JPS6170983A - Novel microorganism and production of l-threonine therewith - Google Patents

Abstract

PURPOSE:The DNA coding for L-threonine production is cut off from a microorganism in Serratia and recombined into the vector plasmid and transferred into a Serratia microorganism to prepare a new microorganism with high L- threonine productivity. CONSTITUTION:A chromosome fragment coding for L-threonine production, which is collected from a microorganism in Serratia capable of producing L- thereonine is recombined to form a hybrid plasmid and it is transferred into a microorganism in Serratia. The resultant organism is cultured in a medium to collect the L-threonine accumulated in the medium.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a novel microorganism and a method for producing L-threonine using the same.

[Prior art]

L-threonine is one of the essential amino acids and is useful as a pharmaceutical, food, and feed additive.

As a method for producing L-threonine by a fermentation method, methods using mutant strains of various microorganisms are known. As methods for using microorganisms of the genus Serratia, various mutant strains 9 are known, such as threonine-degrading enzyme-deficient and threonine-degrading enzyme-deficient. Mutant strains resistant to antimetabolites, or mutant strains in which such microorganisms are endowed with resistance to lysine antimetabolites and/or methionine antimetabolites are used (Japanese Patent Publication No. 48195/1989, Japanese Patent Application Laid-open No. 56-198) No. 134993)
.

[Technical issues to be solved]

However, the L-threonine production ability of these mutant strains cannot be said to be high enough for industrial use, and there is a need for a method that can more efficiently produce L-threonine using microorganisms with higher L-threonine-producing ability. Ta.

[Structure and effects of the invention]

The present invention solves these problems, and L-
The deoxyribonucleic acid (hereinafter referred to as DNA) that controls L-threonine production possessed by a microorganism belonging to the genus Serratia that has threonine-producing ability is excised and incorporated into a vector plasmid, and then transferred to a microorganism belonging to the genus Serratia to produce L-threonine. The present invention was completed by successfully preparing a microorganism with high productivity and by discovering that L-threonine can be industrially advantageously produced by using the microorganism. The present invention provides a microorganism in which a microorganism belonging to the genus Serratia contains a hybrid plasmid that incorporates a chromosomal fragment responsible for L-threonine production collected from a microorganism belonging to the genus Serratia that has the ability to produce L-threonine, and a microorganism that is cultured in a medium. to produce and accumulate L-threonine in the medium,
This is a method for producing L-threonine, which is characterized by collecting this.

Preparation of the present B microorganism (chromosomal DNA and its preparation) In the present invention, the chromosomal fragment that controls L-threonine production (hereinafter referred to as chromosomal DNA) is an enzyme that synthesizes L-threonine from L-aspartic acid, that is, aspartic acid. Refers to a substance containing all or part of deoxyliponucleic acid (hereinafter referred to as DNA) that carries the genetic information of kinase, homoserine dehydrogenase, homoserine kinase, and threonine synthase, especially aspartokinase and homoserine dehydrogenase.

DNA carrying genetic information for homoserine kinase and threonine synthase, or DNA carrying genetic information for aspartokinase and homoserine dehydrogenase is preferable. Moreover, these DNAs are preferably mutant types that are free from feedback inhibition.

Such chromosomal DNA includes chromosome D, which belongs to the genus Serratia and is collected from a microorganism capable of producing L-threonine.
Chromosomal DNA collected from any microorganism as long as it is NA
In addition to the wild strain, known L-threonine-producing mutant strains 9 such as those requiring various amino acids (isoleucine, lysine-2-methionine, diaminopimelic acid, etc.), threonyl-degrading enzymes (L-threonine dehydrogenase, L-threonine deaminase, etc.) deficiency?

Alternatively, it may be a mutant strain in which the feedback inhibition by threonine to aspartokinase and homoserine dehydrogenase is released and the metabolism of threonine biosynthesis is regulated, or a strain further mutated from this strain. Specific examples of such chromosome DNA donating microorganisms include Serratia marcescens HNr.
lOO1 (Feikoken Microbiology No. 3121), Serratia marcescens HNr21 (Applied and Invironmental Microbiology (Appl,
Environ.

Microbiol, ) 35. 834 (1978
)), Serratia marcescens P-103 (Feikoken Bacterium No. 5413), Serratia marcescens D-6
0 [Journal of Bacteriology (J.

Bacteriol, ) 135. 318 (1
Serratia marcescens TLr155, which is derived from 97B) and is not inhibited by L-threonine in the presence of L-lysine, and Serratia marcescens T-1111, which is derived from this strain by a transduction procedure.
Another example is Serratia marcescens T-1164, which is derived from T-1111 by a transduction method.

To collect chromosomal DNA from these microorganisms, for example, the microbial cells are treated with lysozyme or a surfactant (SDS, sarcosyl (sodium N-lauroyl sarcosinate), etc.), followed by deproteinization, followed by ethanol precipitation. [Journal of Molecular Biology (J.

Mo1. Biol, ) 3,208. (1961);
(Biosimi e biofici acta (Bioch
im, Bihys.

Acta, ) 12, 619 (1963)).

(Vector plasmid DNA and its preparation) The vector plasmid into which the chromosomal DNA as described above is incorporated is not particularly limited as long as it can be transferred to a microorganism belonging to the genus Serratia and can be replicated in the transferred microorganism. Dinges of
National Academy of Sciences (PROC, Natl, Acad,
Sci, USA) 1 construction, 3240 (19
73)), pLG339 (Gene, ) 1
8.332 (1982)).

pBR322 (Gene, ) 2.95 (
(1977)), pBR325 (Gene, ).

121, (1978)), pAcYc177 (Journal of Bacteriology (J.

Bacteriol, ) 134. 1141 (
1978) ).

pKP1155 (EcoRI f5 fragment of doblasmid (Proceedings of the National Academy of Sciences)
of science (Proc, Natl, Aca
d.

A plasmid in which the B, C, and A2 fragments containing the replication origin were excised from Sct, ) ) ■, 64 (1976), and ampicillin resistance, chloramphenicol resistance, spectinomycin resistance, and streptomycin resistance genes were ligated to this fragment] can be used. These plasmids were obtained from cleared lysate from the bacterial cells of N. coli that carry them.
)-method [Gene Manipulation Experimental Methods, p. 125 (Yasutaka Takagi, Kodansha, 1980)]; Molecular Cloning (Mo
1ecular CloningP, 86 (Man
iatis et al, + ed., Cold Spring Harbor Laboratory.
Harbor Laboratory ) (1982
) ) etc. can be prepared by conventional methods.

(Hybrid Plasmid I) Preparation of NA) To prepare hybrid plasmid DNA from the chromosomal DNA and vector plasmid DNA obtained above, restriction endonuclease (e.g. HindII [, Pstl, Ba
After cleaving the chromosomal DNA and plasmid DNA strand using ligase (e.g. mflI, 5a11, etc.)
T4 DNA ligase, large intestine W DNA ligase, etc.) or, depending on the cut ends, treated with terminal transferase, DNA polymerase, etc. and then using dust gauze to join the DNA strands [Methods in Enzymology] Menthods
inEnzymology) 69. 41 (
1979), Gene Manipulation Experimental Methods, p. 135 (Yasutaka Takagi, Kodansha, (1980)).

The vibriso and doblasmid DNA thus obtained can be used as is for transformation, or the hybrid plasmid DNA can be transferred into mutant strains having various mutations and a strain carrying the desired hybrid plasmid DNA can be selected in advance. It is also possible to extract a hybrid plasmid from a bacterial strain by a conventional method and transfer it into a host microorganism.

Such a method is preferred because it facilitates the selection of transformed strains. Examples of mutant strains used for this purpose include microorganisms belonging to the genus Lacsetia and Nizierisia, such as L-
Examples include microorganisms that do not have the ability to produce threonine, or mutant strains that have some or all of the mutations that make them sensitive to ampicillin or kanamycin.
Cori K12, C600r"'m-(ATC33525
) can be suitably used.

(Host microorganism) The host microorganism containing the hybrid plasmid DNA may be any microorganism that belongs to the genus Serratia and is capable of transformation, and any of the microorganisms exemplified as the donor microorganism for the chromosomal DNA can be used.

(Transformation with hybrid plasmid DNA) The transformation method is, for example, a method in which host microorganism cells are treated with a calcium chloride solution at low temperature to increase the membrane permeability of the bacterial cells and the hybrid plasmid DNA is incorporated into the host microorganism [Journal] of Molecular Biology (J, Mol.

B161. ) Tateyu, 159 (1970)) can be adopted.

Among the transformed strains obtained in this way, the strain containing the desired hybrid plasmid was selected by fishing out the seedlings that required the ability to secrete L-threonine from among the grown colonies.
This can be done by separating.

In this way, it is possible to obtain a microorganism according to the present invention, that is, a microorganism belonging to the genus Serratia containing a hybrid plasmid incorporating a chromosomal fragment in charge of L-threonine production collected from a microorganism capable of producing L-threonine. I can do it.

Specifically, such microorganisms include, for example, Serratia marcescens.
) TA5011 (Fiber Technology Research Institute No. 7817), Serratia marcescens (5erratia marce
5erratia m.
arcescens) TA5013 (Feikoken Bacteria No. 7
No. 819), etc.

L-threonine '1 method The microorganism thus obtained is cultured in a medium, and the L-threonine produced and accumulated in the medium is collected.
Threonine can be produced.

The L-threonine production medium used in the present invention uses glucose and sucrose as a carbon source.

Sugars such as molasses, organic acids such as fumaric acid and citric acid,
10-20% of alcohols such as glycerol, 1-2% of organic ammonium salts such as ammonium acetate, ammonium sulfate, inorganic ammonium salts such as ammonium hydrochloride, urea, etc. as nitrogen sources, cornstarch liquor as organic nutrients, 0-1 peptone, yeast extract, etc.
A culture medium containing an appropriate amount within a range of % can be suitably used. In addition to these, a small amount of potassium phosphate, magnesium sulfate, etc. may be added, and calcium acetate or ammonia may be added as necessary to maintain the pH of the medium at 6 to 8. Furthermore, substances involved in threonine biosynthesis 2 such as L-aspartic acid, L-
A medium to which homoserine, L-IJ gin, L-methionine, L-isoleucine, etc. are appropriately added can also be used.

According to the present invention, the above-mentioned medium is inoculated with the microorganism of the present invention,
L-
A significant amount of threonine can be accumulated. After the culture is completed, the L-threonine produced can be easily separated and purified by normal separation and purification operations such as adsorption on an ion exchange resin, elution with aqueous ammonia, and crystallization. can be collected.

Hereinafter, the present invention will be explained with reference to Examples and Reference Examples. In the Examples, L-threonine was confirmed by ninhydrin reaction on paper chromatogram, and its quantification was performed by bioassay using Leuconostoc mesenteroides P-60. went.

Example I Tl+ staining DNA preparation 11 Serratia marsenoscens TLr1 obtained in Reference Example 1 described later
55 in L-broth (1% peptone, 0.5% Seungbo extract)
, sodium chloride 0.5%, pH 7, 2)■7! The cells were inoculated and cultured with shaking at 30°C for 4 hours, and the cells in the logarithmic growth phase were collected by centrifugation. This strain was treated with lysozyme and SD
The cells were lysed by S treatment, protein was removed by phenol treatment, and then ethanol treatment was performed to precipitate chromosomal DNA. Then, RNase (final concentration 10 μg/-) was added and incubated at 37°C.
Chromosome 7.2■ was obtained by purifying the chromosomal DNA by treating the cells for 1 hour.

(2) Preparation of vector plasmid DNA Plasmid pLG33 to N. coli strain 12G600
9, [Gene 18,3
35 (1982)) It was inoculated into 800ml of shiitake broth containing 0.2% L-glucose, cultured with shaking at 37°C for 7 hours, and then the bacterial cells were collected by centrifugation. Next, the obtained bacterial cells were lysed by lysozyme treatment and SDS treatment, and then sodium chloride was added to give the final LM.
Centrifugation was performed at 0°000X for 9.30 minutes. The supernatant was then collected and treated with phenol, followed by addition of ethanol and centrifugation to precipitate DNA. The precipitated DNA was dissolved in a 10mM Tris-HCl-1mM EDTA solution (pH 7.5).
) and separated and purified by cesium chloride ethidium bromide equilibrium density gradient centrifugation.
A vector plasmid pLG 33'9 DNA was obtained.

A-tz<t+t-y knee=pregnancy 2. 20 μg of the chromosomal DNA obtained in step 11 was treated with restriction endonuclease Sal I under normal conditions.
The chain was partially cut. Furthermore, 5all was applied to 7 μg of the vector plasmid DNA obtained in (2) above under normal conditions to completely cleave DNAI. After heat treatment at 65° C. for 10 minutes, both reaction solutions were mixed and DNA ligase derived from T4 phage was applied under normal conditions to link the DNA strands.

Restriction endonuclease deficient and homoserine kinase deficient N. coli 12C600r''I
11'''' (ATC33525) was inoculated into L-broth 50-, cultured with shaking at 37°C, and the bacterial cells that had grown to the middle of the logarithmic growth phase were collected.

Then, 50 mL of ice-cooled 0.1M magnesium chloride solution was added.
- 0, 1 M calcium chloride solution, collected after suspension in ice-cooled solution 5. & was confused. The DNA solution obtained in (3) was added to this cell suspension, cooled on ice for 30 minutes, and then heat-treated at 42°C for 2 minutes to incorporate the DNA into the cells. Next, add 50% of L-broth to this suspension.
was added and cultured with shaking at 37°C for 1 hour, centrifuged, and the bacterial cells were suspended in physiological saline 5-. A small amount of this suspension was added to the culture medium (glucose 0.5%, potassium dibasic phosphate 0.7%).
%, potassium phosphate 0.3%, ammonium sulfate 0
．． 1%, magnesium sulfate heptahydrate 0.01%, L-
Leucine 0.001M, thiamine hydrochloride 0.0005%
, DL-β-hydroxynorvaline 0.01%, kanamycin sulfate 0.01%, agar 1.5%).
The cells were cultured at ℃ for 3 days. The resulting colonies were isolated using the halo method [Applied and Invironmental
Microbiology (Appl.

Environ, Microbiol, ) 38.
1045 (1979)], seedlings having the ability to secrete threonine were selected. Hybrid plasmid DNA was extracted from the thus obtained bacterial strain in the same manner as in (2) above.
was extracted, purified and separated.

(5) Preparation of transformed strain The hybrid plasmid DNA obtained in (4) above and extracellular nuclease-deficient and restriction endonuclease-deficient Serratia marcescens TT392 [Biochemistry, AE, 1024 (1983)] were used as (4). The hybrid plasmid DNA was incorporated into TT392 by the same treatment. This strain was then treated with 100μg/kanamycin sulfate.
The colonies were cultured on a broth agar medium containing -, and the resulting colonies were isolated. This strain was inoculated into Pros 11 containing 0.2% glucose, cultured at 37°C for 118 hours with shaking, harvested, and treated in the same manner as in (2) above to obtain hybrid plasmid DNA (pTA509)1.
．． I got 1■.

The obtained pTA509j mutant strain Serratia marcescens D-60, which is deficient in threonine degrading enzyme and has no ability to produce threonine (Journal of Bacteriology)
(J, Bacteriol, 5.318 (1
978)) in the same manner as in (4) above, and cultured on an L-broth agar medium containing 100 μg/- of kanamycin sulfate. Bacillus no. 7817) was obtained.

Serratia marcescens TA5011 obtained above was cultured overnight in a broth slant medium containing 0.01% kanamycin sulfate, and then cultured in a fermentation medium [urea 1.5%, ammonium sulfate 0.0
5%, dibasic calcium phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, L-inleucine 0.1%6 corn stave liquor 0.1% and calcium carbonate 1
A solution containing 15% was poured into a 500-capacity shake flask and sterilized by heat, and 1 g of Sea I was separately heat-sterilized and added to a concentration of 15%. A platinum loop was inoculated at pH 7.0). Also, the parent stock Serratia marcefuscens D
-60 was inoculated into the same nutrient medium as above. The amount of L-threonine produced in the medium when each strain was cultured with reciprocating shaking at 27° C. for 96 hours was as shown in Table 1 below.

Table 1 In addition, the TA5011 cells obtained above were treated in the same manner as in (2) above, and the hybrid plasmid D was extracted from the pro-cough body.
The DNA was extracted and cut with Sal I, and the resulting DNA fragments were examined by agarose gel electrophoresis. As a result, in addition to the approximately 6.2 kb DNA fragment generated when vector pLG339 was cut with Sal I, approximately 4
．． DNA fragments of 1 kb, approximately 1.5 kb, and approximately 0.9 kb were observed, and the hybrid plasmid DNA contained in TA5011 is a hybrid plasmid in which approximately 6.5 kb of chromosomal DNA was integrated into the Sat 1 cleavage site of vector pLG339. This was confirmed.

Example 2 pTA509 obtained in Example 1 (5) and Serratia marcescens T-1111 obtained in Reference Example 2 described later were combined in Example 1 (
Transform in the same manner as in 5) and add 100 μg of kanamycin sulfate.
/- by culturing on an L-broth agar medium containing the desired transformed strain Serratia marcescens rA5012.
(m1 Research Library No. 7818) was obtained. ' When the obtained Serratia marcefucens TA5012 and the parent strain Serratia marcefucens T-1111 were cultured under the same conditions as in Example 1 (6), the L-threon production amount was as shown in Table 2 below. .

Table 2 Example 3 pTA509 obtained in Example 1 (5) and Serratia marcescens T-1164 obtained in Reference Example 3 described later were combined in Example 1 (
Transform in the same manner as in 5) and add 100 μg of kanamycin sulfate.
/- by culturing on an L-broth agar medium containing the desired transformed strain Serratia marcefuscens TA5013
(Feikoken Bibori No. 7819) was obtained.

The obtained TA5013 was cultured overnight in an L-broth slant medium containing 0.01% kanamycin sulfate, and then cultured in a pre-incubation medium [
Sucrose 15%, urea 1.5%, ammonium sulfate 0.0
5%, potassium diphosphate 0.1%, magnesium sulfate heptahydrate 0.1%, L-isoleucine 0.04%, L
- 0.04% methionine, 0.04% cornstarch liquor.
1%, yeast extract 0.2%, kanamycin sulfate 0.01
% and a solution containing 1% calcium carbonate 15-500
mj) The mixture was poured into a shake flask and sterilized (however, sucrose was added after heat sterilization and kanamycin sulfate was added after sterile filtration. po7.0) and inoculated with a platinum loop. The cells were then cultured at 27°C for 24 hours. The thus obtained preculture solution 60- was mixed with a fermentation medium [sucrose 15%, urea 1.
5%, ammonium sulfate 0.05%, dibasic potassium phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, L
-Isoleucine 0.04%, L-methionine 0.04%
, corn staple liquor 0.1%.

Solution 1.21 containing 0.1% yeast extract and 2% calcium carbonate (however, sucrose was added separately after heat sterilization).

The main culture was carried out in a 2.41-volume jar fermentor at 27°C with agitation at an aeration rate of 1.2β/min. c.
, 1 hour) and then 1.2 g of aspartic acid was added. This was repeated three times.

Also, as a control, Serratia marcescens T-1164
The above preculture medium (however, kanamycin sulfate was not used) was inoculated with a platinum loop, and the culture solution obtained by culturing at 27°C for 24 hours was inoculated into the same fermentation medium as above, and under the same conditions as above. Main culture was performed.

The amount of L-threonine produced in each medium after 120 hours was as shown in Table 3 below.

Example 4 The main culture solution 11 of TA5013 obtained in Example 3 was collected and sterilized, and then filtered to remove insoluble materials. Then, the filtrate was treated with cation exchange resin Amberlite IR-12.
The column was passed through a column packed with 0B(H"). After washing with water, the absorbed L-threonine was eluted with 5% aqueous ammonia. The eluate was concentrated under reduced pressure, cooled by adding methanol, and the precipitated crystals were collected by filtration. L-threonine 51J was obtained by recrystallizing the obtained crystals from aqueous methanol.

Reference Example 1 Preparation of Serratia marcescens TLr155 Serratia marcescens D-60 (Journal of Bactiology 153.318 (1978)) was diluted with N-methyl-N゛-
Mutation induction treatment was performed with nitro-N nitrosoguanidine, and a nutrient agar medium (glucose 0.5%, peptone 1.0%, meat extract O 93%, yeast extract 1.0%) was used.

0.5% sodium chloride, 1.5% agar) so that 100 to 300 colonies were formed per plate. Among the resulting colonies, a strain whose growth is significantly retarded by L-threonine, that is, a threonine-sensitive strain, Serratia marcesoscens D-600, was isolated using the replica method [Manual of Methods for General Bactiology].
Manual of Methods for Gen
eral Bacteriology, Page22
American Society for Microbacteriology (Gerhardtet)
at, + ed, + Amerlcan 5ociet
(1981)). The obtained Serratia marcescens D-,
600 was subjected to mutagenesis treatment with N-methyl-N-nitro-N-nitrosoguanidine, and then L-threonine 100mM
, L-lysine 100mM and L-isoleucine 100mM
Minimal agar medium containing mM (glucose 0.5%, ammonium sulfate 0.1%, monopotassium phosphate 0.3%, dibasic potassium phosphate 0.7%, magnesium sulfate heptahydrate 0.01% , agar 1.5%) 1 x 10' per plate
cells were plated and cultured at 30°C for 4 days. A threonine-resistant strain that is a strain that is not inhibited by L-threonine in the presence of L-lysine by isolating large colonies that occur.

Serratia marcescens TLr155 strain was obtained.

Reference Example 2 Preparation Reference Example 1 of Serratia marcescens T-1111
Serratia marcescens TLr155 obtained with PS2
0 Phage [Japanese Journal Op Microbiology (Jpn.

J, Microbiol, ) 17.473 (1
973)) [Applied and Invironmental Microbiology (Appl, E.
nviron.

Microbiol, ) 38. 1045 (1
979)) A phage solution was prepared. Then Serratia
marcescens E-60 (Applied and Invironmental Microbiology (Appl,
Environ, Microbiol, ) 3
8. 1045 (1979)] and the phage solution were mixed and left at 30°C for 30 minutes to allow the phage DNA to be incorporated into the bacterial cells. The bacterial cells were washed twice with physiological saline to prepare bacterial cells. This suspension was applied to a minimum agar medium (Reference Example 1) containing 1 mM of L-isoleucine so that the number of bacteria per plate was 3×10'.

Serratia marcescens T-1111 was obtained by culturing at 30° C. for 4 days and separating the resulting colonies.

Reference Example 3 Preparation of Serratia marcescens T-1164 Serratia marcescens N-16 (Applied and Invironmental Microbiology (Appl.
Environ, Microbiol, ) Soil.

1445 (1983)) was subjected to mutagenesis treatment with N-methyl-N'-nitro-N-nitrosoguanidine.

Nutrient agar medium (Reference example 1) 1 to 3 x 102 per plate
Apply so that several colonies are formed. From the resulting colonies, Serratia furcefuscens strain N-31, which is an isoleucine-, threonine-, arginine-, and methionine-requiring strain, was selected by the replica method in the same manner as in Reference Example 1.

Serratia marcescens T-1111 obtained in Reference Example 2
Irradiate the strain with ultraviolet light [Bacteria, phage genetic experiment method, 64
A phage solution is prepared using the following method (Kyoritsu Shuppan (1972)). Next, the phage solution and the culture solution of the N-31 strain obtained above were mixed, and the phage DNA was incorporated into the N-31 strain in the same manner as in Reference Example 2. After centrifuging the bacterial cells,
A bacterial cell suspension was prepared using physiological saline. This suspension was mixed with 1mM L-isoleucine and 1mM L-arginine.
Serratia marcescens T -11
I got 29.

Serratia marcescens ETr17 (Applied and Invironmental Microbiology)
Appl, Environ, Microbiol,
) 4th Engineering, 1445 (1983)) A phage solution was prepared by infecting the ps20 phage in the same manner as in Reference Example 2, and a bacterial cell suspension was prepared by incorporating T-1129 in the same manner as in Reference Example 2. . This suspension was applied to each plate of a minimal agar medium containing 1 mM of L-isoleucine and 1 mM of L-methionine at a density of 3×10”.

After culturing at 30°C for 4 days, the resulting colonies were isolated. Thus, Serratia marcescens T.
-1164 was obtained.

Procedural amendment (method) 1. Indication of the case 2. Name of the invention
・'enhancement.

3. Relationship with the case of the person making the amendment Patent applicant 3-21 Doshomachi, Higashi-ku, Osaka-shi, Osaka (a6541)
(295) Tanabe Pharmaceutical Co., Ltd. Representative Matsubara - Department 4, Agent

Claims (4)

[Claims] (1) A microorganism in which a microorganism belonging to the genus Serratia is made to contain a hybrid plasmid into which a chromosomal fragment in charge of L-threonine production collected from a microorganism belonging to the genus Serratia having the ability to produce L-threonine is contained. (2) Hybrid plasmids incorporating chromosomal fragments contain deoxyribonucleic acid that carries the genetic information of aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine synthase, or deoxyribonucleic acid that carries the genetic information of aspartokinase and homoserine dehydrogenase, and microorganisms belonging to the genus Serratia. The microorganism according to claim 1, which is a hybrid plasmid consisting of a replicable vector plasmid. (3) Deoxyribonucleic acid and vector plasmid p carrying genetic information for aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine synthase
The microorganism according to claim 1 or 2, which is a microorganism containing a hybrid plasmid consisting of LG339. (4) A microorganism belonging to the genus Serratia containing a hybrid plasmid that incorporates a chromosomal fragment responsible for L-threonine production collected from a microorganism belonging to the genus Serratia that has the ability to produce L-threonine is cultured in a medium. produces and accumulates L-threonine in
A method for producing L-threonine, which comprises collecting the L-threonine.