JPH0716429B2 - Method for producing L-threonine - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing L-threonine by a transformant having a recombinant DNA containing a gene controlling the production of L-threonine.

[Prior Art] Providencia redtteri (Providencia rettgeri,
An L-threonine fermentation method using a mutant strain of P. rettgeri) is known. If genetic engineering is applied to these L-threonine fermenting strains, the productivity of L-threonine is expected to be improved.

[Problems to be Solved by the Invention] The inventors of the present invention devised an original idea in view of such a situation,
A gene controlling L-threonine fermentation production is self-cloned using a plasmid of ttgeri to prepare a composite plasmid having a gene controlling L-threonine fermentation production,
With this composite plasmid, Providencia
A bacterium of the genus or a bacterium of the genus Echerihia was transformed, and the fermentative productivity of L-threonine was successfully improved using this transformant, thus completing the present invention.

[Means for Solving Problems] That is, the present invention provides L-derived strains of Providencia.
A transformant of a bacterium of the genus Providencia or Escherichia having a recombinant DNA of the plasmid pYU300 represented by the restriction map of FIG. 1 containing a gene controlling threonine production was cultured, and L produced and accumulated in the medium was cultured. -Providing a method for producing L-threonine, which comprises collecting threonine.

The present invention will be sequentially described in detail below.

A plasmid derived from P. rettgeri is used as a vector. For example, P.rettgeri ATCC25932, ATCC991
9 、 It can be extracted from ATCC 29944, but P.rettgeri ATCC25932
Plasmid pYU300 extracted from P. rettgeri 21118
Obtained by transforming P. rettgeri ATCC21118 (pYU30
It is more convenient to extract and use the plasmid pYU300 from (0) (Microtechnology Research Institute No. 1174).

As a donor of a gene that controls L-threonine fermentation production,
Bacteria of the genus Providencia can be used, for example P.re
Although ttgeri ATCC21118 can be used, it is not limited to this strain, and various mutant strains such as L-threonine regulatory mutant strain can also be used. Extraction of the chromosomal DNA can be carried out according to a usual method such as Reference 1. In this case, if the lysis process is performed at 30 to 65 ° C. for 20 to 60 minutes, the yield of DNA can be increased, but it is not limited thereto.

The self-cloning of the gene that controls L-threonine fermentation production is performed by, for example, the shot gun method using the usual auxotrophy complementation of Reference 2. An L-threonine-requiring mutant strain of P. rettgeri can be used as a host, and the fragmentation of chromosomal DNA or the cleavage for vectorization of a plasmid can be carried out using a conventional enzyme using a restriction enzyme such as Hind III. It can be carried out by reaction. The ligation of the DNA fragment and the vector can be carried out by an ordinary ligation reaction using an enzyme such as T ₄ DNA ligase, and the host is made competent and transformed by an ordinary ligation reaction such as that of Reference 3. The method can be applied. Screening of transformants can be carried out using the marker rescue of L-threonine auxotrophy of the host as an index, but it is efficient if selective pressure for vector drug resistance is also applied. If the restriction ends of the vector are treated with alkaline phosphatase as described in Reference 4, for example, self-cyclization of the vector can be prevented.

A recombinant plasmid is extracted from the obtained transformant by, for example, the method of Reference 6, and the recombinant plasmid is used to transform a P. rettgeri strain having L-threnion fermentability. Transformants are selected by using drug resistance or high L-threonine fermentability of the plasmid as an index. The host in this case is an auxotrophic mutant strain, a strain in which feedback inhibition or suppression of the L-threonine synthesis system is reduced, or L.
-L-threonine fermentability can be further enhanced by using a strain having a reduced threonine degrading ability or a strain having these traits.

By culturing the retransformant thus obtained in an L-threonine fermentation medium, it becomes possible to accumulate L-threonine in the medium in good yield. The medium may be a normal medium containing a carbon source, a nitrogen source, inorganic ions, and optionally an amino acid. However, in order to prevent loss of the plasmid, it is desirable to add a drug and apply selective pressure during the preculture. As the carbon source, glucose, sucrose and the like, and starch hydrolysates and sugar-condensed hydrolysates containing them are used. Ammonia water, ammonia salt, etc. can be used as the nitrogen source. The culture may be performed under aerobic conditions while appropriately adjusting the pH and temperature of the medium.

A usual method can be applied to the decomposition and purification of L-threonine produced and accumulated in the culture solution.

By using the transformant microorganism of the present invention, not only the accumulated concentration of L-threonine is high but also the culture time can be shortened as compared with the case of using the conventionally known L-threonine-producing bacterium of P. rettgeri.

Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1 (1) Extraction and isolation of plasmid P.rettgeri ATCC21118 (pYU300)
No.) was aerobically cultivated in LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, pH 7.5) at 37 ° C for 17 hours in 1 ml of 3 ml of 0.5 MN containing 15 mg lysozyme hydrochloride.
Suspend in aCl-0.1M EDTA-50mM Tris · HCl (pH8), 37
Incubate for 15 minutes at ℃, freeze and thaw, then 25ml
Add 0.1M Tris-HCl (pH9) -1% SDS-0.1M NaCl 6
It was dissolved by incubating at 0 ° C for 20 minutes. Lysis solution 30,000r
Centrifuge at pm for 30 minutes and add 10 μg of 10 mg / ml RNase A to the supernatant.
l was added and incubated at 37 ° C. for 1 hour, and then crude DNA was obtained by phenol extraction and ethanol precipitation. This was subjected to biogel column chromatography and cesium chloride-ethidium bromide density gradient equilibrium centrifugation to obtain 55 μg of plasmid DNA (pYU300).

The length of plasmid pYU300 is 4.2 Kb and drug resistance is Tc ^R , Cm ^R
Met. The restriction map of pYU300 is shown in Fig. 1.

(2) Extraction of chromosomal DNA After lysing the cells of P. rettgeri ATCC 21118 cultivated in the LB medium of 1 by the method of the first paragraph, 4 mg of chromosomal DNA was obtained by the method of Saito-Miura (Reference 1). It was

(3) Digestion and fractionation of DNA with restriction enzyme 0.25 μg / μl of the DNA obtained in (2) under the reaction conditions of Reference 5 was 0.12 unit / μ
Degradation product (125 µg in terms of DNA amount) digested with 1 l of Hind III for 2 hours was centrifuged in 12 ml of 10-40% sucrose density gradient.
Fractions of 0.5 ml were collected. Centrifugation was performed using a Hitachi RPS40T rotor at 20 ° C and 25,000 rpm for 24 hours. The DNA length of each fraction was measured by electrophoresis, and the fraction of 2 to 10 Kb was ligated to the plasmid. Takara Shuzo Co., Ltd. was used as the restriction enzyme, and the activity unit was defined in the attached instruction manual.

(4) Preparation of vector DNA The plasmid vector pYU300 was replicated in E. coli MM294. Replication and extraction / purification from bacterial cells are according to Reference 7,
After lysing the plasmid-containing cells cultured in LB medium containing 30 mg / l chloramphenicol with lysozyme / SDS,
It was extracted with phenol and purified by cesium chloride-ethidium bromide density gradient equilibrium centrifugation and biogel column chromatography. 50μ of pYU300 per medium
g was obtained. Since it serves as a vector, the restriction enzyme Hind
After completely decomposing using III under the reaction conditions of Reference 4, Reference 4
Alkaline phosphatase treatment was performed according to the above.

(5) Preparation of host P.rettg in logarithmic phase cultured in 5 ml of LB medium at 37 ° C for 3 hours
According to Reference 8, eri ATCC21118 and E. coli MM294 were mutagenized with 100 μg / ml of NTG in TM buffer, and then threonine-auxotrophic mutants of both strains were isolated.

(6) Ligation and transformation of DNA 10 μg of DNA fragment obtained in (3) and vector D obtained in (4)
NA 10 μg was added to 0.5 unit of T ₄ DNA ligase based on the method of Reference 4 and 100 μl of 6 mM MgCl ₂ -6 mM B-mercaptoethanol-0.5 mM ATP-6 mM Tris · HCl (pH 7.6) at 14 ° C.
After incubating overnight, add 10 μl of this reaction mixture (5).
The P. rettgeri host prepared in 1. was mixed with 200 μl of the bacterial solution that had been made competent by the method of Reference 3, and then mixed at 4 ° C. for 45 minutes, then 42
After incubation at 90 ° C. for 90 seconds and at 4 ° C. for 1 minute, LB medium was added to make 1 ml, and the mixture was shake-cultured at 37 ° C. for 1 hour for transformation.

(7) Screening The transformant mixture fungus prepared in (6) above was used in 7.
Minimal medium (0.2% containing chloramphenicol at 5 μg / ml)
Glucose, 0.1% ammonium sulfate, 0.2% KH ₂ PO ₄ , 0.
7% K ₂ HPO ₄ , 0.01% MgSO ₄ · 7H ₂ O, 0.05% sodium citrate, 20 ppm isoleucine), and the selective culture of the transformant was performed by static culture at 37 ° C. The colonies formed on the 3rd day of culture were transplanted again to the minimal medium containing chloramphenicol to reconfirm the growth ability, and then the plasmid was extracted according to the method of Reference 6 and the digested product of Hind III was electrophoresed. . As shown in Fig. 2, there are two types of existence of insert DNAs in opposite directions to each other in the complex plasmid of pYU300 in which the length of the insert DNA in the plasmid is 8.7Kb. We named them pYU302 and pUY302.

(8) Confirmation of gene The composite plasmid prepared by incorporating the 8.7 Kb Hind III fragment of pYU302 into Hind III of pBR322 is E. coli CGSC5075 and 507.
6 and 5077 strains were transformed, and the threonon-requiring marker of either strain was rescued. Therefore, this composite plasmid was presumed to have the thrA, thrB, and thrC genes.

(9) Threonine fermentation productivity Transformant containing pYU302 prepared by the method of the item (7)
P.rettgeri ATCC21118Thr ^- a (pYU302) was cultured in LB medium containing chloramphenicol 10 mg / l, in the methods of the literature 7, the culture solution 400ml per PYU302 give 10 [mu] g. Using this, P. rettgeri ATCC21118 was transformed by the method of Example 2 (5), and then LB containing 10 mg / l chloramphenicol was added.
A clone that grows on the medium was obtained. It was confirmed by restriction analysis that the plasmid contained in the bacterial cell was pYU302.
I confirmed that 02 entered P.rettgeri ATCC 21118.

A 500 ml Sakaguchi flask containing 10 mg / l chloramphenicol was placed in a medium (8% glucose, 2% ammonium sulfate, 0.1% KH ₂ PO ₄ , 0.04% MgSO ₄ .7H ₂ O, 10 ppm FeSO ₄ .7).
_{H 2 O, 7ppm MnCl 2 ·} 4H 2 O, 50ppm L- isoleucine, 4%
After culturing in 50 ml of CaCO ₃ (pH 7) at 30 ° C for 53 hours, the threonine content in the culture broth was quantified by an amino acid automatic analyzer.
The threonine content of ttgeri ATCC 21118 is 0.20 g / l, while pYU
P. rettgeri ATCC21118 (pYU302) containing 302 (Microtechnology Research Institute Article No. 1175) was 0.68 g / l, and threonine fermentation productivity was 3.4 times.

Example 2 Using the chromosomal DNA of a threonine analog AHV resistant mutant of P. rettgeri ATCC 21118 (Ministry of Industrial Science and Technology No. 8079), based on the orthotrophic complementation method shown in Example 1, the host threnion was used. The gene complementing the requirement was self-cloned. The composite plasmids pYU310 and pUY310 produced at this time had the size and restriction map shown in FIG. Both of these composite plasmids are insert DNA
Were inserted in opposite directions.

PYU31 of AHV resistant bacterium of P. rettgeri (Microtechnology Research Institute No. 8079)
The transformant of 0 was cultured for 45 hours by the method described in Example 1 (9), and the fermentative productivity of threonine was examined.
The productivity of non-transformants is 2.31 g / l, while 3.14 g
/ l was 1.4 times that.

Example 3 (1) Preparation of vector DNA pBR322 manufactured by Takara Shuzo Co., Ltd. was completely digested with a restriction enzyme Hind III, and then treated with alkaline phosphatase according to Reference 4.

(2) Cloning of L-threonine gene According to the method of Example 1, Hind III chromosomal DN of P. rettgeri ATCC 21118
After ligating the A fragment to the vector prepared in section (1), the ligation mixture was used to transform the host E. coli M
After transformation of an L-threonion-requiring mutant of M294,
A transformant clone in which the L-threonine marker was rescued was obtained. The smallest of the intracellular plasmids had a foreign gene fragment of 8.7 Kb.

(3) Preparation of L-threnion fermentation-producing bacterium The 8.7 Kb fragment obtained in the above (2) was inserted into the Hind III site of pYU300 according to the method of Example 1 (6) to form a composite plasmid pYU302, This was introduced into P. rettgeri ATCC 21118.

The transformant having this composite plasmid was cultured for 48 hours by the method described in Example 1 (9), and the fermentation productivity of L-threonine was examined. As a result, the productivity of the non-transformant was 0.
While it was 18 g / l, it was 0.70 g / l, which was 3.9 times.

References 1. Saito Hinata: Protein, nucleic acid, oxygen 11,446〜450 (196
6) 2.V.Hershfield et al: Proc.Natl.Acad.Sci, USA71, p
3455 ~ 3459 (1974) 3. Katsuya Shigetada: Cell Engineering 2,616 ~ 626 (1983) 4. RW Davis et al .: Advanced Bacterial Genetics, A Ma
nual for Genetic Engineering p738〜739 (1980) 5.Ibid p227〜230 6.HC Birnboim and J.Doly: Nucleic Acid Res7,1513〜
1523 (1979) 7. T. Maniatis et al .: Molecular Cloning, A Laboratory
Manual p86-96 (1982) Cold Spring Harbor Laborator
y, New York 8. Tatsuo Ishikawa, Ed., "Microbial Genetics Experimental Method," p86-88 (1982)
Kyoritsu Publishing

[Brief description of drawings]

FIG. 1 shows a restriction map of plasmid pYU300, FIG. 2 shows a restriction map of plasmids pYU302 and pUY302, and FIG. 3 shows a restriction map of plasmids pYU310 and pUY310.

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Claims (3)

[Claims] 1. A transformant of a bacterium of the genus Providencia or Escherichia having a recombinant DNA of a plasmid pYU300 represented by the following restriction map, which contains a gene controlling L-threonine production derived from a strain of the genus Providencia. A method for producing L-threonine, which comprises culturing and collecting L-threonine produced and accumulated in the medium. 2. The method for producing L-threonine according to claim 1, wherein the transformant is a bacterium of the genus Providencia. 3. The method for producing L-threonine according to claim 1, wherein the gene controlling L-threonine production is derived from Providencia red geri.