JPH0544277B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a complex recombinant DNA, a prokaryotic cell containing the DNA, and a method for producing L-dopa using the cell.

[Prior art and its problems] β-tyrosinase is known as an enzyme that performs a reaction to produce L-dopa (L-dihydroxyphenylalanine) from catechol, pyruvic acid, and ammonia or ammonium salt (FEBS, Lett. ., 21 , 39-41, 1972). Various microorganisms having this β-tyrosinase activity are known, including Erwinia herbicola , and methods for producing L-dopa using these microorganisms have also been proposed.

However, there were problems such as inconvenience in handling microorganisms having β-tyrosinase activity and insufficient yield of L-dopa.

On the other hand, Pseudomonas is a microorganism that produces catechol from benzene.
Pseudomonas putida strain 98-136 (FERM P-6970) and the like are known, which belong to the genus Pseudomonas putida and have a deletion or reduced activity of an enzyme that degrades catechols.

The present inventors investigated the use of benzene, which is cheaper and more stably available than catechol, as a raw material for producing L-dopa.

[Means for solving the problem] First, the benzene oxygenase and benzene glycol dehydrogenase genes and the β-tyrosinase gene, which are genes involved in the production of catechol, are isolated, and a composite recombinant DNA in which these genes are linked is obtained. By introducing DNA into Escherichia coli, we succeeded in obtaining a microorganism that simultaneously possesses catechol production ability and β-tyrosinase activity. By using this microorganism, benzene can be used as a raw material instead of catechol, and the target L-dopa can be efficiently produced.

That is, the present invention first provides a region encoding the amino acid sequence of the enzyme protein shown below, and a gene containing β-tyrosinase activity, having a base pair number of 1.7 Kb, and containing the restriction enzyme EcoRI in its DNA strand. The present invention relates to a complex recombinant DNA containing a β-tyrosinase gene derived from Erwinia herbicola, including a site that is cleaved at.

The second invention relates to prokaryotic cells transformed with the above complex recombinant DNA. Although there are various types of prokaryotes, Escherichia coli (Escherichia coli) is preferred.

A third aspect of the present invention is characterized in that benzene is reacted with pyruvic acid and ammonia or an ammonium salt in the presence of the prokaryotic cells or a processed product of the cells, and L-dopa is collected from the reaction solution. -Relating to a method for producing dopa.

The benzene oxygenase and benzene glycol dehydrogenase genes, which are catechol producing genes, need only contain at least the four regions described above, and their sequence order does not matter. Also,
This gene may contain a region encoding the amino acid sequence of the enzyme protein described below as a fifth region.

An example of the base sequence of a catechol-producing gene is shown in FIG. This DNA contains restriction enzymes in its DNA strand.
There are sites that are cleaved with EcoRI, SalI, PstI, and Hind; for example, the cleavage site with SalI is between the 1829th G and 1830th T, and the cleavage site with PstI is between the 2133rd C and 2134th T. between 4025th C and 4026th T and between 5269th C and
It is between the 5270th T. Furthermore, the cleavage site by Hind is between the 2172nd A and the 2173rd A.

This gene can be prepared as follows. Catechol-producing bacteria having benzene oxygenase and benzene glycol dehydrogenase genes, such as mutant strains belonging to the genus Pseudomonas, in which catechol-degrading enzymes are deleted or have low activity (for example, Pseudomonas putida strain 98-136, FERM P-6970) The gene region containing both of the above genes was extracted using a conventional method (for example, J.
Bacteriol. 89 , 1065 (1965), etc.),
Partially cut with the restriction enzyme Sau 3A to obtain the necessary
Obtain a DNA fragment (molecular weight approximately 30 Kb). On the other hand, cosmids derived from broad host range plasmid RSF1010
pMMB33 (Gene 24 , 299 (1983)) etc. is used as a plasmid vector, and this is injected with restriction enzymes SmaI,
They are cut separately with HpaI, mixed, then cut with BamHI, mixed with the DNA fragment, and ligated with T4 DNA ligase. Next, this is packaged in the shell of lambda phage, a virus that infects E. coli (E. coli), and infects E. coli to obtain an E. coli clone with a cosmid that incorporates chromosomal DNA (e.g., E. coli).
SI−300, FERM BP−1358).

The DNA fragment obtained by cutting the cosmid pGR300 held by E. coli with the restriction enzyme EcoRI was used as a plasmid pUC8 (Gene 19 ,
259 (1982)), the E. coli JM83 strain is transformed, and E. coli that retains the ability to produce catechols is selected.

A plasmid is extracted from this E. coli transformant and subjected to cleavage analysis with various restriction enzymes to create a restriction enzyme map. Then, based on this, restriction enzyme cleavage and exonuclease Bal
-31 to create deletion derivatives, and use E. coli strain JM83 carrying each deletion plasmid to determine the region containing the gene of interest.

The base sequence of the gene region obtained in this way is
Determined by M13 phage dideoxy method.

A protein-encoding region was searched from this base sequence, and the existence of regions 4 or 5 was confirmed.

Region 617th to 619th ATG sequence to 1961st to 1963th
A region with a molecular weight of 50208 (448 amino acids) having a sequence base up to the 2070th to 2072nd ATG sequences to the 2631st to 2072nd ATG sequences.
A region with a molecular weight of 22103 (187 amino acids) having a sequence base up to the 2633rd TAG sequence From the 2963rd to 2965th ATG sequences to the 4190th to 4190th
A region with a molecular weight of 42819 (409 amino acids) having a sequence base up to the 4192nd TGA sequence From the 4519th to 4521st ATG sequences to the 5014th to 5014th
A region with a molecular weight of 17221 (165 amino acids) having a sequence base up to the 5016th TGA sequence From the ATG sequence 2688th to 2690th to the 2961st to
Has a sequence base up to the 2963rd TGA sequence
91 amino acid region Furthermore, the β-tyrosinase gene can be prepared from a microorganism that retains β-tyrosinase activity as follows. Note that various microorganisms that retain β-tyrosinase activity are known; for example, Agr.Biol.Chem., 36 , No. 11,
Pages 1861-1868 (1972), Special Publication No. 1972-22275,
It is described in Publication No. 51-5475, etc. However, in the present invention, Erwinia herbicola ( Erwinia
herbicola ). Examples of Erwinia herbicola include ATCC21433 strain and ATCC21434 strain.

First of all, we started with a normal strain of Erwinia herbicola that retains β-tyrosinase activity.
Chromosomal DNA is extracted by the DNA extraction method (J. Mol. Biol., 3 , 208 (1961)) and cut with restriction enzymes to obtain chromosomal DNA fragments. On the other hand, after cutting a suitable vector plasmid with the same restriction enzyme, this is ligated to the above-mentioned chromosomal DNA. Next, this recombinant DNA is infected with a microorganism that does not have β-tyrosinase activity, such as Escherichia coli HB101 strain, using a conventional method (for example, J. Mol. Biol., 53 , 159
(1970)). After cloning is carried out using a conventional method, the isolated gene is confirmed to be the β-tyrosinase gene, and this gene region is miniaturized. That is, plasmid DNA was extracted from the transformed strain, and each of the DNA fragments obtained by partial digestion with restriction enzymes was ligated with a vector as described above, and E. coli strain HB101 was transformed to transform the desired gene region. Select the stocks you own. Next, a plasmid is extracted from the selected strain.
Extract the DNA, cut it with restriction enzymes, and make β
- Obtain the tyrosinase gene. This product is subjected to restriction enzyme cleavage analysis to create a restriction enzyme map (Figure 2).

As a result, Erwinia herbicola
In the case of the ATCC21434 strain, it was found that at least the minimum DNA fragment (1.7 kb) derived from its chromosomal DNA is required to express β-tyrosinase activity.

The catechol production gene and β-tyrosinase gene prepared as described above can be linked, for example, by the method described below to obtain a composite recombinant DNA.

A DNA fragment containing the β-tyrosinase gene obtained by miniaturizing the gene region, a similarly miniaturized DNA fragment containing the catechol production gene, and vector DNA prepared by cutting with a restriction enzyme.
The three fragments are ligated using T4-DNA ligase to obtain a composite recombinant DNA.

Next, this composite recombinant DNA is used to transform prokaryotic cells such as Escherichia coli by a conventional method to obtain a microorganism having the ability to produce catechol from benzene and β-tyrosinase activity.

Furthermore, L-dopa can be efficiently produced by reacting catechol with pyruvic acid and ammonia or an ammonium salt in the presence of the prokaryotic cells or their processed material.

[Effects of the Invention] The present invention provides a composite recombinant DNA containing a catechol production gene and a β-tyrosinase gene,
The DNA can be used to transform prokaryotic cells such as Escherichia coli to obtain a microorganism having the ability to produce catechol from benzene and β-tyrosinase activity.

Furthermore, by using this microorganism, it is possible to produce L-dopa using benzene, which is inexpensive and stably available, as a raw material. L-dopa is a useful drug known as a specific drug for Parkinson's disease.

[Example] Next, the present invention will be explained by examples.

Example 1 (1) A DNA fragment obtained by cutting plasmid pGR300 (held by Escherichia coli SI300, FERM BP-1358) containing benzene oxygenase and benzene glycol dehydrogenase genes with restriction enzyme EcoRI was preliminarily digested with restriction enzyme EcoRI. It was ligated to the cut plasmid pUC8 (Gene 19 , 259 (1982)) and transformed into E. coli JM83 to select E. coli capable of producing catechol. At this time, the restriction element cleavage conditions, ligation reaction conditions, transformation, and other recombinant DNA techniques were determined using conventional methods (e.g.
T. Maniatis, EFFritsh, J. Sambrook,
Molecular Cloning，Cold Spring Harbor
Laboratory, 1982).

A plasmid was extracted from the obtained E. coli transformant, and the plasmid pGR115 (Fig. 3), which was reduced in size by cutting with restriction enzymes, was cut with restriction enzymes EcoRI and BamHI to obtain a DNA fragment.

(2) Chromosomal DNA containing the β-tyrosinase gene
(held by Erwinia herbicola ATCC21434 strain) was cut with the restriction enzyme BamHI (37℃,
15 minutes) and pre-treated with restriction enzymes.
Digested with BamHI (37°C, 2 hours) and ligated to vector pBR322, which was further treated with alkaline phosphatase (44% with T4-DNA ligase in the presence of ATP, dithiothreitol, and _MgCl2 ).
℃ for 24 hours), and E. coli HB101 was transformed by a conventional method (J. Mol. Biol., 53 , 159 (1970)) to select E. coli having β-tyrosinase activity.

Extract plasmid DNA from this strain (Nucleic Acid Research, 7 , 1513 (1973))
Then, it was partially digested with the restriction enzyme Sau3AI (37°C, 15 minutes). The resulting DNA fragment was mixed with vector pBR322 previously cut with BamHI, and T4-DNA
After ligation with ligase, E. coli HB101
The strain was transformed. ampicillin
LB- containing 50μg/ml, L-tyrosine 2mg/ml
A strain that grows on the plate and forms a large clear zone is selected, and plasmid DNA is extracted from this strain (pSI2074) and cut with restriction enzymes SalI and Hind to obtain a DNA fragment (3.7Kb) containing the β-tyrosinase gene. I got it.

This DNA fragment and the restriction enzymes SalI, Hind
The cleaved vector pUC18 was ligated with T4-DNA ligase to obtain plasmid pSI2284. Furthermore, this pSI2284 was digested with the restriction enzymes BamHI and
The DNA fragment containing the β-tyrosinase gene was separated by cutting with Hind.

(3) Next, the above-mentioned catechol production gene DNA fragment, β-tyrosinase gene DNA fragment, and vector pUC18 DNA fragment previously cut with restriction enzymes EcoRI and Hind were ligated with T4-DNA ligase to obtain a composite recombinant DNA. Ta.

Using this composite recombinant DNA, E. coli HB101
A microorganism (E. coli SI-10) having the ability to produce catechol from benzene and β-tyrosinase activity was obtained.

A plasmid possessed by this E. coli strain SI-10 was isolated and cut with restriction enzymes, resulting in a plasmid with the restriction enzyme map shown in Figure 4.
pJ10 was obtained.

Example 2 and Comparative Examples 1 and 2 E. coli transformed with plasmid pJ10
SI-10 strain was grown in LB medium (Bacto-tryptone 10g/
, Bacto yeast extract 5g/, sodium chloride 5g/, pH 7.0) was inoculated into 10ml and cultured at 30°C for 20 hours.

After the culture was completed, the bacterial cells collected from the culture solution were washed with physiological saline and then suspended in 10 ml of the following reaction solution.

(Sodium pyruvate 10g/Ammonium acetate 5g/EDTA 1g/Sodium sulfite 2g/After adjusting the pH to 8.0 with ammonia, add benzene 20μ) Next, after reacting at 30°C for 2 hours, L-
Confirmed production of dopa. The results are shown in Table 1.
For comparison, Table 1 shows the results of similar experiments using Escherichia coli HB101 and Erwinia herbicola ATCC21434.

Table 1 L-Dopa ^* strain (mg/) Erwinia herbicola 0 ATCC21434 Escherichia coli HB101 0 Escherichia coli SI-10 450 *High performance liquid chromatography (Japan Bunko Co., Ltd.,
Calculated by comparison with an L-dopa standard solution using a column (catechol pack).

[Brief explanation of the drawing]

Figure 1 shows the nucleotide sequence of the catechol production gene, Figure 2 shows the β-tyrosinase gene pSI2283A and
Construction method of pSI2283B and its restriction enzyme map, Part 3
The figure shows the restriction enzyme map of the catechol production gene pGR115, and Figure 4 shows the method for constructing the composite recombinant DNA plasmid pJ10 and its restriction enzyme map.

Claims (1)

[Scope of Claims] 1. A region encoding the amino acid sequence of the following enzyme protein, and a gene containing β.
- A complex recombinant containing the β-tyrosinase gene derived from Erwinia herbicola, which has tyrosinase activity, has a base pair number of 1.7 Kb, and contains a site that can be cut with the restriction enzyme EcoRI in its DNA strand.
DNA. 2. A region encoding the amino acid sequence of the enzyme protein shown below, and a gene containing β.
- A complex recombinant containing the β-tyrosinase gene derived from Erwinia herbicola, which has tyrosinase activity, has a base pair number of 1.7 Kb, and contains a site that can be cut with the restriction enzyme EcoRI in its DNA strand.
A prokaryotic cell transformed by DNA. 3 Claim 2 in which the prokaryote is Escherichia coli
Cells described in Section. 4 E. coli is Escherichia coli HB101/SI−
10. The cell according to claim 3, wherein the cell number is 10. 5 The region encoding the amino acid sequence of the enzyme protein shown below, and the gene containing β-
β derived from Erwinia herbicola that has tyrosinase activity, has a base pair number of 1.7 Kb, and contains a site that can be cut with the restriction enzyme EcoRI in its DNA strand.
- Complex recombination containing the tyrosinase gene
L-dopa, which comprises reacting benzene with pyruvic acid and ammonia or an ammonium salt in the presence of prokaryotic cells transformed with DNA or a processed product of the cells, and collecting L-dopa from the reaction solution. manufacturing method. 6. The method according to claim 5, wherein the prokaryotic cell is E. coli. 7 E. coli is Escherichia coli HB101/SI−
10. The method according to claim 6.