JPH0342071B2 - - 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 produce the compound 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid and its derivatives. It is expected to be used as an intermediate for 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. (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 for some time, and as a result, they succeeded in extracting a gene involved in the biphenyl metabolic function derived from Pseudomonas pseudoalcaligenes, and the foreign gene bphA- By recombining C into a vector, we have created a transformed microorganism, Pseudomonas putida, which has a biphenyl metabolic function in a host microorganism with excellent growth efficiency. (C) Structure of the Invention The source of the foreign gene bphA-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 0.7×1.5-2.0μ Polar flagella 1 Pigment production None Oxidase positive Starch hydrolysis Negative Growth at 41℃ Positive Optimal growth temperature 35℃ Assimilation glucose, succinic acid, lactic acid , pyruvate Based on the above mycological properties, a search based on the Virgies Manual of Systematic Bacteriology, 9th edition revealed that Pseudomonas alcaligenes
Pseudoalcaligenes). Next, to excise the gene from this strain, the strain is grown overnight in, for example, L medium (10 g of Bactotryptone, 5 g of yeast extract, 5 g of salt, 1 part of distilled water), and the chromosomal DNA is lysed by the lysozyme-SDS method. The present gene can be obtained by preparing chromosomal DNA (1 μg) and then cutting the chromosomal DNA (1 μg) with the restriction enzyme XhoI. 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 bifuel 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 restriction enzymes.
After cutting with XhoI, ligation is performed with T4 ligase to construct recombinant plasmid pMFB1 (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 XhoI, and the kanamycin promoter can be used. Next, Pseudomonas putida as a host strain.
KF107 (Pseudomonas putida KF107 (FERMP)
−8294) is used. For the transformation method and selection of transformants, the strain was multiplied to the logarithmic growth phase (5 x 10 ⁸ cells/ml), harvested, washed, and placed in a cold buffer (10mM MOPS-10mM
RbCl−100mM MgCl−100mM _MgCl2 , PH7.0)
After centrifugation, suspend in cold buffer (100mM MOPS−10mM RbCl−100mM
Resuspend in CaCl ₂ , pH 6.5) and incubate at 0°C for 30 minutes. Next, after centrifugation, suspend the strain in 1/10 volume of cold buffer. Add the recombinant plasmid (0.5 μg) to the 0.2 ml competent cells and store at 0°C for 1 hour.
After incubation for 1 hour, heat shock for 2 minutes at 42°C. After adding 3 ml of L medium and incubating at 30°C for 3 hours, streptomycin (200μ
Transformants harboring the recombinant plasmid are primarily screened on L-agar medium containing 1.5 g/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. Clones containing biphenyl metabolic genes were confirmed by accumulating a yellow substance from biphenyl and biphenyl-related compounds, and the yellow substance was extracted with ethyl acetate under acidic conditions (PH 1 to 2), trimethylsilylated, and analyzed by GC-MS. (Fig. 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, and washed with 0.1M Tris (PH7.9). Suspended in 1mM 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 mixed with 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 digested with restriction enzyme XhoI and ligated with T4-ligase. The recombinant plasmid was then transferred to the host strain Pseudomonas aeruginosa KF107 (FERM P
-8294). That is, the KF107 strain in the logarithmic growth phase (approximately 4 × 10 ⁸ cells/ml) was harvested, and an equal volume of cold buffer (10mM MOPS, 10mM RbCl,
After washing with cold buffer ( _100mM MOPS, 10mM RbCl, 100mM
CaCl ₂ , PH6.5) and left at 0° C. for 30 minutes. Next, after centrifugation, resuspend in 1/10 volume of cold buffer, and add the 0.2ml cell suspension and purified pMFB1.
(0.5 μg) and incubated at 0° C. for 1 hour.
After heat-shocking at 42°C for 2 minutes, 3 ml of L
A medium was added and cultured at 30°C for 3 hours. Transformants carrying pMFB1 containing the biphenyl metabolic gene cluster (bphA-C) were sprayed with 2,3-dihydroxybiphenyl solution (1 mg/ml) on L-agar medium containing streptomycin (200 μg/ml). Colonies that turned yellow were selected. The transformant was then cultured overnight in L medium containing 200 μg/ml of streptomycin, and a plasmid was prepared by a conventional method. Next, the prepared plasmid pMFB1
When cut with XhoI, a 7.9 kb bph A-C gene was excised. This gene had the restriction enzyme cleavage point shown in FIG. Example 2 Pseudomonas putida KF138 carrying pMFB1 obtained in Example 1 was applied (inoculated) to 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 and covered with vinyl. I sealed it with tape. The bacterial cells that took in biphenyl vapor as the KF138 strain multiplied,
Biphenyl was converted into a compound, and the medium turned bright yellow. The above reaction was performed using the host strain Pseudomonas putida KF107 (FERM P-8294), which does not carry pMFB1.
It was not recognized at all. Regarding the host strain and KF138 carrying pMFB1, Gram staining is negative Flagella >1 Pyocyanin not produced Fluorescent pigment produced Optimal growth temperature 25-30℃ Oxidase + GC content 60% Starch hydrolysis + Due to the above properties, etc., KF138 The strain was confirmed to be Pseudomonas putida. (e) Effects of the invention Foreign gene bphA having biphenyl metabolic function
-C into a microorganism with excellent growth efficiency, and using the transformed microorganism, compound 2-hydroxy-
It becomes possible to produce 6-oxo-6-phenylhexa-2,4-dienoic acid and its derivatives at low cost.

[Brief explanation of drawings]

FIG. 1 shows a restriction enzyme cleavage map of the foreign gene bphA-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 derived from Escherichia coli
A transformed microorganism, Pseudomonas putida KF138 strain, is characterized by having a biphenyl metabolic function, which is obtained by transforming the recombined plasmid pMFB1 into a host microorganism, Pseudomonas putida.