JPH0576352A - Production of l-phenylalanine by fermentation - Google Patents

Abstract

PURPOSE:To produce L-phenylalanine by a fermentation process in high efficiency by culturing a microorganism transformed with a specific recombinant DNA and collecting the L-phenylalanine produced in the medium. CONSTITUTION:A microorganism is transformed with a recombinant DNA containing a gene coding an enzyme free from feed-back inhibition activity and obtained by substituting an amino acid residue in prephenate dehydratase with other amino acid residue or depleting the amino acid residue. The transformed microorganism is cultured in a medium and the produced L- phenylalanine is separated from the medium.

Description

Detailed Description of the Invention

[0001]

[Industrial application] Prephenate dehydratase (P
D) and chorismate mutase (CM) are on the biosynthetic system of L-phenylalanine (Phe). Therefore, the present invention relates to the production of L-phenylalanine, which is a useful amino acid, which is used as a raw material for the sweetener aspartame and whose demand is rapidly increasing.

[0002]

2. Description of the Related Art There are many known methods for producing L-phenylalanine using microorganisms. For example, a method using recombinant Escherichia coli is disclosed in JP-A-56-18.
90, JP-A-58-103398, JP-A-61-925.
65, JP-A-1-104160, International Publication WO87 / 0.
There is 0202. In addition, L-phenylalanine or L-
As a method for producing tyrosine, there is JP-A-61-128897 in which a mutant strain of the genus Corynebacterium is used, and JP-A-60-34197 and JP-A-60 in which a recombinant Corynebacterium is used. No. 24192, JP-A-61-260892, and JP-A-61-124375 are known.

In the aromatic amino acid biosynthetic pathway including L-phenylalanine, the key enzyme, which plays a central role in the biosynthetic pathway, undergoes feedback inhibition by the final product. The above-mentioned conventional technique is based on the principle that a target amino acid is produced by using a microorganism having a key enzyme in which the inhibition of the feedback is released.

[0004] In the prior art, as a key enzyme to be released from the inhibition of fidback inhibition, there is prephenate dehydratase (hereinafter abbreviated as "PD").
Among the microorganisms used in the above-mentioned prior art, in Escherichia coli, chorismate mutase (hereinafter referred to as “C
Abbreviated as "M") and a bifunctional enzyme having PD activity (C
M-PD) is known to exist, and the enzyme activity is subject to feedback inhibition by L-phenylalanine. The enzyme is encoded by a gene called pheA, and the nucleotide sequence of the gene and the amino acid sequence of the enzyme have been reported by Hudson and Davidson [Hudson, G.
S. and Davidson, BE, J. Mol. Biol., 180, 1023 (19
84)]. In order to efficiently produce L-phenylalanine, it is essential to cancel the feedback inhibition of CM-PD by L-phenylalanine, and some of the methods analyzed at the amino acid level are known. There is. For example, Gessing and Davidson
We reported that modifying two tryptophan residues (226th and 338th from the N-terminal) of CM-PD with dimethyl [2-hydroxy-5-nitrobenzylsulfonium bromide] made an enzyme resistant to feedback inhibition. [Gething, MJH and Davidson,
BE, Eur. J. Biochem., 78, 111 (1977)]. In addition, Bacmann et al. Deleted the feedback inhibition by deleting the tryptophan residue at the 338th position from the N-terminal side of CM-PD or by substituting this residue with an arginine-glycine residue. It has been found that this can be done (Japanese Patent Laid-Open No. 1-235597). Furthermore, Edwards et al. Also released feedback inhibition by inserting a tryptophan-arginine-serine-proline amino acid sequence at the position of the tryptophan residue at position 338 (International Patent WO87 / 0020).
2). All of these focused on the 338th tryptophan residue, with no knowledge of the other residues.

On the other hand, in coryneform bacteria, there is an enzyme (PD) having an activity of PD alone, and it is known that this reaction is also feedback-inhibited by phenylalanine.

Among the genes encoding the enzyme, Ozaki et al.
[Ozaki, A. et al., Agric. Biol.chem., 49, 2925 (198
6)], and Ito et al. [Ito, H. et al., Appl. Microbiol. B
iotechnol., 33, 190 (1989)] reported a gene in which feedback inhibition by phenylalanine was released. In addition, Foletti and Sinsky reported the nucleotide sequence of the native PD gene and pointed out the homology with the pheA gene of Escherichia coli K-12 [Follettie, MT and Sinsky, AJ, J. . Bacterio
L., 167, 695 (1986)]. However, there was no knowledge about the nucleotide sequence of the enzyme gene for which feedback inhibition was released in coryneform bacteria. Furthermore, no attempt was made to convert at the base sequence level and to release the inhibition by the accompanying amino acid substitution.

[0007]

The object of the present invention is to provide L
-To provide a method for efficiently fermentatively producing phenylalanine.

[0008]

The present inventors have completed the present invention as a result of repeated studies for the purpose of developing a method for efficiently fermentatively producing L-phenylalanine.

The inventors of the present invention firstly canceled the feedback inhibition of Brevibacterium lactofermentum, which is a typical strain belonging to the genus Brevibacterium that produces L-phenylalanine well, by L-phenylalanine. By determining the nucleotide sequence of the gene for PD and comparing and analyzing it with the natural type, it was found that a single amino acid substitution occurred between the two. Based on this finding, C in Escherichia coli K-12
By carrying out the same amino acid substitution, different amino acid substitution, or subsequent deletion of amino acid residues in the homologous region of M-PD, it is possible to obtain a strain having an enzyme whose feedback inhibition has been released, and The present invention has been completed by confirming the improvement in L-phenylalanine productivity.

That is, the present invention includes a gene encoding an enzyme for which feedback inhibition is released, which is obtained by substituting or deleting at least one amino acid residue in prephenate dehydratase with another amino acid residue. A method for producing L-phenylalanine, which comprises culturing a microorganism transformed with a recombinant DNA and L-phenylalanine produced in a medium by culturing the microorganism. Hereinafter, the present invention will be described in detail.

First, a plasmid pPH1 containing a PD gene cloned from Brevibacterium lactofermentum AJ11957 (FERMP-6673).
4 (Brevibacterium lactofermentum AJ1
2259 (contained in FERM P-3565)), the nucleotide sequence was determined to show that the mutation that cancels feedback inhibition was a single amino acid substitution in which the serine residue at the 235th position from the N-terminus was replaced with a proline residue. I found that. Next, it was confirmed that the amino acid sequence near the 235th serine residue has high homology with the vicinity of the 330th serine residue from the N-terminal of the pheA gene of Escherichia coli K-12,
When a PD in which the 330th serine residue of Escherichia coli K-12 was substituted with a proline residue or an aspartic acid residue and a PD in which the 330th serine residue and thereafter were deleted were constructed, the enzyme fed back. It was found that the inhibition was released, and an enzyme suitable for L-phenylalanine production could be obtained.

The enzyme having PD activity referred to in the present invention is
Those derived from microorganisms such as coryneform bacteria and having the activity alone, and further derived from microorganisms such as Escherichia coli are C
It means one having a dual function activity such as M-PD.

In PD, an amino acid residue substituted with another amino acid means an amino acid residue existing in the amino acid sequence region involved in the mechanism of feedback inhibition by L-phenylalanine. For example, it refers to the 235th serine residue from the N-terminal side in PD of Brevibacterium lactofermentum, and the 330th serine residue from the N-terminal side in CM-PD of Escherichia coli.

In PD, the substitution with another amino acid residue is a substitution with another amino acid that cancels feedback inhibition by L-phenylalanine, for example, substitution with a proline residue or an aspartic acid residue. Point

As a method for performing amino acid substitution and deletion, recombinant PCR (recombinan) is used.
t polymerase chain react
on) method [R. Higuchi, 61, in PCR Technology (Erl
ich HA Eds., Stockton press (1989))], site-directed mutagenesis [Kramer W. and Frits HJ, Meth. in Enz
ymol., 154, 350 (1987); Kunkel TA et al., Meth.
in Enzymol., 154, 367 (1987)], a method for directly treating a target gene on a chromosome or a plasmid with hydroxylamine [Hashimoto T. and Sekiguchi M., J. Bacteri.
Ol., 159, 1039 (1984)] or the cells harboring the DNA are chemically synthesized by an ultraviolet irradiation method or a conventional method by treatment with a chemical agent such as N-methyl-N'-nitrosoguanidine or nitrous acid. Method etc.

The mutant gene thus obtained is introduced as a recombinant DNA into a suitable microorganism and expressed therein, whereby feedback inhibition is substantially eliminated.
The microorganism which possesses is acquired.

The recombinant DNA obtained by the above method is a recombinant DNA in which a useful gene encoding PD for which feedback inhibition has been released is incorporated as a passenger into a plasmid or phage DNA vector. At that time, in order to efficiently carry out the expression of the useful gene, l
You may use the promoter which works in microorganisms, such as ac, trp, and PL. In addition, in the recombinant DNA referred to herein, the useful gene is a transposon [Berg, DE and Berg, C.
M., Bio / Technol., 1, 417 (1983)], Mu phage [JP-A-2-109985] or homologous recombination [Expe
riments in Molecular Genetics, Cold Spring Habor La
b. (1972)].

As a microorganism having a recombinant DNA, a microorganism that expresses a gene encoding the desired enzyme such as PD and has L-phenylalanine productivity by imparting resistance to L-phenylalanine analog and the like can be used. , Escherichia genus, Brevibacterium genus, Corynebacterium genus, Bacillus genus, Serratia genus, Pseudomonas genus and the like, any species and strain may be used. Specifically, there are the following. Escherichia coli K-12 ATCC10798 Brevibacterium lactofermentum ATCC13869 Brevibacterium flavum ATCC13826 Corynebacterium glutamicum ATCC13032 Corynebacterium glutamicum ATCC13060

As a method for transforming the above-mentioned microorganism, for microorganisms belonging to Escherichia, Serratia, etc., a method using calcium chloride [Mandel M. and Higa A.,
J. Mol. Biol., 53, 154 (1970)] and a method using electric pulse [Taketo A., Biochem. Biophys. Acta, 949, 318.
(1988)] is possible. For microorganisms belonging to Brevibacterium, Corynebacterium, etc., a method using protoplasts [Miwa K. et al., Gene, 39, 281 (1985)]
And method using electric pulse (JP-A-2-207791)
Etc. In the case of Bacillus, there are methods such as the competent method, electric pulse, and protoplast method.

Recombinant D containing the gene encoding PD for which feedback inhibition is released, obtained by the above method
A microorganism transformed with NA was cultivated, and the desired aromatic amino acid was produced and accumulated in the culture solution, and this was collected.

The medium for L-phenylalanine production used is a conventional medium containing a carbon source, a nitrogen source, inorganic ions and, if necessary, other organic components.

As the carbon source, sugars such as glucose, lactose, galactose, fructose and hydrolysates of starch, alcohols such as glycerol and sorbitol, organic acids such as fumaric acid, citric acid and succinic acid can be used. ..

As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and the like can be used.

As the organic micronutrient source, vitamin B1,
It is desirable to add an appropriate amount of required substances such as L-tyrosine or yeast extract. In addition to these, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions, etc. are added as necessary.

The culture is preferably carried out under aerobic conditions for 16 to 72 hours, the culture temperature is 30 to 45 ° C., and the p
H is controlled to 5-7. An inorganic or organic acidic or alkaline substance, ammonia gas or the like can be used for pH adjustment. Aromatic amino acids can be collected from the fermentation broth by combining ion exchange resin method, precipitation method and other known methods.

[0026]

INDUSTRIAL APPLICABILITY A mutant gene encoding PD in which feedback inhibition is canceled by substituting or deleting at least one amino acid residue is recombinant D
When the transformant introduced as NA was cultured, the productivity of L-phenylalanine was significantly improved.

A more specific description will be given below based on embodiments.

[0028]

【Example】

Example 1 Acquisition of a novel gene encoding PD for which feedback inhibition was released (1) Determination of mutation point of Brevibacterium mutant PD First, known PD gene [Follett of Corynebacterium] [Follett]
ie, MT and Sinsky, AJ, J. Bacteriol., 167, 695
(1986)] as an index, the nucleotide sequence of the NcoI fragment in the plasmid pAJ16 containing the PD gene of Brevibacterium lactofermentum wild strain was determined by the dideoxy method. In addition, the plasmid was used for Brelibacterium lactofermentum AJ12125.
(FERM-P7546). The amino acid sequence encoding the obtained nucleotide sequence is shown in SEQ ID NO: 1.
The amino acid sequence differed from that of Corynebacterium by only 1 amino acid residue.

Next, when the nucleotide sequence of the gene encoding PD of the phenylalanine-producing strain of Brevibacterium lactofermentum, which was also on the plasmid pPH14, was determined, the sequence shown in SEQ ID NO: 2 was obtained. The plasmid is Brevibacterium lactofermentum AJ12259 (FERMBP-356).
5) What was held in the stock was used. Comparing the wild-type strain and the strain in which feedback inhibition was released, the serine residue at position 235 was mutated to a proline residue.

(2) Escherichia coli mutant CM-
Construction of Novel Gene Encoding PD First, chromosomal DNA was extracted from the RR1 strain of Escherichia coli K-12 by a usual method. On the other hand, known literature [Hudson, GS and Davidson, BE, J. Mol. Bi
ol., 180, 1023 (1984)] based on the nucleotide sequence of the pheA gene described below.
Four primers (SEQ ID NO: 3-6) were chemically synthesized by a usual method. SEQ ID NO: 3 TCAACAAGCT GGAACGGACG SEQ ID NO: 4 CGCCGATTTA CCGCCTTGAG SEQ ID NO: 5 CCGTCTGGAA CCACGCCCGA T SEQ ID NO: 6 ATCGGGCGTG ATTCCAGACG G 5 and 6 have homologous sequences upstream and downstream of the pheA gene, respectively. 7 and 8 are complementary to each other, and T
It has homology with the sequence in the vicinity of the 330th serine residue, except that (thymine base) is replaced by C (cytosine base) by one base. CM-P of Escherichia coli K-12
D and PD of Brevibacterium lactofermentum
Has a high homology with Escherichia coli K-1
The serine residue at the 330th position from the N-terminal of CM-PD of 2 corresponds to the 235th serine residue from the N-terminal of Brevibacterium lactofermentum PD. SEQ ID NOS: 7 and 8 are synthesized such that the 330th serine residue becomes a proline residue.

Next, 1 μg of chromosomal DNA and SEQ ID NO: 5
PCR reactions were performed using 300 ng of each of the primers Nos. 8 and 8 or 300 ng of each of the SEQ ID Nos. 6 and 7 to obtain 1.3 Kbp and 0.5 Kbp, respectively.
A Kbp DNA fragment was obtained. The PCR method is the method of Errich et al. [Erlich, HA, ed., PCR Technology, Sto
ckton press (1989)] using a continuous replication reactor (Thermalcycler, Perkin Elmer Cetus) at 94 ° C. for 1 minute, 50 ° C. for 2 minutes, 72 ° C. for 3 minutes.
20 cycles were performed. These DNA
The fragments were subjected to agarose gel electrophoresis and recovered using a DNA recovery kit (Gene Clean, manufactured by Funakoshi Co., Ltd.), and PCR was carried out by using these fragments and the primers of SEQ ID NOs: 5 and 6 to perform 1.8 Kbp DNA. A fragment was obtained. This fragment was digested with restriction enzymes BamHI and PstI, and then a 1.7 Kbp DNA fragment was recovered by agarose gel electrophoresis.
BamHI and PstI digests of SG398 (Takara Shuzo) were ligated using T4 ligase. This was transformed into the Escherichia coli K-12 strain KA197 (pheA), and a plasmid was recovered from the chloramphenicol-resistant strains in which the phenylalanine requirement was lost, and the plasmid was named pPHAB. By determining the nucleotide sequence, it was confirmed that the plasmid carries a mutant CM-PD enzyme gene in which the serine residue at the 330th position was replaced with a proline residue.

(3) ty of Escherichia coli K-12
Construction of rA gene-deficient W3110 strain First, a streptomycin resistant strain was obtained by applying the W3110 strain of Escherichia coli K-12 (obtained from the National Institute of Genetics) to a plate medium containing streptomycin. Next, this strain and Escherichia coli K-
12 ME8424 strains (HfrPO45, thi, re
lA1, tyrA :: Tn10, ung-1, nad
B) Mix the culture solution (obtained from the National Institute of Genetics) and mix 3
After allowing it to stand at 7 ° C for 15 minutes to allow conjugation transfer, it was spread on a plate medium containing streptomycin, tetracycline, and L-tyrosine, and the resulting colonies, that is, Escherichia.
W3110 (tyrA) strain of Kori K-12 was obtained.
The plasmid pPHAB obtained in (2) was introduced into this strain by transformation. The transformed strain of Escherichia coli K-12 [W3110 (tyrA) / pPHAB].
Has been deposited with the Institute of Mechanical Engineering, and the deposit number is FERM BP
It is -3566.

(4) Measurement of PD enzyme activity W3110 (tyrA) of Escherichia coli K-12
/ (PPHAB) strain bacterial cells at 37 ° C using L medium,
The cells were collected by centrifuging the culture solution that had been cultured for 15 hours. Then, the cells were washed twice with physiological saline and suspended in 250 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing 0.5 mM dithiothreitol under ice-cooling, and then 30 times, four times. A crude enzyme solution was prepared by crushing with ultrasonic waves (20 kHz).

The PD enzyme activity is measured by a conventional method [Cotton, RG
H. and Gibson, F., Meth. In Enzymol., 17, 564 (197
0)]. That is, using the crude enzyme solution, 5 mM containing 1 mM barium prephenate and 0.5 mM tyrosine was used.
In the presence of 0 mM Tris-HCl buffer (pH 8.2), 37 ° C
After reacting for 10 minutes and adding 1N sodium hydroxide to stop the reaction, the produced phenylpyric acid was measured at an absorption wavelength of 320 nm. A protein assay kit (Bio Rad) was used for the protein quantification method, and the protocol was followed. As shown in FIG. 4, the results are wild type CM-PD.
The enzyme reaction was strongly inhibited in the presence of 0.5 mM L-phenylalanine, whereas the mutant CM-PD was 5 mM.
L-phenylalanine showed almost no inhibition.

Furthermore, in the case of a plasmid carrying a wild-type enzyme gene in the absence of L-phenylalanine,
3.5 × 10 2 U / mg protein, 1.5 for mutant type
It showed a higher enzyme activity of × 10 4 U / mg protein.
Therefore, by introducing the mutation, L-
Not only was feedback inhibition canceled by phenylalanine, but the enzyme amount or enzyme activity could be increased by 40 times.

Example 2 Fermentative production of L-phenylalanine (1) Construction of Escherichia coli K-12 carrying CM-PD in which feedback inhibition was released CM obtained in Example 1 in which feedback inhibition was released -The transformed strain of Escherichia coli K-12 carrying pPHAB containing the PD gene [W3110
(TyrA) / pPHAB] for L-phenylalanine production medium (glucose 20 g, disodium hydrogen phosphate 29.4 g, potassium dihydrogen phosphate 6 g, sodium chloride 1 g, ammonium chloride 2 g, sodium citrate 1
0 g, 0.4 g of sodium glutamate, 3 g of magnesium sulfate heptahydrate, 0.23 g of calcium chloride, and 2 mg of thiamine hydrochloride in 1 L of water) were used for culturing at 37 ° C. for 24 hours. The results are shown in Table 1. The quantitative determination was carried out by high performance liquid chromatography.

[0037]

[Table 1]

Example 3 Construction of genes encoding CM-PD in which the serine residue at the 330th position is replaced with an aspartic acid residue and CM-PD in which the serine residue at the 330th position and subsequent residues are deleted are the same as in Example 1. , A synthetic DNA of SEQ ID NOs: 7-10 was prepared, and using this, CM-PD in which the 330th serine residue was replaced with an aspartic acid residue and CM-PD in which the 330th serine residue and thereafter were deleted The coding gene was constructed. The plasmids containing these genes were named pPHAD and pPHATerm, respectively.

Furthermore, this plasmid was transformed into W3110 (ty
The rA) strain was introduced using a conventional transformation method. In addition,
W3110 (tyrA) / pPHAD, W3110 (t
yrA) / pPHATerm has been deposited with the Institute of Microtechnology, and the deposit numbers are FERM BP-365.
2 and FERM BP-3653.

When these strains were cultured and the PD enzyme activity was measured, all mutant strains were not inhibited in the presence of 5 mM L-phenylalanine in the same manner as the strains carrying pPHAB. When L-phenylalanine was fermentatively produced using these strains, 0.5 g / l of L-phenylalanine was accumulated in the medium.

[Brief description of drawings]

FIG. 1 shows inhibition of prephenate dehydratase activity of wild-type and mutant forms (substituted by proline residues) of chorismate mutase-prephenate dehydratase by L-phenylalanine. Is.

[Sequence Listing] SEQ ID NO: 1 Sequence Length: 948 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: genomic DNA Origin Biological Name: Brevibacterium lactofermentum (B
revibacteriumlactofermentum) Strain name: AJ12125 (FERM P-7546) Sequence ATG AGC GAC GCA CCA ATT GTT GTG GCC TAT TTG GGG CCT GCC GGA ACC 48 Met Ser Asp Ala Pro Ile Val Val Ala Tyr Leu Gly Pro Ala Gly Thr 1 5 10 15 TTC ACC GAA GAA GCC CTC TAC AAA TTT GCC GAC GCC GGC GTA TTC GGC 96 Phe Thr Glu Glu Ala Leu Tyr Lys Phe Ala Asp Ala Gly Val Phe Gly 20 25 30 GAC GGT GAG ATC GAG CAG CTA CCA GCC AAA TCG CCA CAA GAA GCT GTC 144 Asp Gly Glu Ile Glu Gln Leu Pro Ala Lys Ser Pro Gln Glu Ala Val 35 40 45 GAC GCG GTC CGC CAC GGC ACC GCC CAG TTC GCG GTG GTC GCC ATC GAA 192 Asp Ala Val Arg His Gly Thr Ala Gln Phe Ala Val Val Ala Ile Glu 50 55 60 AAC TTC GTC GAC GGC CCC GTC ACC CCC ACC TTC GAC GCC CTT GAC CAG 240 Asn Phe Val Asp Gly Pro Val Thr Pro Thr Phe Asp Ala Leu Asp Gln 65 70 75 80 GGC TCC AAC GTG CAA ATC ATC GCC GAA GAA GAA CTC GAT ATT GCC TTT 288 Gly Ser Asn Val Gln Ile Ile Ala Glu Glu Glu Leu Asp Ile Ala Phe 85 90 95 TCC ATC ATG GTC CGG CCA GGG ACT TCG CTT GCC GAC GTC AAA ACC CTC 336 Ser Ile Met Val Ar g Pro Gly Thr Ser Leu Ala Asp Val Lys Thr Leu 100 105 110 GCC ACC CAC CCG GTT GGG TAC CAA CAA GTG AAA AAC TGG ATG GCA ACC 384 Ala Thr His Pro Val Gly Tyr Gln Gln Val Lys Asn Trp Met Ala Thr 115 120 125 ACC ATT CCG GAC GCC ATG TAT CTT TCA GCA AGC TCC AAC GGC GCC GGC 432 Thr Ile Pro Asp Ala Met Tyr Leu Ser Ala Ser Ser Asn Gly Ala Gly 130 135 140 GCA CAA ATG GTT GCC GAA GGA ACC GCC GAC GCA GCC GCA GCG CCC TCC 480 Ala Gln Met Val Ala Glu Gly Thr Ala Asp Ala Ala Ala Ala Pro Ser 145 150 155 160 CGC GCA GCC GAA CTC TTC GGA CTG GAA CGC CTT GTT GAT GAT GTC GCC 528 Arg Ala Ala Glu Leu Phe Gly Leu Glu Arg Leu Val Asp Asp Val Ala 165 170 175 GAC GTC CGC GGC GCC CGC ACC CGC TTC GTT GCA GTC CAA GCC CAA GCA 576 Asp Val Arg Gly Ala Arg Thr Arg Phe Val Ala Val Gln Ala Gln Ala 180 185 190 GCC GTT TCC GAA CCG ACC GGC CAC GAC CGC ACC TCC GTC ATT TTC TCC 624 Ala Val Ser Glu Pro Thr Gly His Asp Arg Thr Ser Val Ile Phe Ser 195 200 205 CTA CCG AAT GTG CCA GGC AGC CTC GTG CGC GCC CTC AAC GAA TTC GCC 672 Leu Pr o Asn Val Pro Gly Ser Leu Val Arg Ala Leu Asn Glu Phe Ala 210 215 220 ATC CGT GGC GTC GAC CTC ACC CGC ATC GAA TCC CGC CCC ACC CGC AAA 720 Ile Arg Gly Val Asp Leu Thr Arg Ile Glu Ser Arg Pro Thr Arg Lys 225 230 235 240 GTC TTC GGA ACC TAC CGC TTC CAC CTG GAC ATA TCC GGA CAT ATC CGC 768 Val Phe Gly Thr Tyr Arg Phe His Leu Asp Ile Ser Gly His Ile Arg 245 250 255 GAC ATC CCC GTC GCC GAA GCC CTC CGC GCA CTC CAC CTC CAA GCC GAA 816 Asp Ile Pro Val Ala Glu Ala Leu Arg Ala Leu His Leu Gln Ala Glu 260 265 270 GAA CTC GTA TTC GTC GGT TCC TGG CCC TCC AAC CGT GCA GAA GAC AGC 864 Glu Leu Val Phe Val Gly Ser Trp Pro Ser Asn Arg Ala Glu Asp Ser 275 280 285 ACG CCC CAA ACC GAC CAA CTA GCT AAC GTA CAC AAG GCG GAC GAA TGG 912 Thr Pro Gln Thr Asp Gln Leu Ala Asn Val His Lys Ala Asp Glu Trp 290 295 300 GTT CGC GCA GCA AGC GAA GGA AGG AAA CTT AAC TAG 948 Val Arg Ala Ala Ser Glu Gly Arg Lys Leu Asn 305 310 315 SEQ ID NO: 2 Sequence length: 948 Sequence type: Nucleic acid Number of strands: Double-stranded topology : Linear sequence type: genomic DNA Origin organism name: Brevibacterium lactofermentum (B
revibacteriumlactofermentum) Strain name: AJ12259 (FERM P-3565) Sequence ATG AGC GAC GCA CCA ATT GTT GTG GCC TAT TTG GGG CCT GCC GGA ACC 48 Met Ser Asp Ala Pro Ile Val Val Ala Tyr Leu Gly Pro Ala Gly Thr 1 5 10 15 TTC ACC GAA GAA GCC CTC TAC AAA TTT GCC GAC GCC GGC GTA TTC GGC 96 Phe Thr Glu Glu Ala Leu Tyr Lys Phe Ala Asp Ala Gly Val Phe Gly 20 25 30 GAC GGT GAG ATC GAG CAG CTA CCA GCC AAA TCG CCA CAA GAA GCT GTC 144 Asp Gly Glu Ile Glu Gln Leu Pro Ala Lys Ser Pro Gln Glu Ala Val 35 40 45 GAC GCG GTC CGC CAC GGC ACC GCC CAG TTC GCG GTG GTC GCC ATC GAA 192 Asp Ala Val Arg His Gly Thr Ala Gln Phe Ala Val Val Ala Ile Glu 50 55 60 AAC TTC GTC GAC GGC CCC GTC ACC CCC ACC TTC GAC GCC CTT GAC CAG 240 Asn Phe Val Asp Gly Pro Val Thr Pro Thr Phe Asp Ala Leu Asp Gln 65 70 75 80 GGC TCC AAC GTG CAA ATC ATC GCC GAA GAA GAA CTC GAT ATT GCC TTT 288 Gly Ser Asn Val Gln Ile Ile Ala Glu Glu Glu Leu Asp Ile Ala Phe 85 90 95 TCC ATC ATG GTC CGG CCA GGG ACT TCG CTT GCC GAC GTC AAA ACC CTC 336 Ser Ile Met Val Ar g Pro Gly Thr Ser Leu Ala Asp Val Lys Thr Leu 100 105 110 GCC ACC CAC CCG GTT GGG TAC CAA CAA GTG AAA AAC TGG ATG GCA ACC 384 Ala Thr His Pro Val Gly Tyr Gln Gln Val Lys Asn Trp Met Ala Thr 115 120 125 ACC ATT CCG GAC GCC ATG TAT CTT TCA GCA AGC TCC AAC GGC GCC GGC 432 Thr Ile Pro Asp Ala Met Tyr Leu Ser Ala Ser Ser Asn Gly Ala Gly 130 135 140 GCA CAA ATG GTT GCC GAA GGA ACC GCC GAC GCA GCC GCA GCG CCC TCC 480 Ala Gln Met Val Ala Glu Gly Thr Ala Asp Ala Ala Ala Ala Pro Ser 145 150 155 160 CGC GCA GCC GAA CTC TTC GGA CTG GAA CGC CTT GTT GAT GAT GTC GCC 528 Arg Ala Ala Glu Leu Phe Gly Leu Glu Arg Leu Val Asp Asp Val Ala 165 170 175 GAC GTC CGC GGC GCC CGC ACC CGC TTC GTT GCA GTC CAA GCC CAA GCA 576 Asp Val Arg Gly Ala Arg Thr Arg Phe Val Ala Val Gln Ala Gln Ala 180 185 190 GCC GTT TCC GAA CCG ACC GGC CAC GAC CGC ACC TCC GTC ATT TTC TCC 624 Ala Val Ser Glu Pro Thr Gly His Asp Arg Thr Ser Val Ile Phe Ser 195 200 205 CTA CCG AAT GTG CCA GGC AGC CTC GTG CGC GCC CTC AAC GAA TTC GCC 672 Leu Pr o Asn Val Pro Gly Ser Leu Val Arg Ala Leu Asn Glu Phe Ala 210 215 220 ATC CGT GGC GTC GAC CTC ACC CGC ATC GAA CCC CGC CCC ACC CGC AAA 720 Ile Arg Gly Val Asp Leu Thr Arg Ile Glu Pro Arg Pro Thr Arg Lys 225 230 235 240 GTC TTC GGA ACC TAC CGC TTC CAC CTG GAC ATA TCC GGA CAT ATC CGC 768 Val Phe Gly Thr Tyr Arg Phe His Leu Asp Ile Ser Gly His Ile Arg 245 250 255 GAC ATC CCC GTC GCC GAA GCC CTC CGC GCA CTC CAC CTC CAA GCC GAA 816 Asp Ile Pro Val Ala Glu Ala Leu Arg Ala Leu His Leu Gln Ala Glu 260 265 270 GAA CTC GTA TTC GTC GGT TCC TGG CCC TCC AAC CGT GCA GAA GAC AGC 864 Glu Leu Val Phe Val Gly Ser Trp Pro Ser Asn Arg Ala Glu Asp Ser 275 280 285 ACG CCC CAA ACC GAC CAA CTA GCT AAC GTA CAC AAG GCG GAC GAA TGG 912 Thr Pro Gln Thr Asp Gln Leu Ala Asn Val His Lys Ala Asp Glu Trp 290 295 300 GTT CGC GCA GCA AGC GAA GGA AGG AAA CTT AAC TAG 948 Val Arg Ala Ala Ser Glu Gly Arg Lys Leu Asn 305 310 315 SEQ ID NO: 3 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single-stranded topology Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence TCAACAAGCT GGAACGGACG 20 SEQ ID NO: 4 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence CGCCGATTTA CCGCCTTGAG 20 SEQ ID NO: 5 Sequence Length: 21 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence CCGTCTGGAA CCACGCCCGA T 21 SEQ ID NO: 6 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence ATCGGGCGTG ATTCCAGACG G 21 SEQ ID NO: 7 Sequence length: 21 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence CCGTCTGGAA GACCGCCCCCGA T
21 SEQ ID NO: 8 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATCGGGCGGT CTTCCAGACG G 21 SEQ ID NO: 9 Sequence length : 21 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCGTCTGGAA TGACGCCCGA T 21 SEQ ID NO: 10 Sequence length: 21 Sequence type: Nucleic acid Number: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence ATCGGGCGTC ATTCCAGACG G
21

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1:19) (C12N 1/21 C12R 1:13) (C12P 13/22 C12R 1:19) (C12P 13/22 C12R 1:13) (C12N 15/60 C12R 1:13) (C12N 15/60 C12R 1:19)

Claims (6)

[Claims] 1. Substitution of at least one amino acid residue in prephenate dehydratase with another amino acid residue,
Alternatively, a recombinant D containing a gene encoding an enzyme for which feedback inhibition has been released obtained by deletion
Microorganisms transformed with NA 2. The microorganism according to claim 1, wherein the L-serine residue at the 330th position from the N-terminus of prephenate dehydratase is substituted with another amino acid residue, and the microorganism is Escherichia coli. 3. The microorganism according to claim 2, wherein the other amino acid is a proline residue or an aspartic acid residue. 4. The microorganism according to claim 1, wherein the L-serine residue at the 235th position from the N-terminal of prephenate dehydratase is substituted, and the microorganism is Brevibacterium lactofermentum. 5. The microorganism according to claim 1, wherein the L-serine residue at position 330 from the N-terminal of prephenate dehydratase is deleted, and the microorganism is Escherichia coli. 6. A method for producing L-phenylalanine, which comprises obtaining L-phenylalanine produced in a medium by culturing the microorganism according to any one of claims 1 to 5.