JPS62143691A - Production of amino acid - Google Patents

Abstract

PURPOSE:To produce an amino acid at a low cost, by reacting an amino group- donor with beta-phenylpyruvic acid or 3'-hydroxy-beta-phenylpyruvic acid in an aqueous medium in the presence of microbial cells of a specific transformant. CONSTITUTION:A gene originated from microorganism of Citrobacter genus and coding an enzyme having transaminase activity participating in the formation of L-phenylalanine or L-tyrosine is integrated to obtain a recombinant plasmid. The recombinant plasmid is introduced into a host microorganism to obtain a transformant. beta-Phenylpyruvic acid or 3'-hydroxy-beta-phenylpyruvic acid is made to react with an amino group-donor in the presence of said transformant cell or its treated product and objective L-phenylalanine or L- tyrosine is separated from the produced reaction liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a process for producing amino acids such as L-phenylalanine and L-tyrosine. More specifically, a recombinant plasmid derived from a microorganism belonging to the genus Citrobacter and containing a gene encoding an enzyme having transaminase activity involved in the production of L-phenylalanine or L-tyrosine is introduced into a host microorganism, In the presence of the cells of the resulting transformed strain or its processed material, t-β-phenylpyruvate or 3'-hydroxy-β-phenylpyruvate in an aqueous solution is reacted with an amino group donor, and L is added to the solution. - A method for producing amino acids by producing and collecting phenylalanine or L-tyrosine. L-phenylalanine and L-tyrosine are useful amino acids that can be used in the food and pharmaceutical industries.

Conventional technology Conventionally, a recombinant plasmid containing a gene encoding i.transaminase derived from large intestine atresia [9.Romonsho 59-1
92090. Unexamined Japanese Patent Publication ShoG O-62992.

Journal of Biochemistry (J, Bioc
hemistry) Vol. 97, pp. 993-999 A recombinant plasmid containing a gene encoding an enzyme having transaminase activity derived from a microorganism is not known.

Problems to be Solved by the Invention Transaminase converts α-ketocarboxylic acid into corresponding amino acids, such as L-7 enylalanine, L
- It is an enzyme useful for producing L-amino acids such as tyrosine, L-serine, L-methionine, L-valine, L-isoleucine, L-leucine, and L-tryptophan. Conventionally known methods produce a low amount of transaminase.

Means for Solving the Problems The present inventor extracted a gene encoding an enzyme having transaminase activity involved in the production of mustard phenylalanine or L-tyrosine from a Citrobacter bacterium, incorporated it into a plasmid, and recombined it. It has been discovered that microorganism cells having high transaminase activity can be obtained by creating a transaminase plasmid, introducing the plasmid into a microorganism, and culturing the obtained transformant, and furthermore, the microorganism cells or a processed product thereof can be obtained. The corresponding amino acids such as L-phenylalanine and L-
The present invention was completed by discovering that tyrosine can be produced at low cost.

Structure of the Invention The present invention involves introducing into a host microorganism a recombinant plasmid derived from a microorganism of the genus Citrobacter and incorporating a gene encoding an enzyme having transaminase activity involved in the production of L-phenylalanine or L-tyrosine. The present invention provides a method for producing corresponding amino acids such as L-phenylalanine and L-tyrosine from α-ketocarboxylic acids using the cells of the resulting transformed strain or processed products thereof as an enzyme source.

The gene encoding the enzyme having transaminase activity (hereinafter referred to as transaminase gene) can be obtained from the chromosomal DNA of a microorganism belonging to the genus Citrobacter. The chromosomal DNA donor bacterium may be a wild strain or a mutant strain.

The transaminase gene is transferred to the chromosome D from the donor bacterium.
Extraction of NA, partial cleavage of the DNA with a restriction enzyme, introduction of a genetic DNA fragment into a specific plasmid, transformation of a transaminase-deficient mutant strain using a silk recombinant plasmid, and a trait that complements the amino acid requirement due to transaminase deficiency. It is cloned in a process by selecting convertible strains.

The Citrobacter bacterium used as a transaminase gene donor includes, for example, Citrobacter freundi.
i) ATCC6750 is mentioned.

Chromosome D, donor DNA, from Citrobacter bacteria
The method for isolating NA is the method of Marmur [Journal of Molecular Biology (
J, Molecular Biology) Volume 3,
208, (1961)).

The transaminase-encoding gene DNA obtained from Citrobacter freundii used in the examples has approximately 4.0 kilobases (hereinafter referred to as Kb) and 0.7 kilobases.
Ligation of DNA fragment digested with restriction enzyme PstI of K b (
Figure 1) and about 8. OK b restriction enzyme HindllI
This is a digested DNA fragment (Figure 2).

The vector is not particularly limited as long as it can be replicated within the recipient bacterium, but in the present invention, cloning is carried out in an E. coli host-vector system, so specifically, vectors such as pBR322 and pAcYcl 77 derived from E. coli are used. etc.

If the strain used for transformation to obtain a recombinant plasmid containing the above-mentioned transaminase gene DNA is an auxotrophic mutant strain due to a combination defect and is suitable for the replication of the vector used, Any microorganism may be used. In the present invention, pBR322, pA
Since we use E. coli-derived vectors such as cYc177, the recipient bacteria are E. coli capable of replication.
A transaminase-deficient strain of the strain is used. Specifically, E. coli transaminase A (aspC), B
(i 1vE), D (tyrB)-deficient mutant strain [for example, Escherichia coli DC, strain 30: This strain was sent to the Institute of Microbial Technology (Feikoken), Agency of Industrial Science and Technology on September 4, 1985.
By date, ε5cherichia coli K59
FERM BPloo] is used. Previous studies have shown that in E. coli, transaminase A (EC2,6,1,1) has a corresponding α-
Transaminase B (EC2,6,1,42') catalyzes the biosynthesis of aspartic acid, tyrosine, and phenylalanine from ketocarboxylic acids; similarly, transaminase B (EC2,6,1,42') catalyzes the biosynthesis of phenylalanine, leucine, valine, isoleucine, and transaminase D (EC2,6,]-,
5) is known to catalyze the biosynthesis of aspartic acid, tyrosine, and phenylalanine. Amino acid (aspartic acid.

Tyrosine, phenylalanine, leucine, valine.

It is an auxotrophic mutant strain that requires a combination of isoleucine (isoleucine) for growth. A mutant strain with these properties is used as a recipient strain and grown on a medium containing a combination of the above amino acids (aspartic acid, tyrosine, phenylalanine, leucine, valine, isoleucine) with one type of amino acid removed. By selecting transformants that complement the corresponding requirements, genes encoding transaminase activities involved in the biosynthesis of the corresponding amino acids can be cloned. Favorable results can be obtained by mutating these recipient bacteria to reduce their restriction enzyme activity before use.

When these recipient bacteria are transformed using a recombinant plasmid containing a transaminase gene, they express the information contained in the transaminase gene on the plasmid.

In particular, when transaminase gene DNA is inserted using a multicopy plasmid as a vector, the D''NΔ increases significantly, and the transaminase activity in the bacterial cells increases significantly.

The transaminase gene on the recombinant plasmid possessed by the transformant thus obtained can also be circularly cloned in other host-vector systems.

I transaminase gene DN in the vector plasmid
To insert A, cut the plasmid with an appropriate restriction enzyme depending on the vector plasmid used, and ligate the cut plasmid fragment with genetic DNA cut with the same restriction enzyme that cut the plasmid. be able to.

In order to incorporate a recombinant plasmid containing transaminase gene DNA into E. coli, Citrobacter bacteria, and other recipient bacteria, the calcium chloride method [Advanced Bacterial Genetics (8 advanced
Bacterial Genetics), :]-/
l/Tspring Harbor Publishing 140 pages, 1980
], Lithium Method [Journal of Bacteriology (J, Bacteriology) '+15
3, p. 163.

1983], Method for transforming protoplast cells (Japanese Patent Publication No. 57-186492.
) etc. are used.

Recombinant plasmids ampicillin recipient bacteria.

Since bacteria are transformed into bacteria resistant to drugs such as kanamycin, specdinoma f-cin, streptomycin, and chloradophenicol, the transformed strain containing the recombinant plasmid into which the transaminase gene DNA has been inserted can be tested for drug resistance. Can be easily identified.

L-phenylalanine thus obtained or haL--
F-Involved in the production of rosin. A microorganism into which a recombinant plasmid containing transaminase gene DNA has been introduced can be cultured in a normal medium to accumulate transaminase in the microorganism.

The medium contains carbon sources, nitrogen sources, and inorganic ions, as well as vitamins and amino acids as necessary.

Conventional media containing small amounts of organic nutrients such as organic acids can be used.

As the carbon source, gluco-sucrose-lactose, starch, starch hydrolyzate, blackstrap molasses, etc. are used. Nitrogen sources include gaseous ammonia, liquid ammonia, and ammonia compounds.

Urea and other nitrogen-containing compounds can be used.

Detection of transaminase activity in microbial cells is a method of analytical biochemistry (analytical biochemistry).
l Biochemistry) 1 0 0
This can be carried out by the method (coupled MDH method) described in Vol. 20-24, 1979.

Culture is carried out by aeration and agitation culture at 1Lkp83-9, temperature 20-40°C for 1-40 hours. The bacterial cells with high transaminase activity thus obtained can be used as they are in the reaction, or the treated cells obtained by various treatments of the bacterial cells can be used in the reaction.

Examples of microbial cell-treated products include mechanically ground microbial cells;
Used are sonicated products, freeze-dried products, solvent-treated products, enzyme-treated products, dry-treated products, surfactant-treated products, protein fractions of bacterial cells, and immobilized products of bacterial cells and treated bacterial cells.

The reaction is carried out in an aqueous solution by combining the bacterial cells obtained above or the treated product with β-phenylpyruvic acid or 3'-hydroxy-β-phenylpyruvic acid and the amine group donor L-glutamic acid or L-asparagine. M, - & or fumaric acid and ammonium ion) by the
Phenylalanine or L-tyrosine can be produced.

β-phenylpyruvic acid and 3'-hydroxy-β-phenylpyruvic acid used in the reaction, and the amino group donor used was an ammonia compound and strium salt.

It is used as various salts such as potassium salt and calcium salt.

Ammonium ions include ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium chloride,
Ammonium acetate, ammonium formate.

It is supplied as salts such as ammonium fumarate and ammonium malate, or as ammonia water or ammonia gas.

The concentration of α-ketocarboxylic acid used in the reaction is 0.
01-2 mol, the concentration of amino group donor is 0.01
~4 mol, 0.01 as ammonium ion concentration
It is used in a range of 10 moles.

These raw materials are supplied in bulk or intermittently.

The working conditions include a temperature of 20 to 60°C, preferably 25°C.
~45°C, pH 6-10, preferably 7-9, 10-45°C
Do it for 48 hours.

In this way, L-amino acids are produced in the aqueous liquid. As a method for recovering monoamino acids from the reaction solution, conventional methods used for recovering amino acids, such as an ion exchange resin method and a precipitation method, are used.

Examples are shown below.

Example 1 Cloning of Citrobacter transaminase gene: Cloning was carried out in an E. coli host-vector system. The pΔCYC177 plasmid was used as a vector. The donor DNA, chromosomal DNA, was derived from Citrobacter freundii ΔTCC6750 -r--y
- (Marmur) [Journal of Molecular Biology (J, Mo.
1ecular Biology).

Volume 3, page 208, 1961].

The pAcYcl77 plasmid used as a vector was derived from Adonocyst bacterial geneticus (8advancedBac) from pΔCYC177-carrying bacteria.
terial Genetics), Cold Spring November Publishing, p. 116, 1.980.

Reaction solution for restriction enzyme Pstl (10 g
mMJS hydrochloric acid buffer, 7mM MgCjL, 60mM
NaCff, 7mM 2-mercaptoethanol, pl(8,0) 110μβ in lO units of Pstl
(manufactured by Takara Shuzo Co., Ltd.) was added, and after reacting at a temperature of 37°C for 60 minutes, the reaction was stopped by heating at 65°C for 10 minutes. To the reaction digest, T4 ligase reaction solution (660mM) IJS hydrochloric acid buffer, 66mM MgCl2, 100mM dithiothreyl), pH 7.6) 20μβ, 5mM Δ
20 μA of TP, 0.8 units of T4 ligase (manufactured by Takara Shuzo Co., Ltd.) and 50 μm of water were added, and the mixture was allowed to react at 4° C. for 24 hours.

This ligase reaction product was applied to the E. coli mutant strain 59 (F
ERM BP'-900> was transformed. K5
The 9 strains of competent cells were from Dagert (Dagert).
) et al.'s method [Gene, vol. 6, 2
3, 1979].

Namely, L-medium (Bactodrypton 1%).

Yeast extract 0.5%, NaCj! 0.5%, glucose 0.1%, pH 7,2) 59 to 50ml
The strain was inoculated and cultured at 37°C until the absorbance at 660 nm (hereinafter abbreviated as 0.D) reached 0.6 using a Tokyo Photoden colorimeter. After cooling the culture solution in ice water for 10 minutes, the bacterial cells were
Cells were collected by centrifugation at 8,000×g for 15 minutes at ℃, and cooled to 0.
, suspended in 20 ml of IM calcium chloride at 0°C.
It was left standing for 0 minutes. After that, the bacterial cells were heated to 20,000x at 4°C.
g, Bacteria were collected by centrifugation for 10 minutes, resuspended in 1 ml of 0.1M calcium chloride solution, and left at 0°C for 1 hour. The bacterial liquid 2
20 μl of the ligase reaction product was added to and mixed with 00 μβ, allowed to stand at 0° C. for 30 minutes, and then heated at 37° C. for 5 minutes. Next, 2 ml of L-medium was added and cultured with shaking at 37°C for 2 hours. After centrifugal washing twice with physiological saline, 20 μg/ml of kanamycin and phenylalanine were added.

25 μg/m each of valine, inleucine, leucine, aspartic acid, glutamine, histidine, proline],
Thiamine 0.1 μg/ml was added. M9 minimal agar medium (Na2HPO40.6%, KH2PO40.3%,
NaClO, 05%, NH4cl 0.1%.

Glucose 0.4%, MgSO4・7H200.02%
, Ca Cj! 2 ・21-120 0. O1
%, Batato agar 1.5%) and cultured at 37°C for 3 days.

The resulting transformed strain that was kanamycin resistant and non-tyrosine auxotrophic was isolated as a single colony on an agar medium containing 20 μg/ml of kanamycin.

From the purified transformed strain, the method of An et al. [Journal of Bacteriology (J.
ology) l 4 Q volume, 400 pages, 1979]
The plasmid was isolated by. This plasmid was treated with restriction enzymes Pstl, HinlH, and BamHI.

After digestion with XhoI and EcoRI, agarose gel electrophoresis [Methods in Enzymology]
ods in Enzymology), Volume 53
152 (1979)), the plasmid contained 4. OK
It was found that a PsLT fragment of 0.7 K b and 0.7 K b was inserted. Also, BamHI is present in the insert.

There were no cleavage sites for XhoI and HindIrr. This plasmid was named pT832 (see Figure 1).

Example 2 Vector plasmid pΔCYC177 and chromosomal DNA of Citrobacter freundii ATCC6750
For the digestion, restriction enzyme Hind■ (manufactured by Zenshuzo Co., Ltd.) and reaction solution for HindlI (10mM), Lis-HCl buffer,
7mM MgCjL.

The same procedure as in Example 1 was carried out, except that 50mM NaCC (pH 7.5) was used and ampicillin (50 μg/ml) was used instead of kanamycin as the drug used to select the transformed strain.

As a result, a transformed strain resistant to ampicillin and non-tyrosine auxotrophic was isolated, and analyzed in the same manner as in Example 1.
At the HindllJ cleavage site of Ycl 77, approximately 8. OK
It was found that the Hind■ fragment of b was inserted. In addition, BamHI, XhoI, Hind
There was no cleavage site for llI. Transfer this plasmid to pTA
It was named C33 (see Figure 2).

Example 3 A crude enzyme extract was prepared by disrupting cultured E. coli cells harboring pTAC32 and pTAC33 by ultrasonication and removing cell residue by centrifugation. When the transaminase activity in the crude enzyme extract was measured by the coupled MDH method, pT8 to 32 or p
In Escherichia coli strain 59 (FERMBP'-900) carrying 'rΔC33, transaminase activity 2 to 7 times that of the wild strain was detected. The transaminase activity in the crude enzyme extract of the plasmid-free strain was
was hardly detected (see Table 1).

Table 1 *Protein quantification was performed using a Bio-rad white matter assay kit.

This shows that the transaminase gene derived from Citrobacter freundii is cloned onto the p'rAc32 and pTAC33 plasmids. In addition, in the pTAC32-carrying strain, the improvement in transaminase activity was more pronounced when β-phenylpyruvate or 3'-hydroxy-β-phenylpyruvate was used as a substrate than when α-ketoglucuric acid was used as a substrate. Thank you for your patience. From this point, pTA
It is predicted that the C32+ cloned transaminase gene encodes a transaminase with high specificity for aromatic amino acids.

Example 4 Plasmid pTAC32 or pTΔC described in the previous section was introduced into transaminase-deficient E. coli strain 59 and 59.
33 and four types of E. coli, including the wild strain and -12, were grown in large test tubes containing the following growth medium (lQml).
200 x 22 mm) was inoculated to 1 plant, and incubated at 28°C for 18
After shaking culture on a test tube shaker (21 rpm) for an hour,
8.000. Centrifuge for 15 minutes at rpm to collect the bacterial cells, and add 0.9% NaCj! It was centrifuged and washed once. The total amount of these bacterial cells was mixed with β-phenylpyruvate or 3'-hydroxy-β-phenylpyruvate at 1.0
0mM.

pH 8, containing 200mM sodium L-aspartate, 0.2mM pyridoxal-5'-phosphate,
In a 20 ml Erlenmeyer flask containing 5 ml of 3 aqueous solution, 3
The mixture was allowed to stand for 4 hours at a temperature of 7°C to react.

The mono-phenylalanine and L-tyrosine produced in the reaction solution were developed using paper chromatography (n-butanol:acetic acid:water=4:1:1), air-dried, and added to an acetone solution containing 0.1% ninhydrin. It was quantified according to the conventional method of soaking and developing color. The values are shown in Table 2. As is clear from this table, the plasmid-carrying strain has significantly improved ability to produce 7-phenylalanine and L-tyrosine.

Growth medium composition; glucose 3%, asimonium sulfate 0.
6%, corn steep liquor 0.5%.

K H2P Os 0.05%, K21-I P 0
40.05%.

MgS○s ・71■20 0.025%, CaCO3
2%, pH 6.8 Table 2 * Amount of amino acid produced per bacterial cell corresponding to 0, D, -1, 0 Example 5 Introduction of pTAC32 into Citrobacter freundii: Introducing 32 plasmid into pT8 Citrobacter freundii ATCC 6750 was transformed using the vector. A
Competent cells of TCC6750 were prepared by the calcium chloride method in the same manner as in Example 1. Approximately 4 μg of pTΔC32 was added to 200 μp of competent cell bacterial solution and mixed, and transformation was performed in the same manner as in Example 1. The transformed strain was selected as a kanamycin-resistant strain that grew on an agar medium containing 20 μg/ml of kanamycin at 37° C. for 1 to 2 days.

The reaction was carried out in the same manner as in Example 4, except that the cultured cells of Citrobacter freundii carrying this pTAC32 were suspended in the reaction solution at a concentration of 0.0.1.0, and phenylpyruvic acid was converted to phenylalanine. (see Table 3).

As a result, the conversion activity of phenylpyruvic acid per unit amount of bacterial cells was improved.

Table 3 By the method of the present invention, a recombinant plasmid containing a gene encoding an enzyme having transaminase activity can be obtained. Using the recombinant plasmid,
Since the transaminase activity of Escherichia coli or Citrobacter microorganisms can be enhanced, transaminase can be produced in large quantities at low cost. and,
By using bacteria with enhanced transaminase activity, the corresponding amino acids such as L
- Phenylalanine and L-tyrosine can be efficiently produced.

[Brief explanation of drawings]

FIG. 1 shows the restriction enzyme cleavage map of pTAC32 and its production process. Figure 2 shows the restriction enzyme cleavage map of 33 to pT8 and in the figure, P
is PstI, H is HindIII, B is BamHI, X
represents the cleavage site of xhO■ and E represents the EcoRI cleavage site. The numbers represent the molecular weight of each cleaved fragment. Molecular weights of plasmids are given in kilobases (Kb). Also, Ap
Km represents the site that confers ampicillin resistance, and Km represents the site that confers kanamycin resistance. The manufacturing process is shown below. Figure 1

Claims (3)

[Claims] (1) Characteristics obtained by introducing into a host microorganism a recombinant plasmid derived from a microorganism of the genus Citrobacter and containing a gene encoding an enzyme having transaminase activity involved in the production of L-phenylalanine or L-tyrosine. L-phenylalanine or L-phenylalanine or A method for producing an amino acid, which comprises producing L-tyrosine and collecting it. (2) The method according to claim 1, wherein the host microorganism is a microorganism belonging to the genus Citrobacter or the genus Escherichia. (3) The method according to claim 2, wherein the host microorganism is a microorganism belonging to Citrobacter freundii or Escherichia coli.