JPS61282085A - Exogenote bpha-c having biphenyl metabolic function and novel microorganism having said exogenote bpha-c - Google Patents

Abstract

PURPOSE:To make it possible to take out a gene bphA-C, by preparing an exogenote having 7.9 kilobase molecular weight and specific biphenyl metabolic function with a specific number of cleavages by a specific restriction enzyme. CONSTITUTION:A recombinant plasmid pMFB1 is constructed by taking out pKF330 derived from coliform bacillus Escherichia coli KF637 from a strain thereof by the conventional method, cleaving the resultant pKF330 with a restriction enzyme XhoI, linking the cleaved pKF330 to Pseudomonas pseudoalcaligenes KF107 strain with T4-ligase and introducing the resultant recombinant plasmid into a host strain. Pseudomonas putida KF107 or Pseudomonas aeruginosa KF204, etc., having high multiplication ability may be used as the host strain. In selecting the transformation method and transformant, the strain, etc. means for cultivating the strain, etc. to the logarithmic growth phase, collecting the bacteria, washing the collected bacteria, suspending the washed bacteria in a cold buffer solution, centrifuging the suspension, resuspending the centrifuged bacteria in a cold buffer solution (II) and incubating the suspension at, e.g. 0 deg.C for 30min, may be adopted.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) The present invention relates to Pseudomonas putida having a biphenyl metabolic function.
ida) and Shutomonas aeruginosa (Pseud
The compound (1) 2-hydroxy-6-oxo-6-phenylhexa-2,4-genoic acid and its derivatives were produced using microorganisms recombinantly transformed into S. omonas aeruginosa), which are known as pathogenic bacteria. It is expected that the present invention can contribute to the production of bactericidal agents and physiologically active substance intermediates against Gram-positive bacteria such as Staphylococcus and Durham-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 gene-by-gene basis are 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 biphenyl metabolic mechanism, and as a result, succeeded in extracting genes bph A-C derived from Pseudomonas pseudoalcaligenes, and completed the present invention.

(c) Structure of the invention KF707) (FERM P-8297) can be exemplified.

The mycological properties of this strain are as follows.

[Mycological properties]

Durham negative bacillus 0.7X1.5~2.0μ polar flagella
1 Pigment production None Oxidase Positive Starch hydrolysis Negative Growth at 41℃ Positive Optimum growth temperature 35℃ Assimilation Glucose, succinic acid, lactic acid,
This strain is a novel microorganism in that it possesses the novel foreign genes bph A-C.

Excision of the gene from the present strain can be performed by converting the strain to, for example, L
Lysozyme-3 was grown late in a medium (10 g of bactryptone, 5 g of yeast extract, 5 g of salt, and 100 g of distilled water).
The present gene can be obtained by preparing chromosomal DNA by lysis using the DS method, and then cleaving the chromosomal DNA (1 μg) with the restriction enzyme XhoT.

The number of cleavages of this gene by various restriction enzymes is shown in Table 1.

(>゛AT-dairy mill) Table 1 Furthermore, the restriction enzyme map of the fragment of this gene is as shown in FIG.

Next, the relationship between the biphenyl metabolic mode of this gene and the gene group is as shown below.

! ■ ■
■P iP & eight kk6 611
(?In the above reaction formula, A, B, and C represent enzymes that control the reaction, A represents biphenyl oxygenase, B represents dihydroxydiol dehydrogenase, and 90 represents phenylcatechol oxygenase.

In addition, bphA is a gene corresponding to these enzymes.
,bphB.

bphC exists, and these genes form an operon. Moreover, P represents a promoter.

When using this gene, for example, Escherichia coli 'KF637 (Escherichia coli
coli KF637)' (FERM P-829
6) The derived pKF330 was extracted from the strain using a conventional method, cut with the restriction enzyme Xhol, and ligated with T4 ligase. Also, the promoter of kanamycin can be used.

Next, we used Psaudomonas putida KF107 (Psaudomonas putida), which has high growth ability, as a host strain.
KF107) (FERM P-8294) and Pseudomonas aeruginosa KF204 (Pseudomonas aeruginosa)
nas aeruginosa KF204). For the transformation method and selection of transformants, the strain was cultured to the logarithmic growth phase (5 x 10" cells/ml!), harvested, washed, and then incubated with cold buffer (10mM MOPS-10mM RbCf).
-100mM MgC1z, pH 7,0), then centrifuged, and then suspended in cold buffer y-II (100mM MgClz, pH 7,0).
OPS-10mM RbC1-100mM CaC
1z, pH 6.5) and incubate for 30 minutes at 0°C. Next, after centrifugation, the bacterial strain is suspended in 1/10 volume of cold buffer ■. This 0. 2mj! Inject the recombinant plasmid (0,5,'-) into competent cells and perform the primary screening for transformants carrying pKF330 and the recombinant plasmid on the ground. is 2
．． 3-dihydroxybiphenyl solution ('4mg/ml)
- Select colonies that turn yellow by spraying the colonies generated in the next screening. Clones containing biphenyl metabolism genes accumulate more yellow substances than biphenyl and biphenyl-related compounds, and the yellow substances accumulate under acidic conditions (pH
After extraction with ethyl acetate in steps 1 and 2), the mixture was tritylsilylated and analyzed by GC-MS to confirm that it was compound (1) (FIG. 4) and its derivatives.

(D) Examples Example 1 The biphenyl-assimilating bacterium Shutomonas schoed alcaligenes strain KF707 (FERM P-8297) was
After culturing overnight in a medium, collecting bacteria and washing, 0.1M Tris (p
H7,9), suspend lysozyme in 1mM EDTA buffer at a maximum P-concentration of 2μg/m
12 (-), incubated at room temperature for 5 minutes, and lysed by adding 10% SDS to a total volume of 50μ417ml.

Next, after treatment with pronase and RNase, phenol extraction was performed, and phenol was removed with ether.

The DNA thus prepared was prepared in 10mM Tris, 1mM
Dialyzed into MEDTA buffer.

On the other hand, Escherichia harboring plasmid pKF330
Cori KF6. The recombinant plasmid was then transferred to the host strain Shutomonas aeruginosa KF204 (FE
RM P-8295). i.e., KF204 in logarithmic growth phase (approximately 4 X 10' cells/m j!)
Harvest the strain and add an equal volume of cold buff y-(10mM MOP
S, 10mM RbCj! .

After washing with 100mM MgClt, pH 7,0),
Cold buff Y-11 (100mM MOPS, 10mM
RbCj! , 100mM CaC1z.

pH 6.5) and left at 0°C for 30 minutes.

Next, after centrifugation, resuspend in 1/10 volume of cold buffer ■,
The 0.2 ml cell suspension and purified pMFBl (0,
5 μg) at 0° C. for 1 hour. After heat shock at 42°C for 2 minutes, 3ml of medium was added and cultured at 30°C for 3 hours. Biphenyl metabolism gene group (
Transformants carrying pMFBl integrated with bph A-C) were treated with streptomycin (200 μg/mf).
When a 2,3-dihydroxybiphenyl solution (1 mg/rrl) was sprayed on an L-agar medium containing 7
．． The 0 genes from which the 9 kb bph A-C gene was excised had the restriction enzyme cleavage points shown in FIG.

・,) Example 2 '+1,' Using the recombinant plasmid pMFB1 prepared in Example 1, "domonas putida KF107 (F
ERM P-8294) was transformed by the method of Example 1. As a result, 1X10' cells/μg DN was applied on a BSM agar medium containing succinic acid (1 mg/mj!) as a carbon source (tFIl). Hyphenyl powder was placed on the lid of a Petri dish and sealed with vinyl tape. KF
As the 257 strain grew, the bacterial cells that took in the biphenyl vapor converted biphenyl to compound (■), and the medium turned bright yellow.

The above reaction was performed using the host strain Shutomonas aeruginosa KF204 (FERM P-8), which does not carry pMFBl.
295>, it was not recognized at all. The above reaction is pM
A similar finding was observed in Shutomonas putida KF138, which preserves FBl.

In addition, for the host strain and KF257 strain carrying pMFBl, Durham staining negative flagellum 1, cell size 0.5-0.7X2.0
Optimum growth salinity: 37°C Blue pigment (biocyanin): Generated oxidase + GC content: 67% or more.The KF257 strain was confirmed to be Shutomonas aeruginosa.

In addition, for the host strain and KF138 strain carrying pMFBT, Durham staining Negative flagella 〉1 Biocyanin No fluorescent pigment formation Optimum growth temperature 25-30℃ Oxidase + GCC content 60% Starch hydrolysis KF138 due to more than 10 properties, etc. The strain Shutomonas putida makes it possible to produce hydroxy-6-oxo-6-phenylhexa-2,4-genoic acid and derivatives thereof 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 plasmid pKF3 derived from Escherichia coli.
30 structures are shown. FIG. 3 shows the procedure for producing recombinant plasmid pMFB1 and its structure. Patent applicant Tatsu Todoroki, Director of the Agency of Industrial Science and Technology)
[6°L -1111111---Pst Engineering EcoR Engineering Sac Engineering

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: Genes Pst I, 6 BglII, 1 Xho I 1 EcoR I, 2 BamH I, 1 Xba I 0 Sma I, 1 Sac I, 1 HindIII 0 Sal I, 0 Hpa I, 0 2) Genes derived from Pseudomonas pseudoalcaligenes Foreign genes with biphenyl metabolism function, which have a molecular weight of 7.9 kilobases and are characterized by the number of bases cleaved with the following restriction enzymes: Pst I, 6 BglII, 1 Xho I 1 EcoR I, A novel microorganism Pseudomonas pseudoalcaligenes KF707 strain harboring 2 BamH I , 1 Xba I 0 Sma I , 1 Sac I , 1 HindIII 0 Sal I , 0 Hpa I , 0 .