JPH0847397A - Production of l-isoleucine by fermentation methtod - Google Patents

Abstract

PURPOSE:To inexpensively obtain L-isoleucine useful as a medicine such as nutrient preparation by culturing a bacterium belonging to the genus Escherichia coli, having an operon containing a specific gene, capable of producing L- isoleucine in a medium and collecting a culture product in the culture. CONSTITUTION:A bacterium belonging to the genus Escherichia having thrABC operon containing thrA gene coding for aspartokinase I-homoserine dehydrogenase released from block with L-threonine and ilvGMEDA operon containing ilvA gene encoding threonine deaminase released from block with L-isoleucine and deficient in 953rd to 1160th of a DNA sequence as a region required for attenuation is cultured in a medium, L-isoleucine is formed and accumulated in the medium and L-isoleucine is collected from the medium to inexpensively obtain the subject L-isoleucine which is an essential amino acid and is useful as a medicine, mainly as nutrient preparation in high yield.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing L-isoleucine by a fermentation method and a bacterium belonging to the genus Escherichia used in the method. L-isoleucine is an essential amino acid for humans and animals, and is useful mainly for medicinal purposes including nutritional supplements.

[0002]

2. Description of the Related Art Since isoleucine has two asymmetric carbon atoms, it is difficult to industrially inexpensively produce only L-form by a method by chemical synthesis. Also known is a method for producing L-isoleucine by bioconversion using a precursor of L-isoleucine biosynthesis such as α-aminobutyric acid and α-ketobutyric acid as a raw material. However, these methods require expensive raw materials and cannot be said to be advantageous methods for industrially inexpensively producing L-isoleucine.

The following is known as a method for producing L-isoleucine by a direct fermentation method using an inexpensive carbon source and nitrogen source. 1) A method of using a mutant strain of the genus Corynebacterium, Serratia or Escherichia resistant to an L-isoleucine antagonist as an L-isoleucine producing strain (Japanese Patent Publication No. 51-21077,
Japanese Patent Publication No. 52-4629, Japanese Patent Publication No. 56-29998
2), 2) Threonine deaminase or acetohydroxy acid synthase, which is a key enzyme for L-isoleucine biosynthesis, of Escherichia genus or Corynebacterium genus enhanced by recombinant DNA technology. A method of using a microorganism as an L-isoleucine-producing strain (JP-A-2-458, JP-A-2-42988, etc.).

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing L-isoleucine in a higher yield at a lower cost by using a fermentation method.

[0005]

SUMMARY OF THE INVENTION The present inventors have coded for aspartokinase I-homoserine dehydrogenase I, which is substantially free of inhibition by L-threonine.
The thrABC operon containing the hrA gene, and the ilvGME containing the ilvA gene encoding threonine deaminase whose inhibition by L-isoleucine has been substantially removed, and the region necessary for attenuation was removed.
Using a bacterium belonging to the genus Escherichia having L-isoleucine productivity, which is characterized by retaining a DA operon,
We have succeeded in producing L-isoleucine at a higher yield and at a lower cost.

The present invention comprises a thrA gene coding for aspartokinase I-homoserine dehydrogenase I which is substantially desensitized by L-threonine.
The L-characterized by retaining the hrABC operon and the ilvGMEDA operon containing the ilvA gene encoding threonine deaminase whose inhibition by L-isoleucine is substantially eliminated and removing the region necessary for attenuation. The present invention provides an Escherichia bacterium having isoleucine productivity. The Escherichia bacterium of the present invention can retain the thrABC operon and the ilvGMEDA operon on a plasmid. The Escherichia bacterium of the present invention comprises a plasmid (A) carrying the thrABC operon,
Two types of plasmids, the plasmid (B) carrying the ilvGMEDA operon, can be retained. Specific examples of the ilvGMEDA operon include:
One having a DNA sequence in which 953 to 1160 of the DNA sequence described in SEQ ID NO: 1 of the Sequence Listing is deleted can be mentioned.

Specific examples of the plasmid (A) include pVI
C40 is pM as a specific example of the plasmid (B).
WD5 is mentioned. The preferred Escherichia bacterium is Escherichia coli. A further preferred Escherichia bacterium of the present invention is deficient in the thrC gene, can grow in the presence of 5 mg / ml of L-threonine, is deficient in threonine dehydrogenase activity, and has ilv
A host strain having a leaky mutation in the A gene has the above-mentioned thr
It is Escherichia coli into which the ABC operon and the ilvGMEDA operon are introduced. A specific example thereof is Escherichia coli AJ12919 strain.

The present invention also relates to the thrABC operon containing the thrA gene encoding aspartokinase I-homoserine dehydrogenase I, which is substantially desensitized by L-threonine, and the inhibition by L-lysine. And a lysC gene encoding aspartokinase III, and an ilvGMEDA operon containing an ilvA gene encoding threonine deaminase whose inhibition by L-isoleucine is substantially removed and having a region necessary for attenuation removed. A bacterium belonging to the genus Escherichia having L-isoleucine productivity is provided. The Escherichia bacterium of the present invention can retain the thrABC operon, the lysC gene, and the ilvGMEDA operon on a plasmid.

The Escherichia bacterium of the present invention is the above-mentioned th
Two types of plasmids, a plasmid (C) carrying the rABC operon and the lysC gene and a plasmid (B) carrying the ilvGMEDA operon, can be retained. Specific examples of the ilvGMEDA operon include those having a DNA sequence in which 953 to 1160 of the DNA sequence shown in SEQ ID NO: 1 of the Sequence Listing are deleted. Specific examples of the plasmid (C) include pVICLC * 80A, and specific examples of the plasmid (B) include pMWD5. The preferred Escherichia bacterium is Escherichia coli. A more preferred Escherichia bacterium of the present invention lacks the thrC gene and contains 5 mg / ml of L-.
The above-mentioned thrABC operon and the above-mentioned ly were added to a host strain capable of growing in the presence of threonine, lacking threonine dehydrogenase activity, and having a leaky mutation in the ilvA gene.
Escherichia coli into which the sC gene and the ilvGMEDA operon have been introduced. A specific example thereof is Escherichia coli AJ13100 strain.

The present invention further comprises culturing the above-mentioned Escherichia bacterium in a medium to cause L-isoleucine to be produced and accumulated in the medium, and collecting L-isoleucine from the medium, and L-isoleucine by a fermentation method. The present invention provides a manufacturing method of. Hereinafter, in the specification of the present application, the thrA gene encoding aspartokinase I-homoserine dehydrogenase I whose inhibition by L-threonine is substantially released is referred to as “released thrA gene”.
Sometimes called. In addition, the thrABC operon containing the thrA gene encoding aspartokinase I-homoserine dehydrogenase I whose inhibition by L-threonine is substantially released may be referred to as “released thrABC operon”.

In the present specification, the ilvA gene coding for threonine deaminase whose inhibition by L-isoleucine is substantially released may be referred to as "released ilvA gene". In addition, the ilvGMEDA operon containing the ilvA gene (released ilvA gene) encoding threonine deaminase whose inhibition by L-isoleucine has been substantially released is referred to as “released ilvGMED”.
Sometimes called "A operon". The ilvGMEDA operon containing the released ilvA gene and having the region necessary for attenuation removed was designated as "completely released ilvGM".
Sometimes called the "EDA operon". In the present specification, aspartokinase I-homoserine dehydrogenase I may be abbreviated as “AKI-HDI”. Further, threonine deaminase may be abbreviated as “TD”.

In the present specification, aspartokinase I-in which inhibition by L-threonine is substantially released
Homoserine dehydrogenase I was added to "released AKI-HD
Sometimes referred to as "I". Further, threonine deaminase whose inhibition by L-isoleucine is substantially released may be referred to as “released TD”. Hereinafter, the present invention will be described in detail.

<1. ThrABC operon containing the thrA gene substantially free of inhibition by L-threonine>

ThrABC containing the thrA gene coding for aspartokinase I-homoserine dehydrogenase I substantially desensitized by L-threonine
The operon, that is, the released thrABC operon, is a wild-type thr in that the contained thrA gene has a mutation.
Different from the ABC operon. The mutation means a mutation that cancels the feedback inhibition of AKI-HDI encoded by the thrA gene by L-threonine.

The method for obtaining such a release type thrABC operon is as follows. First, wild type thr
In vitro mutation treatment of DNA containing the ABC operon, and vector DN compatible with the DNA after the mutation treatment and the host
Ligation with A gives recombinant DNA. The recombinant DNA is introduced into a host microorganism to obtain a transformant, and if a transformant capable of expressing the released AKI-HDI is selected, the transformant is released from the thrABC operon. Holding In addition, a DNA containing the wild-type thrABC operon is used as a vector DNA compatible with the host.
To obtain a recombinant DNA, which is then subjected to in vitro mutagenesis, and the mutagenized recombinant DNA is introduced into a host microorganism to obtain a transformant. Even when the one that has reached the expression level of AKI-HDI is selected, the transformant retains the released thrABC operon.

The wild type AKI-HDI producing microorganism may be subjected to a mutation treatment to create a mutant strain producing the released AKI-HDI, and then the released thrABC operon may be obtained from the mutant strain. Alternatively, a transformant into which a recombinant DNA in which the wild-type thrABC operon is ligated is introduced is subjected to mutation treatment to create a mutant strain producing a mutant AKI-HDI, and then the recombinant DNA is transferred from the mutant strain. If recovered, the release type thrABC operon may be created on the same DNA.

Examples of agents for in vitro mutation treatment of DNA include hydroxylamine and the like. Hydroxylamine is a chemical mutagen that causes a mutation from cytosine to thymine by converting cytosine into N4-hydroxycytosine. When the microorganism itself is subjected to mutation treatment, it is treated with ultraviolet rays or a mutagen which is usually used for artificial mutation such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG). thrA
The BC operon is derived from a bacterium belonging to the genus Escherichia, particularly thrAB derived from Escherichia coli (hereinafter sometimes referred to as “E. coli”).
C operon may be mentioned.

When the thrABC operon derived from a bacterium belonging to the genus Escherichia is used, any donor microorganism for the DNA containing the wild type thrABC operon may be used as long as it is a microorganism belonging to the genus Escherichia. Specifically, the book by Neidhardt, F.
C. et.al., Escherichia coli and Salmonella Typhim
urium, American Society for Microbiology, Washingt
on DC, 1208, table 1) can be used. Examples include E. coli JM109 strain and MC1061 strain. When a wild strain is used as a donor of a DNA containing the thrABC operon, wild type thrAB
DNA containing the C operon can be obtained.

(1) Acquisition of wild-type thrABC operon An example of preparing a DNA containing the thrABC operon will be described below. First, E. coli having wild type AKI-HDI, for example, MC1061 strain is cultured to obtain a culture.
In order to cultivate the above-mentioned microorganism, it may be cultivated by an ordinary solid culturing method, but it is preferable to adopt the liquid culturing method in consideration of efficiency at the time of collecting cells. Also, as the medium,
A medium in which sodium chloride (NaCl) is added to yeast extract, peptone, tryptone, or meat extract is used. Specifically, L-broth (Bacto tryptone 1%, Bacto yeast extract 0.5%, NaCl
0.5%, glucose 0.1%, pH = 7.2).
The initial pH of the medium is appropriately adjusted to 6-8. Culturing is carried out at 30 to 42 ° C., preferably around 37 ° C. for 4 to 24 hours by aeration and stirring deep culture, shaking culture, static culture or the like.

The culture thus obtained is, for example,
The cells of E. coli MC1061 strain are obtained by centrifugation at 3,000 rpm for 5 minutes. From this cell, for example, the method of Saito and Miura (Biochem. Biophys. Acta., 72, 619, (1963)), K.
Chromosomal DNA can be obtained by the method of S. Kirby (Biochem. J., 64, 405, (1956)) or the like.

From the chromosomal DNA thus obtained, thr
A chromosomal DNA library is created to isolate the ABC operon. First, chromosomal DNA is partially digested with an appropriate restriction enzyme to obtain a mixture of various fragments. For example, S
au3AI is allowed to act on chromosomal DNA for various times (1 minute to 2 hours) at a temperature of 30 ° C. or higher, preferably 37 ° C., and an enzyme concentration of 1 to 10 units / ml for digestion.

Then, the cut chromosomal DNA fragment is
Vector D capable of autonomous replication in Escherichia bacterial cells
Ligate to NA to make recombinant DNA. In particular,
A restriction enzyme, such as BamH, which produces the same terminal nucleotide sequence as the restriction enzyme Sau3AI used for the cleavage of chromosomal DNA
I at a temperature of 30 ° C or higher and an enzyme concentration of 1 to 100 units /
Under the condition of ml, the vector DNA is allowed to act for 1 hour or more, preferably 1 to 3 hours to be completely digested and cleaved and cleaved. Then, the chromosomal DNA fragment mixture obtained as described above is mixed with the vector DNA which has been cleaved and cleaved, and DNA ligase, preferably T4 DNA ligase, is added at a temperature of 4 to 16 ° C. and an enzyme concentration of 1 to 100 units / ml. The recombinant DNA is obtained by allowing it to act for 1 hour or longer, preferably 6 to 24 hours, under the conditions.

Using the obtained recombinant DNA, a microorganism of the genus Escherichia, such as Escherichia coli K-12 strain, or E. col.
i Gif106M1 strain (thrA1101, metLM1000, lysC1001, ilvA,
Aspartokinase-deficient mutant strains such as argH) are transformed to prepare a chromosomal DNA library. This transformation is performed by DM Morrison (Methods in Enzymology
68, 326, 1979) or a method of treating recipient cells with calcium chloride (Mandel, M. and Higa, A., J. Mol. Bio.
l., 53, 159 (1970)) and so on. The E. coli Gif106M1 strain is an E. coli Genetic Stock Ce.
It is available from nter (Connecticut, USA).

From the obtained chromosomal DNA library, a strain having an increased aspartokinase activity or a strain complemented with an auxotrophy due to the aspartokinase gene deficiency is selected, and a recombinant DNA containing the thrA gene is selected.
To obtain a strain having

To confirm whether the candidate strain having the recombinant DNA containing the thrA gene retains the recombinant DNA in which the thrA gene has been cloned, a cell extract was prepared from the candidate strain, and This can be achieved by preparing an enzyme solution and confirming that the aspartokinase activity is increased. The enzymatic activity of aspartokinase can be measured by the method of Stattman et al. (Stadtman, ER, Cohen, GN, LeBras, G., and Rob
ichon-Szulmajster, H., J. Biol. Chem., 236, 2033
(1961)).

The aspartokinase-deficient mutant strain is L
-Since it requires lysine, L-threonine, L-methionion and diaminopimelic acid, it does not contain L-lysine, L-threonine, L-methionion and diaminopimelic acid when an aspartokinase-deficient mutant is used as a host. A DNA fragment containing the thrA gene can be obtained by isolating a strain that has become capable of growing on a minimal medium or on a medium containing neither homoserine nor diaminopimelic acid and recovering recombinant DNA from the strain. .

However, there are three types of aspartokinase in E. coli and three types of genes encoding aspartokinase. Therefore, in order to confirm whether or not the target thrA gene has been isolated, it is preferable to analyze and confirm the nucleotide sequence or the restriction enzyme cleavage pattern. The nucleotide sequence of the thrA gene of E. coli has already been reported (Katinka, M et al., Pr.
oc. Natl. Acad. Sci. USA, 77, 5730 (1980)).

From the above strains, recombinant D in which the DNA containing the thrA gene is inserted into the vector DNA
For example, the method of P. Guerry et al. (J. Bacteriol., 1
16, 1064, (1973)), DB Clewell's method (J. Bacter
iol., 110, 667, (1972)) and the like.

In order to isolate the full-length thrABC operon, it is necessary to reselect a transformant carrying a recombinant DNA containing the full-length thrABC operon from the chromosomal DNA library. That is, the obtained thrA
Selection is performed by the colony hybridization method using the gene as a probe. The colony hybridization method follows the standard method (Molecular Cloning, 2nd editio
n, Cold Spring Harbor Press (1989)).

To confirm whether or not the full-length thrABC operon was isolated, the isolated candidate DNA was thr.
It was confirmed that the deletion of the thrB gene was recovered by introducing it into the strain deficient in the B gene, and the isolated candidate DN
A was introduced into a strain deficient in the thrC gene, and thrC
It is sufficient to confirm that the gene defect is recovered. thr
E. coli YA73 strain (thrB
1000, thi-1, relA1, λ ー, spoT1). A strain lacking the thrC gene is an E. coli Gif41 strain (thrC100
1, thi-1, relA1, λ ー, spoT1). E. coli
YA73 strain and E. coli Gif41 strain are E. coli Genetic Stock
Available from Center (Connecticut, USA).

In addition to this, to confirm whether the full-length thrABC operon was isolated, isolated candidate DN was isolated.
The nucleotide sequence of A or the restriction enzyme cleavage pattern is analyzed and confirmed. The nucleotide sequence of the thrB gene of E. coli has already been reported (Cossart, P et al., Nucleic A
cids Res., 9, 339 (1981)), and the nucleotide sequence of the thrC gene of E. coli has also been reported (Parsot, C et a.
L., Nucleic Acids Res., 11, 7331 (1983)).

The wild-type thrABC operon can be obtained from a strain having the wild-type thrABC operon on its chromosome.
Chromosomal DNA was prepared by the method of Saito and Miura and the polymerase chain reaction method (PCR: polymerase) was prepared.
chain reaction; White, TJ et al; Trends Genet.
5, 185 (1989)), the amplification of the thrABC operon can also be performed. DN used for amplification reaction
As the A primer, one complementary to both 3'ends of the DNA duplex containing the entire region or a partial region of the thrABC operon is used. When only a partial region of the thrABC operon is amplified, it is necessary to screen a DNA fragment containing the entire region from a chromosomal DNA library using the DNA fragment as a probe. When the whole region of the thrABC operon was amplified, the amplified thrAB
After the PCR reaction solution containing the DNA fragment containing the C operon is subjected to agarose gel electrophoresis, the target DNA fragment is extracted to recover the DNA fragment containing the thrABC operon.

As the DNA primer, for example, E. col
It may be appropriately created based on the known sequence in i. The base sequence of the E. coli thrA gene is Kati
nka, M et al., Proc. Natl. Acad. Sci. USA, 77, 5
730 (1980), and the nucleotide sequence of the thrB gene of E. coli is Cossart, P et al., Nucleic Acids.
Res., 9, 339 (1981) reported that t of E. coli.
The nucleotide sequence of the hrC gene is Parsot, C et al., Nucleic
Acids Res., 11, 7331 (1983).

The primer DNA is synthesized by the phosphoamidide method (see Tetrahedron Letters, 22,1859 (1981)).
A commercially available DNA synthesizer (for example, Appl
It can be synthesized using a DNA synthesizer model 380B manufactured by ied Biosystems. For the PCR reaction, a commercially available PCR reaction device (DNA Thermal Cycler PJ2000 type manufactured by Perkin Elmer Co., Ltd.) is used, and Taq DNA polymerase (supplied by Takara Shuzo Co., Ltd.) is used, and the method specified by the supplier is used. Can be done according to.

The thrABC operon amplified by the PCR method is ligated to a vector DNA capable of autonomous replication in Escherichia bacterium cells, and introduced into Escherichia bacterium cells to carry out operations such as introduction of mutations into the thrA gene. Easier to do. Vector D used
The NA, the transformation method, and the method for confirming the presence of the thrABC operon are the same as those described above.

(2) Introduction of Mutation into thrABC Operon As a method for carrying out mutations such as substitution, insertion and deletion of amino acid residues in the thrABC operon obtained as described above, the recombinant PCR method (Higuchi , R.,
61, PCR Technology (Erlich, HA Eds., Stockton p
ress (1989))), site-directed mutagenesis (Kramer, W. and F
rits, HJ, Meth. in Enzymol., 154, 350 (1987); Kun
kel, TA et.al., Meth. in Enzymol., 154, 367 (198
7)) etc. Using these methods, it is possible to cause a desired mutation at a target site.

Further, according to the method of chemically synthesizing a target gene, a mutation or a random mutation can be introduced into a target site. Further, a method of directly treating the thrABC operon on a chromosome or a plasmid with hydroxylamine (Hashimoto, T. and Sekiguchi, M., J. Ba).
cteriol., 159, 1039 (1984)). Also, thrABC
A method of irradiating Escherichia bacteria having an operon with ultraviolet rays or a method of treating with a chemical agent such as N-methyl-N′-nitro-N-nitrosoguanidine or nitrous acid may be used. According to these methods, mutations can be introduced randomly.

As a method of selecting the release type thrABC operon, first, a recombinant DNA consisting of a DNA fragment containing the thrABC operon and a vector DNA is directly mutated with hydroxylamine or the like, and then, for example, E. col.
i Gif106M1 strain is transformed. Next, the transformant is cultured in a minimal medium such as M9 containing an L-threonine antagonist such as α-amino-β-hydroxyvaleric acid (AHV). The strain carrying the recombinant DNA containing the wild type thrABC operon was able to synthesize L-homoserine and diaminopimelic acid (DAP) because AKI-HDI expressed by the recombinant DNA was inhibited by AHV. It disappears and growth is suppressed. On the other hand, L-
A strain carrying a recombinant DNA containing the thrABC operon containing the thrA gene encoding AKI-HDI derepressed by threonine is the same recombinant DNA.
-Type AKI-HD encoded by the thrA gene in milk
Since I is not inhibited by AHV, it should allow growth on minimal medium supplemented with AHV. Utilizing this phenomenon, strains whose growth is resistant to AHV,
That is, it is possible to select a strain having a recombinant DNA containing the thrABC operon containing the released thrA gene whose inhibition has been released.

The release type thrABC operon thus obtained is introduced as a recombinant DNA into an appropriate host microorganism and expressed to carry AKI-HDI from which feedback inhibition is released, and the LA encoded by the thrABC operon is retained. -It is possible to obtain a microorganism in which the expression of enzymes in the threonine biosynthesis system is enhanced. The host is preferably a microorganism belonging to the genus Escherichia, and examples thereof include Escherichia coli (E. coli).

From the recombinant DNA, the release type thrAB
The C operon taken out and inserted into another vector DNA may be used. The vector DNA that can be used in the present invention includes plasmid vector DN
A is preferred, for example pUC19, pUC18, pBR322, pHSG299.
, PHSG298, pHSG399, pHSG398, RSF1010, pMW11
9, pMW118, pMW219, pMW218 and the like. Alternatively, a phage DNA vector can be used.

Further, in order to efficiently carry out the expression of the release type thrABC operon, the transcription control region (operator or attenuator) located upstream of the release type thrABC operon may be removed. Alternatively,
Other promoters that work in microorganisms such as lac, trp, and PL may be linked, and a promoter specific to the thrABC operon may be amplified and used.

Further, as described above, the release type thrABC
Extrachromosomal DNA such as plasmid introduced into host by inserting operon into autonomously replicable vector DNA
The thrABC operon may be retained by the host as transfection, transposon (Berg, D.
E. and Berg, CM, Bio / Technol., 1, 417 (1983)),
Mu phage (JP-A-2-109985) or homologous recombination (Experiments in Molecular Genetics, Cold Spr
ing Habor Lab. (1972)) may be incorporated into the chromosome of the host microorganism.

<2. The ilvA gene containing the ilvA gene whose inhibition by L-isoleucine has been substantially eliminated and in which the region required for attenuation has been removed> The ilvA gene encoding threonine deaminase whose inhibition by L-isoleucine has been substantially eliminated Containing the ilvGMEDA operon, that is, the release type il
The vGMEDA operon differs from the wild-type ilvGMEDA operon in that the contained ilvA gene has a mutation. This mutation means TD encoded by the ilvA gene.
It means a mutation that cancels the feedback inhibition by L-isoleucine against.

IlvA which encodes threonine deaminase substantially free from inhibition by L-isoleucine
The ilvGMEDA operon containing a gene and having the region necessary for attenuation removed, that is, the completely released ilvGMEDA operon is a released ilvGMED
In addition to the A operon, a wild-type ilvGMEDA is introduced in that a mutation is introduced so that attenuation does not occur.
Different from the operon or the release type ilvGMEDA operon. This mutation includes not only the deletion of all or part or the vicinity of the attenuator existing in the 5'upstream region of the ilvGMEDA operon, but also the insertion of a new DNA fragment inside the attenuator.

The method for obtaining the release type ilvGMEDA operon is as follows. First, wild type ilvGME
In vitro mutation treatment of DNA containing DA operon, vector DN compatible with the DNA after mutation treatment and host
Ligation with A gives recombinant DNA. Recombinant DNA is introduced into a host microorganism to obtain a transformant, and if a transformant capable of expressing the released TD is selected, the transformant retains the released ilvGMEDA operon. are doing. Further, a DNA containing the wild-type ilvGMEDA operon is ligated with a vector DNA compatible with a host to obtain a recombinant DNA, and then the recombinant DNA is subjected to an in vitro mutation treatment, and the recombinant DNA after the mutation treatment is used as a host microorganism. The transformant retains the released ilvGMEDA operon even when the introduced transformant is obtained to select a transformant that expresses the released TD among the transformants.

The wild-type TD-producing microorganism may be subjected to a mutation treatment to create a mutant strain that produces a released TD, and then the released ilvGMEDA operon may be obtained from the mutant strain. Alternatively, after transforming a transformant into which a recombinant DNA in which the wild-type ilvGMEDA operon is ligated has been introduced to create a mutant strain that produces a mutant TD, the recombinant DNA may be recovered from the mutant strain. For example, the same DNA
An unlocked ilvGMEDA operon may be created above.

Examples of agents for in vitro mutation treatment of DNA include hydroxylamine and the like. Hydroxylamine is a chemical mutagen that causes a mutation from cytosine to thymine by converting cytosine into N4-hydroxycytosine. When the microorganism itself is subjected to mutation treatment, it is treated with ultraviolet rays or a mutagen which is usually used for artificial mutation such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG). ilvG
The MEDA operon is derived from a bacterium belonging to the genus Escherichia, particularly ilv derived from Escherichia coli (E. coli).
The GMEDA operon can be mentioned.

IlvGMED from Escherichia bacterium
When the A operon is used, as the donor bacterium of the DNA containing the wild-type ilvGMEDA operon, any microorganism may be used as long as it is a microorganism belonging to the genus Escherichia. Specifically, the book by Neidhardt (Neidhard
t, FC et.al., Escherichia coli and SalmonellaTyp
himurium, American Society for Microbiology, Washi
Those listed in ngton DC, 1208, table 1) can be used. When a wild strain is used as a donor of DNA containing the ilvGMEDA operon, wild-type ilvGM
DNA containing the EDA operon can be obtained.

However, when E. coli is used as a DNA donor of the wild-type ilvGMEDA operon, the K12 wild-type strain expresses an active acetohydroxy acid synthase isozyme II (AHASII) because the ilvG gene has a frameshift mutation. Not done (Proc. Na
tl. Acad. Sci. USA 78, 922, 1981). Therefore, when the K12 strain is used as a DNA donor, a mutant in which the frame is restored so that the activity of acetohydroxy acid synthase II encoded by the ilvG gene is restored is prepared, and then the mutant is used as a DNA donor. There is a need. Or K12
Ilv as a DNA-donating strain other than the strain-derived strain E. coli
Only the G gene was isolated and the ilvGM derived from the K12 strain was isolated.
It may be incorporated into the EDA operon. That is, the ilvMEDA portion was isolated using the K12 strain as a DNA donor bacterium,
Ilv as a DNA-donating strain other than the strain-derived strain E. coli
Isolate G gene only and ligate both to form full-length ilv
The GMEDA operon. The acetohydroxy acid synthase isozyme II (AHASII) is composed of two large and small subunits, and the large subunit is encoded by the ilvG gene. The small subunit is encoded by the ilvM gene.

The method for obtaining the completely released ilvGMEDA operon is as follows. The position and nucleotide sequence of the attenuator existing in the 5'upstream region of the ilvGMEDA operon have been reported by RP Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)). Release type il
By using the vGMEDA operon as the starting material and preparing the ilvGMEDA operon from which the attenuator has been completely removed, the completely released ilvGMEDA operon can be obtained.

The nucleotide sequence described as SEQ ID NO: 1 in the Sequence Listing discloses the region required for attenuation.
The DNA sequence from 999th to 1007th encodes a continuous portion of valine residues present in the leader peptide,
The DNA sequence from 1008 to 1016 encodes a contiguous stretch of isoleucine residues present in the leader peptide. The DNA sequence from the 1081st position to the 1104th position encodes a part of the rhe-independent terminator-like stem / loop in the attenuator.

When L-isoleucine is present in a sufficient amount in cells, RNA transcribed from the DNA sequence from 1081st to 1104th forms a rhe-independent terminator-like stem / loop, which results in transcription by RNA polymerase. Upon termination, the ilvGMEDA operon is not expressed.

When L-isoleucine is deficient in cells, tRNA to which isoleucine is bound is deficient in cells, and translation by ribosome is stalled at a continuous portion of isoleucine residues present in the region encoding the leader peptide.
For this reason, the mRNA starts to form a new three-dimensional structure, and as a result, DNs from 1081 to 1104
The rhe-independent terminator-like stem / loop formed by RNA transcribed from the A sequence is no longer formed. Therefore, transcription by RNA polymerase continues and the ilvGMEDA operon is expressed. Therefore, in order to remove the region necessary for attenuation, the DNA sequence from the 1008th position to the 1016th position of the sequence disclosed in SEQ ID NO: 1 of the sequence listing, or from the 1081th position
It suffices if the 1104th DNA sequence is removed.

By the way, removing a region necessary for attenuation means that a mutation is introduced so that attenuation does not occur. Therefore, the mutation is not limited to the one that removes all attenuators existing in the 5'upstream region of the ilvGMEDA operon. In short, it may be a mutation that renders the attenuator unable to form a rhe-independent terminator-like system / loop. It may be a mutation that does not include a continuous portion of isoleucine residues in the leader peptide. This is because the attenuation will not work in either case.

That is, this mutation is ilvGMEDA
In addition to the one that removes all or part or the vicinity of the attenuator existing in the 5'upstream region of the operon, the one that inserts a new DNA fragment inside the attenuator is also included.

It is to be noted that i which encodes threonine deaminase whose inhibition by L-isoleucine is substantially released.
The ilvGMEDA operon containing the lvA gene and having the region necessary for attenuation removed, that is, the completely released ilvGMEDA operon is a wild-type ilvG.
After the mutation was introduced into the MEDA operon to prevent the occurrence of attenuation, a mutation was introduced into the ilvA gene contained in the ilvGMEDA operon.
It can also be prepared so that the inhibition of threonine deaminase encoded by the vA gene by L-isoleucine is substantially eliminated.

(1) Acquisition of wild-type ilvGMEDA operon To acquire the DNA containing the ilvGMEDA operon, a method of acquiring each of the ilvGM gene, ilvE gene, ilvD gene and ilvA gene and ligating these can be considered.

First, E. coli having a wild-type TD, for example, E. coli K12 strain, E. coli W3110 strain, E. coli MC106.
1 strain (the above 3 strains have ilvG frameshifted), E. coli MI162 strain (thr-10, car-94, λ-, r
elA1, ilvG603, thi-1) and E. coli B strain (the above two strains have normal ilvG) to obtain a culture. In order to cultivate the above-mentioned microorganism, it may be cultivated by an ordinary solid culturing method, but it is preferable to adopt the liquid culturing method in consideration of efficiency at the time of collecting cells. As the medium, a medium obtained by adding sodium chloride (NaCl) to yeast extract, peptone, tryptone, or meat extract is used.
Specifically, L-broth (Bact tryptone 1%,
Bact yeast extract 0.5%, NaCl
0.5%, glucose 0.1%, pH = 7.2).
The initial pH of the medium is appropriately adjusted to 6-8. Culturing is carried out at 30 to 42 ° C., preferably around 37 ° C. for 4 to 24 hours by aeration and stirring deep culture, shaking culture, static culture or the like. E. coli MI162 strain is
E. coli Genetic Stock Center (Connecticut, USA)
More available. The index number for this strain is CGSC5919. The detailed characteristics of the strain are described in Mol. Gen. Genet. 143, 243.
(1976), J. Bacteriol., 149, 294 (1982).

The culture thus obtained is, for example,
Centrifuge at 3,000 rpm for 5 minutes to obtain E. coli cells. From this cell, for example, the method of Saito and Miura (Biochem.
Biophys. Acta., 72, 619 (1963)), KS Kirby's method (Biochem. J., 64, 405 (1956)) and the like to obtain chromosomal DNA.

From the chromosomal DNA thus obtained, ilv
A chromosomal DNA library is created to isolate the GMEDA operon. First, chromosomal DNA is partially digested with an appropriate restriction enzyme to obtain a mixture of various fragments. For example, Sau3AI is used at a temperature of 30 ° C. or higher, preferably 37
The chromosomal DNA is digested by allowing it to act on the chromosomal DNA for various times (1 minute to 2 hours) at an enzyme concentration of 1 to 10 units / ml.

Then, the cut chromosomal DNA fragment is
Vector D capable of autonomous replication in Escherichia bacterial cells
Ligate to NA to make recombinant DNA. In particular,
A restriction enzyme, such as BamH, which produces the same terminal nucleotide sequence as the restriction enzyme Sau3AI used for the cleavage of chromosomal DNA
I at a temperature of 30 ° C or higher and an enzyme concentration of 1 to 100 units /
Under the condition of ml, the vector DNA is allowed to act for 1 hour or more, preferably 1 to 3 hours to be completely digested and cleaved and cleaved. Then, the chromosomal DNA fragment mixture obtained as described above is mixed with the vector DNA which has been cleaved and cleaved, and DNA ligase, preferably T4 DNA ligase, is added at a temperature of 4 to 16 ° C. and an enzyme concentration of 1 to 100 units / ml. The recombinant DNA is obtained by allowing it to act for 1 hour or longer, preferably 6 to 24 hours, under the conditions.

Using the obtained recombinant DNA, a microorganism of the genus Escherichia, for example, E. coli MI262 strain (leuB6, ilv
I614, ilvH612, λ-, relA1, spoT1, ilvB619, ilvG603,
Acetohydroxy acid synthase-deficient mutant strain such as ilvG605 (am), thi-1), E. coli AB2070 strain (proA2, trp-
3, hisG4, ilvE12, metE46, thi-1, ara-9, lacY1 orla
cZ4, galK2, malA1, mtl-1, rpsL8 or rpsL9, ton-1, t
transaminase B such as sx-3, λR, λ-, supE44)
Defective mutant strain or E. coli AB1280 strain (hisG1, ilv
D16, metB1, argH1, thi-1, ara-13, lacY1 or lacZ4,
gal-6, xyl-7, mtl-2, malA1, rpsL8,9 or 17, tonA2,
λR, λ-, supE44) dihydroxy acid dehydratase deficient mutant strain, E. coli AB1255 strain (thi-1, ilvA201, ar
gH1, metB1, hisG1, lacY1 or lacZ4, malA1, mtl-2, x
yl-7, ara-13, gal-6, rpsL8,9 or 17, tonA2, tsx-5,
A threonine deaminase-deficient mutant strain such as λR, λ-, supE44) is transformed to prepare a chromosomal DNA library. This transformation is performed by DM Morrison (Method
In Enzymology 68, 326, 1979) or a method of increasing the permeability of DNA by treating recipient cells with calcium chloride (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1
970)) etc. E. coli MI26
Two strains are available from the E. coli Genetic Stock Center (Connecticut, USA). The index number of this strain is CGSC57
69. The detailed characteristics of the strain are described in Mol. Gen. Genet., 15.
6, 1 (1977). The E. coli AB2070 strain is an E. coli strain.
Available from coli Genetic Stock Center (Connecticut, USA). The index number for this strain is CGSC2070.
The detailed characteristics of the strain are described in J. Bacteriol., 109, 730 (1972).
It is described in. The E. coli AB1280 strain is an E. coli Gene
Available from tic Stock Center (Connecticut, USA). The index number for this strain is CGSC1280. E. coli AB1
255 strains are available from the E. coli Genetic Stock Center (Connecticut, USA). The index number of the stock is CGSC
It is 1255. For detailed characteristics of the strain, see J. Bacteriol., 10
9, 730 (1972).

By the way, the entire nucleotide sequence of the ilvGMEDA operon has been clarified (Nucleic Acids Res. 1
5, 2137 (1987)), a specific restriction enzyme is selected and the chromosomal DNA is digested to make a DNA fragment having a target gene of a specific length. Only the DNA fragment having this specific length is ligated to the vector DNA to prepare a recombinant DNA.
When a chromosomal DNA library is prepared by using NA, a DNA fragment containing the target gene can be obtained more efficiently.

From the obtained chromosomal DNA library, a strain having an increased acetohydroxy acid synthase activity or a strain complemented with an auxotrophy resulting from the acetohydroxy acid synthase gene deficiency was selected, and a group containing the ilvGM gene was selected. A strain having the altered DNA is obtained. From the obtained chromosomal DNA library, a strain with increased transaminase B activity or a strain with complementary auxotrophy due to transaminase B gene deficiency is selected,
A strain having a recombinant DNA containing the ilvE gene is obtained.

From the obtained chromosomal DNA library, a strain having an increased dihydroxy acid dehydratase activity or a strain to which the auxotrophy due to the dihydroxy acid dehydratase gene deficiency is complemented is selected, and a recombinant DNA containing the ilvD gene is selected. To obtain a strain having

From the obtained chromosomal DNA library, a strain having an increased threonine deaminase activity or a strain to which the auxotrophy resulting from the deletion of the threonine deaminase gene is complemented is selected and has a recombinant DNA containing the ilvA gene. Obtain the strain.

Recombinant DNA containing the ilvGM gene
To confirm whether or not the candidate strain having the gene retains the recombinant DNA in which the ilvGM gene was cloned, a cell extract was prepared from the candidate strain and a crude enzyme solution was prepared from it to prepare acetohydroxy acid synthase activity. Can be achieved by making sure that is increasing. The method for measuring the enzyme activity of acetohydroxy acid synthase can be performed by the method of MD Felice et al. (Methods in Enzymology
166, 241).

Since the mutant strain deficient in acetohydroxy acid synthase is isoleucine, leucine and valine auxotrophic, when the mutant strain deficient in acetohydroxy acid synthase is used as a host, it is possible to grow on the minimal medium containing no valine. Isolated strain, and recombinant DNA from the strain
Of the ilvGM gene by recovering D
NA fragments can be obtained.

Alternatively, a DNA containing the ilvGM gene
Has been reported by RP Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)). Therefore, it can be confirmed by isolating the recombinant DNA from the candidate strain, decoding its base sequence, and comparing it with the base sequence reported in the same report.

As described above, ilvG of E. coli K12 strain
Mutations occur within the open reading frame of the gene. As a result, a frame shift occurs, and translation is terminated because a stop codon appears in the middle. That is, ATG at the start codon of the ilvG gene
The stop codon appears at 982 to 984th counting from (1st to 3rd). Therefore, ilv acquired from the same strain
When using the GM gene, it is necessary to restore the mutated part to the normal state by the site-directed mutagenesis method. For example, in the ilvG gene (ilvG603) of the Escherichia coli MI162 strain, two base pairs of TG are inserted before the stop codon TGA at the 982 to 984th positions,
The frame is back to normal. J. Ba for others
It is described in detail in Fig. 2 of cteriol. 149, 294 (1982).

The method for confirming whether the candidate strain having the recombinant DNA containing the ilvE gene retains the recombinant DNA in which the ilvE gene has been cloned is as follows. Since the transaminase B-deficient mutant strain is isoleucine-requiring, when the transaminase B-deficient mutant strain is used as a host, a strain that can grow on a minimal medium containing no isoleucine is isolated, and recombinant DNA is isolated from the strain. A DNA fragment containing the ilvE gene can be obtained by recovering

Alternatively, the nucleotide sequence of DNA containing the ilvE gene has already been reported by RP Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)). Therefore, it can be confirmed by isolating the recombinant DNA from the candidate strain, decoding its base sequence, and comparing it with the base sequence reported in the same report.

The method for confirming whether the candidate strain having the recombinant DNA containing the ilvD gene retains the recombinant DNA in which the ilvD gene has been cloned is as follows. Since the dihydroxy acid dehydratase-deficient mutant strain is isoleucine, leucine and valine auxotrophic,
When a dihydroxy acid dehydratase-deficient mutant strain is used as a host, a strain capable of growing on a medium containing no valine is isolated, and a recombinant DNA is recovered from the strain to obtain a DNA fragment containing the ilvD gene. Can be obtained.

Alternatively, the nucleotide sequence of DNA containing the ilvD gene has already been reported by RP Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)). Therefore, it can be confirmed by isolating the recombinant DNA from the candidate strain, decoding its base sequence, and comparing it with the base sequence reported in the same report.

To confirm whether the candidate strain having the recombinant DNA containing the ilvA gene retains the recombinant DNA in which the ilvA gene was cloned, a cell extract was prepared from the candidate strain, and This can be achieved by preparing an enzyme solution and confirming that threonine deaminase activity is increased. The method for measuring the enzyme activity of threonine deaminase is Methods in Enzymology 17B, 555.
(1971).

Since the threonine deaminase deficient mutant strain is isoleucine auxotrophic, when the threonine deaminase deficient mutant strain is used as a host, a strain that can grow on a minimal medium containing no isoleucine is isolated, By recovering the recombinant DNA from the strain, i
A DNA fragment containing the lvA gene can be obtained.

Alternatively, the nucleotide sequence of DNA containing the ilvA gene has already been reported by RP Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)). Therefore, it can be confirmed by isolating the recombinant DNA from the candidate strain, decoding its base sequence, and comparing it with the base sequence reported in the same report. Recombinant D from each of the above strains
For example, the method of P. Guerry et al. (J. Bacteriol., 1
16, 1064 (1973)), DB Clewell's method (J. Bacteri
ol., 110, 667 (1972)) and the like.

To isolate the full-length ilvGMEDA operon, a DNA fragment containing the ilvGM gene, ilvE
DNA fragment having gene, D having ilvD gene
The NA fragment and the DNA fragment having the ilvA gene are ligated. When linked, it was previously reported by RP Lawther et al. (Nucleic Acids Res. 15, 2137).
(1987)) The entire nucleotide sequence of the ilvGMEDA operon can be referred to.

To obtain the wild-type ilvGMEDA operon, chromosomal DNA is prepared from the strain having the wild-type ilvGMEDA operon on the chromosome by the method of Saito and Miura, and the polymerase chain reaction method (PCR:
polymerase chain reaction; White, TJ et al; Tren
ds Genet. 5, 185 (1989)), ilvGME
This can also be done by amplifying the DA operon. The DNA primer used in the amplification reaction is complementary to both 3'ends of the DNA duplex containing the entire region or a partial region of the ilvGMEDA operon. ilvGMED
When only a part of the A operon is amplified, the DN
It is necessary to screen a DNA fragment containing the entire region from the chromosomal DNA library using the A fragment as a probe. When the entire region of the ilvGMEDA operon was amplified, the PCR reaction solution containing the amplified DNA fragment containing the ilvGMEDA operon was subjected to agarose gel electrophoresis, and the target DNA fragment was extracted to contain the ilvGMEDA operon. It is possible to recover the DNA fragment that does. To make DNA primers, use RP La
Already reported by wther et al. (Nucleic Acid
s Res. 15, 2137 (1987)) ilvGMEDA of E. coli
The entire base sequence of the operon can be referred to.

The synthesis of the primer DNA is carried out by the phosphoamidide method (see Tetrahedron Letters, 22,1859 (1981)).
A commercially available DNA synthesizer (for example, Appl
It can be synthesized using a DNA synthesizer model 380B manufactured by ied Biosystems. For the PCR reaction, a commercially available PCR reaction device (DNA Thermal Cycler PJ2000 type manufactured by Perkin Elmer Co., Ltd.) is used, and Taq DNA polymerase (supplied by Takara Shuzo Co., Ltd.) is used, and the method specified by the supplier is used. Can be done according to.

IlvGMED amplified by PCR method
The A operon is ligated to a vector DNA capable of autonomous replication in Escherichia bacterium cells and introduced into Escherichia bacterium cells to introduce mutations into the ilvA gene and remove regions necessary for attenuation. It becomes easier to peel. Vector DNA used
The transformation method and the method for confirming the presence of the ilvGMEDA operon are the same as those described above.

(2) Introduction of Mutation into ilvGMEDA Operon The recombinant iVGMEDA operon may be subjected to mutation such as amino acid residue substitution, insertion and deletion by the recombinant PCR method (Higuch).
i, R., 61, PCR Technology (Erlich, HA Eds., Sto
ckton press (1989))), site-directed mutagenesis (Kramer,
W. and Frits, HJ, Meth. In Enzymol., 154, 350
(1987); Kunkel, TA et.al., Meth. In Enzymol., 1
54, 367 (1987)). Using these methods, it is possible to cause a desired mutation at a target site. Further, according to the method of chemically synthesizing a target gene, a mutation or random mutation can be introduced into a target site.

Furthermore, i on the chromosome or plasmid
Method for directly treating the lvGMEDA operon with hydroxylamine (Hashimoto, T. and Sekiguchi, M., J. Bacter
iol., 159, 1039 (1984)). Also, ilvGMEDA
A method of irradiating Escherichia bacteria having an operon with ultraviolet rays or a method of treating with a chemical agent such as N-methyl-N′-nitro-N-nitrosoguanidine or nitrous acid may be used. According to these methods, mutations can be introduced randomly.

The method for selecting the DNA fragment having the released ilvA gene is as follows. First, a recombinant DN composed of a DNA fragment having the ilvA gene and a vector DNA
A is directly mutated with hydroxylamine or the like, and this is used to transform E. coli W3110 strain, for example. Then an antagonist of L-isoleucine, such as an appropriate amount of glycyl-
When the transformant was cultured in a minimal medium containing L-leucine, for example, M9, recombinant D having a wild-type ilvA gene was produced.
A strain having NA may not be able to synthesize L-isoleucine and its growth may be suppressed (it is preferable to use a small copy of the vector because the influence of the gene-dose effect can be reduced). In contrast, i encoding TD that has been deinhibited by L-isoleucine
The strain carrying the recombinant DNA having the lvA gene synthesizes an excessive amount of L-isoleucine even in the presence of glycyl-L-leucine, and therefore grows on the minimal medium supplemented with glycyl-L-leucine. It should be possible. By utilizing this phenomenon, it is possible to select a strain that grows resistant to glycyl-L-leucine, that is, a strain that retains a recombinant DNA having a desensitized deactivated ilvA gene.

The deactivated ilvGMEDA operon containing the deactivated ilvA gene thus obtained was recombinant DNA
As a result, by introducing it into a suitable host microorganism and expressing it, TD possesses TD for which feedback inhibition is released
It is possible to obtain a microorganism in which the expression of the enzyme group of the L-isoleucine biosynthesis system encoded by the vGMEDA operon is enhanced. The host is preferably a microorganism belonging to the genus Escherichia, and examples thereof include Escherichia coli (E. coli).

In addition, the release type ilvGM from recombinant DNA
The EDA operon may be taken out and inserted into another vector DNA. The vector DNA that can be used in the present invention is preferably a plasmid vector DNA such as pUC19, pUC18, pBR322, pH.
SG299, pHSG298, pHSG399, pHSG398, RSF1010, pM
Examples include W119, pMW118, pMW219, pMW218 and the like. Alternatively, a phage DNA vector can be used.

Furthermore, in order to efficiently carry out the expression of the release type ilvGMEDA operon, the release type ilvGME is expressed.
The transcription control region (operator or attenuator) located upstream of the DA operon may be removed. Alternatively, other promoters that work in microorganisms such as lac, trp, and PL may be linked, and a promoter specific to the ilvGMEDA operon may be amplified and used.

Further, as described above, the release type ilvGME is used.
The DA operon inserted into a vector DNA capable of autonomous replication is introduced into a host, and extrachromosomal D like a plasmid is introduced.
Although it may be retained in the host as NA, ilvGMED
A operon, transduction, transposon (Berg, DE and Berg, CM, Bio / Technol., 1, 41
7 (1983)), Mu phage (JP-A-2-109985).
Or homologous recombination (Experiments in Molecular Genet
ics, Cold Spring Habor Lab. (1972)) may be incorporated into the chromosome of the host microorganism.

(3) Removal of Region Required for Attenuation of ilvGMEDA Operon To remove a region required for attenuation from the ilvGMEDA operon, that is, to introduce a mutation into the ilvGMEDA operon that does not cause attenuation. , The ilvGMEDA operon 5'upstream of the attenuator existing in the upstream region, in addition to removing all or part of the attenuator, a new DN inside the attenuator
Those which insert the A fragment are also included. The term "attenuator" as used herein refers to a base sequence that forms a row-independent terminator-like stem / loop. For example, from the 1081th position in the base sequence shown in SEQ ID NO: 1 in the Sequence Listing
It is the 1104th and every part.

To remove the attenuator il
A DNA fragment upstream of the attenuator portion of the vGMEDA operon was prepared, and a DNA fragment downstream of the attenuator portion of the ilvGMEDA operon was prepared.
A fragment may be prepared and both may be ligated. For example ilvG
The DNA fragment in the upstream region of the attenuator part of the MEDA operon can be prepared by cleaving the DNA fragment containing the full-length ilvGMEDA operon with an appropriate restriction enzyme. Alternatively, a DNA fragment in the upstream region of the attenuator portion of the ilvGMEDA operon may be amplified by the PCR method. The primer DNA used in the PCR method has already been reported by RP Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)).
The base sequence may be chemically synthesized based on the base sequence already reported by G. Coppola et al. (Gene 97, 21 (1991)). Furthermore, a DNA fragment in the upstream region of the attenuator portion of the ilvGMEDA operon may be chemically synthesized. The same applies to the method of preparing a DNA fragment in the downstream region of the attenuator portion of the ilvGMEDA operon.

The completely released ilvGMEDA operon may be obtained by preparing the ilvGMEDA operon in which a part or the vicinity of the attenuator is removed using the released ilvGMEDA operon as a starting material. The position and nucleotide sequence of the attenuator is R.
Already reported by P. Lawther et al. (Nucleic
Acids Res. 15, 2137 (1987)), so the DNA to be removed is determined based on this sequence.

The DNA to be removed encodes a DNA portion necessary for the attenuator to form a rhein-independent terminator-like stem / loop or a continuous isoleucine residue located upstream of the stem / loop. A suitable DNA portion containing the region or a DNA portion containing both is preferred. In order to remove a part or the vicinity of the attenuator, a DNA fragment in the upstream region of the DNA portion of the ilvGMEDA operon to be removed is prepared, and a DNA fragment in the downstream region of the DNA portion of the ilvGMEDA operon is removed. A DNA fragment may be prepared and both may be ligated. For example, in the ilvGMEDA operon, a DNA fragment upstream of the DNA portion to be removed is a DNA containing the full-length ilvGMEDA operon.
It can be prepared by cleaving the fragment with an appropriate restriction enzyme. Alternatively, a DNA fragment in the upstream region of the removed DNA portion of the ilvGMEDA operon may be amplified by the PCR method. Primer D used in PCR method
NA is a nucleotide sequence previously reported by RP Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)),
Already reported by G. Coppola et al. (Gene 97,
21 (1991)) Chemical synthesis may be performed based on the nucleotide sequence.
Furthermore, a DNA fragment in the upstream region of the removed DNA portion of the ilvGMEDA operon may be chemically synthesized. DN removed from the ilvGMEDA operon
The same applies to the method of preparing a DNA fragment in the downstream region from the A portion.

The completely released ilvGMEDA operon may be obtained by preparing the ilvGMEDA operon in which a new DNA fragment is inserted in the attenuator using the released ilvGMEDA operon as a starting material. The position and base sequence of the attenuator is
Since it has already been reported by RP Lawther et al. Or G. Coppola et al., The insertion position of a new DNA fragment to be inserted and the nucleotide sequence of the inserted DNA fragment are determined based on this sequence.

The new DNA fragment to be inserted is the terminator-like stem /
It is preferably inserted into a DNA portion necessary for forming a loop or a DNA portion encoding a continuous isoleucine residue located upstream of the stem / loop. As a result of the insertion, the attenuator may not be able to form a row-independent terminator-like stem / loop, which is expected to cause the attenuator to fail.

The nucleotide sequence of the new DNA fragment to be inserted is designed so that the attenuator does not form a rhein-independent terminator-like system / loop when inserted, and the inserted sequence It is preferably designed so that no consecutive isoleucine residues are located upstream of the loop.

In order to insert a new DNA fragment into the attenuator, a DNA fragment of the ilvGMEDA operon upstream of the DNA portion into which the new DNA fragment is inserted is prepared, and the ilvGMEDA operon It is only necessary to prepare a DNA fragment in the downstream region of the DNA portion into which a new DNA fragment is inserted, prepare a new DNA fragment to be inserted, and ligate the three. For example, in the ilvGMEDA operon, a DNA fragment upstream of the DNA portion into which a new DNA fragment is inserted can be prepared by cleaving a DNA fragment containing the full-length ilvGMEDA operon with an appropriate restriction enzyme. Alternatively, of the ilvGMEDA operon, a DNA fragment in the upstream region of the DNA portion into which a new DNA fragment is inserted may be amplified by the PCR method. The primer DNA used in the PCR method has already been reported by RP Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)).
The base sequence may be chemically synthesized based on the base sequence already reported by G. Coppola et al. (Gene 97, 21 (1991)). Furthermore, a DNA fragment in the upstream region of the DNA portion of the ilvGMEDA operon into which a new DNA fragment is inserted may be chemically synthesized. ilvGMEDA
The same applies to the method of preparing a DNA fragment in the downstream region of the DNA portion of the operon into which a new DNA fragment is inserted.

The new DNA fragment to be inserted can be prepared by chemical synthesis.

In addition, a DNA fragment in the upstream region of the ilvGMEDA operon that inserts a new DNA fragment or a DNA fragment in the downstream region of the ilvGMEDA operon that inserts a new DNA fragment is selected. A new DNA fragment to be inserted into the primer DNA can be ligated at the time of amplification by the PCR method. For example, a 3'-side DNA used for amplifying a DNA fragment in the upstream region of the DNA portion into which a new DNA fragment is inserted in the ilvGMEDA operon.
One strand of the new DNA fragment to be inserted is ligated to the primer. Similarly, the complementary strand of the new DNA fragment to be inserted is ligated to the 5′-side DNA primer used to amplify the DNA fragment in the downstream region of the DNA portion into which the new DNA fragment of the ilvGMEDA operon is inserted. . Two kinds of DNA fragments to be amplified are ligated with these primers.

The complete release type ilvGM thus obtained
The EDA operon is introduced as a recombinant DNA into an appropriate host microorganism and expressed to carry TD that has been desensitized to feedback inhibition, and L is encoded by the ilvGMEDA operon because no attenuation occurs.
-It is possible to obtain a microorganism in which the expression of the enzymes in the isoleucine biosynthesis system is further enhanced. The host is preferably a microorganism belonging to the genus Escherichia, and examples thereof include Escherichia coli (E. coli).

[0100] In addition, a complete release type ilv from recombinant DNA
The GMEDA operon may be taken out and inserted into another vector DNA. The vector DNA that can be used in the present invention is preferably a plasmid vector DNA such as pUC19, pUC18 and pBR32.
2, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010
, PMW119, pMW118, pMW219, pMW218 and the like.
Alternatively, a phage DNA vector can be used.

Furthermore, in order to efficiently carry out the expression of the complete release type ilvGMEDA operon, the complete release type il
Upstream of the vGMEDA operon, other promoters such as lac, trp, and PL that work in microorganisms may be linked,
A promoter specific to the ilvGMEDA operon may be amplified and used.

As described above, the complete release type ilvG
The MEDA operon, which is inserted into a vector DNA capable of autonomous replication, may be introduced into a host and retained as extrachromosomal DNA such as a plasmid in the host.
EDA operon, transduction, transposon (Berg, DE and Berg, CM, Bio / Technol., 1,
417 (1983)), Mu phage (JP-A-2-10998).
5) or homologous recombination (Experiments in Molecular G
enetics, Cold Spring Habor Lab. (1972)).

As the donor bacterium of the DNA containing the gene (lysC) encoding AKIII, any microorganism may be used as long as it is a microorganism belonging to the genus Escherichia. Specifically, the book by Neidhardt, et al.
FC et, al., Escherichia coliand Salmonella Typhim
urium, American Society for Microbiology, Washingt
on DC, 1208, Table 1) can be used. Examples include E. coli JM109 strain and MG1061 strain.

When a wild strain is used as a donor bacterium of a DNA containing a gene encoding AKIII (lysC), a DNA containing a wild-type AKIII gene can be obtained. To obtain a AKIII gene (lysC ^* ) in which feedback inhibition by L-lysine is released by introducing a mutation into this gene, DNA is directly treated with hydroxylamine.
The in vitro mutation treatment method may be adopted. Hydroxylamine is a chemical mutation treating agent that causes C → T mutation by changing cytosine to N ⁴ -hydroxycytosine.
Further, to obtain DNA containing the AKIII gene in which feedback inhibition by L-lysine is released, AK
It can be obtained by using a mutant strain in which feedback inhibition of III activity by L-lysine is released as a DNA donor bacterium. The mutant strain is obtained, for example, from a cell group of mutant strains that have been subjected to a normal mutagenesis method, ultraviolet irradiation, or a mutagen treatment such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG). be able to.

Next, the gene encoding AKIII (ly
Preparation of DNA containing sC) will be described. First, E. coli having wild-type lysC, for example, MC1061 strain is cultured to obtain a culture. In order to cultivate the above-mentioned microorganism, it may be cultivated by an ordinary solid culturing method, but it is preferable to cultivate by adopting a liquid culturing method from the viewpoint of collection efficiency. As the medium, for example, yeast extract, peptone, meat extract, corn sweep liquor, soybean or wheat exudate, etc.
To one or more kinds of nitrogen sources, one or more inorganic salts such as potassium dibasic phosphate, dibasic potassium phosphate, magnesium sulfate, sodium chloride, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate are added. In addition, a material to which a sugar raw material, vitamins and the like are appropriately added may be used. The initial pH of the medium is appropriately adjusted to 7-8. The culture is carried out at 30 to 42 ° C., preferably 37.
4 to 24 hours at around ℃, usually agitated deep culture, shake culture,
It is performed by static culture or the like. The culture thus obtained is centrifuged, for example, at 3,000 rpm for 5 minutes to obtain E. coli.
Obtain the cells of MC1061 strain. From this cell, for example, the method of Saito et al. (Biochem. Biophys. Acta., 72, 619, (1963)), K.
Chromosomal DNA can be obtained by the method of S. Kirby (Biochem. J., 64, 405, (1956)) or the like.

As a method for isolating the lysC gene, the chromosomal DNA obtained by the above method is cleaved with an appropriate restriction enzyme. Then, a vector DNA that can grow in Escherichia bacterium cells is ligated, and the obtained recombinant DNA is used to aspartokinase I, II of Escherichia microorganism,
III An all-defective mutant strain, for example, GT3 was transformed (gene library), and a strain capable of growing on a minimal medium containing no lysine was isolated, from which recombinant DNA of the lysC gene can be isolated.

Specifically, chromosomal DNA is treated with a restriction enzyme such as Sau3AI at a temperature of 30 ° C. or higher, preferably 37
℃, enzyme temperature 1-10 units / ml for various times (1
(Minutes to 2 hours), digest it, and partially decompose to obtain various chromosomal DNA fragment mixtures. A restriction enzyme, such as BamHI, which produces the same terminal nucleotide sequence as the restriction enzyme Sau3AI used for the cleavage of chromosomal DNA, is added to a vector DNA that can grow in Escherichia bacterium cells at a temperature of 30 ° C. or higher at an enzyme concentration of 1 to 100 units / unit / unit. more than 1 hour in ml,
Preferably, it is allowed to act for 1 to 3 hours for complete digestion to obtain a cleaved and cleaved DNA. Then E. coli obtained as described above.
A mixture derived from Escherichia coli strain MC1061 and containing a DNA fragment containing the lysC gene was mixed with vector DNA which had been cleaved and cleaved, and this was mixed with DNA ligase, preferably T4 DNA.
A ligase is allowed to act at a temperature of 4 to 16 ° C. and an enzyme concentration of 1 to 100 units for 1 hour or more, preferably 6 to 24 hours to obtain a recombinant DNA.

The vector DNA that can be used in the present invention is preferably a psuramid vector DNA, for example, pUC19, pUC18, pBR322, pHSG299, pHSG.
Examples include 399 and RSF1010. Other phage DNA
Vector can also be used. In order to efficiently carry out the expression of the useful gene of interest, a promoter such as lac, trp, PL or the like which operates in the microorganism may be used. In addition, in the recombinant DNA referred to here, the useful gene is 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 Genet
ics, Cold Spring Habor Lab. (1972)).

Using this recombinant DNA, for example, E. coli
K-12 strain, preferably GT3 strain, is transformed to a strain with increased AK activity or a strain with auxotrophy complemented.
A strain having a recombinant DNA of the lysC gene is obtained. This transformation is carried out by the method of DM Morrison (Methods in Enzymol
ogy 68, 326, 1979) or a method of increasing the permeability of DNA by treating recipient cells with calcium chloride (Mandel, M.).
and Higa, A., J. Mol. Biol., 53 , 159 (1970)). A recombinant DNA obtained by inserting a DNA containing the lysC gene into a vector DNA from the above strains can be prepared by, for example, the method of P. Guerry et al. (J. Bacterio
l., 116, 1064, (1973)), DB Clewell's method (J. Bact.
eriol., 110, 667, (1972)) and the like.

To confirm whether the candidate strain actually carries the recombinant DNA in which lysC was cloned,
This can be achieved by preparing a cell extract, and then preparing a crude enzyme solution therefrom and confirming the aspartokinase activity.
The enzymatic activity of aspartokinase can be measured by the method of Stattman et al. (Stadtman, ER, Coh
en, GN, LeBras, G., and Robichon-Szulmajster,
H., J. Biol. Chem., 236 , 2033 (1961)).

Alternatively, the lysC gene can be obtained by PCR (pS) from the chromosomal DNA obtained by the method of Saito et al.
olymerase chain reaction; White, TJ et al; Tren
It can also be carried out by amplifying the lysC gene by ds Genet., 5, 185 (1989)). The DNA primer used for amplification is one that is complementary to both 3'ends of the DNA duplex containing the entire region or a partial region of the lysC gene. When only a partial region of the lysC gene is amplified, it is necessary to screen a DNA fragment containing the entire region from a gene library using the DNA fragment as a primer. When the entire region is amplified, the DNA fragment containing the lysC gene can be recovered by subjecting the DNA fragment to agarose gel electrophoresis and then cutting out the band of interest.

As the DNA primer, for example, E. c
Sequences known in oti (Cassan, M., Parso
t, C., Cohen, GN, and Patte, JC, J. Biol. Che
m., 261 , 1052 (1986)).
2 of 5'-CTTCCCTTGTGCCAAGGCTG-3 '(SEQ ID NO: 6 in the sequence listing below) and 5'-GAATTCCTTTGCGAGCAG-3' (SEQ ID NO: 7 in the sequence listing below) capable of expanding the 1347 base region encoding the lysC gene Species primers are suitable. The DNA is synthesized by Applied Biosystems "DNA
Synthesizer model 3808 "and using the phosphoramidide method (see Tetrahedron Letters, 22, 1859 (1981))
It can be carried out according to a conventional method. TaqD was used for PCR reaction using "DNA thermal cycler PJ2000 type" manufactured by Takara Shuzo.
NA polymerase can be used according to the method specified by the supplier.

The lysC gene amplified by the PCR method is a vector D that can grow in Escherichia bacterial cells.
It is connected to NA and introduced into Escherichia bacterial cells. Vector DNA used, transformation method, and ly
The confirmation method of sC is the same as the above-mentioned method. Got ly
As a method for carrying out mutations such as amino acid substitution, insertion and deletion in sC, a recombinant PCR method (Higuchi, R.,
61, PCR Technology (Erlich, HA Eds., Stockton P
ress (1989)), site-directed mutagenesis (Kramer, W. and Fri.
ts, HJ, Meth. in Enzymol., 154, 350 (1987)) and Kunkel, TA et.al., Meth. in Enzymol., 154, 36.
7 (1987)). When these methods are used, the desired mutation can be made at the target site. further,
A method for directly treating a target gene DNA on a chromosome or a plasmid with hydroxylamine (Hashimoto, T. and
Sekiguchi, M., J. Bacteriol., 159, 1039 (1984)) or the bacterial cells carrying the DNA are subjected to an ultraviolet irradiation method or N
Mutations can be randomly introduced by using a conventional method by treatment with a chemical agent such as -methyl-N'-nitro-N-nitrosoguanidine or nitrous acid, or a method of chemically synthesizing a target gene.

As a method of obtaining the deblocking mutant gene lysC ^* , first, the mutated recombinant DNA is transformed into an AK completely deficient strain, for example, E. coli GT3 strain. Then, the transformant is cultured in a minimal medium containing a considerable amount of lysine, for example, M9. Strains carrying the plasmid with wild-type lysC are threonine, because only AK is blocked by lysine.
Isoleucine, methionine, and diaminopimelic acid (D
AP) cannot be synthesized and growth is suppressed. On the other hand, a plasmid-carrying strain having lysC ^* deinhibited by lysine should be able to grow on a minimal medium supplemented with a significant amount of lysine. Utilizing this phenomenon, the growth is S-2-, which is lysine or an analog of lysine.
Strains resistant to aminoethyl cysteine (AEC),
That is, it is possible to select a plasmid-carrying strain having lysC ^* in which inhibition is released.

By introducing the mutant gene thus obtained into an appropriate microorganism (host) as recombinant DNA and expressing it, a microorganism carrying AK for which feedback inhibition is released can be obtained. Obtained lysC gene or mutant lysC gene to produce threonine
Examples of the host into which (lysC ^* ) is introduced and amplified include wild strains of the bacterium belonging to the genus Escherichia described above. Gene (lysC ^*
) Is functional, the recombinant vector DNA is replicable and
Any bacterium capable of enhancing the ysC gene or the mutant lysC gene (lysC ^* ) can be used as a host. The most preferred host is E. coli strain B-3996.

<3. Production of L-isoleucine according to the present invention> Escherichia bacterium transformed by introducing the release type thrABC operon obtained as described above and further retaining the release type ilvGMEDA operon is cultured in a suitable medium, By producing and accumulating L-isoleucine in the culture and collecting L-isoleucine from the culture, L-isoleucine can be efficiently produced. That is, the release type thrABC
L-isoleucine can be efficiently produced by allowing the bacterium belonging to the genus Escherichia to hold both the operon and the release type ilvGMEDA operon.

Furthermore, a bacterium belonging to the genus Escherichia transformed by introducing the release type thrABC operon obtained as described above and further retaining the complete release type ilvGMEDA operon is cultured in a suitable medium, and the culture is performed. L-isoleucine was produced and accumulated in the product, and L-isoleucine was collected from the culture to obtain L-isoleucine.
-Isoleucine can be produced more efficiently.
That is, the release type thrABC operon and the complete release type i
L-isoleucine can be produced more efficiently by allowing the Escherichia bacterium to hold the lvGMEDA operon together.

[0118] Examples of Escherichia bacterium which retains the release type thrABC operon include Escherichia bacterium transformed by integrating DNA containing the release type thrABC operon into chromosomal DNA, or autonomously replicating in the Escherichia bacterium and the DNA. Examples thereof include Escherichia bacteria transformed by introducing into the cells a recombinant DNA formed by ligating with a possible vector DNA.

On the other hand, in order to transform the Escherichia bacterium by introducing the DNA containing the released ilvGMEDA operon into the bacterium, the DNA containing the released ilvGMEDA operon may be incorporated into the chromosomal DNA for transformation. DNA containing operon
Transformation may be carried out by introducing into the cell a recombinant DNA obtained by ligating Escherichia bacterial cells with a vector DNA capable of autonomous replication.

Furthermore, in order to transform the Escherichia bacterium by introducing a DNA containing the completely released ilvGMEDA operon, the completely released ilvGMED can be used.
The DNA containing the A operon may be incorporated into a chromosomal DNA for transformation, and a recombinant DNA obtained by ligating the DNA containing the completely released ilvGMEDA operon with a vector DNA capable of autonomous replication in Escherichia bacterial cells You may transform by introducing in.

Both the released thrABC operon and the released ilvGMEDA operon, or the released thr
When both the ABC operon and the completely released ilvGMEDA operon are introduced into Escherichia bacteria,
Both operons may be integrated and retained on the chromosomal DNA of Escherichia bacterium, or may be retained as extrachromosomal DNA on a single or separate plasmid in the cell. In addition, one of each operon is chromosome D
It may be integrated and retained on NA, and the other operon may be retained intracellularly on a plasmid as extrachromosomal DNA. Release type thrABC operon and release type ilvGMEDA operon or complete release type ilvGM
When the Escherichia bacterium and the EDA operon are provided in the form of separate plasmids, it is preferable that the two plasmids have different origins of replication so that the two plasmids do not show incompatibility.

DNA containing release type thrABC operon
When introducing a DNA containing the release type ilvGMEDA operon into a bacterium belonging to the genus Escherichia, the order of introducing both DNAs does not matter. When the DNA containing the released thrABC operon and the DNA containing the completely released ilvGMEDA operon are introduced into a bacterium belonging to the genus Escherichia, the order of introduction of both DNAs does not matter.

The Escherichia bacterium used as a host includes intracellular release type thrABC operon, release type ilvGMEDA operon and complete release type ilvG.
When the promoter of the MEDA operon or another promoter for expressing these genes functions, and when any of the operons is to be introduced into a plasmid as extrachromosomal DNA, vector D used for the introduction
Any NA origin of replication that can function and replicate in cells can be used. Release type thrABC operon and release type ilvGMEDA operon or complete release type ilvG
When the Escherichia bacterium has the MEDA operon in the form of separate plasmids, the origins of replication of the two plasmids need to function together.

The production of L-isoleucine by the microorganism of the present invention can be carried out by an ordinary microorganism culture method. The medium to be used may be a synthetic medium or a natural medium as long as it contains a carbon source, a nitrogen source, an inorganic substance and, if necessary, an appropriate amount of a nutrient source required for growth of the strain to be used.

Examples of the carbon source include various carbohydrates such as glucose and various organic acids such as pyruvic acid. Further, alcohol such as ethanol or glycerol can be used depending on the assimilation property of the microorganism used. As the nitrogen source, ammonia, ammonium salts of various acids such as ammonium sulfate, nitrogen compounds such as amines, and natural nitrogen sources such as peptone, soybean hydrolyzate, and decomposed product of fermentation cells can be used. As the inorganic substance, potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate and the like are used.

The culture is carried out under aerobic conditions such as shaking culture, aeration stirring culture, etc., and the culture temperature is 20 to 40 ° C.
It is preferably in the range of 30 to 38 ° C. PH of medium is 5
In the range of -9, the range of 6.5-7.2 is preferable. The pH of the medium is adjusted with ammonia, calcium carbonate, various acids, various bases, buffer solutions and the like. Usually, after culturing for 1 to 3 days, L-isoleucine is accumulated in the culture solution.
After completion of the culture, solids such as bacterial cells are removed from the culture solution by centrifugation or membrane separation, and L-isoleucine can be recovered by an ion exchange method, a concentration method, a crystallization method or the like.

[0127]

Examples of the present invention will be described below. (Example 1 Construction of pMWD5) Escherichia coli M
Chromosomal DNA was extracted from the I162 strain. The chromosome D
NA was cleaved with the restriction enzyme HindIII. The HindIII-HindIII DNA fragment containing the ilvGM gene has been found to be 4.8 kb in length. Therefore, HindIII-Hind having a length of about 4.8 kb
The III DNA fragment was ligated with the DNA fragment obtained by cleaving the plasmid vector pBR322 purchased from Takara Shuzo with HindIII. The resulting DNA mixture was transformed into Escherichia coli, which is a strain lacking acetohydroxy acid synthase.
It was introduced into E. coli strain MI262. A strain which was transformed to complement the acetohydroxy acid synthase-deficient trait was selected, and the plasmid carried by the strain was isolated. As a result of analyzing the structure of the same plasmid, HindII of pBR322
A 4.8 kb DNA fragment containing the ilvGM gene was inserted at the I site. The plasmid was named pBRGM7.

Gene 97, 21, (1991), Proc. Natl. Aca
d. Sci. USA 78, 922, (1981) and J. Bacteriol. 14
Synthetic oligonucleotide DNAs described in SEQ ID NO: 2 and SEQ ID NO: 3 in the Sequence Listing were synthesized with reference to the nucleotide sequence reported in 9, 294, (1982). Using both DNAs as primers and the chromosomal DNA of MI162 strain as a template,
DNA was amplified by the PCR method. Amplified DNA
The fragment is 2 of the nucleotide sequences shown in SEQ ID NO: 1 in the Sequence Listing.
It is a DNA fragment having the 5th to 952nd sequences. The same fragment is referred to as fragment (A).

Similarly, Gene 97, 21, (1991), Pro
c. Natl. Acad. Sci. USA 78, 922, (1981) and J. B.
Synthetic oligonucleotide DNAs described in SEQ ID NO: 4 and SEQ ID NO: 5 of the Sequence Listing were synthesized with reference to the nucleotide sequences reported in acteriol. 149, 294, (1982). Both DN
A was amplified by the PCR method using A as a primer and the chromosomal DNA of the MI162 strain as a template. The DNA fragment to be amplified is a DNA having a sequence from the 1161st position to the 2421st position in the nucleotide sequence shown in SEQ ID NO: 1 of the Sequence Listing.
It is a fragment. The same fragment is referred to as fragment (B).

A large fragment obtained by digesting the fragment (A) with SmaI was ligated with a DNA fragment obtained by digesting pUC18 (Takara Shuzo) with SmaI to prepare a plasmid pUCA. A large fragment obtained by digesting the fragment (B) with KpnI and a large fragment obtained by digesting pHSG399 (Takara Shuzo) with HincII and KpnI were ligated to prepare a plasmid pHSGB.

The plasmid pUCA was digested with KpnI,
The blunt end was blunt-ended with the large fragment of DNA polymerase I (Klenow fragment) and Ps
After digestion with tI, a DNA fragment containing the fragment (A) was finally isolated. The plasmid pHSGB was digested with HindIII, the blunt end was blunt-ended with the large fragment of DNA polymerase I (Klenow fragment), and further digested with PstI to finally isolate the DNA fragment containing the fragment (B). . Both DNA fragments are ligated and the plasmid pH
SGSK was created.

The SmaI-KpnI fragment derived from fragment (A) and fragment (B) mounted on pHSGSK was designated as fragment (C). Fragment (C) is a 4.8 kb HindIII-HindIII fragment containing the ilvGM gene.
It corresponds to a fragment obtained by digestion with maI and KpnI, and contains the promoter, SD sequence and upstream region of the ilvG gene, but lacks the sequence of about 0.2 kb from the leader sequence to the attenuator region. The procedure for constructing pHSGSK is summarized in FIG.

The plasmid pHSGSK was cloned into SmaI and Kp
A fragment (C) was obtained by digesting with nI, and a large DNA fragment was obtained by digesting the plasmid pBRGM7 with SmaI and KpnI, which were ligated together. The resulting plasmid was named pdGM1. 4.6 kb HindIII containing the ilvGM gene carried in pdGM1
The -HindIII fragment lacks the region required for attenuation. The ilvGM gene lacking the region necessary for attenuation is expressed as “ΔattGM” in this example and the drawings. The procedure for constructing pdGM1 is summarized in FIG.

The plasmid pDRIA4 described in Japanese Patent Application Laid-Open No. 2-458 is capable of autonomously replicating in Escherichia bacteria and autonomously replicating in Brevibacterium bacteria, and its inhibition by L-isoleucine. BamH containing the ilvA gene encoding a substantially released threonine deaminase
I-BamHI fragment is ligated and prepared. The BamHI-BamHI fragment is described as 2.3 kb in Japanese Patent Laid-Open No. 2-458, but it is currently 2.
It has been found to be 75 kb. Plasmid pDR
IA4 is Brevibacterium flavum AJ12358
(FERM BP-5087) or Brevibacterium flavum AJ12359 (FERM BP-50
88) outside the chromosomal DNA. The plasmid pDRIA4 can be prepared from these strains by a conventional method.

BamHI-on plasmid pDRIA4
HindII containing the ilvA gene encoding threonine deaminase whose inhibition by L-isoleucine is substantially eliminated in a BamHI 2.3 kb DNA fragment.
The I-BamHI fragment was prepared and the vector pMW119
(Manufactured by Nippon Gene Co., Ltd.) with HindIII and BamHI
It was ligated with the DNA fragment obtained by cutting with. The plasmid thus prepared was designated as pMWA1. The ilvA gene encoding threonine deaminase, which has been substantially released from the inhibition by L-isoleucine, may be expressed as “A *” in this example and the drawings.

The DNA fragment obtained by cleaving the plasmid pMWA1 with HindIII and the DNA fragment containing the ilvGM gene obtained by cleaving the plasmid pdGM1 with HindIII were ligated. By analyzing the position of the restriction enzyme recognition site existing on the plasmid, ilv
The one in which the transcription direction of the GM gene and the transcription direction of the ilvA gene were the same was selected, and this was selected as plasmid pMW.
It was named GMA2. Above, the procedure for constructing pMWGMA2 is summarized in FIG.

A chromosomal DNA of Escherichia coli MI162 strain was prepared and cleaved with SalI and PstI to prepare a DNA fragment mixture. On the other hand, the vector pUC
Cut 19 (Takara Shuzo) with SalI and PstI
The NA fragment was prepared. The DNA fragment mixture was ligated with the DNA fragment obtained by cleaving pUC19 to obtain a DNA mixture. This DNA mixture is treated with transaminase B
A strain that was introduced into the defective strain AB2070 and was transformed to restore the branched chain amino acid requirement was selected. When a plasmid was prepared from this strain, the plasmid pUC1
DNA fragment obtained by cleaving 9 with SalI and PstI, and SalI-PstI DN containing the ilvE gene
The A fragment was ligated. This plasmid was designated as pUCE1
I named it.

A DNA fragment mixture was prepared by partially digesting pMWGMA2 with HindIII. On the other hand, pUCE1 was cleaved with HindIII and part of the ilvE gene and ilvD
1.7 kb HindIII-Hi containing part of the gene
An ndIII DNA fragment was prepared. A dihydroxy acid dehydratase (ilvD gene product) -deficient strain AB1280 strain was transformed with the DNA mixture obtained by ligating the two, and a transformant strain in which the requirement for branched chain amino acids had disappeared was selected. When a plasmid was prepared from the transformant, a DNA fragment obtained by cleaving pMWGMA2 only at the HindIII site existing between ΔattGM and A *, and an ilv derived from pUCE1
1.7 containing part of E gene and part of ilvD gene
The HindIII-HindIII DNA fragment of kb was ligated, and the ilvGMEDA operon was regenerated. The plasmid thus obtained was named pMWD5. The procedure for constructing pMWD5 is summarized in FIG.

Plasmid pMW obtained as described above
D5 is a plasmid that uses pMW119 as a vector, contains the ilvA gene encoding TD that has been substantially desensitized to inhibition by L-isoleucine, and carries the ilvGMEDA operon from which the region required for attenuation has been removed.

(Example 2 Construction of L-isoleucine-producing strain) The plasmid pMWD5 obtained in Example 1 was prepared by using the plasmid pMWD5 disclosed in JP-A-3-501682 and USP 5,175,1.
L-threonine producing bacterium Escherichia.
It was introduced into Escherichia coli strain B-3996 by the conventional rubidium chloride method. Among the strains transformed by pMWD5, streptomycin 100 μg / ml and ampicillin 1
Transformed strains that simultaneously became resistant to 00 μg / ml were selected.
The resulting transformant was named AJ12919 strain.

The L-threonine producing bacterium Escherichia coli B-3996 strain is the former USSR Antibiotics Research Institute.
Arch Institute) under the registration number 1867. The host strain of the B-3996 strain is the Escherichia coli TDH-6 strain, which lacks the thrC gene, has saccharose-assimilating ability, can grow in the presence of 5 mg / ml of L-threonine, and lacks threonine dehydrogenase. However, the ilvA gene has a leaky mutation. Then, the B-3996 strain has a wide host range vector R in which the thrABC operon containing the thrA gene encoding AKI-HDI in which the inhibition by L-threonine is substantially released.
It holds the plasmid pVIC40 obtained by mounting it on a vector derived from SF1010. Therefore, the Escherichia coli AJ12919 strain carrying the plasmid pVIC40 and the plasmid pNWD5 has a thrABC operon containing a thrA gene encoding AKI-HDI in which inhibition by L-threonine is substantially released, and
TD in which inhibition by L-isoleucine is substantially released
The ilvGMEDA operon, which contains the ilvA gene coding for and has the region necessary for attenuation removed, and a strain carrying the ilvGMEDA operon.

Example 3 L- by AJ12919 strain
Isoleucine production test (1)) AJ obtained in Example 2
L-Isoleucine fermentation production test of 12919 strain was conducted. Bactryptone 1%, yeast extract 0.5%, Na
Cl 0.5%, agar 1.5%, streptomycin 10
AJ12919 strain is applied to a medium having a composition of 0 μg / ml and ampicillin 100 μg / ml, and the medium is applied at 37 ° C. for 18
After culturing for 24 hours, a portion of the fermentation broth was treated with a platinum loop to prepare 20 ml of fermentation medium (glucose 4%, ammonium sulfate 1.6%).
%, Potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 0.001%, manganese sulphate pentahydrate 0.001%, yeast extract 0.2% , Calcium carbonate 3%, pH 7.0), and cultured at 37 ° C. for 24 hours with shaking. The L-isoleucine and L-threonine concentrations in the culture supernatant from which the cells had been removed were measured by high performance liquid chromatography. The results are shown in Table 1.

[0143]

[Table 1] strain L- isoleucine concentration (g / L) L- threonine concentration (g / L) AJ12919 10 0

Example 4 L- by AJ12919 strain
Isoleucine production test (2)) AJ12919 strain was cultivated in the same agar medium as in Example 3 at 37 ° C. for 24 hours, and then inoculated into a 1-liter fermentor containing 300 ml of fermentation medium having the same composition as in Example 3, A seed culture was obtained by culturing at 37 ° C. for 16 hours at a stirring rate of 400 rpm and an aeration rate of 300 ml / min. 30 ml of the obtained seed culture broth were glucose 6%, ammonium sulfate 1.6%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 0.001%, Manganese sulfate pentahydrate 0.0
Inoculate a 1-liter fermentor containing 250 ml of fermentation medium having the composition of 01%, yeast extract 0.2%, pH 7.0,
Glucose is appropriately supplied while controlling the stirring number so that the oxygen concentration in the medium becomes 5% or more at 37 ° C. and an aeration rate of 300 ml / min.
Culturing was carried out for 23 hours while maintaining H at around 7.0.
The L-isoleucine concentration in the culture supernatant from which the cells had been removed was measured by high performance liquid chromatography. As a result, the amount of L-isoleucine accumulated in the culture supernatant was 39 g / L.

Example 5 Construction of L-Isoleucine-Producing Strain (2) In the international publication WO94 / 11517, the former USSR Antibiotics Research Institute (USSR Antibiotics Research Institute) is described.
te) under the registration number 1867, the plasmid p carried by the L-threonine-producing strain B-3996 strain
VIC 40 and the Institute of Biotechnology, Institute of Biotechnology, Japan Institute of Industrial Technology have been deposited under the registration number FERM P-13136 (international deposit number FERM BP-4462),
AKIII substantially free of inhibition by L-lysine
The lysC gene-containing plasmid (pLLC)
* 80), the thrABC operon containing the thrA gene encoding AKI-HDI substantially desensitized by L-threonine, and lysC encoding AKIII substantially desensitized by L-lysine.
A method for constructing a plasmid pVICLC * 80A in which a gene is mounted on a vector derived from the broad host range vector RSF1010, and the plasmid pVICLC * 80A
L-threonine producing bacterium introduced into host B-399 strain (TDH-6 strain) of -3996 strain is disclosed.

Plasmid pVICLC * 80A was added to B-3
The plasmid pMWD5 obtained in Example 1 was introduced into the L-threonine-producing bacterium introduced into the 996 strain host B-399 strain (TDH-6 strain) by the conventional method of rubidium chloride. Among the strains transformed by pMWD5, streptomycin 100 μg / ml and ampicillin 100 μg / ml
A transformant strain that simultaneously became resistant to was selected. The resulting transformant was named AJ13100 strain.

The host strain of the AJ13100 strain is the Escherichia coli TDH-6 strain (B-399 strain), and the thrC
It lacks the gene, has sucrose assimilation, and contains 5 mg /
It can grow in the presence of ml of L-threonine, lacks threonine dehydrogenase, and has a leaky mutation in the ilvA gene. And the AJ13100 strain is AKI-HDI in which the inhibition by L-threonine is substantially released.
ThrABC operon containing the thrA gene encoding AKI and AKI substantially free from inhibition by L-lysine
The lysC gene encoding II carries the plasmid pVICLC * 80A mounted on a vector derived from the broad host range vector RSF1010 and the plasmid pMWD5 obtained in Example 1. Therefore, the Escherichia coli AJ13100 strain encodes AKI-HDI in which the inhibition by L-threonine is substantially eliminated.
the thrABC operon containing the rA gene, the lysC gene encoding AKIII whose inhibition by L-lysine is substantially eliminated, and the ilvA gene encoding TD whose inhibition by L-isoleucine is substantially eliminated, and I where the area required for nuations has been removed
It is a strain carrying the lvGMEDA operon with a plasmid vector.

Example 6 L- by AJ13100 strain
Isoleucine production test) AJ131 obtained in Example 5
The L-isoleucine fermentation production test of 00 strain was performed. Bactryptone 1%, yeast extract 0.5%, NaCl0.
5%, agar 1.5%, streptomycin 100 μg /
ml, and a medium having a composition of 100 μg / ml ampicillin, the AJ13100 strain was applied to the medium, and the medium was fed at 37 ° C.
After culturing for 4 hours, a portion of the fermentation medium (platinum loop 20 ml) was used (glucose 4%, ammonium sulfate 1.6%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.
1%, ferrous sulfate heptahydrate 0.001%, manganese sulfate 5
Aqueous salt 0.001%, yeast extract 0.2%, calcium carbonate 3%, pH 7.0) was inoculated and shake-cultured at 37 ° C. for 24 hours. The AJ12919 strain obtained in Example 2 was also cultured in the same manner. The L-isoleucine and L-threonine concentrations in the culture supernatant from which the cells had been removed were measured by high performance liquid chromatography. Table 2 shows the results.

[0149]

[Table 2] strain L- isoleucine concentration (g / L) L- threonine concentration (g / L) AJ12919 10 0 AJ13100 12 0

[0150]

INDUSTRIAL APPLICABILITY The Escherichia bacterium having L-isoleucine-producing ability of the present invention is a thrABC operon containing a thrA gene encoding aspartokinase I-homoserine dehydrogenase I whose inhibition by L-threonine is substantially released. Is carried on a plurality of copies of the plasmid and amplified, so that a sufficient amount of L-threonine, which is a precursor substance of L-isoleucine, is biosynthesized. In addition, ilvA encoding threonine deaminase that is substantially desensitized by L-isoleucine
Since the ilvGMEDA operon containing the gene and having the region necessary for attenuation removed, is mounted on a multiple copy plasmid and amplified, L-threonine is efficiently converted to L-isoleucine.

IlvA Encoding Threonine Deaminase Substantially Removed from Inhibition by L-Isoleucine
Using the ilvGMEDA operon containing the gene and having the region necessary for attenuation removed, ilv
GM gene, ilvE gene, ilvD gene and il
The L-isoleucine biosynthesis enzyme group derived from the vA gene is produced in a balanced manner. Transcription is performed efficiently by the original promoter.

In general, increasing the gene dosage of the ilvGMEDA operon containing the ilvA gene encoding threonine deaminase substantially free from inhibition by L-isoleucine using a multicopy plasmid, the transcription level of the ilvGMEDA operon is increased. The negative control of the above is released, and the productivity of L-isoleucine is considered to be sufficiently improved. However, the present invention surprisingly succeeded in further improving the productivity of L-isoleucine by further removing the region necessary for attenuation.

[0153]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 2841 Sequence type: Nucleic acid Number of strands: Double Topology: Linear Sequence type: Genomic DNA Origin organism Name: Escherichia coli Strain name: MI162 Feature of sequence Characteristic symbol: CDS Location: 957..1055 Method of determining feature: S Characteristic symbol: attenuator Location: 1081..1104 Characteristic Determined method: S Characteristic symbol: CDS Location: 1195..2841 Characteristic determined method: S Characteristic symbol: cleavage-site (SmaI) Location: 52..57 Characteristic determined method: S Characteristic symbol: cleavage-site (KpnI) Location: 2395..2400 Method of determining feature: S sequence CTCGCTTTCC TTGTTCCTGA CCGATAACAT CACTGAGATC ATGTTGTAGC GCCCGGGATA 60 CTGCATGTCCTG TGGTTTCGGG AGT ACTTAATAGC T TCAGTTGCA CTGGAGATTG 240 CGCCGGGAAC GCCACGCAGA GCGCCTGTAA GCGCCAGTTC TCCGACTAAT TCATATTCAT 300 CTAACTTATT GGCTGTAAGC TGTTCTGAGG CCGCCAGCAA CGCAATGGCG ATAGGTAAAT 360 CATATCGTCC CCCTTCTTTT GGCAGATCAG CTGGAGCCAG GTTGATGGTG ATTTTTTTCG 420 CCGGATATTC ATATCCGCTA TTGATAATGG CGCTGCGCAC GCGATCGCGA GCTTCTTTTA 480 CCGTTGTTTC TGGTAAGCCC ACCATCGTTA AGCCGGGTAG ACCTTTACTG ATATGTACCT 540 CAACAGTGAT CGGGGGCGCA TTTACTCCCA GGGCTGCGCG GGTATGAACA ATTGACAGTG 600 ACATAAGCCC TCCTTGAGTC ACCATTATGT GCATAAGATA TCGCTGCTGT AGCCCGCTAA 660 TTCGTGAATT TTAGTGGCTG ATTCCTGTTT ATTTGTGCAA GTGAAGTTGA GTTGTTCTGG 720 CGGTGGAATG ATGCTCGCAA AAATGCAGCG GACAAAGGAT GAACTACGAG GAAGGGAACA 780 ACATTCATAC TGAAATTGAA TTTTTTTCAC TCACTATTTT ATTTTTAAAA AACAACAATT 840 TATATTGAAA TTATTAAACG CATCATAAAA ATCGGCCAAA AAATATCTTG TACTATTTAC 900 AAAACCTATG GTAACTCTTT AGGCATTCCT TCGAACAAGA TGCAAGAAAA GACAAA 956 ATG ACA GCC CTT CTA CGA GTG ATT AGC CTG GTC GTG ATT AGC GTG GTG 1004 Met Thr Ala Leu Leu Arg Val Ile Ser Leu Val Val Ile Ser Val Val 1 5 10 15 GTG ATT ATT ATC CCA CCG TGC GGG GCT GCA CTT GGA CGA GGA AAG GCT 1052 Val Ile Ile Ile Pro Pro Cys Gly Ala Ala Leu Gly Arg Gly Lys Ala 20 25 30 TAGAGATCAA GCCTTAACGA ACTAAGACCC CCGCACCGAA AGGTCCGG12 AC 1172 GATTCAGGAC GGGGAACTAA CT ATG AAT GGC GCA CAG TGG GTG GTA CAT GCG 1224 Met Asn Gly Ala Gln Trp Val Val His Ala 1 5 10 TTG CGG GCA CAG GGT GTG AAC ACC GTT TTC GGT TAT CCG GGT GGC GCA 1272 Leu Arg Ala Gla Val Asn Thr Val Phe Gly Tyr Pro Gly Gly Ala 15 20 25 ATT ATG CCG GTT TAC GAT GCA TTG TAT GAC GGC GGC GTG GAG CAC TTG 1320 Ile Met Pro Val Tyr Asp Ala Leu Tyr Asp Gly Gly Val Glu His Leu 30 35 40 CTA TGC CGA CAT GAG CAG GGT GCG GCA ATG GCG GCT ATC GGT TAT GCT 1368 Leu Cys Arg His Glu Gln Gly Ala Ala Met Ala Ala Ile Gly Tyr Ala 45 50 55 CGT GCT ACC GGC AAA ACT GGC GTA TGT ATC GCC ACG TCT GGT CCG GGC 1416 Arg Ala Thr Gly Lys Thr Gly Val Cys Ile Ala Thr Ser Gly Pro Gly 60 65 70 GCA ACC AAC CTG ATA ACC GG G CTT GCG GAC GCA CTG TTA GAT TCC ATC 1464 Ala Thr Asn Leu Ile Thr Gly Leu Ala Asp Ala Leu Leu Asp Ser Ile 75 80 85 90 CCT GTT GTT GCC ATC ACC GGT CAA GTG TCC GCA CCG TTT ATC GGC ACT 1512 Pro Val Val Ala Ile Thr Gly Gln Val Ser Ala Pro Phe Ile Gly Thr 95 100 105 GAC GCA TTT CAG GAA GTG GAT GTC CTG GGA TTG TCG TTA GCC TGT ACC 1560 Asp Ala Phe Gln Glu Val Asp Val Leu Gly Leu Ser Leu Ala Cys Thr 110 115 120 AAG CAT AGC TTT CTG GTG CAG TCG CTG GAA GAG TTG CCG CGC ATC ATG 1608 Lys His Ser Phe Leu Val Gln Ser Leu Glu Glu Leu Pro Arg Ile Met 125 130 135 GCT GAA GCA TTC GAC GTT GCC TGC TCA GGT CGT CCT GGT CCG GTT CTG 1656 Ala Glu Ala Phe Asp Val Ala Cys Ser Gly Arg Pro Gly Pro Val Leu 140 145 150 GTC GAT ATC CCA AAA GAT ATC CAG TTA GCC AGC GGT GAC CTG GAA CCG 1704 Val Asp Ile Pro Lys Asp Ile Gln Leu Ala Ser Gly Asp Leu Glu Pro 155 160 165 170 TGG TTC ACC ACC GTT GAA AAC GAA GTG ACT TTC CCA CAT GCC GAA GTT 1752 Trp Phe Thr Thr Val Glu Asn Glu Val Thr Phe Pro His Ala Glu Val 175 180 185 GAG CAAGCG CGC CAG ATG CTG GCA AAA GCG CAA AAA CCG ATG CTG TAC 1800 Glu Gln Ala Arg Gln Met Leu Ala Lys Ala Gln Lys Pro Met Leu Tyr 190 195 200 GTT GGC GGT GGC GTG GGT ATG GCG CAG GCA GTT CCG GCT TTG CGT GAA 1848 Val Gly Gly Gly Val Gly Met Ala Gln Ala Val Pro Ala Leu Arg Glu 205 210 215 TTT CTC GCT GCC ACA AAA ATG CCT GCC ACC TGT ACG CTG AAA GGG CTG 1896 Phe Leu Ala Ala Thr Lys Met Pro Ala Thr Cys Thr Leu Lys Gly Leu 220 225 230 GGC GCA GTA GAA GCA GAT TAT CCG TAC TAT CTG GGC ATG CTG GGG ATG 1944 Gly Ala Val Glu Ala Asp Tyr Pro Tyr Tyr Leu Gly Met Leu Gly Met 235 240 245 250 CAC GGC ACC AAA GCG GCA AAC TTC GCG GTG CAG GAG TGT GAC CTG CTG 1992 His Gly Thr Lys Ala Ala Asn Phe Ala Val Gln Glu Cys Asp Leu Leu 255 260 265 ATC GCC GTG GGC GCA CGT TTT GAT GAC CGG GTG ACC GGC AAA CTG AAC 2040 Ile Ala Val Gly Ala Arg Phe Asp Asp Arg Val Thr Gly Lys Leu Asn 270 275 280 ACC TTC GCG CCA CAC GCC AGT GTT ATC CAT ATG GAT ATC GAC CCG GCA 2088 Thr Phe Ala Pro His Ala Ser Val Ile His Met Asp Ile Asp Pro Ala 285 290 295 GAA ATG AAC AAG CTG CGT CAG GCA CAT GTG GCA TTA CAA GGT GAT TTA 2136 Glu Met Asn Lys Leu Arg Gln Ala His Val Ala Leu Gln Gly Asp Leu 300 305 310 AAT GCT CTG TTA CCA GCA TTA CAG CAG CCG TTA AAT CAA TGT GAC TGG 2184 Asn Ala Leu Leu Pro Ala Leu Gln Gln Pro Leu Asn Gln Cys Asp Trp 315 320 325 330 CAG CAA CAC TGC GCG CAG CTG CGT GAT GAA CAT TCC TGG CGT TAC GAC 2232 Gln Gln His Cys Ala Gln Leu Arg Asp Glu His Ser Trp Arg Tyr Asp 335 340 345 CAT CCC GGT GAC GCT ATC TAC GCG CCG TTG TTG TTA AAA CAA CTG TCG 2280 His Pro Gly Asp Ala Ile Tyr Ala Pro Leu Leu Leu Lys Gln Leu Ser 350 355 360 GAT CGT AAA CCT GCG GAT TGC GTC GTG ACC ACA GAT GTG GGG CAG CAC 2328 Asp Arg Lys Pro Ala Asp Cys Val Val Thr Thr Asp Val Gly Gln His 365 370 375 CAG ATG TGG GCT GCG CAG CAC ATC GCC CAC ACT CGC CCG GAA AAT TTC 2376 Gln Met Trp Ala Ala Gln His Ile Ala His Thr Arg Pro Glu Asn Phe 380 385 390 ATC ACC TCC AGC GGT TTA GGT ACC ATG GGT TTT GGT TTA CCG GCG GCG 2424 Ile Thr Ser Ser Gly Leu Gly Thr Met Gly Phe G ly Leu Pro Ala Ala 395 400 405 410 GTT GGC GCA CAA GTC GCG CGA CCG AAC GAT ACC GTT GTC TGT ATC TCC 2472 Val Gly Ala Gln Val Ala Arg Pro Asn Asp Thr Val Val Cys Ile Ser 415 420 425 GGT GAC GGC TCT TTC ATG ATG AAT GTG CAA GAG CTG GGC ACC GTA AAA 2520 Gly Asp Gly Ser Phe Met Met Asn Val Gln Glu Leu Gly Thr Val Lys 430 435 440 CGC AAG CAG TTA CCG TTG AAA ATC GTC TTA CTC GAT AAC CAA CGG TTA 2568 Arg Lys Gln Leu Pro Leu Lys Ile Val Leu Leu Asp Asn Gln Arg Leu 445 450 455 GGG ATG GTT CGA CAA TGG CAG CAA CTG TTT TTT CAG GAA CGA TAC AGC 2616 Gly Met Val Arg Gln Trp Gln Gln Leu Phe Phe Gln Glu Arg Tyr Ser 460 465 470 GAA ACC ACC CTT ACT GAT AAC CCC GAT TTC CTC ATG TTA GCC AGC GCC 2664 Glu Thr Thr Leu Thr Asp Asn Pro Asp Phe Leu Met Leu Ala Ser Ala 475 480 485 490 TTC GGC ATC CAT GGC CAA CAC ATC ACC CGG AAA GAC CAG GTT GAA GCG 2712 Phe Gly Ile His Gly Gln His Ile Thr Arg Lys Asp Gln Val Glu Ala 495 500 505 GCA CTC GAC ACC ATG CTG AAC AGT GAT GGG CCA TAC CTG CTT CAT GTC 2760 Ala Leu Asp Thr Met Le u Asn Ser Asp Gly Pro Tyr Leu Leu His Val 510 515 520 TCA ATC GAC GAA CTT GAG AAC GTC TGG CCG CTG GTG CCG CCT GGC GCC 2808 Ser Ile Asp Glu Leu Glu Asn Val Trp Pro Leu Val Pro Pro Gly Ala 525 530 535 AGT AAT TCA GAA ATG TTG GAG AAA TTA TCA TGA 2841 Ser Asn Ser Glu Met Leu Glu Lys Leu Ser 540 545

SEQ ID NO: 2 Sequence Length: 22 Sequence Type: Nucleic Acid Number of Strands: Single Topology: Linear Sequence Origin: Other Nucleic acid of synthetic DNA sequence TAACATCACT GAGATCATGT TG 22

SEQ ID NO: 3 Sequence Length: 21 Sequence Type: Nucleic Acid Number of Strands: Single Topology: Linear Sequence Origin: Other Nucleic acid of Synthetic DNA sequence TCTTTTCTTG CATCTTGTTC G 21

SEQ ID NO: 4 Sequence Length: 22 Sequence Type: Nucleic Acid Number of Strands: Single Topology: Linear Sequence Origin: Other Nucleic acid of Synthetic DNA sequence TCTGTTTCTC AAGATTCAGG AC 22

SEQ ID NO: 5 Sequence Length: 19 Sequence Type: Nucleic Acid Number of Strands: Single Topology: Linear Sequence Origin: Other Nucleic Acids Synthetic DNA Sequence CGCCGGTAAA CCAAAACCC 19

SEQ ID NO: 6 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Topology: Linear Sequence Origin: Other Nucleic acid synthetic DNA sequence CTTCCCTTGT GCCAAGGCTG 20

SEQ ID NO: 7 Sequence Length: 18 Sequence Type: Nucleic Acid Number of Strands: Single Topology: Linear Sequence Origin: Other Nucleic acid synthetic DNA sequence GAATTCCTTT GCGAGCAG 18

[Brief description of drawings]

FIG. 1 shows the procedure for constructing plasmid pHSGSK. Among the abbreviations used in FIG. 1, H indicates a HindIII cleavage site existing on DNA. Similarly, P is PstI
Cleavage site, K for KpnI cleavage site, B for BamHI
Cleavage site, X for XbaI cleavage site, Sm for SmaI
Indicates the cleavage site. & Indicates that re-cutting by a restriction enzyme is impossible. P → indicates a promoter. SD indicates Shine-Dalgarno sequence. ATG represents a translation initiation codon. TGA indicates a translation termination codon. Ap is a marker gene that imparts ampicillin resistance to the transformed strain. Cm is a marker gene that imparts chloramphenicol resistance to the transformed strain.

FIG. 2 shows the construction of the ilvGM gene from which the region required for attenuation of the ilvGMEDA operon has been removed.
The procedure for constructing the plasmid pdGM1 is shown. The abbreviations used in FIG. 2 have the same meanings as the abbreviations used in FIG.

FIG. 3 shows the construction procedure of plasmid pMWGMA2.
The abbreviations used in FIG. 3 have the same meanings as the abbreviations used in FIG.

FIG. 4 shows the procedure for constructing plasmid pMWD5. FIG.
The abbreviations used in Table 1 have the same meanings as the abbreviations used in FIG. 1, but in addition, S represents a SalI cleavage site. In addition, par indicates the stabilization region of the plasmid.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location C12N 9/12 15/09 ZNA // (C12P 13/06 C12R 1:19) (C12N 1/21 (C12R 1:19) (C12N 9/12 C12R 1:19) (C12N 15/09 ZNA C12R 1:19) C12R 1:19) (72) Inventor Hiroshi Matsui 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Central Research Laboratory of Nomoto Co., Ltd.

Claims (18)

[Claims] 1. A thrAB gene containing a thrA gene encoding aspartokinase I-homoserine dehydrogenase I substantially desensitized by L-threonine.
Ilv, which encodes the C operon and threonine deaminase that has been substantially de-inhibited by L-isoleucine
A bacterium belonging to the genus Escherichia having L-isoleucine productivity, which retains the ilvGMEDA operon containing the A gene and having a region necessary for attenuation removed. 2. The thrABC operon and the il
The Escherichia bacterium according to claim 1, which holds the vGMEDA operon on a plasmid. 3. The Escherichia bacterium according to claim 2, which holds two types of plasmids, a plasmid (A) carrying the thrABC operon and a plasmid (B) carrying the ilvGMEDA operon. . 4. The ilvGMEDA operon is the DNA sequence Nos. 953 to 1 of SEQ ID NO: 1 in the Sequence Listing.
The bacterium of the genus Escherichia according to any one of claims 1 to 3, which has a deficient DNA sequence up to number 160. 5. The Escherichia bacterium according to claim 4, wherein the plasmid (A) is pVIC40 and the plasmid (B) is pMWD5 among the two types of plasmids. 6. The Escherichia bacterium according to any one of claims 1 to 5, wherein the Escherichia bacterium is Escherichia coli. 7. The thrABC operon and the il
vGMEDA operon lacks the thrC gene and
It is capable of growing in the presence of mg / ml of L-threonine, is deficient in threonine dehydrogenase activity, and has the ilvA gene introduced into a host strain having a leaky mutation.
The Escherichia bacterium according to claim 6. 8. The bacterium belonging to the genus Escherichia according to claim 7, which is Escherichia coli AJ12919 strain. 9. A method for culturing the Escherichia bacterium according to any one of claims 1 to 8 in a medium to allow L-isoleucine to be produced and accumulated in the medium, and collecting L-isoleucine from the medium. A method for producing L-isoleucine by a fermentation method, which is characterized. 10. A thrA gene containing a thrA gene encoding aspartokinase I-homoserine dehydrogenase I substantially free from inhibition by L-threonine.
BC operon and lysC that encodes aspartokinase III substantially free of inhibition by L-lysine
L-, which contains a gene and the ilvGMEDA operon containing the ilvA gene encoding threonine deaminase substantially free from inhibition by L-isoleucine and having a region necessary for attenuation removed. A bacterium belonging to the genus Escherichia having isoleucine productivity. 11. The thrABC operon and the l
The bacterium belonging to the genus Escherichia according to claim 10, which has a ysC gene and the ilvGMEDA operon on a plasmid. 12. The thrABC operon and the ly.
a plasmid (C) carrying the sC gene, and
The Escherichia bacterium according to claim 11, which holds two types of plasmids, a plasmid (B) carrying the vGMEDA operon. 13. The ilvGMEDA operon has a DNA sequence in which 953 to 1160 of the DNA sequence shown in SEQ ID NO: 1 of the Sequence Listing is deleted. The bacterium belonging to the genus Escherichia. 14. The bacterium of the genus Escherichia according to claim 13, wherein the plasmid (C) is pVICLC * 80A and the plasmid (B) is pMWD5 among the two types of plasmids. 15. The Escherichia bacterium according to any one of claims 10 to 14, wherein the Escherichia bacterium is Escherichia coli. 16. The thrABC operon and the l
The ysC gene and the ilvGMEDA operon are t
The Escherichia coli according to claim 15, wherein the hrC gene is deficient, can grow in the presence of 5 mg / ml L-threonine, is deficient in threonine dehydrogenase activity, and has introduced the ilvA gene into a host strain having a leaky mutation. Genus bacteria. 17. The Escherichia bacterium according to claim 16, which is Escherichia coli AJ13100 strain. 18. A method of culturing the Escherichia bacterium according to any one of claims 10 to 17 in a medium to allow L-isoleucine to be produced and accumulated in the medium, and collecting L-isoleucine from the medium. A method for producing L-isoleucine by a fermentation method, which is characterized.