JPH0336510B2 - - Google Patents

Description

[Detailed description of the invention]

(b) The present invention relates to a transformed microorganism having a biphenyl metabolic function, which is used to synthesize the compound 2-hydroxyxine-6-oxo-6.
- Phenylhexa-2,4-dienoic acid and its derivatives are produced and used as bactericidal agents and physiologically active substances against Gram-positive bacteria such as Staphylococcus spp. and Gram-negative bacteria such as Escherichia coli, which are known as pathogenic bacteria. It is expected to be used as an intermediate. (b) Conventional technology Several types of bacteria that assimilate biphenyl have been isolated from nature, and their metabolic patterns have been studied, but nothing is known about transformed microorganisms. Therefore, the present inventors have been conducting research on genes related to the biphenyl metabolic mechanism, and as a result, succeeded in extracting a gene involved in the biphenyl metabolic function derived from Pseudomonas pseudoalcaligenes, and this foreign gene bph A -C
This led to the creation of a transformed microorganism, Pseudomonas aeruginosa, in which a host microorganism with excellent growth efficiency was given the ability to metabolize biphenyl. (C) Structure of the Invention The source of the foreign genes bph A-C is Pseudomonas pseudoalcaligenes KF707, which is a microorganism that carries genes involved in biphenyl metabolism in its body, and which the present inventor isolated from nature.
(Pseudomonas Pseudoalcaligenes KF707)
(FERM P-8297) is an example. The mycological properties of this strain are as follows. [Mycological properties] Gram-negative bacillus 0.7 x 1.5-2.0 μ Polar flagellum 1 Pigment production None Oxidase Positive starch hydrolysis Negative Growth at 41℃ Positive Optimum growth temperature 35℃ Qualifying glucose, succinic acid, lactic acid, Pyruvic acid Based on the above mycological properties, we searched based on the 9th edition of Virgies Manual of Systemic Bacteriology, and found that Pseudomonas alcaligenes.
Pseudoalcaligenes). Next, the gene is excised from the present strain by growing the strain overnight, for example, in L medium (10 g of Bactotryptone, 5 g of yeast extract, 5 g of salt, 1 part of distilled water) and lysing the chromosomal DNA using the lysozyme-SDS method. The present gene can be obtained by preparing chromosomal DNA (1 μg) and then cleaving the chromosomal DNA (1 μg) with the restriction enzyme Xho. The number of cleavages of this gene by various restriction enzymes is shown in Table 1.

[Table] The restriction enzyme map of the fragment of this gene is shown in FIG. Next, the relationship between the biphenyl metabolic mode of this gene and the gene group is as shown below. In the above reaction formula, A, B, and C represent enzymes that control the reaction, and A is biphenyl oxygenase B.
indicates dihydroxydiol dehydrogenase, and C indicates phenylcatechol oxygenase. In addition, genes corresponding to these enzymes include
There are bphA, bphB, and bphC, and these genes form an operon. Moreover, P represents a promoter. When using this gene, for example, Escherichia coli KF637 (Escherichia coli
KF637) (FERM P-8296)-derived pKF330 was extracted from the strain using a conventional method, and then treated with the restriction enzyme Xho.
After cleavage, the recombinant plasmid pMFB1 was constructed by ligating with T4 ligase (Fig. 3). Furthermore, pKF330 has kanamycin resistance and streptomycin resistance as shown in Figure 2.
It is a 12.6kb plasmid. The kanamycin resistance gene site has an insertion inactivation site such as Xho, and the kanamycin promoter can be used. Next, we used Pseudomonas aeruginosa KF204 as a host strain.
(FERM P-8295) is used. For the transformation method and selection of transformants, the strain was cultured to the logarithmic growth phase (5 x 10 ⁸ cells/ml), harvested, washed, and then placed in a cold buffer (10mM MOPS-
10mM RbCl-100mM _MgCl2 , PH7.0), then centrifuged, and then suspended in cold buffer (100mM
MOPS-10mM RbCl-100mM _CaCl2 , PH
6.5) and incubate at 0°C for 30 minutes.
Next, after centrifugation, suspend the strain in 1/10 volume of cold buffer. The recombinant plasmid (0.5 μg) was added to the 0.2 ml competent cells, incubated at 0°C for 1 hour, and then heat-shocked at 42°C for 2 minutes. 3
After adding ml of L medium and incubating at 30° C. for 3 hours, transformants carrying the recombinant plasmid are primarily screened on L-agar medium containing streptomycin (200 μg/ml). The target transformant (recombinant microorganism) carrying the biphenyl gene is selected by spraying a 2,3-dihydroxybiphenyl solution (1 mg/ml) onto the colonies generated in the primary screening to select colonies that turn yellow. After confirming that clones containing biphenyl metabolic genes accumulate yellow substances from biphenyl and biphenyl-related compounds, the yellow substances were collected under acidic conditions (PH1
After extraction with ethyl acetate in ~2), trimethylsilylation and analysis by GC-MS confirm that this is the compound (Figure 4) and its derivatives. (d) Examples Example 1 The biphenyl-assimilating bacterium Pseudomonas pseudoalcaligenes KF707 strain (FERM P-8297) was cultured overnight in L medium, collected, washed, and then cultured in 0.1M Tris (PH7.9) 1mM. Suspended in EDTA buffer,
Add lysozyme (final concentration 2 μg/ml) and incubate for 5 min at room temperature, then add 50 μg of 10% SDS.
/ml and lysed. Next, the mixture was treated with pronase and RNase, followed by phenol extraction, and the phenol was removed with ether. The DNA thus prepared was prepared in 10mM Tris, 1mM
Dialyzed into EDTA buffer. On the other hand, Escherichia coli KF637 (FERM P-8296) containing plasmid pKF330 was cultured overnight in L medium, and pKF330 was prepared by the alkaline-SDS method. Chromosomal DNA and plasmid pKF330
was cleaved with restriction enzyme Xho and ligated with T4-ligase. The recombinant plasmid was then transferred to the host strain Pseudomonas aeruginosa KF204 (FERM
P-8295). That is, the KF204 strain in the logarithmic growth phase (approximately 4 × 10 ⁸ cells/ml) was collected, and an equal volume of cold buffer (10mM MOPS, 10mM
After washing with RbCl, 100mM MgCl ₂ , PH7.0), cool buffer (100mM MOPS, 10mM
Resuspend in RbCl, 100mM _CaCl2 , PH6.5) and
It was left at ℃ for 30 minutes. After centrifugation, the cells were resuspended in 1/10 volume of cold buffer and incubated with 0.2 ml of the cell suspension and purified pMFB1 (0.5 μg) at 0° C. for 1 hour. After heat-shocking at 42°C for 2 minutes, 3 ml of L medium was added and cultured at 30°C for 3 hours. Transformants carrying pMFB1 containing the biphenyl metabolic gene cluster (bph A-C) were incubated with 2,3-hydroxybiphenyl solution (1
(mg/ml) was sprayed and the colonies that turned yellow were selected. Then the transformant streptomycin
After culturing overnight in L medium containing 200 μg/ml, a plasmid was prepared by a conventional method. Next, the prepared plasmid pMFB1 was cut with XhoI, resulting in a 7.9kb bph
The A-C gene was excised. This gene had the restriction enzyme cleavage point shown in FIG. Example 2 Pseudomonas aeruginosa KF257 carrying pMFB1 obtained in Example 1 was applied (inoculated) on a BSM agar medium containing succinic acid (1 mg/ml) as a carbon source, and biphenyl powder was placed on the lid of a Petri dish. I sealed it with vinyl tape. KF257
As the strain multiplied, the bacterial cells that took in biphenyl vapor converted biphenyl into a compound, and the medium turned bright yellow. The above reaction was performed using the host strain Pseudomonas aeruginosa KF204 (FERM P-
8295) was not recognized at all. In addition, the host strain and KF257 carrying pMFB1 were Gram-stained negative flagella 1 Pyocyanin Produced fluorescent pigment Optimum growth temperature for production 37℃ Oxidase + GC content 67% Starch hydrolysis - Due to the above properties, the KF257 strain is Pseudomonas aeruginosa. It was confirmed that (e) Effects of the invention Foreign gene bph having biphenyl metabolic function
A-C is recombined into a microorganism with excellent growth efficiency, and the transformed microorganism is used to inexpensively produce the compound 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid and its derivatives. becomes possible.

[Brief explanation of the drawing]

FIG. 1 shows a restriction enzyme cleavage map of the foreign genes bph A-C. Figure 2 shows the structure of plasmid pKF330 derived from Escherichia coli. Figure 3 shows the procedure for producing recombinant plasmid pMFB1 and its structure.

Claims (1)

[Scope of Claims] 1. A foreign gene derived from Pseudomonas pseudoalcaligenes and having a biphenyl metabolic function, which has a molecular weight of 7.9 kilobases and is characterized by the number of bases cleaved by the following restriction enzymes: Vector pKF330 from Escherichia coli
A transformed microorganism Pseudomonas aeruginosa strain KF257 is characterized in that it has a biphenyl metabolic function, which is obtained by transforming the recombined plasmid pMFB1 into a host microorganism Pseudomonas aeruginosa.