JP3225597B2 - Method for producing L-phenylalanine by fermentation - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION L-Phenylalanine is an amino acid whose demand has rapidly increased in recent years as a raw material for sweetener aspartame. The present invention relates to the production of L-phenylalanine.

[0002]

2. Description of the Related Art As a production method for producing L-phenylalanine by fermentation using a microorganism, a recombinant Escherichia.
Japanese Patent Application Laid-Open Nos. 56-1890 and 5
7-170184, JP-A-58-103398, JP-A-61-92565, JP-A-1-104160, and International Publication WO87 / 00202. As a method for producing L-phenylalanine or L-tyrosine, a method using a mutant strain of the genus Corynebacterium may be used.
897, and those using recombinant Corynebacterium are disclosed in JP-A-60-34197 and JP-A-60-2419.
2, JP-A-61-260892, JP-A-61-1243
75 are known.

[0003] In the L-phenylalanine biosynthetic pathway, a key enzyme which plays a central role is usually subject to feedback inhibition by the final product. In the above-mentioned conventional technique, L-type is reduced by using a microorganism having a key enzyme whose feedback inhibition has been released.
The principle is to produce phenylalanine.

[0004] In the prior art, the key enzymes to be released from feedback inhibition include 3-deoxy-D-arabinohepturonic acid-7-phosphate synthase (hereinafter abbreviated as "DS") and prephenate. Dehydratase (hereinafter abbreviated as “PD”) and the like.

[0005] Regarding DS first, it is known that DS has three types of isozymes in Escherichia coli. These are ar o
Are encoded by genes called F , aroG , and aroH , respectively, L-tyrosine, L-phenylalanine,
-Receive feedback inhibition by tryptophan.

[0006] The nucleotide and amino acid sequences of these genes have already been reported [ aroF : Hudson,
GS and Davidson, BE, J. Mol. Biol., 180, 1023
(1984) / aroG : Davies, WD and Davidson, BE,
Nucleic Acids Res., 13, 4045 (1982) / aroH : Ra
y, JM et al, J. Bacteriol., 170, 5500 (1988)].

[0007] In order to efficiently produce aromatic amino acids, it is essential to improve these DSs. Of the three types of DS gene, for DS encoded by aroH, variant a ROH which feedback inhibition by L- tryptophan is released have been reported [Ray,
JM et al, J. Bacteriol., 170, 5500 (1988)]. However, originally, the DS activity derived from aroH is different from other D
Very low compared to the S activity, unsuitable for improvement by recombinant DNA techniques, aroF, considered use those DS feedback inhibition released encoded by the aro G is more efficient.

[0008] As an example of a mutation deactivating feedback inhibition of aroF by L-tyrosine, a report by Weber and Herman [Weaver, LM and Herrmann, KM, J. Bact.
eriol., 172, 6581 (1990)], and 148 from the N-terminus.
The second L-proline residue replaces the L-leucine residue.

Among the DSs whose feedback inhibition has been released, those in which the mutation site is clearly specified are applied to fermentative production of aromatic amino acids, and there are only a few examples shown below. Edwards et al. Added an L-glutamine residue at position 152 of DS encoded by aroF to L-glutamine.
The feedback inhibition by L-tyrosine is released by substituting the isoleucine residue, and it is utilized for the fermentative production of L-phenylalanine [WO 87/0020].
2]. In addition, DS Shinenki et al., Which is encoded in aroG
By substituting the L-leucine residue at position 76 with L-valine residue, DS ( aroG ) in which feedback inhibition by L-phenylalanine was released was obtained and L- leucine was obtained.
Used for fermentative production of phenylalanine
8-103398]. However, in this report, L-
Data on the enzymatic activity of DS in which feedback inhibition by phenylalanine has been released and production of L-phenylalanine are not described.

Concerning PD, in Escherichia coli, chorismate mutase (hereinafter referred to as "C
M ") and a bifunctional enzyme having PD activity (C
M-PD) is known, and the enzyme activity is feedback inhibited by phenylalanine. The enzyme is
It 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, GSa
nd Davidson, BE, J. Mol. Biol., 180, 1023 (198
Four)]. To produce phenylalanine efficiently,
It is important to release the feedback inhibition of CM-PD by phenylalanine, and several methods analyzed at the amino acid level are known. For example, Gessing and Davidson are CM-
It was reported that modification of two tryptophan residues (226th and 338th from the N-terminus) of PD with dimethyl [2-hydroxy-5-nitrobenzylsulfonium bromide] resulted in an enzyme resistant to feedback inhibition. [Gething, MJH and Davidson, BE, Eu
rJ Biochem., 78, 111 (1977)]. In addition, Bachmann et al. Found that an enzyme whose feedback inhibition had been released could be obtained by deleting the tryptophan residue at position 338 from the N-terminal side of CM-PD or by substituting this residue with arginine-glycine. [Japanese Patent Laid-Open No. 1-235997]. Further, Edwards et al. Also released feedback inhibition by inserting the amino acid sequence of tryptophan-arginine-serine-proline at the position of tryptophan residue at position 338 [International Patent WO87 / 000020]. All of these focused on the tryptophan residue at position 338, and there was no knowledge of the other residues.

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

[0012] Among the genes encoding the enzyme, Ozaki et al. [Ozaki, A. e t al. , Agric. Biol.chem., 49, 2925 (1
986)], and Ito et al . [Ito, H. et al ., Appl. Microbio
l. Biotechnol., 33, 190 (1989)] report a gene in which feedback inhibition by phenylalanine has been released. In addition, Foretie and Sinsky reported the nucleotide sequence of the native PD gene and pointed out homology with the pheA gene of Escherichia coli K-12 [Follettie, MT and Sinsky, AJ, J. . Bac
teriol., 167, 695 (1986)]. However, there was no finding about the nucleotide sequence of the enzyme gene whose feedback inhibition was released in coryneform bacteria. Furthermore, no attempt at conversion at the nucleotide sequence level and concomitant release of inhibition by amino acid substitution has been made.

Another L-phenylalanine biosynthesis system
One of the key enzymes is shikimate kinase (hereinafter abbreviated as SK), but the SK gene of Escherichia coli is used.
aroL was cloned by Defaiter et al. [J.
Bacteroil., 165, 226 (1986)] and its nucleotide sequence has been determined [J. Bacteriol., 165, 233 (1986)]. However, a specific example of using SK for fermentative production of L-phenylalanine in Escherichia coli has not been reported yet, and only a report has been reported for a coryneform bacterium (Japanese Patent Application Laid-Open No. 62-143682).

In Escherichia coli, when an aromatic amino acid is excessively produced, a protein called TyrR is activated, and two kinds of DS ( aroF and aroG as genes) and SK in the aromatic amino acid biosynthetic pathway are used.
( AroL as a gene) and tyrosine aminotransferase ( tyrB as a gene) are known to have a so-called feedback suppression mechanism that suppresses the expression of a gene [J. Bacteriol., 108, 400 (1
971)]. This TyrR protein gene ( tyrR )
As a specific example of fermentative production of L-phenylalanine using Escherichia coli lacking E. coli, Choi and Tribe et al. [Biotechnol. Lett., 4, 223 (198
2)] and [JP-A-57-170184] are known.
In this example, only two genes, aroF and pheA , are specifically mentioned as genes to be introduced into Escherichia coli in which the tyrR gene has been deleted.

[0015]

SUMMARY OF THE INVENTION The object of the present invention is to
-To provide a method for efficiently producing phenylalanine by fermentation.

[0016]

Means for Solving the Problems The inventors of the present invention have conducted studies for the purpose of developing a method for efficiently producing L-phenylalanine by fermentation, and as a result, have completed the present invention.

That is, the present invention encodes DS and PD which have been obtained by adding a mutation such as substituting or deleting one or more amino acid residues with another amino acid, and in which feedback inhibition has been released. A tyrR and tyrA gene-deleted microorganism belonging to the genus Escherichia transformed with a DNA fragment and a recombinant vector containing a DNA fragment encoding SK, and a large amount of L-phenylalanine can be obtained by culturing the microorganism. A method for producing L-phenylalanine, which comprises producing and obtaining.

Furthermore, the present invention relates to a microorganism derived from the genus Escherichia, which is a microorganism derived from the genus Escherichia, wherein the host species is deficient in the tyrR and tyrA genes, and the feedback inhibition of which is eliminated, Wherein the aspartic acid residue at position 147 from the N-terminus is replaced by an asparagine residue, aroF
Wherein the serine residue at position 181 from the N-terminus is substituted with a phenylalanine residue, aroG
DS in which the proline residue at the 150th position from the N-terminus is substituted with a leucine residue, whereas DS in which the alanine residue at the 202nd position from the N-terminus is substituted with a threonine residue, which is encoded by aroG, 146th aspartic acid residue from the N-terminus of those encoded by the DS substituted with an asparagine residue, 147th methionine residue from the N-terminus of those encoded by aroG is 332 th are replaced with an isoleucine residue DS glutamic acid residue is replaced with a lysine residue, 147th methionine residue from the N-terminus of those encoded by aroG is DS substituted with an isoleucine residue, the 157th from the N-terminus of those encoded by aroG A methionine residue is replaced with an isoleucine residue, and an alanine residue at position 219 is replaced with threonine. A DNA fragment encoding any one selected from among the DS, which is substituted in residues, derived from Escherichia which is desensitized to feedback inhibition CM-PD
CM-PD in which the 330th serine residue has been replaced with a proline residue, CM-PD in which the 330th serine residue has been replaced with an aspartic acid residue, and CM-PD after the 330th serine residue. A DNA fragment encoding any one of the lost CM-PDs, and a recombinant vector obtained by inserting the DNA fragment encoding SK into a vector for Escherichia bacteria,
And a method for producing L-phenylalanine by a fermentation method using the microorganism.

The present inventors first cloned a natural DS gene of Escherichia coli and mutated it to obtain a novel gene encoding DS in which feedback inhibition was released. In addition, a natural PD gene of Brevibacterium lactofermentum was cloned, and a gene encoding PD in which feedback inhibition was released from L-phenylalanine-producing bacteria was obtained, and the mutation point was determined. Furthermore, a novel gene encoding a CM-PD gene whose feedback inhibition has been released by cloning a natural CM-PD gene of Escherichia coli and mutating it based on a mutation point in Brevibacterium lactofermentum is disclosed. I got it. The gene encoding SK was cloned by a usual method.

Next, the gene encoding DS and the gene encoding PD are combined to form the tyrR gene, tyr
The fermentative production of L-phenylalanine is better when a gene encoding DS and a gene encoding PD and further a gene encoding SK are combined than when the gene is introduced into Escherichia coli lacking the A gene. This led to the completion of the present invention.

Hereinafter, the present invention will be described in detail.

In the present invention, the acquisition of a novel gene encoding DS in which feedback inhibition has been released can be carried out as follows.

First, the MC of Escherichia coli K-12
The aroF and aro G genes were cloned from the chromosomal DNA of strain 1061 (ATCC 53338) by PCR and the target gene was mutated using hydroxylamine.

The terms aroF and aroG refer to genes encoding DSs that are each subject to feedback inhibition by L-tyrosine and L-phenylalanine, and include mutants due to genetic polymorphism and the like. Genetic polymorphism is a phenomenon in which the amino acid sequence of a protein is partially changed due to a spontaneous mutation in a gene.

To generate a mutation in a gene, a recombinant PCR method [PCR Technology, Stockton press (198)
9)], site-directed mutagenesis [Kramer, W. and Frits, H.
J., Methods in Enzymology, 154, 350 (1987)], a method of irradiating a strain having the gene with ultraviolet rays or a treatment with a chemical agent (N-methyl-N′-nitrosoguanidine,
Nitrite) and the method of chemically synthesizing the target gene.

In DS, the amino acid residue substituted with another amino acid refers to an amino acid residue present in an amino acid sequence region involved in the mechanism of feedback inhibition by L-tyrosine, L-phenylalanine, or L-tryptophan. For example, DS encoded by aroF refers to the 147th aspartic acid residue and the 181st serine residue from the N-terminal side, and these are shown in Table 1.
It is summarized in.

The substitution with another amino acid residue in DS means L-tyrosine, L-phenylalanine, or L-tyrosine.
-Refers to substitution with another amino acid residue such that the feedback inhibition by tryptophan is released, as shown in Table 1.

[0028]

[Table 1]

In the present invention, a novel gene encoding PD with feedback inhibition released, that is, PD in Brevibacterium lactofermentum, and CM-PD in Escherichia coli is obtained as follows. Was.

First, L-phenylalanine-producing Brevibacterium lactofermentum L-
-By determining and analyzing the nucleotide sequence of the PD gene from which the feedback inhibition by phenylalanine was released, it was found that the production strain had one amino acid substitution compared to the wild strain. Next, based on the present finding, the same amino acid substitution in the homologous region of CM-PD in Escherichia coli K-12, that is, when the serine residue at position 330 was substituted with a proline residue, CM-PD from which feedback inhibition was released was obtained. Was able to get. Alternatively, when CM-PD in which the 330th serine residue is substituted with an aspartic acid residue and CM-PD in which the 330th serine residue and subsequent residues are deleted are constructed, the feedback inhibition of the enzyme is released. There was found.

The enzyme having PD activity referred to in the present invention is:
Those having the activity alone derived from a microorganism such as a coryneform bacterium; and those derived from a microorganism such as Escherichia coli
It refers to a substance having two functions such as M-PD.

In PD, the amino acid residue substituted or deleted by another amino acid refers to an amino acid residue present in an amino acid sequence region involved in the mechanism of feedback inhibition by L-phenylalanine.
For example, in the case of Brevibacterium lactofermentum PD, the serine residue is substituted at the 235th position from the N-terminal side, and in the CM-PD of Escherichia coli, the substitution is at the 330th position from the N-terminal side. It refers to the deletion after the 330th serine residue from the N-terminal side of CM-PD.

In PD, the substitution with another amino acid residue means a substitution with another amino acid such that feedback inhibition by L-phenylalanine is released. For example, the PD of Brevibacterium lactofermentum
Refers to substitution of a serine residue at position 235 from the N-terminal side with a proline residue in CM-PD of Escherichia coli at position 330 from the N-terminal side. Furthermore, it refers to a protein in which the 330th serine residue of Escherichia coli K-12 is substituted with an aspartic acid residue.

For the SK gene, the aroL gene was cloned from the chromosomal DNA of the MC1061 strain of Escherichia coli K-12 by PCR. The cloning method may be a method other than PCR, which is conventionally obtained from a gene library of Escherichia coli.

The recombinant DNA obtained by the above-mentioned method includes DS, PD and S which have released feedback inhibition.
It means that a useful gene encoding K is incorporated into a plasmid or phage DNA vector as a passenger. At that time, a promoter that works in Escherichia coli such as lac , trp , or PL may be used in order to efficiently express the useful gene. The useful gene is added to the recombinant DNA herein by a transposon [Be
rg, DE and Berg, CM, Bio / Technol., 1, 417 (198
3)], Mu phage [JP-A-2-109985] or homologous recombination [Experiments in Molecular Genetics, C
old Spring Habor Lab. (1972)].

The Escherichia coli lacking the tyrR gene
E. coli was first obtained by cloning the tyrR gene. The tyrR gene was cloned from the chromosomal DNA of MC1061 strain of Escherichia coli K-12 by PCR. This tyrR gene is treated with an appropriate restriction enzyme to construct a deleted tyrR gene,
This deletion ty rR gene that replace the normal tyrR on the chromosome of E. coli, was constructed the deleted Escherichia coli ty rR in other words by the homologous recombination technique of gene substitution using. In addition, deletion tyrR
For the construction of the gene, in addition to deletion by restriction enzyme treatment, the aforementioned chemical agent treatment, recombinant PCR, site-directed mutagenesis, and a method of chemically synthesizing the deleted tyrR gene can be considered. Also, a method of irradiating Escherichia coli with ultraviolet rays or a chemical treatment (N-methyl-N ′)
-Nitrosoguanidine , nitrite, etc.) to directly construct Escherichia coli lacking the tyrR gene.

Escherichia coli lacking the tyrA gene
Acquisition of stiffness is also achieved in the same manner as in the case of tyrR .
The tyrA gene is a gene encoding prephenate dehydrogenase. The enzyme catalyzes a reaction in a pathway for synthesizing L-tyrosine from prephenic acid. In a strain lacking the enzyme, prephenic acid is efficiently converted to L-phenylalanine.

The tyrR , ty transformed with the recombinant DNA containing the DS and PD genes and the SK gene, which have been released from the feedback inhibition, obtained by the above method.
Escherichia coli deficient in the rA gene was cultured, and L-phenylalanine was produced and accumulated in the culture solution, and collected.

The medium for producing L-phenylalanine used is an ordinary medium containing a carbon source, a nitrogen source, inorganic ions and other organic components as required.

As the carbon source, saccharides such as glucose, lactose, galactose, hydrolysates of fructose and starch, alcohols such as glycerol and sorbitol, and 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 organic trace nutrients, vitamin B1,
It is desirable that a required substance such as L-tyrosine or a yeast extract be contained in an appropriate amount.

In addition to these, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions and the like are added as required.

The cultivation is preferably carried out under aerobic conditions for 16 to 72 hours.
H is controlled to 5-7. In addition, an inorganic or organic acidic or alkaline substance, furthermore, ammonia gas or the like can be used for pH adjustment.

The collection of L-phenylalanine from the fermentation broth can be usually carried out by a combination of an ion exchange resin method, a precipitation method and other known methods.

According to the above-described method, DS and PD, and SK having feedback inhibition are released.
When the Escherichia coli transformant lacking the tyrR and tyrA genes was cultured, a significant improvement in L-phenylalanine productivity was observed. This demonstrates the usefulness of the present invention.

Hereinafter, the present invention will be described more specifically based on embodiments.

[0048]

【Example】

Example 1 Acquisition of a novel gene encoding DS in which feedback inhibition has been released (1) Acquisition of a mutated DS gene derived from aroF of Escherichia coli From the MC1061 strain of Escherichia coli K-12, chromosomal DNA was obtained according to a conventional method. Extracted. On the other hand, two synthetic DNA primers as shown in SEQ ID NOs: 1 and 2 were prepared based on the base sequence of the aroF gene described in a known document [J. Mol. Biol., 180, 1023 (1984)]. Synthesized by the method. SEQ ID NO: 1 GCTAACCAGT AAAGCCAACA SEQ ID NO: 2 CCCACTTCAG CAACCAGTTC These have homologous sequences upstream and downstream of the aroF gene, respectively. Using this chromosomal DNA and DNA primers, the method of Elrich et al. [PCR Technology, Stockto
n press (1989)].
A Kbp DNA fragment was obtained. Hereinafter, as shown on the left side of FIG. 1, this fragment was ligated with the restriction enzymes Eco RV and Eco 47II.
After cutting with I, Sm of pHSG398 (Takara Shuzo)
The aI digest was ligated using T4 DNA ligase. In this reaction mixture, JM10 of Escherichia coli K-12 was used.
Nine strains (manufactured by Takara Shuzo Co., Ltd.) were transformed, and plasmids were extracted from the chloramphenicol-resistant strains that had grown and had the plasmid into which the aroF gene was inserted, thereby obtaining plasmid pHSG- aroF . Furthermore, pHSG-
The aroF gene-containing DNA fragment obtained by cutting the aroF with restriction enzymes Eco RI and Hind III,
Plasmid pTS1 using T4 DNA ligase (Japanese Patent Application No.
192162) with EcoRI and HindIII digested fragments. In this reaction mixture, Escherichia
DS loss of E. coli K-12 (aroF, aroG, ar o
H) Transformation of strain AB3257 (AB3257 strain is Escherichia coli genetic stock)
Obtained from the Center). Plasmids were extracted from the grown ampicillin-resistant strains in which the requirement for L-tyrosine, L-phenylalanine, and L-tryptophan was lost, and plasmid pTS- aroF was obtained.

Next, the plasmid pTS- aroF was prepared by a method using hydroxylamine [J. Mol. Biol., 175, 33].
1 (1984)], followed by AB325
After transforming into 7 strains and obtaining ampicillin-resistant strains,
Two strains that grew on the minimal medium supplemented with M L-tyrosine were selected, and the plasmid pTS- aro containing the aroF gene whose feedback inhibition was released from these strains was selected.
F15 and pTS- aroF33 were obtained. The AB3257 strain having a plasmid containing aroF in which the feedback inhibition was not released , when 1 mM L-tyrosine was added to the minimal medium as shown in FIG. 2, the DS activity was subjected to feedback inhibition, and L-phenylalanine and L-tyrosine were inhibited. Aromatic amino acids such as tryptophan cannot be synthesized and cannot grow.

(2) Escherichia coliaroGOrigin
Acquisition of mutant DS genearoF As in the case of the gene,aroGRemains
I got a gene. Known literature [Nucleic Acids Res., 1
0, 4045 (1982)]aroGGene salt
Synthetic D as shown in SEQ ID NOs: 3 and 4 based on the base sequence
Two NA primers were synthesized. SEQ ID NO: 3 GTATTTACCC CGTTATTGTC SEQ ID NO: 4 ACTCCGCCGG AAGTGACTAA Using the primers and chromosomal DNA of MC1061 strain
And perform a PCR reaction to obtain a 2.1 kbp DNA fragment.
Obtained. Hereinafter, as shown on the right side of FIG. 1, this fragment is restricted.
enzymeSalI andEcoAfter cleavage with 47III, pHS
G398 (manufactured by Takara Shuzo)SalI andSmaI cut
Ligation was performed using T4 DNA ligase. This reaction mixture
Chlorampheni grown by transforming the JM109 strain with
Among call resistant strainsaroGPlus with gene inserted
Plasmids were extracted from the strains containing
PHSG-aroGI got Furthermore, pHSG-a
roGThe restriction enzymeEcoWith RIH indCut at III
Obtained byaroGDNA fragment containing
Was ligated with pTS1 using T4 DNA ligase.EcoRI,H
indLigation with the III cleavage fragment. This reaction mixture
AB3257 strain (aroF,aroG,aro
H) Was transformed and grown in ampicillin resistant strains.
L-tyrosine, L-phenylalanine, L-tryptophan
Extract plasmids from strains that have lost the requirement for
And the plasmid pTS-aroGI got

Next, this plasmid was mutated with hydroxylamine in the same manner as in the case of aroF , and then transformed into strain AB3257 to obtain an ampicillin-resistant strain. From these strains, 6 strains grown on a minimal medium supplemented with 10 mM L-phenylalanine were selected,
Feedback inhibition than these strains has been released a r
a plasmid pTS- aroG4 containing the oG gene,
pTS- aroG8 , pTS- aroG15 , pTS-
aroG17 , pTS- aroG29 , pTS- aro
G40 was obtained. Feedback inhibition has not been released
As shown in FIG. 3, when 3 mM L-phenylalanine was added to the minimal medium, the DS activity was feedback-inhibited and the aromatic amino acids such as L-tryptophan and L-tyrosine were synthesized in the AB3257 strain carrying the plasmid containing aroG . And cannot grow.

(3) Measurement of DS enzyme activityaroF(2 types) and variantsaroG
(6 types) containing a plasmid having no DS activity.
And transformed into Escherichia coli AB3257 strain
Converted strains were obtained, and each was transferred to AJ12598 (AB325).
7 / pTS-aroF15), AJ12599 (AB3
257 / pTS-aroF33), AJ12562 (A
B3257 / pTS-aroG4), AJ12600
(AB3257 / pTS-aroG8), AJ1256
3 (AB3257 / pTS-aro G15), AJ12
601 (AB3257 / pTS-aroG17), AJ
12602 (AB3257 / pTS-aroG29)
And AJ12603 (AB3257 / pTS-aroG4
0). Among them, AJ125 was used as a representative strain.
63 and AJ12603, respectively, Escherichia coli
 FERM BP-3567, FERM BP-35
Deposited at 68 as a 68. For comparison, the natural type
A plasmid containing this gene was also introduced into the strain.

These strains were transformed into a known L-phenylalanine production medium [Sugimoto, S. et al., J. Biotechnol., 5,
237 (1989)]. A crude enzyme solution was prepared from the cultured cells by sonication, and a conventional method was used [Gollub, E. et al., Methods Enzymol., 17, 349].
In the case of aroF, in the presence of L-tyrosine, a
In the case of roG , the enzymatic activity of DS was measured in the presence of L-phenylalanine. As a result, as shown in FIGS. 2 and 3, the natural type (Escherichia coli AB3257 /
In pTS- aroF ), the enzyme activity was strongly inhibited in the presence of L-tyrosine, whereas in each mutant, the feedback inhibition by L-tyrosine was released. Similarly, in the other natural type (Escherichia coli AB3257 / pTS- aroG ),
While the enzyme activity was strongly inhibited in the presence of L-phenylalanine, the feedback inhibition by L-phenylalanine was released in each mutant. Further, DS of AJ12562 strain among the mutant types is L
Not only the feedback inhibition by -phenylalanine was released, but also the enzyme activity increased according to the concentration of L-phenylalanine.

(4) D in which feedback inhibition has been released
Determination of S mutation point a which is a DS gene from which feedback inhibition has been released
roF15 , aroF33 , aroG4 , aroG8 ,
aroG15 , aroG17 , aroG29 , aro G
The 40 nucleotide sequence was prepared by the usual method [Molecular Cloning (Sec.
ond Edition), Cold Spring Harbor Press (1989)]. Table 1 shows specific substitution sites on the amino acid sequence and the corresponding mutation points on the base sequence. All of these sequences have not been previously reported.

Example 2 Obtaining a New Gene Encoding a PD from Which Feedback Inhibition has been Released (1) Determination of Mutation Point of Brevibacterium Mutant PD First, a known Corynebacterium PD gene [Follet
tie, MT and Sinsky, AJ, J. Bacteriol., 167, 69
5 as an indicator homology with (1986)], and the nucleotide sequence of the Nco I fragment in plasmid pAJ16 including PD gene of Brevibacterium lactofermentum wild strain was determined by the dideoxy method. The sequence is represented by SEQ ID NO: 5 in the sequence listing.
Shown in In addition, this plasmid was used for brilliobacterium lactofermentum AJ12125 (FERM-P75).
46). The amino acid sequence differs from that of Corynebacterium by only one amino acid residue. Next, the nucleotide sequence of the gene encoding PD of the phenylalanine-producing strain of Brevibacterium lactofermentum on the plasmid pPH14 was determined, and a sequence as shown in SEQ ID NO: 6 in the sequence listing was obtained. The plasmid was used for Brevibacterium lactofermentum AJ12259 (FERM P-3).
565) What was held in the strain was used. Comparison between the wild strain and the strain for which feedback inhibition was released 23
The fifth serine residue was mutated to a proline residue.

(2) Escherichia coli mutant CM-
Construction of New Gene Encoding PD First, chromosomal DNA was extracted from Escherichia coli K-12 strain RR1 according to a conventional method.

On the other hand, a known document [Hudson, GS and Dav
idson, BE, J. Mol. Biol., 180, 1023 (1984)], based on the base sequence of the pheA gene, four synthetic DNA primers as shown below (SEQ ID NO: 7).
-10) was chemically synthesized by an ordinary method. SEQ ID NO: 7 TCAACAAGCT GGAACGGACG SEQ ID NO: 8 CGCCGATTTA CCGCCTTGAG SEQ ID NO: 9 CCGTCTGGAA CCACGCCCGA T SEQ ID NO: 10 ATCGGGCGTG ATTCCAGACG G 7 and 8 have homologous sequences upstream and downstream of the pheA gene, respectively. 9 and 10 are complementary to each other and T
(Thymine base) differs from C (cytosine base) only by one base, and has homology to the sequence near the serine residue at position 330. CM-P of Escherichia coli K-12
D and Brevibacterium lactofermentum PD
Has high homology with Escherichia coli K-1
The serine residue at position 330 from the N-terminus of CM-PD No. 2 corresponds to the serine residue at position 235 from the N-terminus of Brevibacterium lactofermentum PD. SEQ ID NOS: 9 and 10 have been synthesized so that the serine residue at position 330 becomes a proline residue.

Next, 1 μg of chromosomal DNA and SEQ ID NO: 7
PCR reaction was performed using 300 ng of each of the primers of Nos. 10 and 10 or 300 ng of each of the primers of SEQ ID Nos. 8 and 9, and the reaction was performed at 1.3 Kbp and 0.3 Kbp, respectively.
A 5 Kbp DNA fragment was obtained. The PCR method is the method of Elrich et al. [Erlich, HA, ed., PCR Technology,
According to Stockton press (1989)], a continuous replication reaction apparatus (Thermal cycler, manufactured by Perkin Elmer Cetus) was used to carry out 20 cycles of 94 ° C for 1 minute, 50 ° C for 2 minutes, and 72 ° C for 3 minutes as one cycle. . These DNs
The A fragment was subjected to agarose gel electrophoresis, recovered using a DNA recovery kit (Gene Clean, manufactured by Funakoshi), and further subjected to a PCR reaction using these fragments and the primers of SEQ ID NOs: 7 and 8 to perform 1.8 Kbp. A DNA fragment was obtained. After digesting this fragment with the restriction enzymes Bam HI and Pst I, a 1.7 Kbp DNA fragment was recovered by agarose gel electrophoresis.
Bam HI and Pst I digests of SG398 (Takara Shuzo) were ligated using T4 ligase. This was transformed into a KA197 strain ( pheA ) of Escherichia coli K-12, and a plasmid was recovered from a chloramphenicol-resistant strain in which phenylalanine auxotrophy was lost, and named pPHAB. By determining the nucleotide sequence, it was confirmed that the plasmid had a mutant CM-PD enzyme gene in which the serine residue at position 330 was replaced with a proline residue.

(3) Escherichia coli K-12ty
rAConstruction of Gene-Deficient W3110 Strain First, the Escherichia coli K-12 W3110 strain (country
Obtained from the National Institute of Genetics)
Streptomycin resistant by plating on plate medium
Acquired shares. Next, this strain and Escherichia coli K-
12 ME8424 strains (HfrPO45,thi,re
lA1,tyrA:: Tn10,ung-1,nad
B) (Obtained from National Institute of Genetics)
After leaving it at 7 ° C for 15 minutes to allow the joint to transfer,
Including ptomycin, tetracycline, L-tyrosine
After plating on a plate medium, the resulting colonies, ie, Escherichia
Coli K-12 W3110 (tyrA) Acquired shares.
This strain was transformed with the plasmid pPHAB obtained in (2).
Introduced by exchange. In addition, Escherichia coli K-12
The transformed strain [W3110 (tyrA) / PPHAB]
Has been deposited with the microfabrication laboratory and the deposit number is FERM BP
-3566.

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

The PD enzyme activity was measured by a conventional method [Cotton, RG
H. and Gibson, F., Meth. In Enzymol., 17, 564 (197
0)]. That is, using a crude enzyme solution, 5 mM containing 1 mM barium prephenate and 0.5 mM tyrosine was used.
37 ° C. in the presence of 0 mM Tris-HCl buffer (pH 8.2)
After 10 minutes of reaction, 1N sodium hydroxide was added to stop the reaction, and the generated phenylpyrivic acid was measured at an absorption wavelength of 320 nm. The protein quantification method used a protein assay kit (manufactured by Bio Rad) and followed the protocol. The results are as shown in FIG.
In contrast, 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 was hardly inhibited.

Further, 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
It showed a higher enzyme activity of × 10 ⁴ U / mg protein.
Thus, by introducing the mutation, L-
Not only the feedback inhibition by phenylalanine was released, but also the enzyme amount or enzyme activity could be increased by 40 times.

(5) Escherichia coli mutant CM-
Construction of Novel Gene Encoding PD Similarly, (2) similarly, CM-PD and 330 in which the serine residue at position 330 was replaced with an aspartic acid residue by preparing a synthetic DNA of SEQ ID NOS: 11 to 14 and using this DNA A gene encoding CM-PD with the deletion after the second serine residue was constructed. Plasmids containing these genes were named pPHAD and pPHATerm, respectively. SEQ ID NO: 11 CCGTCTGGAA GACCGC
CCGA T SEQ ID NO: 12 ATCGGGCGGT CTTCCA
GACG G SEQ ID NO: 13 CCGTCTGGAA TGACGCCCGA T SEQ ID NO: 14 ATCGGGCGTC ATTCCAGACG G

Further, this plasmid was transferred to W3110 ( ty
rA ) strain was introduced using conventional transformation methods. In addition,
W3110 ( tyrA ) / pPHAD, W3110 ( t
yrA ) / pPHATerm has been deposited with the Microtechnical Laboratory, respectively, and their deposit numbers are FERM BP-365, respectively.
2 and FERM BP-3653.

(6) Measurement of PD enzyme activity W3110 ( tyrA ) of Escherichia coli K-12
/ PPHAD strain, W3110 ( tyrA ) / pPHAT
The cells of the erm strain were collected by centrifuging a culture solution cultured at 37 ° C. for 15 hours using an L medium. Next, the PD enzyme activity was measured in the same manner as in (4).
The results are the same as in (4), and the mutant CM-PD
L-phenylalanine was hardly inhibited.

Furthermore, the activity of these two mutant enzymes in the absence of L-phenylalanine was 1.5 × 10 ⁴ U
/ Mg protein. As a result, by introducing the mutation, not only the feedback inhibition by L-phenylalanine was released, but also the amount or activity of the enzyme could be increased by 40 times.

Example 3 Acquisition of SK Gene First, chromosomal DNA was extracted from the MC1061 strain of Escherichia coli K-12 according to a conventional method.

On the other hand, a known document [J. Bacteriol., 165,
233 (1986)] and the following two synthetic DNA primers (SEQ ID NOS: 15 to 16) were chemically synthesized by an ordinary method based on the nucleotide sequence of the aroL gene described in US Pat . SEQ ID NO: 15 GCGGAGCTCG AGAAGTGGTG SEQ ID NO: 16 ACTCAGAATT CCTTCCGAGC 15 and 16 have substantially homologous sequences upstream and downstream of the aroL gene, respectively. Using this chromosomal DNA and DNA primers, the method of Elrich et al. [PCR Technolog
y, Stockton press (1989)] to obtain a DNA fragment of 1.0 Kbp. Hereinafter, as shown in FIG. 5, this fragment was digested with restriction enzymes Eco RI and Sac I, and then the Eco R of pHSG398 (manufactured by Takara Shuzo Co., Ltd.) was digested.
I and Sac I digests were ligated using T4 DNA ligase to obtain pHSG- aroL .

Example 4 Construction of W3110 strain of Escherichia coli K-12 deficient in tyrR and tyrA genes Chromosomal DNA was extracted from the MC1061 strain of Escherichia coli K-12 according to a conventional method. On the other hand, two synthetic DNA primers as shown in SEQ ID NOs: 17 and 18 were prepared based on the nucleotide sequence of the tyrR gene described in a known document [J. Biol. Chem., 261, 403 (1986)]. Synthesized by the method. SEQ ID NO: 17 GGATTAAAGC TTTGGAGCTT SEQ ID NO: 18 GTGGATGAAT TCACCACCGA These have almost homologous sequences upstream and downstream of the tyrR gene, respectively. Using this chromosomal DNA and DNA primers, the method of Elrich et al. [PCR Technology, St.
ockton press (1989)].
A 1.9 Kbp DNA fragment was obtained. This fragment is
After cutting with Eco RI and Hin dIII, of pTS1
To obtain a ligated PTS tyrR with truncations and T4DNA ligase Eco RI and Hin dIII.

[0070] Next limit the pTS- tyrR enzyme Bgl I
I, a fragment of about 300 bp in the tyrR gene was deleted, the remaining fragment was blunt-ended with a DNA blunting kit (manufactured by Takara Shuzo Co., Ltd.), and then circularized again to obtain pTS
-ΔtyrR was constructed. The constructed pTS-Δ tyr R
Was introduced into the Escherichia coli K-12 strain W3110 (obtained from the National Institute of Genetics), homologous recombination took place, and the normal tyrR gene on the chromosome and the introduced Δ ty
The strain in which the rR gene was replaced was selected based on 3- fluorotyrosine resistance, and the E. coli strain in which the tyrR gene had been deleted was selected.
E. coli K-12 strain W3110 was constructed.

Next, a streptomycin-resistant strain was obtained by applying the constructed strain Escherichia coli K-12 W3110 lacking the tyrR gene to a plate medium containing streptomycin. Next, this strain and the ME8424 strain of Escherichia coli K-12 (HfrPO
45, thi , relA1 , tyrA :: Tn10, u
After ng-1, n adB) (mixed culture of availability) from the National Genetics Institute, was carried out left to conjugal transfer 15 min at 37 ° C., streptomycin, tetracycline, L
- applying the plate medium containing tyrosine, resulting colonies That tyrR, E. lacking of ty rA gene
E. coli K-12 strain W3110 was obtained.

Example 5 Fermentative production of L-phenylalanine (1) DS and CM in which feedback inhibition was released
-T3R carrying PD and SK, W3110 of Escherichia coli K-12 lacking the tyrA gene
Construction of strain pTS- aroG4 containing the DS gene in which feedback inhibition was released and obtained in Example 1 was replaced with restriction enzyme E
cut out aroG4 part in co RI and Hin dIII, Eco RI of this fragment pBR322, Hin dI
The plasmid pBR- aroG4 was inserted into the II cleavage site.
(Ampicillin resistance marker) was obtained. Further, the CM- in which the feedback inhibition obtained in Example 2 was released
The pPHAB containing PD gene, restriction enzyme Bam H
Was digested with I and Hin dIII cut the CM-PD gene-containing fragments, of this fragment pBR- aroG4
Plasmid pBGA1 was constructed by insertion into Bam HI and Hind III cleavage sites. Furthermore, pBGA1 was replaced with Eco.
Was digested with RI and Bam HI aroG4 - cut out pheA fragment, this fragment pMW19 of (manufactured by Wako Pure Chemical Industries, Ltd.)
Plasmid pMGAl was constructed by insertion into Eco RI and Bam HI cleavage sites.

Further, pHSG- aroL was replaced with Eco RI.
The aroL fragment was cut out with Sac I, and this fragment was
A1 of Eco RI, to construct a plasmid pMGAL1 and inserted into Sac I cleavage site.

Next, pMGAL or pMGAL1 is replaced with ty.
Escherichia coli K lacking rR and tyrA genes
-12 was introduced into the W3110 strain, and the transformed strain W
3110 ( tyrR , tyrA ) / pMGAl and transformant W3110 ( tyrR , tyrA ) / pMGA
L1 was created. The transformants were each AJ1274.
No. 0 strain and AJ12741 strain, and deposited at the microfabrication laboratory (FE
RM P-12999, FERM P-13000).

(2) L-phenylalanine productivity The transformants AJ12740 and AJ1 described in the preceding section
2741 is a medium for producing L-phenylalanine (glucose 20 g, disodium hydrogen phosphate 29.4 g, potassium dihydrogen phosphate 6 g, sodium chloride 1 g, ammonium chloride 2 g, sodium citrate 10 g, sodium glutamate 0.4 g, magnesium sulfate 7) 3 g of hydrate,
0.23 g of calcium chloride, 2 mg of thiamine hydrochloride,
(75 mg of tyrosine in 1 L of water) at 37 ° C. for 24 hours. Table 2 shows the results. The quantification was performed by high performance liquid chromatography.

[0076]

[Table 2]

[0077]

EFFECTS OF THE INVENTION In the fermentative production of L-phenylalanine, an efficient novel production bacterium and a method for producing L-phenylalanine using this strain are provided.

[Sequence list]

SEQ ID NO: 1 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GCTAACCAGT AAAGCCAACA 20 SEQ ID NO: 2 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CCCACTTCAG CAACCAGTTC 20 SEQ ID NO: 3 Sequence length: 20 Sequence type: nucleic acid Number of strands: 1 Single-stranded topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GTATTTACCC CGTTATTGTC 20 SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type : Other nucleic acid Synthetic DNA sequence ACTCCGCCGG AAGTGACTAA 20 SEQ ID NO: 5 Sequence length: 948 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Brevibacterium・ Lactofermentum (B
revibacterium lactofermentum) 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: 6 Sequence length: 948 Sequence type: Nucleic acid Number of strands: Double strand Topology : Linear Sequence type: Genomic DNA Origin Organism: Brevibacterium lactofermentum (B
revibacterium lactofermentum) 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: 7 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology Linear Sequence type: Other nucleic acid Synthetic DNA sequence TCAACAAGCT GGAACGGACG 20 SEQ ID NO: 8 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CGCCGATTTA CCGCCTTGAG 20 SEQ ID NO: 9 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: 10 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: 11 Sequence length: 21 Sequence Type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCGTCTGGAA GACCGCCCGA T
21 SEQ ID NO: 12 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: 13 Sequence length : 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCGTCTGGAA TGACGCCCGA T 21 SEQ ID NO: 14 Sequence length: 21 Sequence type: Nucleic acid Number: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence ATCGGGCGTC ATTCCAGACG G 21 SEQ ID NO: 15 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Type Sequence type: Other nucleic acid Synthetic DNA sequence GCGGAGCTCG AGAAGTGGTG 20 SEQ ID NO: 16 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence ACTCAGAATT CCTTCCGAGC 20 SEQ ID NO: 17 Sequence length: 20 Sequence type: Nucleic acid Number: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GGATTAAAGC TTTGGAGCTT 20 SEQ ID NO: 18 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Type Sequence type: Other nucleic acid Synthetic DNA sequence GTGGATGAAT TCACCACCGA
20

[Brief description of the drawings]

FIG. 1 Construction of pTS- aroF and pTS- aroG

FIG. 2 shows the degree of L-tyrosine inhibition of DS activity of native and mutant aroF .

FIG. 3 shows the degree of inhibition of DS activity of native and mutant aroG by L-phenylalanine.

FIG. 4 shows inhibition of prephenate dehydratase activity by wild-type and mutant chorismate mutase-prephenate dehydratase by phenylalanine.

FIG. 5. Construction of pMGAL1 and pMGAL1.

Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI (C12P 13/22 C12R 1:19) C12R 1:19) C12N 15/00 ZNA (56) References JP-A-63-105688 (JP, A JP-A-57-170184 (JP, A) JP-A-61-92565 (JP, A) JP-A-62-143682 (JP, A) Mol. Biol. 180, p. 1023-1051 (1984) Nucleic Acids Res. 13 p. 4045-4058 (1982) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 C12P 13/22

Claims (3)

(57) [Claims] 1. A microorganism belonging to the genus Escherichia,
The host is one in which the tyrR and tyrA genes have been deleted, and 3-deoxy-D-arabinohepturonic acid derived from the genus Escherichia whose feedback inhibition has been released.
7-phosphate synthase (hereinafter abbreviated as DS), comprising: a
roF- encoded D in which the aspartic acid residue at position 147 from the N-terminus is substituted with an asparagine residue
S, which is encoded by aroF but has a 181 serine residue from the N-terminal substituted with a phenylalanine residue;
S, DS in which the proline residue at the 150th position from the N-terminus is replaced by a leucine residue but encoded by aroG , a
DS of 202 th alanine residue from the N-terminus of those encoded roG is substituted with a threonine residue, aro
A DS encoded by G in which the aspartic acid residue at position 146 from the N-terminus is substituted with an asparagine residue, a
a DS encoded by roG in which the 147th methionine residue from the N-terminus is replaced by an isoleucine residue and the 332nd glutamic acid residue is replaced by a lysine residue, a
a DS encoded by ro G in which the 147th methionine residue from the N-terminus is replaced with an isoleucine residue, a
encodes any one selected from DS encoded by roG , wherein the 157th methionine residue from the N-terminus is replaced by an isoleucine residue and the 219th alanine residue is replaced by a threonine residue A DNA fragment and a chorismate mutase-prephenate dehydratase (hereinafter abbreviated as CM-PD) derived from the genus Escherichia whose feedback inhibition has been released, wherein CM-PD in which the serine residue at position 330 is replaced by a proline residue. , 33
CM-PD in which the 0th serine residue has been replaced with an aspartic acid residue, C in which the 330th serine residue and onwards have been deleted
A DNA fragment encoding any one of M-PD and shikimate kinase (hereinafter abbreviated as SK)
Which has a recombinant vector obtained by inserting a DNA fragment encoding the above into a vector for a bacterium belonging to the genus Escherichia. 2. The microorganism according to claim 1, wherein the microorganism is Escherichia coli AJ12741. 3. A method for producing L-phenylalanine by a fermentation method, wherein the microorganism according to claim 1 is used.