JPH05184366A - Gene DNA encoding aspartokinase and use thereof - Google Patents

Abstract

(57) [Summary] [Structure] Brevibacterium flavum MJ-233
DNA encoding aspartokinase was isolated from
The base sequence of this gene was determined. [Effect] Brevibacterium flavum MJ-233 strain transformed with a plasmid capable of replicative growth in a coryneform bacterium into which this gene DNA encoding aspartokinase has been introduced has increased L-lysine production. ..

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a coryneform bacterium-derived gene DNA containing a gene encoding aspartokinase (EC 2.7.2.4.), And a recombinant plasmid containing the gene DNA. , A coryneform bacterium transformed with the plasmid, and a method for producing L-lysine using the coryneform bacterium.

L-lysine is known to exist in proteins as an essential amino acid and is used as a drug or food additive.

[0003]

Conventionally, as an industrial production method of L-lysine, L-lysine has been used by using various auxotrophic strains, various drug-resistant strains and various drug-sensitive strains of coryneform bacteria which are glutamine-producing bacteria.
Methods for producing lysine are known [see, for example, Japanese Patent Publication No. 51-21078, Japanese Patent Publication No. 53-1833, Japanese Patent Publication No. 62-8692]. Further, a production method using a recombinant bacterium has also been proposed [Japanese Patent Application Laid-Open No. 56-1
No. 60997, JP-A-60-62994, JP-A-62-79788]. However,
In the method for producing L-lysine by the conventionally proposed method, the sugar yield is low and / or the accumulation of L-lysine is limited, and from a new point of view, improvement of strains by genetic engineering techniques and the like are included. , There is a strong need to provide a method for producing L-lysine more efficiently.

On the other hand, aspartokinase (EC 2.7.
The gene encoding 2.4.) Is Escherichia
A gene derived from Escherichia coli [Journal of B
iological Chemistry, 256 , p10228 to p10
230, 1981] is well studied. Also,
Aspartokinase (E.C. 2.
As for 7.2.4., Bacillus subtilis (Bacillus)
subtilis), Corynebacterium glutamicum (Cory
neform glutamicum) is known [Journal of Bi
ological Chemistry, 262 , p8787-p879.
8, 1987; Molecular Microbiology, 5 , p119.
7-p1204, 1991]. However, Brevibacterium flavum
No previously reported example has been found for the gene encoding the derived aspartokinase (EC 2.7.2.4.).

[0005]

DISCLOSURE OF THE INVENTION An object of the present invention is to obtain aspartokinase (EC 2.7.2.
4.) is isolated, the gene is introduced into a coryneform bacterium of the same species, and the coryneform bacterium is used to
It is to efficiently produce L-lysine from a new viewpoint.

[0006]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have isolated an aspartokinase gene from a coryneform bacterial chromosome and introduced the gene into an appropriate vector plasmid. Then, it was found that L-lysine can be efficiently produced by transforming a coryneform bacterium and using the transformed coryneform bacterium, and completed the present invention.

Thus, according to the present invention, (1) a gene DNA encoding aspartokinase derived from a coryneform bacterium; (2) a recombinant plasmid into which the gene DNA is introduced; (3) a trait with the recombinant plasmid A transformed coryneform bacterium; and (4) a method for producing L-lysine using glucose as a raw material, using the transformed coryneform bacterium.

The present invention will be described in more detail below.

The "gene DNA encoding aspartokinase" of the present invention is an enzyme that adds phosphate to L-aspartic acid, that is, aspartokinase (EC).
It means a gene DNA encoding 2.7.2.4.).

Although a DNA fragment containing a gene encoding aspartokinase (hereinafter, may be abbreviated as "A fragment") can be synthesized after its nucleotide sequence is determined. In many cases, it is usually cloned from an aspartokinase-producing microorganism, and the microorganism that serves as the source is a coryneform bacterium, especially Brevibacterium flavu.
m) MJ233 (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 this vector is used to transform Escherichia coli mutant strain CGSC507 lacking the aspartokinase gene.
4 [Escherichia coli Genetic Stock Center, Department of Biology, Yale University; POB
ox 6666 New Haven, CT 06511-744, US
A. The preserved strain] is transformed and smeared on a selective medium to obtain a transformed strain.

From the transformant obtained, plasmid DNA is obtained.
And the inserted A fragment derived from the Brevibacterium flavum MJ-233 strain chromosome can be confirmed and obtained.

The thus-obtained A fragment is further cleaved with an appropriate 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 Escherichia coli mutant strain deficient in aspartokinase 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.
And the inserted A fragment derived from the Brevibacterium flavum MJ-233 strain chromosome can be confirmed and obtained.

One of the A fragments thus obtained is
An example is a DNA fragment of about 1.7 kb obtained by excising the chromosomal DNA of the Brevibacterium flavum MJ-233 strain by complete digestion with the restriction enzyme EcoRI and further cutting it with the restriction enzyme NruI. it can.

The number of recognition sites and the size of the cleaved fragment when the DNA fragment containing the gene encoding aspartokinase of about 1.7 kb was cleaved with various restriction enzymes are shown in Table 1 below.

[0019]

[Table 1] 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 gel electrophoresis according to a method known per se. And subjected to 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 the same as those of Escherichia coli lambda phage (λphage) when agarose gel electrophoresis is used.
Based on the standard line drawn by the migration distance on the same agarose gel of a DNA fragment of known molecular weight obtained by cleaving the DNA of E. coli with the restriction enzyme Hind III, and when using polyacrylamide gel electrophoresis, Escherichia Phi-X174 phage (φx174
The size of each digested DNA fragment or each DNA fragment of the plasmid, 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 digesting the DNA of phage) with the restriction enzyme Hae III. To calculate. The size of the plasmid is determined by adding the sizes of the respective cut fragments. In the determination of 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 for the size of fragments of about 0.1 kb to less than 1 kb, The results obtained by 4% polyacrylamide gel electrophoresis were adopted.

On the other hand, the chromosomal DNA of Brevibacterium flavum MJ-233 described above was digested with the restriction enzyme NruI-E.
Regarding the DNA fragment having a size of about 1.7 kb obtained by cleaving with coRI, its nucleotide sequence was determined by the dideoxynucleotide enzymatic method (dideoxyc) using plasmid pUC118 or pUC119 (Takara Shuzo).
hain termination method, Sanger, F.et.al., Proc.Natl.A
cad.Sci.USA, 74 , p5463, 1977). The gene encoding aspartokinase determined from the presence of the open reading frame of the nucleotide sequence of the above-mentioned about 1.7 kb DNA fragment thus determined has the sequence shown below and has 421 amino acids. It is composed of 1263 base pairs that encode.

[0022]

## STR00003 ## The DNA fragment containing the gene encoding the aspartokinase of the present invention, which includes the above-mentioned nucleotide sequence, is not only isolated from natural coryneform bacterial chromosomal DNA, but also a commonly used DNA synthesizer, For example, it may be one synthesized by using System-1 Plus manufactured by Beckman.

The DNA fragment of the present invention obtained from the chromosomal DNA of Brevibacterium flavum MJ-233 as described above has a nucleotide sequence of the nucleotide sequence as long as it does not substantially impair the function of encoding aspartokinase. Some bases may be substituted with other bases or deleted, or new bases may be inserted, and further, a part of the base sequence is rearranged. All of these derivatives are included in the DNA fragment containing the gene encoding the aspartokinase of the present invention.

The size detailed above is about 1.7 kb D
A map of the cleavage points of the NA fragment by the restriction enzyme is shown in FIG.

The DNA fragment (A fragment) containing the gene encoding aspartokinase of the present invention is introduced into an appropriate plasmid, for example, a plasmid vector containing at least a gene that controls the replication / proliferation function of the plasmid in coryneform bacteria. By doing so, a recombinant plasmid capable of highly expressing aspartokinase in a coryneform bacterium can be obtained.

The promoter for expressing the gene encoding the aspartokinase of the present invention may be the promoter of the gene itself possessed by the coryneform bacterium, but is not limited thereto and the aspartokinase is not limited thereto. Any promoter may be used as long as it is a prokaryotic base sequence for initiating gene transcription.

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 stabilizing function of the plasmid in the coryneform bacterium. Preferred are, for example, plasmids pCRY30, p
CRY21, pCRY2KE, pCRY2KE, pCR
Y2KX, pCRY31, pCRY3KE and pCRY
3KX and the like are preferably used.

As a method for preparing the above-mentioned plasmid vector pCRY30, Brevibacterium stationis IFO12144 (F
ERM BP-2515) to the plasmid pBY503.
(For details of this plasmid, see Japanese Patent Application Laid-Open No. 1-9578
(See Japanese Patent Laid-Open No. 5) DNA is extracted, and a DNA fragment containing a gene responsible for the replication / proliferation function of a plasmid having a size of about 4.0 kb is cut out with a restriction enzyme XhoI to obtain a restriction enzyme EcoR.
With I and KpnI, a DNA fragment having a size of about 2.1 kb and containing a gene responsible for the stabilizing function of the plasmid is excised. By incorporating both of these fragments into the EcoRI, KpnI and SalI sites of the plasmid pHSG298 (Takara Shuzo), the plasmid vector pCRY30
Can be prepared.

Next, the introduction of the A fragment of the present invention into the above plasmid vector is carried out, for example, by cleaving a restriction enzyme site existing at only one site in the plasmid vector 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 allows the plasmid pCRY30 to be digested 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 aspartokinase thereto with DNA ligase.

The plasmid pC constructed in this way
The recombinant plasmid in which the A fragment of the present invention having a size of about 1.7 kb was introduced into RY30 is one of the recombinant plasmids that can be preferably used for the production of L-lysine, and the present inventors Was designated as plasmid pCRY30-AK. Details of the method for constructing the plasmid pCRY30-AK will be described in Example 4 below.

A plasmid capable of replicative growth in a coryneform bacterium containing the aspartokinase gene constructed in this manner is introduced into a host microorganism, and L-lysine is stably and efficiently used by using a culture of the microorganism. It becomes possible to produce.

Host microorganisms which can be transformed with the plasmid according to the present invention include coryneform bacteria such as Brevibacterium flavum MJ-233 (FERM BP-1).
497), Brevibacterium flavum MJ-233.
-AB-41 (FERM BP-1498), Brevibacterium flavum MJ-233-ABT-11 (F
ERM BP-1500), Brevibacterium flavum MJ-233-ABD-21 (FERM BP-1)
499) and the like.

The above-mentioned FERM BP-1498 strain is an ethanol-assimilating microorganism which is positively endowed with DL-α-aminobutyric acid resistance using the FERM BP-1497 strain as a parent strain (Japanese Patent Publication No. 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 FERM BP-
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 ammoniagene
s) ATCC 6871, ATCC 13745, AT
CC13746; Brevibacterium divaricatum ATCC14020;
Brevibacto lactofermentum
terium lactofermentum) ATCC13869; Corynebacterium glutam
icum) ATCC31831 etc. can also be used as a 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 (1972)]. 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. Each of the emerged colonies is independently subjected to a plasmid extraction operation to select a strain from which the plasmid pBY502 has been removed. By this operation, plasmid pB
Brevibacterium flavum M with Y502 removed
A strain derived from J-233 is obtained.

As a method for transforming the above-mentioned plasmid into the Brevibacterium flavum MJ-233-derived strain thus obtained, as known for Escherichia coli and Erwinia carotovora [Calvi
n, NM. and Hanawalt, PC, Journal of Bacteriolog
y, 170 , 2796 (1988); Ito, K., Nishid
a, T. and Izaki. K., Agricultural and Biological C
hemistry, 52 , 293 (1988)], pulse wave energization to DNA recipient bacteria [Satoh, Y. et al., Journal of
Industrial Microbiology, 5 , 159 (1990)] can introduce the plasmid.

A method for culturing a coryneform bacterium having an aspartase-producing ability obtained by transformation by the above method, for example, a Brevibacterium flavum MJ-233-derived strain is described below.

Cultivation can be carried out in an ordinary nutrient medium containing a carbon source, a nitrogen source, an inorganic salt and the like. Examples of the carbon source include glucose, ethanol, methanol, molasses, etc.
As the nitrogen source, for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea, etc. are used alone or in combination. Moreover, as the inorganic salt, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used. In addition to this, nutrients such as various vitamins such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid, and biotin can be added to the medium.

Culturing is usually carried out under aerobic conditions such as aeration and shaking, shaking at about 20 to about 40 ° C., preferably at about 25 ° C. to about 3.
It can be carried out at a temperature of 5 ° C. PH during culture is 5
The pH can be adjusted to about 10, preferably about 7 to 8, and pH adjustment during culture can be performed by adding an acid or an alkali.

The carbon source concentration at the start of culture is preferably 1
-5% by volume, more preferably 2-3% 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-lysine production reaction.

In the L-lysine production reaction, these cells can be used as they are, or a crushed cell product subjected to ultrasonic treatment or the like, or as a crude enzyme or a purified enzyme separated and recovered from the microbial cells, or by using them. It can be used after being immobilized on a suitable 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 a "treated bacterial cell product".

According to the present invention, however, glucose is
There is provided a method for producing L-lysine, which comprises contacting the cultured cells or a treated product of the cells to produce L-lysine.

The contact between glucose and the above-mentioned cultured bacterial cells or treated product of bacterial cells is preferably about 20 to about 40 ° C., particularly about 25 to about 3 in an aqueous medium, as in the usual enzyme reaction.
It can be carried out at 5 ° C.

The L-lysine produced can be separated and recovered from the reaction solution by means such as high performance liquid chromatography.

[0049]

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 Cloning of a DNA fragment (A fragment) containing a gene encoding aspartokinase derived from Brevibacterium flavum MJ-233 (A) of Brevibacterium flavum MJ-233
Extraction of total DNA Semi-synthetic medium A medium [Composition: urea 2 g, (NH ₄ ) ₂ SO ₄ 7
g, K ₂ HPO ₄ 0.5 g, KH ₂ PO ₄ 0.5 g, MgS
_{O 4 0.5g, FeSO 4 · 7H} 2 O 6mg, 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 20g, distilled water 1l] 1l, Brevibacterium flavum MJ-233 (FERM BP-14
97) was cultured until the late logarithmic growth phase and the bacterial cells were collected. The obtained cells were 10 mM NaCl-20 mM Tris buffer containing lysozyme at a concentration of 10 mg / ml (pH 8.
The suspension was suspended in 15 ml of 0) -1 mM EDTA-2Na solution. Next, proteinase K was added at a final concentration of 100 μg / m 2.
It was added so as to be 1 and kept at 37 ° C. for 1 hour. Further, sodium dodecyl sulfate was added so that the final concentration was 0.5%, and the mixture was kept warm at 50 ° C. for 6 hours to inoculate cells. To this lysate, an equal amount of phenol / chloroform solution was added and gently shaken at room temperature for 10 minutes, and then the whole was centrifuged (5,000 xg, 20 minutes, 10 to 12 ° C),
The supernatant fraction was collected, sodium acetate was added to 0.3 M, and then twice the amount of ethanol was slowly added. The DNA existing between the aqueous layer and the ethanol layer was scattered with a glass rod, washed with 70% ethanol, and then air-dried. 10 mM Tris buffer (pH 7.
5) Add 5 ml of -1 mM EDTA / 2Na solution and add 4
It was left still overnight at 0 ° C. and used for 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 added to the EcoRI-degraded DNA as a restriction enzyme Ec.
After digestion with oRI, dephosphorylated material was mixed and mixed with 50 mM Tris buffer (pH 7.6), 10 mM dithiothreitol, 1 mM ATP, 10 mM MgCl 2.
Each component of ₂ and 1 unit of T ₄ DNA ligase was added (the concentration of each component is the final concentration), reacted at 4 ° C. for 15 hours, and allowed to bind.

(C) Encodes aspartokinase
Selection of Plasmid Containing Gene The Aspartokinase-deficient Escherichia coli mutant strain used for selection of the above-mentioned gene was Escherichia coli CGSC 5074.
(Thr A1101, lys C1001, met L
1000) [inside () indicates aspartokinase genotype]. Using the plasmid mixture obtained in the above (B), calcium chloride method (Journal of Mo
lecular Biology, 53 , 159, 1970) to transform the Escherichia coli CGSC5074 strain, and select medium containing 50 mg of chloramphenicol [K
₂ HPO ₄ 7 g, KH ₂ PO ₄ 2 g, (NH ₄ ) ₂ SO ₄ 1
g, MgSO ₄ .7H ₂ O 0.1 g, glucose 20 g and agar 16 g were dissolved in distilled water 1 l].

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 of about 3.8 kb in length was observed.

This plasmid was named pHSG399-AK.

(D) encodes aspartokinase
Subcloning of DNA fragment containing gene (A) fragment In order to downsize the DNA insert fragment contained in the plasmid pHSG399-AK obtained in the above (C) to only a necessary part, plasmid pUC119 (commercially available from Takara Shuzo)
DNA containing a gene that coats hair aspartokinase
The fragment was subcloned as follows.

Plasmid pHSG3 obtained in the above (C)
99-AK digested with restriction enzymes EcoRI and NruI, and plasmid pUC119 with restriction enzymes EcoR.
I, cut with SmaI was mixed, and 50 mM Tris buffer (pH 7.6), 10 mM dithiothreitol,
Each component of 1 mM ATP, 10 mM MgCl ₂ and 1 unit of T4 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.

Using the obtained plasmid mixture, the calcium chloride method (Journal of Molecular Biology, 53 , 15) was used.
9, 1970) said Escherichia coli CGSC
Strain 5074 was transformed and a selective medium containing 50 mg of ampicillin [K ₂ HPO ₄ 7 g, KH ₂ PO ₄ 2 g, (NH
₄ ) ₂ SO ₄ 1 g, MgSO ₄ .7H ₂ O 0.1 g, glucose 20 g and agar 16 g were dissolved in distilled water 1 l].

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, and it was found that the length of plasmid pUC119 was long. In addition to the DNA fragment of 3.2 kb, an inserted DNA fragment of about 1.7 kb in length was observed. The number of restriction enzyme recognition sites and the size of the cleaved fragment of a DNA fragment of about 1.7 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 digested with various restriction enzymes, and the size of the digested fragment was measured. The results are shown in Table 2 below.

[0060]

[Table 2] The plasmid characterized by the above restriction enzymes was designated as pU
It was named C119-AK.

Based on the above, a DNA fragment (E of about 1.7 kb in size containing the gene encoding aspartokinase
coRI-BamHI fragment) was obtained.

Example 2 Determination of Nucleotide Sequence of Gene Encoding Aspartokinase About the DNA fragment of about 1.7 kb containing the gene encoding aspartokinase obtained in the item (D) of Example 1 , Its nucleotide sequence was determined using the plasmid pUC118 or pUC119 (Takara Shuzo) in the dideoxynucleotide termination method (Sahg
er, F. et al., Proc. Nat. Acad. Sci. USA 74 , 546
3, 1977) according to the strategy diagram shown in FIG.

Because of the presence of the open reading frame in the base sequence, the gene encoding aspartokinase is composed of 1263 base pairs encoding 421 amino acids having the base sequence shown in the following sequence. found.

[0064]

## STR00004 ## Example 3 Plasmid vector pC which is stable and replicates 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 6m
g, 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 l] 1 l
And Brevibacterium stationis IFO1214
4 was cultured until the late logarithmic growth phase and the bacterial cells were collected. A buffer solution containing lysozyme at a concentration of 10 mg / ml [25 mM tris (hydroxymethyl) aminomethane, 10 mM EDTA, 50 mM glucose] 20 m
It was suspended in 1 and reacted at 37 ° C. for 1 hour. Alkali-SDS solution [0.2N NaOH, 1% (W / V) S]
DS] 40 ml was added, mixed gently and mixed at room temperature for 1
Let stand for 5 minutes. Next, to this reaction solution, potassium acetate solution [60 ml of 5M potassium acetate solution, 11.5 ml of acetic acid,
30 ml of a mixed solution of 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.

An equal amount of phenol-chloroform solution (phenol: chloroform = 1: 1 admixture) was added to and suspended in the suspension, and the suspension was transferred to a centrifuge tube at room temperature for 5 minutes at 15,000.
The aqueous layer was collected by centrifugation at 0 × g. Add twice the amount of ethanol to the aqueous layer, leave at -20 ° C for 1 hour, and then at 4 ° C.
The precipitate was recovered by centrifugation at 15,000 xg for 10 minutes at room temperature.

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 placed at 12 ° C for 42 hours for 11 hours.
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 4 times with an equal amount of isoamyl alcohol 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 dialysis solution containing the plasmid pBY503 thus obtained, and then 2-fold amount of ethanol was added, and the mixture was allowed to stand at -20 ° C for 1 hour. This solution is centrifuged at 15,000 xg to obtain D
NA was precipitated to obtain 50 μg of plasmid pBY503.

(B) Plasmid vector pCRY30
The plasmid pHSG298 (Takara Shuzo) 0.5 μg was reacted with the restriction enzyme SalI (5 units) at 37 ° C. for 1 hour,
The plasmid DNA was completely degraded.

Plasmid pBY prepared in the above (A)
Restriction enzyme XhoI (1 unit) was added to 2 μg of 503 at 37 ° C.
And the plasmid DNA was partially decomposed. Both plasmid DNA digests were mixed and heat-treated at 65 ° C. for 10 minutes to inactivate the restriction enzyme, and then the final concentration of each component in the inactivation solution was 50 mM.
Tris buffer pH 7.6, 10 mM MgCl ₂ , 10 m
M dithiothreitol, 1 mM ATP and T4 DNA
Strengthen each component to make 1 unit of ligase, 16 ℃
It was kept warm for 15 hours. Escherichia
Kori JM109 competent cells (Takara Shuzo) were transformed.

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 1 l, 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 each plasmid was subjected to the alkaline-SDS method [T. Maniatis, EFFritsch, J. Sambrook, "Molecular.
cloning "(1982) p90-91].

As a result, S of plasmid pHSG298
About 4.0k derived from the plasmid pBY503 at the alI 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 section (A) was digested with the restriction enzyme Kpn.
A DNA fragment of about 2.1 kb obtained by treating with I and EcoRI was cloned into the KpnI and EcoRI sites of the above plasmid pHSG298-ori to prepare a plasmid vector pCRY30.

Example 4 Construction of plasmid pCRY30-AK and introduction into coryneform bacterium The plasmid pHSG39 obtained in section (C) of Example 1
5 μg of 9-AK was digested with 5 units of each restriction enzyme EcoRI-NruI at 37 ° C. for 1 hour and decomposed, and 1 μl of BamHI linker (commercially available from Takara Shuzo) was mixed, and 50 mM Tris buffer (pH 7.6), 10 mM was added.
Dithiothreitol, 1 mM ATP, 10 mM MgC
adding the components of l ₂ and T4 DNA ligase 1unit (concentration of each component is the final concentration), it was coupled reacted for 15 hours at 12 ° C..

This DNA was treated with the restriction enzyme BamHI 3 units
And the plasmid pCRY30 1 obtained in item (B) of Example 3 after being decomposed by reacting at 37 ° C. for 1 hour.
μg was added to 1 unit at 37 ° C using BamHI 1 unit restriction enzyme.
After the reaction for a period of time and decomposition, the mixture was mixed, 50 mM Tris buffer (pH 7.6), 10 mM dithiothreitol, 1
mM ATP, 10 mM MgCl ₂ and T4 DNA
Each component of 1 unit of ligase was added (the concentration of each component is the final concentration), and the mixture was allowed to react at 12 ° C for 15 hours for binding. This plasmid was used to transform the Escherichia coli CGSC5074 strain according to the method described above, and a selective medium [K ₂ HPO ₄ containing 50 μg / ml of kanamycin was used.
7 g, KH ₂ PO ₄ 2 g, (NH ₄ ) ₂ SO ₄ 1 g, MgS
O ₄ · 7H ₂ O0.1g, glucose 20g and agar 16
g dissolved in 1 l of distilled water].

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 of 1.7 kb in size was observed.

Plasmid DNA prepared as described above
Was transformed into a coryneform bacterium.

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 medium A until the initial logarithmic growth, penicillin G was added to 1 unit / ml, and the mixture was further cultivated with shaking for 2 hours, and the cells were collected by centrifugation to collect the cells. 20 ml pulse solution (272 mM
Sucrose, 7 mM KH ₂ PO ₄ , 1 mM MgCl ₂ ; p
It was washed with H7.4). Further, the cells were collected by centrifugation, suspended in 5 ml of the pulse solution, and 0.75 ml of the cells were mixed with 50 μl of the plasmid DNA solution obtained above and left standing in water for 20 minutes. Using Gene Pulser (manufactured by Bio-Rad), 2500 V, 25 μ
After setting to FD and applying a pulse, the plate was allowed to stand in ice for 20 minutes. The whole amount was transferred to 3 ml of the A medium and cultured at 30 ° C. for 1 hour, then 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, the above-mentioned Example 3 was selected.
A plasmid was obtained using the method described in section (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.

[0081]

[Table 3] The plasmid characterized by the above restriction enzymes was designated as pCR
It was named Y30-AK.

Brevibacterium flavum MJ2 transformed with the plasmid pCRY30-AK.
33-AK was sent to the Institute of Microbial Technology, Institute of Industrial Science, 1-3-1 Higashi, Tsukuba-shi, Ibaraki Prefecture, on December 16, 1991: No. 12658, Microtechnology Research Institute (FERM P-12).
658).

Example 5 Stability of the plasmid pCRY30-AK The 100 ml of the above-mentioned A medium was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes. Umm flabham M
After inoculating J233-AK with shaking culture at 30 ° C. for 24 hours, 100 ml of A medium prepared in the same manner was added to 5 ml.
The mixture was dispensed into a 00 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes, and the cells were subcultured at a rate of 50 cells per ml, and shake culture was carried out at 30 ° C. for 24 hours. Then, the cells were collected by centrifugation and washed, and then a fixed amount was spread on a plate medium prepared by using A medium supplemented with kanamycin at a rate of 15 μg / ml and A medium not supplemented, and the temperature was raised to 30 ° C. After culturing for 1 day, the growing colonies were counted.

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

Example 6 Production of L-lysine Medium (urea 0.4%, ammonium sulfate 1.4%, 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, NaCl 2
ppm, biotin 200 μg / l, thiamine / HCl
100 μg / l, casamino acid 0.1%, yeast extract 0.1
%) 100 ml was dispensed into a 500 ml Erlenmeyer flask and sterilized (pH 7.0 after sterilization), and then Brevibacterium flavum MJ233-AK
(FERM P-12658) was inoculated, glucose was added aseptically to a concentration of 5 g / l, and shake culture was performed at 30 ° C. for 2 days.

Next, the main culture medium (glucose 5%, ammonium sulfate 2.3%, KH ₂ PO ₄ 0.05%, K ₂ HP
_{O 4 0.05%, MgSO 4 ·} 7H 2 O 0.05%, Fe
_{SO 4 · 7H 2 O 20ppm,} MnSO 4 · 4~6H 2 O
20ppm, biotin 200μg / l, thiamine / HC
1000 μl of 100 μg / l, casamino acid 0.3%, yeast extract 0.3%) was placed in a 2 l aeration and agitation tank, sterilized (120 ° C., 20 minutes), and
Add 0 ml, rotation speed 1000 rpm, aeration amount 1v
Culturing was performed for 24 hours at a temperature of 33 ° C. and a pH of 7.6.

After completion of the culturing, the cells were collected from 500 ml of the culture by centrifugation and washed twice with desalted distilled water to prepare a reaction solution [(NH ₄ ) ₂ SO ₄ 2 g / l; KH ₂ PO ₄ 0.5 g /
l; KH ₂ PO ₄ 0.5 g / l; MgSO ₄ .7H ₂ O 0.
5 g / l; FeSO ₄ .7H ₂ O 20 ppm; MnSO ₄
・ 4 to 6H ₂ O 20 ppm; thiamine hydrochloride 100 μg
/ L; pH 7.6] after being suspended in 1000 ml, the suspension was charged into a 2 l aeration and stirring tank, 9 g of glucose was added, and the rotation speed was 300 rpm, aeration amount was 0.1 vvm, and temperature was 3
The reaction was carried out at 3 ° C. and pH 7.6 for 24 hours.

After completion of the reaction, centrifugation (4000 rpm,
L-lysine in the supernatant liquid, which had been sterilized at 4 ° C for 15 minutes, was quantified. As a result, the amount of L-lysine produced in the supernatant was
It was 1.5 g / l.

After the completion of this reaction, 500 ml of the culture broth was passed through a column of a strongly acidic cation exchange resin (H ⁺ type) to give L-.
After adsorbing lysine, washing with water and eluting with 0.5N aqueous ammonia, the L-lysine fraction was concentrated, and crystals of L-lysine were precipitated with cold ethanol. As a result, 400 mg
The L-lysine crystal of was obtained.

As a comparative example, Brevibacterium flavu (Brevibacterium flavu) was prepared under the same conditions.
m) MJ-233 (FERM BP-1497) was cultured, reacted under the same conditions, and L-lysine in the supernatant was quantified. As a result, the amount of L-lysine produced in the supernatant liquid was 0.6 g / l.

[0091]

[Chemical 5]

[Brief description of drawings]

FIG. 1 is a restriction map of a DNA fragment containing a gene encoding aspartokinase of the present invention, which is digested with a restriction enzyme.

FIG. 2 is a schematic diagram for determining the nucleotide sequence of a DNA fragment of the present invention having a size of about 1.7 kb.

[Chemical 2 Part 1]

[Chemical 2 Part 2]

[Chemical 2 Part 3]

[Chemical formula 1]

[Chemical formula 2]

[Chemical 3 Part 3]

[Chemical 4]

[Chemical 3 Part 5]

[Chemical 4 part 1]

[Chemical 4 part 2]

[Chemical 4 part 3]

[Chemical 4]

[Chapter 4 of 5]

[Chemical 5 part 1]

[Chemical 5 part 2]

[Chemical 5 part 3]

[Chemical 4]

[Chemical 5]

Front page continuation (72) Inventor Hideaki Yukawa 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Sanryo Yuka Co., Ltd. Tsukuba Research Institute

Claims (8)

[Claims] 1. A gene DNA encoding aspartokinase (EC 2.7.2.4.) Derived from a coryneform bacterium. 2. The gene DNA according to claim 1, wherein the coryneform bacterium is Brevibacterium flavum MJ233. 3. The following DNA base sequence: Aspartokinase (EC 2.7.2.4.)
A gene DNA that encodes. 4. An aspartokinase (EC 2.7. 1) represented by the following amino acid sequence:
Gene DNA encoding 2.4.). 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. 8. A method for producing L-lysine, which comprises contacting glucose with the cultured bacterial cell of Coryneform bacterium according to claim 7 or a treated product of the bacterial cell to produce L-lysine.