JP2003284579A - Method of producing 2-ketobutyric acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To produce 2-ketobutyric acid more economically. <P>SOLUTION: A microorganism into which threonine deaminase is incorporated is cultured and an adequate amount of cell bodies or extract from crushed cell bodies are added to a reaction mixture including threonine and whereby 2-ketobutyric acid is effectively produced from threonine. The resultant 2- ketobutyric acid formed in the reaction mixture is separated and purified in usual manners. When the gene is introduced, the lac promoter or T7 promoter is employed. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing 2-ketobutyric acid, 2-ketobutyric acid ester, and 2-ketobutyric acid salt. Keto acids including 2-ketobutyric acid are used as synthetic raw materials for pharmaceutical intermediates and the demand for them is increasing.

[0002]

2. Description of the Related Art Conventionally, as a method for industrially synthesizing 2-ketobutyric acid, a method is known in which diethyl propionate and diethyl oxalate are condensed and then hydrolyzed with concentrated sulfuric acid. Further, as a method for synthesizing 2-ketobutyric acid using a microorganism, a method for producing 2-ketobutyric acid from 1,2-butanediol using a microorganism of the genus Rhodococcus (Japanese Patent Application Laid-Open No. 2-207783), Pseudomonas (Pseudo)
A method for producing 2-ketobutyric acid from 2,3-butanediol using a microorganism belonging to the genus monas (JP-A-3-183476) has been reported. In addition, it was reported that threonine deaminase was expressed by incorporating threonine deaminase and a modified pheA promoter into Escherichia coli (Journal of Molecular Cat.
alysisB: 199, (2000), US Pat. No. 6,1975,58).

[0003]

[Problems to be Solved by the Invention]
It is hard to say that -butanediol and 2,3-butanediol are industrially inexpensive, and the concentration of 2-ketobutyric acid produced was also unsatisfactory. Furthermore, the above report (Journal of Molecular Catalysis B: 199, (200
0) and US Pat. No. 6,1975,58), the method described in 2-
Not enough ketobutyric acid accumulated.

An object of the present invention is to provide a highly efficient industrial production method of 2-ketobutyric acid by a microorganism.

[0005]

In order to solve the above-mentioned problems, the inventors of the present invention have conducted diligent research on a method for producing 2-ketobutyric acid, which has higher productivity, and as a result, have incorporated the threonine deaminase gene into microorganisms. The present inventors have found that the concentration of 2-ketobutyric acid accumulated and the production yield are improved by enzymatically converting threonine using the microorganism, and arrived at the present invention.

[0006] That is, the present invention "uses a microorganism or an extract of the microorganism in which a gene encoding threonine deaminase is inserted so that the gene is expressed under the control of the lac promoter or T7 promoter, and 2-ketobutyric acid is extracted from threonine. 2 characterized by producing
-Method for producing ketobutyric acid. , Culturing and recovering a microorganism incorporating a gene encoding threonine deaminase, adding the microorganism or an extract thereof to a reaction solution containing threonine, and accumulating 2-ketobutyric acid in the reaction solution, A method for producing 2-ketobutyric acid, which comprises recovering 2-ketobutyric acid from the reaction solution.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Threonine is deaminated by the action of threonine deaminase to produce 2-ketobutyric acid.

The host microorganism used in the present invention is not particularly limited as long as it is a microorganism, but bacteria are preferably used, and Escherichia coli (Esherichia c) is particularly preferable.
oli).

In addition, microorganisms may have other properties such as drug resistance and auxotrophy.

The gene encoding threonine deaminase to be incorporated into the host is not particularly limited as long as it is expressed by the host and retains the activity as a threonine deaminase, but is not limited to Escherichia coli.
i), Salmonella typh
imurium), Pseudomonasputi
da), Corynebacterium glutamicum (Corynebact
erium glutamicum), Saccharomyces cerevisiae, Candida utilis, and other genes are known.
These are preferably used.

The method for obtaining the threonine deaminase gene is not particularly limited, and a PCR method, a screening method from a genomic library or a cDNA library and the like are used.

The obtained gene is incorporated into pBR322 having a ColE1 type origin of replication, pSTV28 having a pACYC type origin of replication and their derivative vectors. Particularly preferably
pUC18 / 19 and pET16b vectors (Novagen) are used. As a promoter that controls the expression of threonine deaminase, the lac promoter or T7 promoter is used.

The introduction method is not particularly limited, but for example, in the case of Escherichia coli, the calcium chloride method, electroporation method or the like is used.

By the method as described above, bacterial cells expressing a large amount of the threonine deaminase gene can be obtained.

The method for evaluating 2-ketobutyric acid accumulation in the present invention will be described. 2-Ketobutyric acid is evaluated by high performance liquid chromatography according to a conventional method. The culture method in the present invention will be described. The medium for expressing threonine deaminase is an ordinary medium containing a carbon source, a nitrogen source, inorganic ions and, if necessary, other organic trace components, but is not particularly limited as long as threonine deaminase is expressed.

The culture is usually carried out under aerobic conditions, but the conditions can be appropriately changed depending on the expression of threonine deaminase. The culture temperature is 15 ° C to 45 ° C, preferably 20 ° C to 35 ° C.

[0024] Under these conditions, preferable results can be obtained by shaking or stirring culture for 24-120 hours. When an inducible promoter is used, it is desirable to add an inducer appropriately during culture.

The bacterial cells expressing threonine deaminase are used in an enzymatic reaction after collecting the bacteria. At this time, it may be used as it is, or an extract obtained by crushing the cells may be used.

The cells or the extract are appropriately added to a buffer containing an appropriate amount of threonine, and the mixture is shaken or stirred to
-The formation of ketobutyric acid is seen. Any buffer may be used as long as it has a pH buffering action, and potassium phosphate buffer, sodium phosphate buffer, sodium acetate buffer and the like are used.

The amount of threonine added is not particularly limited, but is preferably 10 mM to 1M.

The 2-ketobutyric acid formed in the reaction solution can be used as it is without being isolated and purified, or can be isolated and purified by a conventional method.

The 2-ketobutyric acid thus obtained can be converted into an ester or salt by a known method.

[0023]

EXAMPLES The present invention will be specifically described below with reference to examples.

Example 1 (1) Cloning of the threonine deaminase gene In order to produce 2-ketobutyric acid from threonine, the threonine deaminase gene was cloned.

The known nucleotide sequence of the threonine deaminase gene is a PC for Esherichia coli based on the sequence in the database (GenBank).
The R primer was designed. The sequences of the primers used for PCR are shown in Sequence Listing 1 and Sequence Listing 2.

Using these primers, Esherichia c
PCR was performed using the genomic DNA of the oli K12 strain as a template, and 1578
A base-pair amplified fragment was obtained. This amplified fragment was subcloned into the HincII cleavage site of pUC18, which is an E. coli vector, and the base sequence was confirmed.
It was confirmed that it encodes the threonine deaminase gene of herichia coli.

(2) Preparation of expression vector After digesting the DNA fragment amplified in (1) with NdeI and BamHI,
PET16b vector with T7 promoter (Novagen)
Was inserted into the NdeI-BamHI site of pETTDA to construct a pETTDA plasmid (Fig.).

(3) Introduction of the expression vector into the host The expression vector pETTDA prepared in (2) was transformed into Esherichia col.
It was introduced into the iBL21 (DE3) strain (Novagen) using the calcium chloride method.

After the introduction of the expression vector, the recombinant strain was selected with the resistance to the antibiotic ampicillin as an index to obtain a transformant. This transformant was named Esherichia coli TDA strain.

Example 2 (Expression of threonine deaminase in transformant) Esherichia coli BL21 (DE3) strain and Esherichia coli TDA strain were cultured. That is, each of these strains was added to LB medium 5
1 platinum loop was inoculated in ml and precultured by shaking at 37 ° C. for 16 hours.

Next, the preculture liquid was added to 10 ml of a fresh LB medium.
Was inoculated with 1% and cultured at 37 ° C. for 6 hours with shaking. Isopropylthio-β-D-galactopyranoside (IPT
G) was added to a final concentration of 1 mM, and then 37 ° C.
It was shaken for 16 hours. Subsequently, the culture solution was centrifuged to obtain bacterial cells.

Example 3 (Production of 2-ketobutyric acid using transformant) To a solution obtained by adding 0.3 g of threonine to 10 ml of 50 mM potassium phosphate buffer solution, 10 ml of the obtained bacterial cell culture solution was added. Three
The enzymatic reaction was carried out at 0 ° C. for 24 hours.

After completion of the reaction, quantification was performed using high performance liquid chromatography. Column is SCR-101H (Shimadzu)
The buffer solution was 0.1% phosphoric acid, and the absorbance at 210 nm was measured. The results are shown in Table 1 below.

[0034]

[Table 1]

Control Esherichia coli BL21
Escherichia coli TDA, which is a recombinant strain compared to the (DE3) strain
The strain showed a significant accumulation of 2-ketobutyric acid.

Example 4 (2-using a transformant extract)
Ketobutyric acid production) To a solution obtained by adding 0.6 g of threonine to 10 ml of 50 mM potassium phosphate buffer solution, the supernatant obtained by ultrasonically crushing 1 ml of the obtained bacterial cell culture solution was added, and the enzyme reaction was performed at 30 ° C for 24 hours. went.

After the reaction was completed, the amount was quantified by high performance liquid chromatography. Column is SCR-101H (Shimadzu)
The buffer solution was 0.1% phosphoric acid, and the absorbance at 210 nm was measured. The results are shown in Table 2 below.

[0038]

[Table 2]

Control Esherichia coli BL21
Escherichia coli TDA, which is a recombinant strain compared to the (DE3) strain
The strain showed a significant accumulation of 2-ketobutyric acid.

[0040]

EFFECT OF THE INVENTION Using the microorganism of the present invention, the enzyme method 2
-Production of ketobutyric acid in the reaction liquid enables more economical production of 2-ketobutyric acid as compared to existing methods.

[0041]

[Sequence list]                               SEQUENCE LISTING <110> TORAY <120> Method for producing 2-ketobutyric acid <130> 51E16560 <160> 2 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR primer to obtain Threonine deaminase from Esherichia coli <400> actggggggt tacatatggc tgactcgcaa <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR primer to obtain Threonine deaminase from Esherichia coli <400> gctatcaggc atggatccct aacccgccaa

[Brief description of drawings]

Figure 1: Threonine deaminase expression vector pETTDA
It is a figure which shows the physical map of.

Claims (6)

[Claims] 1. A method of producing 2-ketobutyric acid from threonine using a microorganism or an extract of the microorganism in which a gene encoding threonine deaminase is expressed so as to be expressed under the control of lac promoter or T7 promoter. A method for producing 2-ketobutyric acid, which is characterized. 2. A gene encoding threonine deaminase is a genus of Esherichia, Salmonella (Sa).
Lmonella), Pseudomonas, Corynebacterium, Saccharomyces, Candida
The method for producing 2-ketobutyric acid according to claim 1, which is derived from any of the genera. 3. The method for producing 2-ketobutyric acid according to claim 1 or 2, wherein the microorganism is a bacterium. 4. The method for producing 2-ketobutyric acid according to claim 3, wherein the microorganism is Escherichia coli. 5. The obtained in any one of claims 1 to 4.
-A method for producing a 2-ketobutyric acid ester, which comprises esterifying ketobutyric acid. 6. The obtained in any one of claims 1 to 4.
-A method for producing 2-ketobutyric acid salt, which comprises neutralizing ketobutyric acid to form a salt.