTC CTG CCA GAC GTC 2531 Gln Glu Phe Ala Glu Ala Gly Ala Asp Ser Ile Leu Leu Pro Asp Val 535 540 545 CCA GTC CGC GAA GGC GCA CCG TTT TCT GCA GCA GCT GCA GCA GCC GGA 2579 Pro Val Arg Glu Gly Ala Pro Phe Ser Ala Ala Ala Ala Ala Ala Gly 550 555 560 ATT GAT CCC ATT TAC ATC GCT CCG GCC AAC GCC AGC GAG AAA ACC CTC 2627 Ile Asp Pro Ile Tyr Ile Ala Pro Ala Asn Ala Ser Glu Lys Thr Leu 565 570 575 580 GAG GGT GTC TCC GCC GCA TCA AAG GGC TAC ATC TAC GCC ATC TCC CGC 2675 Glu Gly Val Ser Ala Ala Ser Lys Gly Tyr Ile Tyr Ala Ile Ser Arg 585 590 595 GAC GGC GTC ACC GGC ACC GAA CGT GAA TCA TCC ACC GAC GGC CTG TCC 2723 Asp Gly Val Thr Gly Thr Glu Arg Glu Ser Ser Thr Asp Gly Leu Ser 600 605 610 GCA GTG GTG GAC AAT ATC AAg AAA TTT GAT GGC GCA CCC ATC TTC TTG 2771 Ala Val Val Asp Asn Ile Lys Lys Phe Asp Gly Ala Pro Ile Phe Leu 615 620 625 GGC TTC GGC ATC TCA TCC CCT CAG CAC GTG GCA GAC GCG ATT GCA GCG 2819 Gly Phe Gly Ile Ser Ser Pro Gln His Val Ala Asp Ala Ile Ala Ala 630 635 640 GGT GCT TCC GGT gCG ATC ACG GGT TCC GCG ATC ACC AAG ATC ATT GCT 2867 Gly Ala Ser Gly Ala Ile Thr Gly Ser Ala Ile Thr Lys Ile Ile Ala 645 650 655 660 TCC CAC TGC GAA GGT GAG CAC CCG AAC CCG TCC ACC ATT CGA GAT ATG 2915 Ser His Cys Glu Gly Glu His Pro Asn Pro Ser Thr Ile Arg Asp Met 665 670 675 GAC GGT TTG AAG AAG GAT CTC ACT GAG TTC ATC TCT GCG ATG AAG GCA 2963 Asp Gly Leu Lys Lys Asp Leu Thr Glu Phe Ile Ser Ala Met Lys Ala 680 685 690 GCG ACC AAG AAG GTT TAGGCCTTTA AATGTGGCAA TGTTTCACGT GAAACATTCT 3018 Ala Thr Lys Lys Val 695 GAGAGAATGT AGAAACATCA AAGAAGCCGC CTCCTAGCTC TCGGGCTGGG AGGCGGCTTC 3078 TTTGTTTGCG GTTTAGGAAA TCTCAGGGTT TTG

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1:13) (C12N 9/88 C12R 1:13) C12R 1:13) (72) Inventor Hideaki Yukawa 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Mitsubishi Petrochemical Co., Ltd. Tsukuba Research Institute

Claims (1)

[Claims] 1. A gene encoding the TrpB protein represented by the amino acid sequence from amino acid number 1 to amino acid number 417 in the sequence of sequence number 1 and the amino acid sequence from amino acid number 418 to amino acid number 697. DNA fragment containing a gene encoding the TrpA protein.