JPH0336515B2 - - Google Patents

Description

[Detailed description of the invention]

(a) The present invention relates to a novel foreign gene pph A-C derived from Pseudomonas Pseudoalcaligenes having a biphenyl metabolic function, and a novel microorganism Pseudomonas Pseudoalcaligenes KF707 strain harboring the same. This gene can be used, for example, in Pseudomonas putida and Pseudomonas aeruginosa, which have high proliferation ability.
The compound 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid and its derivatives are produced using microorganisms that have been recombinantly transformed into A. aeruginosa), etc. It is expected that it can contribute to the production of bactericidal agents and physiologically active substance intermediates against Gram-positive bacteria such as Staphylococcus and Gram-negative bacteria such as Escherichia coli. (b) Conventional technology Several types of bacteria that assimilate biphenyl have been isolated and their metabolic modes have been studied, but no examples of studies on individual genes have been known. Furthermore, there has been no study of specific intermediate metabolites of biphenyl on a gene-by-gene basis, including the metabolic mode. Therefore, the present inventors conducted research on genes related to the biphenyl metabolic mechanism, and found that the gene derived from Pseudomonas pseudomonas alcaligenes
They succeeded in extracting bph A-C and completed the present invention. (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 0.7×1.5-2.0 μ Polar flagellum 1 Pigment production None Oxidase Positive starch hydrolysis Negative growth at 41℃ Positive optimum growth temperature 35℃ Assimilation ability Glucose, succinic acid, lactic acid, pyruvin Based on the above mycological properties, a search based on Virgies, Manual of Systematics of Bacteriology, 9th edition revealed that Pseudomonas alcaligenes
pseudoalcaligenes). This strain contains a novel foreign gene bph A-
It is a novel microorganism in that it possesses C. The gene can be excised from this strain by growing the strain overnight in L medium (10 g of bactryptone, 5 g of yeast extract, 5 g of salt, 1 part of distilled water), and lysing the chromosome by the lysozyme-SDS method.
The present gene can be obtained by preparing DNA and then cleaving chromosomal DNA (1 μg) with 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 is dihydroxydiol dehydrogenase,
C represents 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 is 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, as a host strain, Pseudomonas putida KF107 has a high growth ability.
(FERM P-8294) and Pseudomonas aeruginosa KF204
KF204), etc. In the transformation method and selection of transformants, the strain is cultured to the logarithmic growth phase (5 x 10 ⁸ cells/ml), harvested,
After washing, cool buffer (10mM MOPS-10m
suspended in M RbCl-100mM _MgCl2 , PH7.0),
After centrifugation, cool 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. Add the recombinant plasmid (0.5 μg) to the 0.2 ml competent cells, incubate at 0°C for 1 hour, and then heat-shock at 42°C for 2 minutes. 3
After adding ml of L medium and incubating for 3 hours at 30°C, transformants carrying pKF330 and the recombinant plasmid are screened on an 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. Clones containing biphenyl metabolic genes accumulate yellow substances from biphenyl and biphenyl-related compounds, and the yellow substances are extracted with ethyl acetate under acidic conditions (PH 1 to 2), tritylsilylated, and analyzed by GC-MS. 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). Suspend in 1mM EDTA buffer. Next, lysozyme was added to a final concentration of 2 μg/ml and incubated at room temperature for 5 minutes.
Bacteria were lysed by adding 10% SDS to a concentration of 50μ/ml. Next, the mixture was treated with pronase and RNase, followed by phenol extraction, and the phenol was removed with ether. The DNA prepared in this way was 10mM
Dialyzed into Tris, 1mM 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 KF204 (FERM P
-8295). That is, the KF204 strain in the logarithmic growth phase (approximately 4 x 10 ⁸ cells/ml) was harvested, and an equal amount of cold buffer (10mM MOPS, 10mM RbCl,
After washing with cold buffer ( _100mM MOPS, 10mM RbCl, 100mM
The mixture was resuspended in MCaCl ₂ , PH6.5) and left at 0°C for 30 minutes. Next, after centrifugation, the cells were resuspended in 1/10 volume of cold buffer and purified with the 0.2ml cell suspension.
It was incubated with pMFB1 (0.5 μg) at 0° C. for 1 hour. After heat-shocking at 42℃ for 2 minutes,
ml of L medium was added and cultured at 30°C for 3 hours. The biphenyl metabolism gene group (bph A-C) was integrated.
Transformants harboring pMFB1 were grown on L-agar medium containing streptomycin (200 μg/ml).
Colonies that turned yellow were selected by spraying a 3-dihydroxybiphenyl solution (1 mg/ml). The transformants were then treated with 200 μg/streptomycin.
After culturing overnight in L medium containing ml, plasmids were prepared by a conventional method. The parasmid then prepared
When pMFB1 is cut with Xho, 7.9kb bph A-
The C gene was excised. This gene had the restriction enzyme cleavage point shown in FIG. Example 2 Recombinant plasmid pMFB1 prepared in Example 1
using Pseudomonas putida KF107
(FERM P-8294) was transformed by the method of Example 1. As a result, 1×10 ³ cells/μg
A transformant Pseudomonas putida KF138 carrying pMFB1 was obtained at a DNA frequency. Example 3 Pseudomonas aeruginosa KF257 carrying pMFB1 prepared according to Example 1 was applied (ingested) 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 with vinyl tape. I sealed it. As the KF257 strain grew, 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. The above reaction is
Pseudomonas putida harboring pMFB1
The same was observed in KF138. In addition, the host strain and the KF257 strain carrying pMFB1 were analyzed by Gram staining Negative flagella Single cell size 0.5-0.7×2.0 Optimal growth temperature 37℃ Blue pigment (pyocyanin) Generated oxidase + GC content 67% Due to the above properties, etc., KF257 The strain was confirmed to be Pseudomonas aeruginosa. In addition, the host strain and the KF138 strain carrying pMFBI were tested for Gram staining Negative flagella >1 No pyocyanin produced Fluorescent dye Optimum growth temperature for production 25-30℃ Oxidase + GC content 60% Starch hydrolysis + Due to the above properties, etc., the KF138 strain was confirmed to be Pseudomonas putida. (e) Effects of the invention Foreign gene bph having biphenyl metabolic function
A-C was recombined with a microorganism with excellent growth efficiency, and the transformed microorganism was used to produce the compound 2-hydroxy-6-oxo-6-phenylhex-2,4-
It becomes possible to produce dienetic acid and its derivatives at low cost.

[Brief explanation of drawings]

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 having a biphenyl metabolic function derived from Pseudomonas pseudoalcaligenes, which has a molecular weight of 7.9 kilobases and is characterized by the number of bases cleaved with the following restriction enzymes. 2.・A novel microorganism, Pseudomonas syudo, which possesses a foreign gene with a biphenyl metabolic function derived from Pseudomonas alcaligenes, has a molecular weight of 7.9 kilobases, and is characterized by the number of bases cleaved by the following restriction enzymes. Alcaligenes KF707 strain.