JP3001694B2 - Benzene dioxygenase gene - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a DNA containing a gene that confers an enzyme activity on a host microorganism to produce cisbenzene glycol by oxidizing benzene, a plasmid containing the DNA, and a microorganism transformed with the plasmid. About. Further, the present invention relates to DNA containing a gene encoding benzenedioxygenase, and a polypeptide constituting benzenedioxygenase.

[0002]

2. Description of the Related Art Cisbenzene glycol (cis-1,2)
-Dihydroxy-cyclohexa-3,5-diene) is
It is a compound useful as a raw material for polyparaphenylene, which has recently attracted particular attention as an engineering plastic. Biochemically, it is an important compound that is metabolized by the TCA cycle via ortho- or meta-pathways via catechol. is there.

Conventionally, as a method for producing cis benzene glycol using a microorganism, there is a method of culturing a microorganism belonging to the genus Pseudomonas in a medium containing benzene and collecting cis benzene glycol from the culture (Japanese Patent Laid-Open No. Sho.
No. 8-71891), but the method is inefficient and not practical. On the other hand, as microorganisms having a foreign gene having a function of metabolizing biphenyl by a metabolic process similar to the biological metabolic process of cisbenzene glycol, Pseudomonas putida KF138 strain (JP-A-61-282068) and Pseudomonas -Elginosa KF257 strain (Japanese Unexamined Patent Publication No. 61-28206)
No. 9) and Pseudomonas pseudoalcaligenes KF707 strain (Japanese Patent Application Laid-Open No. Sho 61-282850).
Although these microorganisms have an activity of assimilating biphenyl, they did not produce cisbenzene glycol by exhibiting a metabolic effect on benzene.

[0004]

DISCLOSURE OF THE INVENTION The present inventors encode a benzenedioxygenase from a microorganism having a function of generating cisbenzene glycol by oxidizing benzene by encoding the enzyme. As a result of diligent efforts to isolate the gene, the inventors have succeeded in isolating a gene encoding benzenedioxygenase from a microorganism belonging to Pseudomonas aeruginosa, and have completed the present invention. That is, the present invention relates to an approximately 7.2 Kb DNA containing a benzenedioxygenase gene, a cisbenzeneglycol dehydrogenase gene, and a catechol 2,3-oxygenase gene, which has a restriction enzyme map shown in FIG. DNA, recombinant vector DNA having the DNA, benzenedioxygenase, cisbenzeneglycol dehydrogenase, and catechol transformed with the vector
A DNA producing an oxygenase-producing microorganism and a DNA of about 5.6 Kb containing a benzenedioxygenase gene and having a restriction map shown in FIG.
A, a recombinant vector DNA having the DNA, and a microorganism which is transformed with the vector and produces benzenedioxygenase. The present invention also provides a DNA encoding benzenedioxygenase and a polypeptide constituting benzenedioxygenase.

[0005] The benzenedioxygenase gene (BD
As a source of the DNA containing O), a microorganism having the ability to produce cisbenzene glycol by assimilating benzene can be used. Among microorganisms having such properties, for example, microorganisms belonging to the genus Pseudomonas can be used. Can be used. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas aeruginosa JI10
Preferably, 4 is used. Pseudomonas aeruginosa JI104 is a microorganism isolated from soil collected in Tokyo, and has accession number No. 12180 to the Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology on April 6, 1991.
(FERM P-12180),
The mycological properties are as follows:

The bacterium is a gram-negative bacillus having polar flagella, which is motile, absolutely aerobic, and non-fermenting sugar.
From the above properties and observation with an electron microscope, it was confirmed that the bacterium is a microorganism belonging to the genus Pseudomonas. Therefore, Pseudomonas was classified as follows by the analysis of bacterial fatty acids of H. Oyaizu and K. Komagata (J. Gen. Appl. Microbiol., 29 , 17-14 (1983)).

[0007] First, the freeze-dried microbial cells were treated with 5% NaOH / 50% Me.
After saponification with OH at 100 ° C. for 1 hour, the extracted fatty acid was methylesterified with 15% BCl ₃ -MeOH at 85 ° C. for 5 minutes, and then subjected to GLC analysis. Hydroxy acids were confirmed by TLC analysis. GLC analysis: 15% EGS (3.0 φmm × 2.1 m), 180 ° C. TLC analysis: silica gel 60 TLC (diethyl ether: n-hexane = 1: 4) As a result of the above analysis, the fatty acid component of the bacterial cell was , 12: 0,
14: 0, 16: 0, 16: 1, 18: 0, 18: 1,
Δ17, Δ19, 3-OH · 10: 0, 3-OH · 12: 0
(Trace), 2-OH.12: 0.
In addition, trace amounts of 15: 0, 17: 0, and 19: 0 were also detected. Based on these analysis results, this bacterium was found to be H. Oyaizu and K.
Komagata (J. Gen. Appl. Microbiol., 29 , 17-14 (19
83)) It was confirmed to belong to Group 1. This group includes Pseudomonas aeruginosa (Pseudomonas
aeruginosa), P. putida, P. aureofaciens, P. chlororafis (P. ch)
lororaphis), P. floresces,
P. stutzeri and P. mendocina
mendocina) and the like. Then, 2-OH · 12: 0 is detected from the fatty acid component of the bacterial cell,
The characteristics such as OH · 16: 0 not being detected and Δ19 being detected in a significant amount were the most similar to the bacterial fatty acid pattern of P. aeruginosa in Group 1 described above. DNA is extracted from the cells of this bacterium and the GC content of the DNA is determined by T
As measured by the m method, the GC content was 66.3 mol%. This value is the GC content of P. aeruginosa = 67.2-
This was slightly lower than 68.0 mol%, which was clearly different from the GC content of P. putida = 62.5 mol%. Therefore, cis-1,2-dihydroxy-cyclohexa-3,
The results of Table 1 were obtained by comparing the mycological properties of P. putida, which is known as a 5-diene-producing bacterium, with this bacterium.

Table 1 {Isolated Bacteria} Morphology Gram stain Negative motility Flagella flagella-1 II II. Physiological properties Dye formation Fluorescent dye-pyocyanin-carotenoid pink Growth (41 ° C) ± oxidase reaction + denitrification reaction + gelatin hydrolysis-starch hydrolysis-──────────────── III. Growth glucose + maltose ± sucrose-mannitol-glycerol + L-malic acid + tartaric acid-succinic acid + malonic acid-betaine + sarcosine + geraniol + β-alanine + DL-arginine + L-serine-tyrosine + ──── ──────────────── However, +: positive-: negative As is clear from these results, this bacterium is clearly distinguished from P. putida and is the most similar bacterium. Is P. aeruginosa. However, this isolated bacterium is different from P. aeruginosa particularly in the behavior of gelatin for hydrolysis, mannitol, malonic acid, etc., has a slightly lower GC content than P. aeruginosa, and has a slightly pink cell. This strain was identified as a new variant of P. aeruginosa, since subtle differences were also observed.

The following method can be used to cut out a gene containing the benzenedioxygenase gene (BDO) from a microorganism capable of producing cis benzene glycol by utilizing benzene. The above-mentioned microorganisms having benzene assimilation ability are converted into an appropriate medium, for example, an LB medium (1.
After culturing with 10 g of tryptone / 5 g of yeast extract / 5 g of sodium chloride in a liter, for example, the cells are thawed using, for example, lysozyme to prepare chromosomal DNA by a conventional method, and digested with, for example, a restriction enzyme XhoI. Approximately 7.2 Kb of D containing the benzenedioxygenase gene
An NA fragment (NKKI1) can be obtained. The DNA fragment thus obtained is digested with a restriction enzyme XhoI, for example, a plasmid vector pHSG39.
6 and ligated with E. coli HB101 transformed into competent cells by the Hanahan method to obtain a transformant, for example, followed by culturing in an LB medium containing chloramphenicol by appropriate selection means, for example, chloramphenicol. To select a transformant.

To select a transformant having the recombinant vector containing the benzenedioxygenase gene from the above transformants, cultivating the transformant and spraying catechol thereon to select a yellow colony. This selects catechol 2,3-oxygenase in the same gene region as the benzene oxygenase gene, and 2-hydroxymuconic acid 6-semialdehyde produced as a result of catechol being oxidized by catechol 2,3-oxygenase. This is to utilize the appearance of yellow. As a transformant selected in this way,
For example, Escherichia coli HB101 / pXCY-Bp can be mentioned. E. coli HB101 / pXCY-Bp is a DNA of about 7.2 Kb containing a benzenedioxygenase gene.
Recombinant plasmid pXCY containing fragment (NKKI1)
-Bp transformed microorganism, and submitted to the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology on April 6, 1991 under the accession number of Microtechnical Laboratory No. 12178 (FERM P-1217).
8).

The N inserted into the plasmid pXCY-Bp
KKI1 is a benzenedioxygenase gene (BD
O), about 7.2 Kb of DNA containing the cisbenzene glycol dehydrogenase gene (cBDH) and the catechol 2,3-oxygenase gene (C23O), and the number of restriction enzymes digesting NKKI1 is Pst
I: 6; EcoRI: 2; SmaI: 1; Sal
I: 5; BglII: 1; BamHI: 2; Sa
XhoI: 1; XbaI: 0; Hind
III: 0; KpnI: 0; SphI: 6; and ClaI: 1. The relationship between the restriction enzyme map of NKKI1 and the benzene metabolism mode of the gene and the gene group is as shown in FIG. As the benzenedioxygenase gene, a gene described in Japanese Patent Application No. 62-278985 is known, but the above-mentioned NKKI1 has a restriction enzyme map different from that described in the above publication.

The thus obtained plasmid pXCY
By removing the genes encoding catechol 2,3-oxygenase and cisbenzene glycol dehydrogenase in Bp with exonuclease ExoIII, a DNA fragment containing only the benzenedioxygenase gene, for example, NKKI1-1 (about 5.6 Kb) is obtained. Obtainable. The above-mentioned NKKI1 (about 7.2 Kb) is digested with restriction enzymes KpnI and XbaI, treated with exonuclease ExoIII, treated with mung bean nuclease to remove a single-stranded portion of DNA, and then Klenow fragment is added. Then, the DNA ends are blunted and ligated according to a conventional method to obtain a recombinant plasmid having a benzenedioxygenase gene. Plasmid pBPT8-1 can be mentioned as such a plasmid, and a transformant transformed by a conventional method using the plasmid is selected by the above-described method to obtain Escherichia coli HB101 /
pBPT8-1 can be obtained. Escherichia coli HB101
/ PBPT8-1 is a microorganism transformed with a plasmid pBPT8-1 containing a DNA fragment (NKKI1-1) of about 5.6 Kb containing a benzenedioxygenase gene. Accession No. 12179 (FE)
RM P-12179).

The N inserted into plasmid pBPT8-1
KKI1-1 is a benzenedioxygenase gene (B
DO) and about 5.6 Kb of DNA.
-1 digestion number of restriction enzymes is PstI: 4; E
coRI: 2; SmaI: 1; SalI: 5; B
glII: 1; BamHI: 2; SacI: 1; Xh
oI: 1; XbaI: 0; HindIII: 0;
KpnI: 0; SphI: 6; and ClaI: 0, and the restriction map of NKKI1-1 is as shown in FIG.

By using the transformant thus obtained, cis benzene glycol can be efficiently produced from benzene. For example, a transformed microorganism is cultured in a medium such as M56 with ethanol, acetic acid,
By culturing in the presence of an energy source such as glucose or lactic acid and benzene, preferably in the vapor of benzene, cisbenzene glycol is produced in the medium. The reaction may be performed at 25 to 42 ° C., preferably 37 ° C., usually for 2 to 24 hours, and the concentration of benzene as a raw material may be about 0.07 to 0.2% based on the total amount of the culture solution. After completion of the reaction, cisbenzene glycol can be efficiently produced by extracting the culture solution with ether, methylene chloride or the like.

[0015]

As described above, a gene encoding benzenedioxygenase useful for producing cisbenzene glycol by oxidizing benzene and a microorganism having an ability to efficiently produce cisbenzeneglycol are provided. In addition, it has become possible to produce cis benzene glycol at low cost by utilizing the microorganism.

[0016]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 A Pseudomonas aeruginosa JI104 strain capable of assimilating benzene was added to 100 ml of an LB medium (10 g of tryptone / 5 g of yeast extract / 5 g of sodium chloride per liter).
After overnight culture, the cells were collected, washed with 50 mM phosphate buffer, and 10 ml of STE (10 mM Tris (pH 7.5) / 1 mM EDTA / 100 mM sodium chloride) and 5 mM lysozyme were added to the cells. And incubated at 37 ° C for 30 minutes, followed by freezing at -80 ° C for 30 minutes.
Thaw the cells and add 20 ml of STE containing 0.8% sarkosyl.
And 10 mg of Pronase E were added and incubated at 37 ° C. for 1 hour, followed by phenol treatment and ethanol precipitation. After removing the polysaccharide from the obtained DNA, the DNA was further subjected to phenol treatment and ethanol precipitation to obtain the obtained DNA.
Was suspended in 10 mM Tris / 1 mM EDTA buffer.

The obtained DNA was subjected to restriction enzyme Sau3AI.
(Y-100 buffer: 10 mM Tris (pH 7.5) / 10 mM
The mixture was treated at 37 ° C. for 30 minutes with magnesium chloride / 100 mM sodium chloride / 6 mM mercaptoethanol for partial digestion, and a 5 Kb to 7 Kb DNA fragment was cut out by 0.8% agarose gel electrophoresis. This DNA fragment was ligated with the plasmid pUC19 digested with the restriction enzyme BamHI, and the resulting recombinant plasmid pCY13 was transformed into the host E. coli HB101.
To obtain a transformant capable of growing on an LB medium containing 50 μg / ml of ampicillin.

Catechol 5 was used for colonies on this medium.
A yellowing colony was selected by spraying with 0 mM. Catechol was obtained from the cells obtained by culturing the colonies.
Plasmid pC having 2,3-oxygenase gene
Y13 was extracted and its nucleotide sequence was determined by the dideoxy method. Thus, the orientation of the catechol 2,3-oxygenase gene on the gene was clarified, and the DNA extracted from Pseudomonas aeruginosa JI104 was digested with various restriction enzymes using the 5 ′ upstream of the DNA inserted into pCY13 as a probe. Was subjected to Southern blotting by 1.0% agarose gel electrophoresis. This results in about 7 Kb of D digested by the restriction enzyme XhoI.
It was estimated that the operon containing catechol 2,3-oxygenase and its upstream was contained in the NA fragment.

Pseudomonas aeruginosa JI104
The chromosomal DNA was digested with restriction enzyme XhoI, electrophoresed on a 0.8% agarose gel, a DNA fragment of 6 Kb to 8 Kb was cut out, ligated with plasmid pHSG396 digested with restriction enzyme XhoI, and the obtained recombinant plasmid was transformed into Escherichia coli HB101. And cultured in an LB medium containing 34 μg / ml of chloramphenicol to select a transformant. Catechol 50 was added to the colonies on this medium.
The yellowing colonies were selected by spraying mM. After culturing the selected transformant overnight in 5 ml of LB medium, collecting cells,
Washed, the M56 medium (1 liter of water containing proline 20 [mu] g / ml and succinic acid 5 g / l Na ₂ HPO ₄ .7H ₂ O
_{8.2g / KH 2 PO 4 2.7g /} (NH 2) 2 SO 4 1.0g / FeSO 4 .7H 2 O 0.23mg
) And cultured at 37 ° C. for 2 hours under benzene vapor, and 1 ml was recovered. After removing Escherichia coli by centrifugation and removing polymers having a molecular weight of 10,000 or more by ultrafiltration, the production of cisbenzene glycol and catechol was confirmed by HPLC. Plasmid pXCY-Bp was extracted from the transformant, treated with restriction enzymes, 0.8% agarose gel electrophoresis, and 6% acrylylamide gel electrophoresis to prepare a restriction map of NKKI1 (see FIG. 1).

Further, a plasmid pBPT8-1 incorporating an inserted DNA fragment (NKKI1-1: 5.6 Kb) containing a benzenedioxygenase gene was prepared as follows. 5 μg of plasmid pXCY-Bp was replaced with restriction enzyme Kp
After digestion with nI and XbaI, phenol treatment and ethanol precipitation were performed, suspended in exonuclease ExoIII buffer (50 mM Tris / 1 mM magnesium chloride / 1 mM 2-mercaptoethanol), and 150 units of exonuclease ExoIII was added thereto. Incubation was performed for 5 minutes and then at 65 ° C. for 5 minutes to inactivate the exonuclease ExoIII. Further, an equal amount of mung bean nuclease buffer (60 mM sodium acetate / 100 mM sodium chloride / 2 mM zinc acetate /
10% glycerin) and 5 units of mung bean nuclease and incubate at 37 ° C. for 1 hour to give D
Removal of the single-stranded portion of NA was performed. After phenol treatment and ethanol precipitation, Klenow fragment buffer (67 mM potassium phosphate / 6.7 mM magnesium chloride / 1 mM 2-mercaptoethanol / 33 μM dN
TP), 5 units of Klenow fragment were added, and the mixture was incubated at 37 ° C. for 15 minutes to blunt DNA ends, ligated according to a conventional method, and introduced into E. coli HB101. The restriction map of NKKI1-1 is as shown in FIG. Example 2 Plasmid pB containing benzenedioxygenase gene
5 ml of E. coli HB101 transformed with PT8-1
Was cultured overnight at 37 ° C. using LB medium. 4 × 10 ⁹
Cells / ml of HB101 are collected and washed, and 20 μg / ml
Of proline and 5 ml of M containing 5 g / l of succinic acid
Resuspended in 56 medium. The cells were cultured under benzene vapor for 22 hours, purified by centrifugation and ultrafiltration, and the concentration of cisbenzene glycol was measured by HPLC (OD27
5 nm), and it was confirmed that cisbenzene glycol was produced. Example 3 The nucleotide sequence of NKKI1-1 was determined as follows. NKKI1-1 was excised with each restriction enzyme and then recombined into M13 plasmid or NKKI1-1.
Was cut by the kilosequence method to prepare a sample, and the nucleotide sequence of 4.7 kb encoding benzenedioxygenase was determined by the dideoxy method. The recombinant DNA production method, kilosequence method, and dideoxy method followed conventional methods. As a result, it was revealed that the sequence of the DNA encoding benzenedioxygenase was a 4721 base sequence including five coding regions, as shown below.

[0021]

Embedded image

In the sequence, between the region IV and the region V,
Since the sequence encoding region IV ends at the 3411th to 3413th stop codon, and the sequence encoding region V starts at the 3410th to 3412th start codon, the DNA is partially doubled as described below. Is read.

[0023]

Embedded image

For this reason, in the following sequence listing, 3408
The amino acid encoded by the 3rd to 3409th bases is indicated as Pro.

[0025]

[Sequence list] Sequence length: 7421 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin: Organism name: Shoot {monas erquimonosa} (Pseudomonas aeruginosa) Strain name: JI104 Sequence Features Symbol indicating feature: -10 signal Location: 479..484 Method determining feature: S Symbol indicating feature: -35 signal Location: 455..460 Method determining feature: S array CTCGAGAGCG AATGTACGCC AAAGCGTGCT GAATGGTGAC GCTGAATGGG AGGCGCGGAT 60 CGTCAGTTCG TTTCACCGAC TGTCATTGAT TGAAGAGCCC ACGATGCGGG ATCCGGCTCG 120 CTGGTTTAAT GAGTGGGAGC CAGTCAACCG CCGGTTTTCA CGAAGCTCTT ATCTCTGCCC 180 TGTTCGTCCG TCTGGATCCG GCGGTTCCTG TCCATCCTGT ATGTGCATAT GGAGCGCTAC 240 CGCCGATTGA CTGCTATGCA CAACCCGCCT ACCAGAAACG TACATGAGGA GCATCTAGCA 300 CTTCGCGACA GCCGCTCGCC GGAGATGCCG AGCGCTGTGC TGCGTTGATG GCGGAGCACA 360 TCGAATCATC AATTTCGGTG GTTCGGGAAT TCGGTTTGTT GAGGTGACGC CAACATCCCA 420 CGTGTTTGTT TTCGTCGTGA TGGATGCGGT CGGCATTTTT TC GCCCTACT AAGGGCATTT 480 CAAAGGAGAC GTTGAATC 498 ATG AGC TCA TCA ATC AAA GAA GTG CAG GGA GCC CCT GTG AAG TGG GTT 546 Met Ser Ser Ser Ile Lys Glu Val Gln Gly Ala Pro Val Lys Trp Val 1 5 10 15 ACC AAT TGG ACG CCG GCG ATC CGG GGG TTG GTC GAT CAG GAA AAA 594 Thr Asn Trp Thr Pro Glu Ala Ile Arg Gly Leu Val Asp Gln Glu Lys 20 25 30 GGG CTG CTT GAT CCA CGC ATC TAC GCC GAT CAG AGT CTT TAT GAG CTG 642 Gly Leu Leu Asp Pro Arg Ile Tyr Ala Asp Gln Ser Leu Tyr Glu Leu 35 40 45 GAG CTT GAG CGG GTT TTT GGT CGC TCT TGG CTG TTA CTT GGG CAC GAG 690 Glu Leu Glu Arg Val Phe Gly Arg Ser Trp Leu Leu Leu Gly His Glu 50 55 60 AGT CAT GTG CCT GAA ACC GGG GAC TTC CTG GCC ACT TAC ATG GGC GAA 738 Ser His Val Pro Glu Thr Gly Asp Phe Leu Ala Thr Tyr Met Gly Glu 65 70 75 80 GAT CCG GTG GTT ATG GTG CGA CAG AAA GAC AAG AGC ATC AAG GTG TTC 786 Asp Pro Val Val Met Val Arg Gln Lys Asp Lys Ser Ile Lys Val Phe 85 90 95 CTG AAC CAG TGC CGC GGC ATG CGT ATC TGC CGC TCG GAC GCC GGC AAC 834 Leu Asn Gln Cys Arg Gly Met Arg I le Cys Arg Ser Asp Ala Gly Asn 100 105 110 GCC AAG GCT TTC ACC TGC AGC TAT CAC GGC TGG GCC TAC GAC ATC GCC 882 Ala Lys Ala Phe Thr Cys Ser Tyr His Gly Trp Ala Tyr Asp Ile Ala 115 120 125 GGC AAG CTG GTG AAC GTG CCG TTC GAG AAG GAA GCC TTT TGC GAC AAG 930 Gly Lys Leu Val Asn Val Pro Phe Glu Lys Glu Ala Phe Cys Asp Lys 130 135 140 AAA GAA GGC GAC TGC GGC TTT GAC AAG GCC GAA TGG GGC CCG CTC CAG 978 Lys Glu Gly Asp Cys Gly Phe Asp Lys Ala Glu Trp Gly Pro Leu Gln 145 150 155 160 GCA CGC GTG GCA ACC TAC AAG GGC CTG GTC TTT GCC AAC TGG GAT GTG 1026 Ala Arg Val Ala Thr Tyr Lys Gly Leu Val Phe Ala Asn Trp Asp Val 165 170 175 CAG GCG CCA GAC CTG GAG ACC TAC CTC GGT GAC GCC CGC CCC TAT ATG 1074 Gln Ala Pro Asp Leu Glu Thr Tyr Leu Gly Asp Ala Arg Pro Tyr Met 180 185 190 GAC GTC ATG CTG GAT CGC ACG CCG GCC GGG ACT GTG GCC ATC GGC GGC 1122 Asp Val Met Leu Asp Arg Thr Pro Ala Gly Thr Val Ala Ile Gly Gly 195 200 205 ATG CAG AAG TGG GTG ATT CCG TGC AAC TGG AAG TTT GCC GCC GAG CAG 1170 Met Gln Lys Trp V al Ile Pro Cys Asn Trp Lys Phe Ala Ala Glu Gln 210 215 220 TTC TGC AGT GAC ATG TAC CAC GCC GGC ACC ATG TCG CAC CTG TCC GGC 1218 Phe Cys Ser Asp Met Tyr His Ala Gly Thr Met Ser His Leu Ser Gly 225 230 235 240 ATC CTG GCG GGC ATG CCG CCG GAA ATG GAC CTG TCG CAT GCA CAG GTG 1266 Ile Leu Ala Gly Met Pro Pro Glu Met Asp Leu Ser His Ala Gln Val 245 250 255 CCC ACC AAG GGC AAC CAG TTC CGG GCC GGC TGG GGC GGG CAC GGC TCG 1314 Pro Thr Lys Gly Asn Gln Phe Arg Ala Gly Trp Gly Gly His Gly Ser 260 265 270 GGC TGG TTC GTC GAC GAG CCG GGC ATG CTC ATG GCG GTG ATG GGG CCC 1362 Gly Trp Phe Val Asp Glu Pro Gly Met Leu Met Ala Val Met Gly Pro 275 280 285 AAG GTC ACC CAG TAC TGG ACC GAA GGT CCG GCT GCC GAC CTG GCA GAA 1410 Lys Val Thr Gln Tyr Trp Thr Glu Gly Pro Ala Ala Asp Leu Ala Glu 290 295 300 CAG CGA CTG GGC CAC ACC ATG CCG GTT CGA CGC ATG TTC GGC CAG CAC 1458 Gln Arg Leu Gly His Thr Met Pro Val Arg Arg Met Phe Gly Gln His 305 310 315 320 ATG ACG ATC TTC CCG ACC TGT TCA TTC CTG CCC GCC ATC AAC ACC AT C 1506 Met Thr Ile Phe Pro Thr Cys Ser Phe Leu Pro Ala Ile Asn Thr Ile 325 330 335 CGG ACC TGG CAC CCG CGT GGT CCC AAT GAA ATC GAG GTG TGG GCC TTC 1554 Arg Thr Trp His Pro Arg Gly Pro Asn Glu Ile Glu Val Trp Ala Phe 340 345 350 ACC CTG GTC GAT GCC GAC GCC CCG GCG GAG ATC AAG GAA GAA TAT CGC 1602 Thr Leu Val Asp Ala Asp Ala Pro Ala Glu Ile Lys Glu Glu Tyr Arg 355 360 365 CGG CAC AAC ATC CGC ACC TTC TCC GCA GGC GGC GTG TTT GAG CAG GAC 1650 Arg His Asn Ile Arg Thr Phe Ser Ala Gly Gly Val Phe Glu Gln Asp 370 375 380 GAT GGC GAG AAC TGG GTG GAG ATC CAG AAG GGG CTA CGT GGG TAC AAG 1698 Asp Gly Glu Asn Trp Val Glu Ile Gln Lys Gly Leu Arg Gly Tyr Lys 385 390 395 400 GCC AAG AGC CAG CCG CTC AAT GCC CAG ATG GGC CTG GGT CGG TGC AGA 1746 Ala Lys Ser Gln Pro Leu Asn Ala Gln Met Gly Leu Gly Arg Cys Arg 405 410 415 CCG GAT CAC CCT GAT TTT CCT GGC AAC GTC GGC 1779 Pro Asp His Pro Asp Phe Pro Gly Asn Val Gly 420 425 TAGCTCTACG CCGAAGAAGC GGCGCGGGGT ATGTATCACC ACTGGATGCG CATGATGTCC 1839 GAGCCCAGCT GGGCC ACGCT CAAGCCCTGA TCAAGACGCA ATCGTTAGAT CTGTCAACCG 1899 GAAGAATTCA AC 1911 ATG GTG GGC TGG ACG TGC ATG TGC AGA CGG CGC GCC GAG GTT CCG TCC 1959 Met Val Gly Trp Thr Cys Met Cys Arg Arg Arg Ala Glu Val Pro CT 1 ATT ATT TTG GAG ATA ACT ATT ATG ACA AAT CCA TCC CCG CAT 2007 Pro Asp Ile Tyr Leu Glu Ile Thr Ile Met Thr Asn Pro Ser Pro His 20 25 30 TTT TTC AAA ACA TTT GAA TGG CCA AGC AAG GCG GCT GGC CTT GAG TTG 2055 Phe Phe Lys Thr Phe Glu Trp Pro Ser Lys Ala Ala Gly Leu Glu Leu 35 40 45 CAG AAC GAG ATC GAG CAG TTC TAC TAC CGC GAA GCG CAG TTG CTT GAC 2103 Gln Asn Glu Ile Glu Gln Phe Tyr Tyr Arg Glu Ala Gln Leu Leu Asp 50 55 60 CAC CGG GCC TAC GAG GCC TGG TTT GCC CTG CTG GAC AAA GAT ATC CAC 2151 His Arg Ala Tyr Glu Ala Trp Phe Ala Leu Leu Asp Lys Asp Ile His 65 70 75 80 TAC TTC ATG CCG CTG CGC ACC AAT CGC ATG ATC CGG GAG GGC GAG CTG 2199 Tyr Phe Met Pro Leu Arg Thr Asn Arg Met Ile Arg Glu Gly Glu Leu 85 90 95 GAA TAT TCC GGC GAC CAG GAT ATT GCC CAT TTC GAT GAA ACC CAT GAA 2247 G lu Tyr Ser Gly Asp Gln Asp Ile Ala His Phe Asp Glu Thr His Glu 100 105 110 ACC ATG TAC GGG CGC ATC CGC AAG GTG ACC TCG GAC GTG GGC TGG GCG 2295 Thr Met Tyr Gly Arg Ile Arg Lys Val Thr Ser Asp Val Gly Trp Ala 115 120 125 GAG AAC CCG CCT TCC CGC ACG CGC CAC CTG GTC TCC AAC GTC ATC GTC 2343 Glu Asn Pro Pro Ser Arg Thr Arg His Leu Val Ser Asn Val Ile Val 130 135 140 AAG GAG ACG GCC ACG CCG GAT ACC TTC GAG GTC AAT TCC GCA TTC ATC 2391 Lys Glu Thr Ala Thr Pro Asp Thr Phe Glu Val Asn Ser Ala Phe Ile 145 150 155 160 CTG TAC CGC AAT CGG CTT GAG CGC CAG GTC GAC ATC TTC GCG GGC GAA 2439 Leu Tyr Arg Asn Arg Leu Glu Arg Gln Val Asp Ile Phe Ala Gly Glu 165 170 175 CGC CGG GAC GTG CTG CGC CGC GCC GAC AAC AAC CTT GGT TTC AGC ATC 2487 Arg Arg Asp Val Leu Arg Arg Ala Asp Asn Asn Leu Gly Phe Ser Ile 180 185 190 GCC AAG CGC ACC ATC CTG CTC GAC GCC AGT ACC TTG CTG TCG AAC AAC 2535 Ala Lys Arg Thr Ile Leu Leu Asp Ala Ser Thr Leu Leu Ser Asn Asn 195 200 205 CTG AGC ATG TTC TTC 2550 Leu Ser Met Phe Phe 210 TA GCCCAGCA CGCTGAACCG GCCTCAATGA GGATGCTGCC 2590 ATG AAA AAT GCA AGA CTG TTT TTG ATC GCC ATC GGC GTC TTC TAC ATC 2638 Met Lys Asn Ala Arg Leu Phe Leu Ile Ala Ile Gly Val Phe Tyr Ile 1 5 10 15 ATC AGC CTC CTCTC TTC AGC ACG TTG GGC TTG TTT GGC 2686 Ile Asn Leu Ile Gly Thr Leu Pro Phe Ser Thr Leu Gly Leu Phe Gly 20 25 30 AGG ATG TAT CCA GGC GTA GAA CTG CAC GTG GGT GCG CCG ATT TTC ACC 2734 Arg Met Tyr Pro Gly Val Glu Leu His Val Gly Ala Pro Ile Phe Thr 35 40 45 CTG CTG CAG GAT GCC TGG GCG GTG GTC GGT CTC CAG TTG GGC GCC ATC 2782 Leu Leu Gln Asp Ala Trp Ala Val Val Gly Leu Gln Leu Gly Ala Ile 50 55 60 GGG GCC GTC GCT TTG TGG GGC GCC CGC GAT CCG GGC CGT TAT CGG GCC 2830 Gly Ala Val Ala Leu Trp Gly Ala Arg Asp Pro Gly Arg Tyr Arg Ala 65 70 75 80 GTT ATT CCA GTG GTC ATC GCA ACG GAA GTG GTC GAT GGC CTC TGG GAT 2878 Val Ile Pro Val Val Ile Ala Thr Glu Val Val Asp Gly Leu Trp Asp 85 90 95 TTT TAC AGC ATC GTG TGG AGC CAC GAA GCC TTG TGG TTC GGG CTT GTC 2926 Phe Tyr Ser Ile Val Trp Ser His Glu Ala Leu Trp Phe Gly Leu Val 100 105 110 ACG CTG GTG ATC CAT GTG CTG TGG ATT GGC TGG GGC CTG CAT GCC TGG 2974 Thr Leu Val Ile His Val Leu Trp Ile Gly Trp Gly Leu His Ala Trp 115 120 125 CGT GCC TGG CGT CGA AAT CGC 2995 Arg Ala Trp Arg Arg Asn Arg 130 135 TGAGGACACT TTGAATTACT CTTCAGCCAC CAACAGTGAC TGTTCGCCCC AGGCGATTTA 3055 ACCCTTTTAA CTAATTACAA GAAGCGTT 3083 ATG AAA TTT ACC AGA GTT TGT GAT CGA AGA GAT GAT CGA AGA GAT GAT CGA AGA GAT GGA CGA GGA GAT GGA CGA AGA GAT GGA CGA GGA GAT CGA AGA GAT Asp Arg Arg Asp Val Pro Glu Gly Glu 1 5 10 15 GCC CTG AAG GTC GAA AGT GGA GGC ACC TCC GTC GCG ATT TTC AAT GTG 3179 Ala Leu Lys Val Glu Ser Gly Gly Thr Ser Val Ala Ile Phe Asn Val 20 25 30 GAT GGC GAG CTG TTC GCA ACA CAG GAC CGC TGC ACC CAC GGC GAC TGG 3227 Asp Gly Glu Leu Phe Ala Thr Gln Asp Arg Cys Thr His Gly Asp Trp 35 40 45 TCC CTG TCC GAT GGC GGC TAT CTT GAA GGT GAC GTG GTG GAA TGC TCA 3275 Ser Leu Ser Asp Gly Gly Tyr Leu Glu Gly Asp Val Val Glu Cys Ser 50 55 60 CTG CAC ATG GGG AAG TTT TGC GTT CGC ACG GGC AAG GTC AAA TCA C CG 3323 Leu His Met Gly Lys Phe Cys Val Arg Thr Gly Lys Val Lys Ser Pro 65 70 75 80 CCG CCC TGT GAG GCA CTG AAG ATA TTT CCG ATC CGC ATC GAA GAC AAT 3371 Pro Pro Cys Glu Ala Leu Lys Ile Phe Pro Ile Arg Ile Glu Asp Asn 85 90 95 GAC GTG CTG GTC GAC TTC GAA GCC GGG TAT CTG GCG CC 3409 Asp Val Leu Val Asp Phe Glu Ala Gly Tyr Leu Ala Pro 100 105 ATG ATC GAC ACC ATC GCC ATC ATC GGC GCC GGC CTG GCC GTT CGA CGG 3457 Met Ile Asp Thr Ile Ala Ile Ile Gly Ala Gly Leu Ala Val Arg Arg 1 5 10 15 CTG CGC GCG CAC TGC CGC CAG GGA TAC GAG GGG CGC ATC CAC CTG CTC 3505 Leu Arg Ala His Cys Arg Gln Gly Tyr Glu Gly Arg Ile His Leu Leu 20 25 30 GGG GAT GAG TCG CAT CAG GCC TAT GAC CGG ACC ACG CTG TCC AAG ACG 3553 Gly Asp Glu Ser His Gln Ala Tyr Asp Arg Thr Thr Leu Ser Lys Thr 35 40 45 GTG CTG GCG GGC GAG CAG CCC GAG CCG CCT GCA ATC CTG GAC AGC GCC 3601 Val Leu Ala Gly Glu Gln Pro Glu Pro Pro Ala Ile Leu Asp Ser Ala 50 55 60 TGG TAC GCA TCG GCC CAT GTG GAT GTC CAG CTC GGG CGA CGG GTG AGT 3649 Trp Tyr Ala Ser Ala His Val Asp Val Gln Leu Gly Arg Arg Val Ser 65 70 75 80 TGC CTG GAT CTG GCC AAC CGC CAG ATT CAG TTT GAA TCG GGC GCC CCG 3697 Cys Leu Asp Leu Ala Asn Arg Gln Ile Gln Phe Glu Ser Gly Ala Pro 85 90 95 CTG GCC TAC GAC CGG CTG CTG CTG GCC ACC GGC GCG CGC GCC CGG CGC 3745 Leu Ala Tyr Asp Arg Leu Leu Leu Ala Thr Gly Ala Arg Ala Arg Arg 100 105 110 ATG GCG ATT CGG GGT GGC GAC CTG GCA GGC ATC CAT ACC TTG CGA GAC 3793 Met Ala Ile Arg Gly Gly Asp Leu Ala Gly Ile His Thr Leu Arg Asp 115 120 125 CTC GCC GAC AGC CAG GCG CTG CGG CAG GCG CTG CAA CCG GGC CAG TCG 3841 Leu Ala Asp Ser Gln Ala Leu Arg Gln Ala Leu Gln Pro Gly Gln Ser 130 135 140 CTG GTC ATC GTC GGC GGA GGC CTG ATC GGT TGC GAG GTG GCG ACC ACC 3889 Leu Val Ile Val Gly Gly Gly Gly Leu Ile Gly Cys Glu Val Ala Thr Thr 145 150 155 160 GCC CGC AAG CTG AGT GTC CAT GTC ACG ATT CTG GAA GCC GGC GAC GAG 3937 Ala Arg Lys Leu Ser Val His Val Thr Ile Leu Glu Ala Gly Asp Glu 165 170 175 TTG CTG GTG CGC GTG CTG GGT CAC CGG ACC GGG GCA TGG TGT CGG GCC 3985 Leu Leu Val Arg Val Leu Gly His Arg Thr Gly Ala Trp Cys Arg Ala 180 185 190 GAA CTG GAA CGC ATG GGT GTC CGC GTG GAG CGC AAT GCA CAG GCC GCG 4033 Glu Leu Glu Arg Met Gly Val Arg Val Glu Arg Asn Ala Gln Ala Ala 195 200 205 CGC TTC GAA GGC CAG GGG CAG GTG CGC GCC GTG ATC TGC GCC GAC GGG 4081 Arg Phe Glu Gly Gln Gly Gln Val Arg Ala Val Ile Cys Ala Asp Gly 210 215 220 CGC CGG GTG CCC GCC GAT GTG GTC TTG GTC AGC ATT GGC GCC GAG CCG 4129 Arg Arg Val Pro Ala Asp Val Val Leu Val Ser Ile Gly Ala Glu Pro 225 230 235 240 GCG GAC GAG CTG GCC CGT GCC GCT GGC ATC GCC TGC GCG CGC GGC GTG 4177 Ala Asp Glu Leu Ala Arg Ala Ala Gly Ile Ala Cys Ala Arg Gly Val 245 250 255 CTG GTC GAC GCC ACC GGC GCC ACC TCG TGT CCA GAG GTG TTC GCC GCC 4225 Leu Val Asp Ala Thr Gly Ala Thr Ser Cys Pro Glu Val Phe Ala Ala 260 265 270 270 GGT GAC GTC GCC GCC TGG CCG CTG CGT CAA GGG GGC CAG CGC TCG CTG 4273 Gly Asp Val Ala Ala Trp Pro Leu Arg Gln Gly Gly Gln Arg Ser Leu 275 280 285 GAG ACC TAC CTG AAC AGC CAG ATG GAG GCC GAA ATC GCG G CC AGC GCC 4321 Glu Thr Tyr Leu Asn Ser Gln Met Glu Ala Glu Ile Ala Ala Ser Ala 290 295 300 ATG TTG AGT CAG CCC GTG CCG GCG CCC CAG GTG CCG ACC TCG TGG ACG 4369 Met Leu Ser Gln Pro Val Pro Ala Pro Gln Val Pro Thr Ser Trp Thr 305 310 315 320 GAG ATT GCA GGC CAC CGC ATC CAG ATG ATT GGC GAT GCC GAA GGG CCC 4417 Glu Ile Ala Gly His Arg Ile Gln Met Ile Gly Asp Ala Glu Gly Pro 325 330 335 GGC GAG ATC GTC GTA CGC GGC GAC GCC CAG AGC GGC CAG CCA ATC GTG 4465 Gly Glu Ile Val Val Arg Gly Asp Ala Gln Ser Gly Gln Pro Ile Val 340 345 350 TTG CTC AGG CTG CTT GAT GGC TGC GTC GAG GCC GCG ACG GCG ATC AAT 4513 Leu Leu Arg Leu Leu Asp Gly Cys Val Glu Ala Ala Thr Ala Ile Asn 355 360 365 GCC ACC AGG GAA TTT TCT GTG GCG ACC CGA CTG GTC GGC ACC CGG GTT 4561 Ala Thr Arg Glu Phe Ser Val Ala Thr Arg Leu Val Gly Thr Arg Val 370 375 380 TCT GTT TCC GCC GAG CAA CTG CAG GAC GTC GGC TCG AAC CTG CGG GAT 4609 Ser Val Ser Ala Glu Gln Leu Gln Asp Val Gly Ser Asn Leu Arg Asp 385 390 395 400 400 TTA CTC AAA GCC AAA CCG AAT 46 30 Leu Leu Lys Ala Lys Pro Asn 405 TGATGCGCAT GACCGGCGAA TCGCTTTAAC AATAAAGGGG ATTGGGGATT GGAAAAATGA 4690 AACTGAAAGG TGAACGGGTA CTGTCACGGG G 4721

[Brief description of the drawings]

FIG. 1. Restriction map of NKKI1 and NKKI1
Benzene dioxygenase gene (BD
O), cisbenzene glycol dehydrogenase gene (cBDH), and catechol 2,3-oxygenase gene (C23O) and the metabolic pattern of benzene.

FIG. 2 is a diagram showing a restriction map of NKKI1-1.

FIG. 3 NK containing benzenedioxygenase gene
It is a figure which shows the process of producing the recombinant plasmid pBPT8-1 containing a benzenedioxygenase gene from KI1.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ identification code FI C12R 1:19) (58) Investigated field (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 C12N 1 / 00-1/38 C12N 9/00-9/99 C12P 1/00-41/00 BIOSIS (DIALOG) GenBank / EMBL / DDBJ (GENETYX) WPI (DIALOG)

Claims (17)

(57) [Claims] 1. About 7.2K containing a benzene dioxygenase gene, a cis benzene glycol dehydrogenase gene, and a catechol 2,3-oxygenase gene.
b, wherein the number of cut restriction enzymes is PstI:
6; EcoRI: 2; SmaI: 1; SalI:
5; BglII: 1; BamHI: 2; Sac
XhoI: 1; XbaI: 0; Hind
III: 0; KpnI: 0; SphI: 6; and ClaI: 1, and the following restriction map: A DNA comprising: 2. A recombinant vector DNA having the DNA according to claim 1. 3. The recombinant vector DNA according to claim 2, which is a plasmid pXCY-Bp. 4. A microorganism transformed with the vector according to claim 2 to produce benzenedioxygenase, cisbenzene glycol dehydrogenase, and catechol 2,3-oxygenase. 5. The microorganism according to claim 4, which is Escherichia coli HB101 / pXCY-Bp. 6. A DNA of about 5.6 Kb containing a benzenedioxygenase gene, wherein the number of restriction enzymes cut is Ps.
tI: 4; EcoRI: 2; SmaI: 1; Sa
11: BglII: 1; BamHI: 2; S
acI: 1; XhoI: 1; XbaI: 0; Hin
dIII: 0; KpnI: 0; SphI: 6; and ClaI: 0, and a restriction map shown below. A DNA comprising: 7. A recombinant vector DNA having the DNA according to claim 6. 8. The recombinant vector DNA according to claim 7, which is a plasmid pBPT8-1. 9. A microorganism which is transformed with the vector according to claim 8 and produces benzenedioxygenase. 10. The microorganism according to claim 9, which is Escherichia coli HB101 / pBPT8-1. 11. A D which comprises the following regions I, II, III, IV and V and encodes benzenedioxygenase:
NA. Region I ATG AGC TCA TCA ATC AAA GAA GTG CAG GGA GCC CCT GTG AAG TGG GTT ACC AAT TGG ACG CCG GAG GCG ATC CGG GGG TTG GTC GAT CAG GAA AAA GGG CTG CTT GAT CCA CGC ATC TAC GCC GAT CAG AGT CTT TAT GAG CTG GAG CTT GAG CGG GTT TTT GGT CGC TCT TGG CTG TTA CTT GGG CAC GAG AGT CAT GTG CCT GAA ACC GGG GAC TTC CTG GCC ACT TAC ATG GGC GAA GAT CCG GTG GTT ATG GTG CGA CAG AAA GAC AAG AGC ATC AAG GTG TTC ATC CAG TGC CGC GGC ATG CGT ATC TGC CGC TCG GAC GCC GGC AAC GCC AAG GCT TTC ACC TGC AGC TAT CAC GGC TGG GCC TAC GAC ATC GCC GGC AAG CTG GTG AAC GTG CCG TTC GAG AAG GAA GCC TTT TGC GAC AAG AAA GAGC TGC GGC TTT GAC AAG GCC GAA TGG GGC CCG CTC CAG GCA CGC GTG GCA ACC TAC AAG GGC CTG GTC TTT GCC AAC TGG GAT GTG CAG GCG CCA GAC CTG GAG ACC TAC CTC GGT GAC GCC CGC CCC TAT ATG GAC GTC CTG CTG GAT ACG CCG GCC GGG ACT GTG GCC ATC GGC GGC ATG CAG AAG TGG GTG ATT CCG TGC AAC TGG AAG TTT GCC GCC GAG CAG TTC TGC AGT GAC ATG TAC CAC GCC GGC ACC ATG TCG CAC CTG TCC GGC ATC CTG GCG GGC ATG CCG CCG A ATG GAC CTG TCG CAT GCA CAG GTG CCC ACC AAG GGC AAC CAG TTC CGG GCC GGC TGG GGC GGG CAC GGC TCG GGC TGG TTC GTC GAC GAG CCG GGC ATG CTC ATG GCG GTG ATG GGG CCC AAG GTC ACC CAG TAC TGG ACC GAGA CCG GCT GCC GAC CTG GCA GAA CAG CGA CTG GGC CAC ACC ATG CCG GTT CGA CGC ATG TTC GGC CAG CAC ATG ACG ATC TTC CCG ACC TGT TCA TTC CTG CCC GCC ATC AAC ACC ATC CGG ACC TGG CAC CCG CGT GGT CTC AGA GAG GTG TGG GCC TTC ACC CTG GTC GAT GCC GAC GCC CCG GCG GAG ATC AAG GAA GAA TAT CGC CGG CAC AAC ATC CGC ACC TTC TCC GCA GGC GGC GTG TTT GAG CAG GAC GAT GGC GAG AAC TGG GTG GAG ATC CAG AGT GTA GGG TAC AAG GCC AAG AGC CAG CCG CTC AAT GCC CAG ATG GGC CTG GGT CGG TGC AGA CCG GAT CAC CCT GAT TTT CCT GGC AAC GTC GGC Area II ATG GTG GGC TGG ACG TGC ATG TGC AGA CGG CGC GCC GAG GTT CCG TCCCT ATT TAC TTG GAG ATA ACT ATT ATG ACA AAT CCA TCC CCG CAT TTT TTC AAA ACA TTT GAA TGG CCA AGC AAG GCG GCT GGC CTT GAG TTG CAG AAC GAG ATC GAG CAG TTC TAC TAC CGC GAA GCG CAG TTG CTT GAC CAC GGCC G AG GCC TGG TTT GCC CTG CTG GAC AAA GAT ATC CAC TAC TTC ATG CCG CTG CGC ACC AAT CGC ATG ATC CGG GAG GGC GAG CTG GAA TAT TCC GGC GAC CAG GAT ATT GCC CAT TTC GAT GAA ACC CAT GAA ACC ATG TAC GGG CTC ATC CGC AAG GTG ACC TCG GAC GTG GGC TGG GCG GAG AAC CCG CCT TCC CGC ACG CGC CAC CTG GTC TCC AAC GTC ATC GTC AAG GAG ACG GCC ACG CCG GAT ACC TTC GAG GTC AAT TCC GCA TTC ATC CTG TAC CGC AAT CGG CTT GGC CAG GTC GAC ATC TTC GCG GGC GAA CGC CGG GAC GTG CTG CGC CGC GCC GAC AAC AAC CTT GGT TTC AGC ATC GCC AAG CGC ACC ATC CTG CTC GAC GCC AGT ACC TTG CTG TCG AAC AAC CTG AGC ATG TTC TTC Area III ATG AAA GCA AGA CTG TTT TTG ATC GCC ATC GGC GTC TTC TAC ATC ATC AAC CTC ATT GGC ACG CTT CCC TTC AGC ACG TTG GGC TTG TTT GGC AGG ATG TAT CCA GGC GTA GAA CTG CAC GTG GGT GCG CCG ATT TTC ACC CTG CTG CAG GAT TGG GCG GTG GTC GGT CTC CAG TTG GGC GCC ATC GGG GCC GTC GCT TTG TGG GGC GCC CGC GAT CCG GGC CGT TAT CGG GCC GTT ATT CCA GTG GTC ATC GCA ACG GAA GTG GTC GAT GGC CTC TGG GAT TTT TAC AGC ATC GTG TGC CAC GAA GCC TTG TGG TTC GGG CTT GTC ACG CTG GTG ATC CAT GTG CTG TGG ATT GGC TGG GGC CTG CAT GCC TGG CGT GCC TGG CGT CGA AAT CGC region IV ATG AAA TTT ACC AGA GTT TGT GAT CGA AGA GAT GTG CCC GAAGA GCC CTG AAG GTC GAA AGT GGA GGC ACC TCC GTC GCG ATT TTC AAT GTG GAT GGC GAG CTG TTC GCA ACA CAG GAC CGC TGC ACC CAC GGC GAC TGG TCC CTG TCC GAT GGC GGC TAT CTT GAA GGT GAC GTG GTG GAA TGC TGC ATG GGG AAG TTT TGC GTT CGC ACG GGC AAG GTC AAA TCA CCG CCG CCC TGT GAG GCA CTG AAG ATA TTT CCG ATC CGC ATC GAA GAC AAT GAC GTG CTG GTC GAC TTC GAA GCC GGG TAT CTG GCG CC Area V ATG ATC GAC ACC ATC GCC ATC ATC GGC GCC GGC CTG GCC GTT CGA CGG CTG CGC GCG CAC TGC CGC CAG GGA TAC GAG GGG CGC ATC CAC CTG CTC GGG GAT GAG TCG CAT CAG GCC TAT GAC CGG ACC ACG CTG TCC AAG ACG GTG CTG GCG GGC GAG CAGC GAG CCG CCT GCA ATC CTG GAC AGC GCC TGG TAC GCA TCG GCC CAT GTG GAT GTC CAG CTC GGG CGA CGG GTG AGT TGC CTG GAT CTG GCC AAC CGC CAG ATT CAG TTT GAA TCG GGC GCC CCG CTG GCC TAC GAC CGG CTG CTG CTG CC ACC GGC GCG CGC GCC CGG CGC ATG GCG ATT CGG GGT GGC GAC CTG GCA GGC ATC CAT ACC TTG CGA GAC CTC GCC GAC AGC CAG GCG CTG CGG CAG GCG CTG CAA CCG GGC CAG TCG CTG GTC ATC GTC GGC GGA GGC CTC ATC TGC GAG GTG GCG ACC ACC GCC CGC AAG CTG AGT GTC CAT GTC ACG ATT CTG GAA GCC GGC GAC GAG TTG CTG GTG CGC GTG CTG GGT CAC CGG ACC GGG GCA TGG TGT CGG GCC GAA CTG GAA CGC ATG GGT GTC CGC GTG GAG CGC GCA CAG GCC GCG CGC TTC GAA GGC CAG GGG CAG GTG CGC GCC GTG ATC TGC GCC GAC GGG CGC CGG GTG CCC GCC GAT GTG GTC TTG GTC AGC ATT GGC GCC GAG CCG GCG GAC GAG CTG GCC CGT GCC GCT GGC ATC GCC TCG GGC GTG CTG GTC GAC GCC ACC GGC GCC ACC TCG TGT CCA GAG GTG TTC GCC GCC GGT GAC GTC GCC GCC TGG CCG CTG CGT CAA GGG GGC CAG CGC TCG CTG GAG ACC TAC CTG AAC AGC CAG ATG GAG GCC GAA ATC GCC GGC AGC ATG TTG AGT CAG CCC GTG CCG GCG CCC CAG GTG CCG ACC TCG TGG ACG GAG ATT GCA GGC CAC CGC ATC CAG ATG ATT GGC GAT GCC GAA GGG CCC GGC GAG ATC GTC GTA CGC GGC GAC GCC CAG AGC GGC CAG CCA ATC GTG A GG CTG CTT GAT GGC TGC GTC GAG GCC GCG ACG GCG ATC AAT GCC ACC AGG GAA TTT TCT GTG GCG ACC CGA CTG GTC GGC ACC CGG GTT TCT GTT TCC GCC GAG CAA CTG CAG GAC GTC GGC TCG AAC CTG CGG GAT TTA CTC AAA AAA CCG AAT 12. A DN encoding the following polypeptide:
A. Met Ser Ser Ser Ile Lys Glu Val Gln Gly Ala Pro Val Lys Trp Val Thr Asn Trp Thr Pro Glu Ala Ile Arg Gly Leu Val Asp Gln Glu Lys Gly Leu Leu Asp Pro Arg Ile Tyr Ala Asp Gln Ser Leu Tyr Glu Leu Glu Leu Glu Arg Val Phe Gly Arg Ser Trp Leu Leu Leu Gly His Glu Ser His Val Pro Glu Thr Gly Asp Phe Leu Ala Thr Tyr Met Gly Glu Asp Pro Val Val Met Val Arg Gln Lys Asp Lys Ser Ile Lys Val Phe Leu Asn Gln Cys Arg Gly Met Arg Ile Cys Arg Ser Asp Ala Gly Asn Ala Lys Ala Phe Thr Cys Ser Tyr His Gly Trp Ala Tyr Asp Ile Ala Gly Lys Leu Val Asn Val Pro Phe Glu Lys Glu Ala Phe Cys Asp Lys Lys Glu Gly Asp Cys Gly Phe Asp Lys Ala Glu Trp Gly Pro Leu Gln Ala Arg Val Ala Thr Tyr Lys Gly Leu Val Phe Ala Asn Trp Asp Val Gln Ala Pro Asp Leu Glu Thr Tyr Leu Gly Asp Ala Arg Pro Tyr Met Asp Val Met Leu Asp Arg Thr Pro Ala Gly Thr Val Ala Ile Gly Gly Met Gln Lys Trp Val Ile Pro Cys Asn Trp Lys Phe Ala Ala Glu Gln Phe Cys Ser Asp Met Tyr His Ala Gly Thr Met Ser His Leu Ser Gly Ile Leu Ala Gly Met Pro Pro Glu Met Asp Leu Ser His Ala Gln Val Pro Thr Lys Gly Asn Gln Phe Arg Ala Gly Trp Gly Gly His Gly Ser Gly Trp Phe Val Asp Glu Pro Gly Met Leu Met Ala Val Met Gly Pro Lys Val Thr Gln Tyr Trp Thr Glu Gly Pro Ala Ala Asp Leu Ala Glu Gln Arg Leu Gly His Thr Met Pro Val Arg Arg Met Phe Gly Gln His Met Thr Ile Phe Pro Thr Cys Ser Phe Leu Pro Ala Ile Asn Thr Ile Arg Thr Trp His Pro Arg Gly Pro Asn Glu Ile Glu Val Trp Ala Phe Thr Leu Val Asp Ala Asp Ala Pro Ala Glu Ile Lys Glu Glu Tyr Arg Arg His Asn Ile Arg Thr Phe Ser Ala Gly Gly Val Phe Glu Gln Asp Asp Gly Glu Asn Trp Val Glu Ile Gln Lys Gly Leu Arg Gly Tyr Lys Ala Lys Ser Gln Pro Leu Asn Ala Gln Met Gly Leu Gly Arg Cys Arg Pro Asp His Pro Asp Phe Pro Gly Asn Val Gly 13. A DN encoding the following polypeptide:
A. Met Val Gly Trp Thr Cys Met Cys Arg Arg Arg Ala Glu Val Pro Ser Pro Asp Ile Tyr Leu Glu Ile Thr Ile Met Thr Asn Pro Ser Pro His Phe Phe Lys Thr Phe Glu Trp Pro Ser Lys Ala Ala Gly Leu Glu Leu Gln Asn Glu Ile Glu Gln Phe Tyr Tyr Arg Glu Ala Gln Leu Leu Asp His Arg Ala Tyr Glu Ala Trp Phe Ala Leu Leu Asp Lys Asp Ile His Tyr Phe Met Pro Leu Arg Thr Asn Arg Met Ile Arg Glu Gly Glu Leu Glu Tyr Ser Gly Asp Gln Asp Ile Ala His Phe Asp Glu Thr His Glu Thr Met Tyr Gly Arg Ile Arg Lys Val Thr Ser Asp Val Gly Trp Ala Glu Asn Pro Pro Ser Arg Thr Arg His Leu Val Ser Asn Val Ile Val Lys Glu Thr Ala Thr Pro Asp Thr Phe Glu Val Asn Ser Ala Phe Ile Leu Tyr Arg Asn Arg Leu Glu Arg Gln Val Asp Ile Phe Ala Gly Glu Arg Arg Asp Val Leu Arg Arg Ala Asp Asn Asn Leu Gly Phe Ser Ile Ala Lys Arg Thr Ile Leu Leu Asp Ala Ser Thr Leu Leu Ser Asn Asn Leu Ser Met Phe Phe 14. A DN encoding the following polypeptide:
A. Met Lys Asn Ala Arg Leu Phe Leu Ile Ala Ile Gly Val Phe Tyr Ile Ile Asn Leu Ile Gly Thr Leu Pro Phe Ser Thr Leu Gly Leu Phe Gly Arg Met Tyr Pro Gly Val Glu Leu His Val Gly Ala Pro Ile Phe Thr Leu Leu Gln Asp Ala Trp Ala Val Val Gly Leu Gln Leu Gly Ala Ile Gly Ala Val Ala Leu Trp Gly Ala Arg Asp Pro Gly Arg Tyr Arg Ala Val Ile Pro Val Val Ile Ala Thr Glu Val Val Asp Gly Leu Trp Asp Phe Tyr Ser Ile Val Trp Ser His Glu Ala Leu Trp Phe Gly Leu Val Thr Leu Val Ile His Val Leu Trp Ile Gly Trp Gly Leu His Ala Trp Arg Ala Trp Arg Arg Asn Arg 15. A DN encoding the following polypeptide:
A. Met Lys Phe Thr Arg Val Cys Asp Arg Arg Asp Val Pro Glu Gly Glu Ala Leu Lys Val Glu Ser Gly Gly Thr Ser Val Ala Ile Phe Asn Val Asp Gly Glu Leu Phe Ala Thr Gln Asp Arg Cys Thr His Gly Asp Trp Ser Leu Ser Asp Gly Gly Tyr Leu Glu Gly Asp Val Val Glu Cys Ser Leu His Met Gly Lys Phe Cys Val Arg Thr Gly Lys Val Lys Ser Pro Pro Pro Cys Glu Ala Leu Lys Ile Phe Pro Ile Arg Ile Glu Asp Asn Asp Val Leu Val Asp Phe Glu Ala Gly Tyr Leu Ala Pro 16. D encoding the following polypeptide:
NA. Met Ile Asp Thr Ile Ala Ile Ile Gly Ala Gly Leu Ala Val Arg Arg Leu Arg Ala His Cys Arg Gln Gly Tyr Glu Gly Arg Ile His Leu Leu Gly Asp Glu Ser His Gln Ala Tyr Asp Arg Thr Thr Leu Ser Lys Thr Val Leu Ala Gly Glu Gln Pro Glu Pro Pro Ala Ile Leu Asp Ser Ala Trp Tyr Ala Ser Ala His Val Asp Val Gln Leu Gly Arg Arg Val Ser Cys Leu Asp Leu Ala Asn Arg Gln Ile Gln Phe Glu Ser Gly Ala Pro Leu Ala Tyr Asp Arg Leu Leu Leu Ala Thr Gly Ala Arg Ala Arg Arg Met Ala Ile Arg Gly Gly Asp Leu Ala Gly Ile His Thr Leu Arg Asp Leu Ala Asp Ser Gln Ala Leu Arg Gln Ala Leu Gln Pro Gly Gln Ser Leu Val Ile Val Gly Gly Gly Leu Ile Gly Cys Glu Val Ala Thr Thr Ala Arg Lys Leu Ser Val His Val Thr Ile Leu Glu Ala Gly Asp Glu Leu Leu Val Arg Val Leu Gly His Arg Thr Gly Ala Trp Cys Arg Ala Glu Leu Glu Arg Met Gly Val Arg Val Glu Arg Asn Ala Gln Ala Ala Arg Phe Glu Gly Gln Gly Gln Val Arg Ala Val Ile Cys Ala Asp Gly Arg Arg Val Pro Ala Asp Val Val Leu Val Ser Ile Gly Ala Glu Pro Ala Asp Glu Leu Ala Arg Ala Ala Gly Ile Ala Cys Ala Arg Gly Val Leu Val Asp Ala Thr Gly Ala Thr Ser Cys Pro Glu Val Phe Ala Ala Gly Asp Val Ala Ala Trp Pro Leu Arg Gln Gly Gly Gln Arg Ser Leu Glu Thr Tyr Leu Asn Ser Gln Met Glu Ala Glu Ile Ala Ala Ser Ala Met Leu Ser Gln Pro Val Pro Ala Pro Gln Val Pro Thr Ser Trp Thr Glu Ile Ala Gly His Arg Ile Gln Met Ile Gly Asp Ala Glu Gly Pro Gly Glu Ile Val Val Arg Gly Asp Ala Gln Ser Gly Gln Pro Ile Val Leu Leu Arg Leu Leu Asp Gly Cys Val Glu Ala Ala Thr Ala Ile Asn Ala Thr Arg Glu Phe Ser Val Ala Thr Arg Leu Val Gly Thr Arg Val Ser Val Ser Ala Glu Gln Leu Gln Asp Val Gly Ser Asn Leu Arg Asp Leu Leu Lys Ala Lys Pro Asn 17. The following polypeptides I, II, III, I
V, and benzenedioxygenase containing V. Polypeptide I Met Ser Ser Ser Ile Lys Glu Val Gln Gly Ala Pro Val Lys Trp Val Thr Asn Trp Thr Pro Glu Ala Ile Arg Gly Leu Val Asp Gln Glu Lys Gly Leu Leu Asp Pro Arg Ile Tyr Ala Asp Gln Ser Leu Tyr Glu Leu Glu Leu Glu Arg Val Phe Gly Arg Ser Trp Leu Leu Leu Gly His Glu Ser His Val Pro Glu Thr Gly Asp Phe Leu Ala Thr Tyr Met Gly Glu Asp Pro Val Val Met Val Arg Gln Lys Asp Lys Ser Ile Lys Val Phe Leu Asn Gln Cys Arg Gly Met Arg Ile Cys Arg Ser Asp Ala Gly Asn Ala Lys Ala Phe Thr Cys Ser Tyr His Gly Trp Ala Tyr Asp Ile Ala Gly Lys Leu Val Asn Val Pro Phe Glu Lys Glu Ala Phe Cys Asp Lys Lys Glu Gly Asp Cys Gly Phe Asp Lys Ala Glu Trp Gly Pro Leu Gln Ala Arg Val Ala Thr Tyr Lys Gly Leu Val Phe Ala Asn Trp Asp Val Gln Ala Pro Asp Leu Glu Thr Tyr Leu Gly Asp Ala Arg Pro Tyr Met Asp Val Met Leu Asp Arg Thr Pro Ala Gly Thr Val Ala Ile Gly Gly Met Gln Lys Trp Val Ile Pro Cys Asn Trp Lys Phe Ala Ala Glu Gln Phe Cys Ser Asp Met Tyr His Ala Gly Thr Met Ser His Leu Ser Gly Ile Leu Ala Gly Me t Pro Pro Glu Met Asp Leu Ser His Ala Gln Val Pro Thr Lys Gly Asn Gln Phe Arg Ala Gly Trp Gly Gly His Gly Ser Gly Trp Phe Val Asp Glu Pro Gly Met Leu Met Ala Val Met Gly Pro Lys Val Thr Gln Tyr Trp Thr Glu Gly Pro Ala Ala Asp Leu Ala Glu Gln Arg Leu Gly His Thr Met Pro Val Arg Arg Met Phe Gly Gln His Met Thr Ile Phe Pro Thr Cys Ser Phe Leu Pro Ala Ile Asn Thr Ile Arg Thr Trp His Pro Arg Gly Pro Asn Glu Ile Glu Val Trp Ala Phe Thr Leu Val Asp Ala Asp Ala Pro Ala Glu Ile Lys Glu Glu Tyr Arg Arg His Asn Ile Arg Thr Phe Ser Ala Gly Gly Val Phe Glu Gln Asp Asp Gly Glu Asn Trp Val Glu Ile Gln Lys Gly Leu Arg Gly Tyr Lys Ala Lys Ser Gln Pro Leu Asn Ala Gln Met Gly Leu Gly Arg Cys Arg Pro Asp His Pro Asp Phe Pro Gly Asn Val Gly Polypeptide II Met Val Gly Trp Thr Cys Met Cys Arg Arg Arg Ala Glu Val Pro Ser Pro Asp Ile Tyr Leu Glu Ile Thr Ile Met Thr Asn Pro Ser Pro His Phe Phe Lys Thr Phe Glu Trp Pro Ser Lys Ala Ala Gly Leu Glu Leu Gln Asn Glu Ile Glu Gln Phe Tyr Tyr Arg Glu Ala Gln Leu L eu Asp His Arg Ala Tyr Glu Ala Trp Phe Ala Leu Leu Asp Lys Asp Ile His Tyr Phe Met Pro Leu Arg Thr Asn Arg Met Ile Arg Glu Gly Glu Leu Glu Tyr Ser Gly Asp Gln Asp Ile Ala His Phe Asp Glu Thr His Glu Thr Met Tyr Gly Arg Ile Arg Lys Val Thr Ser Asp Val Gly Trp Ala Glu Asn Pro Pro Ser Arg Thr Arg His Leu Val Ser Asn Val Ile Val Lys Glu Thr Ala Thr Pro Asp Thr Phe Glu Val Asn Ser Ala Phe Ile Leu Tyr Arg Asn Arg Leu Glu Arg Gln Val Asp Ile Phe Ala Gly Glu Arg Arg Asp Val Leu Arg Arg Ala Asp Asn Asn Leu Gly Phe Ser Ile Ala Lys Arg Thr Ile Leu Leu Asp Ala Ser Thr Leu Leu Ser Asn Asn Leu Ser Met Phe Phe polypeptide III Met Lys Asn Ala Arg Leu Phe Leu Ile Ala Ile Gly Val Phe Tyr Ile Ile Asn Leu Ile Gly Thr Leu Pro Phe Ser Thr Leu Gly Leu Phe Gly Arg Met Tyr Pro Gly Val Glu Leu His Val Gly Ala Pro Ile Phe Thr Leu Leu Gln Asp Ala Trp Ala Val Val Gly Leu Gln Leu Gly Ala Ile Gly Ala Val Ala Leu Trp Gly Ala Arg Asp Pro Gly Arg Tyr Arg Ala Val Ile Pro Val Val Ile Ala Thr Glu Val Val Asp Gly Leu Trp Asp Phe Tyr Ser Ile Val Trp Ser His Glu Ala Leu Trp Phe Gly Leu Val Thr Leu Val Ile His Val Leu Trp Ile Gly Trp Gly Leu His Ala Trp Arg Ala Trp Arg Arg Asn Arg Polypeptide IV Met Lys Phe Thr Arg Val Cys Asp Arg Arg Asp Val Pro Glu Gly Glu Ala Leu Lys Val Glu Ser Gly Gly Thr Ser Val Ala Ile Phe Asn Val Asp Gly Glu Leu Phe Ala Thr Gln Asp Arg Cys Thr His Gly Asp Trp Ser Leu Ser Asp Gly Gly Tyr Leu Glu Gly Asp Val Val Glu Cys Ser Leu His Met Gly Lys Phe Cys Val Arg Thr Gly Lys Val Lys Ser Pro Pro Pro Cys Glu Ala Leu Lys Ile Phe Pro Ile Arg Ile Glu Asp Asn Asp Val Leu Val Asp Phe Glu Ala Gly Tyr Leu Ala Pro Polypeptide V Met Ile Asp Thr Ile Ala Ile Ile Gly Ala Gly Leu Ala Val Arg Arg Leu Arg Ala His Cys Arg Gln Gly Tyr Glu Gly Arg Ile His Leu Leu Gly Asp Glu Ser His Gln Ala Tyr Asp Arg Thr Thr Leu Ser Lys Thr Val Leu Ala Gly Glu Gln Pro Glu Pro Pro Ala Ile Leu Asp Ser Ala Trp Tyr Ala Ser Ala His Val Asp Val Gln Leu Gly Arg Arg Val Ser Cys Leu Asp Leu Ala Asn Arg Gln Ile Gln Phe Glu Ser Gly Ala Pro Leu Ala Tyr Asp Arg Leu Leu Leu Ala Thr Gly Ala Arg Ala Arg Arg Met Ala Ile Arg Gly Gly Asp Leu Ala Gly Ile His Thr Leu Arg Asp Leu Ala Asp Ser Gln Ala Leu Arg Gln Ala Leu Gln Pro Gly Gln Ser Leu Val Ile Val Gly Gly Gly Leu Ile Gly Cys Glu Val Ala Thr Thr Ala Arg Lys Leu Ser Val His Val Thr Ile Leu Glu Ala Gly Asp Glu Leu Leu Val Arg Val Leu Gly His Arg Thr Gly Ala Trp Cys Arg Ala Glu Leu Glu Arg Met Gly Val Arg Val Glu Arg Asn Ala Gln Ala Ala Arg Phe Glu Gly Gln Gly Gln Val Arg Ala Val Ile Cys Ala Asp Gly Arg Arg Val Pro Ala Asp Val Val Leu Val Ser Ile Gly Ala Glu Pro Ala Asp Glu Leu Ala Arg Ala Ala Gly Ile Ala Cys Ala Arg Gly Val Leu Val Asp Ala Thr Gly Ala Thr Ser Cys Pro Glu Val Phe Ala Ala Gly Asp Val Ala Ala Trp Pro Leu Arg Gln Gly Gly Gln Arg Ser Leu Glu Thr Tyr Leu Asn Ser Gln Met Glu Ala Glu Ile Ala Ala Ser Ala Met Leu Ser Gln Pro Val Pro Ala Pro Gln Val Pro Thr Ser Trp Thr Glu Ile Ala Gly His Arg Ile Gln Met Ile Gly Asp Ala Glu Gly Pro Gly Glu Ile Val Val Arg Gly Asp Ala Gln Ser Gly Gln Pro Ile Val Leu Leu Arg Leu Leu Asp Gly Cys Val Glu Ala Ala Thr Ala Ile Asn Ala Thr Arg Glu Phe Ser Val Ala Thr Arg Leu Val Gly Thr Arg Val Ser Val Ser Ala Glu Gln Leu Gln Asp Val Gly Ser Asn Leu Arg Asp Leu Leu Lys Ala Lys Pro Asn