JP2003061671A - Strain in which gene of enzyme participating in testosterone metabolism system is broken - Google Patents

Abstract

PROBLEM TO BE SOLVED: To clone a testosterone metabolic pathway system enzyme gene in C. testosteroni TA441 strain, to produce the gene-broken strain, and to analyze its structure. SOLUTION: The testosterone metabolic pathway system enzyme gene is cloned, and its structure is analyzed. Further, the cloned enzyme gene is used to produce the gene-broken strain in which the enzyme gene is broken. A method for using the gene-broken strain to produce a testosterone metabolic intermediate is provided. The gene-broken strain is a gene-broken strain which participates in the testosterone metabolic enzyme and in which a gene encoding either of five specific amino acids existing on the chromosomal DNA of a microorganism cell or encoding either of the amino acids wherein one to several amino acids are deleted, substituted, added and/or inserted and which has the testosterone metabolism activity is broken.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a gene-disrupted strain of an enzyme involved in a testosterone metabolism system, and more specifically, a gram-negative bacterium capable of growing using steroids as its sole carbon source and energy source. Of 1
The present invention relates to a gene-disrupted strain isolated from the seed, Comamonas testosteroni, for the genes of a plurality of enzymes involved in the testosterone metabolic system. The present invention also relates to the use of the gene-disrupted strain.

[0002]

BACKGROUND ART Steroids are widely distributed in the environment as cholesterol, cholic acid, mammalian sex hormones and adrenocortical hormones, and plant phytosterols. It is believed that microorganisms play a central role in the decomposition of steroid compounds released into the environment. In these steroid-degrading bacteria, steroid-metabolizing enzymes are usually induced by steroid compounds which are substrates.

In addition to various aromatic compounds, Comamonas testosteroni is a Gram-negative bacterium that can grow using steroids having 19 and 21 carbon atoms as the sole carbon and energy sources. C. testosteroni metabolizes steroid compounds via a complex metabolic pathway catalyzed by many enzymes, each of which is induced by a specific steroid compound.

Metabolism of testosterone by Comamonas testosteroni is the first dehydrogenation of the 17-β hydroxyl group.
(dehydrogenation) occurred, and a generated by it
It is thought that the A ring of ndrost-4-ene-3, 17-dione is desaturated and further metabolized. 1968 Coulter and Tal
alay is testosterone-induced C. testosteroni
The cell-free extract was analyzed and the degradation pathway of androst-4-ene-3, 17-dione by C. testosteroni was estimated and reported (Fig. 1). In this metabolic system androst-4-ene-3, 17-
dione is 9α-hydroxylation and Δ ¹ −deh
3-hydroxy-9, 10-secoa by ydrogenation
Converted to ndrosta-1, 3, 5 (10) -triene-9, 17-dione. Subsequently, a hydroxyl group was introduced at the 4-position, and 3,4-dihydroxy-
9, 10-secoandrosta-1, 3, 5 (10) -triene-9, 17-dione
It is thought that a meta-cleavage reaction occurs after the conversion to the position 4 and the position 5 is cleaved (Sih et al., J Biol Ch
em 241, 540-550, 1966).

In the 1990s, an enzyme gene involved in testosterone metabolism of C. testosteroni was reported to catalyze the first dehydrogenation of testosterone metabolism. 17
β-dehydrogenase (Abalain et al., J Steroid Bioc
hem Mol Biol 44, 133-139, 1993; Genti-Raimondi et a
l., Gene 105, 43-49, 1991) and androst-4-ene-3, 1
Δ ¹ -dehydrogenase that converts 7-dione to androsta-1,4-diene-3, 17-dione (Florin et al., J Bacter
iol 178, 3322-3330, 1996; Plesiat et al., J Bacter
iol 173, 7219-7227, 1991) was reported. Delta ¹ - Downstream of dehydrogenase, introduce a double bond at the 4-position delta ⁴
(5α) -dehydrogenase has been found. The 4-position of testosterone is a double bond from the beginning (Flor
in etal., J Bacteriol 178, 3322-3330, 1996; Plesia
t et al., J Bacteriol 173,7219-7227, 1991).

In addition to these dehydrogenases, 3α-
hydroxysteroid hydrogenase and Δ ^5-3 ketosteroid
Isomers are also being isolated and analyzed (Abalain et a
l., JSteroid Biochem Mol Biol 55, 233-238, 1995; M
obus & Master, J Biol Chem273, 30888-30896, 1998;
Kulipulos et al., Proc Natl Acad Sci USA. 84, 8893
-8897, 1987; Choi & Benisek, Gene 69, 121-129, 198
8). In addition, N of 10 testosterone inducible proteins (TIPs) has been recently developed.
A terminal sequence was reported (Mobus et al., J Bacteri
ol 179, 5951-5955, 1997). Some of these showed high homology with metabolizing enzymes of aromatic compounds degrading system such as meta-cleaving enzyme BphC for 2,3-dihydroxybiphenyl of biphenyl degrading system (Furukawa et al., J Bact.
eriol 169, 427-429, 1987; Kinbara et al., J Bacter
iol 171, 2740-2747, 1989; Hofer et al., Gene 130,
47-55, 1993).

The C. testosteroni TA441 strain is a Gram-negative bacterium isolated from the termite ecosystem and contains phenol, 3- (3
-Hydroxyphenyl) propionic acid, and the ability to assimilate aromatic compounds such as their derivatives, and also steroid compounds such as testosterone can be assimilated as the sole carbon and energy sources. So far, from the TA441 strain, phenol metabolism enzyme genes (ap
h gene group) (Arai et al., Microbiology 144, 2895-290
3, 1998) and 3-hydroxyphenylpropionate metabolism enzyme genes (mhp genes) (Arai et al., Microbiol
ogy 145, 2813-2820, 1999) have been isolated and analyzed, but the gene for the enzyme of testosterone metabolism has not been clarified.

[0008]

It is very difficult to carry out a specific reaction at a specific position with a compound having a large molecular weight such as a steroid compound by chemical synthesis. In addition, when synthesizing compounds of metabolic intermediates using microorganisms, the metabolism proceeds if strains without gene disruption are used. It's difficult to get it done.

As mentioned above, Comamonas testosteroni
Although the testosterone metabolic pathway of Escherichia coli was revealed in the 1970s, only a small part of the enzyme genes involved in metabolism was elucidated. In the reaction by the enzyme, it is easy to specifically carry out the reaction only at a specific site which is difficult in the chemical synthesis. In addition, when the gene is destroyed, it is considered possible to prepare a strain in which the reaction up to the point of the disruption proceeds and a specific metabolic intermediate accumulates. That is, it is considered that the microbial reaction is useful for substances that are difficult to synthesize, such as steroid compounds.

From the above background, the present invention provides C. testost.
The task to be solved was to clarify the entire metabolic pathway of testosterone in the eroni TA441 strain. More specifically, the present invention has made it a problem to be solved to clone the testosterone metabolic pathway enzyme gene in the C. testosteroni TA441 strain, to prepare a gene-disrupted strain, and to analyze them.

[0011]

[Means for Solving the Problems] C. testoster shown in FIG.
The previously reported metabolic pathway of testosterone in oni goes through the meta-cleavage reaction, but when TA441 strain is actually cultured with testosterone as the sole carbon source, the culture solution turns yellow due to the yellow substance that is presumed to be derived from the meta-cleavage reaction. . From this, it was speculated that the TA441 strain also metabolized testosterone via the meta-cleavage reaction. As mentioned above, the TA441 strain has two meta-cleaving enzyme genes,
The phenol metabolism system aphB and the 3-hydroxyphenylpropionic acid metabolism system mhpB have been isolated (Arai et al., Mi.
crobiology 144, 2895-2903, 1998 & Microbiology 14
5, 2813-2820, 1999). To investigate whether these catalyze the meta-cleavage reaction of testosterone metabolism, aphB and mhpB
When a mutant strain was simultaneously destroyed and cultured with testosterone as the sole carbon source, the mutant strain did not lose its ability to metabolize testosterone. From this, TA441
It was speculated that there was a testosterone-inducible meta-cleaving enzyme other than these two meta-cleaving enzymes in the strain. The meta-cleaving enzyme is relatively easy to clone because the reaction product has a yellow color. Therefore, by trying to obtain the testosterone-inducible meta-cleaving enzyme gene from the TA441 strain and analyzing the genes before and after that,
We succeeded in cloning a novel enzyme gene involved in the testosterone metabolic pathway in C. testosteroni TA441 strain. Furthermore, the present inventors prepared a gene-disrupted strain in which the enzyme gene was cloned using the cloned enzyme gene, cultured it under appropriate conditions, and analyzed the culture solution, and as a result, produced a testosterone metabolic intermediate.・
It was confirmed that it can be accumulated. The present invention has been completed based on these findings.

That is, according to the present invention, there is provided a gene-disrupted strain related to a testosterone-metabolizing enzyme, in which a gene encoding any one of the following amino acid sequences present on the chromosomal DNA of a microbial cell is disrupted. (A) the amino acid sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5; or (b) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. Alternatively, an amino acid sequence having 1 to several amino acids deleted, substituted, added and / or inserted in the amino acid sequence of any one of SEQ ID NO: 5 and having testosterone metabolic activity:

According to another aspect of the present invention, there is provided a gene-disrupted strain relating to a testosterone-metabolizing enzyme, in which a gene having any of the following nucleotide sequences present on the chromosomal DNA of a microbial cell is disrupted. (A) SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or the nucleotide sequence described in any of SEQ ID NO: 10; or (b) SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 Alternatively, a nucleotide sequence in which one to several nucleotides have been deleted, substituted, added and / or inserted in the nucleotide sequence of any one of SEQ ID NO: 10 and which encodes a protein having testosterone metabolic activity ;

Preferably, in the gene-disrupted strain of the present invention, a gene encoding any one of the following amino acid sequences on the chromosome of a microbial cell; (a) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or the amino acid sequence of any one of SEQ ID NO: 5; or (b) 1 to several in the amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. Has a deletion, substitution, addition and / or insertion of the amino acid of, and has a DNA sequence capable of undergoing homologous recombination with an amino acid sequence having testosterone metabolic activity: Loss of homologous recombination DN
By homologous recombination between A and the above gene on the chromosomal DNA of the microbial cell, the enzyme gene involved in the testosterone metabolic system is destroyed.

According to another aspect of the present invention, any one of the gene-disrupted strains described above is cultured in a medium, and a testosterone metabolism intermediate is collected from the culture.
A method for producing a testosterone metabolism intermediate is provided. Preferably, the testosterone metabolism intermediate is 4-androstene-3,17-dione, androsta-1,4-diene-3,17-dione, 3-hydroxy-9,10-.
SECO ANDROSTA-1,3,5 (10) -triene-
9,17-dione, dehydrotestosterone, 3,17
-Dihydroxy-9,10-secoandrosta-1,
3,5 (10) -trien-9-one or 9α-hydroxyandrosta-4-en-3,17-dione.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below. (1) Gene-disrupted strain of the present invention The gene-disrupted strain of the present invention is a gene-disrupted strain related to testosterone-metabolizing enzyme, and more specifically, encodes any of the following amino acid sequences present on the chromosomal DNA of microbial cells. The gene to be used has been destroyed. (A) the amino acid sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5; or (b) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. Alternatively, an amino acid sequence having 1 to several amino acids deleted, substituted, added and / or inserted in the amino acid sequence of any one of SEQ ID NO: 5 and having testosterone metabolic activity:

Specific examples of the above-mentioned genes include genes having any of the following base sequences. (A) SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or the nucleotide sequence described in any of SEQ ID NO: 10; or (b) SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 Alternatively, a nucleotide sequence in which one to several nucleotides have been deleted, substituted, added and / or inserted in the nucleotide sequence of any one of SEQ ID NO: 10 and which encodes a protein having testosterone metabolic activity ;

In the present specification, the range of "1 to several" in the "amino acid sequence in which 1 to several amino acids are deleted, substituted, added and / or inserted in the amino acid sequence" is not particularly limited. , For example 1 to 2
0, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, particularly preferably 1
It means about 3 pieces.

In the present specification, "1 in the nucleotide sequence
The range of "1 to several" in the "base sequence in which several bases are deleted, substituted, added and / or inserted" is not particularly limited, but is, for example, 1 to 60, preferably 1 to 30. It means about 1 to 20, more preferably 1 to 20, still more preferably 1 to 10, particularly preferably 1 to 5.

The term "having testosterone metabolic activity" as used herein means to participate in the testosterone metabolic pathway and contribute to testosterone metabolism, and specifically, to catalyze any reaction in the testosterone metabolic pathway. It means having the same activity as the enzyme.

In the present invention, DNA in which testosterone metabolic activity is destroyed by introducing a mutation and / or a selectable marker into the above-mentioned gene obtained by PCR or cloning (that is, a gene encoding a protein having testosterone metabolic activity). By carrying out homologous recombination using Escherichia coli, a gene-disrupted strain in which the enzyme gene involved in the testosterone metabolic system has been disrupted can be prepared.

As a method for introducing a mutation into a gene encoding a protein having testosterone metabolic activity, a recombinant DNA technique (Sambruc) for manipulating a DNA base sequence is used.
k, J., Fritsch, EF, and Maniatis, T. (1989) Mole
cular Cloning: A Laboratory Manual, Cold Spring Har
bor Laboratory, Cold Spring Harbor, NY), technology applying PCR (Ling MM. and Robinson BH., Anal. Bioch
Many methods are applicable, such as em. 254 (2): 157-78, 1997). For example, a method of inserting an irrelevant gene, preferably a selectable marker gene capable of selecting a strain in which homologous recombination has occurred, after cutting with an appropriate restriction enzyme can be mentioned, and any method capable of introducing an appropriate mutation can be used. However, the method is not limited to this. When there is no suitable restriction enzyme site, an appropriate restriction enzyme site may be inserted by PCR or the like.

The DNA having the disrupted testosterone metabolic activity used for preparing the gene-disrupted strain of the present invention undergoes homologous recombination with the corresponding gene of the testosterone-metabolizing enzyme on the chromosome under physiological conditions, that is, in microbial cells. It is only necessary that the homology is such that the testosterone-metabolizing enzyme gene can be disrupted. Such homology is preferably 80% or more, more preferably 90% or more, and particularly preferably 95%.
The above homology is mentioned. Further, the DNA used for homologous recombination can cause homologous recombination with the corresponding gene for testosterone-metabolizing enzyme on the chromosome under the physical condition, that is, in the microbial cell, thereby destroying the testosterone-metabolizing enzyme gene. As long as it is possible, it may be a part of the testosterone metabolizing enzyme gene. Here, the term "partly" includes those having a length of preferably 50 bases or more, more preferably 100 bases or more.

That is, the gene-disrupted strain of the present invention is preferably a gene encoding any of the following amino acid sequences on the chromosome of a microbial cell; (a) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, sequence Amino acid sequence described in any one of SEQ ID NO: 4 or SEQ ID NO: 5; or (b) 1 to a number in the amino acid sequence described in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. Amino acid sequence in which one amino acid has been deleted, substituted, added and / or inserted, having a DNA sequence capable of undergoing homologous recombination with an amino acid sequence having testosterone metabolic activity: and having a testosterone metabolic activity For homologous recombination losing
It can be produced by disrupting the enzyme gene involved in the testosterone metabolism system by homologous recombination between A and the above gene on the chromosomal DNA of the microbial cell.

The method for obtaining the gene used for homologous recombination in the present invention is not particularly limited. Appropriate probes and primers were prepared based on the information on the nucleotide sequences and amino acid sequences described in SEQ ID NOS: 1 to 11 of the Sequence Listing in the present specification, and using them, Comamonas testostero
The gene of the present invention can be isolated by screening a cDNA library or genomic DNA library such as ni, preferably Comamonas testosteroni TA441 strain.

The gene of the present invention can also be obtained by the PCR method. Comamonas testosteroni, preferably Co
Chromosomal DNA of mamonas testosteroni TA441 strain or c
PCR is performed using the DNA library as a template and a pair of primers designed to amplify the nucleotide sequences shown in SEQ ID NOs: 6 to 10. PCR reaction conditions can be set as appropriate, for example, at 94 ° C. for 3 hours.
Conditions in which a reaction step consisting of 0 seconds (denaturation), 55 ° C. for 30 seconds to 1 minute (annealing) and 72 ° C. for 2 minutes (extension) is defined as one cycle, for example, 30 cycles, and then 72 ° C. for 7 minutes. And so on. The amplified DNA fragment can then be cloned into a suitable vector that can be amplified in a host such as E. coli.

Preparation of the above-mentioned probe or primer,
Operations such as construction of a cDNA library, screening of a cDNA library, and cloning of a target gene are known to those skilled in the art, and for example, Molecular Clon
ing: A laboratory Mannual, 2 ^nd Ed., Cold Spring Har
bor Laboratory, Cold Spring Harbor, NY., 1989 (hereinafter abbreviated as molecular cloning 2nd edition), Cu
rrent Protocols in Molecular Biology, Supplement 1
~ 38, John Wiley & Sons (1987-1997) (hereinafter, abbreviated as Current Protocols in Molecular Biology) and the like.

In the base sequence of any of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 described above, 1 to several bases are deleted or substituted, A gene (mutant gene) having a nucleotide sequence that is an added and / or inserted nucleotide sequence and encodes a protein having testosterone metabolic activity
Can also be made by any method known to those skilled in the art such as chemical synthesis, genetic engineering techniques or mutagenesis.
Specifically, mutant DNAs can be obtained by utilizing DNAs having the nucleotide sequences of SEQ ID NOs: 6 to 10 and introducing a mutation into these DNAs.

For example, the DNA having the nucleotide sequences of SEQ ID NOs: 6 to 10 can be contacted with a drug serving as a mutagen, irradiated with ultraviolet rays, or genetically engineered. . Site-directed mutagenesis, which is one of the genetic engineering methods, is useful because it is a method that can introduce a specific mutation at a specific position. Molecular cloning 2nd edition, Current Protocols in Molecular It can be performed according to the method described in biology and the like.

The gene used for homologous recombination in the present invention can be incorporated into an appropriate vector and used as a recombinant vector. The type of vector can be appropriately selected by those skilled in the art, but it is desirable to select a vector that cannot autonomously replicate outside the chromosome.

In order to prepare a gene-disrupted strain by homologous recombination, first, a DNA encoding the amino acid sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 or Recombinant DNA is constructed by inserting an appropriate selectable marker into the mutant DNA. The selection marker is preferably a drug resistance marker and the like, and examples thereof include a kanamycin resistance gene, an ampicillin resistance gene, and a tetracycline resistance gene. When a drug resistance marker is used as a selection marker, it is necessary to select a drug resistance gene of a drug to which the wild-type strain is susceptible before homologous recombination. By doing so, the homologous recombination strain and the non-homologous recombination strain can be distinguished by the presence or absence of growth in the presence of an antibiotic.

As a method for producing a gene-disrupted strain, DN in which the above-mentioned selection marker is inserted into the gene sequence is used.
A (the gene in the DNA is destroyed),
By introducing into the cells by electroporation etc. and then selecting with a marker,
By homologous recombination, it is possible to obtain a gene-disrupted strain in which the target gene is integrated on the chromosome of the host microorganism.

(2) Test using the gene-disrupted strain of the present invention
Production of Tosterone Metabolism Intermediate In the present invention, the gene-disrupted strain prepared as described above is cultured in the presence of testosterone to generate and accumulate a testosterone metabolism intermediate in the culture, and the metabolism intermediate is produced from the culture. The testosterone metabolism intermediate can be isolated by collecting. The testosterone metabolic intermediates that can be produced by the present invention include 4-androstene-3,17-dione and androsta-1,4-diene-
3,17-dione, 3-hydroxy-9,10-secoandrosta-1,3,5 (10) -triene-9,17
-Dione, dehydrotestosterone, 3,17-dihydroxy-9,10-secoandrosta-1,3,5 (1
0) -trien-9-one, 9α-hydroxyandrosta-4-en-3,17-dione and the like.

When the gene-disrupted strain of the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the medium for culturing these microorganisms includes a carbon source, a nitrogen source, and
Either a natural medium or a synthetic medium may be used as long as it is a medium containing inorganic salts and the like and capable of efficiently culturing the transformant.
A medium containing testosterone is used. Culturing is preferably carried out under aerobic conditions such as shaking culture or deep aeration stirring culture, the culture temperature is usually 15 to 40 ° C., and the culture time is usually 16 hours to 7 days. The pH is maintained at 3.0 to 9.0 during the culture. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like. In addition, if necessary during culturing,
Antibiotics such as ampicillin and tetracycline may be added to the medium.

As a medium for culturing a transformant obtained by using animal cells as host cells, RP which is generally used
M11640 medium [The Journal of the American Medical As
sociation, 199, 519 (1967)], Eagle's MEM medium [Scienc
e, 122, 501 (1952)], DMEM medium (Virology, 8, 396 (19
59)], 199 medium (Proceeding of the Society for the B
iological Medicine, 73, 1 (1950)] or a medium obtained by adding fetal bovine serum or the like to these mediums. Culture is usually pH6~8,30~40 ℃, 1 under conditions such as 5% C0 ₂ presence
Do ~ 7 days. If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture.

As a medium for culturing the gene-disrupted strain obtained by using plant cells as host cells, MS medium, R2P medium and the like, which are generally used depending on the plant species, are used. Culturing is usually carried out for 1 to 21 days under conditions such as pH 6 to 8 and 15 to 35 ° C. If necessary, antibiotics such as kanamycin and hygromycin may be added to the medium during the culture.

In order to isolate and purify the testosterone metabolism intermediate from the culture of the gene-disrupted strain, conventional methods for isolating and purifying organic compounds may be used. For example, after the culture is completed, the cells are collected by centrifugation, suspended in an aqueous buffer solution, and then disrupted by an ultrasonic disrupter, a French press, a Mantongaulin homogenizer, a Dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a conventional method for isolation and purification of organic compounds, that is,
The target compound can be obtained by using extraction with a solvent, various kinds of chromatography, etc., alone or in combination. The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.

[0038]

Examples Example 1: Isolation and analysis of testosterone-inducible meta-cleaving enzyme gene In Example 1, an attempt was made to obtain a meta-cleaving enzyme induced from C. testosteroni TA441 strain when cultured with testosterone. (A) Experimental procedure (1) Medium: LB medium (Polypepton 10 g; NaCl 5 g; Yeast extract
5 g; pH 7.0 (by NaOH); agar (plate) 15 g / 1 LdH
₂ O) Terrific Broth (bacto-tryptone 12 g; bacto-yeast
extract 24 g; glycerol 4 ml; 0.17 M KH ₂ PO ₄・ 0.72
MK ₂ HPO ₄ 100 ml / 1L dH ₂ O) C medium ((NH ₄ ) ₂ SO ₄ 5.0 g; K ₂ HPO ₄ 5.87 g; KH ₂ PO ₄ 2.93
_{g; MgSO 4 · 7H 2 O} (× 100) 10 ml; NaCl 2 g; Yeast e
xtract 0.2 g; 10 g / L CaCl ₂ solution 1 ml (Final10 mg /
L); 10g / L FeSO ₄ solution 1 ml (Final 10 mg / L); Trace el
ement solution (× 100) 20 μl; pH 7.0 (by NaOH)
_{/ 1L dH 2 O; MgSO 4} · 7H 2 O (× 100): MgSO 4 · 7H 2 O 30
g / L ； Trace element solution (× 100): MoO ₃ 0.1
g, ZnSO ₄ / 5H ₂ O 0.7 g, CuSO ₄ / 5H ₂ O 0.0
_{5 g, H 3 BO 3 0.1} g, MnSO 4 · 5H 2 O0.1 g, CoCl 2 · 6H 2 O
0.1 g / 1L dH ₂ O 2 x YT medium: Tryptone 1.6 g; Bacto Yeast extract
1.0 g; NaCl 0.5 g; pH 7.0 to 7.3 / 1 L dH ₂ O

(2) Antibiotics, etc .; × 1000 stock solution (stored at -20 ° C) Ampicillin (Ap): Ampicillin sodium 50 mg /
ml dH ₂ O Kanamycin (Km): Kanamycin sulfate 30 mg / ml d
H ₂ O Carbenicillin (Cb): Carbenicillin disodium 50 m
g / ml dH ₂ O Tetracycline (Tc): Tetracycline hydrochloride 5 mg /
ml Ethanol chloramphenicol (Cm): Chloramphenicol 30
mg / ml ethanol 0.5 M isopropyl-β-D-thiogalactopyranoside (IPT
G): IPTG 119.15 mg / ml dH ₂ O 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X
-gal): X-gal 20 mg / ml N, N-dimethylformamide

(3) Carbon source stock solution (stored at 4 ° C) 10% w / v testosterone (Ts): Ts 1 g / 10 ml DMSO 10% w / v 1,4-androstadiene-3,17-dione (1,
4-): 1, 4- 1 g / 10 ml DMSO 0.2 M p-Hydroxybenzoic acid (pHB): pHB 0.28 g / 10
ml DMSO

(4) Extraction of protein The strain is inoculated into 3 ml of LB and precultured at 30 ° C. Carbon source 0.
1% (1 ml) of the preculture liquid is inoculated into 100 ml of C medium (1 ml of Erlenmeyer flask) containing 1%, and the mixture is shake-cultured at 30 ° C overnight. Dispense the culture solution into two centrifuge tubes and centrifuge (5,000 rpm, 4
℃, 5 minutes). Discard the supernatant. 50 mM KH ₂ PO ₄ (pH 7.
Add 5 ml of 5) and suspend by vortex, and wash. Centrifuge again (5,000 rpm, 4 ° C, 5 minutes) and discard the supernatant. Ten
Add 2 ml of 50 mM KH ₂ PO ₄ (pH 7.5) containing 50% acetone and suspend by vortexing. Combine the suspension in a single tube and add 1/100 volume of 0.1 M dithiothreitol (DDT) (final concentration 1
mM), 1 M FeSO ₄ (50 mM KH ₂ PO ₄ (pH 7.5) dissolved) 1/100
Add 0 volume (final concentration 1 mM). Repeat ultrasonic destruction (out put 7-8, 30 seconds destruction, 1 minute ice cooling) of the cells three times with a sonicator. Return to the centrifuge tube and centrifuge (12,000 rpm, 4
℃, 10 minutes) and collect the supernatant. This was used as a protein solution.

(5) Bio-Rad Protein Assey Kit (BIO R
Quantification of protein using AD) Dilute 0.1% BSA to 1000 μl of diluted solution x 5 (0.1% BSA 20
0 μl + dH ₂ O 800 μl) Adjust. Repeat this (10
20 times. ． ． ) Dilute. Adjusted diluted solution 800 μl
Add 200 μl of staining solution to the blank and (800 μl of dH ₂ O) and mix by vortex to prepare a standard solution. After incubating at room temperature for 5 minutes or more, OD ₅₉₅ is measured. The value obtained by subtracting the blank from the measured value is plotted against the concentration to prepare a calibration curve. Dilute the protein solution 10 times and 100 times to make a sample solution. 800 μl sample solution and 200 stain
Mix 1 μl by vortex. After incubating at room temperature for 5 minutes or more, OD ₅₉₅ is measured. Subtract the blanks,
The amount of protein is calculated from the calibration curve.

(6) Electrophoresis of proteins (native-PAG
E) Prepare the sample. The protein concentration in the sample is 0.
It is 1 to 0.2% (1 to 2 μg / μl). 1 lane up to 30 μl, 2
× Sample buffer (50 mM Tris-HCl 0.6 g (pH 6.5);
Glycerin 10 g; BPB 0.1 g / 100 ml dH ₂ O) is 1/1 of the final amount
0, 1/10 of the final amount of glycerin. Set 10% acrylamide gel in the electrophoresis tank and use 1 x native PAGE buffer (10 x nati
ve PAGE buffer is 0.25 M Tris30 g; 1.92 M glycine
Pour 144 g / L dH ₂ O). 15 mA per gel, approx.
Pre-run for 5 minutes. Load sample and start electrophoresis. After flowing about 1 cm, increase the current to 25 mA and run for 2 to 3 hours. For protein staining, dip the gel in a staining solution (dissolving Coomassie Brilliant Blue in bleaching solution at a ratio of 0.25%) and shake gently. Transfer the gel to a destaining solution (acetic acid: methanol: water = 1: 3: 6), shake gently, and destain until the back surface of the gel becomes almost transparent. In addition, activity staining was performed by immersing the gel in 50 mM KH ₂ PO ₄ (pH 7.5) containing an appropriate amount of 1 M FeSO ₄ and gently shaking it, and then adding 50 μm of 200 μl of 0.2 M 2,3-dihydroxybiphenyl. It is performed by immersing the gel in 20 ml of KH ₂ PO ₄ (pH 7.5) and gently shaking until the activity is expressed.

(7) Crude purification and determination of N-terminal TA441 strain is cultured using testosterone as the sole carbon source. Cells are collected and suspended in 10 mM phosphate buffer (pH 7.5), and the cells are destroyed by sonication (Branson, TW3). The suspension is centrifuged, and the supernatant is charged on a DEAE ion exchange column equilibrated with phosphate buffer and eluted with phosphate buffer containing 150 mM NaCl. When 2,3-dihydroxybiphenyl is added to Buffer, the target meta-cleaving enzyme is observed as a yellow band due to the meta-cleaving substance. Superose-12 gelfiltration colu
mn and Mono-Q HR 5/5 column (Pharmacia LKB Biotec
(hnologyfast protein liquid chromatography system)
To roughly purify. The crude enzyme is separated by native-PAGE on a 12% acrylamide gel. Soak the gel in 0.04% 2,3-dihydroxybiphenyl solution and transfer the yellow band corresponding to the meta-cleavage material to the PVDF membrane. N-terminal sequence is automated Edman degradation (theApplie
d Biosystemd model 492 protein sequencing system)
Determined by

(8) Preparation of plasmid from Escherichia coli (alkaline-SDS method) Escherichia coli having the desired plasmid was LB to which antibiotics were added.
Incubate with shaking at 37 ℃ in 3 ml of medium overnight. 2 ml of culture solution
Transfer to a ml tube, centrifuge (5,000 rpm, 4 ° C, 5 minutes) to collect the cells, and discard the supernatant. Solution1 (50 mM glucose; 25
100 μl of mM Tris-HCl (pH 8.0); 10 mM EDTA / dH ₂ O)
In addition, the cells are suspended by vortexing and left at room temperature for 5 minutes.
Add 200 μl of Solution2 (0.2 N NaOH; 1% SDS), mix gently, and leave on ice for 5 minutes. 150 μl of Solution3 (5 M potassium acetate 60 ml; glacial acetic acid 11.5 ml / 100 ml dH ₂ O)
Add gently. After standing on ice for 5 minutes, centrifuge (15,000
rpm, 4 ° C, 5 minutes). Transfer the supernatant to a 1.5 ml tube.
Add 400 μl of phenol / chloroform solution, mix well by vortexing, and centrifuge (15,000 rpm, 4 ° C, 5 minutes). Transfer the supernatant to a 1.5 μl tube. Add 1 ml of ethanol, mix and centrifuge (15,000 rpm, 4 ° C, 15 minutes). Discard the supernatant, rinse the pellet with 70% ethanol solution and dry. This is dissolved in 100 µl of TE / Rnase solution to make a plasmid solution.

(9) Dephosphorylation treatment After treatment with a restriction enzyme, 5 M NaCl solution (1/200 amount) was added and mixed by vortex, ethanol (2.5 times amount) was added, mixed and centrifuged.
(15,000 rpm, 4 ° C, 15 minutes). Discard the supernatant and pellet
Rinse with 70% ethanol solution and dry. dH ₂ O 178
Dissolve μl in 20 μl of alkaline phosphatase 10 × buffer. Alkaline phosphatase (shrimp alkalin
e phosphatase (Roche Molecular Biochemicals)) 2 μ
Add l and incubate at 37 ° C for 1 hour. Incubate at 65 ° C for 15 minutes to inactivate the enzyme. Add 200 μl of phenol / chloroform solution (equal volume), mix well by vortexing, and centrifuge (15,000 rpm, 4 ° C, 5 minutes). Transfer the supernatant to a 1.5 ml tube. 1 μl of 5 M NaCl solution (1/2
(00 volume) and vortex to mix, then 500 m of ethanol
l (2.5 times volume), mix and centrifuge (15,000 rpm, 4 ° C, 15
Minutes) Discard the ethanol, rinse the pellet with 70% ethanol solution and dry. This is dissolved in TE (appropriate amount).

(10) DNA blunting using the Blunting Kit (TaKaRa). Overhanging DNA (0.1-10 pmol), blunting 10 x buffer (1
μl) and BSA (1 μl), and water to make a total volume of 9 μl. After incubating at 70 ℃ for 5 minutes, transfer to a 37 ℃ constant temperature bath. T4
Add 1 μl of DNA polymerase, mix gently and mix at 37 ° C.
Incubate for 5 minutes. Stir by vortex to inactivate the enzyme. Place on ice to avoid excess reaction and immediately perform ligation reaction.

(11) Ligation reaction This is carried out using DNA ligation kit ver.2 (Takara Shuzo). DNA solution (10 μl) (total amount of gene fragment solution and vector solution) and solution I (Takara DNA Ligation
Mix Kit ver.2) (10 μl) and incubate at 16 ℃ for 30 minutes to overnight. The reaction solution is directly used for transformation of Escherichia coli.

(12) Method for preparing competent cell of E. coli JM109 (calcium treatment) A single colony of E. coli is precultured in 3 ml of LB medium at 37 ° C overnight. Add 500 μl of preculture (1% of medium) to 50 m of LB medium.
Transfer to l and incubate at 37 ° C (120 strokes / min). After 60 minutes and 90 minutes, 1 ml of the culture solution is sampled and the OD ₆₀₀ is measured. When it reaches about 0.4, stop the culture and leave it on ice for 15 to 30 minutes. Transfer to a centrifuge tube and centrifuge (3,000 rp
m, 4 ℃, 5 minutes) and collect the bacteria. Discard the supernatant. Suspend the cells by vortexing in 20 ml of cold calcium solution and centrifuge (3,000
rpm, 4 ° C, 5 minutes). Leave on ice for 30 minutes. Discard the supernatant. Cooled calcium solution with 20% glycerol (10 m
Suspend the microbial cells in 4 ml of M Tris-HCl, pH 7.5; 50 mM CaCl ₂ (cell concentration: 5 × 10 ⁸ to 1 × 10 ⁹ cells / ml). Aliquot and store at -80 ° C.

(13) Transformation of E. coli JM109 100 μl of competent cells are thawed. Used for transformation
Add the DNA solution (max. 20 μl) and leave it on ice for 30 minutes. heat s
After hock (42 ° C, 1 min), cool in ice for 1 min. LB medium 1
Add ml to the culture tube and incubate at 37 ° C for 40-60 minutes with shaking. Spread an appropriate amount on an LB plate containing an appropriate antibiotic and incubate at 37 ° C overnight.

(14) Escherichia coli DH5α COMPETENT high (TO
100 μl of transformation competent cell of YOBO) is thawed, DNA solution for transformation (maximum 20 μl) is added, and the mixture is allowed to stand on ice for 30 minutes. 42 ° C
Heat shock for 1 minute and cool in ice for 1 minute. SOC me
dium (2% Bacto tryptone; 0.5% Bacto yeast extra
ct; 10 mM NaCl; 2.5 mM KCl; 20 mM MgSO ₄ , MgCl ₂ (10
mM each); 20 mM Glucose) 900 μl is added, and the mixture is incubated at 37 ° C. for 1 hour with shaking. Spread an appropriate amount on an LB plate containing appropriate antibiotics and incubate at 37 ° C overnight.

(15) Preparation of E. coli library Total DNA is treated with a restriction enzyme in a 200 μl / 400 μl system. Phenol / chloroform treatment, ethanol precipitation TE 40
Dissolve in μl. To 40 μl of this total DNA, add 10 μl of vector treated with restriction enzyme and dephosphorylated, and perform ligation reaction. E. coli DH5α COMPETENT high (TOYO
BO). Spread an appropriate amount on an LB plate containing appropriate antibiotics and incubate at 37 ° C overnight.

(16) Detection of meta-cleaving activity The grown transformant is directly sprayed with 0.2 M 2,3-dihydroxybiphenyl (2,3-DHBP) (acetone solution). Strains that show a yellow color due to the meta-cleavage substance are selected.

(17) Oligonucleotide 3'end tailin
g DIG Oligonucleotide Tailing Kit. In a tube, 5 × reaction buffer 4 μl (final concentration 1 ×), cobalt chloride solution 4 μl (final concentration 5 mM), DIG-11-dUTP 1
μl (final concentration 0.05 mM), oligonucleotide 100 pmol
(Final concentration 5 μM), dATP 1 μl (final concentration 0.5 mM) and terminal transferase 1 μl (final concentration 2.5 μM)
U / μl) and water to make a total volume of 20 μl. Mix the above reaction mixture and incubate at 37 ℃ for 15 minutes, then transfer to ice. Add 1 μl glycogen solution and 1 μl EDTA to stop the reaction. Precipitate the labeled oligonucleotide by adding 1/10 volume (2.5 μl) of 4 M lithium chloride and 2.5-3 volume (75 μl) of cold (−20 ° C.) 100% ethanol. Mix well and let stand at -80 ℃ for 30 minutes (-20 ℃ for 2 hours). Centrifuge (15,000 rpm, 4 ° C, 5 minutes). Discard the ethanol, wash the pellet with 100 μl of cold 70% ethanol, and centrifuge (15,000 rpm, 4 ° C, 5 minutes). Discard 70% ethanol, dry the pellet and dissolve in 20 μl dH ₂ O.

(18) Probe Labeling Reaction by Random Prime Method DIG DNA Labeling Kit (Roche Molecular Biochemical
s). Boil the template DNA solution for 10 minutes
After that, it is rapidly cooled to denature it into single-stranded DNA. In the tube,
Template DNA 3-10 ng (final concentration 0.5-150 μg / m
l), hexanucleotide mixture 2 μl, dNTP labeling mixture 2 μl, Klenow enzyme 1 μl (final concentration 100 U / m
l) and add water to make a total volume of 20 μl. 1 at 37 ° C
Label for ~ 20 hours. Stop the reaction by adding 2 μl of EDTA. Add 2 μl of 4 M LiCl and 60 μl of chilled ethanol, mix well, leave at -80 ° C for 30 minutes, and then add labeled DNA.
To precipitate. Centrifuge (15,000 rpm, 4 ° C, 15 minutes) and discard the supernatant. After rinsing with cold 70% ethanol and discarding the supernatant completely, vacuum dry. This is dissolved in 50 μl of TE and used as a probe.

(19) Blotting After electrophoresis of DNA, the gel is put in a container and shaken with 0.25 N HCl at room temperature for 15 minutes. Place the gel on the membrane to prevent air bubbles from entering. 0.5 N NaOH-1.5 MN on top of gel
Add aCl solution and vacuum for 15 minutes. 0.25 on the gel
Add N NaOH-1.5M NaCl solution and vacuum for 30 minutes. Membrane (Hybond-N ⁺ (amersham pharmacia biotech))
2 × SSC (20 × SSC is 3.0 M NaCl; 0.3 M Na ₃ Citrate,
Lightly rinse with pH 7.0). Wrap in plastic wrap and irradiate with UV for 5 minutes to fix DNA on the membrane.

(20) Prehybridization and hybridization (reagent) 10% blocking reagent stock solution: blocking reagent 10 g /
buffer1 (0.1 M maleic acid; 0.15 M NaCl, pH7.5) 1
00 ml High SDS hybridization solution (per 500 ml dH ₂ O): SDS 35 g 100% formamide (deionized) 250 ml 30 × SSC 83 ml 1 M sodium phosphate, pH 7.0 25 ml N-lauroyl sarcosine 0.5 g 10% Blocking reagent stock solution 100 ml (operation) Put the membrane in the hybrid bag, add high SDS hybridization solution (20 ml / 100 cm ² ) and seal, and perform prehybridization at 42 ° C for at least 1 hour. 10 minutes when using a double-stranded DNA probe
After boiling, quench on ice to denature the DNA into single strands.
Discard the prehybridization solution and add the SIG hybridization solution containing DIG-labeled probe.
Add (3.5 ml / 100 cm ² ) and perform hybridization at 42 ° C overnight. Wash the membrane twice with 2 x SSC, 0.1% SDS for 5 minutes at room temperature. Wash membrane with 0.1 x wash solution:
Wash 2 times for 15 minutes at the temperature of hybridization with 0.1 x SSC and 0.1% SDS.

(21) Detection (coloring reaction) The washing solution is discarded and the membrane is transferred to a container. Buffer 1 (0.
Lightly rinse with 1 M maleic acid; 0.15 M NaCl, pH 7.5). 1
Block with 00 ml of Buffer 2 (10% (w / v) blocking reagent stock solution diluted 1/10 with buffer 1) at room temperature for 30 minutes with gentle shaking. Gently shake with a diluted labeled antibody solution (alkaline phosphatase-labeled anti-digoxigenin antibody 150 mU / ml buffer2) for 30 minutes at room temperature. Wash twice with sufficient Buffer 1 for 15 minutes each time. 20 ml Buffer 3 (0.1 MTris-HCl, pH9.5; 0.1
Equilibrate with (M NaCl) for 2 minutes. Put the membrane in the hybrid bag and add 45 μl of coloring solution (NBT solution (nitroblue tetrazolium salt 75 mg / ml 70% dimethylformamide)) and X-phosphoric acid solution (5-bromo-4-chloro-3 indolyl phosphate ( X-phosphate), toluidine salt 5
0 mg / ml dimethylformamide) 35 μl / Buffer3 1
Add 0 ml) and leave it in the dark. When the band is detected, wash with 50 ml of TE for 5 minutes to stop the reaction.

(22) Preparation of deletion clone for sequence A Kilo-Sequence Deletion Kit (TaKaRa) is used. First, use plasmid A with restriction enzyme A (insert DNA side: 5'end of cut surface is protruding or blunt end) and restriction enzyme B (annealing side of primer: 3'end of cut surface is protruding). To process. Add an equal amount of phenol / chloroform solution, vortex thoroughly, centrifuge (15,000 rpm, 4 ° C, 5 minutes), transfer the supernatant to a new tube, add 2.5 volumes of ethanol and mix.
Centrifuge (15,000 rpm, 4 ° C, 15 minutes). Discard the supernatant, rinse the pellet with 70% ethanol, and vacuum dry (hereinafter, this operation is referred to as phenol / chloroform extraction and ethanol precipitation). Pellet the ExoIII buffer 100
Dissolve in μl. Add 1 μl of Exonuclease III to 37 ° C
React with. Sampling 10 μl every minute for 20
Add to μl of Mung Bean (MB) nuclease buffer sequentially. Incubate at 65 ° C for 5 minutes to inactivate the enzyme, and cool on ice. MB nuclease buffer mixture (MB nuclease 2 μ
l + MB nuclease buffer 8 μl) in 2 μl aliquots
Incubate at 30 ℃ for 60 minutes. As above, phenol /
Chloroform extraction, ethanol precipitation and pellet
Dissolve in 50 μl of klenow buffer. Add klenow buffer mixture (klenow fragment 2 μl + klenow buffer 8 μl) to 2
Add μl each and incubate at 37 ℃ for 15 minutes. Similar to the above,
Perform phenol / chloroform extraction and ethanol precipitation. Ligation reaction is performed in a 20 μl system, phenol / chloroform extraction and ethanol precipitation are performed in the same manner as above, and the pellet is dissolved in dH ₂ O 17.5 μl. Restriction enzyme A
Buffer 2 μl and restriction enzyme A 0.5 μl were added, and the mixture was incubated at 37 ° C for 3
Let react for 0 minutes. E. coli is transformed with the reaction solution.

(23) Method for preparing plasmid from E. coli
(single step procedure) 2 × Y
Incubate in 1.5 ml of T medium at 37 ° C with shaking overnight. The culture solution is centrifuged (5,000 rpm, 4 ° C, 5 minutes) to collect the cells, and the supernatant is discarded.
Li buffer (2.5 M LiCl; 50 mM Tris-HCl; 4% TritonX
-100; 62.5 mM Na ₂ EDTA, pH 8.0) Add 200 μl and mix by vortexing. Phenol / chloroform solution 200
Add μl, mix well by vortexing, and centrifuge (15,00
0 rpm, 4 ° C, 5 minutes). Transfer the supernatant to a new tube. Add 500 μl of ethanol (2.5 volumes), mix, and centrifuge (15,000 rpm, 4 ° C, 10 minutes). Discard the supernatant, 70%
Add ethanol and centrifuge (15,000 rpm, 4 ° C, 3 minutes).
Thoroughly discard the supernatant and vacuum dry. TE / RNase solution 2
Dissolve in 0 μl.

(24) GPS-1 Genome Priming System (B
Prepare plasmids using io Labs) Wizard mini prep. Wiza
The plasmid solution prepared by rd contains salts, so after ethanol precipitation the pellet is rinsed 3 times with 1.5 ml of 70% ethanol solution and dried. TE 20
Dissolve in μl. Measure A260. Target the tube
DNA 0.08 μg, 10 × GPS buffer 2 μl, pGPS1.1 or pGPS
2.1 Add 1 μl of Donor DNA and add water to make a total volume of 18 μl. Then, add 1 μl of TnsABC transposase, mix, and incubate at 37 ° C for 10 minutes. In addition, Start solution 1
Add μl, mix well by pipetting, incubate at 37 ° C for 1 hour, incubate at 75 ° C for 10 minutes, or treat with phenol / chloroform and ethanol to perform transposase.
Are inactivated and transformed.

(25) Method for preparing plasmid for sequencing from Escherichia coli Wizard Wizard Minipreps Purification System (promega) is used. Inoculate 2.5 ml of Terrific Broth and incubate at 37 ° C with shaking overnight. Transfer the culture medium to a 2 ml tube and centrifuge.
(5,000 rpm, 4 ° C, 5 minutes), collect the cells, and discard the supernatant. C
ell Resuspension Solution (50 mM Tris-HCl; 10 mM ED
Add 200 μl of TA, pH 7.5; 100 μg RNase / ml) and suspend the cells by vortexing. Cell Lysis Solution (0.2
Add 200 μl of N NaOH; 1% SDS) and mix gently. Neutralization Solution (1.32M potassium aceta
te, pH 4.8) 200 μl and gently mix and centrifuge (15,0
00 rpm, 4 ° C, 5 minutes). Transfer the supernatant to a new 2 ml tube, Wizard Minipreps DNAPurification Resin 1 m
Add l and mix well. Vac the column and 2.5 ml syringe
Set to cum manifold, add sample and vacuum. After vacuuming the sample, Column Was
h Solution (80 mM potassium acetate; 8.3 mM Tris-H
Add 2 ml of Cl, pH 7.5; 40 μM EDTA; 55% ethanol) and vacuum. After vacuuming the Column Wash Solution, continue vacuuming for 1-2 minutes to dry the column. Place the column on the tube and centrifuge (15,000 rp
m, 4 ℃, 1 minute) to completely remove water. 1.5 ml new
Transfer the column to a tube, add 20 μl of TE buffer preheated to 65 ° C, and leave it for 1 minute. Centrifugation
(15,000 rpm, 4 ° C, 1 minute) and drop the extracted DNA solution into a tube.

(26) PCR reaction of sequence sample The reaction system was sample DNA (100 ng to 1 μg), 10 × Ex Taq.
buffer (10 μl), 2.5 M dNTPs (8 μl), 20 pmol / μ
l Add water to Primer (1 μl each) and TaKaRa Ex Taq (0.5 μl) to make 100 μl. PCR conditions are 96 ° C for 2 minutes, then 98 ° C for 20 seconds, 55 ° C for 45 seconds and 72 ° C for 1.5 minutes for 30 cycles, and finally 72 ° C for 10 minutes.

(27) Electrophoresis, data analysis, and determination of nucleotide sequence A series of operations was carried out according to the protocol of the same company using 373A DNA Sequencing System ver. 1.2.1 (ABI).・ Analysis of the determined nucleotide sequence DNASIS Mac version 3.7 (Hitachi Software Engineeri
ng Co. Ltd.) was used. Using homology search NCBI BLAST Search, GenBank DNA database,
We searched from SWISS PROT protein database. Amino acid alignment Clustal W software was used.

(28) Preparation of kanamycin resistance gene cassette for gene disruption A 1.5 kb HindIII-SalI fragment encoding a kanamycin resistance (Km ^r ) gene was purified from pSUP5011. pBluescript K
Incorporate into the HindIII + SalI site of S (-). A 1.5 kb SmaI fragment encoding the Km ^r gene was purified from the prepared plasmid to obtain a Km ^r gene cassette.

(29) Method for preparing competent cells for electroporation of TA441 strain TA441 strain is inoculated into 3 ml of LB medium and precultured at 30 ° C. overnight. Inoculate 1 ml (1 ml) of the preculture liquid into 100 ml of LB medium and
Incubate at about 2.5 hours (OD ₆₀₀ , about 0.45). Cool on ice for 20 minutes. Dispense the culture solution into two sterile centrifuge tubes and centrifuge (7,000 rpm, 5 minutes, 4 ° C). Discard the supernatant, add 10 ml of cold sterilized water, suspend by vortexing, and centrifuge (7,000 rp
m, 5 minutes, 4 ° C). Discard the supernatant, add 10 ml of cooled 10% glycerol solution, suspend by vortexing, and centrifuge (7,00
0 rpm, 5 minutes, 4 ° C). Discard the supernatant, add 100 μl of cooled 10% glycerol solution, suspend by vortex, dispense and store at -80 ° C.

(30) Electro-transformation (Escherichia coli, TA441 strain) In the case of homologous recombination, the plasmid was treated with an enzyme that cuts only one vector without cutting the insert, ethanol precipitated and dissolved in dH ₂ O. , Concentrated plasmid solution. When the host bacterium holds the plasmid as it is, a concentrated plasmid solution is prepared by ethanol precipitation without enzymatic treatment. Thaw 40 μl of competent cells for electroporation and add 1 μl of concentrated plasmid solution.
Add. Place in a previously cooled 0.1 cm cuvette and apply voltage using GENE PULSER II (BIO RAD) (conditions: 1.25 kV / 0.1 cm, 200 Ω). Transfer to a culture tube, add 1 ml of LB medium, and shake-culture at 37 ° C for 1 hour for E. coli and at 30 ° C for TA441 strain for 2 hours. Sprinkle an appropriate amount on an LB plate containing an appropriate antibiotic and incubate overnight.

(31) Obtaining a gene-disrupted strain Using electroporation GENE PULSERII (BIO RAD), a Km resistant (Km ^r ) gene was inserted into a gene to be disrupted in a competent cell for TA441 strain electroporation. A plasmid derived from pUC vector is introduced. Transformed strain with Km (400 μl / ml) was added to the LB plate for selection. To select the mutant strain in which only the Km ^r gene was inserted from the obtained Km ^r transformants, LB plate containing Km (800 μl / ml) and Cb (300 μl / ml) were selected.
ml) containing LB plate and culturing by subculture.
^{At r} , a mutant strain that is sensitive to Cb (the resistance gene is encoded in the pUC vector) is selected. These total DNA
On the other hand, hybridization is further carried out using the Km ^r gene and the disrupted gene as a probe, and it is confirmed from the position of the band to be hybridized that the Km ^r gene has been inserted and the gene has been disrupted.

(32) Preparation of growth curve The strain is inoculated in 5 ml of LB medium and precultured overnight at 30 ° C. and 120 strokes / minute. After culturing, centrifuge (5,000 rpm, 10 minutes, 4
(° C) to collect the cells. After removing the supernatant, the cells were washed twice with 2 ml of 50 mM phosphate buffer, centrifuged (5,000 rpm, 5 minutes, 4 ° C.), and then resuspended in 1 ml of 50 mM phosphate buffer. Measure the OD ₆₀₀ of the cell suspension and prepare it so that the OD ₆₀₀ is 10.
Add 0 μl (1% of medium) to 10 ml of C medium (test tube).
Add 0.1% of carbon source to the medium (for testosterone
: 10% stock solution 100 μl, pHB: 0.2 M
stock solution 250 μl). 100 μl of the culture is sampled every 3 hours, diluted appropriately and spread on LB medium.
Number of colonies grown after overnight culture at ℃ (colony formin
g unit, CFU).

(B) Meta-cleaving enzyme induced by culturing TA441 strain with testosterone (method and result) Protein was extracted from each of the cultured TA441 strain and the succinic acid cultured TA441 strain, and
Ive-PAGE was performed. After the electrophoresis, the gel was immersed in 0.04% 2,3-dihydroxybiphenyl solution and observed for the presence or absence of yellow due to meta-cleavage. As a result, a yellow band was observed only when cultured with testosterone.

(C) Purification of meta-cleaving enzyme and determination of N-terminal sequence As a result of Native-PAGE, it was confirmed that there was a meta-cleaving enzyme induced when TA441 strain was cultured with testosterone. Crude purification was performed to determine the N-terminal sequence. The determined meta-cleaving enzyme N-terminal sequence is shown below. This has already been reported by C. testosteroni (ATCC 11996).
(Mobus et al., J Bacteriol 179, 5951-5955, 1997)
Of the testosterone-inducible meta-cleaving enzyme TIP1 was consistent with the N-terminal sequence. N-terminal of metacleaving enzyme of TA441 strain: MMEIRGLAYVVAESSDLDRWVSYAR
DVLG

(D) Acquisition of meta-cleaving enzyme gene An attempt was made to acquire the meta-cleaving enzyme gene by shotgun cloning. With this method, the detection of the cloned meta-cleaving enzyme was performed using 2,3-dihydroxybiphenyl (2,3-D
HBP) is sprayed and the yellow coloration due to the meta-cleaving substance is observed. However, since the substrate specificity of the meta-cleaving enzyme is generally low, it was expected that the meta-cleaving enzyme genes aphB and mhpB already obtained from the TA441 strain could not be distinguished from the target meta-cleaving enzyme gene. So aphB, mhpB
In order to avoid the cloning of T4, TA4 was digested with restriction enzymes (Pstl, Sacl, Sph l) that cleave both aphB and mhpB.
41 strain total DNA was completely decomposed and integrated into pUC19.
An E. coli library was prepared using DH5α COMPETENT high (TOYOBO) as a host. Escherichia coli colonies on the plate were directly sprayed with 0.2 M 2,3-DHBP (acetone solution), and a strain showing a yellow color due to a meta-cleaving substance was selected.

The SacI library contains 500 colonies, SphI,
As a result of examining 2000 colonies of PstI, 3 clones from the Pstl library and 1 clone from the Sacl library were obtained, and clones showing meta-cleaving activity against 2,3-DHBP were obtained.

(E) Structural analysis of meta-cleaving enzyme gene (1) Construction of sub-cloning restriction enzyme map The plasmid of the obtained clone was prepared by using 10 kinds of restriction enzymes (Hindlll, Sphl, Pstl, S) of pUC19 multi-cloning site.
All, Xbal, BamHl, Smal, Kpnl, Sacl, EcoRl) were the same as pCPY or aphB, mhpB except for the plasmid from the Pstl clone designated pCPY. A restriction map of pCPY is shown in Fig. 2 (including the site revealed from the nucleotide sequence in the figure).

Subcloning of pCPY Various deletion plasmids were prepared based on the restriction enzyme map of pCPY (Fig. 2). When Escherichia coli clones holding these were sprayed with 0.2 M 2,3-DHBP, pCP31 exhibited a meta-cleaving activity. Therefore, restriction enzyme A that further cleaves pCP31
The deletion plasmids pCP311 and pCP312 were prepared using pal. Similarly, when sprayed with 2,3-DHBP, about 1.3
Metacleavage activity was observed in pCP311 encoding kb. Therefore, the base sequence of this 1.3 kb was first determined.

Determination of the base sequence of pCP311 Pstl and Sphl, which have a short insertion fragment of pCP311 and many cleavage sites
It seemed that all the subclones necessary for the nucleotide sequence determination were prepared by using E.coli.
Vector, 0.6 kb), Sphl fragment (0.2 kb, 0.8 kb, 0.3 k
A plasmid encoding a b + vector) was prepared, and the nucleotide sequence was determined from both ends. For the parts missing in these clones, synthesize primers pCP31-1 to 3
(FIG. 2), the base sequence was determined.

(2) Analysis of meta-cleaving enzyme gene One ORF was found within the sequence of the insert fragment of pCP311 (about 1.4 kb). N-terminal deduced amino acid sequence of this ORF deduced amino acid sequence (MMEIRGLAYVVAESSDLDRWVSYARDVLG)
Was completely matched with the N-terminal sequence from the purified enzyme, so this gene was designated as tesB. When the putative amino acid sequence of tesB was searched, homology was found with various meta-cleaving enzymes in the bacterial metabolic system of aromatic compounds. TesB is Pseu
domonas sp. strain CA10 (Sato et al., J Bacteriol
179, 4841-4849, 1997) and the highest homology (55% identity) with the meta-cleaving enzyme CarB2.
BphC, a meta-cleaving enzyme of the biphenyl metabolism of As and Rhodococcus, showed 39-42% homology. Dendrograms were created by selecting those with relatively high homology, and various biphenyl metabolic BphC and TesB
The original substrate containing is unknown meta-cleaving enzyme (2, 3-
It shows a meta-cleavage activity for dihydroxybiphenyl). In TesB, 6 amino acids such as the active center conserved in BphC were conserved.

(F) Growth of tesB gene-disrupted strain on testosterone In order to examine whether TesB is essential for TA441 testosterone metabolism, insertion of a kanamycin resistance (Km ^r ) gene
It was decided to create a mutant strain in which the esB gene was disrupted and examine the growth when testosterone was used as the sole carbon source. (1) Preparation of tesB gene-disrupted strain Preparation of tesB gene probe Approximately 650 bp from the Hindlll site derived from pUC19 to the BamHl site in the insert was purified from pCP31, and DIG-High Pri
A tesB probe for hybridization was prepared using me (Roche Molecular Biochemicals) (Fig. 2).

Preparation of tesB gene disruption plasmid pCP311 was treated with EcoRV to dephosphorylate, and a Km ^r gene cassette was inserted into that portion. The clone of interest is Km ^r (30 μ
g / ml). The obtained plasmid is ptesB-Km ^r
(Fig. 2).

Transformation of TA441 strain The ptesB-Km ^r plasmid was treated with Xbal and introduced into TA441 strain competent cells using electroporation GENE PULSERII (BIO RAD), and kanamycin (Km) (400 μg / m
l) was added to the LB plate to select for growth. In order to select the mutant strain in which only the Km ^r gene was inserted from the obtained transformants, seed on LB plate containing Km (800 μg / ml) and LB plate containing carbenicillin (Cb) (300 μl / ml) respectively. Subsequent culture was carried out, and a mutant strain was selected which was susceptible to Cb encoded by the resistance gene in the vector with Km ^r . Hybridization was further performed on these with tesB and the Km ^r gene as probes, and it was confirmed from the position of the band to be hybridized that the Km ^r gene was inserted and tesB was destroyed. The obtained mutant strain was named TesB ⁻ .

[0081] (2) TesB ^- TesB was pre-cultured in the discussion LB medium of growth of the stock to the testosterone ^- and harvested stocks and TA441 strain was washed twice, every three hours by culturing a testosterone as the sole carbon source CFU (colony forming unit) was investigated. The results are shown in Fig. 3. The positive control p-hydroxybenzoic acid (pHB) growth was not observed between TA441 strain and TesB ^- strain, while testosterone growth was comparable to that of TA441 strain, pHB was TesB. ^- strains were comparable to the case of adding no carbon source. From this, TesB was considered to be necessary for the testosterone metabolism of TA441 strain.

The strains and plasmids used in Example 1 are shown in Table 1 below.

[0083]

[Table 1]

(G) Summary of Example 1 The putative metabolic pathway for testosterone in Comamonas testosteroni is via the meta-cleavage reaction. When the N-terminal of the meta-cleaving enzyme induced when TA441 strain was cultured with testosterone was determined, it was consistent with the testosterone-inducible protein TIP1 of C. tesutosteroni ATCC 11996. We obtained a meta-cleaving enzyme gene tesB that matches this N-terminal sequence by shotgun cloning. TesB is Pseudomonas sp. Strain
Highest homology with CA10 meta-cleaving enzyme CarB2 (55%)
In addition, various BphCs that are meta-cleaving enzymes of the biphenyl metabolic system of Pseudomonas and Rhodococcus
Both showed 39-42% homology. Dendrograms were prepared by selecting those with relatively high homology, which resulted in the separation of various BphCs in the biphenyl metabolic system and meta-cleaving enzymes including TesB. Mutants tesB that destroyed tesB gene TA441 strain ^- from losing catabolism of testosterone, tesB is likely to be required for testosterone metabolism.

Example 2: Analysis of the peripheral region of tesB gene It is known that most of the aromatic compound degrading genes are clustered. Therefore, we decided to determine the base sequence of the tesB gene peripheral region. (A) Experimental procedure (1) Colony hybridization Nylon membrane (Hybond-N ⁺ (amersha
m pharmacia biotech)) and inoculate directly on it,
Incubate at 37 ° C overnight. Unwrap the wrap and place the membrane on top of the denatura
Place 2-3 ml of tion buffer (0.5 M NaOH; 1.5 M NaCl) on each well, place the membrane on it and let stand for about 5 minutes to lyse the cells. Transfer to a paper towel to remove water.
Neutratization buffer (1.5 M NaCl;
0.5 M Tris-HCl; 0.001 M EDTA, pH 7.0) is placed on each 2 to 3 ml, and the membrane is placed thereon and left still for about 5 minutes.
After transfer to a paper towel to remove water, dry it for about 60 minutes at room temperature (or 65 ℃, dry in a dry heat oven). Wrap the dry membrane in plastic wrap and irradiate with UV for 5 minutes to fix the DNA. Prehybridization, hybridization, detection is performed as described in the experimental procedure of Example 1.

(2) Promoter activity measuring strain TA441 harboring pRWtesBp (a region predicted as a promoter upstream of tesB and a broad host range vector derived from pRW2)
Use strains. Preculture is LB 2.5 ml + Tc (5 μg / ml) × 2
Book overnight at 30 ° C. The culture solution is collected and dH ₂ O is used for 2
Washed times, suspended in dH ₂ O such that the OD _{6 00} = 10. 0.1% testosterone or Androsta-1, as an inducing substrate
Suspension in 100 ml of C medium containing 4-diene-3, 17-dione 20
Add 0 μl. Using a baffle-free 500 ml Erlenmeyer flask, incubate at 120 rpm and 30 ° C, and sample every 2 hours. The β-galactosidase activity is measured as follows.・ Measure OD ₆₆₀ Take 1 ml of the culture medium in a 2 ml Eppendorf tube and add ethyl acetate.
Add 500 μl and mix well by vortexing. Centrifuge (1
Collect the cells at 5,000 rpm for 5 minutes and remove the supernatant. Open the lid for a while and blow off the remaining ethyl acetate. dH ₂ O
Dissolve in 1 ml and measure OD ₆₆₀ .・ Transfer 2 ml of culture solution for β-galactosidase activity measurement to a 2 ml Eppendorf tube and centrifuge (15,00
Collect the cells at 0 rpm for 5 minutes. Remove the supernatant 1 ml 1 x Z
buffer (2 × Z buffer (10 ml) is 0.5 M phosphate buffer
pH 7.5 (4 ml) (prepared by mixing 0.5 M Na ₂ HPO ₄ and 0.5 M NaH ₂ PO ₄ ), 1 M KCl (0.2 ml), 0.1 M MgSO ₄ (0.2 m
l), dH ₂ O (5.53 ml), and 14.4 M 2-mercaptoethanol (70 μl)). Transfer to a glass tube and add 2 drops of chloroform and 1 drop of 0.1% SDS. Mix well by vortexing and incubate at 30 ° C for 5 minutes. 200 μl of ONPG solution (ο-nitrophenyl-β-D-galactopyranoside (ONPG) 4 mg / 1 ml (1 × Z buffer)) was quickly added using a pipette and mixed, and kept at 37 ° C. To do. If a yellow color is observed, 1 M Na ₂ CO 500 μl
Is added to stop the reaction. Measure the time t (min) until yellow color is observed. OD ₄₂₀ (absorption of ο-nitrophenol and light scattering by cell lysate) and OD
Measure ₅₀₀ (light scattering from cell lysate). Conversion (value in E. coli) 1000 x ([OD ₄₂₀ ]-[OD ₅₀₀ ] x 1.75) / t x [OD ₆₆₀ ] = Mill
er unit of β-galctosidase OD ₄₂₀ light scattering and OD ₅₀₀ light scattering are proportional OD ₄₂₀ light scattering = OD ₅₀₀ light scattering × 1.75

(B) Structural Analysis of Downstream Region of tesB Gene (1) Subcloning and Nucleotide Sequence Determination pCP312 Nucleotide Sequence Determination For pCP312, a deletion mutant was prepared, but there was no suitable restriction enzyme site. Therefore, pCP31
Was treated with ApaI + EcoRI to purify an approximately 2.6 kb fragment, and the ends of the recovered fragment were blunted and inserted into the EcoRV site of pBluescript KS-. This was designated as pCP315, which encodes the same insert fragment as the pCP312 insert fragment in the opposite direction. Restriction enzyme for pCP315
ApaI + HindIII treatment, Kilo-Sequence Deletion Kit
(TaKaRa) was used to create a series of deletion plasmids excised from the upstream of the pCP312 insert. Also, pCP3
12 was also treated with the restriction enzymes BamHI + KpnI to create a series of deletion plasmids deleted from the downstream of the pCP312 insert. In the case of pCP312, since the BamHI site of the insert fragment is used, about 0.6 kb between BamHI and KpnI is lost, but the nucleotide sequence of this part was determined from a subclone using the restriction enzyme site.

PCPY subcloning and determination of nucleotide sequence Treatment with BamHl, Kpnl, Pstl and Sphl was carried out and a BamHl fragment (1.1 kb,
2.8 kb, 0.6 kb), Kpnl fragment (0.4 kb), Pstl fragment (3.0
kb, 1.1kb, 0.8 kb), Sphl fragment (1.1 kb, 0.7 kb, 0.3 k
b, 0.2 kb) was prepared and the nucleotide sequence was determined from both ends. For the part that could not be determined in these clones, primers pCP31-4 to 6 were synthesized (FIG. 4) and the nucleotide sequence was determined.

(2) Gene analysis The nucleotide sequence of pCPY was determined.
Since there were regions considered to be individual ORFs, we selected ORF1, ORF2, and ORF3 from the upstream (Fig. 4). BLAST SEARCH was performed to search for proteins showing homology with respect to the deduced amino acid sequences of these three ORFs, but no particularly significant homology was found to any of them.

(Results of analysis) -The deduced amino acid sequence of ORF1, 2 ORF1 is Acidaminococcus fermentans
(ATCC 25085) glutaconate CoA-transferase α-subun
It was 26.9% homologous to it and ORF2 was 22.4% homologous to β-subunit (Mack et al., Eur J Biochem 226, 41-51, 1994, J.
acob et al., Structure 5, 415-426, 1997). Also ORF2
The N-terminal sequence of C. testosteroni (A
TCC 11996) (Mobus et al., J Bacteriol 179, 5951-595.
5, 1997) N of testosterone-inducible meta-cleaving enzyme TIP6
Only one amino acid differed from the terminal sequence and the others were in agreement.

ORF3 E. coli enoyl CoA hydratase (Eichler et al., Mol M
icrobiol 13, 775-786, 1994) and the like showed about 30% homology. Glutaconate CoA-transferase converts glutaconate to C
Add oA to generate glutaconyl CoA. It is considered that enoyl CoA hydratase catalyzes the reaction of adding a hydroxyl group to glutaconyl CoA. This reaction is C. testoster
In the testosterone metabolic pathway of oni, there is a possibility of reaction after meta-cleavage and hydrolysis.

(C) Growth of ORF1-3 gene-disrupted strain to testosterone The N-terminal sequence of ORF2 has been reported in C. testostero.
ni (ATCC 11996) (Mobus et al., J Bacteriol 179, 59
51-5955, 1997) testosterone-inducible meta-cleaving enzyme TI.
Since it was almost identical to the N-terminal sequence of P6, it is highly likely that the protein encoded by ORF2 is involved in the growth of TA441 strain into testosterone. Therefore, it was decided to investigate whether the ORF1 to 3 genes were destroyed to cause a change in the growth of the mutant strain to testosterone.

(1) Preparation of ORF1 to 3 gene disruption strains Preparation of probe for hybridization for confirming gene disruption ORF1 probe: pCP31, Apal-Sacl fragment 850 bp ORF2 probe: Sacll-EcoT22l fragment 750 bp ORF3 probe: EcoT22l-Kpnl Fragment 1.0 kb purified and labeled for hybridization
It was used as an ORF 1, 2, 3 gene probe (Fig. 4).

Preparation of gene disruption plasmid-ORF1 disruption plasmid Since there was no enzyme that cuts at only one site suitable for gene disruption, a part of the fragment was once transferred to another vector and the resistance gene was inserted into the plasmid for gene disruption. I decided to return to. Purify 850 bp of Apal-Sacll fragment from pCP31 and use Apal + Sa
It was recombined into cll-treated pBluescript KS (+). This recombinant plasmid was treated with EcoRV, dephosphorylated, and a kanamycin resistance (Km ^r ) gene cassette was inserted. The target clone was selected with Km ^r (30 μg / ml), and the obtained plasmid was designated as pORF1-KS +. The Apal-Sacll fragment 2.1 kb was purified from pORF1-KS + and the Apal-Sacll fragment of pCP31 was 6.6 kb (pUC
(Including 19) to obtain plasmid pORF1-Km ^r (FIG. 4).

The ORF2 disruption plasmid pCP31 was treated with Sacll, dephosphorylated, and the Km ^r gene cassette was inserted. Target clone is Km ^r (30 μg / ml)
The obtained plasmid was designated as pORF2-Km ^r (Fig. 4).

The ORF3 disruption plasmid pCP312 was treated with BamH1 and blunt-ended, and the Km ^r gene cassette was inserted. The target clone was selected with Km ^r (30 μg / ml), and the obtained plasmid was designated as pCP312-Km ^r . As a result of the experiment, it was not possible to obtain a gene-disrupted strain with pCP312-Km ^r , but it was speculated that the cause was that the downstream of the BamHl site of the insert fragment was short and homologous recombination did not occur easily.
A plasmid having this portion lengthened was newly prepared. pCP3
A Scal fragment of 5.2 kb (from Scal on the vector to Scal in the insert) was purified from 12-Km ^r , and pCP01 (BamHl-P of pCPY) was purified.
Scal fragment of stl fragment) 2 kb (Scal in insert
To Scal on the vector) and plasmid pOR
Obtained F3-Km ^r . In the case of pORF3-Km ^r , about 1 kb is encoded downstream of the BamHl site where the resistance gene is inserted.

Acquisition of gene-disrupted strain pORF1-Km ^r , pORF2-Km ^r and pORF3-Km ^r were treated with Kpnl to prepare a concentrated plasmid solution. This was introduced into TA441 competent cells by electroporation, and Km ^r
(400 μg / ml) was selected. The transformant obtained was designated as Km (8
LB plate containing 00 μg / ml) and carbenicillin (Cb) (30
(0 μg / ml) was subcultured on each LB plate and cultured, and mutant strains sensitive to Cb with Km ^r were selected. The Km ^r gene and ORF1, ORF2, ORF3 gene were used as probes for hybridization, and the Km ^r gene was inserted from the position of the band to be hybridized.
It was confirmed that was destroyed. The acquired mutant strain
ORF1 ^-, ORF2 ^-, ORF3 ^- was.

(2) Examination of growth of ORF1-3 gene-disrupted strains to testosterone (method) Mutant strains ORF1 ⁻ , ORF2 ⁻ and ORF3 ⁻ were grown on testosterone (0.1% w / v) in the same manner as the TesB ⁻ strain. Examined. (Results) Since all of the mutant strains ORF1 ⁻ , ORF2 ⁻ , and ORF3 ⁻ had decreased growth in testosterone (FIG. 5), ORF1 ~
3 was also considered to be essential for testosterone metabolism. ORF1 ^- ~ORF3 ^- it was confirmed that growing the strain similar p- hydroxybenzoic acid ^- tesB also strains.

(D) Acquisition and analysis of upstream region of tesB gene It was shown that tesB downstream is required for testosterone metabolism
Then, it was decided to analyze further upstream. (Method) TesB^-The strain has a Km^rGene assembled
Because it is embedded, TesB ^-Km if stock is used^rBy
It is thought that cleaning is possible. pORF1-Km^rEcoT22I
Not be cleaved upstream of the meta-cleaving enzyme gene by treatment with
After confirming that, TesB using EcoT22I^-Process strain totalDNA
Then a gene library was prepared. (Result) Km obtained^rExtract the plasmid from the clone
When treated with a restriction enzyme, pCPY was added to this plasmid.
Since the upstream region was cloned, this is designated as pCT
We decided to determine the base sequence.

(E) Structural analysis of upstream region of tesB gene (1) Construction of restriction enzyme map
al, Xhol, Hindlll, Sall, Xbal, BamHl, Smal, Kpnl,
Sacl, EcoRl, EcoRV, Pstl, Sphl) was cut to prepare an approximate restriction enzyme map (Fig. 6; the figure also includes the site revealed from the nucleotide sequence).

(2) Determination of pCT nucleotide sequence Various deletion plasmids were prepared based on the restriction enzyme map of pCT (Fig. 6). Treat pCT with a restriction enzyme to prepare BamHl
Fragment (5.3 kb, 1.4 kb), EcoRl fragment (4.8 kb, 2.0 kb), Eco
RV fragment (2.4 kb), Pstl fragment (2.4 kb, 1.0 kb), Sall fragment
(2.8 kb), Sphl fragment (1.6 kb, 1.2 kb, 0.8 kb, 0.7 kb,
0.5 kb, 0.3 kb) and a plasmid encoding the Smal fragment (0.5 kb) were prepared. When the nucleotide sequences at these ends were determined, the sequences at the upstream and downstream ends of the pCT insert were roughly determined. The central part of the pCT insert that was not determined is the plasmid pCT62 which encodes the entire central part.
Treated with Sacl + Xbal, pCTA6 (from pCT Sall-Apal fragment 1.8 kb
(The plasmid that was purified and integrated into pBluescript KS +)
A deletion mutant was prepared by processing Sacl + Hindlll, and the nucleotide sequence was determined. For the part that could not be determined in these clones, primers pCP31-7 to 12 were synthesized (FIG. 6) and the nucleotide sequence was determined.

(3) Gene analysis Five ORFs were found upstream of tesB.
ORFs 6 to 10 were assigned from the side closer to sB (Fig. 6). (Analysis result) ・ ORF6, 7 The deduced amino acid sequence of ORF6 showed the highest homology with the N-ethylmaleimide reductase NemA of E. coli K12 (37% in identity at the amino acid level). Moreover, although the homology was lower than that of NemA, homology with the bile acid-inducible enzymes BaiH and BaiC of Eubasterium sp. VPI 12708 was found. The deduced amino acid sequence of ORF7 showed homology (19%) with BaiE. Bai
H and BaiC are presumed to be oxidoreductases having an iron-sulfur center (4Fe-4S) from the homology.・ ORF8, 9 The predicted amino acid sequence of ORF8, 9 is short-chain alcohol deh.
They show homology to ydrogenase and are similar to each other (40%). Of the 13 amino acids specifically conserved in short-chain alcohol dehydrogenase, 10 ORF8 and ORF9
Was stored 12 pieces.

(F) Growth of tesB upstream ORF6-9 gene-disrupted strain into testosterone (1) Preparation of ORF6-9 gene-disrupted strain Preparation of gene probe for hybridization to confirm gene disruption ORF6 probe: pCT13, Sphl fragment 500 bp ORF7 probe: pCT02, Pvull fragment 600 bp ORF9 probe: (Stul-Kpnl) KS- (pCT42 Stul-Kpnl fragment 2.1 kb was inserted into pBluescriptKS (-) EcoRV + Kpnl site, Hincll fragment Each 500 bp was purified and labeled to obtain ORF6, 7 and 9 gene probes (FIG. 6) ORF8 was confirmed using ORF9 probe.

Preparation of plasmid for gene disruption ORF6 disruption plasmid: pCT13 was treated with Bglll to make the ends blunt, and the Km ^r gene cassette was inserted. ORF7 disruption plasmid: pCT02 was treated with Apal, blunted and dephosphorylated, and the Km ^r gene cassette was inserted. ORF8 disruption plasmid: pCT02 was treated with Clal to blunt-end and dephosphorylated, and the Km ^r gene cassette was inserted. ORF9 disruption plasmid: A 2.6 kb Pvull fragment was purified from pCT02 and inserted into the pUC19Hincll site to prepare plasmid pCTP1. pCTP1 was treated with Sall to be blunt-ended, dephosphorylated, and the Km ^r gene cassette was inserted. The clones of interest were selected with Km ^r (30 μg / ml), and the obtained plasmids were pORF6-Km ^r , pORF7-Km ^r , pORF8-Km ^r , pORF9-K, respectively.
m ^r (Fig. 6). When the length of both sides of the resistance gene insertion position is short, homologous recombination is unlikely to occur, so the short plasmid pORF6-Km ^r on the upstream side inserted the Stul fragment derived from pCT02 on the upstream side.

Acquisition of gene-disrupted strain pORF6-Km ^r , pORF7-Km ^r , pORF8-Km ^r and pORF9-Km ^r were treated with Kpnl to prepare a concentrated plasmid solution. This was introduced into TA441 strain competent cells by electroporation, and selected with Km ^r (400 μg / ml). The transformants obtained were LB plates containing Km (800 μg / ml) and Cb (300 μg / ml).
ml) containing LB plate and subculture to culture, Km
^{At r} , a mutant strain sensitive to Cb was selected. Further, hybridization was carried out using the Km ^r gene ORF6, 7, 9 gene as a probe, and it was confirmed from the position of the band to be hybridized that the Km ^r gene was inserted and the ORF was destroyed. The obtained mutants ORF6 ^-, ORF7 ^-,
ORF8 ^-, ORF9 ^- was.

[0106] (2) ORF6~9 study of the growth of testosterone to gene-disrupted strain (method) mutant ^{ORF6 -, ORF7 -, ORF8 -} , ORF9 - About TesB
^- similar to the strain, was examined growth for testosterone (0.1% w / v). (Result) first to the result of examining the growth after 24 hours, ORF6 ^-, ORF
7 ^-, ORF8 ^-, ORF9 ^- from the both grown to testosterone (Fig. 7), ORF6~9 was estimated to be likely not involved in testosterone metabolism. Therefore, the study of growth over time was not performed.

(G) Measurement of promoter activity upstream of tesB gene A sequence presumed to be a promoter region was found immediately upstream of the tesB gene. The distance between tesB and the three ORFs downstream thereof is extremely narrow (16 bp between tesB and ORF1, 12 b between ORF1 and ORF2).
p, ORF2-ORF3 is 1 bp), which suggests that it forms an operon. In order to confirm this, it was decided to prepare a plasmid containing this promoter region to form a fusion protein of TesB and LacZ, introduce it into the TA441 strain, and measure LacZ activity.

(1) Construction of Promoter Activity Plasmid Sacl fragment 3.1 kb was purified from pCP31 and self-ligated, and the obtained plasmid was designated as pUCtesBp. pUCtes
Bp is a region predicted to be a promoter upstream of tesB and tesB
It encodes about 40 bp on the N-terminal side. Wide host range vector pR
Although the W2 cloning site includes the Hindlll site, pRW2 used in this laboratory is cleaved by Hindlll at sites other than the cloning site, so we decided to use the Bglll site instead of the Hindlll site for cloning. pUCtesBp was treated with Hindlll to blunt the ends, and Bglll
A Hindlll site was changed to a Bglll site by inserting a linker. The plasmid in which the Hindlll site of pUCtesBp was replaced with the Bglll site was designated as pUCtesBp (Bg). pUCtesBp (Bg)
Was digested with Bglll + EcoRl to purify the 350 bp fragment from a low melting agarose gel and incorporated into Bglll + EcoRl treated pRW2. The resulting plasmid was designated as pRWtesBp.

(2) Acquisition of mutant strain pRWtesBp was introduced into 441 strains by electroporation,
Selection was performed on an LB plate containing tetracycline (Tc) (12.5 μg / ml). Hybridization was performed on these using the tesB gene as a probe, and it was confirmed that the plasmid was introduced from the position of the hybridizing band. The obtained mutant strain was designated as TA441-pRWtesBp strain.

(3) Measurement of lacZ activity (method) When TA 441 strain was cultured in testosterone, TesB
It was already known that L. is induced, but it was unknown whether the inducing substrate was testosterone or an intermediate metabolite. Therefore, testosterone (0.1% w / v) as a substrate and androsta-1,4-diene-3, 17-dione (0.1%
w / v) was used. The TA441-pRWtesBp strain cultivated in LB medium was collected, washed and inoculated into 100 ml of C medium containing each substrate. The culture was performed at 30 ° C. and 120 strokes / min, and sampling was performed every 2 hours to measure β-galactosidase activity. (Results) The experiment was performed 3 times or more, and the typical results are shown in FIG. Testosterone and androsta-1,4-diene-
It was shown that 3, 17-dione induced TesB in the logarithmic growth phase. In addition, since androsta-1,4-diene-3, 17-dione showed a faster and stronger induction, it was speculated that the inducer was not testosterone itself but a metabolic intermediate.

The strains and plasmids used in Example 2 are shown in Tables 2 to 4 below.

[0112]

[Table 2]

[0113]

[Table 3]

[0114]

[Table 4]

(H) Summary of Example 2 In Example 2, the peripheral region of the meta-cleaving enzyme gene tesB was analyzed. It is known that many known aromatic compound-degrading genes are clustered. In contrast,
In the vicinity of tesB, no protein showing significant homology with the aromatic compound-degrading gene was found. In the downstream region of the tesB gene, there were three regions (ORF1 to 3) considered to be ORFs (Fig. 4). Of these, the N-terminal sequence of ORF2 has been reported in C. testosteroni (ATCC 11996) (Mobus
et al., J Bacteriol 179, 5951-5955, 1997), except for one amino acid, which is identical to the N-terminal sequence of the testosterone-inducible meta-cleaving enzyme TIP6. The deduced amino acid sequences of ORF1 and ORF1 are Acidaminococcus fermentans (ATCC 2508
5) glutaconate CoA-transferase (Mack et al., Eur
J Biochem 226, 41-51, 1994, Jacob et al., Structur
e 5, 415-426, 1997) α-subunit and 26.9%, β-subunit
With 22.4% homology. ORF3 is E. coli enoyl CoA
hydratase (Eichleret al., Mol Microbiol 13, 775-78
6, 1994) etc. and showed about 30% homology. Since all the mutant strains that disrupted ORF1 to 3 decreased the growth of testosterone, it was considered that ORF1 to 3 are also essential for testosterone metabolism. Enoyl CoA hydratase is an enzyme involved in β-oxidation of fatty acids. ORF1, 2 also CoA-transfe
Homology to rase is expected to catalyze the relevant reaction. Regarding the reaction involving ORF1-3, considering the results of analysis of other enzyme genes described in Examples 3 and 4, one of the two compounds divided into two compounds after meta-cleavage / hydrolysis was terminated with a carboxylic acid like a fatty acid. The compound
It is most likely to catalyze the metabolism of (compound in Figure 1).

Immediately upstream of tesB, a putative promoter region was found. When the β-galactosidase activity was measured using the TesB :: LacZ fusion protein, testosterone and androsta-1, 4-diene-3, 17-
It was shown that TesB was induced by dione. androsta-1,
Since 4-diene-3 and 17-dione induce faster and stronger induction, it was speculated that the inducer is a metabolic intermediate, not testosterone itself. This result also suggests that genes below tesB are induced when cultured in testosterone and involved in metabolism.

Example 3: Acquisition and analysis of random mutant strains using transposon Usually, in the metabolic system of aromatic compounds, an enzyme gene that catalyzes hydrolysis after meta-cleavage is often present near the meta-cleavage gene. When a hydrolase gene is destroyed, the gene-disrupted strain accumulates a yellow meta-cleavage substance. But,
No such gene was found around tesB ORF1-3
Since the gene-disrupted strain of Escherichia coli did not accumulate yellow substance, it was speculated that other testosterone-metabolizing enzyme genes including the hydrolase gene of the meta-cleaving substance are likely to be distant. Therefore, we created a mutant strain in which the gene was randomly disrupted using a transposon, and analyzed the mutation around the transposon insertion site for the mutant strain with reduced testosterone metabolism, and elucidated the testosterone-metabolizing enzyme gene of TA441 strain. Decided to try.

(A) Experimental procedure (1) Acquisition of mutant strain with reduced testosterone utilization (introduction of transposon mutant pSup5011 (Tn5)) pSup5011
(Tn5) is introduced into the TA441 strain competent cell using electroporation. Kanamycin (Km) (400 μg /
ml) and add to LB plate and incubate at 30 ℃ overnight. No growth is observed by patching to C plate with 1% testosterone (10% DMSO solution)
Select mutant strains whose growth is lower than that of the 1 strain or whose clear zone around the colony is smaller than that of the TA441 strain. (Confirmation of testosterone utilization capacity) Km (400 μg / ml)
Subcultured directly from the colonies on the LB plate to which was added to 10 ml of C medium (test tube) containing 0.1% testosterone,
Incubate at 30 ° C overnight. (Excluding mutants that are suspected to have been destroyed around tesB) Total DNA of testosterone-utilizing mutants was Eco
Rs, Sall treated, tesB gene, kanamycin resistance (Km ^r ).
Hybridization with the gene as a probe,
Mutants presumed to have disrupted already sequenced parts around tesB are excluded.

(2) The total DNA of the cloned testosterone-utilizing mutant 21 around the transposon insertion site was replaced with BamHl.
Treatment and other total DNA are treated with Sall and cloned into pUC19. A part of the nucleotide sequence is determined from the obtained clone, and it is reconfirmed that it is not around tesB already obtained.

(3) Cosmid vector SuperCos l (STRA
Cloning to TAGENE) (Procedure) (Preparation of genomic DNA) TA441 strain Total DNA is partially digested with Bam Hl and dephosphorylated. Measure the concentration after purifying the treated total DNA. SuperCosl is treated with Xba I, dephosphorylated, and then treated with BamHI. After purification of the treated SuperCosl, the concentration is measured. Electrophoresis 0.5 μl of SuperCo
Confirm the cleavage of sl (the bands are 6.5 kb and 1.1 kb). (Ligation) (using ligation kit ver. II) Treated total DNA (2.5 μg) and treated SuperCos l (1 μg)
Add the same amount of solution ll to the mixture and mix well. Add twice the total volume of solution l and stir well. Incubate at 26 ℃ for 5-10 minutes. (Packaging) (Gigapack III XL packaging extra
ct) (STRATAGENE) Melt packaging extracts. Hold in your hand to thaw, and immediately after melting, ligation reaction solution (1 to 4 μl, 0.1
Containing ~ 0.5 μg of DNA). Be careful not to create bubbles and poke with your fingertips or mix gently by pipetting. Flush for 2-3 seconds if necessary. Incubate at 22 ° C for 2 hours. Add 500 μl SM buffer. (Preparation of host E. coli) 20% maltose 40 μl (final concentration
0.2% (w / v)) , 1M MgSO 4 · 7H 2 O 40 μl ( final concentration 10 m
M) was added to 4 ml of LB, and E. coli was subcultured at OD ₆₀₀ at 30 ° C.
Incubate so that it does not exceed 1. Collect the cells by centrifugation at 5000 rpm for 5 minutes. Sterilized to a final concentration of OD ₆₀₀ = 1.0 1
Gently suspended MgSO ₄ · 7H ₂ O of 0 mM. (Infection) 100 μl of packaging reaction solution with SM buffer
Dilute to μl. Add 100 μl of E. coli suspension to the diluted solution and incubate at room temperature for 30 minutes. Add 800 μl of LB medium and incubate at 37 ℃ for 1 hour. Ampicillin (100 μg /
ml) and sprinkle on the LB plate.

(B) Acquisition of mutant strain with reduced testosterone assimilation capacity and cloning around transposon insertion site (1) Acquisition of mutant strain with reduced testosterone assimilation capacity Introduction of transposon Transposon Tn5 was introduced into TA441 strain to improve drug resistance. A mutant strain in which the transposon was integrated into the genome was selected.
The mutants were patched on a plate of C medium in which testosterone was suspended, and strains with reduced growth and 66 strains in which the clear zone caused by assimilation of testosterone was clearly smaller than TA441 were selected. These mutants were cultured in C medium liquid medium containing testosterone as a carbon source and C medium containing pHB, and the growth to pHB was the same as TA441 strain. Selected (FIGS. 9 and 10). Hybridization was performed on these total DNAs using the tesB gene and the Km ^r gene as probes, and one mutant strain, which is presumed to have destroyed the already determined sequence around tesB, was removed. As a result, 9 strains (9, 2) were hypothesized that testosterone assimilating ability was reduced from the first 66 mutants, and that a gene different from the existing gene was disrupted.
1, 26, 29, 42, 46, 47, 54, 65) were obtained. Of these, 26 strains showed a strong yellow coloration, which is presumed to be the accumulation of meta-cleavage substances when cultured with testosterone as a carbon source.

The total DNA of the cloned mutants 9, 26, 29, 42, 46, 47, 54, 65 around the transposon insertion site was Sal
l (There is a site immediately after the Km ^r gene of Tn5, that is, the clone of interest can be selected by Km ^r during cloning), Sa
The mutant strain 21 in which the ll site was in the vicinity of the Tn5 insertion site and became a short fragment was treated with BamHl that cuts further beyond the Sall site, and cloned into pUC19 (Fig. 11). From these clones, Km ^r clones were selected, the size of the plasmid retained therein was examined, and the one with the shortest insert fragment was selected. The obtained plasmids were designated 9-2, 21-2, 26-, respectively.
It was set as 1, 29-4, 42-1, 46-1, 47-2, 54-4, 65. Using these plasmids as templates, the terminal sequences of Tn5 repeats on both sides were used as primers (Tn5: CAG GAC GCT ACT TGT G
TA TA) and the nucleotide sequence was determined. The sequence determined here is a part of the gene disrupted by the insertion of transposon, but the function could not be estimated although the homology search was performed. In addition, the determined sequence showed no homology with the existing tesB periphery. Since a gene may be mutated due to the insertion of a transposon, the portion corresponding to the transposon insertion site from the TA441 strain must be recloned before the entire gene analysis. Since cloning was performed by colony hybridization, it was decided to prepare probes for hybridization from these plasmids.

Preparation of Probe for Colony Hybridization Each plasmid was treated with various restriction enzymes to prepare an approximate restriction enzyme map, and the enzyme for probe preparation was determined with reference to this. For most plasmids, Tn5 IS
Hpal, which cleaves the upstream side (185 bp) of the region, and another kind of the obtained enzyme with an appropriate size (1-2 kb) were used. By doing so, a probe for the disrupted gene can be prepared. For those that could not be combined with Hpal, an enzyme that yielded a fragment of the appropriate size was used. 9 strain probe: 9-2 Pstl + Hpal fragment 1.3 kb 21 strain probe: 21-2 BamHl + Hpal fragment 1.2 kb 26 strain probe: 26-1 EcoRl + Hpal fragment 2.0 kb 29 strain probe: 29-4 EcoRl + Hpal fragment 1.7 kb 42 strain probe: From 42-1 Sacll + Hpal fragment 1.8 kb 46 strain probe: From 46-1 EcoRl + Hpal fragment 1.2 kb 47 strain probe: From 47-2-1-1 Hincll fragment 1.8 kb 54 strain probe: 54-4 Hindlll + Hpal fragment 1.7 kb 65 strain probe: 65 Sphl fragment 2.0 kb

From Comamonas testosteroni, the testosterone-inducible Δ ¹ -dehydrogenase (Δ1-DH) gene,
The Δ ⁴ (5α) -dehydrogenase (Δ4-DH) gene and β-hydroxysteroid dehydrogenase (β-DH) gene have already been reported. When performing colony hybridization, it was decided to try to isolate these enzyme genes as well. For these, forward and reverse primers were prepared from the reported sequences, and the target fragment was amplified using PCR using the TA441 genome as a template. Δ1-DH: 1-F (GTC TAG AGT ATG GCA GAA CAA GAA TAT) 1-R (GGG ATC CGT CAA ACC GTC TTG CCC AGC) Δ4-DH: 4-F (GTC TAG AAG ATG TCC AAT GTC AAA AAG) 4-R (GGG ATC CCT CAC GTA ACG CGA GGA AGC) β-DH: β-F (GTC TAG AGG ATG ACA AAT CGT TTG CAG) β-R (GGG ATC CAT CTA TAG CCC CAT GCC CAG) PCR result, There was a DNA fragment amplified to the expected size. This PCR product was purified from a 1% agarose gel and
Using High Prime (Roche Molecular Biochemicals), Δ1-DH, Δ4-DH and β-DH probes for hybridization were prepared.

(2) Obtaining a cosmid clone encoding the vicinity of the transposon insertion position from the TA441 strain (method) Cosmid vector Su capable of cloning 32 to 42 kb
perCos l (STRATAGENE) (7.6 kb) and Gigapack lll XLPackegingExtr that selectively packages 47-51 kb gene
A cosmid library of TA441 strain genome was prepared using act (STRATAGENE). Fourteen replicas of each type of probe were prepared from the master plate, and colony hybridization was performed using each mutant strain probe. ACCUTRAN REPRICA PLATER (Sch
leicher & Schuell) was used. (Results) Hybridizing clones were obtained from the probes of the 6 strains shown below. Clones hybridized with 21 strain probes: 15-21, 21-42,
24-42, 19-54 Clone hybridized with 26 strain probe: 12-29, 12-26 Clone hybridized with 29 strain probe: 12-29, 12-26 Clone hybridized with 54 strain probe: 15-21, 19 -54 Clones hybridized with the 42 strain probe: 21-42, 24-42 65 Clones hybridized with the probe: 3-65 (Hereinafter, the plasmids retained by these clones should be designated as p15-21 by adding p to the clone name. Shown in.)

(3) Grouping of cosmid clones (method) In order to examine the positional relationship of these transposon insertion sites, plasmids were prepared and treated with restriction enzymes, and tesB
Hybridization was performed using the gene, each mutant strain, and three previously reported probes for dehydrogenase. (Results) From the hybridization results, the obtained clones were divided into the following three groups. (In the same group, there are duplications in some of the inserts.) 1. Mutants 26, 29, and Δ1-DH, Δ4-DH clones 2. Mutants 21, 42, 54 clones, and tesB Peripheral region 3. Mutant 65 We decided to proceed with the analysis by selecting one clone from each group. Since we were able to obtain clones of 26 strains expected to have a hydrolase gene next to meta-cleavage and clones that are thought to be linked to the tesB peripheral region, we prioritized these analyzes, and did not obtain clones of other mutant strains. Put on hold.

(C) Group 1 (mutants 29, 26, Δ1-D
H, Δ4-DH) (1) Determination of the nucleotide sequence of p12-29 insert Cosmid is difficult to analyze as it is because it clones a long fragment of 30-40 kb, so it encodes a fragment of several kb-10 kb. Various subclones were prepared (Fig. 12). 26
P12-29E3 to which both strains and 29 strain probes hybridize
First, a restriction map was prepared by cutting with a suitable restriction enzyme (Fig. 12). Based on this, BamHl, Pst
l, Sphl subclones (Fig. 12) and Genome Pr
The nucleotide sequence was determined using iming System (GPS) (New England Biolabs, Inc.). As a result, in the region of the p12-29E3 insert, a hydrolytic enzyme after metacleavage and a gene that is thought to be involved in its metabolism were found (tesD,
E, F, G; detailed analysis will be described later). So further p12-29E3
The upstream and downstream of the insert were analyzed, and p12-29Sm3 was prepared for the upstream, and the nucleotide sequence was determined using GPS. For downstream, p12-29 was Nrul treated and cloned as is without size fractionation, using primer DH5
(GAT GGC CGA GTA TCT GCG CC), DH11 (ATC TTC CAG CG
PCR reaction was performed directly from the colony using TTTCGCC CT) and the amplified plasmid p12-29Nru38 of about 1.0 kb fragment was amplified.
Was selected. This was treated with Kpnl, Hindlll + Sall and subcloned, and the resulting plasmid p12-29Nru-HdSl, p1
The nucleotide sequence of 2-29Nru-HdSl self was determined using GPS. For the part that could not be determined with these clones, primers (DH4 to 8, DH11 to 16, Nru-HdSl1,
Nru-HdSl 2, Nru-Kp) was synthesized (FIG. 12) and the nucleotide sequence was determined.

(2) Gene analysis of p12-29 insert When the region around the transposon insertion position of 29 strains was analyzed, 7 regions presumed to be Δ1-DH and Δ4-DH genes and ORF were found ( (Fig. 12). This was designated as ORF11-17. The Δ1-DH and Δ4-DH genes have already been reported in C. tes.
tosteroni TA441 gene (Florin et al., J Bacteriol
178, 3322-3330, 1996; Plesiat et al., J Bacteriol
173, 7219-7227, 1991). The results of the homology search for each deduced amino acid sequence are shown in Tables 5 and 6. The determined nucleotide sequence and amino acid sequence are shown in SEQ ID NOS: 1 to 11 of the sequence listing. SEQ ID NOs: 1 and 6: ORF12 (TesA) amino acid sequence and base sequence SEQ ID NOs: 2 and 7: ORF13 (TesD) amino acid sequence and base sequence SEQ ID NOs: 3 and 8: ORF14 (TesE) amino acid sequence and base sequence SEQ ID NO: 4 and 9: Amino acid sequence and base sequence of ORF15 (TesF) SEQ ID NOs: 5 and 10: Amino acid sequence and base sequence of ORF16 (TesG) SEQ ID NO: 11: Complete sequence of DNA fragment containing ORF11 to ORF16

[0129]

[Table 5]

[0130]

[Table 6]

(Analysis Results) ORF11 The predicted amino acid sequence of ORF11 is Rhodococcus erythropolis.
About 33% with hydroxylase such as TA421, R. opacus NCI
About 26% homology with B12038 dioxygenase (iden
tity). Although the homology is low, it shows homology with other oxygen-adding enzymes, so that it may be an oxygen-adding enzyme.・ ORF12 (TesA) ORF12 is a dioxygenase of R. erythropolis TA421 and 35
%, And about 31% homology with the monooxygenase of Chelatobacter heintzii ATCC 29600. This may also be an oxygenase enzyme. Mutant 29 had ORF12 disrupted.・ ORF13 (TesD) ORF13 is a gene that was disrupted in mutant strain 26 that accumulates a yellow metacleavage substance when cultured in testosterone.
The end sequence is C. testosteroni (ATCC 11996) (Mo
bus et al., J Bacteriol 179, 5951-5955, 1997), which was almost the same as the N-terminal sequence of the testosterone-inducible protein TIP5 (Fig. 13). Furthermore, since homology with the meta-cleaving substance hydrolase (hydrolase) BphD in the biphenyl metabolic system of C. testosteroni and Pseudomonas bacteria is also observed, it also catalyzes hydrolysis after meta-cleavage reaction in testosterone metabolism. It was speculated that So we chose ORF13 as tesD. A dendrogram was prepared with TesD and a hydrolase of a meta-cleaving substance in the metabolism of other aromatic compounds such as biphenyl and phenol.
Although it was close to BphD of testosteroni and Pseudomonas bacteria, the amino acid level identity showed only about 30% homology (Fig. 14).

ORF14, 15, 16 (TesE, F, G) ORF14, 15 and 16 are compounds produced after meta-cleavage and hydrolysis of aromatic compound metabolism system (2-oxopent- in the case of phenol metabolism system). It showed high homology with the metabolic enzyme genes of 4-enoic acid). In particular, it showed the highest homology with the phenol metabolism enzymes AphE, F, and G of the same TA441 strain at 61%, 83%, and 90%, respectively, showing that BphH, I, and J of Pseudomonas spp.
Both showed high homology. The reaction catalyzed by AphE, F, G is shown in FIG. The N-terminal sequence of ORF14 is the same as the N-terminal sequence of testosterone-inducible protein TIP8 and ORF16.
Almost coincided with TIP4 (Fig. 13). So ORF14, 15, 16
Were designated as tes E, F, and G, respectively (Fig. 12).

(3) TesD, TesE, ORF11, 12 gene-disrupted strains showed that TesD was a hydrolase after meta-cleavage from the homology search for testosterone.
E, F, and G were presumed to be metabolic enzymes of 2-oxopent-4-enoic acid or its similar compounds. These catalytic reactions are shown in Fig. 1 in the metabolic pathway of testosterone in C. testosteroni.
It is presumed that the reaction shown in 6 is. Considering this metabolic pathway, it is predicted that when a tesD gene-disrupted strain is grown on testosterone, meta-cleavage substances are accumulated and the culture solution turns yellow, and growth is not observed like the tesB gene-disrupted strain. In addition, TesE, F, G speculated reaction (2-oxo-cis-4-h
If it catalyzes the metabolism of exenoic acid, TesE-disrupted strains are 3αα, 4β, 5, 6, 7, 7α-hexahydro-7αβ-met.
It is expected that metabolites of hyl-1, 5-dioxo-4-indanpropionic acid will cause some growth. To confirm these predictions, and to confirm that ORF11, 12 whose function could not be inferred from the homology search is involved in testosterone metabolism, gene disruption strains were prepared for TesD, TesE, OEF11, 12. We decided to investigate the growth on testosterone.

Preparation of gene probe for hybridization for confirmation of gene disruption ORF11 probe: BamHl fragment from p12-29Bm3 1.5 kb TesD probe: Smal-Pstl fragment from p12-29E3 1.5 kb TesE probe: EcoRl-Sacl from p12-29E3 Fragment 1.5 k
b Purified separately, DIG High Prime (Roche Molecular Bi
oChemicals) was used to prepare a probe for hybridization (Fig. 4-5). ORF12 could not select the restriction enzyme site well, so substitute TesD probe,
It was decided to select an appropriate restriction enzyme at the time of hybridization.

Preparation of plasmid for gene disruption ORF11 disruption plasmid: The Sphl-EcoRl fragment 3.1 kb was purified from p12-29E3 and ligated to pUC19. Insert the Km ^r gene cassette into the Nrul site. ORF12 disruption plasmid: Sphl-Nrul fragment from p12-29E3
Purify 2.0 kb and ligate to pUC118 (Sphl + Smal site). Ps
Insert the Km ^r gene cassette into the tl site (in this case ORF12
Approximately 500 bp of the Pstl fragment inside is missing) TesD disruption plasmid: The EcoRl-BamHl fragment 3.7 kb was purified from p12-29E3 and ligated to pUC118. Insert the Km ^r gene cassette into the NspV site. TesE disruption plasmid: The EcoRl-BamHl fragment 3.7 kb was purified from p12-29E3 and ligated to pUC118. Insert the Km ^r gene cassette into the Smal site. The clone of interest is Km (30 μg
/ ml) resistance. The obtained plasmid was designated as pORF11
-Km ^r , pORF12-Km ^r , pTesD-Km ^r , pTesE-Km ^r (Fig. 1
2).

Acquisition of mutant strain pORF11-Km ^r , pTesD-Km ^r , and pTesE-Km ^r were treated with Scal, pORF12
-TA441 by electroporation after Kpnl treatment of Km ^r
It was introduced into a strain-competent cell line and selected for resistance to Km (400 μg / ml). Further, from the obtained Km ^r transformants Km (800
A mutant strain resistant to carbenicillin (300 μg / ml) was selected. In addition to these mutants, ORF11, T
Hybridization was performed using the esD, TesE gene and the Km ^r gene as probes, and it was confirmed that the gene was disrupted by inserting the Km ^r gene from the position of the band to be hybridized (ORF12 was substituted with the TesD probe). . Obtained a mutant strain respectively ^{ORF11 -, ORF12 -, TesD -} , Tes
E ^- and it was.

Examination of Growth of Mutant Strain to Testosterone (Method) The mutant strains ORF11 ⁻ , ORF12 ⁻ , TesD ⁻ , and TesE ⁻ were examined for growth on testosterone (0.1% w / v). ^{(Result) ORF11 -, ORF12 -, TesD} - most growth is no longer observed for, TesE ^- in was seen lagging behind the growth in comparison with the TA441 strain. TesE ^- is finally TA441 (after 24 hours)
It grew to a level very close to the strain (Fig. 17).

(D) Analysis of group 2 (mutant strains 21, 42, 54 and tesB peripheral region) (1) Determination of nucleotide sequence of p15-21 insert For p15-21, several kb to 10 in the same manner as p12-29. It was decided to create a kb subclone and determine the nucleotide sequence. First the hybridizing EcoRl fragment of the 21,54 probe 10.5
A plasmid p15-21E1 derived from pHSG397 encoding kb was prepared (FIG. 18), and the nucleotide sequence was determined using GPS.
Furthermore, the upstream and downstream base sequences were also determined. Downstream to Sphl
The plasmid p15- from pHSG397 encoding the fragment 3.2 kb
21Sp11 was prepared and the base sequence was determined using GPS. Upstream, pUC encoding Stul fragment 4.0 kb from 15-21
A plasmid p15-21Stu11 derived from 19 was prepared. This is Eco
It was treated with Rl and cloned again into pUC19 to prepare a plasmid p15-21StuEl excluding a portion derived from SuperCosl.
About this, the base sequence was determined using GPS. As for the downstream, the operon continued further downstream than p15-21Stu11, so the Kpnl fragment downstream of p15-21Stu11 was approximately 8 kb.
A plasmid p15-21Kpn17 derived from pHSG397 which encodes In order to remove the portion of p15-21Kpn17 whose nucleotide sequence had already been determined, the EcoRV fragment 6.4 kb was subcloned into pUC19 to prepare plasmid p15-21EV1 and its nucleotide sequence was determined using GPS.

(2) Gene analysis of p15-21 insert As a result of gene analysis of p15-21 insert, ORF3 and subsequent ORFs were analyzed.
14 regions (ORF4-5, ORF21-23, ORF25-
28, ORF30-34) existed (Fig. 18). these
When the putative amino acid sequence of ORF was searched, most of the proteins showed homology with enzymes involved in the β-oxidation pathway of fatty acids (Fig. 19).

(3) Since the function could not be estimated from the search for homology of growth of ORF33, 34 gene-disrupted strains to testosterone, it was positioned at the end of the region presumed to be ORF within the range of the nucleotide sequence determined so far. ORF33, which seems to be a regulator ORF34
Regarding to, a gene-disrupted strain was prepared to examine the growth on testosterone.

Preparation of gene probe for hybridization for confirming gene disruption 3.2 kb of Sphl fragment of p15-21Sp11 was ligated to pUC19 to obtain p15-21p
Sp11 was prepared. From this, 1.8 kb of Ncol fragment was purified and labeled to prepare an ORF33 probe for hybridization (FIG. 18). The ORF34 probe was used as a substitute for ORF34, and an appropriate restriction enzyme was selected during hybridization.

Preparation of plasmid for gene disruption ORF33 disruption plasmid: p15-21pSp11 was partially digested with Nael and the Km ^r gene cassette was inserted. ORF34 disruption plasmid: p15-21EV1 is partially digested with Nael and the Km ^r gene cassette is inserted. The target clone is Km
(30 μg / ml) resistance was selected. The obtained plasmids were designated as pORF33-Km ^r and pORF34-Km ^r (Fig. 18).

Acquisition of Mutant Strains pORF33-Km ^r and pORF34-Km ^r were treated with Xbal and introduced into TA441 strain competent cells by electroporation to obtain Km (4
(00 μg / ml) resistance was selected. Furthermore, from the obtained Km ^r transformant, Km (800 μg / ml) resistance and carbenicillin (300
Mutants sensitive to μg / ml) were selected. These mutants were further hybridized with the ORF33 gene and the Km ^r gene as probes, and it was confirmed from the position of the hybridizing band that the Km ^r gene had been inserted and the gene had been destroyed. The obtained mutants ORF33 ^-, ORF3
4 ^- and it was. Study of the growth of testosterone to mutant strain (method) mutant ORF33 ^-, ORF34 ^- testosterone for
Growth was examined for (0.1% w / v). (Results) The results of growth are shown in FIG. Mutants ORF33 ^-, ORF3
4 ^- Both had poor growth. Although growth can be seen for up to about 9 hours, the growth stopped while the crystal of testosterone remained, which is considered to be due to the introduction of nutrients from the preculture performed in the LB medium.

The strains and plasmids used in Example 3 are shown in Tables 7 to 9 below.

[0145]

[Table 7]

[0146]

[Table 8]

[0147]

[Table 9]

(E) Summary of Example 3 In Example 3, a mutant strain in which testosterone metabolizing ability was reduced by random mutation using transposon was prepared, and gene analysis was performed on the transposon peripheral region. As a result, the meta-cleaving enzyme te described in Example 1 and Example 2
A gene group further downstream of sB and its peripheral region and a gene group containing Δ1-DH reported by other groups were obtained. In the gene group containing Δ1-DH, there is one ORF (ORF11) before Δ1-DH and Δ4-DH after Δ1-DH, and the ORF is found further downstream (FIG. 12). Of these, ORF11 was shown to be required for the growth of testosterone by gene disruption experiments, and it is considered that it is probably an oxygenase due to the homology. Since the disrupted strain of ORF11 rarely grows on testosterone, it is likely involved in the early stages of testosterone metabolism.

On the other hand, upstream of ORF11, five ORFs (O
RF12, tesD, E, F, G) were found. As a result of homology search, tesD accumulates a yellow meta-cleavage when TesD gene-disrupted strain accumulates in testosterone.
It was considered to encode the hydrolase after the meta-cleavage reaction by sB. Due to the homology of tesE, F and G as well, 2-oxo-cis-4-hexenoic acid (originally one of the two separated testosterone through meta-cleavage and hydrolysis)
It was presumed to be the metabolic enzyme gene of the part of the A ring). When the tesE gene-disrupted strain was grown using testosterone as a carbon source, the growth was slower than that of the TA441 strain, but eventually it grew to a level close to that of TA441. This supports the speculation that the genes below tesE are only involved in the metabolism of the smaller compound after splitting in two. 2-oxo-cis-4-hexe by tesE, F, G
Noic acid is converted into acetyl-CoA and pyruvic acid, or their compounds with a methyl group.

Example 4: Testosterone metabolic intermediate TL
C analysis As a result of the gene analysis up to Example 3, a large number of ORFs presumed to be involved in testosterone metabolism were found. To determine the structure of the intermediate metabolites that accumulate in culture of gene-disrupted strains to further clarify the function of the protein,
First, it was decided to perform analysis by TLC using the testosterone culture solution of the mutant strain.

(A) Experimental Procedure (Culture Conditions) (Preculture) The strain is inoculated into 5 ml of LB medium and cultured overnight at 30 ° C. and 120 strokes / minute. After culturing, centrifuge (5,000 rp
m, 10 minutes, 4 ℃) to collect the cells. Remove the supernatant, 2 ml
After washing with dH ₂ O and centrifuging (5,000 rpm, 5 minutes, 4 ° C), 1
Resuspend in ml dH ₂ O. Measure the OD _{600 of the} bacterial cell suspension and
Prepare so that the D ₆₀₀ is 10. (Main culture) Add 100 μl of the prepared cell suspension to 10 ml of C medium (test tube). Add testosterone 0.1% w / v to the medium. Incubate overnight at 30 ° C and 120 strokes / min. (Extraction conditions for TLC) Add 1 ml of ethyl acetate to 2 ml of the culture medium and vortex for 1 minute. Collect the ethyl acetate layer. (TLC development conditions) TLC plate: kieselgel 60 F ₂₅₄ (Merck KGaA, Germany) Development solvent: Benzene: Methanol = 95: 5 Detection: UV ₂₄₅ Sample spot amount: 8 μl

(B) TLC analysis (1) TesB ^- strain (method) TA441 strain precultured in LB medium, TesB-disrupted strain TesB ^- strain were collected and washed, and TA441 strain was testosterone,
TesB ^- strain is cultivated in C medium supplemented with 0.1% w / v of testosterone and p-hydroxybenzoic acid (metabolism other than testosterone metabolizing enzyme) as a carbon source, and the start of culturing 0 to 12 hours. TLC analysis was performed by sampling 2 ml of the culture solution every 3 hours and after 24 hours. (Result) Cultured with testosterone + pHB (carbon source)
In the TesB ⁻ strain, at least 4 spots were observed in addition to testosterone (FIG. 21). Compared to the case where the TA441 strain was cultured only with pHB, T4 was presumed to be pHB, not a testosterone intermediate metabolite. When TA441 strain was cultured in testosterone, the amount of intermediate metabolites detected was significantly lower than that of TesB ^- strain. This is because testosterone was successfully metabolized in the TA441 strain, but spots other than testosterone were observed up to 12 hours like the TesB ^- strain. After 24 hours, all spots including the testosterone spot disappeared. When TA441 strain is cultured with testosterone, it enters into the stationary phase after 12 hours (Fig. 3), and it is speculated that testosterone is completely metabolized at this time.

[0153] (2) Gene disruption strain TesD^-, TesE^-, ORF11^-, ORF12^- (Method) In the same procedure as in (1) above, the gene-disrupted strain Tes
D^-, TesE^-, ORF11^-, ORF12^-Testosterone 0.1% w /
 Cultivated in C medium containing v and cultured 24 hours after the start of culture
The liquid was sampled and TLC analysis was performed. (Result) TesB in (1) above^-This time from the analysis results of the stock culture
It was found that there are metabolic intermediates that can be extracted under the conditions
Andros, which is a commercially available compound that seems to be a metabolic intermediate,
t-4-ene-3,17-dione and androsta-1, 4-diene-3, 17-
I used dione. By comparison with these standards, compound T1
Is androst-4-ene-3,17-dione, T2 is androsta-1, 4-dien
It was presumed to be e-3, 17-dione (Fig. 22). Tes
D^-, TesE^-, ORF11 ^-, ORF12^-Of the strains, close to TA441 strain
TesE showing upbringing^-Is almost the same as in TA441
While not seen, otherwise androst-4-ene-3,17
-dione, androsta-1, 4-diene-3, 17-dione, and T4
The compound was found. ORF12^-In addition to that for the stock OR
F12^-A new spot was found only in the strain.

(3) Mutant strain 2 with reduced testosterone assimilation ability
9, 26, 21, 65 (Method) Testosterone-utilizing mutant 29 (ORF12 is destroyed), 26 (TesD is destroyed), 21 (ORF3
2 destroyed), 65 testosterone 0.1% w / v
The cells were cultured in C medium supplemented with, and the TLC analysis was performed by sampling the culture medium 24 hours after the start of the culture. Similarly ORF1 ^- ~OR
An analysis was also performed for the F4 ⁻ strain. (Results) FIG. 23 shows the results of the time when the accumulation of the intermediate was easy to see and the carbon source. As with TesB ^- shares, androst-4-e
ne-3,17-dione and androsta-1, 4-diene-3, 17-dion
e, unknown substance T4 was detected. ORF12 is destroyed 29
Strains, ORF12 in experiments 5-2-2 ^- had accumulated a substance that is found only in strains.

(C) Summary of Example 4 In Example 4, in order to elucidate the function of each gene, TA441
Strains and gene-disrupted strain ^{TesB -, ORF1 -, ORF2 -} , ORF3 -, OR
^{F11 -, ORF12 -, TesD -} , TesE - were cultured testosterone substrate were analyzed culture by TLC. As a result, in most of the mutant strains, at least three spots of compounds considered to be metabolic intermediates were observed in addition to testosterone. Of these, the two with the highest mobility were compared with the standard by a
ndrost-4-ene-3,17-dione and androsta-1,4-diene-
It was presumed to be 3, 17-dione. The structure of the remaining one compound is unknown, but it was found in all strains including the TA441 strain. The ORF12 ^- strain accumulated intermediates that were not observed in other gene-disrupted strains. It is inferred that ORF12 encodes an oxidase from the homology. In particular, since homology with phenol hydroxylase is observed, it is highly possible to catalyze the reaction that produces a catechol body (reaction (6) in FIG. 24).

Example 5: Activity of TesD Gene Product The supernatant of the culture medium of the mutant TesD disrupted in the presence of testosterone was used as the reaction solution. C-medium 1 containing 0.1% testosterone for the mutant that disrupted TesD
Incubated in 0 ml at 30 ° C. for 15 hours. The yellowish culture medium of the meta-cleavage compound was centrifuged at 15000 rpm at 4 ° C and the supernatant transferred to a new tube. have pTesD
E. coli JM109 was cultured in LB medium containing ampicillin and IPTG at 30 ° C. overnight, centrifuged, and washed twice with C-medium,
After homogenization with ultrasonic waves, the cell-free extract was used as an enzyme solution. MhpC (TA441 hydrolase used in 3- (3-hydroxyphenyl) propionic acid degradation) was used as a negative control. Reaction is 30 ℃
And the hydrolase activity was assayed by monitoring the decrease in the level of meta-cleaving compound using a spectrophotometer. The reaction was 450 μg in 4.5 ml of supernatant.
It was started by adding the protein of. The first measurement was performed about 2 minutes after the addition of the enzyme, and the absorbance was measured every 20 minutes.

The results of measuring the change in absorbance are shown in FIG. FIG. 25 shows the change in absorbance measured every 20 minutes at 300 to 500 nm after incubation with TesD and MphC. The absorbance at 320 nm decreased immediately after the addition of TesD, while the decrease in yellow color corresponded to the decrease in absorbance at 440 nm, which was almost zero after 1 hour of incubation. A comparison of the decrease in absorbance at 440 nm after the addition of Tes and MhpC and in the absence of enzyme is shown in Figure 6B. The absorbance at 440 nm was decreased with TesD with TesD and hardly decreased with MhpC or in the absence of enzyme.

Example 6: Accumulation of metabolic intermediates in testosterone metabolizing enzyme gene-disrupted strain <Culture conditions> The strains shown in Table 9 (parent strain TA441, Δ ⁻¹ DH, ORF)
^{1 -, ORF11 -, TesA -} , TesB -, TesD -, TesE -, ORF34 -) was cultured overnight in 120 strokes / min inoculated to 30 ° C. in LB medium 5 ml. After culturing, centrifuge (15,000 rpm, 10 minutes, 4
(° C) to collect the bacterial cells. After removing the supernatant, washing with ₂ ml of dH ₂ O, centrifugation (15,000 rpm, 5 minutes, 4 ° C), and then 1 ml
And resuspended in dH ₂ O of. OD ₆₀₀ of the cell suspension was measured,
It was prepared so that the OD ₆₀₀ was 10. 100 μl of the prepared cell culture was transferred to 10 ml of C medium. Testosterone 0.1% w / v was added to the medium. 120 at 30 ° C
Cultured at stroke / min for 2 days.

<Extraction and analysis conditions> Second day culture solution (pH
7-9) was extracted by adding an equal amount of ethyl acetate. The ethyl acetate layer was separated and dehydrated with anhydrous sodium sulfate. Nitrogen gas was blown into 1 ml of the ethyl acetate extract for concentration. The concentrated residue was dried with a desiccator and then dissolved in 0.5 ml of methanol to prepare an analytical sample. HPLC conditions (Waters 600) HPCL column: Intertsil ODS-3, 4.6 × 250 mm, column temperature: 40 ° C. HPLC solvent: acetonitrile: water (1: 1) 1 ml /
Min, UV: 210 nm, injection volume: 5 μl Peak area / 1000 is shown in Table 10.

[0160]

[Table 10]

<Experimental Results> The TesE-disrupted strain (TesE ⁻ ) grew to the same extent as the parent strain after 24 hours, and, like the parent strain, almost no metabolic intermediate of testosterone (TS) was detected. TESE ^- In, metabolic intermediates other than 2-hydroxyurea-2,4-dienoic acid is believed to have been degraded assimilated.
In the Δ ¹ DH-disrupted strain (Δ ¹ DH ⁻ ), 4-androstene-3,17-dione (4AD) was mainly accumulated, and X4 was slightly accumulated. Since Δ ¹ DH is an enzyme that introduces a double bond into the A ring of TS, accumulation of 4AD is a valid result. In addition, the metabolic pathway of the TA441 strain is bifurcated by the next reaction of 4AD, but since the accumulated amount of X4 in Δ ¹ DH ⁻ is delicate, the main metabolic pathway is dehydrogenation at the 1-position. 1,4-Androstadiene-3,17
-Dione (ADD) was generated, which was shown to be the pathway followed by hydroxylation at the 9-position. In each gene-disrupted strain other than these, accumulation of 4AD and ADD was observed. Among them, the compound X1 accumulated characteristically in the TesA-disrupted strain (TesA ⁻ ). Further, TESD disrupted strain (TESD ^-) in the culture solution showed an intense yellow is characteristic of the meta cleavage mass quality. By using these gene-disrupted strains, it is possible to easily accumulate metabolic intermediates that are difficult to accumulate in the parent strain that efficiently metabolizes TS to accumulate intermediates. Δ4 for 4AD
If DH ⁻ is used, it can be remarkably accumulated.
The TesA ^- is possible in. Moreover, although ADD accumulated in many gene-disrupted strains, ORF11 ⁻ accumulated most remarkably under the present conditions.

<Peak Identification> TS, 4AD and AAD
For, the respective peaks were separated and purified, and GC-MS
As a result of measuring (GCM-QP5050A type, SHIMAZU), TS is the parent peak 288 (molecular formula C ₁₉ H
₂₈ O ₂ ), 4AD showed 286 (molecular formula C ₁₉ H ₂₆ O ₂ ), and ADD showed 284 (molecular formula C ₁₉ H ₂₄ O ₂ ). It was confirmed that these data were consistent with the MS data of each commercial preparation. Also, X1, X2, X3 and X
For 4, the respective peaks were separated and purified, and each was subjected to GC-MS or ESI-MS (API-2000).
Type, Perkin ElmerSciex Instrument)
The substance was identified as shown in Table 10.

[0163]

According to the present invention, first, the Comamonas testostero
Cloning and gene analysis of testosterone metabolizing enzyme gene from ni TA441 strain were performed. Many of the cloned enzyme genes TesA, B, D, E, F, and G had low homology with known enzyme genes. In addition, gene-disrupted strains were prepared based on the gene information, and the substances accumulated by these disrupted strains were analyzed. This allowed the accumulation of metabolic intermediates that were difficult to detect from the original culture medium of TA441 strain. Genes related to testosterone metabolism with unknown functions have been found around these genes, and it is possible to clarify the full range of compounds and reactions shown in the metabolic pathways in the explanatory diagram by proceeding with the analysis.

[0164]

[Sequence list] SEQUENCE LISTING <110> RIKEN <120> A strain which has a defective gene of enzymes which are involved in testosterone metabolism system <130> A11408MA <160> 11

[0165] <210> 1 <211> 325 <212> PRT <213> Comamonas testosteroni <400> 1 Met Gln Ser Thr Asp Ala Thr Phe Asp Pro Lys Asp Phe Arg Arg Ala   1 5 10 15 Leu Gly Met Phe Gly Thr Gly Val Thr Ile Val Thr Thr Arg Ala Glu              20 25 30 Asn Gly Glu Pro Val Gly Ile Thr Ala Asn Ser Phe Asn Ser Val Ser          35 40 45 Leu Glu Pro Pro Met Val Leu Trp Ser Leu Ala Lys Asn Ala Arg Ser      50 55 60 Leu Pro Val Phe Gln Ser Ala Asp Thr Trp Asn Val His Ile Leu Ser  65 70 75 80 Asn Glu Gln Glu Ala Leu Ser Asn Arg Phe Ala Arg Ala Gly Glu Asp                  85 90 95 Lys Phe Ser Gly Leu Pro Leu Asp Ser Glu Ala Ala His Ala Pro Leu             100 105 110 Leu Gln Asp Cys Ser Ala Arg Phe Arg Cys Lys Thr Ala Phe Gln Tyr         115 120 125 Asp Gly Gly Asp His Ile Ile Phe Val Gly Glu Val Thr Asp Tyr Asp     130 135 140 Ala Asn Pro His Pro Pro Leu Leu Tyr Val Thr Gly Gly Tyr Ala Leu 145 150 155 160 Ala Ser Arg Lys Ala Asn Ala Val Ala Ser Glu Pro Ala Ala Asp Thr                 165 170 175 Asn Thr Val Tyr Ser Glu Asn Leu Ile Gly Tyr Leu Met Gly Arg Ala             180 185 190 His Phe Gln Phe Leu Ser Gly Leu Arg Lys Pro Met Ala Ala Tyr Gly         195 200 205 Met Thr Asp Ala Asp Phe Tyr Val Leu Ser Leu Leu Ser Ile Gln Gln     210 215 220 Pro Gln Ser Pro Ala Glu Ile Ala Ser His Met Ala Tyr Thr Gly Thr 225 230 235 240 Asp Ile Gly Ala Val Ala Leu Gln Ser Leu Ile Ser Lys Gly Trp Val                 245 250 255 Glu Glu Ser Arg Glu Arg Ala Glu Ser Leu Gln Leu Thr Pro Leu Gly             260 265 270 Asn Glu Ala Ile Leu His Val Leu Ala Ala Ala Lys Ala Val Glu Thr         275 280 285 Asp Leu Ile Ala Ser Leu Gly Glu Met Glu Ala Ala Thr Leu Arg Asn     290 295 300 Leu Leu Lys Lys Ala Ile Ala Ala Thr Asp Pro Gly Leu Pro Lys Leu 305 310 315 320 Trp Gln Ala Arg Ala                 325

[0166] <210> 2 <211> 279 <212> PRT <213> Comamonas testosteroni <400> 2 Met Ser Gln Phe Ser Ile Pro Glu Gly Lys Tyr Val Asp Ile Gly Asn   1 5 10 15 Val Ala Gly His Pro Gln Arg Val His Tyr His Glu Gln Gly Ser Gly              20 25 30 Asp Pro Val Ile Phe Leu His Gly Ala Gly Thr Gly Ala Ser Gly Tyr          35 40 45 Ser Asn Phe Lys Gly Asn Tyr Pro Glu Phe Ala Ala Ala Gly Phe Arg      50 55 60 Ser Ile Val Pro Asp Leu Leu Gly Tyr Gly Leu Ser Ser Lys Thr Glu  65 70 75 80 Glu Pro Arg Gln Tyr Asp Met Asp Phe Phe Ile Ala Gly Val Lys Gly                  85 90 95 Leu Val Glu Gln Leu Gly Leu Lys Asn Ile Thr Leu Leu Gly Asn Ser             100 105 110 Leu Gly Gly Ala Val Ala Leu Gly Tyr Ala Leu Lys Tyr Pro Glu Asp         115 120 125 Val Lys Ser Leu Ile Leu Met Ala Pro Gly Gly Val Glu Glu Phe Glu     130 135 140 Ala Tyr Met Ala Met Pro Gly Ile Ala Asn Met Phe Asn Val Tyr Lys 145 150 155 160 Ser Gly Lys Thr Gly Pro Glu Ala Met Arg Ala Val Met Ser Met Gln                 165 170 175 Val Val Asp Gln Ser Leu Leu Thr Asp Glu Ile Ile Asn Glu Arg Ala             180 185 190 Pro Ile Ala Leu Thr Gln Thr Glu Ala Ser Arg Gln Arg Leu Tyr Ile         195 200 205 Pro Asn Met Thr Asp Gln Leu Pro Glu Leu Arg Cys Asn Val Leu Gly     210 215 220 Phe Trp Gly Met Gln Asp Ala Phe Asn Pro Val Gly Gly Ala Asp Lys 225 230 235 240 Leu Ala Lys Gly Ile Lys Asn Cys Arg Val Val Leu Val Asn Gln Cys                 245 250 255 Gly His Trp Val Gln Val Glu His Arg Glu Met Phe Asn Arg Ser Cys             260 265 270 Ile Asp Phe Met Lys Asn Gly         275

[0167] <210> 3 <211> 264 <212> PRT <213> Comamonas testosteroni <400> 3 Met Ser Asp Lys Gln Phe Ile Glu Thr Gln Gly Gln Arg Leu Tyr Glu   1 5 10 15 Ala Leu Arg Ser Ala Arg Thr Leu Ala Pro Leu Thr Asp Asn His Pro              20 25 30 Glu Met Thr Val Glu Asp Ala Tyr His Ile Ser Leu His Met Leu Arg          35 40 45 Leu Arg Glu Ala Ser Gly Glu Arg Val Ile Gly Lys Lys Ile Gly Val      50 55 60 Thr Ser Lys Pro Val Gln Asp Met Leu Asn Val His Gln Pro Asp Phe  65 70 75 80 Gly Phe Leu Thr Asp Ser Met Glu Tyr Glu Asp Gly Ala Ala Val Ser                  85 90 95 Leu Lys Ala Ala Gly Leu Ile Gln Pro Arg Ala Glu Gly Glu Ile Ala             100 105 110 Phe Met Leu Lys Lys Asp Leu Gln Gly Pro Gly Val Thr Arg Glu Asp         115 120 125 Val Leu Ala Ala Thr Glu Trp Val Ala Pro Cys Phe Glu Ile Val Asp     130 135 140 Ser Arg Ile Asn Asp Trp Lys Ile Lys Ile Gln Asp Thr Val Ala Asp 145 150 155 160 Asn Ala Ser Cys Gly Val Phe Val Ile Gly Lys Gln His Thr Asp Pro                 165 170 175 Ala Ser Leu Asp Leu Ala Ala Ala Ala Met Gln Met Ser Lys Asn Gly             180 185 190 Gln Pro Ala Gly Ser Gly Leu Gly Ser Ala Val Gln Gly His Pro Ala         195 200 205 Glu Ala Val Ala Trp Leu Ala Asn Thr Leu Gly Ala Phe Gly Ile Pro     210 215 220 Phe Lys Ala Gly Glu Val Ile Leu Ser Gly Ser Leu Ala Pro Leu Val 225 230 235 240 Pro Ala Ala Ala Gly Asp Arg Phe Asp Met Val Ile Glu Gly Met Gly                 245 250 255 Thr Cys Ser Ile Gln Phe Thr Glu             260

[0168] <210> 4 <211> 307 <212> PRT <213> Comamonas testosteroni <400> 4 Met Thr Gln Lys Lys Ile Lys Cys Ala Leu Ile Gly Pro Gly Asn Ile   1 5 10 15 Gly Thr Asp Leu Leu Met Lys Leu Gln Arg Ser Pro Ile Leu Glu Pro              20 25 30 Val Trp Met Val Gly Ile Asp Pro Glu Ser Asp Gly Leu Lys Arg Ala          35 40 45 Arg Glu Met Gly Ile Lys Thr Thr Ala Asp Gly Val Asp Gly Leu Leu      50 55 60 Pro Phe Val Lys Glu Asp Gly Ile Gln Ile Ala Phe Asp Ala Thr Ser  65 70 75 80 Ala Tyr Val His Ala Glu Asn Ser Arg Lys Leu Asn Glu Leu Gly Val                  85 90 95 Leu Met Ile Asp Leu Thr Pro Ala Ala Ile Gly Pro Tyr Cys Val Pro             100 105 110 Ser Val Asn Leu Ala Glu Lys Val Ala Glu Lys Ala Met Asn Val Asn         115 120 125 Met Val Thr Cys Gly Gly Gln Ala Thr Ile Pro Met Val Ala Ala Val     130 135 140 Ser Arg Val Gln Ala Val Ser Tyr Gly Glu Ile Val Ala Thr Val Ser 145 150 155 160 Ser Arg Ser Val Gly Pro Gly Thr Arg Lys Asn Ile Asp Glu Phe Thr                 165 170 175 Arg Thr Thr Ser Gly Ala Val Glu Lys Ile Gly Gly Ala Gln Lys Gly             180 185 190 Lys Ala Ile Ile Val Ile Asn Pro Ala Glu Pro Pro Leu Ile Met Arg         195 200 205 Asp Thr Ile His Cys Leu Thr Val Asp Thr Pro Lys Pro Ala Glu Ile     210 215 220 Glu Ala Ser Val His Ala Met Ile Lys Glu Val Gln Lys Tyr Val Pro 225 230 235 240 Gly Tyr Lys Leu Val Asn Gly Pro Val Ile Asp Gly Asn Arg Val Ser                 245 250 255 Ile Tyr Met Glu Val Glu Gly Leu Gly Asp Tyr Leu Pro Lys Tyr Ala             260 265 270 Gly Asn Leu Asp Ile Met Thr Ala Ala Ala Ala Arg Thr Ala Glu Met         275 280 285 Phe Ala Glu Glu Ile Leu Ala Gly Arg Phe Glu Leu Ala Glu Ala Ala     290 295 300 Val Ala Val 305

[0169] <210> 5 <211> 350 <212> PRT <213> Comamonas testosteroni <400> 5 Met Ser Leu Lys Gly Lys Lys Ile Thr Val His Asp Met Thr Leu Arg   1 5 10 15 Asp Gly Met His Pro Lys Arg His Gln Met Thr Leu Glu Gln Met Lys              20 25 30 Ser Val Ala Thr Gly Leu Asp Ala Ala Gly Val Pro Leu Ile Glu Val          35 40 45 Thr His Gly Asp Gly Leu Gly Gly Ser Ser Val Asn Tyr Gly Phe Pro      50 55 60 Ala His Thr Asp Glu Glu Tyr Leu Ser Thr Val Ile Pro Leu Met Lys  65 70 75 80 Gln Ala Lys Val Ser Ala Leu Leu Leu Pro Gly Ile Gly Thr Val Asp                  85 90 95 His Leu Lys Met Ala His Glu Leu Gly Val Ser Thr Ile Arg Val Ala             100 105 110 Thr His Cys Thr Glu Ala Asp Val Ser Glu Gln His Ile Ala Met Ala         115 120 125 Arg Lys Met Gly Met Asp Thr Val Gly Phe Leu Met Met Ala His Met     130 135 140 Tyr Ser Ala Glu Gly Leu Val Lys Gln Ala Lys Leu Met Glu Gly Tyr 145 150 155 160 Gly Ala Asn Cys Ile Tyr Ile Thr Asp Ser Ala Gly Tyr Met Leu Pro                 165 170 175 Asp Asp Val Lys Glu Arg Leu Ser Ala Val Arg Gln Ala Leu Lys Pro             180 185 190 Glu Thr Glu Leu Gly Phe His Gly His His Asn Leu Ala Met Gly Ile         195 200 205 Ala Asn Ser Leu Ala Ala Val Glu Val Gly Ala Asn Arg Ile Asp Ala     210 215 220 Ala Ala Ala Gly Leu Gly Ala Gly Ala Gly Asn Thr Pro Met Glu Val 225 230 235 240 Leu Val Ala Val Cys Ala Arg Met Gly Ile Glu Thr Gly Val Asp Val                 245 250 255 Phe Lys Ile Gln Asp Val Ala Glu Asp Leu Val Val Pro Leu Met Asp             260 265 270 Phe Pro Ile Arg Ile Asp Arg Asp Ala Leu Thr Leu Gly Tyr Ala Gly         275 280 285 Val Tyr Gly Ser Phe Leu Leu Phe Ala Lys Arg Ala Glu Ala Lys Tyr     290 295 300 Gly Ile Pro Ala Arg Glu Leu Leu Leu Glu Leu Gly Arg Arg Gly Met 305 310 315 320 Val Gly Gly Gln Glu Asp Met Ile Glu Asp Thr Ala Leu Thr Met Val                 325 330 335 Arg Glu Arg Gln Lys Leu Gly His Lys Val Ala Val Ala Ala             340 345 350

[0170] <210> 6 <211> 978 <212> DNA <213> Comamonas testosteroni <400> 6 atg caa agc act gat gca acc ttc gac ccc aaa gac ttt cgc cgc gcc 48 Met Gln Ser Thr Asp Ala Thr Phe Asp Pro Lys Asp Phe Arg Arg Ala   1 5 10 15 ctg ggc atg ttt ggc acc ggg gtc acc atc gtc acc acc cgc gcc gag 96 Leu Gly Met Phe Gly Thr Gly Val Thr Ile Val Thr Thr Arg Ala Glu              20 25 30 aat ggc gag cct gta gga atc act gcc aac agc ttc aac tcg gtt tcg 144 Asn Gly Glu Pro Val Gly Ile Thr Ala Asn Ser Phe Asn Ser Val Ser          35 40 45 ctg gag ccc ccc atg gtt ttg tgg agc ctg gcc aag aac gcc cgc agc 192 Leu Glu Pro Pro Met Val Leu Trp Ser Leu Ala Lys Asn Ala Arg Ser      50 55 60 ctg cct gtg ttc cag agc gcc gac acc tgg aat gtg cac atc ctc tcc 240 Leu Pro Val Phe Gln Ser Ala Asp Thr Trp Asn Val His Ile Leu Ser  65 70 75 80 aac gag caa gaa gcc ctg tcg aac cgc ttt gcc cgt gct ggc gaa gac 288 Asn Glu Gln Glu Ala Leu Ser Asn Arg Phe Ala Arg Ala Gly Glu Asp                  85 90 95 aag ttc tcc ggc ctg ccg ctg gac tcc gag gca gcc cat gcc ccg ctg 336 Lys Phe Ser Gly Leu Pro Leu Asp Ser Glu Ala Ala His Ala Pro Leu             100 105 110 ctg cag gac tgc agc gcg cgc ttc agg tgc aag acg gca ttt cag tac 384 Leu Gln Asp Cys Ser Ala Arg Phe Arg Cys Lys Thr Ala Phe Gln Tyr         115 120 125 gac ggc ggc gac cac atc atc ttt gtg ggc gaa gtg acc gac tac gac 432 Asp Gly Gly Asp His Ile Ile Phe Val Gly Glu Val Thr Asp Tyr Asp     130 135 140 gcc aac cca cac cct ccc ctg ctc tac gtg acc ggc ggt tac gca ctg 480 Ala Asn Pro His Pro Pro Leu Leu Tyr Val Thr Gly Gly Tyr Ala Leu 145 150 155 160 gct tca cgc aag gcc aac gcc gtg gcc agc gaa cct gca gct gac acc 528 Ala Ser Arg Lys Ala Asn Ala Val Ala Ser Glu Pro Ala Ala Asp Thr                 165 170 175 aat acc gtc tac tcg gaa aac ctg atc gga tat ttg atg ggc cgc gcg 576 Asn Thr Val Tyr Ser Glu Asn Leu Ile Gly Tyr Leu Met Gly Arg Ala             180 185 190 cac ttt caa ttt ctg agc ggc ttg cgc aag ccc atg gcg gct tac ggc 624 His Phe Gln Phe Leu Ser Gly Leu Arg Lys Pro Met Ala Ala Tyr Gly         195 200 205 atg acc gat gcc gat ttt tac gtg ctc tcg ctg ctc agc atc cag caa 672 Met Thr Asp Ala Asp Phe Tyr Val Leu Ser Leu Leu Ser Ile Gln Gln     210 215 220 ccc cag tcg cct gcc gaa att gcg tct cac atg gcc tac acc ggc acc 720 Pro Gln Ser Pro Ala Glu Ile Ala Ser His Met Ala Tyr Thr Gly Thr 225 230 235 240 gac atc gga gcc gtg gcc ctg cag tcg ctg atc agc aag ggc tgg gtg 768 Asp Ile Gly Ala Val Ala Leu Gln Ser Leu Ile Ser Lys Gly Trp Val                 245 250 255 gag gaa agc cgc gag cgc gcc gag tcg ctg cag ctc acc ccc ctg ggc 816 Glu Glu Ser Arg Glu Arg Ala Glu Ser Leu Gln Leu Thr Pro Leu Gly             260 265 270 aac gaa gcg atc ttg cat gtg ctg gca gca gcc aaa gcc gtg gaa acc 864 Asn Glu Ala Ile Leu His Val Leu Ala Ala Ala Lys Ala Val Glu Thr         275 280 285 gat ttg ata gcg agc ctt ggc gag atg gaa gca gcc acg cta cgc aat 912 Asp Leu Ile Ala Ser Leu Gly Glu Met Glu Ala Ala Thr Leu Arg Asn     290 295 300 ctg ctc aaa aag gcg att gcc gcc acc gac ccc ggc ctg ccc aag ctc 960 Leu Leu Lys Lys Ala Ile Ala Ala Thr Asp Pro Gly Leu Pro Lys Leu 305 310 315 320 tgg caa gcc agg gct tga 978 Trp Gln Ala Arg Ala                 325

[0171] <210> 7 <211> 840 <212> DNA <213> Comamonas testosteroni <400> 7 atg agc caa ttt tcc atc cca gaa ggc aag tat gtg gac atc ggc aat 48 Met Ser Gln Phe Ser Ile Pro Glu Gly Lys Tyr Val Asp Ile Gly Asn   1 5 10 15 gtg gcc ggt cat cct cag cgc gtt cac tat cat gag caa ggc tct ggc 96 Val Ala Gly His Pro Gln Arg Val His Tyr His Glu Gln Gly Ser Gly              20 25 30 gac ccc gtg atc ttt ttg cac ggc gcc ggc acc ggt gcc agc ggc tac 144 Asp Pro Val Ile Phe Leu His Gly Ala Gly Thr Gly Ala Ser Gly Tyr          35 40 45 agc aac ttc aag ggc aac tac ccc gag ttt gct gcg gcc ggc ttt cgc 192 Ser Asn Phe Lys Gly Asn Tyr Pro Glu Phe Ala Ala Ala Gly Phe Arg      50 55 60 tcc atc gtt ccc gac ctg ctc ggc tac ggc ctc tcg tcc aag acc gag 240 Ser Ile Val Pro Asp Leu Leu Gly Tyr Gly Leu Ser Ser Lys Thr Glu  65 70 75 80 gaa ccc agg cag tac gac atg gat ttc ttc atc gcc ggc gtc aaa ggt 288 Glu Pro Arg Gln Tyr Asp Met Asp Phe Phe Ile Ala Gly Val Lys Gly                  85 90 95 ctg gtg gag caa ctg ggc ctg aaa aac atc acc ctg ctg ggc aac tcg 336 Leu Val Glu Gln Leu Gly Leu Lys Asn Ile Thr Leu Leu Gly Asn Ser             100 105 110 ctg ggc ggt gcc gtg gcg ctg ggc tat gcg ctc aag tac ccc gaa gac 384 Leu Gly Gly Ala Val Ala Leu Gly Tyr Ala Leu Lys Tyr Pro Glu Asp         115 120 125 gtc aag agc ctg atc ctc atg gcg cct ggc ggc gtg gaa gag ttc gaa 432 Val Lys Ser Leu Ile Leu Met Ala Pro Gly Gly Val Glu Glu Phe Glu     130 135 140 gcc tat atg gcc atg ccc ggc att gcc aat atg ttc aat gtc tac aag 480 Ala Tyr Met Ala Met Pro Gly Ile Ala Asn Met Phe Asn Val Tyr Lys 145 150 155 160 tcg ggc aag acc ggc ccc gag gcc atg cgc gcc gtg atg agc atg cag 528 Ser Gly Lys Thr Gly Pro Glu Ala Met Arg Ala Val Met Ser Met Gln                 165 170 175 gtg gtg gat cag tca ttg ctg acc gac gag atc atc aat gaa cgc gcc 576 Val Val Asp Gln Ser Leu Leu Thr Asp Glu Ile Ile Asn Glu Arg Ala             180 185 190 ccg att gcg ctg acc cag acc gag gcc tcg cgc cag cgc ctg tac atc 624 Pro Ile Ala Leu Thr Gln Thr Glu Ala Ser Arg Gln Arg Leu Tyr Ile         195 200 205 ccc aat atg acc gat cag ttg cct gag ctc agg tgc aat gtg ttg ggc 672 Pro Asn Met Thr Asp Gln Leu Pro Glu Leu Arg Cys Asn Val Leu Gly     210 215 220 ttc tgg ggc atg cag gat gcc ttc aac ccc gtg ggc ggc gcc gac aag 720 Phe Trp Gly Met Gln Asp Ala Phe Asn Pro Val Gly Gly Ala Asp Lys 225 230 235 240 ctg gcc aag ggc atc aag aat tgc cgc gtg gtg ctg gtc aac cag tgc 768 Leu Ala Lys Gly Ile Lys Asn Cys Arg Val Val Leu Val Asn Gln Cys                 245 250 255 ggc cac tgg gtg cag gtc gag cat cgc gag atg ttc aac cgc agc tgc 816 Gly His Trp Val Gln Val Glu His Arg Glu Met Phe Asn Arg Ser Cys             260 265 270 atc gac ttc atg aag aac ggc tga 840 Ile Asp Phe Met Lys Asn Gly         275

[0172] <210> 8 <211> 795 <212> DNA <213> Comamonas testosteroni <400> 8 atg agt gac aag cag ttc atc gaa acg caa ggc cag cgc ctt tat gag 48 Met Ser Asp Lys Gln Phe Ile Glu Thr Gln Gly Gln Arg Leu Tyr Glu   1 5 10 15 gcc ctg cgc tcg gcc agg acg ctg gca ccg ctg acc gac aac cac ccc 96 Ala Leu Arg Ser Ala Arg Thr Leu Ala Pro Leu Thr Asp Asn His Pro              20 25 30 gaa atg acg gtg gaa gac gct tat cac atc tcc ctg cac atg ctg cgc 144 Glu Met Thr Val Glu Asp Ala Tyr His Ile Ser Leu His Met Leu Arg          35 40 45 ctg cgc gaa gcg tcg ggc gaa cgt gtg atc ggc aag aag atc ggc gtg 192 Leu Arg Glu Ala Ser Gly Glu Arg Val Ile Gly Lys Lys Ile Gly Val      50 55 60 acc tcc aag ccc gtg cag gac atg ctg aat gtg cac cag ccc gac ttc 240 Thr Ser Lys Pro Val Gln Asp Met Leu Asn Val His Gln Pro Asp Phe  65 70 75 80 ggc ttt ctg acc gac agc atg gaa tac gag gac ggt gca gcc gtc tcg 288 Gly Phe Leu Thr Asp Ser Met Glu Tyr Glu Asp Gly Ala Ala Val Ser                  85 90 95 ctc aag gca gcc ggt ctg atc cag ccc cgc gcc gaa ggc gaa att gcc 336 Leu Lys Ala Ala Gly Leu Ile Gln Pro Arg Ala Glu Gly Glu Ile Ala             100 105 110 ttc atg ctc aag aag gat ctg caa ggc ccg ggg gtg acc cgc gaa gac 384 Phe Met Leu Lys Lys Asp Leu Gln Gly Pro Gly Val Thr Arg Glu Asp         115 120 125 gtg ctg gcc gct acc gaa tgg gtg gcg ccc tgc ttc gag atc gtc gat 432 Val Leu Ala Ala Thr Glu Trp Val Ala Pro Cys Phe Glu Ile Val Asp     130 135 140 tcg cgc atc aac gac tgg aag atc aag atc cag gac acc gtg gcc gac 480 Ser Arg Ile Asn Asp Trp Lys Ile Lys Ile Gln Asp Thr Val Ala Asp 145 150 155 160 aac gcc tcc tgc ggc gtc ttt gtc atc ggc aag cag cac aca gat ccg 528 Asn Ala Ser Cys Gly Val Phe Val Ile Gly Lys Gln His Thr Asp Pro                 165 170 175 gct tcg ctg gat ctg gcc gct gcc gcc atg cag atg agc aag aac ggc 576 Ala Ser Leu Asp Leu Ala Ala Ala Ala Met Gln Met Ser Lys Asn Gly             180 185 190 cag ccc gca ggc tcg ggc ctg ggc agc gcc gtg cag ggc cac ccc gcc 624 Gln Pro Ala Gly Ser Gly Leu Gly Ser Ala Val Gln Gly His Pro Ala         195 200 205 gaa gcc gtg gcc tgg ctg gcc aac aca ttg ggc gct ttc ggc att cct 672 Glu Ala Val Ala Trp Leu Ala Asn Thr Leu Gly Ala Phe Gly Ile Pro     210 215 220 ttc aaa gcc ggt gaa gtg att ctc tct ggc tct ctc gcc ccc ctg gtg 720 Phe Lys Ala Gly Glu Val Ile Leu Ser Gly Ser Leu Ala Pro Leu Val 225 230 235 240 ccc gcc gcc gcc ggc gac cgt ttc gac atg gtc atc gaa ggc atg ggc 768 Pro Ala Ala Ala Gly Asp Arg Phe Asp Met Val Ile Glu Gly Met Gly                 245 250 255 acc tgt tcc att caa ttc acc gag taa 795 Thr Cys Ser Ile Gln Phe Thr Glu             260

[0173] <210> 9 <211> 924 <212> DNA <213> Comamonas testosteroni <400> 9 atg acg caa aaa aag atc aag tgc gca ctg atc ggc ccc ggc aat atc 48 Met Thr Gln Lys Lys Ile Lys Cys Ala Leu Ile Gly Pro Gly Asn Ile   1 5 10 15 ggc acc gat ctg ctc atg aaa ctg cag cgt tcc ccc att ctg gag cct 96 Gly Thr Asp Leu Leu Met Lys Leu Gln Arg Ser Pro Ile Leu Glu Pro              20 25 30 gtg tgg atg gtc ggt atc gac cct gaa tcc gat ggt ctc aag cgt gcc 144 Val Trp Met Val Gly Ile Asp Pro Glu Ser Asp Gly Leu Lys Arg Ala          35 40 45 cgc gag atg ggc atc aag acc acg gcc gac ggt gtg gac ggc ctg ctt 192 Arg Glu Met Gly Ile Lys Thr Thr Ala Asp Gly Val Asp Gly Leu Leu      50 55 60 ccc ttt gtc aag gaa gat ggc atc cag atc gcc ttt gac gcc acc agc 240 Pro Phe Val Lys Glu Asp Gly Ile Gln Ile Ala Phe Asp Ala Thr Ser  65 70 75 80 gcc tat gtc cac gcc gaa aac agc cgc aag ctc aac gag cta ggc gtg 288 Ala Tyr Val His Ala Glu Asn Ser Arg Lys Leu Asn Glu Leu Gly Val                  85 90 95 ctg atg atc gac ctg acg cct gcg gcc atc ggc ccc tac tgc gtg ccc 336 Leu Met Ile Asp Leu Thr Pro Ala Ala Ile Gly Pro Tyr Cys Val Pro             100 105 110 agc gtg aat ctg gcc gag aag gtt gca gaa aag gcc atg aac gtg aat 384 Ser Val Asn Leu Ala Glu Lys Val Ala Glu Lys Ala Met Asn Val Asn         115 120 125 atg gtc acc tgc ggc ggc cag gcc acc att ccc atg gtc gcg gcg gtt 432 Met Val Thr Cys Gly Gly Gln Ala Thr Ile Pro Met Val Ala Ala Val     130 135 140 tcg cgc gtg cag gcc gtg agc tac gga gag atc gtg gcc acg gta tcg 480 Ser Arg Val Gln Ala Val Ser Tyr Gly Glu Ile Val Ala Thr Val Ser 145 150 155 160 agc cgc tcg gtc ggc ccc gga acg cgc aag aac atc gac gaa ttc acc 528 Ser Arg Ser Val Gly Pro Gly Thr Arg Lys Asn Ile Asp Glu Phe Thr                 165 170 175 cgc acc aca tcg ggt gcc gtg gaa aag att ggc ggc gca caa aag ggc 576 Arg Thr Thr Ser Gly Ala Val Glu Lys Ile Gly Gly Ala Gln Lys Gly             180 185 190 aag gcc atc atc gtc atc aac ccc gcc gag ccg ccg ctg atc atg cgc 624 Lys Ala Ile Ile Val Ile Asn Pro Ala Glu Pro Pro Leu Ile Met Arg         195 200 205 gac acc atc cac tgc ctg acg gtg gat acg ccc aag cct gcc gag atc 672 Asp Thr Ile His Cys Leu Thr Val Asp Thr Pro Lys Pro Ala Glu Ile     210 215 220 gaa gcc tcg gtg cac gcc atg atc aag gaa gtg cag aaa tac gtg ccc 720 Glu Ala Ser Val His Ala Met Ile Lys Glu Val Gln Lys Tyr Val Pro 225 230 235 240 ggc tac aag ctg gtc aat ggc ccc gtg atc gac ggc aac cgc gtc tcc 768 Gly Tyr Lys Leu Val Asn Gly Pro Val Ile Asp Gly Asn Arg Val Ser                 245 250 255 atc tat atg gaa gtc gaa ggc ctg ggc gac tac ctg ccc aag tac gcg 816 Ile Tyr Met Glu Val Glu Gly Leu Gly Asp Tyr Leu Pro Lys Tyr Ala             260 265 270 ggc aat ctg gac atc atg acc gcc gct gcg gcg cgc acg gcc gag atg 864 Gly Asn Leu Asp Ile Met Thr Ala Ala Ala Ala Arg Thr Ala Glu Met         275 280 285 ttt gcc gaa gaa atc ctc gcc ggc cgt ttc gaa ttg gcc gaa gcc gct 912 Phe Ala Glu Glu Ile Leu Ala Gly Arg Phe Glu Leu Ala Glu Ala Ala     290 295 300 gtt gct gtc tga 924 Val Ala Val 305

[0174] <210> 10 <211> 1053 <212> DNA <213> Comamonas testosteroni <400> 10 atg tcc ctc aaa ggt aag aag atc acc gtt cac gac atg acg ctg cgt 48 Met Ser Leu Lys Gly Lys Lys Ile Thr Val His Asp Met Thr Leu Arg   1 5 10 15 gac ggc atg cac ccc aag cgc cac cag atg acc ctg gaa cag atg aag 96 Asp Gly Met His Pro Lys Arg His Gln Met Thr Leu Glu Gln Met Lys              20 25 30 tcc gtg gcc acg gga ctg gat gcc gcc ggc gtg ccc ctg att gaa gtg 144 Ser Val Ala Thr Gly Leu Asp Ala Ala Gly Val Pro Leu Ile Glu Val          35 40 45 acc cat ggc gac ggc ctg ggt ggc agc tcg gtc aac tac ggc ttt cct 192 Thr His Gly Asp Gly Leu Gly Gly Ser Ser Val Asn Tyr Gly Phe Pro      50 55 60 gca cat acg gac gag gag tac ctg tcc acc gtc atc ccg ctg atg aag 240 Ala His Thr Asp Glu Glu Tyr Leu Ser Thr Val Ile Pro Leu Met Lys  65 70 75 80 cag gcc aag gtc tcg gcc ctg ttg ctg ccc ggc atc ggc act gtc gat 288 Gln Ala Lys Val Ser Ala Leu Leu Leu Pro Gly Ile Gly Thr Val Asp                  85 90 95 cac ctg aag atg gct cac gag ctg ggc gtc tcc acc atc cgt gtg gcc 336 His Leu Lys Met Ala His Glu Leu Gly Val Ser Thr Ile Arg Val Ala             100 105 110 acg cat tgc acc gag gcc gat gtc tcc gag cag cac atc gcc atg gcc 384 Thr His Cys Thr Glu Ala Asp Val Ser Glu Gln His Ile Ala Met Ala         115 120 125 cgc aag atg ggc atg gat acc gtg ggc ttt ctc atg atg gcg cat atg 432 Arg Lys Met Gly Met Asp Thr Val Gly Phe Leu Met Met Ala His Met     130 135 140 tac agt gcc gaa ggt ctg gtc aag cag gcc aag ctg atg gaa ggc tac 480 Tyr Ser Ala Glu Gly Leu Val Lys Gln Ala Lys Leu Met Glu Gly Tyr 145 150 155 160 ggc gcc aac tgc atc tac atc acg gac tcg gcc ggc tat atg ctg ccg 528 Gly Ala Asn Cys Ile Tyr Ile Thr Asp Ser Ala Gly Tyr Met Leu Pro                 165 170 175 gac gac gtg aaa gag cgt ctg tcc gcc gtg cgc cag gct ctg aag ccc 576 Asp Asp Val Lys Glu Arg Leu Ser Ala Val Arg Gln Ala Leu Lys Pro             180 185 190 gag acc gaa ctg ggt ttc cat ggc cac cac aac ctg gcc atg ggc atc 624 Glu Thr Glu Leu Gly Phe His Gly His His Asn Leu Ala Met Gly Ile         195 200 205 gcc aac tct ctg gct gca gtg gag gtc ggt gcc aac cgc atc gac gct 672 Ala Asn Ser Leu Ala Ala Val Glu Val Gly Ala Asn Arg Ile Asp Ala     210 215 220 gcg gct gcg ggc ctg ggc gcc ggt gca ggc aac acg cct atg gaa gtg 720 Ala Ala Ala Gly Leu Gly Ala Gly Ala Gly Asn Thr Pro Met Glu Val 225 230 235 240 cta gtg gcc gtg tgc gcg cgc atg ggc att gaa acc ggc gtg gac gta 768 Leu Val Ala Val Cys Ala Arg Met Gly Ile Glu Thr Gly Val Asp Val                 245 250 255 ttc aag atc cag gac gtg gcc gaa gac ctg gtc gtg ccg ctg atg gac 816 Phe Lys Ile Gln Asp Val Ala Glu Asp Leu Val Val Pro Leu Met Asp             260 265 270 ttt ccc atc cgc atc gac cgc gac gcg ctg acc ctg ggc tat gca ggc 864 Phe Pro Ile Arg Ile Asp Arg Asp Ala Leu Thr Leu Gly Tyr Ala Gly         275 280 285 gtg tac ggc agc ttc ctg ctg ttt gcc aag cgt gcc gaa gcc aag tac 912 Val Tyr Gly Ser Phe Leu Leu Phe Ala Lys Arg Ala Glu Ala Lys Tyr     290 295 300 ggc att ccc gcg cgc gag ctg ctg ctg gag ctt ggc cgc cgc ggc atg 960 Gly Ile Pro Ala Arg Glu Leu Leu Leu Glu Leu Gly Arg Arg Gly Met 305 310 315 320 gtg ggc ggc cag gaa gac atg atc gaa gac acg gcg ctg acc atg gtg 1008 Val Gly Gly Gln Glu Asp Met Ile Glu Asp Thr Ala Leu Thr Met Val                 325 330 335 cgc gag cgc cag aag ctg gga cac aag gtg gcc gtg gct gcc tga 1053 Arg Glu Arg Gln Lys Leu Gly His Lys Val Ala Val Ala Ala             340 345 350

[0175] <210> 11 <211> 7484 <212> DNA <213> Comamonas testosteroni <400> 11 gaattcaatc agacaaaggg ttcctggttg ggaatgccca gcacatgcga gccataggcg 60 cgcacataag catccgtgtt gttcgcaatg tgggtacgtc cctgatggat gtcgcggaag 120 atgcgctcga tgggattgtt cttgaaagta ccgctggctg cgcaggcacg catcagctca 180 ttggcatgaa agcccgtcag cttgggaacg tatgacgcct gagcacgctg cagcagacgt 240 tcttccagcg tcagctgaat accagtcttg gccgcatcca caatgcgtgc gtaattgcgg 300 aacagaacca tcttgagctg gtccagcgca atcgtggctt cggtcacggc cgtctgggcg 360 ttcacgtcct cggccatccg gttgccatgc tttccaacgt gcgccgtcgc gcgctcggta 420 aaggatgcga tggcgctttc cagcgcgccc aggcaggcgg tggacaccgc acgctggaac 480 acatgggtaa aggggatacg gaacagccag ctggtgttct ccgtgcgacc ggggcagccc 540 acatcgctgt ggtcgttggt gtactggatg cgatgtgccg ggacaaacgc gttttccacc 600 acgatgtcat gactgcctgt gccgcgcagg cccagcacat cccagttgtc ctcgatgcgg 660 tagtcggact ttggcagcag aaaggtcaca tgctccaggc caggcgtgcc atcacgcttg 720 ggcagcaggc cgccgagaaa cagccagtcg caatgcttgg agccggtgga aaagctccag 780 cggccgttga acctgaagcc gccttccacg ggctcggcct tgccggtggg catataggtc 840 gaggcaatgc gcacgcccgt atccttgccc cagacatctt cctgcgcctg ggcgggaaac 900 agcgccagct gccagggatg aacaccgacc acgccataga tccaggcggt ggacatgcaa 960 cccttggcca gctccatctg cacgctgtag aacacacggg gatccatttc atagccgccc 1020 cagcgcttgg gctgcagcac acggaacaga cctgcctcgg ccatctcctg aacggtttcg 1080 tcgctgatct ttccttcgcg atccgcctgc tgggcgcggg ccgccagctt gggtgccaga 1140 gccctggcac gctcgatcac ggccagcgca tcaggggtga ggtagttatc tgtcattgtg 1200 ttcattcagg tctccagttg ccgctccacc ggacggcttg tggtgtggtg ggcaaggcct 1260 tttcgcctat gacagaaatg ctctcaccat gcaattttcc gttcatcgtc cgatcagact 1320 aacgatgaag caggcgtttc cggacctctc caaagcctga tcccgcacag caaaaacact 1380 gagcctagga caaacaccaa gccctgtttt gcgctgctag tccatgtgaa ggattcataa 1440 aagccctgct cgcagcactc ttgcaccaag gagacattca ccccatgcaa agcactgatg 1500 caaccttcga ccccaaagac tttcgccgcg ccctgggcat gtttggcacc ggggtcacca 1560 tcgtcaccac ccgcgccgag aatggcgagc ctgtaggaat cactgccaac agcttcaact 1620 cggtttcgct ggagcccccc atggttttgt ggagcctggc caagaacgcc cgcagcctgc 1680 ctgtgttcca gagcgccgac acctggaatg tgcacatcct ctccaacgag caagaagccc 1740 tgtcgaaccg ctttgcccgt gctggcgaag acaagttctc cggcctgccg ctggactccg 1800 aggcagccca tgccccgctg ctgcaggact gcagcgcgcg cttcaggtgc aagacggcat 1860 ttcagtacga cggcggcgac cacatcatct ttgtgggcga agtgaccgac tacgacgcca 1920 acccacaccc tcccctgctc tacgtgaccg gcggttacgc actggcttca cgcaaggcca 1980 acgccgtggc cagcgaacct gcagctgaca ccaataccgt ctactcggaa aacctgatcg 2040 gatatttgat gggccgcgcg cactttcaat ttctgagcgg cttgcgcaag cccatggcgg 2100 cttacggcat gaccgatgcc gatttttacg tgctctcgct gctcagcatc cagcaacccc 2160 agtcgcctgc cgaaattgcg tctcacatgg cctacaccgg caccgacatc ggagccgtgg 2220 ccctgcagtc gctgatcagc aagggctggg tggaggaaag ccgcgagcgc gccgagtcgc 2280 tgcagctcac ccccctgggc aacgaagcga tcttgcatgt gctggcagca gccaaagccg 2340 tggaaaccga tttgatagcg agccttggcg agatggaagc agccacgcta cgcaatctgc 2400 tcaaaaaggc gattgccgcc accgaccccg gcctgcccaa gctctggcaa gccagggctt 2460 gacaagcctc gtcatccaag atttctcacc cgaaggcagg agacagcctt cgcctatcca 2520 ctccaagcat aaagaacaga gatgagccaa ttttccatcc cagaaggcaa gtatgtggac 2580 atcggcaatg tggccggtca tcctcagcgc gttcactatc atgagcaagg ctctggcgac 2640 cccgtgatct ttttgcacgg cgccggcacc ggtgccagcg gctacagcaa cttcaagggc 2700 aactaccccg agtttgctgc ggccggcttt cgctccatcg ttcccgacct gctcggctac 2760 ggcctctcgt ccaagaccga ggaacccagg cagtacgaca tggatttctt catcgccggc 2820 gtcaaaggtc tggtggagca actgggcctg aaaaacatca ccctgctggg caactcgctg 2880 ggcggtgccg tggcgctggg ctatgcgctc aagtaccccg aagacgtcaa gagcctgatc 2940 ctcatggcgc ctggcggcgt ggaagagttc gaagcctata tggccatgcc cggcattgcc 3000 aatatgttca atgtctacaa gtcgggcaag accggccccg aggccatgcg cgccgtgatg 3060 agcatgcagg tggtggatca gtcattgctg accgacgaga tcatcaatga acgcgccccg 3120 attgcgctga cccagaccga ggcctcgcgc cagcgcctgt acatccccaa tatgaccgat 3240 cagttgcctg agctcaggtg caatgtgttg ggcttctggg gcatgcagga tgccttcaac 3300 cccgtgggcg gcgccgacaa gctggccaag ggcatcaaga attgccgcgt ggtgctggtc 3360 aaccagtgcg gccactgggt gcaggtcgag catcgcgaga tgttcaaccg cagctgcatc 3420 gacttcatga agaacggctg atgaatcatg agtgacaagc agttcatcga aacgcaaggc 3480 cagcgccttt atgaggccct gcgctcggcc aggacgctgg caccgctgac cgacaaccac 3540 cccgaaatga cggtggaaga cgcttatcac atctccctgc acatgctgcg cctgcgcgaa 3600 gcgtcgggcg aacgtgtgat cggcaagaag atcggcgtga cctccaagcc cgtgcaggac 3660 atgctgaatg tgcaccagcc cgacttcggc tttctgaccg acagcatgga atacgaggac 3720 ggtgcagccg tctcgctcaa ggcagccggt ctgatccagc cccgcgccga aggcgaaatt 3780 gccttcatgc tcaagaagga tctgcaaggc ccgggggtga cccgcgaaga cgtgctggcc 3840 gctaccgaat gggtggcgcc ctgcttcgag atcgtcgatt cgcgcatcaa cgactggaag 3900 atcaagatcc aggacaccgt ggccgacaac gcctcctgcg gcgtctttgt catcggcaag 3960 cagcacacag atccggcttc gctggatctg gccgctgccg ccatgcagat gagcaagaac 4020 ggccagcccg caggctcggg cctgggcagc gccgtgcagg gccaccccgc cgaagccgtg 4080 gcctggctgg ccaacacatt gggcgctttc ggcattcctt tcaaagccgg tgaagtgatt 4140 ctctctggct ctctcgcccc cctggtgccc gccgccgccg gcgaccgttt cgacatggtc 4200 atcgaaggca tgggcacctg ttccattcaa ttcaccgagt aagaaaccat gacgcaaaaa 4260 aagatcaagt gcgcactgat cggccccggc aatatcggca ccgatctgct catgaaactg 4320 cagcgttccc ccattctgga gcctgtgtgg atggtcggta tcgaccctga atccgatggt 4380 ctcaagcgtg cccgcgagat gggcatcaag accacggccg acggtgtgga cggcctgctt 4440 ccctttgtca aggaagatgg catccagatc gcctttgacg ccaccagcgc ctatgtccac 4500 gccgaaaaca gccgcaagct caacgagcta ggcgtgctga tgatcgacct gacgcctgcg 4560 gccatcggcc cctactgcgt gcccagcgtg aatctggccg agaaggttgc agaaaaggcc 4620 atgaacgtga atatggtcac ctgcggcggc caggccacca ttcccatggt cgcggcggtt 4680 tcgcgcgtgc aggccgtgag ctacggagag atcgtggcca cggtatcgag ccgctcggtc 4740 ggccccggaa cgcgcaagaa catcgacgaa ttcacccgca ccacatcggg tgccgtggaa 4800 aagattggcg gcgcacaaaa gggcaaggcc atcatcgtca tcaaccccgc cgagccgccg 4860 ctgatcatgc gcgacaccat ccactgcctg acggtggata cgcccaagcc tgccgagatc 4920 gaagcctcgg tgcacgccat gatcaaggaa gtgcagaaat acgtgcccgg ctacaagctg 4980 gtcaatggcc ccgtgatcga cggcaaccgc gtctccatct atatggaagt cgaaggcctg 5040 ggcgactacc tgcccaagta cgcgggcaat ctggacatca tgaccgccgc tgcggcgcgc 5100 acggccgaga tgtttgccga agaaatcctc gccggccgtt tcgaattggc cgaagccgct 5160 gttgctgtct gacccaggag aagaacatgt ccctcaaagg taagaagatc accgttcacg 5220 acatgacgct gcgtgacggc atgcacccca agcgccacca gatgaccctg gaacagatga 5280 agtccgtggc cacgggactg gatgccgccg gcgtgcccct gattgaagtg acccatggcg 5340 acggcctggg tggcagctcg gtcaactacg gctttcctgc acatacggac gaggagtacc 5400 tgtccaccgt catcccgctg atgaagcagg ccaaggtctc ggccctgttg ctgcccggca 5460 tcggcactgt cgatcacctg aagatggctc acgagctggg cgtctccacc atccgtgtgg 5520 ccacgcattg caccgaggcc gatgtctccg agcagcacat cgccatggcc cgcaagatgg 5580 gcatggatac cgtgggcttt ctcatgatgg cgcatatgta cagtgccgaa ggtctggtca 5640 agcaggccaa gctgatggaa ggctacggcg ccaactgcat ctacatcacg gactcggccg 5700 gctatatgct gccggacgac gtgaaagagc gtctgtccgc cgtgcgccag gctctgaagc 5760 ccgagaccga actgggtttc catggccacc acaacctggc catgggcatc gccaactctc 5820 tggctgcagt ggaggtcggt gccaaccgca tcgacgctgc ggctgcgggc ctgggcgccg 5880 gtgcaggcaa cacgcctatg gaagtgctag tggccgtgtg cgcgcgcatg ggcattgaaa 5940 ccggcgtgga cgtattcaag atccaggacg tggccgaaga cctggtcgtg ccgctgatgg 6000 actttcccat ccgcatcgac cgcgacgcgc tgaccctggg ctatgcaggc gtgtacggca 6060 gcttcctgct gtttgccaag cgtgccgaag ccaagtacgg cattcccgcg cgcgagctgc 6120 tgctggagct tggccgccgc ggcatggtgg gcggccagga agacatgatc gaagacacgg 6240 cgctgaccat ggtgcgcgag cgccagaagc tgggacacaa ggtggccgtg gctgcctgat 6300 cgcctgactt cacattctct tgccactttg tatcgggttc agcggctgct tggggcggcc 6360 gttgaaccct tttttacagc atcgtcccga tgcgtcaggc ctctatccat ccagcccgtc 6420 gcccctggca gcggctctga cccaggccgg tcggcccggg ccgtatggca cagcttttct 6480 agaagcgata gcgcgccacc acctgggccg agcgaggagc acccggcagg ttgagattgg 6540 gcgagctgcc atgaccggcc acgatgtagc gacgatccag cagattgttg agtttgagct 6600 gcacatccca ggggcccatg cggtagtagg ccatggcatc cagcgtggtg taaccgggca 6660 aggtcacggt gttgccggga ttggcgaacc gggcaccgac atagttcagc cccccgccca 6720 ggccgaagcc gtggcccagt gccttggtca cccagaggtt ggcgctttgc ctgggggtca 6780 gcgtggggcg cttgccctgg accggctggc cggcatcgac ggccggcgat gtgctcatct 6840 caccgtcgag gaatgcatag ccggcccaga cctgccagct gggtgcaatc tctccggcaa 6900 aggtcaagct ccagcccgtt ggtgcgctgc acgcccagag gcaccacggt attgcttgtg 6960 ggatcgacgc tcttgatatt ggtgcgctcc agcttgaaca gcgaggccgt gaccgaagcc 7020 ttgccatcgg gcagatccca tttgacgccg acttcctggc tggtggtttt ttccggtgcc 7080 agttgcgcat tgttggctgc cagcgcaaag gtttcacccg aaggttggaa ggacttgctc 7140 cacgatgcat agtaagacca tgccgacgag ggctgataga ccagcccgaa gcgcggactc 7200 caggcggtat ccgtgcggcc cagatccggc ttgcccggct ggcgctcatg cgtttcctgc 7260 tggaagcggt catagcgcac gccggccaag gccttccact gagagttgat gctgaccaga 7320 tcctgcagat acaggctggc caccttgaag acgctgtggt tgtccgccga aggcttgcca 7380 tcgatgcgca gcggcaaagt cggcagcacc ggattgaaca gcgagaccgt cgccacattg 7440 gcctggctgt atgacagcag gtccttgctc tggcgaccga attc 7484

[Brief description of drawings]

FIG. 1 shows putative metabolic pathways for testosterone.

FIG. 2 shows the pCPY insert and metacleavage activity.

FIG. 3 shows the growth of TA441 and TesB ⁻ strains on testosterone.

FIG. 4 shows the pCPY insert.

FIG. 5 shows growth of mutant strains ORF1 ⁻ , ORF2 ⁻ , and ORF3 ⁻ to testosterone.

FIG. 6 shows pCT and pCPY inserts.

FIG. 7 shows CFU after 24 hours when the gene-disrupted strain was cultured in testosterone.

FIG. 8 shows promoter activity upstream of tesB.

FIG. 9 shows the growth of Tn5 mutant strains to testosterone.

FIG. 10 shows the growth of Tn5 mutant strains on p-hydroxybenzoic acid.

FIG. 11 shows transposon variants.

FIG. 12 shows the p12-29 insert.

FIG. 13 shows a comparison of N-terminal sequences of testosterone-inducing enzyme TIP corresponding to TesD, E, and G.

FIG. 14 shows a hydrolase having homology with TesD.

[Fig. 15] Fig. 15 shows A in the phenol metabolism pathway of TA441 strain.
The reaction catalyzed by phE, F, and G is shown.

FIG. 16 shows putative testosterone metabolic pathways.

FIG. 17 shows that mutant ORF11 ⁻ , ORF12 ⁻ , TesD ⁻ , T
3 shows the growth of esE ⁻ to testosterone.

FIG. 18 shows the tesB peripheral region and the p15-21 insert.

FIG. 19 shows the homology search results of the tesB peripheral region and the p15-21 insert, and the β-participation pathway of fatty acids.

FIG. 20 shows growth of mutant strains ORF33 ⁻ and ORF34 ⁻ to testosterone.

FIG. 21 shows TL of culture solution of TA441 strain and TesB ⁻ strain.
C analysis is shown.

FIG. 22 shows gene-disrupted strains TesD ⁻ , TesE ⁻ , ORF1.
1 ^-, ORF12 ^- shows a TLC analysis of culture during 24 hours of culture of.

FIG. 23 shows a TLC analysis of a culture solution at the time of culturing a testosterone-utilizing mutant and a gene-disrupted strain.

FIG. 24 shows putative testosterone metabolic pathways.

FIG. 25 shows the results of measuring the hydrolase activity by absorbance in order to investigate the activity of the TesD gene product.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12P 33/00 C12N 15/00 ZNAA // C12N 9/00 5/00 A (72) Inventor Toshiaki Hayashi Saitama 2-1, Hirosawa, Wako-shi, Japan In the Institute of Physical and Chemical Research (72) Inventor Toshiaki Kudo 2-1, Hirosawa, Wako-shi, Saitama F-term in the Institute of Physical and Chemical Research (reference) 4B024 AA01 BA01 CA03 HA03 4B050 CC02 DD02 LL01 LL10 4B064 AH07 CA19 CC24 DA01 4B065 AB01 BA02 CA44 CA60

Claims (5)

[Claims] 1. A gene-disrupted strain relating to a testosterone-metabolizing enzyme, in which a gene encoding any one of the following amino acid sequences present on the chromosomal DNA of a microbial cell is disrupted. (A) the amino acid sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5; or (b) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. Alternatively, an amino acid sequence having 1 to several amino acids deleted, substituted, added and / or inserted in the amino acid sequence of any one of SEQ ID NO: 5 and having testosterone metabolic activity: 2. A gene-disrupted strain relating to a testosterone-metabolizing enzyme, in which a gene having any of the following nucleotide sequences present on the chromosomal DNA of a microbial cell is disrupted. (A) SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or the nucleotide sequence described in any of SEQ ID NO: 10; or (b) SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 Alternatively, a nucleotide sequence in which one to several nucleotides have been deleted, substituted, added and / or inserted in the nucleotide sequence of any one of SEQ ID NO: 10 and which encodes a protein having testosterone metabolic activity ; 3. A gene encoding any of the following amino acid sequences on the chromosome of a microbial cell; (a) any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. Or (b) deletion, substitution, addition of 1 to several amino acids in the amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5; And / or inserted amino acid sequence having a DNA sequence capable of undergoing homologous recombination with an amino acid sequence having testosterone metabolic activity: and DN for homologous recombination having lost testosterone metabolic activity
The enzyme gene involved in the testosterone metabolic system is disrupted by homologous recombination between A and the gene on the chromosomal DNA of the microbial cell.
The gene-disrupted strain described in 1. 4. A testosterone metabolism intermediate, comprising culturing the gene-disrupted strain according to claim 1 in a medium and collecting a testosterone metabolism intermediate from the culture. Method. 5. A testosterone metabolic intermediate is 4-androstene-3,17-dione, androsta-1,4.
-Diene-3,17-dione, 3-hydroxy-9,1
0-secoandrosta-1,3,5 (10) -triene-9,17-dione, dehydrotestosterone, 3,1
7-dihydroxy-9,10-secoandrosta-1,
The method according to claim 4, which is 3,5 (10) -trien-9-one or 9α-hydroxyandrosta-4-en-3,17-dione.