JPH05344893A - Gene capable of coding acetohydroxy acid synthase and its utilization - Google Patents

Abstract

PURPOSE:To obtain the subject gene DNA, isolated from a coryneform bacterium such as Brevibacterium.flavum MJ-233, having a specific base sequence and capable of providing a transformant remarkably improved in productivity of L-isoleucine, L-valine, etc. CONSTITUTION:Brevibacterium.flavum MJ-233 which is a corynebacterium is cultured in a culture medium till the latter period of the logarithmic growth phase and the microbial cell is collected and suspended in a buffer solution containing a lysozyme. Protenase K(R) and sodium dodecyl sulfate are then added to carry out the lysis. The resultant lysate is subsequently extracted with a phenol/chloroform solution. Ethanol is added to the extract solution to recover a DNA, which is then treated with a restriction enzyme, bound to a cloning vector and inserted into Escherichia coli to perform transformation. The obtained transformant is subsequently cloned to sort out a positive clone. A plasmid is recovered from the obtained strain and treated with a restriction enzyme to afford the objective gene DNA, capable of coding an acetohydroxy acid synthase derived from the coryneform bacterium and expressed by the formula, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a coryneform bacterium-derived gene DNA containing a gene encoding acetohydroxy acid synthase (EC 4.1.3.18), and a recombinant plasmid containing the gene DNA. A coryneform bacterium transformed with the plasmid, and L using the coryneform bacterium
-A method for producing isoleucine or L-valine.

[0002] L-isoleucine and L-valine are one of essential amino acids, and as amino acids that play an important role in nutrition of humans and animals, they have been added to pharmaceuticals, foods, and feed additives, and the demand for them has been increasing in recent years. It is increasing rapidly.

[0003]

2. Description of the Related Art L-isoleucine, which has stereoisomerism as in the case of other amino acids, is generally difficult to chemically synthesize only the L-form, and is industrially produced mainly by a fermentation method. It is being appreciated. As the production by the fermentation method, for example, a method using a precursor of L-isoleucine such as DL-α-aminobutyric acid and threonine (Japanese Patent Publication No. 43-870).
No. 9, 40-2880, etc.), a so-called direct fermentation method (see Japanese Patent Publication No. 38-7091 and JP-A No. 49-93586, etc.) in which a precursor is not added is disclosed. There is.

On the other hand, as the production of L-isoleucine by the enzymatic method, for example, in the presence of an ammonium ion or an L- or DL-amino acid other than isoleucine, D-
A method for producing L-isoleucine from-, L-, or DL-α-keto-β-methylvaleric acid (Japanese Patent Publication No. 46-297).
89); in the presence of ammonium ions or L- or DL-amino acids other than isoleucine, D-
A method of producing L-isoleucine by converting isoleucine or D-alloisoleucine alone or in a mixture, or an appropriate mixture of these and optical isomers thereof (see Japanese Patent Publication No. 46-29788); Serratia (Ser
glucose and D using immobilized products of bacteria
-Method for producing L-isoleucine from threonine (Proceedings of the Conference of the Japan Fermentation Industries Association, p.47-48, 1972)
(Year) etc. have been reported.

However, these methods are not fully satisfactory because of problems such as high raw material costs and low yield. In addition, as an industrial production method of L-valine,
Since there are stereoisomers as in the case of isoleucine, it is difficult to produce only the L-form by the chemical synthesis method, and the fermentation method is mainly used. However, a known method for producing L-valine by a fermentation method provides a method for effectively producing L-valine from a new point of view, because the yield of sugar is low and the accumulation of L-valine is limited. Was strongly desired.

[0006]

The object of the present invention is to obtain acetohydroxy acid synthase (E.
C. Isolation of the gene encoding 4.1.3.18),
The gene is introduced into a coryneform bacterium of the same species, and L-isoleucine or L-valine is efficiently produced from a new viewpoint using the coryneform bacterium.

[0007]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have isolated an acetohydroxy acid synthase gene from a coryneform bacterial chromosome,
It was found that L-isoleucine or L-valine can be efficiently produced by introducing the gene into an appropriate vector plasmid to transform a coryneform bacterium, and using the transformed coryneform bacterium. The invention was completed.

Thus, according to the present invention, (1) a gene DNA encoding an acetohydroxy acid synthase derived from a coryneform bacterium, (2) a recombinant plasmid having the gene DNA introduced therein, (3) the recombinant plasmid. (4) L-using glucose as a raw material using the coryneform bacterium transformed with
Methods for producing isoleucine or L-valine are provided.

The present invention will be described in more detail below. The “gene DNA encoding acetohydroxy acid synthase” of the present invention is an enzyme that synthesizes α-aceto-α-hydroxybutyric acid by adding pyruvic acid to α-ketobutyric acid,
Alternatively, a gene DN encoding an enzyme that synthesizes α-acetolactate from two molecules of pyruvate, that is, acetohydroxy acid synthase (EC 4.1.3.18)
Means A.

Although a DNA fragment containing a gene encoding acetohydroxy acid synthase (hereinafter, may be abbreviated as "A fragment") can be synthesized after its nucleotide sequence is determined. , Which can be generally cloned from a microorganism having acetohydroxy acid synthase-producing ability, and as a microorganism which is a source thereof,
Coryneform bacteria, especially Brevibacterium flavum (B
revivalium flavum) MJ-2
33 (FERM BP-1497) and its derived strains are advantageously used.

An example of the basic procedure for preparing the A fragment from these source microorganisms is as follows: The A fragment is the above coryneform bacterium, eg Brevibacterium flavum MJ-233 (FERM). BP-14
97) It is present on the chromosome of the strain and can be isolated and obtained by the method described below from among the cleaved fragments generated by cleaving this chromosome with an appropriate restriction enzyme.

First, Brevibacterium flavum MJ
Chromosomal DNA is extracted from a culture of strain -233. The chromosomal DNA is completely digested with an appropriate restriction enzyme such as EcoRI.

The obtained DNA fragment is inserted into a cloning vector such as pHSG399 (Takara Shuzo), and isoleucine and valine-requiring Escherichia coli MI262 [Escherichia coli MI262 deficient in the acetohydroxy acid synthase gene are used using this vector.
Genetech Stock Center (Escherich
ia coli Genetic Stock Cen
ter), Department of Biology, Yale University (Department of B)
iology, Yale University);
P. O. Box6666 New Haven, CT
06511-744, U.S.P. S. A. The preserved strain] is transformed and smeared on a selective medium to obtain a transformed strain. A derived from chromosome of Brevibacterium flavum MJ-233 strain inserted by extracting plasmid DNA from the obtained transformant and analyzing it with a restriction enzyme.
You can check and obtain fragments.

The thus obtained A fragment is further cleaved with a suitable restriction enzyme, the resulting DNA fragment is inserted into a vector plasmid capable of replicating in Escherichia coli, and this vector plasmid is transformed by a commonly used transformation method, for example, It is introduced into the above isoleucine- and valine-requiring Escherichia coli mutant strain by transformation by the calcium chloride method, electric pulse method or the like, and smeared on a selective medium.

From the obtained transformant, plasmid DNA is obtained.
The A fragment derived from the inserted Brevibacterium flavum MJ-233 strain chromosome can be confirmed and obtained by extracting B. One of the A fragments thus obtained is the chromosomal DNA of the Brevibacterium flavum MJ-233 strain, which is a restriction enzyme E.
A DNA fragment having a size of about 3.4 kb obtained by cleaving out by complete digestion of coRI and further cleaving it with a restriction enzyme SacI can be mentioned.

Table 1 below shows the number of recognition sites and the size of the cleaved fragment when the DNA fragment containing the gene encoding acetohydroxy acid synthase of about 3.4 kb was cleaved with various restriction enzymes.

[0017]

[Table 1] Table 1 Restriction enzymes Number of recognition sites Size of cleavage fragment (kb) KpnI 1 1.75 1.65 NcoI 2 1.7 0.95 0.75 EcoRV 2 1.8 1.3 0.3 0.3 Eco065I 3 1.65 1.55 0.15 0.05

In the present specification, the "number of recognition sites" by a restriction enzyme means that a DNA fragment or a plasmid is completely decomposed in the presence of a restriction enzyme, and those decomposition products are subjected to 1% agarose according to a method known per se. Gel electrophoresis and 5
% Polyacrylamide gel electrophoresis, and the value determined from the number of separable fragments was adopted.

The "size of the cleaved fragment" and the size of the plasmid are such that, when agarose gel electrophoresis is used, Escherichia coli lambda phage (λphag).
Based on the standard line drawn by the migration distance on the same agarose gel of the DNA fragment of known molecular weight obtained by cleaving the DNA of e) with the restriction enzyme HindIII, and when using polyacrylamide gel electrophoresis, Escherichia・ Coli's Phi-X174 phage (φx17
Based on the standard line drawn by the migration distance on the same polyacrylamide gel of the DNA fragment of known molecular weight obtained by cleaving the DNA of 4 phase) with the restriction enzyme HaeIII, the size of each cleaved DNA fragment or each DNA fragment of the plasmid is determined. calculate. The size of the plasmid is determined by adding the sizes of the respective cut fragments. In determining the size of each DNA fragment, the results obtained by 1% agarose gel electrophoresis were used for the size of fragments of 1 kb or more, and the size of fragments of about 0.1 kb to less than 1 kb was used. The results obtained by 4% polyacrylamide gel electrophoresis were adopted.

On the other hand, the chromosomal DNA of Brevibacterium flavum MJ-233 as described above was digested with the restriction enzyme EcoR.
Regarding the DNA fragment having a size of about 3.4 kb obtained by digestion with I and SacI, its base sequence is the plasmid pUC118 and / or pUC119.
(Takara Shuzo Co., Ltd.) using dideoxy nucleotide enzyme method
n method, Sanger, F .; et. al. , Proc. N
atl. Acad. Sci. USA, 74 , p546
3, 1977). The gene encoding acetohydroxy acid synthase determined from the presence of the open reading frame of the nucleotide sequence of the above-mentioned DNA fragment of about 3.4 kb determined in this manner is
It has the sequence shown in SEQ ID NO: 1 in the sequence listing described below, and is composed of 1782 base pairs encoding 594 amino acids.

The above-mentioned DNA fragment containing the gene encoding the acetohydroxy acid synthase of the present invention, which comprises the nucleotide sequence represented by SEQ ID NO: 1 in the Sequence Listing, is isolated from natural coryneform bacterial chromosomal DNA. In addition to the above, it may be one synthesized by using a commonly used DNA synthesizer, for example, System-1 Plus manufactured by Beckman.

Further, as described above, the DNA fragment of the present invention obtained from the chromosomal DNA of Brevibacterium flavum MJ-233 has a nucleotide sequence that does not substantially impair the function of encoding acetohydroxy acid synthase. A part of the bases may be replaced with other bases or deleted, or a new base may be inserted, and further, a part of the base sequence is rearranged. Any of these derivatives may be included in the DNA fragment containing the gene encoding the acetohydroxy acid synthase of the present invention.

The size of D described above is about 3.4 kb.
A restriction enzyme cleavage point map of the NA fragment is shown in FIG. The DNA fragment (A fragment) containing the gene encoding acetohydroxyacid synthase of the present invention is introduced into an appropriate plasmid vector, for example, a plasmid vector containing at least a gene that controls the replication / proliferation function of the plasmid in coryneform bacteria. As a result, a recombinant plasmid capable of highly expressing acetohydroxy acid synthase in a coryneform bacterium can be obtained.

The promoter for expressing the gene encoding the acetohydroxy acid synthase of the present invention may be the promoter of the gene itself possessed by the coryneform bacterium, but the promoter is not limited thereto. Any promoter may be used as long as it is a nucleotide sequence derived from a prokaryote for initiating transcription of the hydroxy acid synthase gene.

The A fragment of the present invention can be introduced,
Examples of the plasmid vector containing at least a gene that controls the replication / proliferation function in coryneform bacteria include, for example, Japanese Patent Application Laid-Open No. Hei 3
Plasmid pCRY3 described in JP-A-210184.
0; plasmids pCRY21, pCRY2KE, pCRY2KX, pC described in JP-A-2-276575.
RY31, pCRY3KE and pCRY3KX; plasmid pCRY2 described in JP-A-1-191686.
And pCRY3; pAM330 described in JP-A-58-67679; pHM1519 described in JP-A-58-77895; pAJ655, pAJ611 and pAJ184 described in JP-A-58-192900.
4; pCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197; pCG4 and pCG described in JP-A-57-183799.
11 etc. can be mentioned.

Among them, the plasmid vector used in the coryneform bacterium host vector system has a gene that controls the replication / proliferation function of the plasmid in the coryneform bacterium and a gene that controls the plasmid stabilizing function in the coryneform bacterium. Are preferred, for example plasmids pCRY30, pCR
Y21, pCRY2KE, pCRY2KE, pCRY2
KX, pCRY31, pCRY3KE and pCRY3K
X and the like are preferably used.

As a method for preparing the above-mentioned plasmid vector pCRY30, Brevibacterium stationii is used.
s) Plasmid pBY503 (for details of this plasmid, see JP-A-1-95785), DNA was extracted from IFO12144 (FERM BP-2515), and a replication-proliferating function of a plasmid having a size of about 4.0 kb with a restriction enzyme XhoI. The DNA fragment containing the gene that controls the plasmid is cut out, and the DNA fragment containing the gene that controls the stabilizing function of the plasmid having a size of about 2.1 kb with the restriction enzymes EcoRI and KpnI is cut out. These two fragments were used as EcoRI and KpnI of plasmid pHSG298 (Takara Shuzo).
The plasmid vector pCRY30 can be prepared by incorporating the site and the SalI site.

Next, the introduction of the A fragment of the present invention into the above-mentioned plasmid vector can be carried out, for example, by introducing 1
Cleaving a restriction enzyme site existing only at the site with the restriction enzyme,
If necessary, the A fragment and the cleaved plasmid vector may be treated with S1 nuclease to make it blunt-ended, or in the presence of an appropriate adapter DNA.
It can be performed by ligating with ligase treatment.

The introduction of the A fragment of the present invention into the plasmid pCRY30 is carried out by using the plasmid pCRY30 with the restriction enzyme Eco.
It can be carried out by cleaving with RI and ligating the DNA fragment (A fragment) containing the gene encoding the acetohydroxy acid synthase thereto with DNA ligase. The plasmid pCRY30 constructed in this way
The recombinant plasmid of the present invention into which the A fragment having a size of about 3.4 kb is introduced is one of the recombinant plasmids that can be preferably used for the production of L-isoleucine and L-valine. And the plasmid pCRY30
-Named AHAS. Plasmid pCRY30-AH
Details of the method of creating the AS will be described in Example 4 below.

A plasmid capable of replicative growth in a coryneform bacterium containing a gene encoding acetohydroxy acid synthase thus produced is introduced into a host microorganism, and L-isoleucine and L-
Valine can be stably and efficiently produced. Host microorganisms that can be transformed with the plasmid according to the present invention include coryneform bacteria such as Brevibacterium flavum MJ-233 (FERM BP-1497), Brevibacterium flavum MJ-233-AB-41.
(FERM BP-1498), Brevibacterium
Flavum MJ-233-ABT-11 (FERM BP
-1500), Brevibacterium flavum MJ-2
33-ABD-21 (FERM BP-1499) and the like.

The above-mentioned FERM BP-1498 strain is an ethanol-assimilating microorganism to which DL-α-aminobutyric acid resistance has been positively imparted, using the FERM BP-1497 strain as a parent strain (Japanese Patent Publication No. 59-59). 28398 gazette column 3-4 reference). In addition, FERM BP-1500
Strain is a highly active mutant of L-α-aminobutyric acid transaminase using the strain of FERM BP-1497 as a parent strain (see JP-A-62-51998). Furthermore, the strain of FERM BP-1499 is FERMBP-
It is a highly active mutant strain of D-α-aminobutyric acid deaminase using the strain 1497 as a parent strain (JP-A-61-177993).
(See the official gazette).

In addition to these microorganisms, Brevibacterium ammoniagenes (Brevibacterium)
ammoniagenes) ATCC 6871, A
TCC13745, ATCC13746; Brevibacterium debaricatum (Brevibacterium)
m divaricatum) ATCC14020; Brevibacterium lactofermentum (Brevi
Bacteriumlactofermentum) A
TCC13869; Corynebacterium glutamicum
um) ATCC31831 and the like can also be used as the host microorganism.

When a strain derived from Brevibacterium flavum MJ-233 is used as a host, the plasmid pBY502 possessed by this strain (JP-A-63-367) is used.
Since it may be difficult to transform because of this, it is desirable to remove the plasmid pBY502 from this strain in such a case. As a method for removing such a plasmid pBY502, for example, it is possible to spontaneously delete it by repeating subculture, or it is possible to remove it artificially [Bact. Rev. , 36 , p. 361-405 (1
972)]. The following is an example of a method for artificially removing the plasmid pBY502.

Host Brevibacterium flavum MJ-
Approximately 10 cells per ml in a medium containing acridine orange (concentration: 0.2 to 50 μg / ml) or ethidium bromide (concentration: 0.2 to 50 μg / ml) at a concentration that incompletely inhibits the growth of 233. Inoculate so that
Incubate at about 35 ° C. for about 24 hours while incompletely inhibiting the growth. After diluting the culture solution, the culture solution is applied to an agar medium and cultured at about 35 ° C. for about 2 days. A plasmid extraction operation is independently performed on each of the emerged colonies to select a strain from which the plasmid pBY502 has been removed. By this operation, plasmid p
A strain derived from Brevibacterium flavum MJ-233 from which BY502 is removed is obtained.

As a method for transforming the above-mentioned plasmid into the strain derived from Brevibacterium flavum MJ-233 obtained in this manner, as known for Escherichia coli and Erwinia carotovora [Cal
vin, N.N. M. and Hanawalt, P. et al.
C. , Journal of Bacterialog
y, 170 , 2796 (1988); Ito, K .; , N
Ishida, T .; and Izaki. K. , Agri
cultural and Biological C
Chemistry, 52 , 293 (1988)],
Pulse wave energization to DNA recipient bacteria [Satoh, Y. et
al. , Journal of Industry
l Microbiology, 5 , 159 (199
It is possible to introduce a plasmid according to 0).

A coryneform bacterium capable of producing acetohydroxy acid synthase, which is obtained by transformation by the above method,
For example, a method for culturing a strain derived from Brevibacterium flavum MJ-233 will be described below. Culture is a carbon source, a nitrogen source,
It can be carried out in a usual nutrient medium containing inorganic salts and the like, carbon sources such as glucose, ethanol, methanol, molasses and the like, and nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea and the like. Are used alone or as a mixture. Further, as the inorganic salt, for example, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used. In addition to this, nutrient sources such as various vitamins such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid, and biotin can be added to the medium.

The culture is usually carried out under aerobic conditions such as aeration and shaking, at about 20 to about 40 ° C, preferably at about 25 ° C to about 3 ° C.
It can be carried out at a temperature of 5 ° C. PH during the culture is 5
The pH can be adjusted to 10, preferably about 7 to 8, and pH can be adjusted during the culture by adding an acid or an alkali. The carbon source concentration at the start of culture is preferably 1 to 5% by volume, more preferably 2 to 3% by volume. The culture period can be usually 1 to 7 days, and the optimum period is 3 days.

The cells can be collected from the culture thus obtained, washed with water or an appropriate buffer, and used in the L-isoleucine or L-valine producing reaction. In the L-isoleucine or L-valine production reaction, these bacterial cells can be used as they are, or as a crushed bacterial cell product subjected to ultrasonic treatment or the like, or a crude enzyme or purified enzyme separated therefrom, or a suitable enzyme. It can be used after being immobilized on a carrier. In the present specification, the crushed product of the bacterial cells, the crude or purified enzyme, the immobilized product, and the like as described above are collectively referred to as "treated bacterial cell product".

According to the present invention, however, L-isoleucine or L-isoleucine is obtained by enzymatically reacting in the presence of the above-mentioned cultured bacterial cells or treated bacterial cell product in an aqueous reaction solution containing at least a carbon source and a nitrogen source. -L-characterized by producing valine
A method for producing isoleucine or L-valine is provided. The above-mentioned enzymatic reaction can be carried out usually in the range of about 20 to about 40 ° C, preferably about 25 to about 35 ° C.

Examples of the carbon source and nitrogen source that can be added to the aqueous reaction solution include those used in the above-mentioned ordinary nutrient medium. Further, to the aqueous reaction solution, an inorganic salt or the like that can be used in the above-mentioned ordinary nutrient medium can be added. In particular, when the host microorganism that can be transformed with the plasmid of the present invention is a biotin-requiring coryneform bacterium, it contains the cultured bacterial cell prepared as described above or its immobilized product, and at least a carbon source and a nitrogen source. However, it is preferable that L-isoleucine or L-valine is produced by enzymatic reaction in an aqueous reaction solution containing no biotin. In this case, the biotin-requiring coryneform bacterium does not grow in an aqueous reaction solution that does not substantially contain biotin, and the carbon source and the nitrogen source in the metabolic system possessed by the cell are enzymes accompanied by energy coupling. The reaction is allowed to proceed to produce L-isoleucine or L-valine.

According to the present invention, therefore, (1) in the presence of the above-mentioned cultured cells or an immobilized product thereof, at least ethanol and a butyric acid derivative are enzymatically reacted in an aqueous reaction solution containing no biotin to give L-. A method for producing L-isoleucine, which is characterized in that isoleucine is produced, (2) L-isoleucine is subjected to an enzymatic reaction in an aqueous reaction solution containing at least glucose in the presence of the above-mentioned cultured cells or its immobilized product. A method for producing L-valine is provided, which comprises producing valine.

The above-mentioned aqueous reaction solution according to the present invention may be water or a buffer solution such as phosphoric acid or Tris-hydrochloric acid which does not substantially contain biotin, but a biotin-free synthetic medium is preferably used. .. This synthetic medium includes an aqueous solution containing an inorganic nitrogen source and / or an inorganic substance having a known chemical structure that does not contain natural nutrients such as yeast extract, peptone, and coast liquor.
Examples of the inorganic nitrogen source of the synthetic medium that can be used in the present invention include ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium phosphate and the like, and examples of the inorganic substance include phosphoric acid-potassium hydrogen and phosphorus. Examples thereof include potassium dihydrogen acid, magnesium sulfate, manganese sulfate, iron sulfate and the like. These inorganic nitrogen sources and inorganic salts may be used alone or in admixture of two or more.

An example of the synthetic medium used in the method for producing L-isoleucine or L-valine according to the present invention is as follows: (NH ₄ ) ₂ SO ₄ 2 g / l; KH
₂ PO ₄ 0.5 g / l; K ₂ HPO ₄ 0.5 g / l; M
_{gSO 4 · 7H 2 O 0.5g /} l; FeSO 4 · 7H
_{2 O 20ppm; MnSO 4 · 4~6H} 2 O 20p
An aqueous solution having a pH of 7.6 containing pm.

The amount of the cultured bacterial cells or treated product of bacterial cells prepared as described above used in the method for producing L-isoleucine or L-valine of the present invention is not particularly limited, but the medium Generally 1 to 50 based on the capacity of
% (Wt / vol), preferably 2 to 20% (wt / v)
ol) can be used at a concentration within the range. The enzymatic reaction using the cultured cells or treated cells in the aqueous reaction solution having the composition described above is generally about 20 to about 50 ° C., preferably about 30 to about 40 ° C.
It can be carried out for 0 to about 72 hours.

In the method for producing L-isoleucine according to the present invention, L-isoleucine is produced by enzymatically reacting ethanol, a butyric acid derivative and a nitrogen source in the above-mentioned aqueous reaction solution containing no biotin. In producing L-isoleucine, the concentration of ethanol in the aqueous reaction solution is usually 0.5 to 40% by volume, preferably 1 to 20% by volume.
Can be within the range. Examples of the butyric acid derivative in the aqueous reaction solution include DL-α-aminobutyric acid, α-ketobutyric acid, and salts thereof. The concentration of the butyric acid derivative in the aqueous reaction solution is usually 0.1 to 20% (wt.
/ Vol) concentration range is suitable, but especially when α-ketobutyric acid or a salt thereof is used, the concentration in the reaction solution should not always exceed 0.3% (wt / vol). When added, the production of by-product norvaline is reduced and L-
The yield of isoleucine can also be improved. The above-mentioned addition of the reaction substrate may be carried out continuously or intermittently as long as the above-mentioned concentration is not exceeded. Examples of the salt of the butyric acid derivative that can be used in the reaction include alkali metal salts such as sodium and potassium; alkaline earth metal salts such as calcium; ammonium salt and the like, and among these, the sodium salt is preferable. ..

Further, in the method for producing L-valine according to the present invention, glucose and a nitrogen source are enzymatically reacted with each other in the above-mentioned aqueous reaction solution containing no biotin.
Valine is produced. The glucose concentration in the aqueous reaction solution during the production of L-valine can be usually within the range of 0.1 to 5.0% by weight. Glucose is preferably added to the aqueous reaction solution continuously or intermittently so as to maintain the concentration within the above range during the reaction.

Separation and purification of L-isoleucine or L-valine thus produced from the aqueous reaction solution can be carried out according to a commonly used method known per se.
For example, a method such as an ion exchange resin treatment method and a crystallization method can be appropriately combined and performed.

[0048]

The present invention has been described above, but the present invention will be described in more detail with reference to the following examples. Example 1 D containing a gene encoding acetohydroxy acid synthase from Brevibacterium flavum MJ-233
Cloning of NA fragment (A fragment)

(A) Brevibacterium flavum MJ
-233 Total DNA extraction Semi-synthetic medium A medium [Composition: Urea 2 g, (HN ₄ ) ₂ SO ₄
7 g, K ₂ HPO ₄ 0.5 g, KH ₂ PO ₄ 0.5 g,
MgSO ₄ 0.5 g, FeSO ₄ .7H ₂ O ₆ mg, M
nSO ₄ 4-6H ₂ O 6 mg, yeast extract 2.5 g,
Casamino acid 5g, biotin 200μg, thiamine hydrochloride 2
Brevibacterium flavum MJ-233 (FERM) in 00 μg, glucose 20 g, distilled water 11] 11
BP-1497) was cultured until the late logarithmic growth phase, and the bacterial cells were collected. The resulting cells were 10mM NaCl-20mM Tris buffer (pH 8.0) -1mM EDTA-2Na solution containing lysozyme at a concentration of 10mg / ml 15m
suspended in 1 l. Next, proteinase K was added to a final concentration of 10
The mixture was added to 0 μg / ml and kept at 37 ° C. for 1 hour. Furthermore, sodium dodecyl sulfate was added to a final concentration of 0.
It was added so as to be 5% and lysed by incubating at 50 ° C. for 6 hours. After adding an equal amount of phenol / chloroform solution to this lysate and gently shaking for 10 minutes at room temperature,
Centrifuge the whole volume (5,000 xg, 20 minutes, 10-1)
2 ° C), collect the supernatant fraction, and add 0.3% of sodium acetate.
After adding so as to have M, double volume of ethanol was slowly added. DN existing between water layer and ethanol layer
A was scattered with a glass rod, washed with 70% ethanol, and then air dried. 5 ml of 10 mM Tris buffer (pH 7.5) -1 mM EDTA / 2Na solution was added to the obtained DNA.
Was added and the mixture was allowed to stand at 4 ° C. overnight and used in the subsequent experiments.

(B) Creation of Recombinant Brevibacterium flavum MJ obtained in the above (A)
-90 μl of the total DNA solution of H-233 with EcoR
Using I 50 units, the reaction was carried out at 37 ° C. for 1 hour for complete decomposition. A cloning vector pHSG399 (commercially available from Takara Shuzo) was cleaved with the EcoRI-decomposed DNA after digestion with the restriction enzyme EcoRI and then dephosphorylated, and then mixed to obtain 50 mM Tris buffer (pH 7.6), 10 mM.
Dithiothreitol, 1 mM ATP, 10 mM Mg
Each component of Cl ₂ and T ₄ DNA ligase 1 unit was added (concentration of each component is the final concentration), reacted at 4 ° C. for 15 hours, and allowed to bind.

(C) Acetohydroxy acid synthase
Selection of Plasmid Containing Gene to be Included The selection of the above-mentioned gene was carried out by using the Escherichia coli mutant strain Escherichia coli MI262. Using the plasmid mixture obtained in (B) above, the calcium chloride method (Journa
l of Molecular Biology, 5
3 , 159, 1970) and transformed into Escherichia coli MI262 to obtain chloramphenicol 50 m.
selective medium containing 7 g of K ₂ HPO ₄ and KH ₂ PO ₄ 2
_{g, (NH 4) 2 SO} 4 1g, MgSO 4 · 7H 2 O
0.1 g, glucose 20 g, leucine 20 mg, thiamine 1 mg and agar 16 g were dissolved in distilled water 11].

The growth strain on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture solution, the plasmid was cleaved with a restriction enzyme and examined by agarose gel electrophoresis to find that the length of plasmid pHSG399 was long. In addition to the 2.2 kb DNA fragment, an inserted DNA fragment having a length of about 5.8 kb was observed. This plasmid is pHSG3
It was named 99-AHAS.

(D) Acetohydroxy acid synthase
Subfragment of DNA fragment (A fragment) containing the gene to be encoded
The plasmid pHSG399-AHA obtained in (C) above
In order to miniaturize the DNA insert contained in S to a necessary part, a DNA fragment containing a gene encoding the plasmid pUC119 (commercially available from Takara Shuzo) acetohydroxy acid synthase was subcloned as follows.

Plasmid pHSG3 obtained in the above (C)
99-AHAS digested with restriction enzymes EcoRI and SacI, and plasmid pUC119 digested with restriction enzymes EcoRI.
Those digested with RI and SacI were mixed and mixed in 50 mM Tris buffer (pH 7.6), 10 mM dithiothreitol, 1 mM ATP, 10 mM MgCl ₂ and T ₄ D.
Each component of NA ligase 1 unit was added (concentration of each component is the final concentration) and reacted at 12 ° C. for 15 hours to bind.

Using the obtained plasmid mixture, the calcium chloride method (Journal of Molecular) was used.
Biology, 53 , 159, 1970) and transformed into Escherichia coli MI262, and a selective medium containing 50 mg of ampicillin [7 g of K ₂ HPO ₄ , KH
_{2 PO 4 2g, (NH 4} ) 2 SO 4 1g, MgSO 4 · 7
H ₂ O 0.1g, glucose 20g, leucine 20m
g, thiamin 1 mg, and agar 16 g were dissolved in distilled water 11].

A strain grown on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture medium, the plasmid was cleaved with a restriction enzyme and examined by agarose gel electrophoresis to find that the length of plasmid pUC119 was long. In addition to the 3.2 kb DNA fragment, an inserted DNA fragment having a length of about 3.4 kb was observed. The number of restriction enzyme recognition sites and the size of the cleaved fragment of a DNA fragment of about 3.4 kb in length when cleaved with various restrictions were as shown in Table 1 above. A map of restriction enzyme cleavage points of this DNA fragment is shown in FIG.

The plasmid obtained above was cleaved with various restriction enzymes, and the size of the cleaved fragment was measured. The results are shown in Table 2 below.

[0058]

[Table 2] Table 2 Plasmid pUC119-AHAS restriction enzyme Number of recognition sites Size of cleavage fragment (kb) KpnI 2 4.85 1.75 NcoI 2 5.65 0.95 EcoRV 2 5.25 1.35 The above restriction The plasmid characterized by the enzyme is pU
It was named C119-AHAS.

Based on the above, a DN of about 3.4 kb containing a gene encoding acetohydroxy acid synthase
A fragment (EcoRI-SacI fragment) could be obtained.

Example 2 Determination of Nucleotide Sequence of Gene Encoding Acetohydroxy Acid Synthase D-containing a gene encoding acetohydroxy acid synthase obtained in item (D) of Example 1 and having a length of about 3.4 kb
Regarding the NA fragment, its nucleotide sequence is shown as plasmid pUC1.
18 and pUC119 dideoxy nucleotide enzymatic method
method) (Sahger, F. et al., Pro
c. Nat. Acad. Sci. USA 74 , 546
3, 1977) according to the strategy diagram shown in FIG.

Due to the presence of the open reading frame in the base sequence, the gene encoding acetohydroxy acid synthase encodes 594 amino acids having the base sequence shown in SEQ ID NO: 1 in the Sequence Listing, which is 178.
It was composed of 2 base pairs.

Example 3 Plasmid vector pC which replicates and is stable in coryneform bacteria
Construction of RY30 (A) Preparation of plasmid pBY503 Plasmid pBY503 was prepared from Brevibacterium statinis IFO12144 (FERM BP-251).
It is a plasmid having a molecular weight of about 10 megadalton isolated from 5) and was prepared as described in JP-A-1-95785.

Semi-synthetic medium A medium [urea 2 g, (NH ₄ ) ₂
SO ₄ 7 g, K ₂ HPO ₄ 0.5 g, KH ₂ PO ₄ 0.
_{5g, MgSO 4 0.5g, FeSO 4} · 7H 2 O 6
mg, MnSO ₄ .4-6H ₂ O 6 mg, yeast extract 2.5 g, casamino acid 5 g, biotin 200 μg, thiamine hydrochloride 200 μg, glucose 20 g and distilled water 1
1] In 11, Brevibacterium stationis IFO
12144 was cultured until the late logarithmic growth phase and the bacterial cells were collected. The obtained bacterial cells were suspended in 20 ml of a buffer solution containing lysozyme at a concentration of 10 mg / ml [25 mM tris (hydroxymethyl) aminomethane, 10 mM EDTA, 50 mM glucose] and reacted at 37 ° C. for 1 hour. Alkali-SDS solution [0.2N NaOH, 1%
(W / V) SDS] 40 ml was added, mixed gently and allowed to stand at room temperature for 15 minutes. Next, a potassium acetate solution [60 ml of a 5M potassium acetate solution, 1 ml of acetic acid was added to the reaction solution.
30 ml of a mixed solution of 1.5 ml and 28.5 ml of distilled water] was added, mixed well, and allowed to stand in ice water for 15 minutes.

The total amount of the lysate was transferred to a centrifuge tube and centrifuged at 4 ° C. for 10 minutes at 15,000 × g to obtain a supernatant. To this, an equal amount of phenol-chloroform solution (phenol: chloroform = 1: 1 mixture) was added and suspended, then, transferred to a centrifuge tube, and 15,000 × g at room temperature for 5 minutes.
Was centrifuged and the aqueous layer was recovered. To the aqueous layer was added twice the amount of ethanol, and the mixture was allowed to stand at -20 ° C for 1 hour, then at 4 ° C for 10
The precipitate was recovered by centrifugation at 15,000 × g for 1 minute.

After drying the precipitate under reduced pressure, TE buffer [Tris 1
0 mM, EDTA 1 mM; adjusted to pH 8.0 with HCl] dissolved in 2 ml. Cesium chloride solution [5]
170 g of cesium chloride in 100 ml of double concentration TE buffer
15 ml and 10 mg / ml ethidium bromide solution 1 ml were added to give a density of 1.392 g /
adjusted to ml. This solution was heated at 12 ° C for 42 hours, 11
Centrifugation at 6,000 xg was performed.

The plasmid pBY503 is found as a lower band in the centrifuge tube by UV irradiation. By pulling this band from the side of the centrifuge tube with a syringe,
A fraction containing the plasmid pBY503 was obtained. Next, this fraction was treated with an equal amount of isoamyl alcohol four times to extract and remove ethidium bromide, and then dialyzed against TE buffer. A 3 M sodium acetate solution was added to the final concentration of 30 mM to the dialysate containing the plasmid pBY503 thus obtained, and then double the amount of ethanol was added, and the mixture was allowed to stand at -20 ° C for 1 hour. This solution 15,
The DNA was precipitated by centrifugation at 000 × g to obtain 50 μg of plasmid pBY503.

(B) Construction of plasmid vector pCRY30
0.5 μg of the prepared plasmid pHSG298 (Takara Shuzo) was reacted with the restriction enzyme SalI (5 units) at 37 ° C. for 1 hour to completely decompose the plasmid DNA. 2 μg of the plasmid pBY503 prepared in the above (A) was reacted with a restriction enzyme XhoI (1 unit) at 37 ° C. for 30 minutes,
The plasmid DNA was partially digested.

Both plasmid DNA digests were mixed,
After heat treatment at 65 ° C. for 10 minutes to inactivate the restriction enzyme, the final concentration of each component in the inactivating solution was 5
0 mM Tris buffer pH 7.6, 10 mM MgC
Each component was fortified so as to have 1 ₂ , 10 mM dithiothreitol, 1 mM ATP and 1 unit of T ₄ DNA ligase, and incubated at 16 ° C. for 15 hours. This solution was used to transform Escherichia coli JM109 competent cells (Takara Shuzo).

Transformed strain is 30 μg / ml (final concentration)
Kanamycin, 100 μg / ml (final concentration) IP
TG (isopropyl-β-D-thiogalactopyranoside) 100 μg / ml (final concentration) of X-gal (5-
L medium containing bromo-4-chloro-3-indolyl-β-D-galactopyranoside) (tryptone 10 g, yeast extract 5 g, NaCl 5 g and distilled water 11, pH 7.
After culturing in 2) at 37 ° C. for 24 hours, it was obtained as a growing strain. Of these growing strains, those that grew in white colonies were selected, and the plasmids were used for the alkaline-SDS method [T. Maniatis, E .; F. Fritsch,
J. Sambrook, “Molecular clo
Ning "(1982) p90-91].

As a result, S of plasmid pHSG298
About 4.0 k derived from the plasmid pBY503 at the aI1 site
Plasmid pHSG298-or in which the fragment of b was inserted
i was obtained. Then, using the same method, the plasmid pBY503DNA obtained in the above (A) was digested with the restriction enzyme Kp.
About 2.1 kb obtained by processing with nI and EcoRI
DNA fragment of the above plasmid pHSG298-ori
Was cloned into the KpnI and EcoRI sites to prepare plasmid vector pCRY30.

Example 4 Construction of plasmid pCRY30-AHAS and introduction into coryneform bacterium Plasmid pHSG39 obtained in section (C) of Example 1
5 μg of 9-AHAS was added with restriction enzymes EcoRI and SacI.
5units of each were reacted at 37 ° C. for 1 hour to decompose, blunt-ended and mixed with 1 μl of EcoRI linker (commercially available from Takara Shuzo), 50 mM Tris buffer (pH 7.6), 10 mM dithiothreitol, 1 mM
Each component of ATP, 10 mM MgCl ₂ and T ₄ DNA ligase 1 unit was added (concentration of each component is the final concentration), and reacted at 12 ° C. for 15 hours to bind.

This DNA was digested with the restriction enzyme EcoRI 3un.
that was decomposed by reacting at 37 ° C for 1 hour using its,
The plasmid pCRY30 obtained in (B) of Example 3
Using 1 μg of restriction enzyme EcoRI 1 unit, 3
The reaction mixture was reacted at 7 ° C for 1 hour and decomposed, and then mixed to 50 mM.
Tris buffer (pH 7.6), 10 mM dithiothreitol, 1 mM ATP, 10 mM MgCl ₂ and T
₄ Each component of 1 unit of DNA ligase was added (the concentration of each component is the final concentration), and the mixture was reacted at 12 ° C. for 15 hours for binding. This plasmid was used to transform the Escherichia coli MI262 strain according to the method described above, and a selective medium [K ₂ HPO containing 50 μg / ml of kanamycin was used.
_{4 7g, KH 2 PO 4 2g} , (NH 4) 2 SO 4 1g, M
gSO ₄ .7H ₂ O 0.1 g, glucose 20 g, leucine 20 mg, thiamine 1 mg and agar 16 g were dissolved in distilled water 11].

The strain grown on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture solution, the plasmid was cleaved with a restriction enzyme and examined by agarose gel electrophoresis to find that the length of plasmid pCRY30 was long. In addition to the 8.6 kb DNA fragment, an inserted DNA fragment with a size of 3.4 kb was observed. The plasmid DNA prepared as described above was transformed into a coryneform bacterium.

The transformation was carried out as follows using the electric pulse method. Brevibacterium flavum MJ-23
3 (FERM BP-1497) plasmid pBY50
The 2 removed strain was cultivated in 100 ml of the above-mentioned medium A until the initial logarithmic growth, penicillin G was added so as to be 1 unit / ml, shake culturing was continued for 2 hours, and the microbial cells were collected by centrifugation to collect the microbial cells. 20 ml pulse solution (272 mM
Sucrose, 7 mM KH ₂ PO ₄ , 1 mM M
It was washed with gCl ₂ ; pH 7.4). The cells were further collected by centrifugation, suspended in 5 ml of the pulse solution,
75 ml of cells and 50 μl of the plasmid DNA solution obtained above were mixed and left standing in water for 20 minutes. Using Gene Pulser (manufactured by Bio-Rad), it was set to 2500 V and 25 μFD, and after applying a pulse, it was put in ice for 20 minutes.
Let stand for a minute. Transfer the whole amount to 3 ml of the above A medium and
After 1 hour of culture, the cells were inoculated into the A agar medium containing 15 μg / ml (final concentration) of kanamycin and cultured at 30 ° C. for 2 to 3 days. From the emerged kanamycin resistant strain, a plasmid was obtained using the method described in the above Example 3 (A). This plasmid was cleaved with various restriction enzymes and the size of the cleaved fragment was measured. The results are shown in Table 3 below.

[0075]

Table 3 Plasmid pCRY30-AHAS restriction enzyme number of recognition sites Size of cleavage fragment (kb) BamHI 1 12.0 EcoRI 2 8.6 3.4 pCR is a plasmid characterized by the above restriction enzymes
It was named Y30-AHAS. This plasmid pCRY
A restriction map of 30-AHAS is shown in FIG.

The plasmid pCRY30-AHAS
Brevibacterium flavum M transformed by
J233-AHAS is 1-3 1-3 East, Tsukuba City, Ibaraki Prefecture.
Issued by the Institute of Microbial Technology, Institute of Industrial Technology, 1994
On October 10th: Microtechnology Research Institute, Microbiology No. 12994 (FERM
(P-12994).

Example 5 Stability of plasmid pCRY30-AHAS 100 ml of the above A medium was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes, and the transformant Brevibacterium obtained in Example 4 was obtained. Umm flabham M
After inoculating J233-AHAS and shaking culture at 30 ° C. for 24 hours, 100 ml of A medium prepared in the same manner
Was dispensed into a 500 ml Erlenmeyer flask and heated at 120 ° C for 15
The cells were sterilized for 1 minute, and the cells were subcultured at a rate of 50 cells per ml, and shaking culture was performed at 30 ° C. for 24 hours. Next, the cells were collected by centrifugation and washed, and then kanamycin was added at a rate of 15 μg / ml.
A fixed amount was smeared on a plate medium prepared by using the medium and the A medium without any addition, and the grown colonies were counted after culturing at 30 ° C. for 1 day.

As a result, it was confirmed that the number of colonies grown on the kanamycin-added medium and the non-added medium was the same, and that all the A-medium-grown colonies grew on the kanamycin-added medium, that is, the high stability of the plasmid.

Example 6 Production of L-isoleucine Medium (0.4% urea, 1.4% ammonium sulfate, KH
₂ PO ₄ 0.05%, K ₂ HPO ₄ 0.05%, MgS
_{O 4 · 7H 2 O 0.05%} , CaCl 2 · 2H 2 O
_{2ppm, FeSO 4 · 7H 2 O} 2ppm, MnSO
_{4 · 4~6H 2 O 2ppm, ZnSO} 4 · 7H 2 O
2 ppm, NaCl 2 ppm, biotin 200 μg /
1, thiamine / HCl 100 μg / 1, casamino acid 0.1%, yeast extract 0.1%) 100 ml 500 m
Dispense into a 1-liter Erlenmeyer flask and sterilize (pH 7.0 after sterilization), and then Brevibacterium flavum (Brevibac
terium flavum) MJ233-AHAS was inoculated, 2 ml of ethanol was added aseptically, and shake culture was performed at 30 ° C. for 2 days.

Next, the main culture medium (ammonium sulfate 2.
3%, KH₂PO_Four0.05%, K ₂HPO_Four0.05
%, MgSO_Four・ 7H₂O 0.05%, FeSO_Four・
7H ₂O 20ppm, MnSO_Four・ 4-6H₂O 2
0 ppm, biotin 200 μg / l, thiamine / HCl
 100 μg / l, casamino acid 0.3%, yeast extract
(1000% of 0.3%) was charged into a 21-volume aeration stirring tank.
After sterilization (120 ° C, 20 minutes), ethanol 20m
1 and 20 ml of the above preculture were added, and the rotation speed was 100
0 rpm, aeration rate 1 vvm, temperature 33 ° C, pH 7.6
For 48 hours.

It should be noted that ethanol has a concentration of medium in the culture.
Intermittently every 1 to 2 hours so as not to exceed 2% by volume
Was added. After culturing, centrifuge from 500 ml of culture
After collecting the cells in, the cells washed twice with desalted distilled water are used as the reaction solution.
[(NH_Four)₂SO_Four2 g / l; KH₂PO_Four0.5 g /
l; KH₂PO_Four0.5 g / l; MgSO_Four・ 7H₂O
 0.5 g / l; FeSO _Four・ 7H₂O 20ppm;
MnSO_Four・ 4-6H₂O 20ppm; thiamine hydrochloric acid
Salt 100 μg / l; α-ketobutyric acid 1.0%; pH 7.
6] was suspended in 1000 ml, and the suspension was aerated with 2 l of aeration.
Charge to a stirring tank, 20 ml of ethanol and sodium pyruvate
Add 10 g / l of lithium and rotate at 300 rpm.
Volume of 0.1 vvm, temperature of 33 ℃, pH 7.6 at 15:00
Reaction was carried out.

After completion of the reaction, centrifugation (4000 rpm,
L-Isoleucine was quantified in the supernatant liquid that had been sterilized for 15 minutes at 4 ° C. As a result, the amount of L-isoleucine produced in the supernatant was 2.3 g / l. After completion of this reaction, 500 ml of the culture solution was added to a strongly acidic cation exchange resin (H ⁺ type).
L-isoleucine was adsorbed through the column of No. 3, washed with water and eluted with 0.5N ammonia water, the L-isoleucine fraction was concentrated, and crystals of L-isoleucine were broken out with cold ethanol. As a result, 730 mg of L-isoleucine crystals were obtained.

As a comparative example, under the same conditions, Brevibacterium flavum MJ-233 (FERM
BP-1497) was cultured, reacted under the same conditions, and L-isoleucine in the supernatant was quantified. as a result,
The amount of L-isoleucine produced in the supernatant was 1.2 g / l.

Example 7 Production of L-valine Medium (0.4% urea, 1.4% ammonium sulfate, KH ₂ PO ₄ 0.
_{05%, K 2 HPO 4 0.05} %, MgSO 4 · 7H 2
O 0.05%, CaCl ₂ · 2H ₂ O 2ppm, F
_{eSO 4 · 7H 2 O 2ppm,} MnSO 4 · 4~6H
_{2 O 2ppm, ZnSO 4 · 7H} 2 O 2ppm, N
aCl 2 ppm, biotin 200 μg / l, thiamine hydrochloride 100 μg / l, casamino acid 0.1%, yeast extract 0.1%) 100 ml was dispensed into a 500 ml Erlenmeyer flask, sterilized and adjusted to pH 7, and then Brevibacterium. Um flavum MJ-233-AHAS was inoculated and glucose was added aseptically to a concentration of 5 g / l, and 30
Shaking culture was carried out at 0 ° C. for 2 days.

Next, the main culture medium (glucose 5%, ammonium sulfate 2.3%, KH ₂ PO ₄ 0.05%, K ₂ HPO ₄₀ .
_{05%, MgSO 4 · 7H 2} O 0.05%, FeSO
_{4 · 7H 2 O 20ppm, MnSO} 4 · 4~6H 2 O
20 ppm, biotin 200 μg / l, thiamine hydrochloride 100 μg / l, casamino acid 0.3%, yeast extract 0.3%) 1000 ml was charged into a 2 l aeration stirring tank,
20m of the above preculture after sterilization (120 ° C, 20 minutes)
1000 rpm, aeration rate 1 vv
Culture was carried out at a temperature of 33 ° C. and a pH of 7.6 for 24 hours.

After the completion of the culture, the cells were collected from 100 ml of the culture by centrifugation and washed twice with demineralized distilled water to prepare a reaction solution (glucose 100 g / l, KH ₂ PO ₄ 0.05 g).
_{/ L, K 2 HPO 4 0.05g} / l, MgSO 4 · 7H
_{2 O 0.5g / l, FeSO 4} · 7H 2 O 20pp
m, MnSO ₄ .4 to 6H ₂ O (20 ppm), thiamine hydrochloride (100 μg / l, pH 8.0) and suspended in 50 ml to carry out the reaction.

Further, in order to adjust the pH, dry heat sterilization (150
Calcium carbonate heated at 5 ° C. for 5 hours) was added at a concentration of 50 g / l. The reaction was carried out by using a 500 ml Erlenmeyer flask and shaking at 33 ° C. and a rotation speed of 220 rpm for 40 hours. After completion of the reaction, centrifugation (4000 rpm,
L-valine was quantified in the supernatant after sterilization for 15 minutes at 4 ° C.

As a result, the amount of L-valine produced in the supernatant was 2.0 g / l. As a comparative example, Brevibacterium flavum MJ-233 under the same conditions.
(FERM BP-1497) was cultured, reacted under the same conditions, and L-valine in the supernatant was quantified. As a result, the amount of L-valine produced in the supernatant was 0.8 g / l.

[0089]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1785 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Origin organism name: Brevibacterium flavum Strain name: MJ233 Sequence features Symbol: peptide Location: 1-1785 Method for determining characteristics: P sequence ATG ACA GGT GCA CAG GCA ATT GTT CGA TCG CTC GAG GAG CTT AAC GCC 48 Met Thr Gly Ala Gln Ala Ile Val Arg Ser Leu Glu Glu Leu Asn Ala 1 5 10 15 GAC ATC GTG TTC GGT ATT CCT GGT GGT GCG GTG CTA CCG GTG TAT GAC 96 Asp Ile Val Phe Gly Ile Pro Gly Gly Ala Val Leu Pro Val Tyr Asp 20 25 30 CCG CTC TAT TCC TCC ACA AAG GTG CGC CAC GTC TTG GTG CGC CAC GAG 144 Pro Leu Tyr Ser Ser Thr Lys Val Arg His Val Leu Val Arg His Glu 35 40 45 CAG GGC GCA GGC CAC GCA GCA ACC GGC TAC GCG CAG GTT ACT GGA CGC 192 Gln Gly Ala Gly His Ala Ala Thr Gly Tyr Ala Gln Val Thr Gly Arg 50 55 60 GTT GGC GTC TGC ATT GCA ACC TCT GGC CCA GGA GCA ACC AAC TTG GTT 240 Val Gly Val Cys Ile Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu Val 65 70 75 80 ACC CCA ATC GCT GAT GCA AAC TTG GAC TCC GTT CCC ATG GTT GCC ATC 288 Thr Pro Ile Ala Asp Ala Asn Leu Asp Ser Val Pro Met Val Ala Ile 85 90 95 ACC GGC CAG GTC GGA AGT GGC CTG CTG GGT ACC GAC GCT TTC CAG GAA 336 Thr Gly Gln Val Gly Ser Gly Leu Leu Gly Thr Asp Ala Phe Gln Glu 100 105 110 GCC GAT ATC CGC GGC ATC ACC ATG CCA GTG ACC AAG CAC AAC TTC ATG 384 Ala Asp Ile Arg Gly Ile Thr Met Pro Val Thr Lys His Asn Phe Met 115 120 125 GTC ACC GAC CCC AAC GAC ATT CCA CAG GCA TTG GCT GAG GCA TTC CAC 432 Val Thr Asp Pro Asn Asp Ile Pro Gln Ala Leu Ala Glu Ala Phe His 130 135 140 CTC GCG ATT ACT GGT CGC CCT GGC CCT GTT CTG GTG GAT ATT CCT AAG 480 Leu Ala Ile Thr Gly Arg Pro Gly Pro Val Leu Val Asp Ile Pro Lys 145 150 155 160 GAT GTC CAG AAC GCT GAA TTG GAT TTC GTC TGG CCA CCA AAG ATC GAC 528 Asp Val Gln Asn Ala Glu Leu Asp Phe Val Trp Pro Pro Lys Ile Asp 165 170 175 CTG CCA GGC TAC CGC CCA GTT TCA ACA CCA CAT GCT CGC CAG ATC GAG 576 Leu Pro Gly Tyr Arg Pro Val Ser Thr Pro His Ala Arg Gln Ile Glu 180 185 190 CAG GCA GTC AAG CTG ATC GGT GAG GCC AAG AAG CCC GTC CTT TAC GTT 624 Gln Ala Val Lys Leu Ile Gly Glu Ala Lys Lys Pro Val Leu Tyr Val 195 200 205 GGA GGC GGC GTT ATC AAG GCT GAC GCA CAC GAA GAG CTT CGT GCG TTC 672 Gly Gly Gly Val Ile Lys Ala Asp Ala His Glu Glu Leu Arg Ala Phe 210 215 220 GCT GAG TAC ACC GGC ATC CCA GTT GTC ACC ACC TTG ATG GCT TTG GGT 720 Ala Glu Tyr Thr Gly Ile Pro Val Val Thr Thr Leu Met Ala Leu Gly 225 230 235 240 ACT TTC CCA GAG TCT CAC GAG CTG CAC ATG GGT ATG CCA GGC ATG CAT 768 Thr Phe Pro Glu Ser His Glu Leu His Met Gly Met Pro Gly Met His 245 250 255 GGC ACT GTG TCC GCT GTT GGT GCA CTG CAG CGC AGC GAC CTG CTG ATT 816 Gly Thr Val Ser Ala Val Gly Ala Leu Gln Arg Ser Asp Leu Leu Ile 260 265 270 GCT ATC GGC TCC CGC TTT GAT GAC CGC GTC ACC GGT GAC GTT GAC ACC 864 Ala Ile Gly Ser Arg Phe Asp Asp Arg Val Thr Gly Asp Val Asp Thr 275 280 285 TTC GCG CCT GAC GCC AAG ATC ATT CAC GCC GAT ATT GAT CCT GCC GAA 912 Phe Ala Pro Asp Ala Lys Ile Ile His Ala Asp Ile Asp Pro Ala Glu 290 295 300 ATC GGA AAG ATC AAG CAG GTT GAG GTT CCA ATC GTG GGC GAT GCC CGC 960 Ile Gly Lys Ile Lys Gln Val Glu Val Pro Ile Val Gly Asp Ala Arg 305 310 315 320 GAA GTT CTT GCT CGT CTG CTG GAA ACC ACC AAG GCA AGC AAG GCA GAG 1008 Glu Val Leu Ala Arg Leu Leu Glu Thr Thr Lys Ala Ser Lys Ala Glu 325 330 335 ACC GAG GAC ATC TCC GAG TGG GTT GAC TAC CTC AAG GGC CTC AAG GCA 1056 Thr Glu Asp Ile Ser Glu Trp Val Asp Tyr Leu Lys Gly Leu Lys Ala 340 345 350 CGT TTC CCA CGT GGC TAC GAC GAG CAG CCA GGC GAT CTG CTG GCA CCA 1104 Arg Phe Pro Arg Gly Tyr Asp Glu Gln Pro Gly Asp Leu Leu Ala Pro 355 360 365 CAG TTT GTC ATT GAA ACC CTG TCC AAG GAA GTT GGC CCC GAC GCA ATT 1152 Gln Phe Val Ile Glu Thr Leu Ser Lys Glu Val Gly Pro Asp Ala Ile 370 375 380 TAC TGC GCC GGC GTC GGA CAG CAC CAA ATG TGG GCA GCT CAG TTC GTT 1200 Tyr Cys Ala Gly Val Gly Gln His Gln Met Trp Ala Ala Gln Phe Val 385 390 395 400 GAC TTT GAA AAG CCA CGC ACC TGG CTC AAC TCC GGT GGA CTG GGC ACC 12 48 Asp Phe Glu Lys Pro Arg Thr Trp Leu Asn Ser Gly Gly Leu Gly Thr 405 410 415 ATG GGC TAC GCA GTT CCT GCG GCC CTT GGA GCA AAG GCT GGC GCA CCT 1296 Met Gly Tyr Ala Val Pro Ala Ala Leu Gly Ala Lys Ala Gly Ala Pro 420 425 430 GAC AAG GAA GTC TGG GCT ATC GAC GGC GAC GGC TGT TTC CAG ATG ACC 1344 Asp Lys Glu Val Trp Ala Ile Asp Gly Asp Gly Cys Phe Gln Met Thr 435 440 445 AAC CAG GAA CTC ACC ACC GCC GCA GTT GAA GGT TTC CCC ATT AAG ATC 1392 Asn Gln Glu Leu Thr Thr Ala Ala Val Glu Gly Phe Pro Ile Lys Ile 450 455 460 GCA CTA ATC AAC AAC GGA AAC CTG GGC ATG GTT CGC CAA TGG CAG ACC 1440 Ala Leu Ile Asn Asn Asn Gly Asn Leu Gly Met Val Arg Gln Trp Gln Thr 465 470 475 480 CTA TTC TAT GAA GGA CGG TAC TCA AAT ACT AAA CTT CGT AAC CAG GGC 1488 Leu Phe Tyr Glu Gly Arg Tyr Ser Asn Thr Lys Leu Arg Asn Gln Gly 485 490 495 GAG TAC ATG CCC GAC TTT GTT GCC CTT TCT GAG GGA CTT GGC TGT GTT 1536 Glu Tyr Met Pro Asp Phe Val Ala Leu Ser Glu Gly Leu Gly Cys Val 500 505 510 GCC ATC CGC GTC ACC AAA GCG GAG GAA GTA CTG CCA GCC ATC CAA AAG 1584 Ala Ile Arg Val Thr Lys Ala Glu Glu Val Leu Pro Ala Ile Gln Lys 515 520 525 GCT CGA GAA ATC AAC GAC CGC CCA GTA GTC ATC GAC TTC ATC GTC GGT 1632 Ala Arg Glu Ile Asn Asp Arg Pro Val Val Ile Asp Phe Ile Val Gly 530 535 540 GAA GAC GCA CAG GTA TGG CCA ATG GTG TCT GCT GGA TCA TCC AAC TCC 1680 Glu Asp Ala Gln Val Trp Pro Met Val Ser Ala Gly Ser Ser Asn Ser 545 550 555 560 GAT ATC CAG TAC GCA CTC GGA TTG CGC CCA TTC TTT GAC GGC GAC GAA 1728 Asp Ile Gln Tyr Ala Leu Gly Leu Arg Pro Phe Phe Asp Gly Asp Glu 565 570 575 TCA GCT GCA GAA GAC CCT GCA GAC ATT CAT GCT TCC GTT GAT TCG CG Ser Ala Ala Glu Asp Pro Ala Asp Ile His Ala Ser Val Asp Ser Thr 580 585 590 GAG GCA TAA 1785 Glu Ala ***

[Brief description of drawings]

FIG. 1 is a map of restriction enzyme cleavage points of a DNA fragment having a size of about 3.4 kb containing a gene encoding acetohydroxy acid synthase of the present invention.

FIG. 2 is a strategy diagram for determining the nucleotide sequence of a DNA fragment of the present invention having a size of about 3.4 kb.

FIG. 3 is a restriction enzyme map of the plasmid pCRY30-AHAS of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication (C12N 15/60 C12R 1:13) (C12N 1/21 C12R 1:13) (C12P 13/06 C12R 1:13) (C12P 13/08 C12R 1:38)

Claims (7)

[Claims] 1. A gene DNA encoding an acetohydroxy acid synthase derived from a coryneform bacterium. 2. The coryneform bacterium is Brevibacterium flavu.
m) The gene DNA according to claim 1, which is MJ-233. Wherein following DNA base sequence ATGACAGGTG CACAGGCAAT TGTTCGATCG CTCGAGGAGC TTAACGCCGA CATCGTGTTC 60 GGTATTCCTG GTGGTGCGGT GCTACCGGTG TATGACCCGC TCTATTCCTC CACAAAGGTG 120 CGCCACGTCT TGGTGCGCCA CGAGCAGGGC GCAGGCCACG CAGCAACCGG CTACGCGCAG 180 GTTACTGGAC GCGTTGGCGT CTGCATTGCA ACCTCTGGCC CAGGAGCAAC CAACTTGGTT 240 ACCCCAATCG CTGATGCAAA CTTGGACTCC GTTCCCATGG TTGCCATCAC CGGCCAGGTC 300 GGAAGTGGCC TGCTGGGTAC CGACGCTTTC CAGGAAGCCG ATATCCGCGG CATCACCATG 360 CCAGTGACCA AGCACAACTT CATGGTCACC GACCCCAACG ACATTCCACA GGCATTGGCT 420 GAGGCATTCC ACCTCGCGAT TACTGGTCGC CCTGGCCCTG TTCTGGTGGA TATTCCTAAG 480 GATGTCCAGA ACGCTGAATT GGATTTCGTC TGGCCACCAA AGATCGACCT GCCAGGCTAC 540 CGCCCAGTTT CAACACCACA TGCTCGCCAG ATCGAGCAGG CAGTCAAGCT GATCGGTGAG 600 GCCAAGAAGC CCGTCCTTTA CGTTGGAGGC GGCGTTATCA AGGCTGACGC ACACGAAGAG 660 CTTCGTGCGT TCGCTGAGTA CACCGGCATC CCAGTTGTCA CCACCTTGAT GGCTTTGGGT 720 ACTTTCCCAG AGTCTCACGA GCTGCACATG GGTATGCCAG GCATGCATGG CACTGTGTCC 780 GCTGTTGGTG CACTGCAGCG CAGCGACCTG CTGATTGCTA TCGGCTCCCG CTTTGATGAC 840 CGCGTCACCG GTGACGTTGA CACCTTCGCG CCTGACGCCA AGATCATTCA CGCCGATATT 900 GATCCTGCCG AAATCGGAAA GATCAAGCAG GTTGAGGTTC CAATCGTGGG CGATGCCCGC 960 GAAGTTCTTG CTCGTCTGCT GGAAACCACC AAGGCAAGCA AGGCAGAGAC CGAGGACATC 1020 TCCGAGTGGG TTGACTACCT CAAGGGCCTC AAGGCACGTT TCCCACGTGG CTACGACGAG 1080 CAGCCAGGCG ATCTGCTGGC ACCACAGTTT GTCATTGAAA CCCTGTCCAA GGAAGTTGGC 1140 CCCGACGCAA TTTACTGCGC CGGCGTCGGA CAGCACCAAA TGTGGGCAGC TCAGTTCGTT 1200 GACTTTGAAA AGCCACGCAC CTGGCTCAAC TCCGGTGGAC TGGGCACCAT GGGCTACGCA 1260 GTTCCTGCGG CCCTTGGAGC AAAGGCTGGC GCACCTGACA AGGAAGTCTG GGCTATCGAC 1320 GGCGACGGCT GTTTCCAGAT GACCAACCAG GAACTCACCA CCGCCGCAGT TGAAGGTTTC 1380 CCCATTAAGA TCGCACTAAT CAACAACGGA AACCTGGGCA TGGTTCGCCA ATGGCAGACC 1440 CTATTCTATG AAGGACGGTA CTCAAATACT AAACTTCGTA ACCAGGGCGA GTACATGCCC 1500 GACTTTGTTG CCCTTTCTGA GGGACTTGGC TGTGTTGCCA TCCGCGTCAC CAAAGCGGAG 1560 GAAGTACTGC CAGCCATCCA AAAGGCTCGA GAAATCAACG ACCGCCCAGT AGTCATCGAC 1620 TTCATCGTCG GTGAAGACGC ACAGGTATGG CCAATGGTGT CTGCTGGAT C ATCCAACTCC 1680 GATATCCAGT ACGCACTCGG ATTGCGCCCA TTCTTTGACG GCGACGAATC AGCTGCAGAA 1740 GACCCTGCAG ACATTCATGC TTCCGTTGAT TCGACCGAGG CATAA 1785 is a gene DNA encoding acetohydroxy acid synthase. 4. The following amino acid sequence Met Thr Gly Ala Gln Ala Ile Val Arg Ser Leu Glu Glu Leu Asn Ala 1 5 10 15 Asp Ile Val Phe Gly Ile Pro Gly Gly Ala Val Leu Pro Val Tyr Asp 20 25 30 Pro Leu Tyr Ser Ser Thr Lys Val Arg His Val Leu Val Arg His Glu 35 40 45 Gln Gly Ala Gly His Ala Ala Thr Gly Tyr Ala Gln Val Thr Gly Arg 50 55 60 Val Gly Val Cys Ile Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu Val 65 70 75 80 Thr Pro Ile Ala Asp Ala Asn Leu Asp Ser Val Pro Met Val Ala Ile 85 90 95 Thr Gly Gln Val Gly Ser Gly Leu Leu Gly Thr Asp Ala Phe Gln Glu 100 105 110 Ala Asp Ile Arg Gly Ile Thr Met Pro Val Thr Lys His Asn Phe Met 115 120 125 Val Thr Asp Pro Asn Asp Ile Pro Gln Ala Leu Ala Glu Ala Phe His 130 135 140 Leu Ala Ile Thr Gly Arg Pro Gly Pro Val Leu Val Asp Ile Pro Lys 145 150 155 160 Asp Val Gln Asn Ala Glu Leu Asp Phe Val Trp Pro Pro Lys Ile Asp 165 170 175 Leu Pro Gly Tyr Arg Pro Val Ser Thr Pro His Ala Arg Gln Ile Glu 180 185 190 Gln Ala Val Lys Leu Ile Gly Glu Ala Lys Lys Pro Val Le u Tyr Val 195 200 205 Gly Gly Gly Val Ile Lys Ala Asp Ala His Glu Glu Leu Arg Ala Phe 210 215 220 Ala Glu Tyr Thr Gly Ile Pro Val Val Thr Thr Leu Met Ala Leu Gly 225 230 235 240 Thr Phe Pro Glu Ser His Glu Leu His Met Gly Met Pro Gly Met His 245 250 255 Gly Thr Val Ser Ala Val Gly Ala Leu Gln Arg Ser Asp Leu Leu Ile 260 265 270 Ala Ile Gly Ser Arg Phe Asp Asp Arg Val Thr Gly Asp Val Asp Thr 275 280 285 Phe Ala Pro Asp Ala Lys Ile Ile His Ala Asp Ile Asp Pro Ala Glu 290 295 300 Ile Gly Lys Ile Lys Gln Val Glu Val Pro Ile Val Gly Asp Ala Arg 305 310 315 320 Glu Val Leu Ala Arg Leu Leu Glu Thr Thr Lys Ala Ser Lys Ala Glu 325 330 335 Thr Glu Asp Ile Ser Glu Trp Val Asp Tyr Leu Lys Gly Leu Lys Ala 340 345 350 Arg Phe Pro Arg Gly Tyr Asp Glu Gln Pro Gly Asp Leu Leu Ala Pro 355 360 365 Gln Phe Val Ile Glu Thr Leu Ser Lys Glu Val Gly Pro Asp Ala Ile 370 375 380 Tyr Cys Ala Gly Val Gly Gln His Gln Met Trp Ala Ala Gln Phe Val 385 390 395 400 Asp Phe Glu Lys Pro Arg Thr Trp Leu Asn Ser Gly Gly Le u Gly Thr 405 410 415 Met Gly Tyr Ala Val Pro Ala Ala Leu Gly Ala Lys Ala Gly Ala Pro 420 425 430 Asp Lys Glu Val Trp Ala Ile Asp Gly Asp Gly Cys Phe Gln Met Thr 435 440 445 Asn Gln Glu Leu Thr Thr Ala Ala Val Glu Gly Phe Pro Ile Lys Ile 450 455 460 Ala Leu Ile Asn Asn Gly Asn Leu Gly Met Val Arg Gln Trp Gln Thr 465 470 475 480 Leu Phe Tyr Glu Gly Arg Tyr Ser Asn Thr Lys Leu Arg Asn Gln Gly 485 490 495 Glu Tyr Met Pro Asp Phe Val Ala Leu Ser Glu Gly Leu Gly Cys Val 500 505 510 Ala Ile Arg Val Thr Lys Ala Glu Glu Val Leu Pro Ala Ile Gln Lys 515 520 525 Ala Arg Glu Ile Asn Asp Arg Pro Val Val Ile Asp Phe Ile Val Gly 530 535 540 Glu Asp Ala Gln Val Trp Pro Met Val Ser Ala Gly Ser Ser Asn Ser 545 550 555 560 Asp Ile Gln Tyr Ala Leu Gly Leu Arg Pro Phe Phe Asp Gly Asp Glu 565 570 575 Ser Ala Ala Glu Asp Pro Ala Asp Ile His Ala Ser Val Asp Ser Thr 580 585 590 Glu Ala is the gene DNA encoding the acetohydroxy acid synthase. 5. A recombinant plasmid into which the gene DNA according to any one of claims 1 to 4 is introduced. 6. A recombinant plasmid having the gene DNA according to any one of claims 1 to 4 and a DNA containing a gene that controls a replication / proliferation function in a coryneform bacterium. 7. A coryneform bacterium transformed with the recombinant plasmid according to claim 6.