JP2001309788A - Enzyme capable of splitting organosulfur compound and gene encoding the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a means for e.g. demulsification of wastewater or the like containing surfactant. SOLUTION: This means comprises utilizing an enzyme capable of splitting alkyl sulfates. The other objective gene encoding the above enzyme is provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a protein involved in the decomposition of an organic sulfur compound and a gene encoding the same. By utilizing the protein or gene of the present invention, sulfur in organic sulfur compounds contained in fossil fuels such as petroleum can be released. The so-called sulfur can be easily removed from the fossil fuel. Further, the protein of the present invention is sodium dodecyl sulfate having a surfactant effect (hereinafter, referred to as “SDS”).
Can decompose the alkyl sulfate represented by
It can be used for demulsification of wastewater and the like.

[0002]

2. Description of the Related Art As a method for desulfurization for removing sulfur from a hydrocarbon fuel such as petroleum, there are known methods such as alkali washing and solvent desulfurization. However, at present, hydrodesulfurization is mainly used. I have. Hydrodesulfurization is a method in which a sulfur compound in a petroleum fraction is reacted with hydrogen in the presence of a catalyst, and removed as hydrogen sulfide to reduce the sulfur content of the product. As the catalyst, a metal catalyst such as cobalt, molybdenum, nickel, and tungsten is used with alumina as a carrier.
In the case of a molybdenum-supported alumina catalyst, cobalt or nickel is usually added as a co-catalyst in order to improve the catalytic performance. There is no doubt that hydrodesulfurization using metal catalysts is an extremely complete process that is currently widely used worldwide. However, there are several issues in terms of the process for making petroleum products in compliance with stricter environmental regulations. The following is a brief description of an example.

[0003] In order to desulfurize organic sulfur compounds in fossil fuels, hydrodesulfurization requires a high reaction temperature and pressure, and thus has a high burden on the environment. More recently, the impact of significant energy consumption on the environment has been identified, and measures have been taken to reduce CO2 emissions. Industry has to take some action on this regulation. Further, in the hydrodesulfurization method, since hydrogen is required for the reaction, there is room for improvement in terms of safety, and capital investment and operation costs for enhancing safety are also required.

On the other hand, the enzymatic reaction performed by living organisms proceeds under relatively mild conditions, and the rate of the enzymatic reaction itself is not inferior to the rate of a reaction using a chemical catalyst. Furthermore, it is necessary to properly cope with a wide variety of biological reactions that occur in vivo, so that there are numerous types of enzymes, and these enzymes generally show very high substrate specificity. Have been. Such a feature is expected to be utilized in a so-called biodesulfurization reaction that removes sulfur in sulfur compounds contained in fossil fuels using microorganisms (Monticello, DJ, Hydrocar
bon Processing 39-45 (1994)).

[0005] There have been many reports on the use of bacteria to remove sulfur from heterocyclic sulfur compounds contained in petroleum products. However, they have disclosed ring-cleavage (CC bond cleavage) type reactions and CS bond cleavage type reactions. They are roughly divided into Bacteria having CC bond attack type desulfurization activity are known. These bacteria are
Metabolism of the type that breaks the CC bond in a heterocyclic sulfur compound represented by dibenzothiophene (hereinafter referred to as “DBT”), decomposes the benzene ring, and releases a sulfur salt through the subsequent oxidation cascade. Is what you do. Furthermore, microorganisms that decompose not only crude oil and coal but also model compounds containing sulfur and selectively remove heteroatom sulfur to produce sulfates and hydroxylated compounds have been reported. This type of reaction is considered to be a reaction that specifically cleaves the CS bond in the sulfur compound and releases sulfur in the form of sulfate, in view of the structure of the metabolite.

[0006] In a bacterium known to cause a CS bond-cleaving type desulfurization reaction, a gene encoding an enzyme activity involved in the DBT decomposition reaction has been identified and its nucleotide sequence determined. To the inventors' knowledge, the dsz gene of the Rhodococcus sp. IGTS8 strain (Denome, S., Oldfleld., C., Nash, LJ and Young,
KDJBacteriol., 176: 6707-6716, 1994; Piddingto
n, CS, Kovacevich, BR and Rambosek, J. Appl. E
nviron. Microbiol., 61: 468-475, 1995) and the tds gene of Paenibacillus sp.
No. 87). The degradation reaction of DBT by IGTS8 strain is from DBT to DBT sulfoxide (hereinafter “DBTO”).
DszC catalyzes the conversion to DBT sulfone (hereinafter referred to as “DBTO2”) via DBTO, 2- (2'-hydroxyphenyl) from DBTO2
DszA and 2- catalyze the conversion to benzenesulfinic acid
(2'-Hydroxyphenyl) benzenesulfinic acid to 2-
Catalyzed by three enzymes of DszB that catalyze the conversion to hydroxybiphenyl (hereinafter “2-HBP”) (Deno
me, S., Oldfield., C., Nash, LJ and Young, KD
J. Bacteriol., 176: 6707-6716, 1994; Gray, KA, Pog
rebinshy, OS, Mrachko, GT, Xi, L. Monticello,
DJ and Squires, CH Nat Biotechnol., 14: 1705-17
09, 1996; Oldfield, C., Pogrebinsky, O., Simmonds,
J., Olson, ESand Kulpa, CF, Microbiology, 14
3: 2961-2973, 1997). The corresponding genes are dszA,
They are called dszB and dszC. DszC and DszA are monooxygenases, both of which are known to require coexistence of NADH-FMN oxidoreductase activity for their oxygenation reactions (Gray, KA, Pogrebinsky, OS, Mrachko, G.
T., Xi, L. Monticello, DJ and Squires, CH Nat B
iotechnol., 14: 1705-1709, 1996; Xi, L. Squires, C.
H., Monticello, DJ and Chids, JD Biochem. Biop
hys. Res Commun., 230: 73-76, 1997). The tds gene of Paenibacillus sp. Is benzothiophene (hereinafter `` BT '').
IGTS8) because it has a DBT and BT decomposition activity specific to CS binding under high temperature conditions of 50 ° C or higher.
It is different from the strain's desulfurization enzyme Dsz.

[0007] Petroleum products such as light oil do not contain sulfur compounds that are relatively easy to remove by hydrodesulfurization such as alkylsulfuric acid. However, a future energy-saving process for minimizing the impact on the environment will be considered. In such cases, it may be possible to replace existing hydrodesulfurization processes. At that time, it is necessary to consider the complex use of enzymes that desulfurize various organic sulfur compounds including not only heterocyclic sulfur compounds but also linear organic sulfur compounds.
By applying to multiple oil species or crude oil before fractionation, the energy saving of the bioprocess can be further utilized.

[0008] As enzymes and genes for decomposing linear organic sulfur compounds, enzymes and genes of Escherichia coli (Esherichia coli) that decompose taurine and generate sulfite ions (Van der Ploeg, JR, Weiss, MA, Sal
ler, E., Nashimoto, H., Saito, N., Kertesz, MA,
Leisinger, T (1996) J. Bacteriol. 178 (18), 5438-4
6), Bacillus subtilis that decomposes aliphatic sulfonic acids, Escherichia coli, Pseudomonas aeruginosa,
Pseudomonas putida enzymes and genes (van derPloeg, JR, Iwanicka-Nowicka, R.,
Bykowski, T., Hryniewicz, M. and Leisinger, T. (1999)
J. Biol. Chem. 274, 29358-29365), which decomposes methanesulfonic acid to produce sulfite ions.
Elginosa enzymes and genes (Kertesz, MA, Schmidt-
Larbig, K., Wuest, T. (1999) J. Bacteriol. 181, 14
64-1473), an enzyme / gene that degrades the alkyl sulfate of Pseudomonas putida and generates sulfate ions (Davison,
J., Brunel, F., Phanopoulos A., Prozzi, D., Terpst
ra, P. (1992) Gene 114 (1), 12-24), and other strains with high ability to degrade polycyclic organic sulfur compounds in fossil fuels, such as Rhodococcus sp. No such enzyme or gene has been disclosed. We have shown that transgenic bacteria with Rhodococcus bacteria as the host have a high desulfurization capacity, and to obtain straight-chain organic sulfur compounds derived from the same species or strains of the same genus that can be easily applied to this host. Degrading enzymes and genes were not previously known.

In addition, it is known that a linear organic sulfur compound having a structure such as alkyl sulfate generally has a surfactant effect. Such surfactants are often used in ordinary households as toothpastes and detergents, and in the industry as detergents and emulsifiers, and are sometimes mixed into rivers as wastewater. In a closed system such as a factory or a wastewater treatment plant, chemical demulsification can be performed using an antifoaming agent such as alcohol, but in an open system such as the natural world, such a method is often not suitable. A method of increasing the ability of conventional microorganisms to degrade, called biostimulation, has been used, waiting for the microorganisms to be degraded by native microorganisms. On the other hand, as a method for treating marine pollution and soil pollution due to oil spill, a method called bioremediation, which sprays microorganisms having a high oil decomposing ability, has recently attracted attention. Bacteria that finally degrade not only oil but also a surfactant used for dispersing the oil are considered useful for environmental purification. Rhodococcus bacteria with high ability to degrade heterocyclic sulfur compounds contained in petroleum are also expected to be applied to bioremediation, but they can be easily applied to this host, or originate from strains of the same species or the same genus.
Enzymes and genes that remove surfactants, ie, degrade linear alkyl sulfates, have not been previously known.

[0010]

SUMMARY OF THE INVENTION An object of the present invention is to isolate genes that decompose organic sulfur compounds, particularly linear organic sulfur compounds, to specify the structure (particularly the base sequence), Is to create a novel desulfurized microorganism by introducing it into a microorganism different from the one from which it was isolated and conferring the ability to degrade organic sulfur compounds. Another object of the present invention is to establish a method of releasing sulfur by causing such a microorganism to actually act on an organic sulfur compound.

[0011]

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that the desulfurized bacterium Rhodococcus erythropolis (Rhodococcus erythropoli) has been developed.
s) The genes involved in the decomposition of organic sulfur compounds were successfully isolated from the KA2-5-1 strain, and the present invention was completed. That is, the first aspect of the present invention relates to a gene encoding a protein constituting the ABC transporter. The second of the present invention is
The present invention relates to a gene encoding a protein having the ability to degrade alkyl sulfate. The third aspect of the present invention relates to a protein constituting the ABC transporter. A fourth aspect of the present invention relates to a protein having alkylsulfate degradability.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. (1) Genes The genes of the present invention include the following first to fifth genes. Hereinafter, each gene will be described. First gene The first gene is a protein represented by the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2. And a gene encoding a protein having the same function as the protein represented by the amino acid sequence of SEQ ID NO: 2.

[0013] The protein represented by the amino acid sequence of SEQ ID NO: 2 is presumed to be a substrate binding protein constituting the ABC transporter. Therefore, “having the same function” as described above means having a function as a substrate binding protein constituting an ABC transporter. Whether a protein has a function as a substrate binding protein constituting an ABC transporter is determined by converting a DNA encoding the protein into a gene encoding another ABC transporter protein (that is, a second, a third, or a third protein). Together with the fourth gene) into an appropriate host, and checking whether or not the substance is being transported by the ABC transporter in that host. Alternatively, vectors such as pK18mobsacB that can be widely used from gram-positive bacteria to gram-negative bacteria (Justin ACPowell
& John AcArcher "Molecular chracterisation of a R
hodococcus ohp operon "Antonie van Leeuwenhoek 74: 1
75-188, 1998), the gene can be disrupted by conjugative transfer, and it can be determined by examining whether the substance transport by the ABC transporter has been impaired.

Among the first genes, the gene encoding the protein represented by the amino acid sequence shown in SEQ ID NO: 2 can be obtained by the method described in Examples of the present specification. The nucleotide sequence of this gene is as shown in SEQ ID NO: 1.
As shown in (1), appropriate primers are synthesized based on these sequences and, for example, PCR is performed using DNA prepared from Rhodococcus erythropolis strain KA2-5-1 as a template. Can also be obtained. Rhodococcus erythropolis KA2-5-1 strain was transferred to FERM P-16277
(Deposit date: June 17, 1997).

In the amino acid sequence of SEQ ID NO: 2,
Alternatively, a gene encoding an amino acid sequence in which a plurality of amino acids have been deleted, substituted or added can be obtained by a technique commonly used at the time of filing the present application, for example, site-directed mutagenesis (Zoller et al., Nucleic Acids Res. 6487-6500,
1982) by modifying the gene encoding the protein represented by the amino acid sequence of SEQ ID NO: 2. In addition, such a gene prepares an appropriate probe or primer based on the amino acid sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 1,
Using this, it can be isolated from microorganisms other than Rhodococcus erythropolis strain KA2-5-1. Examples of microorganisms and the like from which such genes are isolated include, for example,
Rhodococcus rhodochrou
s) IGTS8 strain (ATCC 53968), Mycobacterium (Mycobacterium
bacterium) genus G3 strain (FERM P-16105) (JP-A-10-24379)
1), Corynebacterium genus SY-1 strain
(Ohmori, T., Monna, L., Saiki, Y. and Kodama, T. App
l. Environ. Microbiol., 58, 911-915, 1992).

Since the first gene encodes an ABC transporter-constituting protein, by introducing this gene into a host, the ability to take up a new substance into the host,
Alternatively, a discharge capacity can be provided.

Second Gene The second gene is a protein represented by the amino acid sequence of SEQ ID NO: 4, or one or more amino acids in the amino acid sequence of SEQ ID NO: 4 are deleted, substituted or added. And a gene encoding a protein having the same function as the protein represented by the amino acid sequence set forth in SEQ ID NO: 4.

The protein represented by the amino acid sequence of SEQ ID NO: 4 is presumed to be a permease that constitutes the ABC transporter. Therefore, “having the same function” as described above means having a function as a permease constituting the ABC transporter. Whether a protein has a function as a permease that constitutes the ABC transporter is encoded by the protein
DNA is introduced into an appropriate host together with genes encoding other ABC transporter-constituting proteins (ie, first, third, and fourth genes), and the substance is transported by the ABC transporter in the host. It can be determined by examining whether the Alternatively, it can be determined by disrupting the gene and examining whether the transport of the substance by the ABC transporter has been impaired. The second gene can be produced in the same manner as the first gene, and can be used for the same purpose.

Third Gene The third gene is a protein represented by the amino acid sequence of SEQ ID NO: 6, or the amino acid sequence of SEQ ID NO: 6 in which one or more amino acids are deleted, substituted, or added. And a gene encoding a protein having the same function as the protein represented by the amino acid sequence set forth in SEQ ID NO: 6.

The protein represented by the amino acid sequence of SEQ ID NO: 6 is presumed to be a permease that constitutes the ABC transporter. Therefore, “having the same function” as described above means having a function as a permease constituting the ABC transporter. Whether a protein has a function as a permease that constitutes the ABC transporter is encoded by the protein
DNA is introduced into an appropriate host together with genes encoding other ABC transporter-constituting proteins (ie, first, second, and fourth genes), and the substance is transported by the ABC transporter in the host. It can be determined by examining whether the Alternatively, it can be determined by disrupting the gene and examining whether the transport of the substance by the ABC transporter has been impaired. The third gene can be produced in the same manner as the first gene, and can be used for similar uses.

Fourth Gene The fourth gene is a protein represented by the amino acid sequence of SEQ ID NO: 8, or one or more amino acids in the amino acid sequence of SEQ ID NO: 8 are deleted, substituted or added. And a gene encoding a protein having the same function as the protein represented by the amino acid sequence set forth in SEQ ID NO: 8.

The protein represented by the amino acid sequence of SEQ ID NO: 8 is presumed to be an ATP-binding protein constituting an ABC transporter. Therefore, the above “having the same function” means that ATP constituting the ABC transporter
It means that it has a function as a binding protein. ATP is a component of ABC transporter
Whether a protein has a function as a binding protein is determined by introducing a DNA encoding the protein into an appropriate host together with genes encoding other ABC transporter-constituting proteins (ie, first, second, and third genes). In the host
The determination can be made by checking whether or not the substance is transported by the C transporter. Alternatively, it can be determined by disrupting the gene and examining whether the transport of the substance by the ABC transporter has been impaired. The fourth gene can be produced in the same manner as the first gene, and can be used for the same purpose.

Fifth Gene The fifth gene is a protein represented by the amino acid sequence of SEQ ID NO: 10, or the amino acid sequence of SEQ ID NO: 10 in which one or more amino acids are deleted, substituted, or added. And a gene encoding a protein having the same function as the protein represented by the amino acid sequence of SEQ ID NO: 10 and represented by the following amino acid sequence.

The protein represented by the amino acid sequence of SEQ ID NO: 10 can degrade alkyl sulfate. Therefore, “having the same function” as described above means having the function of decomposing alkyl sulfate. Whether a protein has the function of degrading alkyl sulfate can be determined, for example, by introducing a gene encoding the protein into a microorganism and performing the experiments described in Examples 9 and 10 on the microorganism. Can be.

The fifth gene can be produced by a method similar to that for the first gene. By introducing the fifth gene, resistance to SDS can be imparted, and its utility can be imparted to microorganisms that cannot use SDS as a sulfur source. Therefore, this gene
It can be used to impart these properties to microorganisms.

(2) Protein The protein of the present invention has SEQ ID NO: 2, 4, 6, 8 or 10
A protein represented by the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, or 10, wherein the protein is represented by an amino acid sequence in which one or more amino acids are deleted, substituted, or added; Number 2,
Includes proteins having the same function as the protein represented by the amino acid sequence of 4, 6, 8 or 10.

The protein of the present invention can be produced by preparing an appropriate expression vector containing the above-described gene of the present invention, introducing the vector into an appropriate host cell, and culturing the host cell. An expression vector can be prepared by inserting a DNA fragment containing the gene of the present invention into a known vector. The vector into which the DNA fragment is inserted may be determined according to the host to be transformed, and if Escherichia coli is used as the host, the following vectors are preferably used. As strong promoters, for example, lac, lacUV5, trp, tac, trc, λp
PUR, pGEX, pUC, pET including L, T7, rrnB, etc.
System, pT7 system, pBluescript system, pKK system, pBS system, pBC
It is preferable to use a vector such as a pCAL system. Cells used as hosts for the transformants may be plant cells or animal cells, but microorganisms such as Escherichia coli are preferred. Representative strains include those described in Sambrook et al.
ingLaboratory Mannual 2nd ed., 71 /
18, BB4, BHB2668, BHB2690, BL21 (DE3), BNN102
(C600hflA), C-1a, C600 (BNN93), CES200, CES201, CJ
236, CSH18, DH1, DH5, DH5α, DP50supF, ED865
4, ED8767, HB101, HMS174, JM101, JM105, JM107
, JM109, JM110, K802, KK2186, LE392, LG90, M52
19, MBM7014.5, MC1061, MM294, MV1184, MV1193, M
Z-1, NM531, NM538, NM539, Q358, Q359, R594, RB7
91, RR1, SMR10, TAP90, TG1, TG2, XL1-Blue, X
S101, XS127, Y1089, Y1090hsdR, YK537 and the like.

[0028]

The present invention will be described below in more detail with reference to examples. Experiments related to genetic engineering in the examples were mainly
atis et al. (Sambrook, J., Fritsch, E., F. and Mania
tis, T. 1989. Molecular Cloning. A LaboratoryManua
l. 2nd. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY.).

Example 1 Screening of Organosulfur Compound Degrading Enzyme DBT, dimethyl sulfoxide (hereinafter referred to as "DMSO"), 2-aminoethanesulfonic acid (taurine), or methionine, cysteine, sodium sulfate as sole sulfur sources Was added to 200 mL of the basic medium to a final concentration of 0.14 mM, and the desulfurized bacterium Rhodococcus erythropolis strain KA2-5-1 (hereinafter simply referred to as “KA2-5
-1 strain), and cultured until late logarithmic growth. Regardless of which sulfur source was used, the turbidity (measuring wavelength 660 nm) of the culture solution measured with a spectrophotometer was 4 to 6, indicating that the growth was good.

As the basal medium, the composition described in JP-A-11-181446, "Method for producing desulfurizing active microorganism" was used. The culture solution was washed with a phosphate buffer and then suspended in the same buffer to obtain a reaction bacterial cell solution. Add DBT to this to a final concentration of 150ppm, and add 3
After a shaking reaction at 0 ° C. for 2 hours, a DBT degradation test was performed by a resting cell method. The reaction solution was adjusted to acidic conditions by adding hydrochloric acid and extracted with ethyl acetate to prepare a measurement sample, which was then analyzed by gas chromatography to quantify the amount of 2-HBP, a DBT desulfurization product of strain KA2-5-1.

When the sulfur source was methionine, cysteine or sodium sulfate, almost no DBT decomposition activity was observed, but when the sulfur source was DMSO or 2-aminoethanesulfonic acid, this activity was observed. . Furthermore, when the sulfur source was DBT, high activity (about twice as high as when the sulfur source was DMSO or 2-aminoethanesulfonic acid) was observed.

The cells cultured using the above-mentioned various sulfur sources were suspended in 2 ml of 100 mM phosphorus buffer (pH 7.0) per 1 g of wet cells, sonicated, and then centrifuged (14,000 × g, 4 ° C., Two
0 minutes), and the proteins contained in the centrifuged supernatant were analyzed by two-dimensional electrophoresis. The first dimension is pH4? Using a gel having an immobilized pH gradient of pH 7, isoelectric focusing was performed in the presence of urea.
The second dimension is at 90 degrees to the gel after one-dimensional electrophoresis.
SDS electrophoresis was performed with the Laemli system. The proteins in the gel were visualized by silver staining.

Further, spots of desulfurization enzymes in the two-dimensional electrophoresis image were analyzed by an immunoplot method. In immunoblotting, an anti-DszA rabbit polyclonal antibody, an anti-DszB rabbit polyclonal antibody, an anti-DszC rabbit polyclonal antibody prepared using a synthetic peptide prepared based on a sequence expected to have high antigenicity in desulfurase as an antigen, or desulfurization An anti-DszD rabbit polyclonal antibody prepared using DszD purified from the strain KA2-5-1 as an antigen was used.

FIG. 1 shows the results of two-dimensional electrophoresis. In the figure
a, b, c, and d show electrophoretic images when cultured using DBT, DMSO, methionine, and sodium sulfate as sulfur sources, respectively. Also, b-1, b-2, b-3, b-4 are anti-DszA antibodies,
The results of immunoblotting when using anti-DszB antibody, anti-DszC antibody, and anti-DszD antibody are shown.

The spots whose size and density varied depending on the sulfur source were surrounded by a square, and spots having the same mobility by SDS electrophoresis were grouped. Although the two-dimensional electrophoresis image when 2-aminoethanesulfonic acid was used as the sulfur source was not shown in the figure, the tendency of the size and the density of the grouped spots surrounded by squares coincided with the case of DMSO. Although the two-dimensional electrophoresis image when cysteine was used as the sulfur source was not shown in the figure, the size and the density tendency of the spots grouped by boxes were consistent with those of sodium sulfate.
By two-dimensional electrophoresis, eight groups of spots, which were clearly changed by the sulfur source, were found. From the results of immunoblotting, group 1 was converted to desulfurization enzyme DszA, group 2
Desulfurase DszB, Group 3 Desulfurase DszC, Group 6
Was identified as desulfurization enzyme DszD. Group 4 and Group 5
Was observed as a clear spot under culture using DBT, DMSO or 2-aminoethanesulfonic acid as a sulfur source, that is, under conditions where desulfurization activity was exhibited. On the other hand, these spot groups were not observed under conditions where the sulfur source did not have desulfurization activity such as methionine, cysteine, or sodium sulfate, or was extremely low. group
1 to group 3 spot groups, namely DszA, DszB and D
This tendency was exactly the same for szC. Group 6 (identified as DszD) and Group 7 were observed as large and dense spots under culture using DBT as a sulfur source, that is, under conditions with high desulfurization activity. Also, groups 1-5
Contrary to the tendency, these spot groups were observed even under conditions in which the sulfur source had no or very low desulfurization activity such as methionine, cysteine, or sodium sulfate. Group 8 showed a tendency to become rich and large only when cultured using methionine as a sulfur source. It is considered a spot induced by methionine.

Table 1 shows the molecular weight in the presence of SDS, and the isoelectric point in the presence of urea, of spots whose size and density change depending on the sulfur source in the desulfurizing bacterium strain KA2-5-1. D
Group 4, group in addition to szA, DszB, DszC and DszD
The spot groups shown as 5 and group 7 were also presumed to be involved in the metabolism of DBT or other organic sulfur compounds.

[0037]

[Table 1]

Example 2 Partial amino acid sequence of a 38 kD protein presumed to be an organic sulfur compound-degrading enzyme The spot group 5 controlled by a sulfur source is the most expressed amount among the spot groups found in Example 1. Many,
It was considered that there is a high possibility that the enzyme is an organic sulfur compound degrading enzyme. According to the method described in Example 1, two-dimensional electrophoresis of a cell-free extract (0.5 mg) prepared from desulfurized bacterium strain KA2-5-1 cultured with dibenzothiophene as the sole sulfur source was performed. The spots after the two-dimensional electrophoresis were transferred from the gel to the PVDF membrane using Western blotting, the protein components on the membrane were visualized with Coomassie brilliant blue staining solution, and spots corresponding to group 5 were transferred after decolorization. Each membrane was cut out.

Among the three spots of group 5 having different isoelectric points in two-dimensional electrophoresis, a large and dense spot
The two were designated as spot 1 and spot 2 from the neutral side, and their N-terminal sequences were analyzed. Spot 1 NH ₂ -SITELERVTQTPEQIYAAGGIT Spot 2 NH ₂ -SITELERVTQ (amino acids are indicated by single letter symbols.)

Although both sequences were completely identical and had different isoelectric points, they were determined to be spot groups derived from the same protein having the same amino acid sequence. Although the reason why the isoelectric points are different is not clear, it is considered that the protein is modified so as to change the isoelectric point. It is interesting that only proteins involved in the metabolism of sulfur compounds produce multiple spots with different isoelectric points. If the protein is modified, it may play important roles such as regulating enzyme activity.

The cut PVDF membrane was applied to Iwamatsu et al. (“Systematic sample processing method for trace proteins”, Akihiko Iwamatsu, Cell Engineering Vo.
According to l.16 No.2 269-281, 1997), the resulting peptide was treated with lysyl endopeptidase and trypsin after reductive alkylation, the internal peptide was isolated, and the amino acid sequence of each peptide was determined. Internal sequence 1 -QGDAQHVTNY- Internal sequence 2 -GDASEF- Internal sequence 3 -VTLAGAVP- (amino acids are indicated by single letter symbols.)

Example 3 Cloning of a Gene Fragment Encoding a 38 kD Protein Presumed to be an Organosulfur Compound Degrading Enzyme In the desulfurized bacterium KA2-5-1 described in Example 2, it is assumed to be an organic sulfur compound degrading enzyme. Based on the partial amino acid sequence of the 38 kD protein to be prepared, the following PCR primers were prepared. Sense primer 1 5'-CCNGARCARATHTAYGC-3 'Antisense primer 15'-TGYTGNGCRTCNCCYTG-3' Using these sense primers and antisense primers, PCR was performed using DNA extracted from KA2-5-1 strain as a template.
Was done. Preparation of DNA was performed as follows.

In the basal medium described in Example 1 containing DBT,
The cells of the KA2-5-1 strain cultured at 40 ° C for 40 hours were mixed with 11 ml of B1 buffer (50 mM EDTA, 50 mM Tris-HCl, 0.5% Triton X-100, 0.2%
mg / ml RNaseA, pH 8.0). Add 10 to this suspension
300 μl of 0 mg / ml lysozyme solution and 20 mg / ml of Proteinas
500 μl of the eK solution was added and reacted at 37 ° C. for 1 hour.
Add 4 ml of B2 buffer (800 mM GuHCl, 20% Tween-20,
pH 5.5), stirred and mixed, reacted at 55 ° C. for 30 minutes, and stirred with a mixer for 5 seconds to prepare a cell reaction solution. QIAGEN GENOMIC-TIP filled with anion exchange resin
Use a 500 / G (QIAGEN) column with 10 ml of QBT buffer (750 mM
NaCl, 50mM MOPS, 15% ethanol, 0.15% Triton X-100, p
H7.0), and the cell reaction solution was injected into the column. Use 30 ml of QC buffer (1.0 M NaCl, 50 mM MOPS,
After washing with 15% ethanol, pH 7.0), 15 ml of QF buffer (1.25 M NaCl, 50 mM Tris-HCl, 15% ethanol, pH 8.5)
Eluted the genomic DNA solution. 10.5 ml in genomic DNA solution
After precipitating the DNA by adding isopropanol,
It was wound up with a glass rod and collected. 100 μl of recovered DNA
Was dissolved in a TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to prepare a genomic DNA solution.

The conditions of PCR performed using the prepared KA2-5-1 strain DNA as a template are as follows. Reaction solution composition: 50 mM KCl 1.5 mM MgCl2 0.2 mM each dNTP Mixture 1 μM Sense primer 1 μM Antisense primer 200 ng Template DNA 2.5 U Taq DNA polymerase Annealing temperature: Change the temperature from 46 ° C to 60 ° C at 2 ° C intervals PCR was performed. PCR cycle: 94 ° C 2min once 94 ° C 1min ↓ 46-60 ° C 1min Repeat this process 30 times 72 ° C 3min ↑ 72 ° C 7min 1 time DNA amplifier: RobocyclerTMGRADIENT96 Temperature cycler (manufactured by STRATAGENE)

As a result of performing PCR under the above conditions, it was confirmed that when the annealing temperature was 46 to 54 ° C., an amplified fragment of about 500 bp was obtained. This 500 bp PCR product is
It was cloned into E. coli strain JM109 using pCR-Script SK (+) vector. The amino acid sequence deduced from this sequence was identical to the N-terminal amino acid sequence and internal sequence 3 of the 38 kD protein estimated to be an organic sulfur compound-degrading enzyme in the KA2-5-1 strain described in Example 2.

The partial gene sequence of a 38 kD protein estimated to be an organic sulfur compound-degrading enzyme in the strain KA2-5-1 identified in this example, and the desulfurized bacterium KA2-5- described in Example 2
In one strain, 38k estimated to be an organic sulfur compound-degrading enzyme
Based on the partial amino acid sequence of the protein D, PCR primers as shown below were prepared. Sense primer 2 5'- TACGCTGCCGGCGGAAT -3 'Antisense primer 25'- AAYTCNGANGCRTCNCC -3'

As a result of performing PCR by the above method using these sense primer and antisense primer, it was confirmed that when the annealing temperature was 46 to 60 ° C., an amplified fragment of about 900 bp was obtained. This 900 bp PCR product was transformed into E. coli JM109 using pCR-Script SK (+) vector.
Cloned into the strain. The amino acid sequence predicted from this sequence is, in the KA2-5-1 strain described in Example 2, the N-terminal amino acid sequence of the 38 kD protein predicted to be an organic sulfur compound degrading enzyme, internal sequence 1, internal sequence 2, and It matched internal sequence 3.

Example 4 Preparation of KA2-5-1 Strain, Total DNA Library The method for preparing the total DNA was as follows:
It is the same as the preparation method. Library preparation method A total DNA library of the KA2-5-1 strain was prepared as follows. Approximately 2 μg of the total DNA sample of the KA2-5-1 strain was digested with 0.1 unit of Sau3AI for 20 minutes, 30 minutes, and 40 minutes, respectively, and the digest was extracted with phenol-chloroform, collected by ethanol precipitation, and centrifuged. The obtained DNA fragment was treated with 8 units of alkaline phosphatase derived from calf small intestine at 37 ° C.
Dephosphorylation was performed by treating for 60 minutes. After alkaline phosphatase treatment, DNA was extracted by phenol-chloroform treatment and recovered by ethanol precipitation. About 0.2 μg of the obtained DNA fragment was added to λDASHI / BamHI
The reaction was carried out at 4 ° C. for 18 hours in the presence of about 2 μg of the arm and 2 units of T4 DNA ligase. Gigapack III XL
In vitro packaging was performed by reacting with packaging Extract (STRATAGENE) to prepare a phage library. The titer of the phage solution after packaging was 1.5 × 10 ⁶ pfu.

Example 5 Screening of a Gene Fragment Encoding a 38 kD Protein Presumed to be an Organosulfur Compound Degrading Enzyme DN for Screening a Phage Library
The A probe was prepared as follows. The 900 bp PCR product described in Example 3 was labeled with dioxygenin (DIG) by the random prime method (multiprime method) using the gene fragment cloned into the Escherichia coli JM109 strain using the pCR-Script SK (+) vector as a template. The prepared DSZA probe was prepared. The method for preparing DIG-labeled probes is described in Boehringer Man
The nheim protocol was followed. The method for preparing a DIG-labeled probe is described below.

1 μg (5 μl) of the obtained PCR product was heat denatured in boiling water for 10 minutes and cooled on ice containing salt. To the obtained denatured DNA solution, add 10 μl of a hexanucleotide mixture (0.5 M Tris-HCl, 0.1 M MgCl2, 1 mM
oerythriol, 2mg / ml BSA, 3.143mg / ml Random Primer,
pH 7.2), 10 μl dNTP labeling mixture (1 mM dATP, 1 mM dCT
P, 1mM dGTP, 0.65mM dTTP, 0.35mM DIG-dUTP, pH7.5
), 70 μl of sterile distilled water and 5 μl of Klenow enzyme (10 u
nits) and reacted at 37 ° C. for 18 hours. The reaction was stopped by adding 5 μl of a 0.5 M EDTA solution to the reaction solution. Next, 5 μl of 8 M LiCl and 275 μl of cold ethanol (−
20 ° C) and left at -80 ° C for 30 minutes.
Centrifugation was performed at 00 rpm for 30 minutes to precipitate DNA. The precipitated DNA was washed with cold 70% (w / v) ethanol, dried by suction, and dissolved in 50 μl of TE buffer to prepare a DIG-labeled probe.

38 kD estimated to be an organic sulfur compound-degrading enzyme
Screening of the gene fragment encoding the protein of the above, using the DIG-labeled probe prepared by the method described above, Hybond N
+ Performed by plaque hybridization to plaques transferred to the membrane. For detection of hybridizing clones, DIG-ELISA (Boehringer Mannhei
m) was used. When about 2,000 phage plaques were screened from the genomic library using a DIG-labeled probe, four positive plaques were detected. This 4
Single plaques were separated from each plaque, and plaque hybridization was performed again. As a result, four positive plaques were confirmed. Phage clones were prepared using the DIG-labeled probe-positive plaques detected, and phage clones were prepared from those clones using the QIAGEN Lambda kit.
DNA was extracted. The phage DNA prepared using the four positive plaques was cut with XbaI, SacI, NotI, and KpnI.
A restriction map shown in Fig. 2 was created.

Further, these four types of phage DNAs were
When a DNA fragment obtained by digestion with I or XbaI and KpnI was subjected to Southern blot analysis using a DIG-labeled probe, No. 1 and No. 2 clones
It was confirmed to hybridize to the SacI fragment of b or the XbaI-KpnI fragment of about 10 kb. From the results of these restriction enzyme maps and Southern blot analysis, No. 1 and No. 2 clones contained a 38 kD
It was presumed to include the full length of the gene fragment encoding the protein. Also, it was considered that the restriction map of the phage DNA of No. 4 did not match due to recombination.

Example 6 Analysis of a Gene Fragment Encoding a 38 kD Protein Presumed to be an Organosulfur Compound Degrading Enzyme Presumed to be an Organosulfur Compound Degrading Enzyme Derived from KA2-5-1 Strain
To determine the nucleotide sequence of the DNA fragment encoding the 8 kD protein, an approximately 8.5 kb SacI fragment was ligated with pBluescriptII KS (+)
And cloned into E. coli JM109 strain. For this plasmid, use the GPS-1 Genomic Priming System (BioLabs
), A primer sequence for DNA sequencing was randomly inserted, and the full-length nucleotide sequence of the SacI fragment of about 8.5 kb was analyzed. Specifically, an approximately 8.5 kb SacI fragment was
Using 0.1 μg of plasmid DNA cloned into E. coli JM109 strain using escript II KS (+), transposon Tn7
Add 0.02 μg of pGPS1 Donor plasmid DNA having L and TN7R, and having a kanamycin resistance gene and a DNA sequencing primer sequence between both transposons,
Transposons Tn7L and TN by Tns ABC Transpose
The DNA between the 7Rs was inserted randomly and cloned into E. coli JM109. The sequencing reaction of this clone is Th
ermo Sequenase (Amersham), ALFred (Pharm
acia). The obtained nucleotide sequence data was obtained using GENETYX-MAC / ATSQ v3.0 and GENETYX-MAC v
Analysis was performed using 8.0.

As a result of searching for ORF in the determined sequence,
An ORF with a length of 200b or more is placed at the center of the 8.5kb inserted DNA.
Five were found (Figure 2). These ORFs are ORF 1,
Named 2, 3, 4, 5 In addition, the presence of a short ORF of 180b was confirmed at the 3 'end of ORF5. ORF 1, 2, 3, 4, 5 are each
It encodes 563, 319, 284, 566 and 324 amino acids. The translation termination codon TGA of ORF 2 and the translation initiation codon ATG of ORF 3 partially overlap to form the sequence 5'-ATGA-3 ', With the stop codon of the tauB gene involved
It was confirmed that they had the same configuration as the overlap of the start codon of the tauC gene and the overlap of the stop codon of the tauC gene and the start codon of the tauD gene. TauB and TauC encoded by the above-described genes are ABC transporters involved in taurine membrane permeation, and TauD is an α-ketoglutarate-requiring dioxygenase involved in taurine oxidation, and is a group of genes expressed in E. coli under sulfur limitation. All three are coded for the tau operon, which has been shown to be involved in the utilization of taurine. (Eichhorn, E., van der Ploeg, JR,
Kertesz, MA, Leisinger, T. (1997) J. Biol. Che
m. 272 (37), 23031-23036. and Van der Ploeg, J.
R., Iwanicka-Nowicka, R., Kertesz, MA, Leisinge
r, T., Hyniewicz, MM (1997) .J. Bacteriol. 179 (2
4), 7671-7678.)

Further, such overlap is most likely to occur with Rhodococcus erythropolis, which is best studied as a desulfurizing bacterium.
IGTS8 dsz operon (Piddington CS, Kovacevich
BR, Rambosek J. (1995) Applied and Environmenta
l, Microbiol. 61, 468-475.) and the desulfurization gene tds operon of Paenibacillus sp. A11-2 strain isolated by the group of the present inventors (JP-A-11-341987). Both genes are genes for decomposing organic sulfur compounds, and it has been clarified that their expression is controlled by sulfur compounds. Such overlap of gene sequences can be said to be one feature of the organic sulfur compound decomposition gene.

About these ORFs through the Internet, the US National Center for Biotechnology Inform
server (http://www.ncbi.nlm.nih.gov/) and search database Bl against database nr
A homology search was performed using astp. Fifteen of the top 20 known sequences highly homologous to the amino acid sequence encoded by ORF1 were substrate-binding proteins constituting ABC transporters or proteins presumed to be those.
The other four were heme-binding proteins or their precursors, and the other one was a dipeptide-binding protein. The top 20 known sequences with high homology to the amino acid sequence encoded by ORF2 were all permeases constituting ABC transporters or proteins presumed to be the same. The top 20 known sequences highly homologous to the amino acid sequence encoded by ORF3 were all permeases constituting ABC transporters or proteins presumed to be the same. Eighteen of the top 20 known sequences highly homologous to the amino acid sequence encoded by ORF4 were ATP-binding proteins constituting ABC transporters or proteins presumed to be ATP-binding proteins. The other two were spore-forming proteins.

The ABC transporter is a membrane ATPase that transports a substance by using energy obtained by hydrolysis of ATP as a driving force. ATP-binding cassett
e) is characterized in that the size of the molecule or organic ion to be transported is large. Its substrate specificity is extremely wide,
It spans from ions to peptides. The primary structure of the ATP binding site is well conserved across transporter types and species, including Walker motif A (GXXGXGKS / T) and
B (L / I / FL / IXDE / D) without exception (Walker et al., EMBO
J., 1, 945-951 (1982), Higgins et al., Annu. Rev. Cell.
Biol., 8, 67-113 (1992)). ORF4 in the present invention also contains this Walker motif.

The amino acid sequence of ORF1, ORF2, ORF3 and ORF4 is determined by agropin (agrop) which is specifically expressed in plant tumors.
ABC transporters (AgaA,
ABC transporters involved in the metabolism of mangapine (AgaB, AgaC and AgaD), or similarly expressed mannopine
(MoaA, MoaB, MoaC and MoaD). The homology between the amino acid sequences of ORF1 and AgaA or MoaA was 27.2%, respectively. The homology between the amino acid sequences of ORF2 and AgaB was 36.9%, and the homology between the amino acid sequences of ORF2 and MoaB was 34.4%. The amino acid sequence homology between ORF3 and AgaC is 31.9%, and the amino acid sequence homology between ORF3 and MoaC is 3
It was 4.1%. The amino acid sequence homology between ORF4 and AgaD is 4
The homology between the amino acid sequences of ORF4 and MoaD was 4.1% and 44.6%. The above results suggest that a group of proteins composed of ORF1, ORF2, ORF3 and ORF4 are ABC transporters.

Twelve of the thirteen known sequences having homology to the amino acid sequence encoded by ORF5 were dioxygenases classified as α-ketoglutarate-requiring dioxygenase. The other one was a putative dioxygenase sequence. The amino acid sequence encoded by ORF5 is α-ketoglutarate-requiring dioxygenase TauD encoded by a taurine utilization gene identified in Escherichia coli (Van der Ploeg, JR, Weiss, M.
A., Saller, E., Nashimoto, H., Saito, N., Kertesz,
MA, Leisinger, T (1996) J. Bacteriol. 178 (18),
5438-46) and 34.3% homology. Furthermore, in Pseudomonas putida strain S-313, it showed 53.5% homology to α-ketoglutarate-requiring dioxygenase AtsK encoded by the sulfate ester desulfurization gene. AtsK is an alkylsulfatas
supposed to be e, accession number AAD
It is reported in the amino acid sequence information shown in 31784 and the gene sequence information shown in accession number AF126201. Highest homology to ORF5 amino acid sequence 2
The amino acid sequences of all species are those of organic sulfur compound degrading enzymes. This fact strongly suggests that the 38 kD protein estimated to be the organic sulfur compound degrading enzyme described in Examples 1 to 5, ie, ORF5 in this example, is an organic sulfur compound degrading enzyme.

ORF5 is an agropine or mannopine which is specifically expressed in plant tumors.
Although no homology with the metabolic enzyme of α was observed, the ABC transporter sequence was found upstream of the gene sequence encoding α-ketoglutarate-required dioxygenase TauD, and the sulfate ester encoding α-ketoglutarate-required dioxygenase AtsK was also found. ORF1, ORF2, ORF3, ORF
F4 and ORF5 constitute an operon, ORF1, ORF2, ORF
The ABC transporter composed of F3 and ORF4 is ORF5
It was strongly suggested that this is a transporter that transports a substrate or a reaction product.

Example 7 Preparation of a Plasmid Expressing a 38 kD Protein Presumed to be an Organosulfur Compound Degrading Enzyme Rhodococcus rodochrous
Plasmid pRC4 extracted and purified from IFO3338 strain, digested with ClaI and blunted, and vector obtained from Takara Shuzo Co., Ltd.
pHSG298 was digested with StuI and dephosphorylated, and ligated to construct a Rhodococcus-E. coli shuttle vector pRHK1 (FIG. 3).

An approximately 11 kb DNA fragment containing the dibenzothiophene degradation gene dszABC was cloned from the KA2-5-1 strain by plaque hybridization. This was digested with PvuI, the resulting 4.5 kb DNA fragment was blunted, and pBlues
Insert into the SmaI site of criptIIKS (+) and insert the plasmid pBKP
P and pBKPPR were constructed. EcoRI for pBKPP and pBKPPR
Digested with XbaI and EcoRI-X of shuttle vector pRHK1
Ligation was performed with the baI digest to construct plasmids pRKPP and pRKPPR containing the degradation gene (FIG. 4).

A gene encoding a 38 kD protein, which is presumed to be an organic sulfur compound-degrading enzyme, cloned from E. coli JM109 strain from KA2-5-1 strain using pBluescript II KS (+) was digested with the restriction enzyme SacII. By ligation, the genes encoding ORF1, ORF2, ORF3 and a part of the gene encoding ORF4 were removed, digested with SmaI, and pR
Ligation was performed with the DraI-SnaBI digest of KPP to construct a plasmid pRKI5 containing a gene encoding a protein corresponding to ORF5, which is presumed to be an organic sulfur compound-degrading enzyme (Fig.
Five). This plasmid contains a promoter that expresses the DBT-degrading gene dsz, and does not contain a sequence encoding the DBT-degrading enzyme group Dsz, and is a plasmid that arranges a gene corresponding to ORF5 downstream of the promoter. Can be expressed.

Example 8 Introduction of Gene Expressing a Protein Corresponding to ORF5 Rhodococcus erythropolis (Rhodococcus erythropolis) was placed in 100 ml of sterilized LB medium in a 500 ml Erlenmeyer flask.
olis) The KA2-5-1 strain was inoculated and cultured at 30 ° C. for 24 hours. The obtained culture solution was centrifuged at 4 ° C. and 10,000 rpm for 10 minutes, and the precipitate was washed twice with sterilized water and suspended in a 10% glycerol aqueous solution so that the turbidity at the measurement wavelength of 660 nm became 40. .

1 μl of the pRKI5 or pRHK1 gene solution was added to 80 μl of the suspension, and the suspension was treated with Generader II (Biorad) at 25 μF, 400 Ω, 1.5 kV / cm. For processing solution
0.42 ml of SOC medium was added, and the cells were cultured at 30 ° C. for 3 hours. The obtained culture solution was spread on an LB medium (LBAK medium) containing 100 mg / l of kanamycin sulfate and 15 g / l of agar powder, and was applied at 30 ° C and 48 ° C.
Cultured for hours. Bacteria that formed colonies in the LBAK medium were streaked again on fresh LBAK medium to purify the cells to obtain pRKI5 / KA2-5-1 strain or pRHK1 / KA2-5-1 strain.

The pRKI5 / KA2-5-1 strain or the pRHK1 / KA2-5-1 strain was prepared by the method of Denis-Larose et al. (Appl. Environ. Microbiol. 63: 2
915-2919 (1997)), plasmid DNA was extracted from the cells, purified, and the pR
Transformation with KI5 or pRHK1 was confirmed. Also, a cell-free extract was prepared from pRKI5 / KA2-5-1 strain cultured with dibenzothiophene as the sole sulfur source, and S
By performing DS electrophoresis, it was confirmed that the band corresponding to 38 kD had increased.

Example 9 pRKI5 / KA2-5-1 strain and pRHK1
Sodium dodecyl sulfate degradability of strain / KA2-5-1 0.14 mM DBT (N, N-dimethylformamide solution) and 0.1
A medium containing ~ 0.5 mM SDS (Izumi Y et al., Applied and Env
Ironmental Microbiology, 223-226 (1994)) was inoculated with one white ear of pRKI5 / KA2-5-1 strain or pRHK1 / KA2-5-1 strain and cultured at 30 ° C. for 120 hours. 660 nm of the obtained culture solution
Table 2 shows the results of measurement of the absorbance (OD660) of the sample.

[0068]

[Table 2]

The pRHK1 / KA2-5-1 strain does not grow at an SDS concentration of 0.3 mM or more. In contrast, the pRKI5 / KA2-5-1 strain was 0.4 mM
Although it does not grow at the above SDS concentration, it can be seen that it grows at 0.3 mM. The growth at high concentrations is not possible because the surface-active effect of SDS is toxic to bacterial cells, but the pRKI5 / KA2-5-1 strain has the ability to express large amounts of a 38 kD organic sulfur compound degrading enzyme. , The decomposition speed is remarkably fast, and its toxicity can be easily reduced. On the other hand, in non-genetically modified cells, or in cells that have only been modified with a vector that does not contain the gene,
Since the above-mentioned activity is weak, this toxicity, that is, the surface activity of the culture solution cannot be reduced, and the cells cannot grow in the presence of 0.3 mM SDS.

If this enzyme expressed by the pRKI5 / KA2-5-1 strain acts on the alkyl chain, the product will remain with an alkyl sulfate having a short chain length, so that the surface activity does not decrease and the toxicity can be reduced. Without it, the strain should not be able to grow. Since the pRKI5 / KA2-5-1 strain can grow well in the presence of 0.3 mM SDS, this enzyme is not an alkyl chain,
It can be said that it acts directly on the terminal sulfate group. It is clear that a 38 kD protein controlled by sulfur source is involved in the degradation of alkyl sulfate represented by SDS.

Example 10 Use of SDS as Sulfur Source A medium containing 0.2 mM SDS (Izumi Y et al., Applied and Envir.
onmental Microbiology, 223-226 (1994)), inoculated with one white ear of pRKI5 / KA2-5-1 strain or pRHK1 / KA2-5-1 strain, and cultured at 30 ° C. Absorbance of culture solution after 120 hours (660 n
m) is 4.63 for pRKI5 / KA2-5-1 strain, 0.21 for pRHK1 / KA2-5-1 strain
Met. From these results, it is clear that the KA2-5-1 strain can grow using SDS as a single sulfur source. Also,
Since the growth of the transgenic bacterium transformed with pRKI5 is better, it is clear that the 38 kD protein controlled by the sulfur source is involved in the degradation of alkyl sulfate represented by SDS.

[0072]

Industrial Applicability The present invention provides a novel gene encoding a protein constituting the ABC transporter and a novel gene encoding an enzyme that degrades alkyl sulfates. By introducing these genes, new desulfurized microorganisms can be created.

[0073]

[Sequence List] SEQUENCE LISTING <110> PETROLEUM ENERGY CENTER <120> YUHKIIOUKAGOUBUTSU WO BUNKAI SURU KOUSO OYOBI IDENNSHI <130> P00-0203 <160> 10 <170> PatentIn Ver. 2.0 <210> 1 <211> 8188 <212> DNA <213> Rhodococcus erythropolis <220> <221> CDS <222> (1070) .. (2758) <400> 1 gagctcagtc tatgccgtcg gcgccgaaag tcggacatga gtgacagcga tgggtagggc 60 ggggcgagtc gggtaccccg aggaccgaag cgagagtgaa ggaggagaac cgatgcgtgt 120 gaaagcgatg gtcggaacag gcgcgatggc tgttgcgctg gccgccggcc cagttgccac 180 agcttctgct gaaggcgccg agacggcggg gtcggagggt tcaccggaga tgctgacctg 240 cgaggtggtc ttcgatgcac tggaagacat gagcccggaa tgggcggaag aggggatcga 300 tcaggtcgaa gactggtgtg aggcgatcac cggcgaagaa gactgaacgt cagccgcgcg 360 agatcactcg tcgcgacagt cgccaccgcc cgtcttcgag ttcgaactcg tcggcgtagc 420 tcccgacgcc actgagcttg ggagttgtgg tcgtgtcgac gtagaacgtc cacaccgcat 480 ccgcgcgggc tgtagtttcc gacgtcacgt ccacggcgat cgccgtggtg acgtgacgtg 540 tgaacgtgcc cgggccttgt atgccggcgg ctcgacgctc gattactcca gcagcaattt 600 cttcgcggcc acgtcgtacc tgtgcgggga taccgcgatc gggattggcc ggcatgttcc 660 attcggcctc gcgggtgaac tgctcgacgt agtcctcgac agagccggtg tcagccagat 720 gggcgatccg agcgagcgtt gaacgaatct gttcggcaag gctctcgggt tgcatgctgt 780 tctccgtacg tgtgagtgct gtggtggatc gcaggatatt gcccgtggac tgaccgaaca 840 ccgaggcgtc c cagtcagtg gcagcagttg agagaaattc agatcaccgt gagttcaact 900 atttccccga tatcagggga aatggttgag tatcccctga aggcaaaagc cttaattccg 960 accgggaggt ttcacaccgt gtccgtcgct cctcgatgtc ggctctgact tcttcagcgc 1020 cacaacgact gtcgtagcag ctcgcttttc tgaattcccg ggagtgagc atg tcg gaa 1078 Met Ser Glu 1 cga cac tct ttc cca ttc tcg caa gcg tgt gcc ctg aga gtg cac agc 1126 Arg His Ser Phe Pro Phe Ser Gln Ala Cys Ala Leu Arg Val His Ser 5 10 15 tgc gca cct att acg aag ggc aac acc tcc gtg aac tcg tct ctt tcg 1174 Cys Ala Pro Ile Thr Lys Gly Asn Thr Ser Val Asn Ser Ser Leu Ser 20 25 30 35 acg gcc ggt ctc gac cgc cgt gcc ttc ttc cgt ggc agt gcg atc gcc 1222 Thr Ala Gly Leu Asp Arg Arg Ala Phe Phe Arg Gly Ser Ala Ile Ala 40 45 50 gca ttc gcc atc ttc ggt agc tca ctg gcc gcg tgc tcc tct gca 1270 Ala Phe Ala Ile Phe Gly Ser Gly Ser Leu Ala Ala Cys Ser Ser Ala 55 60 65 gtc ggc gaa caa cgc tcg gac gca ggt gaa tca gtt gag ccg gtt cga 1318 Val Gly Glu Ser Asp Ala Gly Glu Ser Val Glu Pro Val Arg 70 75 80 ggt ggg acg ctc aca ctg gcg ata ccg gac gac gtc agc cct gcg gca 1366 Gly Gly Thr Leu Thr Leu Ala Ile Pro Asp Asp Val Ser Pro Ala Ala 85 90 95 ctt ttg acc agc acc acc gtt gcc ggt gtc acg ata tcc ggg gtg 1414 Leu Leu Thr Ser Thr Thr Val Ala Gly Val Thr Ile Ser Gly Leu Val 100 105 110 115 tac gag acc ctg acc cgc tat cca ctc gat tcg gta gtg cca cag cca 1462 Tyr Glu Thr Leu Thr Arg Tyr Pro Leu Asp Ser Val Val Pro Gln Pro 120 125 130 gtt ctg gcc aag gcc tgg aag ctg tcc gac gac ggg ctg acg ctc acg 1510 Val Leu Ala Lys Ala Trp Lys Leu Ser Asp Asp Gly Leu Thr Leu Thr 135 140 145 ctc aat ctg cgc gat gac gtc acc ttc cac tcc ggc cgg cgc ttc acc 1558 Leu Asn Leu Arg Asp Asp Val Thr Phe His Ser Gly Arg Arg Phe Thr 150 155 160 tcc gct gat gcg gag ttc tcg ctg cgc acc tat gcg gac ccg aaa ttc 1606 Ala Glu Phe Ser Leu Arg Thr Tyr Ala Asp Pro Lys Phe 165 170 175 tcg gcc cag ctc agg agt act gcg gcc gcc atc acc ggc ttc gac tct 1654 Ser Ala Gla Gln Leu Arg Ser Thr Ala Ala Ala Ile Thr Gly Phe Asp Ser 180 185 190 195 tcg gat ccc aat acg ctg gtt ctc acc ctc gca cac cga acc ggg aac 1702 Ser Asp Pro Asn Thr Leu Val Leu Thr Leu Ala His Arg Thr Gly Asn 200 205 210 atc ttc gat ctt ctc gat cta gcg ccg atc ttc gac ccc gaa acg ttc 1750 Ile Phe Asp Leu Leu Asp Leu Ala Pro Ile Phe Asp Pro Glu Thr Phe 215 220 225 gac aag gcg gtg acc ggt gaa tcc ttc gtt ggt acg ggc cct ttc gta 1798 Asp Lys Ala Val Ser Phe Val Gly Thr Gly Pro Phe Val 230 235 240 ttc acc tca cgc acc ccg aac agt tcc atc tcc ttc gat cgg aac ccg 1846 Phe Thr Ser Arg Thr Pro Asn Ser Ser Ile Ser Phe Asp Arg Asn Pro 245 250 255 aac tac tgg gtc gcc gag cgg ccg tac ctc gat cac gtg gaa gcg cgg 1894 Asn Tyr Trp Val Ala Glu Arg Pro Tyr Leu Asp His Val Glu Ala Arg 260 265 270 270 275 atc gtt ccg gac gcc caa gcg cgc ctg acatg cag 1942 Ile Val Pro Asp Ala Gln Ala Arg Leu Thr Ser Leu Lys Ser Gly Gln 280 285 290 gtc tct ctt gtt gct ccg gcg tcg ttg ccc ttc cgt gac tca cag aac 1990 Val Ser Leu Val Ala Pro Ala Ser Leu Pro Phe Arg Asp Ser Gln Asn 295 300 305 ttg acc aag acc agc ggg ttc gtg aca acc tcg atc gag ggt gcg gaa 2038 Leu Thr Lys Thr Ser Gly Phe Val Thr Thr Ser Ile Glu Gly Ala Glu 310 315 320 cta cag ata tac ctg ggc aca aat gtc acc gcc gac ggg ctg acc gat 2086 Leu Gln Ile Tyr Leu Gly Thr Asn Val Thr Ala Asp Gly Leu Thr Asp 325 330 335 gtg cgg gca cga aag gcg ttg gcc tac gcg atc gat cgt gac cgc atc 2134 Val Ala Arg Lys Ala Leu Ala Tyr Ala Ile Asp Arg Asp Arg Ile 340 345 350 355 atc agt gag gtg tat cgc gat acg ggg tat gcg gtc aat ctg ccg tgg 2182 Ile Ser Glu Val Tyr Arg Asp Thr Gly Tyr Ala Val Asn Trp 360 365 370 ccg aag tcg tcg ccc gcc tac gac gct gcc aag aac gcg acc tac act 2230 Pro Lys Ser Ser Pro Ala Tyr Asp Ala Ala Lys Asn Ala Thr Tyr Thr 375 380 385 cgc gat gtc gac aag gcg cgc gcc ctg ggc gat ctg ggt gca ctg 2278 Arg Asp Val Asp Lys Ala Arg Ala Leu Val Gly Asp Leu Gly Ala Leu 390 395 400 ccg acc att ccg ttg acc tac aca gga gcg agc ccg gat ttc gaa gcc 2326 Pro Thr Ile Pro hr Tyr Thr Gly Ala Ser Pro Asp Phe Glu Ala 405 410 415 gcc gcg cag atc gtg cag gcc aat ctc gcc gag gca ggg atc cgc gtc 2374 Ala Ala Gln Ile Val Gln Ala Asn Leu Ala Glu Ala Gly Ile Arg Val 420 425 430 435 gaa ttg gat cct gtc gag gca tcc atc ttc gtc aag cag ttg atc ggc 2422 Glu Leu Asp Pro Val Glu Ala Ser Ile Phe Val Lys Gln Leu Ile Gly 440 445 450 gct gag ttc aaa gga ctt tgg ctg acc atat gcg caa tat 2470 Ala Glu Phe Lys Gly Leu Trp Leu Thr Asn His Thr Phe Ala Gln Tyr 455 460 465 gtt ccg tca acc ctg acg gtc agc gcc tac ccg ttc aac gct gca cac 2518 Val Pro Ser Thr Leu Thr Val Ser Ala Tyr Pro Phe Asn Ala Ala His 470 475 480 aat gcc tcg aag ttc agt tcg ccg gcg tat tcc gcc gca gcg gag ggt 2566 Asn Ala Ser Lys Phe Ser Ser Pro Ala Tyr Ser Ala Ala Ala Glu Gly 485 490 490 495 gcg tcc cag gac ggt tcg agc gat gag gca aag aaa ctg tat 2614 Ala Trp Gln Val Pro Asp Gly Ser Ser Asp Glu Ala Lys Lys Leu Tyr 500 505 510 515 gcc gaa ttg ggc acg caa ctg ttg aac gag ttg ttc ttg atc ttg atc atg atc 2 Ala Glu Leu Gly Thr Gln Leu Leu Asn Glu Leu Phe Leu Ile Glu Ile 520 525 530 ggt gtt gtc gtg ccg cag gta tcg gcg gcc gag acc gtt cgt ggt ctg 2710 Gly Val Val Val Pro Gln Val Ser Ala Ala Glu Thr Val Arg Gly Leu 535 540 545 gcc tgg aca aag ggt cgc cag cca caa ttc gcc aac aca ttc ctg gcg 2758 Ala Trp Thr Lys Gly Arg Gln Pro Gln Phe Ala Asn Thrh Phe Leu Ala 550 555 555 taggggtc gtg gg gtcg gtg ggtc gg gtc gg gtc gg gg gg gatattgatc ttctcggtga tgcgtctggt gcctggcgac cccgcgttgg 2878 ctctcgccgg ccccgatgca actccggaag cgattgcagc cattcgtcat tcgctcggtc 2938 tcgacaggtc gattcccgcg cagtacctga cctggatcgg cgacgtactg acattggatc 2998 tcgggcgttc gttcgttctc ggcggccaga tttccgatct ggtgctggcg gggttgggga 3058 acaccgcagt tctggcggga agtgcgttgc tgctggccgt cgtgttgagt ctggtgctca 3118 gtgttgcggt ggtggtgtgg ccgaagaaat ggctgacctc ggtggtgaac cttctcaaca 3178 ctctctcggt tgctctgccc aacttcgtga ccggtgtcct cctggttctc gtcttcgcgg 3238 tgctgattcc ggttctgcca tccggtggtg ttccaccagg tgggtatctc gctcgtccc g 3298 acatcacgtt ccagtacctg ctgctgccgt cactgtgcct cgcacttcct gtcgcggccg 3358 ctctcaccag gttcctgtcc gaagcgctgc gcaccgagat ggcgcagcag tacgtgatca 3418 ccgctcgcgc agccggtgtg ccacggtgga acttggttac acgcagtgca ttacgcaatg 3478 cactgccaac tatgctcacc gtgctcggaa ttcagaccgg gcaccttctc ggcggagccg 3538 tactggttga ggcgatcttt gcctggcctg gcatcggcca attgatcgag cagggaatcg 3598 gtcggcggga ttacccggtg gtgcaggtgc tgctcctgct gtcggtcacg atcttcgttc 3658 tgatccaact tctgaccgac atcgtgcatg cctacctcga cccacgaatc agaatcggag 3718 ggcagcaatg acggtggccg atcagtctgt tgacgaaagg gttcggcgat ctcgtcctgt 3778 gcgcgctgct ctgacgcacg gtcaggggtt ggcgggtgtg ttgatcctgg tggtcatcgc 3838 agtagcggga attgctgctc cactgttgac tcactacggg cccaacgagc agatcgaggg 3898 tgcaaatctg ttgggggcca gtgcacagca ctggttcgga acagatcagg tcaaccgtga 3958 cgtcttcgcc cgttctcttt acggaatccg gatcaacctt gtcatcgtgc tcgtcgcggt 4018 gccagcgggc gcgatcatcg gttcgctcgc aggcctggtg tcgagtatca attcggtggc 4078 agatgtcatc gcacagcgga tcttcgatgt gattcttgcc ttcccgctgt tgattctggc 4138 gatcacgttg gtcgcgatca cggggccggg agtagcgccg gtgatcgcgg tgatcgtggc 4198 tgctgaaata cccttgttcg ggcgcctggt gcgaacgacg gtgttgaagg tgcgcgaact 4258 cccgttcgtg gagtcggccg aggtaatcgg tgcggggcgg tggtgggtac tgcgtaagca 4318 tgtgctgccc aacgccgtag aaccgctggg tgtgcagatt gcgctctcca tgtcggttgc 4378 ggtgttcgcc gaaagtgcga tgagttttct cggaatcggc gtgcgcccgc cggatccgtc 4438 tctcggttcg atcatcgcgg gcgccattcc gaacctcgat gccaatccgg cctacgcgat 4498 cggacctttg gtgatcgtct cggcgttggt cctgagcttc ctgctgatcg cccagggact 4558 gggcaaggcc cgccgaatct gaaattgcca ccgccaccac cacttcaagg acgtagtcat 4618 gaatgcaaca cccgcacagg cagttctcgc catccgggat ctggtgatcg agttcgatgc 4678 cgacggttcc cgtggctcgt cgacaactgt ggtcgaccac gtcaacctcg aattgggttt 4738 cggtgagata ttggcgttgg tcggcgagtc cggatccggt aagtcgttga ccgcacgcgc 4798 tgttctcggg ttgttaccgg acggcgcgca ggcccgcggg ttgatccaac tcggtggcca 4858 gcaggtgctc ggtgccgacg aggcgacgct caatcaattg cgggggactc gggccgcgat 4918 ggttttccag gagcctcaga ctgcactgaa tccagtgcag aaggtgggtt ggcagatcgc 4978 gcag gcgctg cgggcacacg gcaagatctc gcgtgccgac gccagagttc gcgcaatcaa 5038 tcttctacgg atggtggaga ttccggaacc ggagcgtcgg gtcgactggt atccgcatca 5098 actctcgggt ggacagaagc agcgggttgt catcgcgctg gcgttgtcgg gttctcctga 5158 tttactcatc gccgacgaac cgactactgc gctcgacgtc accgtgcagg ccgagattct 5218 gcaactcctg cgtaatctgc gtgatcgttc gggcactgca atactgctca tcacacacaa 5278 catgggtgtc gttgcggaca tcgcagatcg cgtgctggtg atgcgatcgg gcagggtcgt 5338 cgagcagcag tccgttttcg atctctttgc gacgccgcgt gagtcgtaca cacaggcttt 5398 gcttgcggcc gtgccacgtc tgccggaggt cgaccagccg aagcctgtgg ttgctctggt 5458 cgatagcgaa ggcgacgcgc aagtcccgcc gatcctgcgg ttcgacgcgg cctccatcgt 5518 gtacccggcg aggctgggca acagagagtt tcgtgcggtc gatcaggtga gcctgacggt 5578 tcaggccggc gaggttgtcg gactggtcgg tgaatcggga tcagggaaga caactctcgg 5638 ccgggctgcg ctcggtgtta tcagggccag tagcggcatc gttgctttcg acggaaccga 5698 tctcggaaag atctccgccg cgaatcttcg gggcatccgc aagggaatgg ccttgattca 5758 tcaggaccct gcggcatctc tcgatccgcg tcggacggtc ggacaaagcg tcggcgagcc 5818 actcgaagtc catcgctttg ccaccggagc cctgctgcga aaccgggtgg gggagttact 5878 cgaatcagtt cgcctaccga agtcttttgc tcaccgggca ccaggagaac tttccggtgg 5938 tcaacgccag cgtgttgcgc tggcccgagc acttgccctc ggaccgagac tgctggtggc 5998 cgatgagccg accagcgctc tggatgtttc cgtccaagcc gatgttcttt cactctttgc 6058 cgatctcagg gaggaataca acttcgcatg tctgttcatc agccacgatc ttgcagtcgt 6118 caatcaggta gccgaccggg tagcggtact tcgcagcggg gagttggtcg aagagggccc 6178 cgctgcagag gtattcagct cgccacggca ggactacaca caacagttgg ttgccgcggt 6238 gcctgttcca gatccgcgtc gtcagcgcgc cgggcgggaa cgggaatcac tggttcccga 6298 cctgactgtt gccgtttgac ccgtctcgac atcgaacagc attaccacca cctcaacctt 6358 ttgcagactc ataggagctt gacatgtcca tcaccgaact cgaacgcgtc actcagaccc 6418 cggagcagat ctacgctgcc ggcggaatca ctgtccacaa ggtcggtgag ctgatcggcg 6478 cccgcatcga cggcgtacac ctctctgggg atctgtccga agagaccgcc tacgcgatca 6538 actatgcgct ggcagcgcac aaggttgtgt tcttccgtgg gcagcagcat ctcgacgaca 6598 cgtcgcagta cgaattcgcc ggaaccctcg gcagccagac caccacgcat cccactctca 6658 agtccaagga caaca agctg ttggtgctcg acggcgctgc cagcagctgg cacaccgacg 6718 tcactttcat cgatcgcatc ccgaaggcat cgatcctgcg cgcgaccaca attcctgagt 6778 acggcggagc caccacgtgg gcgtctacca cggctgcgta caaccaactg ccgcattcgc 6838 tcaaggtatt ggtcgagaat ctgcgcgctg tccacaccaa tgcatacgac tacgccgaga 6898 ttatcgacaa ggtgaagcaa ggcgacgctc agcgcgtgac gaactactcc gaattcactc 6958 gcgaaatcta cgagacggag catccggtgg ttcgggtcca tcctgcgaca ggggagaaga 7018 ctctgcttct cggtcacttc gtcaaggagt tcgtcggact gaagccgtcg gagtcggttg 7078 ctctctacca gcttctgcag gcacgaatca tcaagttgga aaacaccgtt cggtggtcat 7138 gggctcccgg tgatctggcg atctgggaca accaggcaac ccagcattac gggatttccg 7198 attacgggac acaggcgcgc agcgtgcatc gcgtgacgtt ggccggtgac gttcccgtcg 7258 acgtccacgg tgagcagagc cgcatcatca agggcgatgc atccgagttc tcgatcgttg 7318 cagacatcga ccggcttccc ggctttgccg caaactgatc agtcctgctg cgggtgatcc 7378 ggcgggaaca tctgtcccca tgagtgattg catgctcggc acgtccattc cggggtgtcc 7438 ggtcccacga cgcagcctgt tccggcgagc cacggtaggt gatcgaatgg atggaccggc 7498 atccccgggg aacactgagc ggttctgtgt gcaccgcaga gtgggcacga cggatttttc 7558 gccagcatgt agtcgatgaa gttggtgtgc ccggacgtgg ccatccttcc ggtgcatttg 7618 cggcacagga cggctgggta gtcgtcacca ggttcagcct gcctgaagat ccgtgatgcg 7678 gaatgaaagt gaaggcgatc gctgctgttg aactttcgca acggcatcgg tgagctgcaa 7738 cctgtgcagt tgccccgctc gttcatcaac gttcgggcgt gggcagctgt ggacgtcgga 7798 tccagcgtgt cgatgtcgcc gaagtagtgc gcatagtgtg actggccggc cgtgagaatc 7858 cggtcggcca gtgcgtcgtc gaccggccca ggatccgtgg gaaacgtgat ggaccatcgt 7918 tgttcgagtt ctggaacggt gggcaaaccg tggagttcgt acgggcgttc atcgacttcc 7978 atccgcgacg tgccgtcgaa gggcgcagcc acggtctcga tgcagagcag cgtgcgagga 8038 gtggtgtcga gaacagtgat cagcaggtcg ccgatggcca tctttccgcg catgatccag 8098 ggatcgacac gggcacgggg ttcgagaaac gcgaccacgg catcgagcca cggcgtgccg 8158 agtgcgatga atacgtgctg accggagctc 8188 <210> 2 <211> 563 <212> PRT <213> Rhodococcus erythropolis <400> 2 Met Ser Glu Arg His Ser Phe Pro Phe Ser Gln Ala Cys Ala Leu Arg 1 5 10 15 Val His Ser Cys Ala Pro Ile Thr Lys Gly Asn Thr Ser Val Asn Ser 20 25 30 Ser Leu Ser Thr Ala Gly Leu Asp Arg Arg Ala Phe Phe Arg Gly Ser 35 40 45 Ala Ile Ala Ala Phe Ala Ile Phe Gly Ser Gly Ser Leu Ala Ala Cys 50 55 60 Ser Ser Ala Val Gly Glu Gln Arg Ser Asp Ala Gly Glu Ser Val Glu 65 70 75 80 Pro Val Arg Gly Gly Thr Leu Thr Leu Ala Ile Pro Asp Asp Val Ser 85 90 95 Pro Ala Ala Leu Leu Thr Ser Thr Thr Val Ala Gly Val Thr Ile Ser 100 105 110 Gly Leu Val Tyr Glu Thr Leu Thr Arg Tyr Pro Leu Asp Ser Val Val 115 120 125 Pro Gln Pro Val Leu Ala Lys Ala Trp Lys Leu Ser Asp Asp Gly Leu 130 135 140 Thr Leu Thr Leu Asn Leu Arg Asp Asp Val Thr Phe His Ser Gly Arg 145 150 155 160 Arg Phe Thr Ser Ala Asp Ala Glu Phe Ser Leu Arg Thr Tyr Ala Asp 165 170 175 Pro Lys Phe Ser Ala Gln Leu Arg Ser Thr Ala Ala Ala Ile Thr Gly 180 185 190 Phe Asp Ser Ser Asp Pro Asn Thr Leu Val Leu Thr Leu Ala His Arg 195 200 205 Thr Gly Asn I le Phe Asp Leu Leu Asp Leu Ala Pro Ile Phe Asp Pro 210 215 220 Glu Thr Phe Asp Lys Ala Val Thr Gly Glu Ser Phe Val Gly Thr Gly 225 230 235 240 Pro Phe Val Phe Thr Ser Arg Thr Pro Asn Ser Ser Ile Ser Phe Asp 245 250 255 Arg Asn Pro Asn Tyr Trp Val Val Ala Glu Arg Pro Tyr Leu Asp His Val 260 265 270 Glu Ala Arg Ile Val Pro Asp Ala Gln Ala Arg Leu Thr Ser Leu Lys 275 280 285 Ser Gly Gln Val Ser Leu Val Ala Pro Ala Ser Leu Pro Phe Arg Asp 290 295 300 Ser Gln Asn Leu Thr Lys Thr Ser Gly Phe Val Thr Thr Ser Ile Glu 305 310 315 320 Gly Ala Glu Leu Gln Ile Tyr Leu Gly Thr Asn Val Thr Ala Asp Gly 325 330 335 Leu Thr Asp Val Arg Ala Arg Lys Ala Leu Ala Tyr Ala Ile Asp Arg 340 345 350 Asp Arg Ile Ile Ser Glu Val Tyr Arg Asp Thr Gly Tyr Ala Val Asn 355 360 365 Leu Pro Trp Pro Lys Ser Ser Pro Ala Tyr Asp Ala Ala Lys Asn Ala 370 375 380 Thr Tyr Thr Arg Asp Val Asp Lys Ala Arg Ala Leu Val Gly Asp Leu 385 390 395 400 Gly Ala Leu Pro Thr Ile Pro Leu Thr Tyr Thr Gly Ala Ser Pro Asp 405 410 415 Phe Glu Ala A la Ala Gln Ile Val Gln Ala Asn Leu Ala Glu Ala Gly 420 425 430 Ile Arg Val Glu Leu Asp Pro Val Glu Ala Ser Ile Phe Val Lys Gln 435 440 445 Leu Ile Gly Ala Glu Phe Lys Gly Leu Trp Leu Thr Asn His Thr Phe 450 455 460 Ala Gln Tyr Val Pro Ser Thr Leu Thr Val Ser Ala Tyr Pro Phe Asn 465 470 475 480 Ala Ala His Asn Ala Ser Lys Phe Ser Ser Pro Ala Tyr Ser Ala Ala 485 490 495 Ala Glu Gly Ala Trp Gln Val Pro Asp Gly Ser Ser Asp Glu Ala Lys 500 505 510 510 Lys Leu Tyr Ala Glu Leu Gly Thr Gln Leu Leu Asn Glu Leu Phe Leu 515 520 525 Ile Glu Ile Gly Val Val Val Pro Gln Val Ser Ala Ala Glu Thr Val 530 535 540 Arg Gly Leu Ala Trp Thr Lys Gly Arg Gln Pro Gln Phe Ala Asn Thr 545 550 555 560 Phe Leu Ala <210> 3 <211> 8188 <212> DNA <213> Rhodococcus erythropolis <220> <221> CDS <222> (2770) .. (3726) <400> 3 gagctcagtc tatgccgtcg gcgccgaaag tcggacatga gtgacagcga tgggtagggc 60 ggggcgagtc gggtaccccg aggaccgaag cgagagtgaa ggaggagaac cgatgcgtgt 120 gaaagcgatg gtcggaacag gcgcgatggc tgttgcgctg gccgccggcc cagttgccac 180 agcttctgct gaaggcgccg agacggcggg gtcggagggt tcaccggaga tgctgacctg 240 cgaggtggtc ttcgatgcac tggaagacat gagcccggaa tgggcggaag aggggatcga 300 tcaggtcgaa gactggtgtg aggcgatcac cggcgaagaa gactgaacgt cagccgcgcg 360 agatcactcg tcgcgacagt cgccaccgcc cgtcttcgag ttcgaactcg tcggcgtagc 420 tcccgacgcc actgagcttg ggagttgtgg tcgtgtcgac gtagaacgtc cacaccgcat 480 ccgcgcgggc tgtagtttcc gacgtcacgt ccacggcgat cgccgtggtg acgtgacgtg 540 tgaacgtgcc cgggccttgt atgccggcgg ctcgacgctc gattactcca gcagcaattt 600 cttcgcggcc acgtcgtacc tgtgcgggga taccgcgatc gggattggcc ggcatgttcc 660 attcggcctc gcgggtgaac tgctcgacgt agtcctcgac agagccggtg tcagccagat 720 gggcgatccg agcgagcgtt gaacgaatct gttcggcaag gctctcgggt tgcatgctgt 780 tctccgtacg tgtgagtgct gtggtggatc gcaggatatt gcccgtggac tgaccgaaca 840 ccgaggcgtc cc agtcagtg gcagcagttg agagaaattc agatcaccgt gagttcaact 900 atttccccga tatcagggga aatggttgag tatcccctga aggcaaaagc cttaattccg 960 accgggaggt ttcacaccgt gtccgtcgct cctcgatgtc ggctctgact tcttcagcgc 1020 cacaacgact gtcgtagcag ctcgcttttc tgaattcccg ggagtgagca tgtcggaacg 1080 acactctttc ccattctcgc aagcgtgtgc cctgagagtg cacagctgcg cacctattac 1140 gaagggcaac acctccgtga actcgtctct ttcgacggcc ggtctcgacc gccgtgcctt 1200 cttccgtggc agtgcgatcg ccgcattcgc catcttcggt agcggctcac tggccgcgtg 1260 ctcctctgca gtcggcgaac aacgctcgga cgcaggtgaa tcagttgagc cggttcgagg 1320 tgggacgctc acactggcga taccggacga cgtcagccct gcggcacttt tgaccagcac 1380 caccgttgcc ggtgtcacga tatccgggct cgtgtacgag accctgaccc gctatccact 1440 cgattcggta gtgccacagc cagttctggc caaggcctgg aagctgtccg acgacgggct 1500 gacgctcacg ctcaatctgc gcgatgacgt caccttccac tccggccggc gcttcacctc 1560 cgctgatgcg gagttctcgc tgcgcaccta tgcggacccg aaattctcgg cccagctcag 1620 gagtactgcg gccgccatca ccggcttcga ctcttcggat cccaatacgc tggttctcac 1680 cctcgcacac cgaaccggga acatcttcga tcttctcgat ctagcgccga tcttcgaccc 1740 cgaaacgttc gacaaggcgg tgaccggtga atccttcgtt ggtacgggcc ctttcgtatt 1800 cacctcacgc accccgaaca gttccatctc cttcgatcgg aacccgaact actgggtcgc 1860 cgagcggccg tacctcgatc acgtggaagc gcggatcgtt ccggacgccc aagcgcgcct 1920 gacatcgctg aagtccggac aggtctctct tgttgctccg gcgtcgttgc ccttccgtga 1980 ctcacagaac ttgaccaaga ccagcgggtt cgtgacaacc tcgatcgagg gtgcggaact 2040 acagatatac ctgggcacaa atgtcaccgc cgacgggctg accgatgtgc gggcacgaaa 2100 ggcgttggcc tacgcgatcg atcgtgaccg catcatcagt gaggtgtatc gcgatacggg 2160 gtatgcggtc aatctgccgt ggccgaagtc gtcgcccgcc tacgacgctg ccaagaacgc 2220 gacctacact cgcgatgtcg acaaggcgcg cgccctggtc ggcgatctgg gtgcactgcc 2280 gaccattccg ttgacctaca caggagcgag cccggatttc gaagccgccg cgcagatcgt 2340 gcaggccaat ctcgccgagg cagggatccg cgtcgaattg gatcctgtcg aggcatccat 2400 cttcgtcaag cagttgatcg gcgctgagtt caaaggactt tggctgacca atcacacttt 2460 tgcgcaatat gttccgtcaa ccctgacggt cagcgcctac ccgttcaacg ctgcacacaa 2520 tgcctcgaag ttcagttcgc cggc gtattc cgccgcagcg gagggtgcgt ggcaggttcc 2580 cgacggttcg agcgatgagg caaagaaact gtatgccgaa ttgggcacgc aactgttgaa 2640 cgagttgttc ttgatcgaga tcggtgttgt cgtgccgcag gtatcggcgg ccgagaccgt 2700 tcgtggtctg gcctggacaa agggtcgcca gccacaattc gccaacacat tcctggcgta 2760 ggggattcg atg att cgt tac att gcg ggg cga cta ccg tcg gcg gtg gtg 2811 Met Ile Arg Tyr Ile Ala Gly Arg Leu Pro Ser Ala Val Val 1 5 10 gtt ctc ttc ctc gcg tcg ata ttg atc ttc tcg gtg atg cgt ctg gtg 2859 Val Leu Phe Leu Ala Ser Ile Leu Ile Phe Ser Val Met Arg Leu Val 15 20 25 30 cct ggc gac ccc gcg ttg gct ctc gcc ggc ccc gat gca act ccg gaa 2907 Pro Gly Asp Pro Ala Leu Ala Leu Ala Gly Pro Asp Ala Thr Pro Glu 35 40 45 gcg att gca gcc att cgt cat tcg ctc ggt ctc gac agg tcg attccc Ile Ala Ala Ile Arg His Ser Leu Gly Leu Asp Arg Ser Ile Pro 50 55 60 gcg cag tac ctg acc tgg atc ggc gac gta ctg aca ttg gat ctc ggg 3003 Ala Gln Tyr Leu Thr Trp Ile Gly Asp Val Leu Thr Leu Asp Leu Gly 65 70 75 cgt tcg ttc gtt ctc ggc ggc cag att t cc gat ctg gtg ctg gcg ggg 3051 Arg Ser Phe Val Leu Gly Gly Gln Ile Ser Asp Leu Val Leu Ala Gly 80 85 90 ttg ggg aac acc gca gtt ctg gcg gga agt gcg ttg ctg ctg gcc gtc 3099 Leu Val Asn Leu Ala Gly Ser Ala Leu Leu Leu Ala Val 95 100 105 110 gtg ttg agt ctg gtg ctc agt gtt gcg gtg gtg gtg tgg ccg aag aaa 3147 Val Leu Ser Leu Val Leu Ser Val Ala Val Val Val Trp Pro Lys Lys 115 120 125 tgg ctg acc tcg gtg gtg aac ctt ctc aac act ctc tcg gtt gct ctg 3195 Trp Leu Thr Ser Val Val Asn Leu Leu Asn Thr Leu Ser Val Ala Leu 130 135 140 ccc aac ttc gtg acc ggt gtc ctc ctg gtt ctc gc gtt ctg 3243 Pro Asn Phe Val Thr Gly Val Leu Leu Val Leu Val Phe Ala Val Leu 145 150 155 att ccg gtt ctg cca tcc ggt ggt gtt cca cca ggt ggg tat ctc gct 3291 Ile Pro Val Leu Pro Ser Gly Gly Val Pro Pro Gly Gly Tyr Leu Ala 160 165 170 cgt ccc gac atc acg ttc cag tac ctg ctg ctg ccg tca ctg tgc ctc 3339 Arg Pro Asp Ile Thr Phe Gln Tyr Leu Leu Leu Pro Ser Leu Cys Leu 175 180 185 190 gca ctt cct g gcc gct ctc acc agg ttc ctg tcc gaa gcg ctg 3387 Ala Leu Pro Val Ala Ala Ala Leu Thr Arg Phe Leu Ser Glu Ala Leu 195 200 205 cgc acc gag atg gcg cag cag tac gtg atc acc gct cgc gca gcc ggt 3 Thr Glu Met Ala Gln Gln Tyr Val Ile Thr Ala Arg Ala Ala Gly 210 215 220 gtg cca cgg tgg aac ttg gtt aca cgc agt gca tta cgc aat gca ctg 3483 Val Pro Arg Trp Asn Leu Val Thr Arg Ser Ala Leu Arg Asn Ala Leu 225 230 235 cca act atg ctc acc gtg ctc gga att cag acc ggg cac ctt ctc ggc 3531 Pro Thr Met Leu Thr Val Leu Gly Ile Gln Thr Gly His Leu Leu Gly 240 245 250 gga gcc gta ctg gtt gag gcg atc ttt gcc tgg cct ggc atc ggc caa 3579 Gly Ala Val Leu Val Glu Ala Ile Phe Ala Trp Pro Gly Ile Gly Gln 255 260 265 270 ttg atc gag cag gga atc ggt cgg cgg gat tac ccg gtg gtg cag gtg 3627 Leu Ile Glu Gly Arg Arg Asp Tyr Pro Val Val Gln Val 275 280 285 ctg ctc ctg ctg tcg gtc acg atc ttc gtt ctg atc caa ctt ctg acc 3675 Leu Leu Leu Leu Ser Val Thr Ile Phe Val Leu Ile Gln Leu Leu Thr 290 295 30 0 gac atc gtg cat gcc tac ctc gac cca cga atc aga atc gga ggg cag 3723 Asp Ile Val His Ala Tyr Leu Asp Pro Arg Ile Arg Ile Gly Gly Gly Gln 305 310 315 caa tgacggtggc cgatcagtct gttgcgc gcgttcgc gcgttcgc gagttcgc gagttcgc gagttcgc gagttcgc gagttcgg ggtggtcatc 3836 gcagtagcgg gaattgctgc tccactgttg actcactacg ggcccaacga gcagatcgag 3896 ggtgcaaatc tgttgggggc cagtgcacag cactggttcg gaacagatca ggtcaaccgt 3956 gacgtcttcg cccgttctct ttacggaatc cggatcaacc ttgtcatcgt gctcgtcgcg 4016 gtgccagcgg gcgcgatcat cggttcgctc gcaggcctgg tgtcgagtat caattcggtg 4076 gcagatgtca tcgcacagcg gatcttcgat gtgattcttg ccttcccgct gttgattctg 4136 gcgatcacgt tggtcgcgat cacggggccg ggagtagcgc cggtgatcgc ggtgatcgtg 4196 gctgctgaaa tacccttgtt cgggcgcctg gtgcgaacga cggtgttgaa ggtgcgcgaa 4256 ctcccgttcg tggagtcggc cgaggtaatc ggtgcggggc ggtggtgggt actgcgtaag 4316 catgtgctgc ccaacgccgt agaaccgctg ggtgtgcaga ttgcgctctc catgtcggtt 4376 gcggtgttcg ccggagcc ccggagtcg ccggagcc ccggagtc tcggtt cgatcatcgc gggcgccatt ccgaacctcg atgccaatcc ggcctacgcg 4496 atcggacctt tggtgatcgt ctcggcgttg gtcctgagct tcctgctgat cgcccaggga 4556 ctgggcaagg cccgccgaat ctgaaattgc caccgccacc accacttcaa ggacgtagtc 4616 atgaatgcaa cacccgcaca ggcagttctc gccatccggg atctggtgat cgagttcgat 4676 gccgacggtt cccgtggctc gtcgacaact gtggtcgacc acgtcaacct cgaattgggt 4736 ttcggtgaga tattggcgtt ggtcggcgag tccggatccg gtaagtcgtt gaccgcacgc 4796 gctgttctcg ggttgttacc ggacggcgcg caggcccgcg ggttgatcca actcggtggc 4856 cagcaggtgc tcggtgccga cgaggcgacg ctcaatcaat tgcgggggac tcgggccgcg 4916 atggttttcc aggagcctca gactgcactg aatccagtgc agaaggtggg ttggcagatc 4976 gcgcaggcgc tgcgggcaca cggcaagatc tcgcgtgccg acgccagagt tcgcgcaatc 5036 aatcttctac ggatggtgga gattccggaa ccggagcgtc gggtcgactg gtatccgcat 5096 caactctcgg gtggacagaa gcagcgggtt gtcatcgcgc tggcgttgtc gggttctcct 5156 gatttactca tcgccgacga accgactact gcgctcgacg tcaccgtgca ggccgagatt 5216 ctgcaactcc tgcgtaatct gcgtgatcgt tcgggcactg caatactgct catcacacac 5276 aacatgggt g tcgttgcgga catcgcagat cgcgtgctgg tgatgcgatc gggcagggtc 5336 gtcgagcagc agtccgtttt cgatctcttt gcgacgccgc gtgagtcgta cacacaggct 5396 ttgcttgcgg ccgtgccacg tctgccggag gtcgaccagc cgaagcctgt ggttgctctg 5456 gtcgatagcg aaggcgacgc gcaagtcccg ccgatcctgc ggttcgacgc ggcctccatc 5516 gtgtacccgg cgaggctggg caacagagag tttcgtgcgg tcgatcaggt gagcctgacg 5576 gttcaggccg gcgaggttgt cggactggtc ggtgaatcgg gatcagggaa gacaactctc 5636 ggccgggctg cgctcggtgt tatcagggcc agtagcggca tcgttgcttt cgacggaacc 5696 gatctcggaa agatctccgc cgcgaatctt cggggcatcc gcaagggaat ggccttgatt 5756 catcaggacc ctgcggcatc tctcgatccg cgtcggacgg tcggacaaag cgtcggcgag 5816 ccactcgaag tccatcgctt tgccaccgga gccctgctgc gaaaccgggt gggggagtta 5876 ctcgaatcag ttcgcctacc gaagtctttt gctcaccggg caccaggaga actttccggt 5936 ggtcaacgcc agcgtgttgc gctggcccga gcacttgccc tcggaccgag actgctggtg 5996 gccgatgagc cgaccagcgc tctggatgtt tccgtccaag ccgatgttct ttcactcttt 6056 gccgatctca gggaggaata caacttcgca tgtctgttca tcagccacga tcttgcagtc 6116 gtcaatcagg tagc cgaccg ggtagcggta cttcgcagcg gggagttggt cgaagagggc 6176 cccgctgcag aggtattcag ctcgccacgg caggactaca cacaacagtt ggttgccgcg 6236 gtgcctgttc cagatccgcg tcgtcagcgc gccgggcggg aacgggaatc actggttccc 6296 gacctgactg ttgccgtttg acccgtctcg acatcgaaca gcattaccac cacctcaacc 6356 ttttgcagac tcataggagc ttgacatgtc catcaccgaa ctcgaacgcg tcactcagac 6416 cccggagcag atctacgctg ccggcggaat cactgtccac aaggtcggtg agctgatcgg 6476 cgcccgcatc gacggcgtac acctctctgg ggatctgtcc gaagagaccg cctacgcgat 6536 caactatgcg ctggcagcgc acaaggttgt gttcttccgt gggcagcagc atctcgacga 6596 cacgtcgcag tacgaattcg ccggaaccct cggcagccag accaccacgc atcccactct 6656 caagtccaag gacaacaagc tgttggtgct cgacggcgct gccagcagct ggcacaccga 6716 cgtcactttc atcgatcgca tcccgaaggc atcgatcctg cgcgcgacca caattcctga 6776 gtacggcgga gccaccacgt gggcgtctac cacggctgcg tacaaccaac tgccgcattc 6836 gctcaaggta ttggtcgaga atctgcgcgc tgtccacacc aatgcatacg actacgccga 6896 gattatcgac aaggtgaagc aaggcgacgc tcagcgcgtg acgaactact ccgaattcac 6956 tcgcgaaatc tacgagacgg agcatccggt ggttcgggtc catcctgcga caggggagaa 7016 gactctgctt ctcggtcact tcgtcaagga gttcgtcgga ctgaagccgt cggagtcggt 7076 tgctctctac cagcttctgc aggcacgaat catcaagttg gaaaacaccg ttcggtggtc 7136 atgggctccc ggtgatctgg cgatctggga caaccaggca acccagcatt acgggatttc 7196 cgattacggg acacaggcgc gcagcgtgca tcgcgtgacg ttggccggtg acgttcccgt 7256 cgacgtccac ggtgagcaga gccgcatcat caagggcgat gcatccgagt tctcgatcgt 7316 tgcagacatc gaccggcttc ccggctttgc cgcaaactga tcagtcctgc tgcgggtgat 7376 ccggcgggaa catctgtccc catgagtgat tgcatgctcg gcacgtccat tccggggtgt 7436 ccggtcccac gacgcagcct gttccggcga gccacggtag gtgatcgaat ggatggaccg 7496 gcatccccgg ggaacactga gcggttctgt gtgcaccgca gagtgggcac gacggatttt 7556 tcgccagcat gtagtcgatg aagttggtgt gcccggacgt ggccatcctt ccggtgcatt 7616 tgcggcacag gacggctggg tagtcgtcac caggttcagc ctgcctgaag atccgtgatg 7676 cggaatgaaa gtgaaggcga tcgctgctgt tgaactttcg caacggcatc ggtgagctgc 7736 aacctgtgca gttgccccgc tcgttcatca acgttcgggc gtgggcagct gtggacgtcg 7796 gatccagcgt gtcgatgtcg ccgaa gtagt gcgcatagtg tgactggccg gccgtgagaa 7856 tccggtcggc cagtgcgtcg tcgaccggcc caggatccgt gggaaacgtg atggaccatc 7916 gttgttcgag ttctggaacg gtgggcaaac cgtggagttc gtacgggcgt tcatcgactt 7976 ccatccgcga cgtgccgtcg aagggcgcag ccacggtctc gatgcagagc agcgtgcgag 8036 gagtggtgtc gagaacagtg atcagcaggt cgccgatggc catctttccg cgcatgatcc 8096 agggatcgac acgggcacgg ggttcgagaa acgcgaccac ggcatcgagc cacggcgtgc 8156 cgagtgcgat gaatacgtgc tgaccggagc tc 8188 <210> 4 <211> 319 <212> PRT <213> Rhodococcus erythropolis <400> 4 Met Ile Arg Tyr Ile Ala Gly Arg Leu Pro Ser Ala Val Val Val Leu 1 5 10 15 Phe Leu Ala Ser Ile Leu Ile Phe Ser Val Met Arg Leu Val Pro Gly 20 25 30 Asp Pro Ala Leu Ala Leu Ala Gly Pro Asp Ala Thr Pro Glu Ala Ile 35 40 45 Ala Ala Ile Arg His Ser Leu Gly Leu Asp Arg Ser Ile Pro Ala Gln 50 55 60 Tyr Leu Thr Trp Ile Gly Asp Val Leu Thr Leu Asp Leu Gly Arg Ser 65 70 75 80 Phe Val Leu Gly Gly Gln Ile Ser Asp Leu Val Leu Ala Gly Leu Gly 85 90 95 Asn Thr Ala Val Leu Ala Gly Ser Ala Leu Leu Leu Ala Val Val Leu 100 105 110 Ser Leu Val Leu Ser Val Ala Val Val Val Trp Pro Lys Lys Trp Leu 115 120 125 Thr Ser Val Val Asn Leu Leu Asn Thr Leu Ser Val Ala Leu Pro Asn 130 135 140 Phe Val Thr Gly Val Leu Leu Val Leu Val Phe Ala Val Leu Ile Pro 145 150 155 160 Val Leu Pro Ser Gly Gly Val Pro Pro Gly Gly Tyr Leu Ala Arg Pro 165 170 175 Asp Ile Thr Phe Gln Tyr Leu Leu Leu Pro Ser Leu Cys Leu Ala Leu 180 185 190 Pro Val Ala Ala Ala Leu Thr Arg Phe Leu Ser Glu Ala Leu Arg Thr 195 200 205 Glu Met Ala Gl n Gln Tyr Val Ile Thr Ala Arg Ala Ala Gly Val Pro 210 215 220 Arg Trp Asn Leu Val Thr Arg Ser Ala Leu Arg Asn Ala Leu Pro Thr 225 230 235 240 Met Leu Thr Val Leu Gly Ile Gln Thr Gly His Leu Leu Gly Gly Ala 245 250 255 Val Leu Val Glu Ala Ile Phe Ala Trp Pro Gly Ile Gly Gln Leu Ile 260 265 270 Glu Gln Gly Ile Gly Arg Arg Asp Tyr Pro Val Val Gln Val Leu Leu 275 280 285 285 Leu Leu Ser Val Thr Ile Phe Val Leu Ile Gln Leu Leu Thr Asp Ile 290 295 300 Val His Ala Tyr Leu Asp Pro Arg Ile Arg Ile Gly Gly Gln Gln 305 310 315 <210> 5 <211> 8188 <212> DNA <213> Rhodococcus erythropolis <220> <221> CDS <222> (3726) .. (4577) <400> 5 gagctcagtc tatgccgtcg gcgccgaaag tcggacatga gtgacagcga tgggtagggc 60 ggggcgagtc gggtaccccg aggaccgaag cgagagtgaa ggaggagaac cgatgcgtgt 120 gaaagcgatg gtcggaacag gcgcgatggc tgttgcgctg gccgccggcc cagttgccac 180 agcttctgct gaaggcgccg agacggcggg gtcggagggt tcaccggaga tgctgacctg 240 cgaggtggtc ttcgatgcac tggaagacat gagcccggaa tgggcggaag aggggatcga 300 tcaggtcgaa gactggtgtg aggcgatcac cggcgaagaa gactgaacgt cagccgcgcg 360 agatcactcg tcgcgacagt cgccaccgcc cgtcttcgag ttcgaactcg tcggcgtagc 420 tcccgacgcc actgagcttg ggagttgtgg tcgtgtcgac gtagaacgtc cacaccgcat 480 ccgcgcgggc tgtagtttcc gacgtcacgt ccacggcgat cgccgtggtg acgtgacgtg 540 tgaacgtgcc cgggccttgt atgccggcgg ctcgacgctc gattactcca gcagcaattt 600 cttcgcggcc acgtcgtacc tgtgcgggga taccgcgatc gggattggcc ggcatgttcc 660 attcggcctc gcgggtgaac tgctcgacgt agtcctcgac agagccggtg tcagccagat 720 gggcgatccg agcgagcgtt gaacgaatct gttcggcaag gctctcgggt tgcatgctgt 780 tctccgtacg tgtgagtgct gtggtggatc gcaggatatt gcccgtggac tgaccgaaca 840 ccgaggcgtc cc agtcagtg gcagcagttg agagaaattc agatcaccgt gagttcaact 900 atttccccga tatcagggga aatggttgag tatcccctga aggcaaaagc cttaattccg 960 accgggaggt ttcacaccgt gtccgtcgct cctcgatgtc ggctctgact tcttcagcgc 1020 cacaacgact gtcgtagcag ctcgcttttc tgaattcccg ggagtgagca tgtcggaacg 1080 acactctttc ccattctcgc aagcgtgtgc cctgagagtg cacagctgcg cacctattac 1140 gaagggcaac acctccgtga actcgtctct ttcgacggcc ggtctcgacc gccgtgcctt 1200 cttccgtggc agtgcgatcg ccgcattcgc catcttcggt agcggctcac tggccgcgtg 1260 ctcctctgca gtcggcgaac aacgctcgga cgcaggtgaa tcagttgagc cggttcgagg 1320 tgggacgctc acactggcga taccggacga cgtcagccct gcggcacttt tgaccagcac 1380 caccgttgcc ggtgtcacga tatccgggct cgtgtacgag accctgaccc gctatccact 1440 cgattcggta gtgccacagc cagttctggc caaggcctgg aagctgtccg acgacgggct 1500 gacgctcacg ctcaatctgc gcgatgacgt caccttccac tccggccggc gcttcacctc 1560 cgctgatgcg gagttctcgc tgcgcaccta tgcggacccg aaattctcgg cccagctcag 1620 gagtactgcg gccgccatca ccggcttcga ctcttcggat cccaatacgc tggttctcac 1680 cctcgcacac cgaaccggga acatcttcga tcttctcgat ctagcgccga tcttcgaccc 1740 cgaaacgttc gacaaggcgg tgaccggtga atccttcgtt ggtacgggcc ctttcgtatt 1800 cacctcacgc accccgaaca gttccatctc cttcgatcgg aacccgaact actgggtcgc 1860 cgagcggccg tacctcgatc acgtggaagc gcggatcgtt ccggacgccc aagcgcgcct 1920 gacatcgctg aagtccggac aggtctctct tgttgctccg gcgtcgttgc ccttccgtga 1980 ctcacagaac ttgaccaaga ccagcgggtt cgtgacaacc tcgatcgagg gtgcggaact 2040 acagatatac ctgggcacaa atgtcaccgc cgacgggctg accgatgtgc gggcacgaaa 2100 ggcgttggcc tacgcgatcg atcgtgaccg catcatcagt gaggtgtatc gcgatacggg 2160 gtatgcggtc aatctgccgt ggccgaagtc gtcgcccgcc tacgacgctg ccaagaacgc 2220 gacctacact cgcgatgtcg acaaggcgcg cgccctggtc ggcgatctgg gtgcactgcc 2280 gaccattccg ttgacctaca caggagcgag cccggatttc gaagccgccg cgcagatcgt 2340 gcaggccaat ctcgccgagg cagggatccg cgtcgaattg gatcctgtcg aggcatccat 2400 cttcgtcaag cagttgatcg gcgctgagtt caaaggactt tggctgacca atcacacttt 2460 tgcgcaatat gttccgtcaa ccctgacggt cagcgcctac ccgttcaacg ctgcacacaa 2520 tgcctcgaag ttcagttcgc cggc gtattc cgccgcagcg gagggtgcgt ggcaggttcc 2580 cgacggttcg agcgatgagg caaagaaact gtatgccgaa ttgggcacgc aactgttgaa 2640 cgagttgttc ttgatcgaga tcggtgttgt cgtgccgcag gtatcggcgg ccgagaccgt 2700 tcgtggtctg gcctggacaa agggtcgcca gccacaattc gccaacacat tcctggcgta 2760 ggggattcga tgattcgtta cattgcgggg cgactaccgt cggcggtggt ggttctcttc 2820 ctcgcgtcga tattgatctt ctcggtgatg cgtctggtgc ctggcgaccc cgcgttggct 2880 ctcgccggcc ccgatgcaac tccggaagcg attgcagcca ttcgtcattc gctcggtctc 2940 gacaggtcga ttcccgcgca gtacctgacc tggatcggcg acgtactgac attggatctc 3000 gggcgttcgt tcgttctcgg cggccagatt tccgatctgg tgctggcggg gttggggaac 3060 accgcagttc tggcgggaag tgcgttgctg ctggccgtcg tgttgagtct ggtgctcagt 3120 gttgcggtgg tggtgtggcc gaagaaatgg ctgacctcgg tggtgaacct tctcaacact 3180 ctctcggttg ctctgcccaa cttcgtgacc ggtgtcctcc tggttctcgt cttcgcggtg 3240 ctgattccgg ttctgccatc cggtggtgtt ccaccaggtg ggtatctcgc tcgtcccgac 3300 atcacgttcc agtacctgct gctgccgtca ctgtgcctcg cacttcctgt cgcggccgct 3360 ctcaccaggt tcctgtccga agcgctgcgc accgagatgg cgcagcagta cgtgatcacc 3420 gctcgcgcag ccggtgtgcc acggtggaac ttggttacac gcagtgcatt acgcaatgca 3480 ctgccaacta tgctcaccgt gctcggaatt cagaccgggc accttctcgg cggagccgta 3540 ctggttgagg cgatctttgc ctggcctggc atcggccaat tgatcgagca gggaatcggt 3600 cggcgggatt acccggtggt gcaggtgctg ctcctgctgt cggtcacgat cttcgttctg 3660 atccaacttc tgaccgacat cgtgcatgcc tacctcgacc cacgaatcag aatcggaggg 3720 cagca atg acg gtg gcc gat cag tct gtt gac gaa agg gtt cgg cga tct 3770 Met Thr Val Ala Asp Gln Ser Val Asp Glu Arg Val Arg Arg Ser 1 5 10 15 cgt cct gtg cgc gct gct ctg acg cac ggt cag ggg ttg gcg ggt gtg 3818 Arg Pro Val Arg Ala Ala Leu Thr His Gly Gln Gly Leu Ala Gly Val 20 25 30 ttg atc ctg gtg gtc atc gca gta gcg gga att gct gct cca ctg ttg 3866 Leu Ile Leu Val Val Ile Ala Val Ala Gly Ile Ala Ala Pro Leu Leu 35 40 45 act cac tac ggg ccc aac gag cag atc gag ggt gca aat ctg ttg ggg 3914 Thr His Tyr Gly Pro Asn Glu Gln Ile Glu Gly Ala Asn Leu Leu Gly 50 55 60 gcc agt gca cag cac tgg ttc gga aca gat cag gtc aac cgt gac gtc 3962 Ala Ser Ala Gln His Trp Phe Gly Thr Asp Gln Val Asn Arg Asp Val 65 70 75 ttc gcc cgt tct ctt tac gga atc cgg atc aac ctt gtc atc gtg ctc 4010 Phe Ala Arg Ser Leu Tyr Gly Ile Arg Ile Asn Leu Val Ile Val Leu 80 85 90 95 gtc gcg gtg cca gcg ggc gcg atc atc ggt tcg ctc gca ggc ctg gtg 4058 Val Ala Val Pro Ala Gly Ala Ile Ile Gly Ser Leu Ala Gly Leu Val 100 105 110c agt atc aat tcg gtg gca gat gtc atc gca cag cgg atc ttc gat 4106 Ser Ser Ile Asn Ser Val Ala Asp Val Ile Ala Gln Arg Ile Phe Asp 115 120 125 gtg att ctt gcc ttc ccg ctg ttg att ctg gcg atc ac gcg 4154 Val Ile Leu Ala Phe Pro Leu Leu Ile Leu Ala Ile Thr Leu Val Ala 130 135 140 atc acg ggg ccg gga gta gcg ccg gtg atc gcg gtg atc gtg gct gct 4202 Ile Thr Gly Pro Gly Val Ala Pro Val Ile Ala Val Ile Val Ala Ala 145 150 155 gaa ata ccc ttg ttc ggg cgc ctg gtg cga acg acg gtg ttg aag gtg 4250 Glu Ile Pro Leu Phe Gly Arg Leu Val Arg Thr Thr Val Leu Lys Val 160 165 170 175 cgc gac ctc cc g gag tcg gcc gag gta atc ggt gcg ggg cgg 4298 Arg Glu Leu Pro Phe Val Glu Ser Ala Glu Val Ile Gly Ala Gly Arg 180 185 190 tgg tgg gta ctg cgt aag cat gtg ctg ccc aac gcc gta gaa ccg Trp ctg 4346 Val Leu Arg Lys His Val Leu Pro Asn Ala Val Glu Pro Leu 195 200 205 ggt gtg cag att gcg ctc tcc atg tcg gtt gcg gtg ttc gcc gaa agt 4394 Gly Val Gln Ile Ala Leu Ser Met Ser Val Ala Val Phe Ala Glu Ser 210 215 220 gcg atg agt ttt ctc gga atc ggc gtg cgc ccg ccg gat ccg tct ctc 4442 Ala Met Ser Phe Leu Gly Ile Gly Val Arg Pro Pro Asp Pro Ser Leu 225 230 235 ggt tcg atc atc gcg ccc gcc atc gat gcc aat ccg gcc 4490 Gly Ser Ile Ile Ala Gly Ala Ile Pro Asn Leu Asp Ala Asn Pro Ala 240 245 250 255 tac gcg atc gga cct ttg gtg atc gtc tcg gcg ttg gtc ctg agc ttc 4538 Tly Ala Ila Val Ile Val Ser Ala Leu Val Leu Ser Phe 260 265 270 ctg ctg atc gcc cag gga ctg ggc aag gcc cgc cga atc tgaaattgcc 4587 Leu Leu Ile Ala Gln Gly Leu Gly Lys Ala Arg Arg Ile 275 280 accgccacca ccactca g gacgtagtca tgaatgcaac acccgcacag gcagttctcg 4647 ccatccggga tctggtgatc gagttcgatg ccgacggttc ccgtggctcg tcgacaactg 4707 tggtcgacca cgtcaacctc gaattgggtt tcggtgagat attggcgttg gtcggcgagt 4767 ccggatccgg taagtcgttg accgcacgcg ctgttctcgg gttgttaccg gacggcgcgc 4827 aggcccgcgg gttgatccaa ctcggtggcc agcaggtgct cggtgccgac gaggcgacgc 4887 tcaatcaatt gcgggggact cgggccgcga tggttttcca ggagcctcag actgcactga 4947 atccagtgca gaaggtgggt tggcagatcg cgcaggcgct gcgggcacac ggcaagatct 5007 cgcgtgccga cgccagagtt cgcgcaatca atcttctacg gatggtggag attccggaac 5067 cggagcgtcg ggtcgactgg tatccgcatc aactctcggg tggacagaag cagcgggttg 5127 tcatcgcgct ggcgttgtcg ggttctcctg atttactcat cgccgacgaa ccgactactg 5187 cgctcgacgt caccgtgcag gccgagattc tgcaactcct gcgtaatctg cgtgatcgtt 5247 cgggcactgc aatactgctc atcacacaca acatgggtgt cgttgcggac atcgcagatc 5307 gcgtgctggt gatgcgatcg ggcagggtcg tcgagcagca gtccgttttc gatctctttg 5367 cgacgccgcg tgagtcgtac acacaggctt tgcttgcggc cgtgccacgt ctgccggagg 5427 tcgaccagcc gaagcctgtg gttg ctctgg tcgatagcga aggcgacgcg caagtcccgc 5487 cgatcctgcg gttcgacgcg gcctccatcg tgtacccggc gaggctgggc aacagagagt 5547 ttcgtgcggt cgatcaggtg agcctgacgg ttcaggccgg cgaggttgtc ggactggtcg 5607 gtgaatcggg atcagggaag acaactctcg gccgggctgc gctcggtgtt atcagggcca 5667 gtagcggcat cgttgctttc gacggaaccg atctcggaaa gatctccgcc gcgaatcttc 5727 ggggcatccg caagggaatg gccttgattc atcaggaccc tgcggcatct ctcgatccgc 5787 gtcggacggt cggacaaagc gtcggcgagc cactcgaagt ccatcgcttt gccaccggag 5847 ccctgctgcg aaaccgggtg ggggagttac tcgaatcagt tcgcctaccg aagtcttttg 5907 ctcaccgggc accaggagaa ctttccggtg gtcaacgcca gcgtgttgcg ctggcccgag 5967 cacttgccct cggaccgaga ctgctggtgg ccgatgagcc gaccagcgct ctggatgttt 6027 ccgtccaagc cgatgttctt tcactctttg ccgatctcag ggaggaatac aacttcgcat 6087 gtctgttcat cagccacgat cttgcagtcg tcaatcaggt agccgaccgg gtagcggtac 6147 ttcgcagcgg ggagttggtc gaagagggcc ccgctgcaga ggtattcagc tcgccacggc 6207 aggactacac acaacagttg gttgccgcgg tgcctgttcc agatccgcgt cgtcagcgcg 6267 ccgggcggga acgggaatca ctggttccc g acctgactgt tgccgtttga cccgtctcga 6327 catcgaacag cattaccacc acctcaacct tttgcagact cataggagct tgacatgtcc 6387 atcaccgaac tcgaacgcgt cactcagacc ccggagcaga tctacgctgc cggcggaatc 6447 actgtccaca aggtcggtga gctgatcggc gcccgcatcg acggcgtaca cctctctggg 6507 gatctgtccg aagagaccgc ctacgcgatc aactatgcgc tggcagcgca caaggttgtg 6567 ttcttccgtg ggcagcagca tctcgacgac acgtcgcagt acgaattcgc cggaaccctc 6627 ggcagccaga ccaccacgca tcccactctc aagtccaagg acaacaagct gttggtgctc 6687 gacggcgctg ccagcagctg gcacaccgac gtcactttca tcgatcgcat cccgaaggca 6747 tcgatcctgc gcgcgaccac aattcctgag tacggcggag ccaccacgtg ggcgtctacc 6807 acggctgcgt acaaccaact gccgcattcg ctcaaggtat tggtcgagaa tctgcgcgct 6867 gtccacacca atgcatacga ctacgccgag attatcgaca aggtgaagca aggcgacgct 6927 cagcgcgtga cgaactactc cgaattcact cgcgaaatct acgagacgga gcatccggtg 6987 gttcgggtcc atcctgcgac aggggagaag actctgcttc tcggtcactt cgtcaaggag 7047 ttcgtcggac tgaagccgtc ggagtcggtt gctctctacc agcttctgca ggcacgaatc 7107 atcaagttgg aaaacaccgt tcggtggtca tggg ctcccg gtgatctggc gatctgggac 7167 aaccaggcaa cccagcatta cgggatttcc gattacggga cacaggcgcg cagcgtgcat 7227 cgcgtgacgt tggccggtga cgttcccgtc gacgtccacg gtgagcagag ccgcatcatc 7287 aagggcgatg catccgagtt ctcgatcgtt gcagacatcg accggcttcc cggctttgcc 7347 gcaaactgat cagtcctgct gcgggtgatc cggcgggaac atctgtcccc atgagtgatt 7407 gcatgctcgg cacgtccatt ccggggtgtc cggtcccacg acgcagcctg ttccggcgag 7467 ccacggtagg tgatcgaatg gatggaccgg catccccggg gaacactgag cggttctgtg 7527 tgcaccgcag agtgggcacg acggattttt cgccagcatg tagtcgatga agttggtgtg 7587 cccggacgtg gccatccttc cggtgcattt gcggcacagg acggctgggt agtcgtcacc 7647 aggttcagcc tgcctgaaga tccgtgatgc ggaatgaaag tgaaggcgat cgctgctgtt 7707 gaactttcgc aacggcatcg gtgagctgca acctgtgcag ttgccccgct cgttcatcaa 7767 cgttcgggcg tgggcagctg tggacgtcgg atccagcgtg tcgatgtcgc cgaagtagtg 7827 cgcatagtgt gactggccgg ccgtgagaat ccggtcggcc agtgcgtcgt cgaccggccc 7887 aggatccgtg ggaaacgtga tggaccatcg ttgttcgagt tctggaacgg tgggcaaacc 7947 gtggagttcg tacgggcgtt catcgacttc catccgcgac gtgccgtcga agggcgcagc 8007 cacggtctcg atgcagagca gcgtgcgagg agtggtgtcg agaacagtga tcagcaggtc 8067 gccgatggcc atctttccgc gcatgatcca gggatcgaca cgggcacggg gttcgaggac 8g cgggcgag cggggcgagc 8g <210> 6 <211> 284 <212> PRT <213> Rhodococcus erythropolis <400> 6 Met Thr Val Ala Asp Gln Ser Val Asp Glu Arg Val Arg Arg Ser Arg 1 5 10 15 Pro Val Arg Ala Ala Leu Thr His Gly Gln Gly Leu Ala Gly Val Leu 20 25 30 Ile Leu Val Val Ile Ala Val Ala Gly Ile Ala Ala Pro Leu Leu Thr 35 40 45 His Tyr Gly Pro Asn Glu Gln Ile Glu Gly Ala Asn Leu Leu Gly Ala 50 55 60 Ser Ala Gln His Trp Phe Gly Thr Asp Gln Val Asn Arg Asp Val Phe 65 70 75 80 Ala Arg Ser Leu Tyr Gly Ile Arg Ile Asn Leu Val Ile Val Leu Val 85 90 95 Ala Val Pro Ala Gly Ala Ile Ile Gly Ser Leu Ala Gly Leu Val Ser 100 105 110 Ser Ile Asn Ser Val Ala Asp Val Ile Ala Gln Arg Ile Phe Asp Val 115 120 125 Ile Leu Ala Phe Pro Leu Leu Ile Leu Ala Ile Thr Leu Val Ala Ile 130 135 140 Thr Gly Pro Gly Val Ala Pro Val Ile Ala Val Ile Val Ala Ala Glu 145 150 155 160 Ile Pro Leu Phe Gly Arg Leu Val Arg Thr Thr Val Leu Lys Val Arg 165 170 175 Glu Leu Pro Phe Val Glu Ser Ala Glu Val Ile Gly Ala Gly Arg Trp 180 185 190 Trp Val Leu Arg Lys His Val Leu Pro Asn Ala Val Glu Pro Leu Gly 195 200 205 Val Gln Ile A la Leu Ser Met Ser Val Ala Val Phe Ala Glu Ser Ala 210 215 220 Met Ser Phe Leu Gly Ile Gly Val Arg Pro Pro Asp Pro Ser Leu Gly 225 230 235 240 Ser Ile Ile Ala Gly Ala Ile Pro Asn Leu Asp Ala Asn Pro Ala Tyr 245 250 255 Ala Ile Gly Pro Leu Val Ile Val Ser Ala Leu Val Leu Ser Phe Leu 260 265 270 Leu Ile Ala Gln Gly Leu Gly Lys Ala Arg Arg Ile 275 280 <210> 7 <211> 8188 <212> DNA <213> Rhodococcus erythropolis <220> <221> CDS <222> (4617) .. (6314) <400> 7 gagctcagtc tatgccgtcg gcgccgaaag tcggacatga gtgacagcga tgggtagggc 60 ggggcgagtc gggtaccccg aggaccgaag cgagagtgaa ggaggagaac cgatgcgtgt 120 gaaagcgatg gtcggaacag gcgcgatggc tgttgcgctg gccgccggcc cagttgccac 180 agcttctgct gaaggcgccg agacggcggg gtcggagggt tcaccggaga tgctgacctg 240 cgaggtggtc ttcgatgcac tggaagacat gagcccggaa tgggcggaag aggggatcga 300 tcaggtcgaa gactggtgtg aggcgatcac cggcgaagaa gactgaacgt cagccgcgcg 360 agatcactcg tcgcgacagt cgccaccgcc cgtcttcgag ttcgaactcg tcggcgtagc 420 tcccgacgcc actgagcttg ggagttgtgg tcgtgtcgac gtagaacgtc cacaccgcat 480 ccgcgcgggc tgtagtttcc gacgtcacgt ccacggcgat cgccgtggtg acgtgacgtg 540 tgaacgtgcc cgggccttgt atgccggcgg ctcgacgctc gattactcca gcagcaattt 600 cttcgcggcc acgtcgtacc tgtgcgggga taccgcgatc gggattggcc ggcatgttcc 660 attcggcctc gcgggtgaac tgctcgacgt agtcctcgac agagccggtg tcagccagat 720 gggcgatccg agcgagcgtt gaacgaatct gttcggcaag gctctcgggt tgcatgctgt 780 tctccgtacg tgtgagtgct gtggtggatc gcaggatatt gcccgtggac tgaccgaaca 840 ccgaggcgtc c cagtcagtg gcagcagttg agagaaattc agatcaccgt gagttcaact 900 atttccccga tatcagggga aatggttgag tatcccctga aggcaaaagc cttaattccg 960 accgggaggt ttcacaccgt gtccgtcgct cctcgatgtc ggctctgact tcttcagcgc 1020 cacaacgact gtcgtagcag ctcgcttttc tgaattcccg ggagtgagca tgtcggaacg 1080 acactctttc ccattctcgc aagcgtgtgc cctgagagtg cacagctgcg cacctattac 1140 gaagggcaac acctccgtga actcgtctct ttcgacggcc ggtctcgacc gccgtgcctt 1200 cttccgtggc agtgcgatcg ccgcattcgc catcttcggt agcggctcac tggccgcgtg 1260 ctcctctgca gtcggcgaac aacgctcgga cgcaggtgaa tcagttgagc cggttcgagg 1320 tgggacgctc acactggcga taccggacga cgtcagccct gcggcacttt tgaccagcac 1380 caccgttgcc ggtgtcacga tatccgggct cgtgtacgag accctgaccc gctatccact 1440 cgattcggta gtgccacagc cagttctggc caaggcctgg aagctgtccg acgacgggct 1500 gacgctcacg ctcaatctgc gcgatgacgt caccttccac tccggccggc gcttcacctc 1560 cgctgatgcg gagttctcgc tgcgcaccta tgcggacccg aaattctcgg cccagctcag 1620 gagtactgcg gccgccatca ccggcttcga ctcttcggat cccaatacgc tggttctcac 1680 cctcgcacac cgaaccggg a acatcttcga tcttctcgat ctagcgccga tcttcgaccc 1740 cgaaacgttc gacaaggcgg tgaccggtga atccttcgtt ggtacgggcc ctttcgtatt 1800 cacctcacgc accccgaaca gttccatctc cttcgatcgg aacccgaact actgggtcgc 1860 cgagcggccg tacctcgatc acgtggaagc gcggatcgtt ccggacgccc aagcgcgcct 1920 gacatcgctg aagtccggac aggtctctct tgttgctccg gcgtcgttgc ccttccgtga 1980 ctcacagaac ttgaccaaga ccagcgggtt cgtgacaacc tcgatcgagg gtgcggaact 2040 acagatatac ctgggcacaa atgtcaccgc cgacgggctg accgatgtgc gggcacgaaa 2100 ggcgttggcc tacgcgatcg atcgtgaccg catcatcagt gaggtgtatc gcgatacggg 2160 gtatgcggtc aatctgccgt ggccgaagtc gtcgcccgcc tacgacgctg ccaagaacgc 2220 gacctacact cgcgatgtcg acaaggcgcg cgccctggtc ggcgatctgg gtgcactgcc 2280 gaccattccg ttgacctaca caggagcgag cccggatttc gaagccgccg cgcagatcgt 2340 gcaggccaat ctcgccgagg cagggatccg cgtcgaattg gatcctgtcg aggcatccat 2400 cttcgtcaag cagttgatcg gcgctgagtt caaaggactt tggctgacca atcacacttt 2460 tgcgcaatat gttccgtcaa ccctgacggt cagcgcctac ccgttcaacg ctgcacacaa 2520 tgcctcgaag ttcagttcgc cggc gtattc cgccgcagcg gagggtgcgt ggcaggttcc 2580 cgacggttcg agcgatgagg caaagaaact gtatgccgaa ttgggcacgc aactgttgaa 2640 cgagttgttc ttgatcgaga tcggtgttgt cgtgccgcag gtatcggcgg ccgagaccgt 2700 tcgtggtctg gcctggacaa agggtcgcca gccacaattc gccaacacat tcctggcgta 2760 ggggattcga tgattcgtta cattgcgggg cgactaccgt cggcggtggt ggttctcttc 2820 ctcgcgtcga tattgatctt ctcggtgatg cgtctggtgc ctggcgaccc cgcgttggct 2880 ctcgccggcc ccgatgcaac tccggaagcg attgcagcca ttcgtcattc gctcggtctc 2940 gacaggtcga ttcccgcgca gtacctgacc tggatcggcg acgtactgac attggatctc 3000 gggcgttcgt tcgttctcgg cggccagatt tccgatctgg tgctggcggg gttggggaac 3060 accgcagttc tggcgggaag tgcgttgctg ctggccgtcg tgttgagtct ggtgctcagt 3120 gttgcggtgg tggtgtggcc gaagaaatgg ctgacctcgg tggtgaacct tctcaacact 3180 ctctcggttg ctctgcccaa cttcgtgacc ggtgtcctcc tggttctcgt cttcgcggtg 3240 ctgattccgg ttctgccatc cggtggtgtt ccaccaggtg ggtatctcgc tcgtcccgac 3300 atcacgttcc agtacctgct gctgccgtca ctgtgcctcg cacttcctgt cgcggccgct 3360 ctcaccaggt tcctgtccga agcgctgcg c accgagatgg cgcagcagta cgtgatcacc 3420 gctcgcgcag ccggtgtgcc acggtggaac ttggttacac gcagtgcatt acgcaatgca 3480 ctgccaacta tgctcaccgt gctcggaatt cagaccgggc accttctcgg cggagccgta 3540 ctggttgagg cgatctttgc ctggcctggc atcggccaat tgatcgagca gggaatcggt 3600 cggcgggatt acccggtggt gcaggtgctg ctcctgctgt cggtcacgat cttcgttctg 3660 atccaacttc tgaccgacat cgtgcatgcc tacctcgacc cacgaatcag aatcggaggg 3720 cagcaatgac ggtggccgat cagtctgttg acgaaagggt tcggcgatct cgtcctgtgc 3780 gcgctgctct gacgcacggt caggggttgg cgggtgtgtt gatcctggtg gtcatcgcag 3840 tagcgggaat tgctgctcca ctgttgactc actacgggcc caacgagcag atcgagggtg 3900 caaatctgtt gggggccagt gcacagcact ggttcggaac agatcaggtc aaccgtgacg 3960 tcttcgcccg ttctctttac ggaatccgga tcaaccttgt catcgtgctc gtcgcggtgc 4020 cagcgggcgc gatcatcggt tcgctcgcag gcctggtgtc gagtatcaat tcggtggcag 4080 atgtcatcgc acagcggatc ttcgatgtga ttcttgcctt cccgctgttg attctggcga 4140 tcacgttggt cgcgatcacg gggccgggag tagcgccggt gatcgcggtg atcgtggctg 4200 ctgaaatacc cttgttcggg cgcctggtgc gaac gacggt gttgaaggtg cgcgaactcc 4260 cgttcgtgga gtcggccgag gtaatcggtg cggggcggtg gtgggtactg cgtaagcatg 4320 tgctgcccaa cgccgtagaa ccgctgggtg tgcagattgc gctctccatg tcggttgcgg 4380 tgttcgccga aagtgcgatg agttttctcg gaatcggcgt gcgcccgccg gatccgtctc 4440 tcggttcgat catcgcgggc gccattccga acctcgatgc caatccggcc tacgcgatcg 4500 gacctttggt gatcgtctcg gcgttggtcc tgagcttcct gctgatcgcc cagggactgg 4560 gcaaggcccg ccgaatctga aattgccacc gccaccacca cttcaaggac gtagtc atg 4619 Met 1 aat gca aca ccc gca cag gca gtt ctc gcc atc cgg gat ctg gtg atc 4667 Asn Ala Thr Pro Ala Gln Ala Val Leu Ala Ile Arg Asp Leu Val Ile 5 10 15 gag ttc gat gcc gac ggt tcc cgt ggc tcg tcg aca gt gac 4715 Glu Phe Asp Ala Asp Gly Ser Arg Gly Ser Ser Thr Thr Val Val Asp 20 25 30 cac gtc aac ctc gaa ttg ggt ttc ggt gag ata ttg gcg ttg gtc ggc 4763 His Val Asn Leu Glu Leu Gly Phe Gly Glu Leu Ala Leu Val Gly 35 40 45 gag tcc gga tcc ggt aag tcg ttg acc gca cgc gct gtt ctc ggg ttg 4811 Glu Ser Gly Ser Gly Lys Ser Leu Thr Ala Arg Ala Val Leu Gly Leu 50 55 60 65 tta ccg gac ggc gcg cag gcc cgc ggg ttg atc caa ctc ggt ggc cag 4859 Leu Pro Asp Gly Ala Gln Ala Arg Gly Leu Ile Gln Leu Gly Gly Gln 70 75c g gtg ct gcc gac gag gcg acg ctc aat caa ttg cgg ggg act 4907 Gln Val Leu Gly Ala Asp Glu Ala Thr Leu Asn Gln Leu Arg Gly Thr 85 90 95 cgg gcc gcg atg gtt ttc cag gag cct cag act gca ctg aat cca gtg 4955 Ala Ala Met Val Phe Gln Glu Pro Gln Thr Ala Leu Asn Pro Val 100 105 110 cag aag gtg ggt tgg cag atc gcg cag gcg ctg cgg gca cac ggc aag 5003 Gln Lys Val Gly Trp Gln Ile Ala Gln Ala Leu Arg Ala His Gly Lys 115 120 125 atc tcg cgt gcc gac gcc aga gtt cgc gca atc aat ctt cta cgg atg 5051 Ile Ser Arg Ala Asp Ala Arg Val Arg Ala Ile Asn Leu Leu Arg Met 130 135 140 145 gtg gag att ccg gaa ccg gag cgg gtc gac tgg tat ccg cat caa 5099 Val Glu Ile Pro Glu Pro Glu Arg Arg Val Asp Trp Tyr Pro His Gln 150 155 160 ctc tcg ggt gga cag aag cag cgg gtt gtc atc gcg ctg gcg ttg tcg 5147 Leu Ser Gly Gly Gln Arg Val Val Ile Ala Leu Ala Leu Ser 165 170 175 ggt tct cct gat tta ctc atc gcc gac gaa ccg act act gcg ctc gac 5195 Gly Ser Pro Asp Leu Leu Ile Ala Asp Glu Pro Thr Thr Ala Leu Asp 180 185 190 gtc acc gtg cag gcc gag att ctg caa ctc ctg cgt aat ctg cgt gat 5243 Val Thr Val Gln Ala Glu Ile Leu Gln Leu Leu Arg Asn Leu Arg Asp 195 200 205 cgt tcg ggc act gca ata ctg ctc atc aca cac gac atg 5291 Arg Ser Gly Thr Ala Ile Leu Leu Ile Thr His Asn Met Gly Val Val 210 215 220 225 gcg gac atc gca gat cgc gtg ctg gtg atg cga tcg ggc agg gtc gtc 5339 Ala Asp Ile Ala Asp Arg Val Leu Val Met Arg Gly Arg Val Val 230 235 240 gag cag cag tcc gtt ttc gat ctc ttt gcg acg ccg cgt gag tcg tac 5387 Glu Gln Gln Ser Val Phe Asp Leu Phe Ala Thr Pro Arg Glu Ser Tyr 245 250 255 aca cag gct ttg ctt gcg gcc gtg cca cgt ctg ccg gag gtc gac cag 5435 Thr Gln Ala Leu Leu Ala Ala Val Pro Arg Leu Pro Glu Val Asp Gln 260 265 270 270 ccg aag cct gtg gtt gct ctg gtc gat agc gaa ggc gac gcg Caa gtc 5a Pro Val Val Ala Leu Val Asp Ser Glu Gly Asp Ala Gln Val 275 280 285 ccg ccg atc ctg cgg ttc gac gcg gcc tcc atc gtg tac ccg gcg agg 5531 Pro Pro Ile Leu Arg Phe Asp Ala Ala Ser Ile Val Tyr Pro Ala Arg 290 295 300 305 ctg ggc aac aga gag ttt cgt gcg gtc gat cag gtg agc ctg acg gtt 5579 Leu Gly Asn Arg Glu Phe Arg Ala Val Asp Gln Val Ser Leu Thr Val 310 310 320 cag gcc ggc gag gtt gtc gg gct gaa tcg gga tca ggg aag 5627 Gln Ala Gly Glu Val Val Gly Leu Val Gly Glu Ser Gly Ser Gly Lys 325 330 335 aca act ctc ggc cgg gct gcg ctc ggt gtt atc agg gcc agt agc ggc 5675 Thr Thr Leu Gly Arg Ala Leu Gly Val Ile Arg Ala Ser Ser Gly 340 345 350 atc gtt gct ttc gac gga acc gat ctc gga aag atc tcc gcc gcg aat 5723 Ile Val Ala Phe Asp Gly Thr Asp Leu Gly Lys Ile Ser Ala Ala Asn 355 360 365 ctt cgg ggc atc cgc aag gga atg gcc ttg att cat cag gac cct gcg 5771 Leu Arg Gly Ile Arg Lys Gly Met Ala Leu Ile His Gln Asp Pro Ala 370 375 380 385 gca tct ctc gat ccg cgt cgg acg gtc gga cag c gag cca 5819 Ala Ser Leu Asp Pro Arg Arg Thr Val Gly Gln Ser Val Gly Glu Pro 390 395 400 ctc gaa gtc cat cgc ttt gcc acc gga gcc ctg ctg cga aac cgg gtg 5867 Leu Glu Val His Arg Phe Ala Thr Gly Ala Leu Leu Arg Asn Arg Val 405 410 415 ggg gag tta ctc gaa tca gtt cgc cta ccg aag tct ttt gct cac cgg 5915 Gly Glu Leu Leu Leu Glu Ser Val Arg Leu Pro Lys Ser Phe Ala His Arg 420 425 430 430 gca cca gga gactt tcc ggt ggt caa cgc cag cgt gtt gcg ctg gcc 5963 Ala Pro Gly Glu Leu Ser Gly Gly Gln Arg Gln Arg Val Ala Leu Ala 435 440 445 cga gca ctt gcc ctc gga ccg aga ctg ctg gtg gcc gat gag gacc gat gag acc Leu Ala Leu Gly Pro Arg Leu Leu Val Ala Asp Glu Pro Thr 450 455 460 465 agc gct ctg gat gtt tcc gtc caa gcc gat gtt ctt tca ctc ttt gcc 6059 Ser Ala Leu Asp Val Ser Val Gln Ala Asp Val Leu Ser Leu Phe Ala 470 475 480 gat ctc agg gag gaa tac aac ttc gca tgt ctg ttc atc agc cac gat 6107 Asp Leu Arg Glu Glu Tyr Asn Phe Ala Cys Leu Phe Ile Ser His Asp 485 490 495 ctt gca gtc gat gac gat cag cgg gta gcg gta ctt cgc agc 6155 Leu Ala Val Val Asn Gln Val Ala Asp Arg Val Ala Val Leu Arg Ser 500 505 510 ggg gag ttg gtc gaa gag ggc ccc gct gca gag gta ttc agc tcg cca 6203 Gly Glu Leu Gly Pro Ala Ala Glu Val Phe Ser Ser Pro 515 520 525 cgg cag gac tac aca caa cag ttg gtt gcc gcg gtg cct gtt cca gat 6251 Arg Gln Asp Tyr Thr Gln Gln Leu Val Ala Ala Val Pro Val Pro Asp 530 535 540 540 545 ccg cgt cgt cag cgc gcc ggg cgg gaa cgg gaa tca ctg gtt ccc gac 6299 Pro Arg Arg Gln Arg Ala Gly Arg Glu Arg Glu Ser Leu Val Pro Asp 550 555 560 ctg act gtt gcc gtt tgacccgta ccacat Acacacacacagca trca gcat Val 565 ccttttgcag actcatagga gcttgacatg tccatcaccg aactcgaacg cgtcactcag 6414 accccggagc agatctacgc tgccggcgga atcactgtcc acaaggtcgg tgagctgatc 6474 ggcgcccgca tcgacggcgt acacctctct ggggatctgt ccgaagagac cgcctacgcg 6534 atcaactatg cgctggcagc gcacaaggtt gtgttcttcc gtgggcagca gcatctcgac 6594 gacacgtcgc agtacgaatt cgccggaacc ctcggcagcc agaccaccac gcatcccact 6654 ctc aagtcca aggacaacaa gctgttggtg ctcgacggcg ctgccagcag ctggcacacc 6714 gacgtcactt tcatcgatcg catcccgaag gcatcgatcc tgcgcgcgac cacaattcct 6774 gagtacggcg gagccaccac gtgggcgtct accacggctg cgtacaacca actgccgcat 6834 tcgctcaagg tattggtcga gaatctgcgc gctgtccaca ccaatgcata cgactacgcc 6894 gagattatcg acaaggtgaa gcaaggcgac gctcagcgcg tgacgaacta ctccgaattc 6954 actcgcgaaa tctacgagac ggagcatccg gtggttcggg tccatcctgc gacaggggag 7014 aagactctgc ttctcggtca cttcgtcaag gagttcgtcg gactgaagcc gtcggagtcg 7074 gttgctctct accagcttct gcaggcacga atcatcaagt tggaaaacac cgttcggtgg 7134 tcatgggctc ccggtgatct ggcgatctgg gacaaccagg caacccagca ttacgggatt 7194 tccgattacg ggacacaggc gcgcagcgtg catcgcgtga cgttggccgg tgacgttccc 7254 gtcgacgtcc acggtgagca gagccgcatc atcaagggcg atgcatccga gttctcgatc 7314 gttgcagaca tcgaccggct tcccggcttt gccgcaaact gatcagtcct gctgcgggtg 7374 atccggcggg aacatctgtc cccatgagtg attgcatgct cggcacgtcc attccggggt 7434 gtccggtccc acgacgcagc ctgttccggc gagccacggt aggtgatcga atggatggac 7494 cggcatcc cc ggggaacact gagcggttct gtgtgcaccg cagagtgggc acgacggatt 7554 tttcgccagc atgtagtcga tgaagttggt gtgcccggac gtggccatcc ttccggtgca 7614 tttgcggcac aggacggctg ggtagtcgtc accaggttca gcctgcctga agatccgtga 7674 tgcggaatga aagtgaaggc gatcgctgct gttgaacttt cgcaacggca tcggtgagct 7734 gcaacctgtg cagttgcccc gctcgttcat caacgttcgg gcgtgggcag ctgtggacgt 7794 cggatccagc gtgtcgatgt cgccgaagta gtgcgcatag tgtgactggc cggccgtgag 7854 aatccggtcg gccagtgcgt cgtcgaccgg cccaggatcc gtgggaaacg tgatggacca 7914 tcgttgttcg agttctggaa cggtgggcaa accgtggagt tcgtacgggc gttcatcgac 7974 ttccatccgc gacgtgccgt cgaagggcgc agccacggtc tcgatgcaga gcagcgtgcg 8034 aggagtggtg tcgagaacag tgatcagcag gtcgccgatg gccatctttc cgcgcatgat 8094 ccagggatcg acacgggcac ggggttcgag aaacgcgacc acggcatcga gccacggcgt 8154 gccgagtgcg atgaatacgt gctgaccgga gctc 8188 <210> 8 <211> 566 <212> PRT <213> Rhodococcus erythropolis <400> 8 Met Asn Ala Thr Pro Ala Gln Ala Val Leu Ala Ile Arg Asp Leu Val 1 5 10 15 Ile Glu Phe Asp Ala Asp Gly Ser Arg Gly Ser Ser Thr Thr Val Val 20 25 30 Asp His Val Asn Leu Glu Leu Gly Phe Gly Glu Ile Leu Ala Leu Val 35 40 45 Gly Glu Ser Gly Ser Gly Lys Ser Leu Thr Ala Arg Ala Val Leu Gly 50 55 60 Leu Leu Pro Asp Gly Ala Gln Ala Arg Gly Leu Ile Gln Leu Gly Gly 65 70 75 80 Gln Gln Val Leu Gly Ala Asp Glu Ala Thr Leu Asn Gln Leu Arg Gly 85 90 95 Thr Arg Ala Ala Met Val Phe Gln Glu Pro Gln Thr Ala Leu Asn Pro 100 105 110 Val Gln Lys Val Gly Trp Gln Ile Ala Gln Ala Leu Arg Ala His Gly 115 120 125 Lys Ile Ser Arg Ala Asp Ala Arg Val Arg Ala Ile Asn Leu Leu Arg 130 135 140 Met Val Glu Ile Pro Glu Pro Glu Arg Arg Val Asp Trp Tyr Pro His 145 150 155 160 Gln Leu Ser Gly Gly Gln Lys Gln Arg Val Val Ile Ala Leu Ala Leu 165 170 175 Ser Gly Ser Pro Asp Leu Leu Ile Ala Asp Glu Pro Thr Thr Ala Leu 180 185 190 Asp Val Thr Val Gln Ala Glu Ile Leu Gln Leu Leu Arg Asn Leu Arg 195 200 205 Asp Arg Ser G ly Thr Ala Ile Leu Leu Ile Thr His Asn Met Gly Val 210 215 220 Val Ala Asp Ile Ala Asp Arg Val Leu Val Met Arg Ser Gly Arg Val 225 230 235 240 Val Glu Gln Gln Ser Val Phe Asp Leu Phe Ala Thr Pro Arg Glu Ser 245 250 255 Tyr Thr Gln Ala Leu Leu Ala Ala Val Pro Arg Leu Pro Glu Val Asp 260 265 270 Gln Pro Lys Pro Val Val Ala Leu Val Asp Ser Glu Gly Asp Ala Gln 275 280 285 Val Pro Pro Ile Leu Arg Phe Asp Ala Ala Ser Ile Val Tyr Pro Ala 290 295 300 300 Arg Leu Gly Asn Arg Glu Phe Arg Ala Val Asp Gln Val Ser Leu Thr 305 310 315 320 Val Gln Ala Gly Glu Val Val Gly Leu Val Gly Glu Ser Gly Ser Gly 325 330 335 Lys Thr Thr Leu Gly Arg Ala Ala Leu Gly Val Ile Arg Ala Ser Ser 340 345 345 350 Gly Ile Val Ala Phe Asp Gly Thr Asp Leu Gly Lys Ile Ser Ala Ala 355 360 365 Asn Leu Arg Gly Ile Arg Lys Gly Met Ala Leu Ile His Gln Asp Pro 370 375 380 Ala Ala Ser Leu Asp Pro Arg Arg Thr Val Gly Gln Ser Val Gly Glu 385 390 395 400 Pro Leu Glu Val His Arg Phe Ala Thr Gly Ala Leu Leu Arg Asn Arg 405 410 415 Val Gly Glu L eu Leu Glu Ser Val Arg Leu Pro Lys Ser Phe Ala His 420 425 430 Arg Ala Pro Gly Glu Leu Ser Gly Gly Gln Arg Gln Arg Val Ala Leu 435 440 445 Ala Arg Ala Leu Ala Leu Gly Pro Arg Leu Leu Val Ala Asp Glu Pro 450 455 460 Thr Ser Ala Leu Asp Val Ser Val Gln Ala Asp Val Leu Ser Leu Phe 465 470 475 480 Ala Asp Leu Arg Glu Glu Tyr Asn Phe Ala Cys Leu Phe Ile Ser His 485 490 495 Asp Leu Ala Val Val Asn Gln Val Ala Asp Arg Val Ala Val Leu Arg 500 505 510 Ser Gly Glu Leu Val Glu Glu Gly Pro Ala Ala Glu Val Phe Ser Ser 515 520 525 Pro Arg Gln Asp Tyr Thr Gln Gln Leu Val Ala Ala Val Pro Val Pro 530 535 540 Asp Pro Arg Arg Gln Arg Ala Gly Arg Glu Arg Glu Ser Leu Val Pro 545 550 555 560 Asp Leu Thr Val Ala Val 565 <210> 9 <211> 8188 <212> DNA <213> Rhodococcus erythropolis <220> <221> CDS <222> (6382) .. (7353) <400> 9 gagctcagtc tatgccgtcg gcgccgaaag tcggacatga gtgacagcga tgggtagggc 60 ggggcgagtc gggtaccccg aggaccgaag cgagagtgaa ggaggagaac cgatgcgtgt 120 gaaagcgatg gtcggaacag gcgcgatggc tgttgcgctg gccgccggcc cagttgccac 180 agcttctgct gaaggcgccg agacggcggg gtcggagggt tcaccggaga tgctgacctg 240 cgaggtggtc ttcgatgcac tggaagacat gagcccggaa tgggcggaag aggggatcga 300 tcaggtcgaa gactggtgtg aggcgatcac cggcgaagaa gactgaacgt cagccgcgcg 360 agatcactcg tcgcgacagt cgccaccgcc cgtcttcgag ttcgaactcg tcggcgtagc 420 tcccgacgcc actgagcttg ggagttgtgg tcgtgtcgac gtagaacgtc cacaccgcat 480 ccgcgcgggc tgtagtttcc gacgtcacgt ccacggcgat cgccgtggtg acgtgacgtg 540 tgaacgtgcc cgggccttgt atgccggcgg ctcgacgctc gattactcca gcagcaattt 600 cttcgcggcc acgtcgtacc tgtgcgggga taccgcgatc gggattggcc ggcatgttcc 660 attcggcctc gcgggtgaac tgctcgacgt agtcctcgac agagccggtg tcagccagat 720 gggcgatccg agcgagcgtt gaacgaatct gttcggcaag gctctcgggt tgcatgctgt 780 tctccgtacg tgtgagtgct gtggtggatc gcaggatatt gcccgtggac tgaccgaaca 840 ccgaggcgtc cc agtcagtg gcagcagttg agagaaattc agatcaccgt gagttcaact 900 atttccccga tatcagggga aatggttgag tatcccctga aggcaaaagc cttaattccg 960 accgggaggt ttcacaccgt gtccgtcgct cctcgatgtc ggctctgact tcttcagcgc 1020 cacaacgact gtcgtagcag ctcgcttttc tgaattcccg ggagtgagca tgtcggaacg 1080 acactctttc ccattctcgc aagcgtgtgc cctgagagtg cacagctgcg cacctattac 1140 gaagggcaac acctccgtga actcgtctct ttcgacggcc ggtctcgacc gccgtgcctt 1200 cttccgtggc agtgcgatcg ccgcattcgc catcttcggt agcggctcac tggccgcgtg 1260 ctcctctgca gtcggcgaac aacgctcgga cgcaggtgaa tcagttgagc cggttcgagg 1320 tgggacgctc acactggcga taccggacga cgtcagccct gcggcacttt tgaccagcac 1380 caccgttgcc ggtgtcacga tatccgggct cgtgtacgag accctgaccc gctatccact 1440 cgattcggta gtgccacagc cagttctggc caaggcctgg aagctgtccg acgacgggct 1500 gacgctcacg ctcaatctgc gcgatgacgt caccttccac tccggccggc gcttcacctc 1560 cgctgatgcg gagttctcgc tgcgcaccta tgcggacccg aaattctcgg cccagctcag 1620 gagtactgcg gccgccatca ccggcttcga ctcttcggat cccaatacgc tggttctcac 1680 cctcgcacac cgaaccggga acatcttcga tcttctcgat ctagcgccga tcttcgaccc 1740 cgaaacgttc gacaaggcgg tgaccggtga atccttcgtt ggtacgggcc ctttcgtatt 1800 cacctcacgc accccgaaca gttccatctc cttcgatcgg aacccgaact actgggtcgc 1860 cgagcggccg tacctcgatc acgtggaagc gcggatcgtt ccggacgccc aagcgcgcct 1920 gacatcgctg aagtccggac aggtctctct tgttgctccg gcgtcgttgc ccttccgtga 1980 ctcacagaac ttgaccaaga ccagcgggtt cgtgacaacc tcgatcgagg gtgcggaact 2040 acagatatac ctgggcacaa atgtcaccgc cgacgggctg accgatgtgc gggcacgaaa 2100 ggcgttggcc tacgcgatcg atcgtgaccg catcatcagt gaggtgtatc gcgatacggg 2160 gtatgcggtc aatctgccgt ggccgaagtc gtcgcccgcc tacgacgctg ccaagaacgc 2220 gacctacact cgcgatgtcg acaaggcgcg cgccctggtc ggcgatctgg gtgcactgcc 2280 gaccattccg ttgacctaca caggagcgag cccggatttc gaagccgccg cgcagatcgt 2340 gcaggccaat ctcgccgagg cagggatccg cgtcgaattg gatcctgtcg aggcatccat 2400 cttcgtcaag cagttgatcg gcgctgagtt caaaggactt tggctgacca atcacacttt 2460 tgcgcaatat gttccgtcaa ccctgacggt cagcgcctac ccgttcaacg ctgcacacaa 2520 tgcctcgaag ttcagttcgc cggc gtattc cgccgcagcg gagggtgcgt ggcaggttcc 2580 cgacggttcg agcgatgagg caaagaaact gtatgccgaa ttgggcacgc aactgttgaa 2640 cgagttgttc ttgatcgaga tcggtgttgt cgtgccgcag gtatcggcgg ccgagaccgt 2700 tcgtggtctg gcctggacaa agggtcgcca gccacaattc gccaacacat tcctggcgta 2760 ggggattcga tgattcgtta cattgcgggg cgactaccgt cggcggtggt ggttctcttc 2820 ctcgcgtcga tattgatctt ctcggtgatg cgtctggtgc ctggcgaccc cgcgttggct 2880 ctcgccggcc ccgatgcaac tccggaagcg attgcagcca ttcgtcattc gctcggtctc 2940 gacaggtcga ttcccgcgca gtacctgacc tggatcggcg acgtactgac attggatctc 3000 gggcgttcgt tcgttctcgg cggccagatt tccgatctgg tgctggcggg gttggggaac 3060 accgcagttc tggcgggaag tgcgttgctg ctggccgtcg tgttgagtct ggtgctcagt 3120 gttgcggtgg tggtgtggcc gaagaaatgg ctgacctcgg tggtgaacct tctcaacact 3180 ctctcggttg ctctgcccaa cttcgtgacc ggtgtcctcc tggttctcgt cttcgcggtg 3240 ctgattccgg ttctgccatc cggtggtgtt ccaccaggtg ggtatctcgc tcgtcccgac 3300 atcacgttcc agtacctgct gctgccgtca ctgtgcctcg cacttcctgt cgcggccgct 3360 ctcaccaggt tcctgtccga agcgctgcgc accgagatgg cgcagcagta cgtgatcacc 3420 gctcgcgcag ccggtgtgcc acggtggaac ttggttacac gcagtgcatt acgcaatgca 3480 ctgccaacta tgctcaccgt gctcggaatt cagaccgggc accttctcgg cggagccgta 3540 ctggttgagg cgatctttgc ctggcctggc atcggccaat tgatcgagca gggaatcggt 3600 cggcgggatt acccggtggt gcaggtgctg ctcctgctgt cggtcacgat cttcgttctg 3660 atccaacttc tgaccgacat cgtgcatgcc tacctcgacc cacgaatcag aatcggaggg 3720 cagcaatgac ggtggccgat cagtctgttg acgaaagggt tcggcgatct cgtcctgtgc 3780 gcgctgctct gacgcacggt caggggttgg cgggtgtgtt gatcctggtg gtcatcgcag 3840 tagcgggaat tgctgctcca ctgttgactc actacgggcc caacgagcag atcgagggtg 3900 caaatctgtt gggggccagt gcacagcact ggttcggaac agatcaggtc aaccgtgacg 3960 tcttcgcccg ttctctttac ggaatccgga tcaaccttgt catcgtgctc gtcgcggtgc 4020 cagcgggcgc gatcatcggt tcgctcgcag gcctggtgtc gagtatcaat tcggtggcag 4080 atgtcatcgc acagcggatc ttcgatgtga ttcttgcctt cccgctgttg attctggcga 4140 tcacgttggt cgcgatcacg gggccgggag tagcgccggt gatcgcggtg atcgtggctg 4200 ctgaaatacc cttgttcggg cgcctggtgc gaacg acggt gttgaaggtg cgcgaactcc 4260 cgttcgtgga gtcggccgag gtaatcggtg cggggcggtg gtgggtactg cgtaagcatg 4320 tgctgcccaa cgccgtagaa ccgctgggtg tgcagattgc gctctccatg tcggttgcgg 4380 tgttcgccga aagtgcgatg agttttctcg gaatcggcgt gcgcccgccg gatccgtctc 4440 tcggttcgat catcgcgggc gccattccga acctcgatgc caatccggcc tacgcgatcg 4500 gacctttggt gatcgtctcg gcgttggtcc tgagcttcct gctgatcgcc cagggactgg 4560 gcaaggcccg ccgaatctga aattgccacc gccaccacca cttcaaggac gtagtcatga 4620 atgcaacacc cgcacaggca gttctcgcca tccgggatct ggtgatcgag ttcgatgccg 4680 acggttcccg tggctcgtcg acaactgtgg tcgaccacgt caacctcgaa ttgggtttcg 4740 gtgagatatt ggcgttggtc ggcgagtccg gatccggtaa gtcgttgacc gcacgcgctg 4800 ttctcgggtt gttaccggac ggcgcgcagg cccgcgggtt gatccaactc ggtggccagc 4860 aggtgctcgg tgccgacgag gcgacgctca atcaattgcg ggggactcgg gccgcgatgg 4920 ttttccagga gcctcagact gcactgaatc cagtgcagaa ggtgggttgg cagatcgcgc 4980 aggcgctgcg ggcacacggc aagatctcgc gtgccgacgc cagagttcgc gcaatcaatc 5040 ttctacggat ggtggagatt ccggaaccgg agcgtcgggt cgactggtat ccgcatcaac 5100 tctcgggtgg acagaagcag cgggttgtca tcgcgctggc gttgtcgggt tctcctgatt 5160 tactcatcgc cgacgaaccg actactgcgc tcgacgtcac cgtgcaggcc gagattctgc 5220 aactcctgcg taatctgcgt gatcgttcgg gcactgcaat actgctcatc acacacaaca 5280 tgggtgtcgt tgcggacatc gcagatcgcg tgctggtgat gcgatcgggc agggtcgtcg 5340 agcagcagtc cgttttcgat ctctttgcga cgccgcgtga gtcgtacaca caggctttgc 5400 ttgcggccgt gccacgtctg ccggaggtcg accagccgaa gcctgtggtt gctctggtcg 5460 atagcgaagg cgacgcgcaa gtcccgccga tcctgcggtt cgacgcggcc tccatcgtgt 5520 acccggcgag gctgggcaac agagagtttc gtgcggtcga tcaggtgagc ctgacggttc 5580 aggccggcga ggttgtcgga ctggtcggtg aatcgggatc agggaagaca actctcggcc 5640 gggctgcgct cggtgttatc agggccagta gcggcatcgt tgctttcgac ggaaccgatc 5700 tcggaaagat ctccgccgcg aatcttcggg gcatccgcaa gggaatggcc ttgattcatc 5760 aggaccctgc ggcatctctc gatccgcgtc ggacggtcgg acaaagcgtc ggcgagccac 5820 tcgaagtcca tcgctttgcc accggagccc tgctgcgaaa ccgggtgggg gagttactcg 5880 aatcagttcg cctaccgaag tcttttgctc accgggcacc aggag aactt tccggtggtc 5940 aacgccagcg tgttgcgctg gcccgagcac ttgccctcgg accgagactg ctggtggccg 6000 atgagccgac cagcgctctg gatgtttccg tccaagccga tgttctttca ctctttgccg 6060 atctcaggga ggaatacaac ttcgcatgtc tgttcatcag ccacgatctt gcagtcgtca 6120 atcaggtagc cgaccgggta gcggtacttc gcagcgggga gttggtcgaa gagggccccg 6180 ctgcagaggt attcagctcg ccacggcagg actacacaca acagttggtt gccgcggtgc 6240 ctgttccaga tccgcgtcgt cagcgcgccg ggcgggaacg ggaatcactg gttcccgacc 6300 tgactgttgc cgtttgaccc gtctcgacat cgaacagcat taccaccacc tcaacctttt 6360 gcagactcat aggagcttga c atg tcc atc acc gaa ctc gaa cgc gtc act 6411 Met Ser Ile Thr Glu Leu Glu Arg Val Thr 1 5 10 cag acc ccg gag cag atc tac gct gcc ggc gga atc act gtc Gac Prog ag 59 Gln Ile Tyr Ala Ala Gly Gly Ile Thr Val His Lys 15 20 25 gtc ggt gag ctg atc ggc gcc cgc atc gac ggc gta cac ctc tct ggg 6507 Val Gly Glu Leu Ile Gly Ala Arg Ile Asp Gly Val His Leu Ser Gly 30 35 40 gat ctg tcc gaa gag acc gcc tac gcg atc aac tat gcg ctg gca gcg 6555 Asp Leu Ser Glu Glu Thr Ala Tyr Ala Ile Asn Tyr Ala Leu Ala Ala 45 50 55 cac aag gtt gtg ttc ttc cgt ggg cag cag cat ctc gac gac acg tcg 6603 His Lys Val Val Phe Phe Arg Gly Gln Gln His Leu Asp Asp Thr Ser 60 65 70 cag tac gaa ttc gcc gga acc ctc ggc agc cag acc acc acg cat ccc 6651 Gln Tyr Glu Phe Ala Gly Thr Leu Gly Ser Gln Thr Thr Thr His Pro 75 80 85 90 act ctc aag tcc aag gac aac aag ctg ttg gtg ctc gac ggc gct gcc 6699 Thr Leu Lys Ser Lys Asp Asn Lys Leu Leu Val Leu Asp Gly Ala Ala 95 100 105 agc agc tgg cac acc gac gtc act ttc atc gat cgc atc ccg aag gca 6747 Ser Ser Trp His Thr Asp Val Thr Phe Ile Asp Arg Ile Pro Lys Ala 110 115 120 tcg atc ctg cgc gcg acc aca att cct gag tac ggc gga gcc acc acg 6795 Ser Ile Leu Arg Ala Thr Thr Ile Pro Glu Tyr Gly Gly Ala Thr Thr 125 130 135 tgg gcg tct acc acg gct gcg tac aac caa ctg ccg cat tcg ctc aag 6843 Trp Ala Ser Thr Thr Ala Ala Tyr Asn Gln Leu Pro His Ser Leu Lys 140 145 150 gta ttg gtc gag aat ctg cgc gct gtc cac acc aat gca tac gac tac Val Leu Val Glu Asn Leu Arg Ala Val His Thr Asn Ala Tyr Asp Tyr 155 160 165 170 gcc gag att atc gac aag gtg aag caa ggc gac gct cag cgc gtg acg 6939 Ala Glu Ile Ile Asp Lys Val Lys Gln Gly Asp Ala Gln Arg Val Thr 175 180 185 aac tac tcc gaa ttc act cgc gaa atc tac gag acg gag cat ccg gtg 6987 Asn Tyr Ser Glu Phe Thr Arg Glu Ile Tyr Glu Thr Glu His Pro Val 190 195 200 gtt cgg gtc cat cct gcg aca ggg gag aag act ctg ctt ctc ggt cac 7035 Val Arg Val His Pro Ala Thr Gly Glu Lys Thr Leu Leu Leu Gly His 205 210 215 ttc gtc aag gag ttc gtc gga ctg aag ccg tcg gag tcg gtt gct ctc 7083 Phe Val Lys Glu Gly Leu Lys Pro Ser Glu Ser Val Ala Leu 220 225 230 tac cag ctt ctg cag gca cga atc atc aag ttg gaa aac acc gtt cgg 7131 Tyr Gln Leu Leu Leu Gln Ala Arg Ile Ile Lys Leu Glu Asn Thr Val Arg 235 240 245 250 tgg tca tgg gct ccc ggt gat ctg gcg atc tgg gac aac cag gca acc 7179 Trp Ser Trp Ala Pro Gly Asp Leu Ala Ile Trp Asp Asn Gln Ala Thr 255 260 265 cag cat tac ggg att tcc gat tac ggg aca cag gcg c gc agc gtg cat 7227 Gln His Tyr Gly Ile Ser Asp Tyr Gly Thr Gln Ala Arg Ser Val His 270 275 280 cgc gtg acg ttg gcc ggt gac gtt ccc gtc gac gtc cac ggt gag cag 7275 Arg Val Thr Leu Ala Gly Asp Val Pro Val Asp Val His Gly Glu Gln 285 290 295 agc cgc atc atc aag ggc gat gca tcc gag ttc tcg atc gtt gca gac 7323 Ser Arg Ile Ile Lys Gly Asp Ala Ser Glu Phe Ser Ile Val Ala Asp 300 305 310 atc gac cgg ctt ccc ggc ttt gcc gca aac tgatcagtcc tgctgcgggt 7373 Ile Asp Arg Leu Pro Gly Phe Ala Ala Asn 315 320 gatccggcgg gaacatctgt ccccatgagt gattgcatgc tcggcacgtc cattccgggg 7433 tgtccggtcc cacgacgcag cctgttccgg cgagccacgg taggtgatcg aatggatgga 7493 ccggcatccc cggggaacac tgagcggttc tgtgtgcacc gcagagtggg cacgacggat 7553 ttttcgccag catgtagtcg atgaagttgg tgtgcccgga cgtggccatc cttccggtgc 7613 atttgcggca caggacggct gggtagtcgt caccaggttc agcctgcctg aagatccgtg 7673 atgcggaatg aaagtgaagg cgatcgctgc tgttgaactt tcgcaacggc atcggtgagc 7733 tgcaacctgt gcagttgccc cgctcgtcca tgcgtcg cgctcgtcca tgcgtcgt ggatccag cgtgtcgatg tcgccgaagt agtgcgcata gtgtgactgg ccggccgtga 7853 gaatccggtc ggccagtgcg tcgtcgaccg gcccaggatc cgtgggaaac gtgatggacc 7913 atcgttgttc gagttctgga acggtgggca aaccgtggag ttcgtacggg cgttcatcga 7973 cttccatccg cgacgtgccg tcgaagggcg cagccacggt ctcgatgcag agcagcgtgc 8033 gaggagtggt gtcgagaaca gtgatcagca ggtcgccgat ggccatcttt ccgcgcatga 8093 tccagggatc gacacgggca cggggttcga gaaacgcgac cacggcatcg agccacggcg 8153 tgccgagtgc gatgaatacg tgctgaccgg agctc 8188 <210> 10 <211> 324 <212> PRT <213> Rhodococcus erythropolis <400> 10 Met Ser Ile Thr Glu Leu Glu Arg Val Thr Gln Thr Pro Glu Gln Ile 1 5 10 15 Tyr Ala Ala Gly Gly Ile Thr Val His Lys Val Gly Glu Leu Ile Gly 20 25 30 Ala Arg Ile Asp Gly Val His Leu Ser Gly Asp Leu Ser Glu Glu Thr 35 40 45 Ala Tyr Ala Ile Asn Tyr Ala Leu Ala Ala His Lys Val Val Phe Phe 50 55 60 Arg Gly Gln Gln His Leu Asp Asp Thr Ser Gln Tyr Glu Phe Ala Gly 65 70 75 80 Thr Leu Gly Ser Gln Thr Thr Thr His Pro Thr Leu Lys Ser Lys Asp 85 90 95 Asn Lys Leu Leu Val Leu Asp Gly Ala Ala Ser Ser Trp His Thr Asp 100 105 110 Val Thr Phe Ile Asp Arg Ile Pro Lys Ala Ser Ile Leu Arg Ala Thr 115 120 125 Thr Ile Pro Glu Tyr Gly Gly Ala Thr Thr Trp Ala Ser Thr Thr Ala 130 135 140 Ala Tyr Asn Gln Leu Pro His Ser Leu Lys Val Leu Val Glu Asn Leu 145 150 155 160 Arg Ala Val His Thr Asn Ala Tyr Asp Tyr Ala Glu Ile Ile Asp Lys 165 170 175 Val Lys Gln Gly Asp Ala Gln Arg Val Thr Asn Tyr Ser Glu Phe Thr 180 185 190 Arg Glu Ile Tyr Glu Thr Glu His Pro Val Val Arg Val His Pro Ala 195 200 205 Thr Gly Glu L ys Thr Leu Leu Leu Gly His Phe Val Lys Glu Phe Val 210 215 220 Gly Leu Lys Pro Ser Glu Ser Val Ala Leu Tyr Gln Leu Leu Gln Ala 225 230 235 240 Arg Ile Ile Lys Leu Glu Asn Thr Val Arg Trp Ser Trp Ala Pro Gly 245 250 255 Asp Leu Ala Ile Trp Asp Asn Gln Ala Thr Gln His Tyr Gly Ile Ser 260 265 270 Asp Tyr Gly Thr Gln Ala Arg Ser Val His Arg Val Thr Leu Ala Gly 275 280 285 Asp Val Pro Val Asp Val His Gly Glu Gln Ser Arg Ile Ile Lys Gly 290 295 300 Asp Ala Ser Glu Phe Ser Ile Val Ala Asp Ile Asp Arg Leu Pro Gly 305 310 315 320 Phe Ala Ala Asn

[Brief description of the drawings]

[Fig. 1] 2 of the proteins contained in the lysate of KA2-5-1 strain
3 shows a two-dimensional electrophoresis image.

FIG. 2 shows a restriction map of a gene fragment of a phage clone showing a positive result in the screening of Example 5.

FIG. 3. Rhodococcus-E. Coli shuttle vector pRHK1
Is shown.

FIG. 4 shows the structures of plasmids pRKPP and PRKPPR.

FIG. 5 shows the structure of plasmid pRKI5.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Masanori Suzuki 136-1 Nishikubo Apartment 1-134 Nishikubo, Shimizu-shi, Shizuoka (72) Inventor Kenji Maruhashi 1-6-1, Nishikubo, Shimizu-shi, Shizuoka Yoneju A-201 F term (reference) 4B024 AA17 BA08 BA80 CA04 DA06 EA04 GA11 HA01 4B050 CC03 DD02 LL10 4H045 AA10 BA10 CA11 EA61 FA74

Claims (10)

[Claims] 1. A gene encoding the following protein (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 2; (b) a protein represented by the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids are deleted, substituted, or added; and A protein having the same function as the protein of a). 2. A gene encoding the following protein (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 4; (b) a protein represented by the amino acid sequence of SEQ ID NO: 4 in which one or more amino acids are deleted, substituted, or added; and A protein having the same function as the protein of a). 3. A gene encoding the following protein (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 6; (b) represented by an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 6, and ( A protein having the same function as the protein of a). 4. A gene encoding the following protein (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 8; (b) a protein represented by the amino acid sequence of SEQ ID NO: 8 in which one or more amino acids have been deleted, substituted, or added; and A protein having the same function as the protein of a). 5. A gene encoding the following protein (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 10; (b) represented by an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 10, and ( A protein having the same function as the protein of a). 6. A protein represented by the following (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 2; (b) a protein represented by the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids are deleted, substituted, or added; and A protein having the same function as the protein of a). 7. A protein represented by the following (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 4; (b) a protein represented by the amino acid sequence of SEQ ID NO: 4 in which one or more amino acids are deleted, substituted, or added; and A protein having the same function as the protein of a). 8. A protein represented by the following (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 6; (b) represented by an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 6, and ( A protein having the same function as the protein of a). 9. A protein represented by the following (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 8; (b) a protein represented by the amino acid sequence of SEQ ID NO: 8 in which one or more amino acids have been deleted, substituted, or added; and A protein having the same function as the protein of a). 10. A protein represented by the following (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 10; (b) represented by an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 10, and ( A protein having the same function as the protein of a).