JP2001095577A - New polypeptide, dna encoding the polypeptide, and their use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biological method for hydroxylating position 7 of indole. SOLUTION: Cloning of DNA having a sequence shown by sequence 1 or DNA which hybridizes to the DNA under a stringent condition or the like from a microorganism which has an ability of producing 7-hydroxyindole and belongs to the genus Xanthomonas, and providing polypeptides having an enzymatic activity of hydroxylating position 7 of indole, DNA encoding the DNA, a vector consisting of the DNA, and a microorganism carrying the vector allowed producing 7-hydroxyindole and 7-hydroxytryptophan.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a polypeptide having an enzymatic activity for hydroxylating the 7th position of indole, a DNA encoding the polypeptide, a recombinant DNA obtained by incorporating the DNA, and a recombinant DNA obtained by incorporating the DNA. Useful microorganisms as a starting material for medicines, dyes and reagents using microorganisms that carry the enzyme, enzymes that produce enzymes that hydroxylate the 7th position of indole, or microorganisms that carry the recombinant DNA.
-Hydroxyindole and 7-hydroxytryptophan.

[0002]

2. Description of the Related Art Conventionally, 7-hydroxyindole has been produced by a synthesis method, but requires several steps and is expensive to produce. As a production method by a microbial conversion method, a method of adding 3-hydroxy-2-amino-benzoic acid to genetically engineered Escherichia coli and using a tryptophan-anthranilic acid cycle (WO 9534657) is known. There is no known production method by hydroxylating indole at position 7 using a microorganism or an enzyme. On the other hand, as a method for producing 7-hydroxytryptophan, in addition to the above-described microorganism conversion method, 7-hydroxyindole produced by a synthetic method is converted to 7-hydroxytryptophan by a microorganism in addition to the aforementioned microorganism conversion method. However, a method for directly producing 7-hydroxytryptophan from indole using a microorganism is not known.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to reduce the number of positions of indole to 7-hydroxyindole and 7-hydroxytryptophan, which are regarded as important as raw materials for medicines, dyes and reagents. It is an object of the present invention to provide a group of polypeptides having a hydroxylating enzyme activity and a gene encoding the polypeptide, and to provide a method for producing 7-hydroxyindole and 7-hydroxytryptophan using a microorganism having the gene. .

[0004]

Means for Solving the Problems In view of the above problems, the present inventors searched for a microorganism that hydroxylates the 7th position of indole, and added a microorganism belonging to the genus Xanthomonas isolated from soil to a medium. It was discovered that it has the ability to hydroxylate indole at position 7 to produce 7-hydroxyindole, and also to produce 7-hydroxytryptophan. Next, the present inventors attempted to clone a gene involved in hydroxylation of indole at position 7 from the microorganism, succeeded in cloning the genes, and completed the present invention.

Hereinafter, the present invention will be described in detail. As the polypeptide of the present invention, any polypeptide having an activity of hydroxylating the 7th position of indole can be used. For example, in the base sequence shown in SEQ ID NO: 1,
(1) a polypeptide having an amino acid sequence encoded by DNA having a sequence from the 2602nd position to the 2796th position, (2) 2825
A polypeptide having an amino acid sequence encoded by DNA having a sequence from position 3829 to position (3) from position 3847 to position 4
A polypeptide having an amino acid sequence encoded by a DNA having the sequence at position 116, (4) a polypeptide having an amino acid sequence encoded by a DNA having the sequence from position 4145 to position 5704, and (5) from position 5740 to position 6099. (6) a polypeptide having an amino acid sequence encoded by DNA having the sequence of
A polypeptide having an amino acid sequence encoded by NA, (7) a polypeptide having an amino acid sequence encoded by DNA having a sequence of 7190 to 7528, or an amino acid sequence represented by (1) to (7). Examples of the amino acid sequence of the group of polypeptides include polypeptides having an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and having an activity of hydroxylating the 7-position of indole.

The present invention includes (1) the DNA represented by SEQ ID NO: 1 and a DNA which hybridizes with the DNA under stringent conditions, and (2) 2602 in the nucleotide sequence represented by SEQ ID NO: 1. A DNA having a sequence from the 2nd to 2796th or a DNA that hybridizes with the DNA under stringent conditions;
A DNA having the ninth sequence or a DNA that hybridizes with the DNA under stringent conditions, (4) 3
DNA having the sequence of positions 847 to 4116 or DNA that hybridizes with the DNA under stringent conditions, (5) D having the sequence of positions 4145 to 5704
DNA that hybridizes under stringent conditions with NA or its DNA, (6) DNA having the sequence of positions 5740 to 6099 or DNA that hybridizes with its DNA under stringent conditions, (7) from position 6126 DNA having the sequence at position 7181 or DN which hybridizes with the DNA under stringent conditions
A, (8) DNA having the sequence from position 7190 to position 7528 or DNA that hybridizes with the DNA under stringent conditions, (9) DNA represented by SEQ ID NO: 2 or the DNA under stringent conditions Hybridizing DNA is included.

The present invention also relates to a recombinant DN obtained by incorporating at least one kind of the above-mentioned DNAs (1) to (9) and any combination of two to eight kinds of DNAs.
A, expressing a microorganism carrying the recombinant DNA, at least one of the above (1) to (7), any combination of two to seven, and a polypeptide (polypeptide group) containing all of them The above-mentioned microorganism is cultured under conditions for causing the reaction, and 7-hydroxyindole or 7-hydroxytryptophan is obtained by adding and reacting indole or an indole derivative to the culture solution or a solution containing cells separated from the culture solution. And producing the 7-hydroxyindole or 7-hydroxytryptophan.

[0008] Microorganisms suitable for carrying out the present invention include, for example, Xanthomonas malthilia Me
r-H108 strain can be mentioned. This strain is a strain isolated from the soil of the field in Fujisawa City, and its form is 0.4 μm in width.
Motility by polar flagellum, yeast extract 0.1
%, Peptone 0.1%, KH ₂ PO ₄ 0.1%, glucose 0.
Agar medium consisting of 1% and 2% agar (pH 6.
8) and grown aerobically and cultured at 31 ° C for 1 day
It is a Gram-stained negative bacterium that forms a pale yellow flat colony with a dot size of 0.5 mm or less. Based on these properties, this strain was identified as Xanthomonas malthilia based on the Barge's Manual of Systematic Bacteriology, and Xanthomonas malthilia Me was identified.
It was named r-H108. However, the strain that can be used in the present invention is not limited to the present strain, and any strain that can achieve the object can be used. This strain Mer-H108 has been deposited with the National Institute of Bioscience and Human Technology as Xanthomonas malthilia H108 (Accession No. FERM P-17359).

Next, Xanthomonas maltohilia Me
The cloning of a gene related to hydroxylation at position 7 of indole from the r-H108 strain will be described in detail. 1) Acquisition of a non-converted strain An Xanthomonas malthilia Mer-H108 strain was isolated on a separation medium (Yeast Ex.
tract (Difco) 0.1%, Bacto Peptone (Difco) 0.1%,
_{KH 2 PO 4 0.1%, FeSO} 4 · 7H 2 O 0.01%, pH
6.8) Inoculate 50 ml of a platinum loop from a slant medium at 28 ° C.
With shaking overnight. Using this as a seed bacterium, the culture solution 50
0 μl was inoculated into 50 ml of the separation medium, and cultured with shaking at 28 ° C. for 6 hours. The cells were collected from the culture by centrifugation at 5000 × g for 10 minutes, and 0.1 M phosphate buffer (pH 7.
Wash once with 0) and add 0.1M phosphate buffer (pH 7.0)
It was suspended in 5 ml. NTG was added to the cell suspension at a final concentration of 1%.
mg / ml, and shake-cultured at 28 ° C. for 30 minutes. The cells collected from this culture solution were transferred to a 0.1 M phosphate buffer (p
H 7.0), and the whole amount was inoculated into 50 ml of a separation medium and cultured with shaking at 28 ° C. overnight. 500 μl of this culture
The resulting solution was dispensed in aliquots and stored frozen at -70 ° C to obtain a mutant stock solution.

[0010] This mutant stock solution was diluted 1000-fold with sterile water, and separated on an agar medium (Yeast Extract (Difco) 0.1%, Bacto) containing 100 µg / ml of indole in an 8 cm Petri dish.
Peptone (Difco) 0.1%, KH ₂ PO ₄ 0.1%, FeSO
_{4 · 7H 2 O 0.01%,} 1.5% agar, 100 microns to pH 6.8)
Each of them was applied and cultured at 28 ° C. for one week. Among the colonies that grew, white colonies were separated by 1 m from the separation medium.
1 and incubating with shaking at 28 ° C. for 2 days. 20 μl of this culture was spotted on each lane of a silica gel TLC plate and dried. This plate was developed with a solvent system consisting of chloroform: diethyl ether (3: 2), and Ehrl
ich was developed and the presence or absence of 7-hydroxyindole was checked in each lane. Thus, no indole is converted to 7-hydroxyindole from the mutant strains.
Nine mutants were isolated. This mutant strain has been deposited at the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology as Xanthomonas malthilia mutant 18-9 (accession number FE).
RM P-17420).

2) Construction of Host-Vector System and Transformation System Xanthomonas malthilia Mer-H108 strain was inoculated into 3 ml of a separation medium and cultured with shaking at 32 ° C. overnight.
Using this as a seed bacterium, 100 μl of the culture solution is separated into 50
The mixture was inoculated into a ml, and cultured with shaking at 32 ° C for 5 hours. Cells are collected from this culture by centrifugation at 5000 × g for 10 minutes,
After washing once with a 10% glycerol solution, the suspension in 200 μl of a 10% glycerol solution was used as an electrocell. In this electrocell, Broad Host Range Vector pBBR12
2 (Mo Bi Tec) 1 μl of 200 μg / ml TE solution was added. This is a 0.2 cm electro cuvette (Elect
rocuvette) (BIORAD) and put in Gene Pulser (Gen
e Pulser) II (BIORAD), 2.5KV, 200Ω,
One electric pulse was applied under the condition of 25 μF. The cell was placed in a Falcon tube, and 1 ml of an ice-cooled separation medium was added, followed by shaking culture at 32 ° C. for 2 hours. This culture solution was spread on a separated agar medium containing 20 μg / ml of kanamycin sulfate, and cultured at 32 ° C. for 3 days. 1.0 × 10 ⁴
Transformants were obtained.

3) Construction of plasmid pCF704 A primer having an EcoRI site and an NcoI site attached to its ends was synthesized (Pharmacia), and Taq polymerase (Taq polymerase) (Pharmacia) and a PCR thermal cycler (Thermal Cycler) PERSONAL (TaKaRa)
Was used to amplify the multi-cloning site of pUC18 and its 95 bp peripheral region. This DNA fragment was digested with restriction enzymes EcoRI and NcoI, and pBBR122
Ligation Kit version 2 (EcoRI, NcoI site) (TaK
aRa). E.col with this reaction solution
Plasmid pCF704 was transformed from a transformant obtained by transforming i-competent cells JM109 (TaKaRa) into Qiagen Plasmid Midi Kit (QIAGEN Plasmid).
Midi Kit).

4) Construction of Plasmid pIZ311 The genomic DNA of Xanthomonas malthilia Mer-H108 strain was ligated to Qiagen Brad and Cell.
Culture DNA Kit (QIAGEN Blood and Cell Cult)
ure DNA Kit). This genome DN
A is partially digested with a restriction enzyme SauIIIAI, and its 4 to 8 Kbp
Cut the fragments from agarose gel and use Ultra Free C
DNA was recovered and purified using 3 units of 0.45 μm (Millipore) and dissolved in a TE solution to obtain an insert DNA solution. On the other hand, pCF704 was digested with the restriction enzyme BamHI and dephosphorylated with alkaline phosphatase. This and the insert DNA solution are combined with Ligation Kit version 2 (Ligation Kit version 2) (TaKaR
A DNA library was obtained by ligation reaction using (Company a).

This DNA library was added to an electrocell of the 18-9 mutant strain, and an electric pulse was applied. The cell was placed in a Falcon tube, and 1 ml of an ice-cooled separation medium was added, followed by shaking culture at 32 ° C. for 2 hours. The whole amount of this culture solution was applied 100 μl each to a separated agar medium in an 8 cm petri dish containing 20 μg / ml of kanamycin sulfate and 100 μg / ml of indole, and cultured at 32 ° C. for 3 weeks. Among the colonies that grew, those colonies showing red were kanamycin 20 μg / ml and indole 10
It was spread on an 8 cm Petri dish containing 0 μg / ml of the separated agar medium and cultured at 32 ° C. for 5 days. The grown cells are cut out of the agar with a cork borer, extracted with methanol,
HPLC analysis (mobile phase: 50% CH ₃ CN, flow rate: 0.5 m)
L / min, detection wavelength: 280 nm, column temperature: 35 ° C.
Column: YMC-Pack ODS A-312 S-5120A, 6 x 150m
In 7), 7-hydroxyindole was detected, and it was confirmed that the insert DNA was complementary to the 7-hydroxylation activity mutation of indole. A plasmid was prepared from the complemented strain using a QIAGEN Plasmid Midi Kit, and E. coli JM109 was transformed with the plasmid. The re-prepared plasmid was designated as pIZ311. Approximately 9.
A 5 Kbp insert DNA had been inserted. pIZ
Xanthomonas malthilia H108 transformed in 311 was sent to Xanthomonas malthilia H108 (pIZ31)
No. 1) (Accession No. FERM P-17421).

E. coli transformed with pIZ311
L medium containing 20 μg / ml of kanamycin sulfate (JM109) (Polypepton 1.0%, Yeast extract 0.5%, N
aCl 0.5%, Glucose 0.1%, pH 7.2)
Shaking culture was performed at 32 ° C. overnight. HPLC analysis of this culture supernatant revealed that 7-hydroxyindole was about 20 μg /
ml was detected. This is because the gene related to hydroxylation at the 7-position of indole derived from the introduced Xanthomonas malthilia Mer-H108 strain is expressed in E. coli JM109, maintains its activity, and is produced by E. coli tryptophan synthesis or degradation pathway. Indore 7
It is considered that the hydroxylation is performed. This indicates that the gene relating to hydroxylation of indole at position 7 exists on the cloned pIZ311.

5) Determination of gene base sequence of pIZ311 insert region ABIPRISM 377 XL for pIZ311 insert region
The nucleotide sequence was determined by a primer walking method using a DNA Sequencer (Perkin Elmer) (SEQ ID NO: 1). Further, the sequence of SEQ ID NO: 1 was examined for open reading frame (ORF) according to the method of Bibb et al. (Gene, 30 , 157 (1984)). As a result, IoxA (No. 2602-2796 in SEQ ID NO: 1), IoxB (Sequence No. 2) 28 in number 1
No. 25-3829), IoxC (3847-4116 in SEQ ID NO: 1)
No.), IoxD (No. 4145-5704 in SEQ ID NO: 1), Io
xE (sequence number 1, 5740-6099), IoxF (sequence number 1, 6126-7181), IoxG (sequence number 1,
7190-7528), IoxH (7258-8496 in SEQ ID NO: 1)
No.), and a part of ORFs named IoxR (Nos. 2287 to 580 in SEQ ID NO: 1 (reverse direction)) and a part of IoxI (Nos. 8544 to 9320 in SEQ ID NO. 1) were found. The amino acid sequences considered to be encoded by these nucleotide sequences are also shown. Also, since IoxR is encoded in the reverse direction, it is shown in SEQ ID NO: 2 from 2730 bases of SacI site.
In addition, in SEQ ID NO: 2591 to 2596, 2815 to 2820, 3837 to
3849, 4134-4139, 5739-5740, 6114-6118, 7182-71
85, 7551 to 7555, and 8553 to 8536 contain a sequence that seems to be a Shine-Dalgarno sequence.

The above IoxA, IoxB, IoxC, Io
xD, IoxE, IoxF, IoxG, IoxH, Io
Table 1 shows the results of a FASTA search performed on amino acid sequences thought to be encoded by xR and IoxI.

[0018]

[Table 1]

As is clear from Table 1, the pIZ311 insert region has a high homology with various phenol hydroxylases throughout, and particularly with Pseol hydroxylase.
High homology with phenolic hydroxylase of udomonas sp. Strain CF600.

From these results, ORFs involved in indole-7-hydroxylase activity were found to be IoxA, B,
C, D, E, F, G and IoxR were considered. Upstream of IoxA, as with various phenol hydroxylases, σ54-dependent -24 / -12 type promoter
A consensus sequence of TGGC-N8-TGCA (2548 to 2563 in SEQ ID NO: 1), and transcription of the Iox operon is regulated by the transcription regulatory protein IoxR, DNA binding protein IHF, and σ54 it is conceivable that.

6) Examination of the gene (ORF) region required for hydroxylation of indole at position 7 using various deletion mutants (i) Iox deletion mutant
Preparation of t) First, specific sense primers of SEQ ID NOs: 3 to 4 (Sense primers) and antisense primers of SEQ ID NOs: 5 to 7 (Antisense primers) (each 2
8mer) (underlined part is a recognition sequence of restriction enzyme XbaI).

IoxRRF: CT TCTAGA GGCA
TTCGGTGAAGGCATGC (SEQ ID NO: 3), ioxAF: TT TCTAGA CTTGTCTCTCTC
CTGCGTGCC (SEQ ID NO: 4), comp. R: GA TCTAGA TCCAGTGGGCC
GCATTTGCCG (SEQ ID NO: 5) ioxGR: GA TCTAGA TTATGCTTGTT
TGGTACAGC (SEQ ID NO: 6), ioxFR: GT TCTAGA CTAGATGCCGCT
TGAACAGCG (SEQ ID NO: 7).

The above primer and TaKaRa LA Taq (TaKaRa
PCR reaction (reaction conditions: 96 ° C. for 3 minutes, then 98 ° C. for 20 seconds−60) using pIZ311 as a template.
C. for 20 seconds-68.degree. C. for 10 minutes for 25 cycles). After confirming that a PCR product has been formed by agarose gel electrophoresis, the reaction solution was purified using QIAquick PCR Purificat
(Ion Kit, manufactured by QIAGE). Purified product is DN
A Ligation Kit Version 2 (Ligation
Kit Ver.2, manufactured by TaKaRa) using the E. coli vector pT7
-Blue (Novagen) and E. coli JM109
Strain (TaKaRa) and transformed with 50 μg / ampicillin /
ml of LB agar medium (polyheptone 1.0%, yeast extract 0.5%, NaCl 0.5%, glucose 0.1%,
(Agar 1.5%). The next morning, it was confirmed that a colony had been formed, and the plasmid was extracted after inoculation into an LB medium (polypeptone 1.0%, yeast extract 0.5%, NaCl 0.5%, glucose 0.1%).
After treatment with the restriction enzyme XbaI (manufactured by TaKaRa), agarose gel electrophoresis was performed to confirm the presence or absence of the fragment. ioxR
RF primer and comp. R primers and plasmid pIOXRComp. And the plasmid pIOX using the ioxRRF and ioxFR primers.
RF, the plasmid pIOXRG using the ioxRRF primer and the ioxGR primer, and the plasmid pIORG using the ioxAF and ioxFR primers.
OXAF was used to construct plasmid pIOXAG using the ioxAF and ioxGR primers.

(Ii) Culture test of the deletion mutant The plasmid pIOXRComp. , PIOX
E. coli JM109 strain was transformed with R, pIOXRG, pIOXAF and pIOXAG to obtain respective clones. Each clone was inoculated into an LB medium supplemented with 50 μg / ml of ampicillin, and cultured at 37 ° C. overnight with shaking. 500 μl of the culture solution was inoculated into an LB medium (50 ml / 250 ml Erlenmeyer flask) supplemented with 50 μg / ml of ampicillin, and shaking culture was started at 37 ° C. on a rotary shaker. Indole and FeSO ₄ so that the concentration becomes 100 μg / ml.
· 7H ₂ O were added, respectively. Thereafter, 2 ml of the culture solution was sampled over time, and the cells were removed from the culture solution by centrifugation to determine indole and 7-position indole hydroxide in the supernatant by HPLC (column: YMC-PackA-31).
2ODS (4.6 × 150 mm), mobile phase: 50% acetonitrile, flow rate: 0.5 ml / min, detection: 280 nm). As a result, 7
The accumulated amount of hydroxylated indole was 1 in the pIOXAF-converted strain.
1.8 μM, 66.6 μm for pIOXAG-converted strain, pIOXR
21.4 μM for the F transformant, 59.4 μM for the pIOXRG transformant,
pIOXRComp. It was 98 μM in the converted strain. From this, OR necessary to hydroxylate the 7th position of indole
F is IoxA, IoxB, IoxC, IoxD, Iox
E, IoxF.

From the above results and the results in Table 1, the expected role of the polypeptide group encoded by the nucleotide sequence of SEQ ID NO: 1 is shown in FIG.

Next, a method for culturing a microorganism transformed with the gene cloned by the above method or a microorganism having the gene on the chromosome, and a method for producing the produced 7-hydroxylated indole and 7-hydroxylated tryptophan Will be described in detail. The medium composition used in the present invention includes a carbon source, a nitrogen source, an inorganic salt, a natural organic nutrient, etc., which are suitable for the microorganism used to grow well and exhibit the activity of hydroxylating the 7th position of the indole derivative. It consists of As the carbon source, glucose, fructose, glycerol, sorbitol, alcohols, acetic acid, starch and the like can be used alone or in combination. The concentration of the carbon source is not particularly limited, and approximately 1 to 10% is appropriate. Nitrogen sources include ammonia, urea, ammonium sulfate, ammonium nitrate,
One or more compounds such as ammonium acetate can be used. As the inorganic salt, salts such as monopotassium phosphate, dipotassium phosphate, magnesium sulfate, manganese sulfate, and ferrous sulfate can be used. Furthermore, peptone, meat extract, yeast extract, corn steep liquor, casamino acid and the like are used as organic nutrients having a growth promoting effect of the used bacteria, and vitamins and nucleic acids can be contained in a small amount of medium.

The indole for hydroxylating the 7th position of the indole may be added either at the beginning of the growth of the cells or after the cells have grown. Alternatively, the cells may be recovered from the culture solution and the cells may be added to an appropriate aqueous solution. May be added to the suspension. Indole can be used at various concentrations, but is
It is desirable to use it at a concentration of g / liter to 1 g / liter, and it may be added all at once or in portions. Culture is performed under aerobic conditions such as aeration stirring culture and shaking culture.
Alternatively, stationary culture may be used. The pH during the culturing is preferably neutral to weakly alkaline. As the pH neutralizing agent for this, ammonia, sodium hydroxide, potassium hydroxide,
Known materials such as calcium carbonate and hydrochloric acid can be used. The cultivation temperature can be in the range of 20 to 40 ° C. and can be cultivated at a temperature optimum for the growth of the used bacteria. The cultivation time varies depending on the strain used, the method of adding the raw materials, the concentration added, and the like.
In 7 days, 7-hydroxyindole and 7-hydroxytryptophan are produced and accumulated.

The generated and accumulated 7-hydroxyindole and 7-hydroxytryptophan can be isolated and purified according to a method known per se. That is,
Adsorb to a hydrophobic carrier or ion-adsorbing resin, elute with an organic solvent or alkaline water, acidic water, concentrate,
It is recovered by crystallization and the like.

[0029]

The present invention will be described in detail with reference to the following examples. Example 1: 0.1% yeast extract, 0.1% peptone, KH ₂
Xanthomonas malthilia Mer-H108 strain grown on a slope agar medium (adjusted to pH 6.8 before heat sterilization) consisting of 0.1% PO _4, 0.1% glucose and 2% agar was picked up with a platinum loop, yeast extract 0.1%, peptone 0.1%,
Medium containing 0.1% KH ₂ PO ₄ (pH 6.8 before heat sterilization)
This was inoculated into a 250-ml Erlenmeyer flask containing 50 ml and cultured at 28 ° C. for 19 hours on a rotary shaker. 15 ml of this seed culture was used for 0.01% indole, 0.1% yeast extract, 0.1% peptone.
%, KH ₂ PO ₄ 0.1% medium (pH before heat sterilization)
(Adjusted to 6.8) Inoculated in a 3 liter jar fermenter containing 1.5 liters, aerated and agitated at 28 ° C. for 21 hours (aerated amount of 0.75 liter / min, agitated at 400 rpm), and 7-hydroxyindole was added to the culture solution. 25 μg /
ml was generated and accumulated. After removing the cells from the culture solution by centrifugation, a Diaion HP-20 column filled with 1.4 liters of the culture supernatant with water (resin was manufactured by Mitsubishi Chemical, 7φ ×
12-cm), 7-hydroxyindole was adsorbed on the resin, washed with 1 liter of water, and then 7-hydroxyindole was eluted from the column with 500 ml of methanol. Fractions eluted with 7-hydroxyindole were collected from the eluate, concentrated under reduced pressure using an evaporator, and water and methanol were distilled off to obtain 23 mg of 7-hydroxyindole as crystals. FAB-MS and ¹ H-NMR of the thus obtained 7-hydroxyindole were measured.

FAB-MS (matrix; glycerin): m / z 133 (M ⁺ ), ¹ H-NMR (400 MHz, CD ₃ OD): δ 6.39 (1
H, d, J = 3.3Hz), 6.49 (1H, d, J = 7.7Hz), 6.79 (1H, d
d, J = 7.7Hz, J = 7.7Hz), 7.03 (1H, d, J = 7.7Hz), 7.15
(1H, d, J = 3.3Hz).

Example 2: 0.1% yeast extract, peptone
0.1%, KH_TwoPO_Four 0.1%, glucose 0.1%, agar 2
% Agar medium (adjusted to pH 6.8 before heat sterilization)
Xanthomonas malthyilia Me grown on
Take one platinum loop of the r-H108 strain, 0.1% yeast extract,
Puton 0.1%, KH_TwoPO_Four Medium consisting of 0.1%
250 ml volume containing 50 ml
Inoculate Lurenmeier flask and incubate at 28 ° C for 19 hours.
The culture was performed on a rotary shaker. This seed culture
150 ml of the solution is indole 0.01%, yeast extract 0.1
%, Peptone 0.1%, KH_TwoPO_Four0.1%, 0.01% FeS
O_Four・ 7H_TwoMedium consisting of O (adjusted to pH 6.8 before heat sterilization)
30) 30 liter jar ferment including 15 liter
And incubate at 28 ° C with aeration and agitation (aeration rate of 7.5 l).
Torr / min, stirring 200 rpm). Culture 15:00
In the meantime, 7.5 g of L-serine was added and the culture was continued for up to 26 hours.
No, 9 μl of 7-hydroxytryptophan in culture
g / ml was generated and accumulated. After the culture is completed,
Was removed by centrifugation, and 14 liters of the obtained culture supernatant was removed.
To a strong basic ion exchange resin DIAION PA306s
Pass through a column (resin made by Mitsubishi Kasei, 7φ × 15cm)
Then, 7-hydroxytryptophan is adsorbed on the resin and
After washing with 1 liter of water, 7-
Hydroxytryptophan was eluted. 7 from the eluate
1N combined with the eluted fractions of hydroxytryptophan
Adjust the pH to 6.0 with NaOH, and reduce the pressure with an evaporator.
After concentration, freeze-drying was performed to obtain 21.3 g of a crude product.
This was dissolved in 10 ml of 20 mM phosphate buffer (pH 6.8).
Preparative HPLC (column: Inertsil PREP-ODS (2
× 25 cm, gas chromatography industry), mobile phase: 5% MeOH
20 mM phosphate buffer (pH 6.8), flow rate: 7.0 ml /
Min, detection: UV 210 nm) retention time 7 / 34.6
-The peak of hydroxytryptophan was collected. Sorting
Same as above except that the mobile phase was water to desalinate the fractions
Preparative HPLC of the condition of 7-hydroxytryptophan
The peak was collected, lyophilized, and dried.
86 mg was obtained as the sodium salt of the compound. Thus obtained
FAB-MS of 7-hydroxytryptophan and ¹
1 H-NMR was measured.

FAB-MS (matrix; NBA):
m / z 221 ((M + H) ⁺ ), ¹ H-NMR (400 MHz, CD ₃ OD): δ 3.05 (1
H, dd, J = 9.5Hz, J = 15.0Hz), 3.45 (1H, dd, J = 3.3Hz,
J = 15.0Hz), 3.79 (1H, dd, J = 4.0Hz, J = 9.5Hz), 6.57 (1
H, d, J = 7.7Hz), 6.85 (1H, dd, J = 7.7Hz, J = 7.7Hz),
7.13 (1H, s), 7.19 (1H, d, J = 7.7Hz).

Example 3 E. coli JM109 transformed with pIZ311 was used in an amount of 20 μg of kanamycin sulfate.
/ Ml of L medium (polypeptone 1.0%, yeast extract 0.5%, NaCl 0.5%, glucose
(0.1%, pH 7.2) and cultured with shaking at 32 ° C overnight. After removing the cells from this culture by centrifugation,
The culture supernatant was subjected to HPLC (column: ODS-A (0.6 × 15
cm), YMC), mobile phase: 50% acetonitrile, flow rate: 0.5 ml / min, detection: UV 280 nm) to detect a peak of 7-hydroxyindole with a retention time of 10.08 minutes. About 20 μg / ml of 7-hydroxyindole
was detected.

[Sequence List] SEQUENCE LISTING <110> Mercian Corporation <120> NOVEL POLYPEPTIDE, DNA ENCODING THE SAME AND USE THEREOF <130> MEP3078 <160> 7 <210> 1 <211> 9320 <212> DNA <213> Xanthomonas maltophilia <220> <221> CDS <222> (2602) .. (2793) <220> <221> CDS <222> (2825) .. (3826) <220> <221> CDS <222> (3847) .. (4113) <220> <221> CDS <222> (4145) .. (5701) <220> <221> CDS <222> (5740) .. (6096) <220> <221> CDS <222> (6126) .. (7178) <220> <221> CDS <222> (7190) .. (7525) <220> <221> CDS <222> (7558) .. (8493) <220> <221> CDS <222> (8544) .. (9320) <400> 1 gatcctggtg tgctcttcat gcaccttgcg cggcctggac gaatgcacga tggtcaggta 60 gcgatagcgc tccgcctgcc ggtacagcaa gccgatcatg taacgcagcc agcgtgacgg 120 gcaggcgccg atcagcgcct cgtggaactg ccggttgcag gcttcccact ccggcgcatg 180 gtctttcttc acactgcccc tgctcttctc catccggtcc agcctgtggt atgcagcgac 240 cagccccgcc tcccagccgt cgtccccggc cgcgatcgac tgcctcagcg cctcgcattc 300 gagcaggatg cgctgccgcg tgatgtcgcg caggtcgtcc agcgatgtcg ggctgacgta 360 aaagccgcgc tgcccctcgc tgacgaccag cgcatcggag agcagcagcg acaaggcttc 420 gcgcagcgtg ccggcgccga tctcgtagtc gttcttcagg tgctcgacgc gcagcttggt 480 gcccggcgcc agcctgccct cgatgatgtc gcggcgcagg cgcaggtagg ccgcttcgac 540 cagcgtgcgc ggctcggacg ggcattcggt gaaggcatgc tcaaatttcc gcatggtgcc 600 cgccggtctc gcaggtgccg atctcgcagc gcttcaggcg gtagctgagc tgggggcggg 660 tcatgccgag caggcgggcg gcgccggcga ggttgccctt gcagcgcgcc acggcttcgc 720 gcagcatcat gttctccatt tgttccagcg tgagcccgct ggcaaggaaa cgctcgcaca 780 atgccgattc gcgctcgtac ccgggcccgt gctcgtcgag tgcgccggta tcgccgatgc 840 tggggcgctc gt ccgccacc gccacgaagt tcgggaacag gtcatcgcat tctatcgagt 900 cgccctgggc cgccaggatc acgccgcgct cgatcatgtt ctccagctcg cgcacattgc 960 ccggccaggt gtgcgccagc aaggcctgca tggcccggtc ggtgacgccc gagacccgct 1020 tctggtgcaa agcgcagaac ttctcgacca tcgttgccac catcgacggg atgtcgctgc 1080 gacgctcgcg cagcggtgga atcagaatcg gatagacatt caagcgatag tagaggtcag 1140 cgcggaagcg ccctgccttc accgcctccg gcaggtcgac gttggtggcc gcgacgatcc 1200 gcacgtccac cttgcgcggc cggtcgtcgc cgaggcgctc gatctcgcct tcctgcagcg 1260 cgcgcagcag cttggcctgg gaggccagcg gaagctcgcc cagctcgtcc aggaacagcg 1320 taccgccatg cgcgcgctcg aacttgccgg gacgcgaatt ggtggcgccg gtataggcgc 1380 ccttctccac cccgaacagt tcggcctcga tcaggtcgtg cggcagcgcg gcgcagttga 1440 tcgccacgaa gggcttgtcg gcgcgccggc tgcggctgtg gatggcgcgt gcgaaacgct 1500 ccttgccgac cccggtctcg cccgacagca gcacggtcac gctggtctcg gcagccttct 1560 cgaccagccg gtaagcgcgc cggaacgcag gcgactcgcc caccatgtct tcgagattgc 1620 ggcatttctt caggctgcag cgcagcacct cgacctggga cgacagctcc agcaactgcg 1680 aaacgatgga ttcaggttc g taccaggccg cgtactcatc gccgtccggc cactcctcga 1740 ccggcttgcc gacgatgcgg cactcctcgt gtgaacaggc gatgcacctg atttccctga 1800 acaggacgaa gcgtcccatg aaggccgaag tatagccgga ggcatagccg agcaggctcc 1860 agcagaccgg atcgtgctgc ggtccgaagg ccttaacgtg ggcctcggcc tcccaggaat 1920 tacgccaaat gaattcgccg ttgaaaagtc ccttctcgac atccatctcg accgccaccg 1980 ggatcacctg tacaccgccc tcgagcgcat gcatctgtgg cccggtcgcg aacgcctcct 2040 cggggctcat gttgccgcgg atcttgcgag ccagctcggc gtcgcgtttg ccggaggcga 2100 agcccatgcg cgtgaacatg cgccgcgtgg cggcttcgcc caccgagtgc atcatctctt 2160 cgcgcaggct gccgagcgag gctgcgtgca gcagcagcat ccggctttcc gacagccaga 2220 tcgagccatc ctccgccgaa aagcggatca gccggcgcag atccaggtct tccggatatt 2280 tgatcatctt gtctcctcct gcgtgcctat ggaggcagcc gtaaaaatct cgatattttt 2340 tgttttctcg attttaacca tttcatcaac tcatctggca agcaaaaaat gtgaattgac 2400 caaatgatga atgtttaatt gaggaaacat tcggcgctgc agtgcagcac ttgaaaccct 2460 gtaacgcccg tgttcatggg cgtaggcgcg ctgttgcgcc gcgggagccg ggcctcccga 2520 atggtccgga cgagcgcctg cgcg tactgg catggcgctt gcaaacatcc tgtgcagaac 2580 acctacacac aggagacaag g atg gtt atc ccc acc ccc gcc gcg gcc ggg 2631 Met Val Ile Pro Thr Pro Ala Ala Ala Gly 1 5 10 atg gat gtc agc aag cgc tac gtg gcc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc cg Asp Val Ser Lys Arg Tyr Val Arg Ile Thr Gly Val His Asp Gly 15 20 25 ttc gtc gag ttc gat ttc gcc atc ggc gac ccg gaa atc aac gtc gag 2727 Phe Val Glu Phe Asp Phe Ala Ile Gly Asp Pro Glu Ile Asn Val Glu 30 35 40 ctc atc ctc ccc aag ccc gcc ttc gac gag ttc tgc cgc gca aac gcg 2775 Leu Ile Leu Pro Lys Pro Ala Phe Asp Glu Phe Cys Arg Ala Asn Ala 45 50 55 gtg gtg cag ctg gaa ggagac gacg cc atg 2827 Val Val Gln Leu Glu Ala Met 60 65 acg atc gaa ctc aag acc cgc aac atc aag ccg ctg cgc cat acc ttt 2875 Thr Ile Glu Leu Lys Thr Arg Asn Ile Lys Pro Leu Arg His Thr Phe 70 75 80 gcg cac gtg gcg cgc cat atc ggc ggc gac aag ccg gct tcg cgc tac 2923 Ala His Val Ala Arg His Ile Gly Gly Asp Lys Pro Ala Ser Arg Tyr 85 90 95 cag gag gcg acg ctg gag acc cag ccg acc ggc ccc ttc cac tac cgg 2971 Gln Glu Ala Thr Leu Glu Thr Gln Pro Thr Gly Pro Phe His Tyr Arg 100 105 110 ccg atc tgg gcg ccg gaa cac gag atc ttc gac gcc gcg cgc acc gcg 3019 Pro Ile Trp A Pro Glu His Glu Ile Phe Asp Ala Ala Arg Thr Ala 115 120 125 ctg aag atg aac aac tgg tac gcc ttc aag gat ccg cgc cag tac tac 3067 Leu Lys Met Asn Asn Trp Tyr Ala Phe Lys Asp Pro Arg Gln Tyr Tyr 130 135 140 145 tac ggg aac tgg acc cag acc cgg gca cgc cag cag gag acg atg gaa 3115 Tyr Gly Asn Trp Thr Gln Thr Arg Ala Arg Gln Gln Glu Thr Met Glu 150 155 160 gcg aac ttc gag ttc gtc gag tcg cgc ggc tct cag ttg ccg 3163 Ala Asn Phe Glu Phe Val Glu Ser Arg Gly Leu Val Ser Gln Leu Pro 165 170 175 gag gcg gtc ggc acg gcg gcc ctc gag ctg ctg atg ccg ctg cgc cac 3211 Glu Ala Val Gly Thr Alu Leu Leu Met Pro Leu Arg His 180 185 190 gtg gcc tgg ggc gcc aac atg aac aac tcg acc atc tgc gcc tat ggc 3259 Val Ala Trp Gly Ala Asn Met Asn Asn Ser Thr Ile Cys Ala Tyr Gly 195 200 205 tac ggc acc gca atc acc gca ccc gca atg ttc tgc gcg atg gac cag 3307 Tyr Gly Thr Ala Ile Thr Ala Pro Ala Met Phe Cys Ala Met Asp Gln 210 215 220 225 ctt ggc atc gcg cag tac ttg acc cgc gtc ggc ctg ccg gac 3355 Leu Gly Ile Ala Gln Tyr Leu Thr Arg Val Gly Leu Leu Leu Ala Asp 230 235 240 ccg gaa gcg ctc gac cag ggc aag cgc gac tgg ctg gaa gcg ccg cgc 3403 Pro Glu Ala Leu Asp Gg Lys Glu Ala Pro Arg 245 250 255 tgg cag gcg ctg cgc cgc tat gtc gag gac agc ttc gtc gtc gag gac 3451 Trp Gln Ala Leu Arg Arg Tyr Val Glu Asp Ser Phe Val Val Glu Asp 260 265 270 tgg ttc cc gc ctc gacct cag aac ttc gcc ctc gac ggc atc ctg 3499 Trp Phe Glu Leu Phe Val Ala Gln Asn Phe Ala Leu Asp Gly Ile Leu 275 280 285 tat ccg ctg gtc tac gac gcc atc gtc gtc aac gag cac ctg tcg ccg yr Tyr Asp Ala Ile Val Asn Glu His Leu Ser Pro Lys 290 295 300 305 aac ggc aca gcg atc gcg atc gtc acg tcc ttc atg agc gac tgg ttt 3595 Asn Gly Thr Ala Ile Ala Ile Val Thr Ser Phe Met Ser Asp Trp P he 310 315 320 gcc gag caa agc aag tgg gtc gac gcc cag ctc aag acc gcg gcc acg 3643 Ala Glu Gln Ser Lys Trp Val Asp Ala Gln Leu Lys Thr Ala Ala Thr 325 330 335 gag tcg gaa cag aac cat gcg tta ctc act gcc tgg acc ggc gcc tgg 3691 Glu Ser Glu Gln Asn His Ala Leu Leu Thr Ala Trp Thr Gly Ala Trp 340 345 350 cgc gac cgc gct ctg gcc gcc ctg cgc ccg gtc gcc gac cac gtc tac 3739 Arg Asp Arla Ala Ala Lea Leu Arg Pro Val Ala Asp His Val Tyr 355 360 365 ggc gac gcc ggc cac gcg gac agc gcg atg gag gaa gcc gcc gaa cga 3787 Gly Asp Ala Gly His Ala Asp Ser Ala Met Glu Glu Ala Ala Glu Arg 370 375c 380 380 385 aac gcc cgc gcc gcc aag ctc gga ctg acc ctg aac tgacctgaca 3836 Phe Asn Ala Arg Ala Ala Lys Leu Gly Leu Thr Leu Asn 390 395 aggacaccgc atg tcg acc gta ttc atc gcc ttc cag gcc aac Met gat aac Met Ala Phe Gln Ala Asn Glu Asp 400 405 410 acc cgc ccg atc gtg aac gcc atc ctg cgc gac aac ccc ggt gcc cag 3933 Thr Arg Pro Ile Val Asn Ala Ile Leu Arg Asp Asn Pro Gly Ala Gln 415 420 425 ctc gag gaa gcc ccg gcg ctg gtc aag atc aac gcc gag ggc cgc ctc 3981 Leu Glu Glu Ala Pro Ala Leu Val Lys Ile Asn Ala Glu Gly Arg Leu 430 435 440 acg gtg cgc cgc gag gg gg gc gg gg gg gc gg gg gg gg gg gc gg gc gag gg acc ccg ttc aac 4029 Thr Val Arg Arg Glu Thr Val Val Glu Glu Glu Leu Gly Arg Pro Phe Asn 445 450 455 ctg cag gaa atg cac gtc aac ctg atc acc atc acg ggc aac atc gag 4077 Leu Gln Glu Met His Val Asn Leu Ile Thr Ile Thr Gly Asn Ile Glu 460 465 470 475 gaa gac gac gac caa ttc acg ctc acc tgg gcg cac tgagacgcat 4123 Glu Asp Asp Asp Gp. ctg 4174 Met Asp Met Lys Ala Thr Ala Lys Lys Leu 490 495 ggc ctg aag gaa cgc tat gcc tac atg acc cgc ggc ctg gac tgg gaa 4222 Gly Leu Lys Glu Arg Tyr Ala Tyr Met Thr Arg Gly Lelu Asp Trp 510 acc agc tac cag ccg atg gac gcc gtc tac ccc ttc gac aag ttc gaa 4270 Thr Ser Tyr Gln Pro Met Asp Ala Val Tyr Pro Phe Asp Lys Phe Glu 515 520 525 ggc atc aag atc cac gac tgg g ac aag tgg gaa gac cca ttc cgc ctg 4318 Gly Ile Lys Ile His Asp Trp Asp Lys Trp Glu Asp Pro Phe Arg Leu 530 535 535 540 545 acc atg gac gcc tac tgg aag tac cag ggc gag aag gaa aag aag ctg 4366 Thr Ala Tyr Trp Lys Tyr Gln Gly Glu Lys Glu Lys Lys Leu 550 555 560 tac gcc gtg atc gac gcc ttc gcc cag aac aac ggc cac ctg aac gtg 4414 Tyr Ala Val Ile Asp Ala Phe Ala Gln Asn Asn Gly His Leu 570 575 agc gat gcc cgc tac ctg aac gcg atc aag ctg ttc atc cag gcc gtc 4462 Ser Asp Ala Arg Tyr Leu Asn Ala Ile Lys Leu Phe Ile Gln Ala Val 580 585 590 agc ccg ctc gg cc gc tc gc tc gc tc gc tc gc tc gc cat gtc ggc cgc 4510 Ser Pro Leu Glu Tyr Met Ala His Arg Gly Phe Ala His Val Gly Arg 595 600 605 cag ttc cgc ggc gcc ggc gcg cgc gtg gcc tgc cag atg cag gcc gtg 4558 Gln Phe Arg Gly Aly Ala Cys Gln Met Gln Ala Val 610 615 620 625 625 gac gaa ctg cgc cac gcc cag acc cag atc cac acg atc tcg aac tac 4606 Asp Glu Leu Arg His Ala Gln Thr Gln Ile His Thr Ile Ser Asn Tyr 630 635 640 aac aa g tac tac aac ggc atg cac tcc tgg cgc gac tgg tac gag aag 4654 Asn Lys Tyr Tyr Asn Gly Met His Ser Trp Arg Asp Trp Tyr Glu Lys 645 650 650 655 gtg tgg tac ctg tcg gtg ccc aag tcc ttc gcc gcc acc 4702 Val Trp Tyr Leu Ser Val Pro Lys Ser Phe Phe Asp Asp Ala Ile Thr 660 665 670 gcc ggg ccc ttc gaa ttc atc gtg agc gtc agt ttc gcc ttt gaa tac 4750 Ala Gly Pro Phe Glu Phe Ile Val Ser Val Ser Phe Ala Phe Glu Tyr 675 680 685 gtg ctg acc aac ctg ttg ttc gtc ccc ttc atg tcc ggc gcg gcc tac 4798 Val Leu Thr Asn Leu Leu Phe Val Pro Phe Met Ser Gly Ala Ala Tyr 690 695 700 705 aac gcg acc at gag acc ttc ggc ttc tcg gcc cag tcc gac 4846 Asn Gly Asp Met Ser Thr Glu Thr Phe Gly Phe Ser Ala Gln Ser Asp 710 715 720 gag tcg cgc cac atg acg ctg ggc ctg gaa gcc atc aag ttc ctg ctggg Serg His Met Thr Leu Gly Leu Glu Ala Ile Lys Phe Leu Leu 725 730 735 gag cag gac gcg gcc aat gta ccg atc gtg cag ggc tgg ctc gac aag 4942 Glu Gln Asp Ala Ala Asn Val Pro Ile Val Gln Gly Trp Leu Asp Lyc s 740 745 750 tgg ttc tgg cgc ggc tac cgc ctg ctg acc ctg gtc ggc atg atg atg 4990 Trp Phe Trp Arg Gly Tyr Arg Leu Leu Thr Leu Val Gly Met Met Met 755 760 765 gac tac atg cgg gcc ag cg gc ag cg gc gc gc gc gc gc ag tgg aaa gag gcc tgg gag 5038 Asp Tyr Met Leu Pro Lys Arg Val Met Ser Trp Lys Glu Ala Trp Glu 770 775 780 785 atc tac gcc gag cag aac ggc ggc gcc ctg ttc aag gac ctg gcg cgc 5086 Ile Tyr Ala Gly Gly Ala Leu Phe Lys Asp Leu Ala Arg 790 795 800 tac ggc atc cgc ccg ccg atg ggc tgg gac cag gct tgc aag gag aag 5134 Tyr Gly Ile Arg Pro Pro Met Gly Trp Asp Gln Ala Cys Lys Glu Lys 805 cac atc acc cac cag gcc tgg gca gcc ttc tac aac tat acg ggc 5182 Asp His Ile Thr His Gln Ala Trp Ala Ala Phe Tyr Asn Tyr Thr Gly 820 825 830 gcg gcc gcc ttc cac acc tgg atc ccg ggc gag gag gag atg tgg 5230 Ala Ala Ala Phe His Thr Trp Ile Pro Gly Glu Asp Glu Met Ala Trp 835 840 845 ctg tcg gaa aaa tac ccg aac agc ttt gac cag ctg tac cgc ccg cgc 5278 Leu Ser Glu Lys Tyr Pro Asnln Phe Leu Tyr Arg Pro Arg 850 855 860 865 ctg gag cac tgg gac cag cag gaa aaa gcc ggc aag cgc ttc tac aac 5326 Leu Glu His Trp Asp Gln Gln Glu Lys Ala Gly Lys Arg Phe Tyr Asn 870 875 880 gcc acct ctg tgc acg acc tgc acc atc ccc atg ttc ttc 5374 Gly Thr Leu Pro Met Leu Cys Thr Thr Cys Thr Ile Pro Met Phe Phe 885 890 895 acc gag ccg gac gac ccg aca aaa atc tgc tac cgc cgc tcc gag tac 5422 Thr Pro Asp Asp Pro Thr Lys Ile Cys Tyr Arg Arg Ser Glu Tyr 900 905 910 aag ggc gac cag tac cac ttc tgt tcg gac ggc tgc aag cac atc ttc 5470 Lys Gly Asp Gln Tyr His Phe Cys Ser Asp Gly Cys Lys His Ile Phe 915 920 925 gac cag gag ccg gaa aag tac gtg cag gcg ctg ctg ccc acc cac cag 5518 Asp Gln Glu Pro Glu Lys Tyr Val Gln Ala Leu Leu Pro Thr His Gln 930 935 940 945 atc tac cag ggc aag aac ttcag gcc gat ccc gcc gcg ccg 5566 Ile Tyr Gln Gly Lys Asn Phe Lys Pro Asp Ala Asp Pro Ala Ala Pro 950 955 960 ggc ttc gat ccg ctg gcc gcc gta ctg gac ttc tac ggc ctc gtc Lep Pro Gly Ala Ala Val Leu Asp Phe Tyr Gly Leu Val Val 965 970 975 ggc cgc gac aac ttc gac ttc aac ggt tcg gaa gac cag cgg cac ttc 5662 Gly Arg Asp Asn Phe Asp Phe Asn Gly Ser Glu Asp Gln Arg His P980 gcg gac tgg agc ggc aag ggc aag ccc gcc aat gca gtt tgaaccacgc 5711 Ala Asp Trp Ser Gly Lys Gly Lys Pro Ala Asn Ala Val 995 1000 1005 cgaaccacga caacacgagg agacaatc atg cca gtc aag gct ctc Aca gcc ctc Aca gcc ctc Aca gc ctc Act gc ctc Aca gc ctc Aca gc ctc gc ccla gca cc Pro 1010 tat gca ttc gcg ccg cag gac agc gcc gac cgc ttc cat ggc aag cag 5811 Tyr Ala Phe Ala Pro Gln Asp Ser Ala Asp Arg Phe His Gly Lys Gln 1015 1020 1025 1030 cta ctg aac atc ggc tgg gac gac ttc tgc gcc ccg cac 5859 Leu Leu Asn Ile Gly Trp Asp Asp His Leu Met Phe Cys Ala Pro His 1035 1040 1045 acc ctg cca cta ccg ccg gag atg ccg ttc gcg gcc ctg gtc ggc gag 5907 Thr Leu Prou Pro Phe Ala Ala Leu Val Gly Glu 1050 1055 1060 gtc ctg ccg gcg gtc tac ggc agc cac ccc gac ttc gcg cag gtc gac 5955 Val Leu Pro Ala Val Tyr Gly Ser His Pro As p Phe Ala Gln Val Asp 1065 1070 1075 tgg gac gcg gtc gag tgg atc aag tcc ggc cag ccc tgg acg ccg gac 6003 Trp Asp Ala Val Glu Trp Ile Lys Ser Gly Gln Pro Trp Thr Pro Asp 1080 1085 1090 ccg ggc cgc ag gcc ggc aac ggg ctg cgc cac aag gac gtc ctg 6051 Pro Gly Arg Ser Leu Ala Gly Asn Gly Leu Arg His Lys Asp Val Leu 1095 1100 1105 1110 cgc ttc cgc acg ccc ggc ctg cgc ggc agc cgg agcgc agcgc agcgc Arg Thr Pro Gly Leu Arg Gly Leu Gln Gly Ser Cys Asn 11 15 1 120 1 125 atc ctc gac 6197 Pro Leu Gly Val Thr Ile Pro Ile Glu Glu Gly Gln Asn Ile Leu Asp 1135 1140 1145 gcc gcc ctg cgc aac ggc atc tac ctg cca cat gcc tgc tgc cac ggt 6245 Ala Ala Leu Arg Asn Gly Ileyr His Ala Cys Cys His Gly 1150 1155 1160 1165 ctg tgc gcg act tgc aag gtg cag gtc acc cac ggc gag atc gag cac 6293 Leu Cys Ala Thr Cys Lys Val Gln Val Thr His Gl y Glu Ile Glu His 1170 1175 1180 ggc gac gcc tca ccc ttc gcc ctg atg gac ttc gag cgc gac gaa ggc 6341 Gly Asp Ala Ser Pro Phe Ala Leu Met Asp Phe Glu Arg Asp Glu Gly 1185 1190 1195 aag gcc gccg gcc acc ctg tcc agc gac gcc acc ctg gaa 6389 Lys Ala Leu Ala Cys Cys Ala Thr Leu Ser Ser Asp Ala Thr Leu Glu 1200 1205 1210 gcc gag atc gac gaa gaa ccg gac gcc gag acg att ccg gta cgt gacle 6437 Ala Asp Glu Glu Pro Asp Ala Glu Thr Ile Pro Val Arg Asp 1215 1220 1225 ctg cag gga acg gtt ctc aag ctg gaa cag ctc acg ccc acc gtg aag 6485 Leu Gln Gly Thr Val Leu Lys Leu Glu Gln Leu Thr Pro Thr Val Lys 1230 1235 1240 1245 ggg gtg ttc atc cag ctg gac gag ccg ctc cgc ttc cag gcc ggc cag 6533 Gly Val Phe Ile Gln Leu Asp Glu Pro Leu Arg Phe Gln Ala Gly Gln 1250 1255 1260 tac gtc aac ccg gac atg agc cgc gcg ttc tcg 6581 Tyr Val Asn Leu His Ile Pro Gly Ala Asn Cys Ser Arg Ala Phe Ser 1265 1270 1275 gtg gcc agc gcc ggc agc gac ccg agc ctc atc gaa ctg aac gtg cgt 6629 Val Ala Ser Ala Gly Ser Asp Pro Ser Leu Ile Glu Leu Asn Val Arg 1280 1285 1290 atc gtg ccg ggc ggc atc ggc act agc tac gtc cac gaa cag ctc cag 6677 Ile Val Pro Gly Gly Ile Gly Thr Ser Tyr Val His Glu Gln Le Gln 1295 1300 1305 gtg ggc gag cgg gtg acg atc tcc ggc cca tat ggg cgc ttc ttc gta 6725 Val Gly Glu Arg Val Thr Ile Ser Gly Pro Tyr Gly Arg Phe Phe Val 1310 1315 1320 1325 cgc aag tcg gcg cg cg acg ttc atg gcc ggc ggc tcg ggc 6773 Arg Lys Ser Ala Arg Thr Pro Met Leu Phe Met Ala Gly Gly Ser Gly 1330 1335 1340 ctg tcg agc ccg cgc gcc atg atc cac gag ctg ctg cgc gaa Lecca gc Serc 821 Met Ile His Glu Leu Leu Arg Glu Ala Cys 1345 1350 1355 gcg ctg ccg atc acg ctg gtc tac ggc cag cgc agc cgc cag gag ctg 6869 Ala Leu Pro Ile Thr Leu Val Tyr Gly Gln Arg Ser Arg Gln Glu 1370 tac cac gat gag ttc ctc gac ctg gcc gcg aag cac ccg aac ttc 6917 Tyr Tyr His Asp Glu Phe Leu Asp Leu Ala Ala Lys His Pro Asn Phe 1375 1380 1385 acc tac gtg ccg gca ctc tcg gat gcg gcc ggc gat ccg gac tgg gac 6965 Thr Tyr Val Pro Ala Leu Ser Asp Ala Ala Gly Asp Pro Asp Trp Asp 1390 1395 1400 1405 ggc tac cgc ggt ttc gtg cac gac gcg gcc agg gcg cac ttc gac aac70 Gc Phe Val His Asp Ala Ala Arg Ala His Phe Asp Asn 1410 1415 1420 gac ttc cgc gga cac aag gcc tac ctg tgc ggc ccg ccg ccg atg atc 7061 Asp Phe Arg Gly His Lys Ala Tyr Leu Cys Gly Pro ProMet Ile 14 1435 gat gcc tgc ctg tcc acc ctg atg cag ggt cag ctg tac gaa cgc gac 7109 Asp Ala Cys Leu Ser Thr Leu Met Gln Gly Gln Leu Tyr Glu Arg Asp 1440 1445 1450 atc cat acc gag aag ttc atc tcg gcc gcc gcc cag gta cgc 7157 Ile His Thr Glu Lys Phe Ile Ser Ala Ala Asp Ala Gln Gln Val Arg 1455 1460 1465 agc ccg ctg ttc aag cgc atc taggaggcca c atg gat ccc gcg atc agg 7207 Ser Pro Leu Phe Lys Arg Ile Aset Ile Arg 1470 1475 1480 tcc cgg ccg ctg acg gtc agc ctg gtg gag acc agc gaa agc ttc cag 7255 Ser Arg Pro Leu Thr Val Ser Leu Val Glu Thr Ser Glu Ser Phe Gln 1485 1490 1495 t gc cgc gac ggc gag acc ctg ctg cag ggg atg gcg cgc ctc ggg cgc 7303 Cys Arg Asp Gly Glu Thr Leu Leu Gln Gly Met Ala Arg Leu Gly Arg 1500 1505 1510 aaa ggc atc ccg acc gc tcc gg gc tcc gg ag tgc aag 7351 Lys Gly Ile Pro Thr Gly Cys Leu Asn Gly Gly Cys Gly Ile Cys Lys 1515 1520 1525 1530 gtg cat atc ctg gcc ggc gag ttc gag tgc ggg gcg atg agc cgg gcc 7399 Val His Ile Leu Glu Glu Plu Gly Ala Met Ser Arg Ala 1535 1540 1545 cac gtg tgc gag gac gac gtc tgc cat ggc gtt gtg ctg gca tgc agg 7447 His Val Cys Glu Asp Asp Val Cys His Gly Val Val Leu Ala Cys Arg 1550 1555 1560 gca cgg cca gac gtg cag ctg aaa gtg gtc ggc aag atg tgc 7495 Ala Arg Pro Arg Thr Asp Val Gln Leu Lys Val Val Gly Lys Met Cys 1565 1570 1575 aac tcg gtt ttc cgc tgt acc aaa caa gca taagaacaat ctcacacac 7545 Thr Lys Gln Ala 1580 1585 aaaggaggag tc atg gga gtc atg aga atc ggc cat atc agc ctg cgg gtc 7596 Met Gly Val Met Arg Ile Gly His Ile Ser Leu Arg Val 1590 1595 1600 atg gat atg gaa gcc gcc gtc cgg cac tac gag aac atc ctg gga atg 7644 Met Asp Met Glu Ala Ala Val Arg His Tyr Glu Asn Ile Leu Gly Met 1605 1610 1615 aag gtc gtg cac tgg gaa ggc ag gag acg ag gag ag gag ag gag gag ag gag ag gag gag ag gag ag tgc tgg 7692 Lys Val Val His Trp Glu Gly Asn Glu Thr Val Tyr Leu Lys Cys Trp 1620 1625 1630 gac gaa tgg gac aag tat tcg atc cag ctg acc gcc tcg gac acg gca 7740 Asp Glu Trp Asp Lys Tyr Ser Ile Gln Leu Thr Ala Ser Asp Thr Ala 1635 1640 1645 ggg atg aac cac atg gcc tac aag gtc gag cac gat gct gac ctg gac 7788 Gly Met Asn His Met Ala Tyr Lys Val Glu His Asp Ala Asp Leu Asp 1650 1655 1660 1665 gcc atc aag cag cg atc gaa gcc cat ggc gtg acg acc gag atg ctg 7836 Ala Ile Lys Gln Arg Ile Glu Ala His Gly Val Thr Thr Glu Met Leu 1670 1675 1680 ccc gag ggc cag ctg ccg ttc tgc ggc cgc tca ttg cgccc ttg cgctt Gly Gln Leu Pro Phe Cys Gly Arg Ser Leu Arg Phe Thr Ile 1685 1690 1695 ccc agc ggc cac ctg atg cac ctg tac gcc agg aag gac ttc gtc ggc 7932 Pro Ser Gly His Leu Met His Leu Tyr A la Arg Lys Asp Phe Val Gly 1700 1705 1710 aag gac gtc ggc atc gtc gac ccc gag ccc tgg ccg gat ggt ctc aag 7980 Lys Asp Val Gly Ile Val Asp Pro Glu Pro Trp Pro Asp Gly Leu Lys 1715 1720 1725 ggc gc gc cac tgg ctc gac cac tgc ctg ctg atg tgc gag ctg 8028 Gly Val Gly Ala His Trp Leu Asp His Cys Leu Leu Met Cys Glu Leu 1730 1735 1740 1745 aac ccg gac acc ggc atc aac aag gtg gcc gag aacgt Asn Pro Asp Thr Gly Ile Asn Lys Val Ala Glu Asn Val Lys Phe Phe 1750 1755 1760 aag gag acc atg gac ttc cac ttg gcc gag cag gtc gtg gcc ggc ccg 8124 Lys Glu Thr Met Asp Phe His Leu Ala Glu Gln Val Ala Gly Pro 1765 1770 1775 gac ggt tct atc cag gcc gtc gcc ttc atg ttc cgc acc acc acg ccg 8172 Asp Gly Ser Ile Gln Ala Val Ala Phe Met Phe Arg Thr Thr Thr Pro 1780 1785 1790 cac gac att gcc ttc gcc ggc ggc cgc tcg gga ctg cac cac ctg 8220 His Asp Ile Ala Phe Ala Gly Gly Pro Arg Ser Gly Leu His His Leu 1795 1800 1805 tcc ttc ttc ctc gac agc tgg cac gac atc ctg aag tgc ggc gac atc 8 268 Ser Phe Phe Leu Asp Ser Trp His Asp Ile Leu Lys Cys Gly Asp Ile 1810 1815 1820 1825 ctg gcc aag aac cgg gtc cgg atc gac gtg gcc ccg acc cgc cac ggc 8316 Leu Ala Lys Asn Arg Val Arg Ile Asp Ala Thr Arg His Gly 1830 1835 1840 atc acc cgc ggc gag acg atc tat ttc ttc gac ccg tcc ggc aac cgc 8364 Ile Thr Arg Gly Glu Thr Ile Tyr Phe Phe Asp Pro Ser Gly Asn Arg 1845 1850 1855 aac gag acc tttgcc gg ggt tac ctg gcc cag ccg gat atg ccg 8412 Asn Glu Thr Phe Ala Gly Leu Gly Tyr Leu Ala Gln Pro Asp Met Pro 1860 1865 1870 gcg atc acc tgg acc gac ccg gcg cgc ggc atc ttc tat cac acg ggg Trp 8460 Ala Thr Asp Pro Ala Arg Gly Ile Phe Tyr His Thr Gly 1875 1880 1885 aac atg gac gag acc ttc ctg gca gtc cat acc tgaggtcgcg ttgcctttgc 8513 Asn Met Asp Glu Thr Phe Leu Ala Val His Thr 1890 1895 1900 attcaaggag ctactaggagca gag ttacta agc aat ctc gaa 8567 Met Ala Glu Ala Ser Asn Leu Glu 1905 atc gcc cgc agc gtg gtc gcc aac ggc atc cgc acg aac tac cac gac 8615 Ile Ala Arg Se r Val Val Ala Asn Gly Ile Arg Thr Asn Tyr His Asp 1910 1915 1920 gcc ggc gac ggt gcc ccg gtc ctc atg atc cac ggc tcc ggc cct ggc 8663 Ala Gly Asp Gly Ala Pro Val Leu Met Ile His Gly Ser Gly Pro Gly 1925 1930 1935 1940 gta agc gcc tgg gcc aac tgg cgc ctg acc atg ccc gag ctg gcc acg 8711 Val Ser Ala Trp Ala Asn Trp Arg Leu Thr Met Pro Glu Leu Ala Thr 1945 1950 1955 cgc ttc cgc gtc atc gcc ccc gacg atg ttc ggg tat tcc cag 8759 Arg Phe Arg Val Ile Ala Pro Asp Met Val Gly Phe Gly Tyr Ser Gln 1960 1965 1970 cgg ccg gag ggc atc cac tac agc ctc gac acc tgg gtc aac cag gcc 8807 Arg Pro Glu Gly Ile His Tyr Ser Leu Asp Thr Trp Val Asn Gln Ala 1975 1980 1985 gtc gcc ctg ctg gat gcc ctc gac atc gaa cag gcc agc gtg gtg ggc 8855 Val Ala Leu Leu Asp Ala Leu Asp Ile Glu Gln Ala Ser Val Val Gly 1990 1995tt aac t ggc ggc gcg atc gcg ctg gcg ctc gcc atc cgc cac ccg 8903 Asn Ser Phe Gly Gly Ala Ile Ala Leu Ala Leu Ala Ile Arg His Pro 2005 2010 2015 2020 aaa cgc gtc aag cgt ctg gtg ctg atg ggc agc gtc ggg gtg tcc ttc 8951 Lys Arg Val Lys Arg Leu Val Leu Met Gly Ser Val Gly Val Ser Phe 2025 2030 2035 ccg atc acc gaa gga ctg gat gcc gtc tgg ggc tac cag ccc agc gtg 8lu Pro Leu Thr Asp Ala Val Trp Gly Tyr Gln Pro Ser Val 2040 2045 2050 gag aac atg cgg gcg ctg ctc gac att ttc gcc tac gac cgc aag ctg 9047 Glu Asn Met Arg Ala Leu Leu Asp Ile Phe Ala Tyr Asp Arg Lys Leu 2055 20 aac gac gag ctg gcg caa atg cgc tac aag gcc agc atc cag cct 9095 Val Asn Asp Glu Leu Ala Gln Met Arg Tyr Lys Ala Ser Ile Gln Pro 2070 2075 2080 gga ttc cag gaa tcg ttc tcg gcc atg gcc cc gcc c aac 9143 Gly Phe Gln Glu Ser Phe Ser Ala Met Phe Pro Ala Pro Arg Gln Asn 2085 2090 2095 2100 ggc gtg gag atg atg gcc agt ccg atc gag cag atc cgc ggc atc gag 9191 Gly Val Glu Met Met Ala Ser Pro Ile Glu Ile Arg Gly Ile Glu 2105 2110 2115 cac cag acc ctg gtc gtc cac ggc cgc gag gac aag gtc atc ccg ctg 9239 His Gln Thr Leu Val Val His Gly Arg Glu Asp Lys Val Ile Pro Leu 2120 2125 2130 caa aac tcc tac gag ctg ctg cag gcg atc ccg aac gcc cag ctg cac 9287 Gln Asn Ser Tyr Glu Leu Leu Gln Ala Ile Pro Asn Ala Gln Leu His 2135 2140 2145 gtg ttc ggc aaa tgc ggc cc tgg accc Phe Gly Lys Cys Gly His Trp Thr Gln Ile 2150 2155 <210> 2 <211> 2730 <212> DNA <213> Xanthomonas maltophilia <220> <221> CDS <222> (444) .. (2147) <400> 2 gagctcgacg ttgatttccg ggtcgccgat ggcgaaatcg aactcgacga aaccgtcatg 60 gacgccagtg atgcgcacgt agcgcttgct gacatccatc ccggccgcgg cgggggtggg 120 gataaccatc cttgtctcct gtgtgtaggt gttctgcaca ggatgtttgc aagcgccatg 180 ccagtacgcg caggcgctcg tccggaccat tcgggaggcc cggctcccgc ggcgcaacag 240 cgcgcctacg cccatgaaca cgggcgttac agggtttcaa gtgctgcact gcagcgccga 300 atgtttcctc aattaaacat tcatcatttg gtcaattcac attttttgct tgccagatga 360 gttgatgaaa tggttaaaat cgagaaaaca aaaaatatcg agatttttac ggctgcctcc 420 ataggcacgc aggaggagac aag atg atc aaa tat ccg gaa gac ctg gat ctg 473 Met Ile Lys Tyr Pro Glu Asp Leu Asp Leu 1 5 10 cgc cgg ctg atc cgc Arg Ile Arg Phe Ser Ala Glu Asp Gly Ser Ile Trp Leu Ser 15 20 25 gaa agc cgg atg ctg ctg ctg cac gca gcc tcg ctc ggc agc ctg cgc 569 Glu Ser Arg Met Leu Leu Leu His Ala Ala Ser Leg Gly Seru 35 40 gaa gag atg atg cac tcg gtg ggc gaa gcc gcc acg cgg cgc atg ttc 617 Glu Glu Met Met His Ser Val Gly Glu Ala Ala Thr Arg Arg Met Phe 45 50 55 acg cgc atg ggc ttc gcc tcc ggc aaa cgc gac gcc gag ctg gct cgc 665 Thr Arg Met Gly Phe Ala Ser Gly Lys Arg Asp Ala Glu Leu Ala Arg 60 65 70 aag atc cg aac atg agc ccc gag gag gcg ttc gcg acc ggg cca 713 Lys Ile Arg Gly Asn Met Ser Pro Glu Glu Ala Phe Ala Thr Gly Pro 75 80 85 90 cag atg cat gcg ctc gag ggc ggt gta cag gtg atc ccg gtg gg Gln Met His Ala Leu Glu Gly Gly Val Gln Val Ile Pro Val Ala Val 95 100 105 gag atg gat gtc gag aag gga ctt ttc aac ggc gaa ttc att tgg cgt 809 Glu Met Asp Val Glu Lys Gly Leu Phe Asn Gly Glu Phe Ile Trp Arg 110 115 120 aat tcc tgg gag gcc gag gcc cac gtt aag gcc ttc gga ccg cag cac 857 Asn Ser Trp Glu Ala Glu Ala His Val Lys Ala Phe Gly Pro Gln His 125 130 135 gat ccg gtc tgc tgg agc ctg ctc gg tat gcc tcc ggc tat act tcg 905 Asp Pro Val Cys Trp Ser Leu Leu Gly Tyr Ala Ser Gly Tyr Thr Ser 140 145 150 gcc ttc atg gga cgc ttc gtc ctg ttc agg gaa atc agg tgc atc gcc 953 Ala Phe Met Gly Val Leu Ph e Arg Glu Ile Arg Cys Ile Ala 155 160 165 170 tgt tca cac gag gag tgc cgc atc gtc ggc aag ccg gtc gag gag tgg 1001 Cys Ser His Glu Glu Cys Arg Ile Val Gly Lys Pro Val Glu Glu Trp 175 180 185 ccg ggc gat gag tac gcg gcc tgg tac gaa cct gaa tcc atc gtt 1049 Pro Asp Gly Asp Glu Tyr Ala Ala Trp Tyr Glu Pro Glu Ser Ile Val 190 195 200 tcg cag ttg ctg gag ctg tcg tcc cag gtc gag gcg gc gg gcg gcg gcg gcg gc gg gc gc gg gc 1097 Ser Gln Leu Leu Glu Leu Ser Ser Gln Val Glu Val Leu Arg Cys Ser 205 210 215 ctg aag aaa tgc cgc aat ctc gaa gac atg gtg ggc gag tcg cct gcg 1145 Leu Lys Lys Cys Arg Asn Leu Glu Asp Met Gly Ser Pro Ala 220 225 230 ttc cgg cgc gct tac cgg ctg gtc gag aag gct gcc gag acc agc gtg 1193 Phe Arg Arg Ala Tyr Arg Leu Val Glu Lys Ala Ala Glu Thr Ser Val 235 240 245 250 acc gtg ctg ctg tcg gg acc ggg gtc ggc aag gag cgt ttc gca 1241 Thr Val Leu Leu Ser Gly Glu Thr Gly Val Gly Lys Glu Arg Phe Ala 255 260 265 cgc gcc atc cac agc cgc agc cgg cgc gcc gac aag ccc ttc gtg gcg Alg A289g e His Ser Arg Ser Arg Arg Ala Asp Lys Pro Phe Val Ala 270 275 280 atc aac tgc gcc gcg ctg ccg cac gac ctg atc gag gcc gaa ctg ttc 1337 Ile Asn Cys Ala Ala Leu Pro His Asp Leu Ile Glu Ala Glu 285 290 295 ggg gtg gag aag ggc gcc tat acc ggc gcc acc aat tcg cgt ccc ggc 1385 Gly Val Glu Lys Gly Ala Tyr Thr Gly Ala Thr Asn Ser Arg Pro Gly 300 305 310 aag ttc gag cgc gcg cat ggc gtt acg ct ctg gac gag ctg ggc 1433 Lys Phe Glu Arg Ala His Gly Gly Thr Leu Phe Leu Asp Glu Leu Gly 315 320 325 330 gag ctt ccg ctg gcc tcc cag gcc aag ctg ctg cgc gcg ctg cag gaa 1481 Glu Leu Ala Lys Leu Leu Arg Ala Leu Gln Glu 335 340 345 ggc gag atc gag cgc ctc ggc gac gac cgg ccg cgc aag gtg gac gtg 1529 Gly Glu Ile Glu Arg Leu Gly Asp Asp Arg Pro Arg Lys Val Asp Val 350 355 gtc gcg gcc acc aac gtc gac ctg ccg gag gcg gtg aag gca 1577 Arg Ile Val Ala Ala Thr Asn Val Asp Leu Pro Glu Ala Val Lys Ala 365 370 375 ggg cgc ttc cgc gct gac ctc tac tat cgc ttg atg att 1625 Gly Arg Phe Arg Ala Asp Leu Tyr Tyr Arg Leu Asn Val Tyr Pro Ile 380 385 390 ctg att cca ccg ctg cgc gag cgt cgc agc gac atc ccg tcg atg gtg 1673 Leu Ile Pro Pro Leu Arg Aslu Arg Arg Pro Ser Met Val 395 400 405 410 gca acg atg gtc gag aag ttc tgc gct ttg cac cag aag cgg gtc tcg 1721 Ala Thr Met Val Glu Lys Phe Cys Ala Leu His Gln Lys Arg Val Ser 415 420 425 ggc gtc acc gac cgg gcc atg cag gcc ttg ctg gcg cac acc tgg ccg 1769 Gly Val Thr Asp Arg Ala Met Gln Ala Leu Leu Ala His Thr Trp Pro 430 435 440 ggc aat gtg cgc gag ctg gag aac atg atc gag cgc ggc gtg atg atg ctg g Arg Glu Leu Glu Asn Met Ile Glu Arg Gly Val Ile Leu 445 450 455 gcg gcc cag ggc gac tcg ata gaa tgc gat gac ctg ttc ccg aac ttc 1865 Ala Ala Gla Gln Gly Asp Ser Ile Glu Cys Asp Asp Lephe 460 465 470 gtg gcg gtg gcg gac gag cgc ccc agc atc ggc gat acc ggc gca ctc 1913 Val Ala Val Ala Asp Glu Arg Pro Ser Ile Gly Asp Thr Gly Ala Leu 475 480 485 490 gac gag cac ggg ccc ggg gac cag aa tcg gca ttg tgc gag cgt 1961 Asp Glu His Gly Pro Gly Tyr Glu Arg Glu Ser Ala Leu Cys Glu Arg 495 500 505 ttc ctt gcc agc ggg ctc acg ctg gaa caa atg gag aac atg atg ctg 2009 Phe Leu Ala Thr Leu Glu Gln Met Glu Asn Met Met Leu 510 515 520 cgc gaa gcc gtg gcg cgc tgc aag ggc aac ctc gcc ggc gcc gcc cgc 2057 Arg Glu Ala Val Ala Arg Cys Lys Gly Asn Leu Ala Arg 530 A ctc ggc atg acc cgc ccc cag ctc agc tac cgc ctg aag cgc tgc 2105 Leu Leu Gly Met Thr Arg Pro Gln Leu Ser Tyr Arg Leu Lys Arg Cys 540 545 550 gag atc ggc acc tgc gag acc ggc ggg cac cat Glu Ile Gly Thr Cys Glu Thr Gly Gly His His Ala Glu Ile 555 560 565 tgagcatgcc ttcaccgaat gcccgtccga gccgcgcacg ctggtcgaag cggcctacct 2207 gcgcctgcgc cgcgacatca tcgagggcag gctggcgccg ggcaccaagc tgcgcgtcga 2267 gcacctgaag aacgactacg agatcggcgc cggcacgctg cgcgaagcct tgtcgctgct 2327 gctctccgat gcgctggtcg tcagcgaggg gcagcgcggc ttttacgtca gcccgacatc 2387 gctggacgac ctgcgcgaca tcacgcggca gcgcatcctg ctcgaatgcg aggcgctgag 2447 gcagtcgatc gcggccgggg acgacggctg ggaggcgggg ctggtcgctg cataccacag 2507 gctggaccgg atggagaaga gcaggggcag tgtgaagaaa gaccatgcgc cggagtggga 2567 agcctgcaac cggcagttcc acgaggcgct gatcggcgcc tgcccgtcac gctggctgcg 2627 ttacatgatc ggcttgctgt accggcaggc ggagcgctat cgctacctga ccatcgtgca 2687 ttcgtccagg ccgcgcaagg tgcatgaaga gcacaccagg atc 2730 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Sense primer ioxRRF <400> 3 cttctagagg cattcggtga aggcatgc 28 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Sense primer ioxAF <400> 4 tttctagact tgtctcctcc tgcgtgcc 28 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Antisense primer comp.R <400> 5 gatctagatc cagtggccgc atttgccg 28 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Antisense primer ioxGR <400> 6 gatctagatt atgcttgttt ggtacagc 28 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Antisense primer ioxFR <400> 7 gttctagact agatgcgctt gaacagcg 28

[Brief description of the drawings]

FIG. 1 shows the expected role of the group of polypeptides encoded by the genes according to the invention. In the figure, thick arrows indicate primers, and thin arrows indicate sizes and directions of genes.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12N 15/09 ZNA (C12N 1/21 C12R 1:64) C12R 1:64) (C12N 1 / 21 (C12N 1/21 C12R 1:64) C12R 1:19) (C12N 1/21 (C12P 13/22 Z C12R 1:19) C12R 1:64) (C12P 13/22 (C12P 13/22 Z C12R 1 : 64) C12R 1:19) (C12P 13/22 (C12P 17/10 C12R 1:19) C12R 1:64) (C12P 17/10 (C12P 17/10 C12R 1:64) C12R 1:19) (C12P 17/10 C12N 15/00 ZNAA C12R 1:19) C12R 1:64) (72) Inventor Shinji Hirosue 2-2-1-212, Nagatakita, Minami-ku, Yokohama, Kanagawa, Japan (72) Inventor Tadashi Fujii, Kanagawa Fujisawa City Shonandai −37-2-215 (72) Inventor Jin Agematsu 3-2-1-419 Minamigaoka, Hadano City, Kanagawa Prefecture (72) Kunio Isshiki 2-2-17 Minami Kurihara, Zama City, Kanagawa Prefecture (72) Inventor Takeo Yoshioka 1782-10 Yoshioka, Yoshioka, Ayase-shi, Kanagawa F-term (reference) 4B024 AA03 BA08 CA04 DA05 DA06 EA04 GA11 HA01 4B050 CC03 DD02 EE02 LL05 4B064 AE03 AE34 AE48 CA02 CA19 CA21 CB12 CC03 CD12 DA16 4B065 AA26X AA56 AB12

Claims (21)

[Claims] 1. A DNA which hybridizes with the DNA represented by SEQ ID NO: 1 or a DNA thereof under stringent conditions. 2. In the base sequence represented by SEQ ID NO: 1, 2602
DNA having the sequence from position 2796 to its DN or its DN
A that hybridizes with A under stringent conditions
NA. 3. The nucleotide sequence represented by SEQ ID NO: 1
DNA having the sequence from the 3rd to the 3829th or its DN
A that hybridizes with A under stringent conditions
NA. 4. In the base sequence represented by SEQ ID NO: 1, 3847
DNA having the sequence from position 4116 to position 4 or its DN
A that hybridizes with A under stringent conditions
NA. 5. The nucleotide sequence of SEQ ID NO: 1, wherein 4145
DNA having the sequence from the 5th to the 5704th or its DN
A that hybridizes with A under stringent conditions
NA. 6. In the base sequence represented by SEQ ID NO: 1, 5740
DNA having the 60th to 6099th sequence or its DN
A that hybridizes with A under stringent conditions
NA. 7. In the base sequence represented by SEQ ID NO: 1, 6126
DNA having the sequence from the 7th to 7181st or its DN
A that hybridizes with A under stringent conditions
NA. 8. In the nucleotide sequence represented by SEQ ID NO: 1, 7190
DNA having the sequence from position 7528 to position DN or its DN
A that hybridizes with A under stringent conditions
NA. 9. A DNA represented by SEQ ID NO: 2 or a DNA that hybridizes with the DNA under stringent conditions. 10. In the base sequence represented by SEQ ID NO: 1, 26
DNA having sequence from position 02 to position 2796 or D
A polypeptide having an amino acid sequence encoded by DNA that hybridizes with NA under stringent conditions and having an enzymatic activity to hydroxylate position 7 of indole, or a deletion of one or more amino acids of the amino acid sequence, A polypeptide comprising a substituted or added amino acid sequence and having an enzymatic activity for hydroxylating position 7 of indole. 11. In the base sequence represented by SEQ ID NO: 1, 28
DNA having the sequence from the 25th position to the 3829th position or its D
A polypeptide having an amino acid sequence encoded by DNA that hybridizes with NA under stringent conditions and having an enzymatic activity to hydroxylate position 7 of indole, or a deletion of one or more amino acids of the amino acid sequence, A polypeptide comprising a substituted or added amino acid sequence and having an enzymatic activity for hydroxylating position 7 of indole. 12. In the base sequence represented by SEQ ID NO: 1, 38
DNA having the sequence from position 47 to position 4116 or its D
A polypeptide having an amino acid sequence encoded by DNA that hybridizes with NA under stringent conditions and having an enzymatic activity to hydroxylate position 7 of indole, or a deletion of one or more amino acids of the amino acid sequence, A polypeptide comprising a substituted or added amino acid sequence and having an enzymatic activity for hydroxylating position 7 of indole. 13. In the base sequence represented by SEQ ID NO: 1, 41
DNA having the sequence from position 45 to position 5704 or its D
A polypeptide having an amino acid sequence encoded by DNA that hybridizes with NA under stringent conditions and having an enzymatic activity to hydroxylate position 7 of indole, or a deletion of one or more amino acids of the amino acid sequence, A polypeptide comprising a substituted or added amino acid sequence and having an enzymatic activity for hydroxylating position 7 of indole. 14. In the base sequence represented by SEQ ID NO: 1, 57
DNA having the sequence from position 40 to position 6099 or its D
A polypeptide having an amino acid sequence encoded by DNA that hybridizes with NA under stringent conditions and having an enzymatic activity to hydroxylate position 7 of indole, or a deletion of one or more amino acids of the amino acid sequence, A polypeptide comprising a substituted or added amino acid sequence and having an enzymatic activity for hydroxylating position 7 of indole. 15. In the base sequence represented by SEQ ID NO: 1, 61
DNA having the sequence from position 26 to position 7181 or its D
A polypeptide having an amino acid sequence encoded by DNA that hybridizes with NA under stringent conditions and having an enzymatic activity to hydroxylate position 7 of indole, or a deletion of one or more amino acids of the amino acid sequence, A polypeptide comprising a substituted or added amino acid sequence and having an enzymatic activity for hydroxylating position 7 of indole. 16. In the base sequence represented by SEQ ID NO: 1, 71
DNA having the sequence from the 90th position to the 7528th position or its D
A polypeptide having an amino acid sequence encoded by DNA that hybridizes with NA under stringent conditions and having an enzymatic activity to hydroxylate position 7 of indole, or a deletion of one or more amino acids of the amino acid sequence, A polypeptide comprising a substituted or added amino acid sequence and having an enzymatic activity for hydroxylating position 7 of indole. 17. The DNA according to claim 1, or at least one of the DNAs according to claims 2 to 9, or 2
A recombinant DNA obtained by incorporating any combination of at least eight kinds of DNAs. 18. The recombinant DNA according to claim 17,
Holding microorganisms. 19. A condition for expressing a polypeptide group containing at least one kind of the polypeptides according to claim 10 to 16 or two to seven kinds of polypeptides in any combination. By culturing the microorganism according to the above, adding and reacting indole or indole derivative to the culture solution or a solution containing cells separated from the culture solution to produce and accumulate 7-hydroxyindole or 7-hydroxytryptophan, A method for producing 7-hydroxyindole or 7-hydroxytryptophan, comprising collecting these. 20. A method for culturing a microorganism producing an enzyme that hydroxylates the 7th position of indole, and adding and reacting indole or a derivative of indole to the culture solution or a solution containing cells separated from the culture solution. A method for producing 7-hydroxyindole or 7-hydroxytryptophan, which comprises producing and accumulating 7-hydroxyindole or 7-hydroxytryptophan, and collecting them. 21. The method for producing 7-hydroxyindole or 7-hydroxytryptophan according to claim 20, wherein the microorganism is a microorganism belonging to the genus Xanthomonas or the genus Escherichia.