Benzene oxygenase gene

BACKGROUND OF THE INVENTION

1,2-Dihydroxy-cyclohexa-3,5-diene is of high utility value and has been used as a starting material for preparing engineering plastics (See: Japanese Patent Application Kokai Koho No. 71903/1983).

Only one method has been known for the production of 1,2-dihydroxy-cyclohexa-3,5-diene, which is described in the specification of Japanese Patent Application Kokai Koho No. 71891/1983.

In this known method, an aromatic compound is treated with a mutant of Pseudomonas putida, and is converted to 1,2-­dihydroxy-cyclohexa-3,5-diene.

This method, however, has some disadvantages. It is diffi­cult to handle the cells of Pseudomonas putida and a long time is required for the production of the desired compound.

There has been made no attempt either to isolate benzene oxygenase gene or to transform microorganisms with this gene­tic information.

The present inventors, after extensive research, have succeeded in isolating a DNA sequence that corresponds to a part of the gene which is contained in a microorganism capable of producing catechol from benzene or its derivative (See: Japanese Patent Application No. 68700/1986). At least gene DNA segments Nos. I, II, III and IV were found to be the base sequences, in combination, coding for an amino acid se­quence of one particular enzyme protein. Said gene DNA is occasionally isolated in such a form that a DNA segment de­signated as V is contained together with the DNA segments Nos. I, II, III and IV.

In the further progress of the research, the present in­ventors have now succeeded in the isolation of a DNA sequence that contains at least DNA segments Nos. I, II and III loca­lized therein, but is devoid of the segment No. IV,and encodes a polypeptide having benzene oxygenase activity. The present inventors have also succeeded in the preparation of a recom­binant DNA consisting of said DNA sequence and a vector, and in the transformation of prokaryotic cells by the use of said recombinant DNA. The present inventors have found that the transformed prokaryotic cells produce 1,2-dihydroxy-cyclohexa-­3,5-diene from benzene.

SUMMARY OF THE INVENTION

This invention relates to a DNA sequence (gene) coding for a polypeptide having benzene oxygenase activity, and to a prokaryotic cell transformed with said gene. This invention also relates to a process for the production of 1,2-dihydroxy-cyclohexa-3,5-diene by use of said transformed cells. The invention is defined in the claims.

BRIEF DESCRIPTION OF THE DRAWING

Fig. 1 shows a restriction enzyme cleavage map of DNA (nucleotide) segments contained in pGR115 and pGR1153, which are derived from a microorganism having benzene-oxidizing ability.

DETAILED DESCRIPTION OF THE INVENTION

According to the first aspect of this invention, there is, therefore, provided a DNA sequence encoding a polypeptide having benzene oxygenase activity which contains at least DNA segments Nos. I, II and III in combination. These sequences have the following formulae:

The invention also relates to DNA sequences which are degenerate as a result of the genetic code to any of the fore­going DNA sequences and which code for a polypeptide having benzene oxygenase activity.

According to the second aspect of this invention, there is provided a prokaryotic cell which has been transformed with a recombinant DNA consisting of the DNA sequence as defined above and a vector DNA.

Further, according to the third aspect of this invention, there is provided a process for the production of 1,2-dihydroxy-­cyclohexa-3,5-diene, which comprises treating an aromatic com­pound with the transformed prokaryotic cells as defined above.

The DNA sequence encoding a polypeptide having benzene oxy­genase activity (benzene oxygenase gene) may be prepared by the following procedure:

Cosmid pGR300 prepared from an E. coli strain (SI-300, FERM BP-1358) which contains the benzene oxygenase gene and the benzene glycol dehydrogenase gene, was subjected to a known treatment such as the one described in "T. Maniatis, E.F. Fritsch, J. Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1982" to prepare a mini-plasmid containing the desired gene. The plasmid thus obtained was submitted to prepare transformed cells of a E. coli strain. Plasmid pGR115 was isolated from the transfor­med cells of an Escherichia coli strain, and was cut with se­veral restriction enzymes, so that the restriction enzyme cleavage map as shown in Fig. 1 was prepared. DNA fragments obtained from the plasmid pGR115 by cleavage with several restriction enzymes as mentioned above was subjected to agarose gel electrophoresis, thereby obtaining a DNA frag­ment of about 4.2 Kb. This DNA fragment was combined with a vector plasmid and the combined product was introduced (trans­ferred) into cells of E. coli strain, so that transformant cells were obtained. Plasmids thus obtained from transfor­mant cells of the E. coli strain prepared as above were cut with several restriction enzymes and analyzed for identifica­tion, so that the E. coli JM109 strain which carries plasmid pGR1153 containing DNA fragments derived from plasmid pGR115 was selected. The segments of the base sequence which corres­pond to the desired gene were thus selected. The base sequences of the DNA segments thus obtained were determined according to M13 phage-dideoxy method. In the base sequence as determined, there are contained at least three DNA segments in combination coding for a polypeptide having benzene oxygenase activity, namely DNA segments designated as I, II and III.

The benzene oxygenase gene thus isolated may be introduced, in a known manner, into cells of microorganisms, particularly of prokaryotic microorganisms which lack the benzene oxygenase activity. Various prokaryotic microorganisms, preferably Escherichia coli may be used in this invention.

1,2-Dihydroxy-cyclohexa-3,5-diene can be prepared effective­ly in a high yield by allowing an aromatic compound to react, in contact with transformed cells as described above of a pro­karyotic microorganism to effect the conversion of said aroma­ tic compound into the desired compound, namely 1,2-dihydroxy-­cyclohexa-3,5-diene. Preferred examples of aromatic compounds are benzene, toluene, biphenyl or naphthalene. The reaction is carried out in the presence of assimilable carbon sources such as ethanol, acetic acid, glucose and lactic acid, where­by the aromatic compound is converted into 1,2-dihydroxy-­cyclohexa-3,5-diene. The reaction may be conducted at a tem­perature from 10 to 45°C, preferably from 25 to 35°C. After the completion of the reaction, the reaction solution is concentra­ted, and the resulting concentrate is extracted with an appro­priate organic solvent such as diethyl ether or methylene chloride. From the extract, after having been concentrated if necessary, there is recovered 1,2-dihydroxy-cyclohexa-3,5-­diene in a high yield.

1,2-Dihydroxy-cyclohexa-3,5-diene finds various practical uses such as in the production of engineering plastics.

This invention will be illustrated in more detail with respect to the following examples. It should be understood that these examples should not be construed as limiting the scope of this invention.

Example 1

Cosmid pGR300 derived from the cells of Escherichia coli SI-300 (FERM BP-1358), which contains the benzene oxygenase gene and the benzene glycol dehydrogenase gene was cut with restriction enzyme EcoRI to obtain a cleaved DNA fragment. The DNA fragment thus obtained was ligated with plasmid pUC8 cut with EcoRI (See: Gene, 19, 259 (1982)) and the ligated product was introduced into the cells of the Escherichia coli JM83 strain.

The cleavage of cosmid pGR300 with the restriction enzyme EcoRI, the ligation of the DNA fragment with the cleaved plasmid pUC8 and the transformation of the cells of Escherichia coli was performed in a known manner according, for example, to "T. Maniatis, E.F. Fritsch, J. Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1982".

The transformant cells of Escherichia coli were subjected to a known isolation procedure for plasmids. The isolated plas­mids were cut with restriction enzyme and analyzed in a con­ventional manner, so that a restriction enzyme cleavage map was prepared. Through cleavage with restriction enzyme and treatment with exonuclease Bal-31 in a conventional manner which was carried out based on the restriction enzyme cleavage map mentioned above, a deletion product, i.e. plasmid pGR115, was prepared.

The plasmid pGR115 was cleaved with restriction enzyme PvuII to obtain a DNA fragment which was then submitted to agarose gel electrophoresis, whereby a DNA fragment of about 4.2 Kb was selectively isolated from the gel. The DNA frag­ment thus obtained was ligated to plasmid pUC18 (a product of Takara Shuzo Co., Ltd.) cleaved with restriction enzyme SmaI, and the ligated DNA fragments were introduced (trans­ferred) into the cells of the Escherichia coli JM109 strain, to obtain transformant cells which grow in a LB medium containing Ampicillin 100 µg/ml. The cleavage of plasmid pGR115 as well as of plasmid pUC18 with the corresponding restriction enzymes, agarose-gel electrophoresis of DNA frag­ments, isolation of DNA fragments from gel, ligation of DNA fragments with plasmid pUC18 and transformation of the cells of Escherichia coli were carried out in a conventional manner as described, for example in "T. Maniatis, E.F. Fritsch, J. Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1982 ".

From the transformant cells of Escherichia coli were iso­lated plasmids (7.0 Kb) which were then cut and analyzed in a conventional manner to identify the base sequence. As a re­sult, Escherichia coli JM109 (FERM BP-1353) has been selected, which bears plasmid pGR1153 containing a DNA fragment of 4.2 Kb derived from plasmid pGR115.

It was found that the DNA fragment of 4.2 Kb thus obtained contains the DNA (nucleotide) segments Nos. I, II, III and a part of the DNA (nucleotide) segment No. IV. These four DNA (nucleotide) segments Nos. I, II, III and IV have been des­cribed as encoding respectively four particular proteins (See: Japanese Patent Application No. 68700/1986). The re­striction enzyme cleavage map of the DNA segments contained in plasmids pGR115 and pGR1153 and isolated from the cells of microorganisms having a benzene-oxidizing ability, such as Escherichia coli , as shown in Fig. 1.

Example 2

The cells of the Escherichia coli JM109 strain which contain plasmids pGR115, pGR1153 and pUC18 were inoculated in a LB medium (100 ml) containing Ampicillin 100 µg/ml and cultured at 30°C for 16 hours. After the cultivation, the culture liquid was centrifuged to recover the cells which were then washed with phosphate buffer (pH 7.0) (50 mH) and further suspended in the phosphate buffer (50 ml). The sus­pension was placed in a 500 ml flask (Sakaguchi), admixed with benzene (100 µl) and ethanol (100 µl), and the resultant mixture was stirred at 30°C to effect the conversion of ben­zene. After a reaction time of 7 hours, the content of 1,2-­dihydroxy-cyclohexa-3,5-diene in the reaction solution was determined by means of high-speed liquid chromatography (a product of Nihon Bunko Co., Ltd.), on the column of Finepac C-18, and detected by the absorption at 265nm.

The result is shown in the Table 1 below;

Example 3

The cells of Escherichia coli (FERM BP-1353) containing plasmids pGR1153 were treated in the same manner as in the Example 2, to obtain a reaction solution.

The reaction solution (200 ml) thus obtained was concen­trated to a volume of about 30 ml by a rotary evaporator. The concentrate was extracted successively with portions (each 50 ml) of diethyl ether. The extracts were combined and dried and evaporated to remove the diethyl ether. The solid residue was recrystallized from a mixture of methylene chloride and pentane, to obtain crystalls which were submitted to the fol­lowing test for identification of the product.

• (1) Analysis by NMR (proton) (270 MHz); NMR (deuterium) shows signals at 4.2 ppm (C-H, 2H), 4.6 ppm (O-H, 2H) and 5.9 ppm (C=C, 4H), which identifies the sample as cis-1,2-dihydroxy-­cyclohexa-3,5-diene.
• (2) Analysis by GC-MS (Gas Chromatography-Mass Spectrometry) GC-MS conducted on a column SE-30 (2 m), at 100°C (tem­perature on injection: 250°C), identified the sample as cor­responding to cis-1,2-dihydroxy-cyclohexa-3,5-diene, in view of its molecular weight of 112.