JP2001008689A - Polyester polymerase and gene encoding the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a novel polyester polymerase which comprises a protein containing a specific amino acid sequence and exhibiting polyester polymerase activity and is useful in the production or the like of a polyester that is a soft polymeric material having plasticity as a biodegradable plastic. SOLUTION: This protein is a novel polyester polymerase protein comprising a protein containing the amino acid sequence represented by the formula or a protein which contains an amino acid sequence in which one or several numbers of amino acids are deleted, substituted or added to the amino acid sequence represented by the formula and exhibits polyester polymerase activity and the polymerase is useful in the production or the like of a polyester, e.g. poly-3- hydroxy-alkane acid or the like, which is a soft polymeric material having plasticity as a biodegradable plastic. This protein is obtained by separating a chromosomal DNA from Pseudomonas sp. strain 61-3, preparing a library by a conventional method, screening out the library by a probe comprising the partial base sequence and integrating the resultant gene into an expression system to express.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a polyester polymerizing enzyme, a gene encoding the enzyme, a recombinant vector containing the gene, a transformant transformed by the vector, and a polyester polymerization using the transformant. The present invention relates to a method for producing an enzyme.

[0002]

2. Description of the Related Art Polyesters (e.g., poly-3-hydroxyalkanoic acid) biosynthesized by microorganisms are biodegradable plastics having a wide variety of properties from thermoplastic to hard to viscoelastic rubber. Poly-3-hydroxybutanoic acid composed of monomer units having 4 carbon atoms (P (3H
B)) is a typical polyester, but a rigid and brittle polymeric material, which limits its use. For this reason, various copolymerized polyesters including P (3HB-co-3HV) copolymerized with 3-hydroxyvaleric acid (3HV), a monomer unit having 5 carbon atoms, by adding propionic acid or the like to the medium. And attempts have been made to change the physical properties of the polyester. On the other hand, polyester comprising a monomer unit having 6 or more carbon atoms is a soft polymer material having plasticity.

[0003] Polyester-synthesizing bacteria can be roughly classified into two types: those that synthesize polyesters having 4 to 5 carbon atoms as monomer units and those that synthesize polyesters having 6 to 12 carbon atoms as monomer units. it can. The former bacterium has a polyester synthase using a C4-5 monomer unit as a substrate, and the latter bacterium has a polyester synthase using a C6-12 monomer unit as a substrate. Therefore, polyesters having different properties are synthesized by each microorganism. However, since the polyester synthases from the known microorganisms as described above have different substrate specificities, the monomer units having 4 to 5 carbon atoms and the monomer units having 6 to 12 carbon atoms are copolymerized by one kind of enzyme. It is difficult to synthesize a modified polyester.

[0004]

SUMMARY OF THE INVENTION The present invention relates to a polyester synthase using a monomer unit having a wide range of carbon atoms as a substrate.
It is an object of the present invention to provide a gene encoding the enzyme, a recombinant vector containing the gene, a transformant transformed with the vector, and a method for producing a polyester synthase using the transformant.

[0005]

Means for Solving the Problems As a result of intensive studies based on the above problems, the present inventors succeeded in cloning the gene for polyester synthase from a microorganism belonging to the genus Pseudomonas isolated from soil, The present invention has been completed.

That is, the present invention relates to the following protein (a) or (b): (a) a protein containing the amino acid sequence represented by SEQ ID NO: 2 (b) an amino acid sequence represented by SEQ ID NO: 2 containing an amino acid sequence in which one or several amino acids are deleted, substituted or added, and a polyester polymer Protein with enzymatic activity

Further, the present invention relates to a polyester synthase gene encoding the following protein (a) or (b). (a) a protein containing the amino acid sequence represented by SEQ ID NO: 2 (b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and a polyester synthase Active protein

Further, the present invention relates to the following (a) or (b):
It is a gene containing A. (a) a DNA containing the nucleotide sequence represented by SEQ ID NO: 1 (b) a protein that hybridizes under stringent conditions to a DNA containing the nucleotide sequence represented by SEQ ID NO: 1 and has a polyester synthase activity DNA to encode

Further, the present invention is a recombinant vector containing the gene. Furthermore, the present invention is a transformant containing the recombinant vector. Further, the present invention provides
A method for producing a polyester synthase, which comprises culturing the transformant and collecting a polyester synthase protein from the resulting culture. Further, the present invention is a method for producing a polyester, comprising culturing the transformant and collecting the polyester from the resulting culture. Hereinafter, the present invention will be described in detail.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Cloning of Polyester Synthase Gene The polyester synthase gene of the present invention is isolated from the cells of a microorganism belonging to the genus Pseudomonas. First, chromosomal DNA is prepared from a strain having a polyester synthase gene. Examples of the strain include Pseudomonas sp. Strain 61-3 (Pseudomonas sp. JCM100
15). Known methods can be used for the preparation of chromosomal DNA. For example, Pseudomonas sp.
After culturing the 61-3 strain in a bouillon medium, the hexadecyltrimethylammonium bromide method (Currnt Protocols in Mole
Chromosomal DNA is prepared according to the method described in Vol. 1, pp. 2.4.3, John Wiley & Sons, 1994).

The DNA obtained by the above method is partially digested with an appropriate restriction enzyme (eg, Sau3AI, BamHI, BglII, etc.). This is cleaved with a restriction enzyme (for example, BamHI, BglII, etc.), treated with alkaline phosphatase, and ligated with a dephosphorylated vector to prepare a library. A phage or plasmid capable of autonomous propagation in a host microorganism is used as the vector. Examples of phage vectors include EMBL3, M13, λgt11 and the like,
As plasmid vectors, for example, pBR322, pUC18, p
Bluescript II (manufactured by STRATAGENE) and the like. In addition, Escherichia coli and Bacillus brevis
(Bacillus brevis) or other vectors capable of autonomous propagation in two or more host microorganisms, as well as various shuttle vectors. Such a vector can also be cleaved with the restriction enzyme to obtain a fragment thereof.

A known DNA ligase is used to ligate the DNA fragment and the vector fragment. Then, the DNA fragment and the vector fragment are annealed and then ligated to prepare a recombinant vector. The introduction of the recombinant vector into the host microorganism can be performed by a known method. For example, when the host microorganism is Escherichia coli, the calcium chloride method (Le
derberg, EM et al., J. Bacteriol. 119, 1072 (197
4)) and electroporation (Current Protocols
in Molecular Biology, 1, 1.8.4, 1994), and when the host microorganism is phage DNA, the in vitro packaging method (Current Protocols in
Molecular Biology, Vol. 1, p. 5.7.1, 1994). In the present invention, an in vitro packaging kit (Gigapack II; manufactured by STRATAGENE or the like) may be used.

Next, a probe for obtaining a DNA fragment containing the polyester synthase gene of Pseudomonas sp. Strain 61-3 is prepared. Several types of amino acid sequences of polyester synthase are already known (Peoples, OP and Sinskey, AJ, J. Biol. Che.
m., 264, 15293 (1989); Huisman, GW et al., JB
iol. Chem., 266, 2191 (1991); Pieper, U. et al., F.
EMS Microbiol. Lett., 96, 73 (1992); Timm, A. and
Steinbuchel, A., Eur.J. Biochem., 209, 15 (1992)
other). Therefore, a well-conserved region is selected from these amino acid sequences, and a nucleotide sequence encoding the region is estimated to design an oligonucleotide. These oligonucleotides include Timm, A. and Steinbuchel,
The following sequence reported by A., Eur. J. Biochem., 209, 15 (1992): 5'-CC (G / C) CAGATCAACAAGTT (C / T) TA (C / G)
GAC-3 ′ (SEQ ID NO: 5), but is not limited thereto.

Next, the synthetic oligonucleotide is labeled with an appropriate reagent, and colony hybridization is performed from the chromosomal DNA library (Currnt Prototype).
(Tocols in Molecular Biology, Vol. 1, p. 6.0.3, 1994)
. Escherichia coli screened by colony hybridization was analyzed using the alkaline method (Currnt Protocols i).
The DNA fragment containing the polyester synthase gene can be obtained by recovering the plasmid according to Molecular Biology, Vol. 1, page 1.6.1 (1994). The nucleotide sequence of the DNA fragment is determined by a known method such as the Sanger method (Molecula method).
r Cloning, vol. 2, p. 13.3, 1989) and the like, and can be carried out using an automatic base sequence analyzer, for example, a 373A DNA sequencer (Applied Biosystems).

SEQ ID NO: 1 shows the nucleotide sequence of the polyester synthase gene of the present invention, and SEQ ID NO: 2 shows the amino acid sequence of the polyester synthase of the present invention. A protein containing these amino acid sequences has the activity of a polyester synthase. As long as one or several amino acids are mutated in the amino acid sequence, mutations such as deletion, substitution, and addition may occur.

For example, one of the amino acid sequences represented by SEQ ID NO: 2, preferably 10 to 20, more preferably 5
~ 10 amino acids may be deleted, or one in the amino acid sequence represented by SEQ ID NO: 2, preferably 10-20,
More preferably, 5 to 10 amino acids may be added, or 1 of the amino acid sequence represented by SEQ ID NO: 2 may be added.
, Preferably 10-20, more preferably 5-10 amino acids may be replaced by other amino acids. In addition, DNAs capable of hybridizing with the above genes under stringent conditions are also included in the genes of the present invention. The stringent conditions are, for example, a temperature of 50 to 68 ° C,
Preferably 55-60 ° C, sodium concentration 50-150 m
M, preferably at 80-100 mM.

In order to introduce a mutation into a gene, Kunk
For example, a kit for mutagenesis using a site-directed mutagenesis method (for example, Mutan-K (manufactured by TAKARA)) by a known method such as an el method, a gapped duplex method or a method equivalent thereto,
Mutan-G (TAKARA)) or TAKAR
It can be performed using LA PCR in vitro Mutagenesis series kit from Company A. After the nucleotide sequence is determined by the above method, by chemical synthesis or chromosome DN
The gene of the present invention can be obtained by a PCR method using A as a template or by hybridization using a DNA fragment having the nucleotide sequence as a probe.

2. Preparation of Recombinant Vector and Transformant The recombinant vector of the present invention can be obtained by ligating (inserting) the gene of the present invention into an appropriate vector. It can be obtained by introducing a recombinant vector into a host so that the gene of the present invention can be expressed.

The host is not particularly limited as long as it can express the gene of interest. For example, a bacterium belonging to the genus Ralstonia such as Ralstonia eutropha, Pseudomonas putida (Pseudom
onas putida), bacteria belonging to the genus Pseudomonas, bacteria belonging to the genus Bacillus such as Bacillus subtilis, Escherichia coli (Escherichia col.
Bacteria belonging to the genus Escherichia such as i), yeast belonging to the genus Saccharomyces such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida maltosa (Candida maltosa) and Candida (Candid maltosa)
a) Yeast belonging to the genus, animal cells such as COS cells, CHO cells and the like.

Further, after introducing a plasmid containing the cloned polyester synthase gene into a bacterium deficient in polyester polymerization ability, the resulting transformant is cultured, and the polyester accumulated in the cells is analyzed. The activity of the polymerizing enzyme can be examined. Bacteria deficient in polyester polymerization ability include Ralstonia eutropha PHB ^- 4 and Pseudomonas putida GPp104.

When a bacterium belonging to the genus Ralstonia or Escherichia coli is used as a host, the recombinant DNA of the present invention is used.
Is preferably capable of autonomously replicating in the host, and also contains a promoter, the DNA of the present invention, and a transcription termination sequence. As an expression vector, pLA2917 (AT
(CC 37355) or pJRD215 (ATCC 37
533) and the like.

Any promoter can be used as long as it can be expressed in the host. For example, trp promoter, lac promoter, PL promoter, P
Promoters derived from Escherichia coli and phages, such as the R promoter and T7 promoter, are used. As a method for introducing the recombinant DNA into bacteria, for example, a method using calcium ions (Current Protocols in Molecular Bio
Logic, 1, 1.8.1, 1994) and electroporation (Current Protocols in Molecular Biology, 1).
Vol. 1.8.4, 1994).

When yeast is used as a host, examples of expression vectors include YEp13 and YCp50. As the promoter, for example, gal 1 promoter, gal 10
Promoters and the like. Methods for introducing recombinant DNA into yeast include, for example, electroporation (Methods Enzymol., 194, 182-187 (1990)) and spheroplast method (Proc. Natl. Acad. Sci. USA, 84, 1929-). 193
3 (1978)), lithium acetate method (J. Bacteriol., 153, 163).
-168 (1983)). When using an animal cell as a host, for example, pcDNAI, pcDN
AI / Amp (Invitrogen) or the like is used. Examples of a method for introducing a recombinant DNA into animal cells include an electroporation method and a calcium phosphate method.

3. Production of Polyester Synthase The polyester synthase of the present invention is obtained by culturing the transformant of the present invention in a medium, producing and accumulating the polyester synthase of the present invention in a culture (cultured cells or culture supernatant). It is performed by collecting the polyester synthase from the culture. The method for culturing the transformant of the present invention in a medium is performed according to a usual method used for culturing a host. Examples of a medium for culturing a transformant obtained by using a bacterium such as Escherichia coli as a host include a complete medium or a synthetic medium such as LB medium and M9 medium. The culture temperature is 25-37
Polyester synthase is accumulated in the microbial cells by aerobically culturing for 12 to 48 hours in a temperature range of ℃ and recovered.

The carbon source is necessary for the growth of microorganisms, and examples thereof include carbohydrates such as glucose, fructose, sucrose, and maltose. Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, as well as peptone, meat extract, yeast extract, corn steep liquor, and the like. Further, examples of the inorganic substance include monobasic potassium phosphate, dibasic potassium phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride.

The cultivation is usually carried out under aerobic conditions such as shaking culture at 25 to 37 ° C. for 2 hours or more after the induction of expression. During the culture,
Antibiotics such as kanamycin, ampicillin and tetracycline may be added to the medium. When culturing a microorganism transformed with an expression vector using an inducible promoter, an inducer can be added to the medium. For example, isopropyl-β-D-thiogalactopyranoside (IPTG), indoleacrylic acid (IAA) and the like can be added to the medium.

As a medium for culturing the transformant obtained using animal cells as a host, for example, RPMI-1640, DMEM medium or a medium obtained by adding fetal bovine serum to these mediums is used. Cultivation is usually performed in the presence of 5% CO ₂ at 30-37 ° C.
Perform for ~ 7 days. During culture, antibiotics such as kanamycin and penicillin may be added to the medium. To purify the polyester synthase, the obtained culture is collected by centrifugation (cells are disrupted with a sonicator), and affinity chromatography, cation or anion exchange chromatography, gel filtration, etc. are used alone or in an appropriate combination. Can be done by Confirmation that the obtained purified substance is the target enzyme is carried out by a usual method, for example, S
It is performed by DS polyacrylamide gel electrophoresis, western blotting or the like.

4. Production of Polyester Polyester can be produced by culturing the transformant obtained in the above item 2 in a suitable medium. As a medium, SM medium containing gluconic acid, octanoic acid, dodecanoic acid, tetradecanoic acid, etc. as a sole carbon source (Kato, M. et al., Appl. Microbiol. Biotechnol. 4)
5, 363 (1996)). The intracellular content of polyester accumulated in cells and the composition of polyester are as follows:
Kato et al. (Kato, M. et al., Appl. Microbiol. Bi
otechnol. 45, 363 (1996); Kato, M. et al., Bull. Ch.
According to em. Soc. Jpn. 69, 515 (1996)), after extraction from cells with an organic solvent such as acetone, the extract can be measured and analyzed by subjecting it to gas chromatography, NMR, etc. . In addition, 3-hydroxybutanoic acid (3H
B) A polyester having a high fraction can be synthesized, and a flexible polyester can be obtained.

[0029]

The present invention will be described more specifically with reference to the following examples. However, the present invention does not limit the technical scope to these examples. Example 1 Cloning of Polyester Synthase Gene of Pseudomonas sp. 61-3 Strain First, a chromosomal DNA library of Pseudomonas sp. 61-3 was prepared. Pseudomonas sp. 61-3 strain (Pseudomona
s sp. JCM 10015) in 100 ml of bouillon medium (1% meat extract, 1% peptone, 0.5% sodium chloride, pH 7.2)
After culturing at 30 ° C. overnight, the hexadecyltrimethylammonium bromide method (Currnt Protocols in Molecular Biolog
y, Vol. 1, 2.4.3., 1994; published by John Wiley & Sons)
As a result, chromosomal DNA was obtained.

The obtained chromosomal DNA was partially digested with the restriction enzyme Sau3AI. As a vector plasmid, cosmid vector pLA2917 (ATCC 37355) was used.
This plasmid is digested with the restriction enzyme BglII and dephosphorylated (Molecular Cloning, Vol. 1, 5.7.2, 1989; Col.
d Spring Harbar Laboratory publication), and ligated to a partially degraded fragment of chromosomal DNA using DNA ligase. In vitro packaging method using this ligated DNA fragment (Current Protocolsin Molecular Biology, 1
Volume, p. 5.7.2, 1994) by Escherichiaco
li) Transform S17-1 strain and use Pseudomonas sp.
A chromosomal DNA library of 61-3 strains was obtained.

Next, a probe for obtaining a DNA fragment containing a polyester synthase gene of Pseudomonas sp. Strain 61-3 was prepared. Timm, A. and Steinbuchel,
A ', Eur. J. Biochem., 209, 15 (1992), 5'-CC (G / C) CAGATCAACAAGTT (C / T) TA (C / G) GAC-3' (SEQ ID NO: An oligonucleotide having the base sequence of 5) was synthesized. The oligonucleotide was labeled with digoxigenin using a DIG oligonucleotide tailing kit (Boehringer Mannheim) to obtain a probe.

Using the obtained probe, Escherichia coli having a plasmid containing a polyester synthase gene was isolated from a chromosomal DNA library of Pseudomonas sp. Strain 61-3 by colony hybridization.
DNA was prepared from the obtained Escherichia coli, digested with EcoRI and PstI, and subjected to Southern hybridization with the aforementioned probe. As a result, a band was detected at about 6.0 kbp.
The full-length nucleotide sequence of this EcoRI-PstI fragment was determined by the Sanger method. The determined nucleotide sequence is shown in SEQ ID NO: 6.

As shown in FIG. 1 (a), several genes were found in this sequence.
Pseudomonas oleovor
anns) and Pseudomonas aeruginosa (Pseudomona
s aeruginosa), similar to those found in the pha locus. It is thought that this encodes a polyester synthase (SEQ ID NO: 2) consisting of 560 amino acids.
phaC2 _Ps gene (SEQ ID NO: 1) and 559 amino phaC1 believed to encode a polyester synthase (SEQ ID NO: 4) consisting of an amino acid _Ps gene (SEQ ID NO: 3) were present. The homology between the amino acid sequences of these two polyester synthases was 53.2%.

Example 2 Production of Polyester Using the Polyester Synthase Gene-Introduced Strain of the Present Invention The polyester synthase gene obtained in Example 1 was introduced into a polyester deficient strain, and the polyester accumulated in cells was removed. Measured and analyzed. The DNA fragment in the plasmid used for introducing the polyester synthase gene is shown in FIG. 1 (b). The measurement of the intracellular polyester content was performed by extracting the polyester from the cells with acetone and then subjecting the extract to gas chromatography, and the composition analysis of the polyester was performed by NMR analysis of the extract.

That is, plasmid pJASc60 (containing the phaC1 _Ps gene and phaC2 _Ps gene) containing both of the two polyester synthase genes obtained in Example 1 or plasmid pJASc22 containing one of the genes
(Containing the phaC1 _Ps gene) or pJASc60dC1Z (phaC2 _Ps
Ralstonia eutropha PHB ^- 4 (Ralstonia eutropha DSM541 strain)
Was transformed by the conjugative transfer method, and the resulting transformant was transformed with M containing 2% gluconic acid, 0.5% octanoic acid, 0.5% dodecanoic acid or 0.5% tetradecanoic acid as the sole carbon source.
S medium (Kato, M. etal., Appl. Microbiol. Biotechno
l. 45, 363 (1996)) and cultured with shaking at 30 ° C. for 72 hours to examine the polyester accumulated in the cells. The results are shown in Table 1.

[0036]

[Table 1]

As shown in Table 1, it was confirmed that polyester was accumulated in the cells of all the strains. When gluconic acid was cultured using carbon as the carbon source, a polyester containing only 4-hydroxybutanoic acid (3HB) having 4 carbon atoms as a monomer unit was accumulated in each strain, and octanoic acid, decanoic acid, or tetradecanoic acid was converted to carbon. When cultured as a source, in addition to 3HB as a monomer unit, 3-hydroxyhexanoic acid (3HHx) having 6 carbon atoms, 3-hydroxyoctanoic acid (3HO) having 8 carbon atoms, and 3-hydroxydecane having 10 carbon atoms. acid
(3HD), a copolyester containing 12-carbon 3-hydroxydodecanoic acid (3HDD) and the like were accumulated. From the above results, it was found that the enzyme of the present invention can use a monomer unit having a wide carbon number of 4 to 12 as a substrate.

[0038]

Industrial Applicability According to the present invention, there are provided a gene encoding a polyester synthase, a recombinant vector containing the gene, and a transformant transformed with the vector. INDUSTRIAL APPLICABILITY The gene of the present invention encodes a polyester synthase using a monomer having a wide range of carbon numbers as a substrate, and is useful for producing a copolymer polyester having various physical properties.

[0039]

[Sequence List] SEQUENCE LISTING <110> The Institute of Physical and Chemical Research; Japan Science and Technology Corporation <120> Polyester Synthase and a Gene Coding for the Same <130> RJH11-031 <160> 6 <210> 1 < 211> 1683 <212> DNA <213> Pseudomonas sp.strain 61-3 <220> <221> CDS <222> (1) .. (1680) <400> 1 atg aga gag aaa cca acg ccg ggc ttg ctg ccc aca ccc gcg acg ttc 48 Met Arg Glu Lys Pro Thr Pro Gly Leu Leu Pro Thr Pro Ala Thr Phe 1 5 10 15 atc aac gct cag agt gcg att acc ggt ctg cgc ggc cgg gat ctg ttc 96 Ile Asn Ala Gln Ser Ala Ile Thr Gly Leu Arg Gly Arg Asp Leu Phe 20 25 30 tcg acc ctg cgc agc gtg gcc gcc cac ggc ctg cgt cac ccg gtg cgc 144 Ser Thr Leu Arg Ser Val Ala Ala His Gly Leu Arg His Pro Val Arg 35 40 45 agc gcc cgt cat gtt ctg gca ctg ggc ggc cag ttg ggc cgc gtg ctg 192 Ser Ala Arg His Val Leu Ala Leu Gly Gly Gly Gln Leu Gly Arg Val Leu 50 55 60 ctg ggc gaa acg ctg cac acg ccg aac ccg gac ac gc gcac gc gc 240 Leu Gly Glu Thr Leu His Thr Pro Asn Pro Lys Asp Asn Arg Phe Ala 65 70 75 80 gac ccg acc tgg aga ctg aat ccg ttt tac cgg cgc agc ctg cag gcc 288 Asp Pro Thr Trp Arg Leu Asn Pro Phe Tyr Arg Arg Ser Leu Gln Ala 85 90 95 tat ctg agc tgg cag aaa cag gtc aaa agc tgg atc gat gaa agc ggc 336 Tyr Leu Ser Trp Gln Lys Gln Val Lys Ser Trp Ile Asp Glu Ser Gly 100 105 110 atg agt gac gat gac cgc gcc cgc gcg cat ttc gtc ttc gca ctg Ascp Asp 384 Asp Arg Ala Arg Ala His Phe Val Phe Ala Leu Leu 115 120 125 aat gac gcc gtg tcc ccc tcc aat acc ctg ctc aac ccg cta gcg atc 432 Asn Asp Ala Val Ser Pro Ser Asn Thr Leu Leu Asn Pro Leu Ala Ile 130 135 140 aag gag ctg ttc aac tcc ggt ggc aac agc ctg gtc cgc ggt ctc agc 480 Lys Glu Leu Phe Asn Ser Gly Gly Asn Ser Leu Val Arg Gly Leu Ser 145 150 155 160 cat tta ttc gac gac ctg atg gac ag ccc agt cag gtc 528 His Leu Phe Asp Asp Leu Met His Asn Asn Gly Leu Pro Ser Gln Val 165 170 175 acc aaa cac gcc ttc gag att ggc aag acc gtg gca acc acc gcc ggg 576 Thr Lys His Ala Phe Glu Ile Gly Lys Thr Val Ala Thr Thr Ala Gly 180 185 190 tcc gtg gtg ttt cgc aac gag ctg ctc gag ctg atg cag tac aag ccg 624 Ser Val Val Phe Arg Asn Glu Leu Leu Glu Leu Met Gln Tyr Lys Pro 195 200 205 atg agc gaa aaa cag gcc aag ccg ttg ctg atc gtc ccg ccg cag 672 Met Ser Glu Lys Gln Tyr Ala Lys Pro Leu Leu Ile Val Pro Pro Gln 210 215 220 att aac aag tac tac att ttc gac ctc agc ccg ggt aac agc ttcgt Lyc Tyr Tyr Ile Phe Asp Leu Ser Pro Gly Asn Ser Phe Val 225 230 235 240 cag tac gca ttg aag aat ggt ctg cag gtg ttc gtg gtc agc tgg cgt 768 Gln Tyr Ala Leu Lys Asn Gly Leu Gln Val Phe Val Ser Trp Arg 245 250 255 aac ccg gat gtt cgc cac cgc gaa tgg ggc ctg tcc agt tac gtt gag 816 Asn Pro Asp Val Arg His Arg Glu Trp Gly Leu Ser Ser Tyr Val Glu 260 265 270 270 gca ctg gaa gaa gca ctg aat gttgc atc acc ggc gcg cgc 864 Ala Leu Glu Glu Ala Leu Asn Val Cys Arg Ala Ile Thr Gly Ala Arg 275 280 285 gac gtc aat ctg atg ggc gcc tgt gct ggc ggc ctg acc atc gcg gct 912 Asp Val Asn Le Mu Ala Gly Gly Leu Thr Ile Ala Ala 290 295 300 ctg caa ggt cat ctg caa gcc aag cgg caa ctg cgg cgg gtc tcc agc 960 Leu Gln Gly His Leu Gln Ala Lys Arg Gln Leu Arg Arg Val Ser Ser 305 310 315 320 gcc ac tac ctg gtc agc ctg ctg gat agc cag ata gac agc ccg gcg 1008 Ala Ser Tyr Leu Val Ser Leu Leu Asp Ser Gln Ile Asp Ser Pro Ala 325 330 335 acg ttg ttc gcc gat gag cag acg ctg gag gcc gcc gcc gcc 1056 Thr Leu Phe Ala Asp Glu Gln Thr Leu Glu Ala Ala Lys Arg His Ser 340 345 350 tat caa cga ggt gtg ctc gag ggg cgc gac atg gcg aaa atc ttc gcc 1104 Tyr Gln Arg Gly Val Leu Glu Gly Arg Asp Mla Lyg Ile Phe Ala 355 360 365 tgg atg cgc ccc aat gac ctg atc tgg aac tac tgg gtc aac aac tac 1152 Trp Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr Trp Val Asn Asn Tyr 370 375 380 ctg ctg ggc cc gaga ttc gac att ctg tat tgg aac agt 1200 Leu Leu Gly Lys Glu Pro Pro Ala Phe Asp Ile Leu Tyr Trp Asn Ser 385 390 395 400 gac aac acg cgc ctg cca gcg gca ttc cat ggc gac ctg ctg gac Astc 1248 As hr Arg Leu Pro Ala Ala Phe His Gly Asp Leu Leu Asp Phe 405 410 415 ttc aag cac aat ccg ctg act cac ccc ggc ggg ctg gag gtc tgt ggc 1296 Phe Lys His Asn Pro Leu Thr His Pro Gly Gly Leu Glu Val Cys Gly 420 425 430 acg cct atc gat ttg cag aag gtc aac gta gac agc ttc agc gtg gcc 1344 Thr Pro Ile Asp Leu Gln Lys Val Asn Val Asp Ser Phe Ser Val Ala 435 440 445 445 ggc atc aac gac cac atc act ccg tgg gac gtg tac cgc tcg acc 1392 Gly Ile Asn Asp His Ile Thr Pro Trp Asp Ala Val Tyr Arg Ser Thr 450 455 460 ctg ctg ctg ggt ggc gac cgg cgc ttc gta ctg tcc aac agc ggg cat 1440 Leu Leu Leu Gly Gly Asp Phe Val Leu Ser Asn Ser Gly His 465 470 475 480 480 atc cag agc atc ctc aac ccg ccg agc aac ccc aag tcc aac tac atc 1488 Ile Gln Ser Ile Leu Asn Pro Pro Ser Asn Pro Lys Ser Asn Tyr Ile 485 490 495 gag aac ccc aag ctc agt ggc gat cca cgc gcc tgg tat tac gac ggc 1536 Glu Asn Pro Lys Leu Ser Gly Asp Pro Arg Ala Trp Tyr Tyr Asp Gly 500 505 510 acc cat gtc gaa ggt agc tgg tgg cca cgt tgg ggggc cag 1584 Thr His Val Glu Gly Ser Trp Trp Pro Arg Trp Leu Ser Trp Ile Gln 515 520 525 gag cgc tcc ggt acc caa cgc gaa acc ctg atg gcc ctt ggt aac cag 1632 Glu Arg Ser Gly Thr Gln Arg Glu Thr Leu Met Ala Leu Gly Asn Gln 530 535 540 aac tat cca ccg atg gag gcg gcg cca ggt acc tac gtg cgc gtg cgc 1680 Asn Tyr Pro Pro Met Glu Ala Ala Pro Gly Thr Tyr Val Arg Val Arg 545 550 555 555 560 tga 1683 <210> 2 <211> 560 <212> PRT <213> Pseudomonas sp.strain 61-3 <400> 2 Met Arg Glu Lys Pro Thr Pro Gly Leu Leu Pro Thr Pro Ala Thr Phe 1 5 10 15 Ile Asn Ala Gln Ser Ala Ile Thr Gly Leu Arg Gly Arg Asp Leu Phe 20 25 30 Ser Thr Leu Arg Ser Val Ala Ala His Gly Leu Arg His Pro Val Arg 35 40 45 Ser Ala Arg His Val Leu Ala Leu Gly Gly Gln Leu Gly Arg Val Leu 50 55 60 Leu Gly Glu Thr Leu His Thr Pro Asn Pro Lys Asp Asn Arg Phe Ala 65 70 75 80 Asp Pro Thr Trp Arg Leu Asn Pro Phe Tyr Arg Arg Ser Leu Gln Ala 85 90 95 Tyr Leu Ser Trp Gln Lys Gln Val Lys Ser Trp Ile Asp Glu Ser Gly 100 105 110 Met Ser Asp Asp Asp Arg Ala Arg Ala His Phe Val Phe Ala Leu Leu 115 120 125 Asn Asp Ala Val Ser Pro Ser Asn Thr Leu Leu Asn Pro Leu Ala Ile 130 135 140 Lys Glu Leu Phe Asn Ser Gly Gly Asn Ser Leu Val Arg Gly Leu Ser 145 150 155 160 His Leu Phe Asp Asp Leu Met His Asn Asn Gly Leu Pro Ser Gln Val 165 170 175 Thr Lys His Ala Phe Glu Ile Gly Lys Thr Val Ala Thr Thr Ala Gly 180 185 190 Ser Val Val Phe Arg Asn Glu Leu Leu Glu Leu Met Gln Tyr Lys Pro 195 200 205 Met Ser Glu Lys Gln Tyr Ala Lys Pro Leu Leu Ile Val Pro Pro Gln 210 215 220 Ile Asn Lys Tyr Tyr Ile Phe Asp Leu Ser Pro Gly Asn Ser Phe Val 225 230 235 240 Gln Tyr Ala Leu Lys Asn Gly Leu Gln Val Phe Val Val Ser Trp Arg 245 250 255 Asn Pro Asp Val Arg His Arg Glu Trp Gly Leu Ser Ser Tyr Val Glu 260 265 270 Ala Leu Glu Glu Ala Leu Asn Val Cys Arg Ala Ile Thr Gly Ala Arg 275 280 285 Asp Val Asn Leu Met Gly Ala Cys Ala Gly Gly Leu Thr Ile Ala Ala 290 295 300 Leu Gln Gly His Leu Gln Ala Lys Arg Gln Leu Arg Arg Val Ser Ser 305 310 315 320 Ala Ser Tyr Leu Val Se r Leu Leu Asp Ser Gln Ile Asp Ser Pro Ala 325 330 335 Thr Leu Phe Ala Asp Glu Gln Thr Leu Glu Ala Ala Lys Arg His Ser 340 345 350 Tyr Gln Arg Gly Val Leu Glu Gly Arg Asp Met Ala Lys Ile Phe Ala 355 360 365 Trp Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr Trp Val Asn Asn Tyr 370 375 380 Leu Leu Gly Lys Glu Pro Pro Ala Phe Asp Ile Leu Tyr Trp Asn Ser 385 390 395 400 400 Asp Asn Thr Arg Leu Pro Ala Ala Phe His Gly Asp Leu Leu Asp Phe 405 410 415 Phe Lys His Asn Pro Leu Thr His Pro Gly Gly Leu Glu Val Cys Gly 420 425 430 Thr Pro Ile Asp Leu Gln Lys Val Asn Val Asp Ser Phe Ser Val Ala 435 440 445 Gly Ile Asn Asp His Ile Thr Pro Trp Asp Ala Val Tyr Arg Ser Thr 450 455 460 Leu Leu Leu Gly Gly Asp Arg Arg Phe Val Leu Ser Asn Ser Gly His 465 470 475 480 Ile Gln Ser Ile Leu Asn Pro Pro Ser Asnn Pro Lys Ser Asn Tyr Ile 485 490 495 Glu Asn Pro Lys Leu Ser Gly Asp Pro Arg Ala Trp Tyr Tyr Asp Gly 500 505 510 Thr His Val Glu Gly Ser Trp Trp Pro Arg Trp Leu Ser Trp Ile Gln 515 520 525 Glu Arg Ser Gly Thr Gln Ar g Glu Thr Leu Met Ala Leu Gly Asn Gln 530 535 540 Asn Tyr Pro Pro Met Glu Ala Ala Pro Gly Thr Tyr Val Arg Val Arg 545 550 555 560 <210> 3 <211> 1680 <212> DNA <213> Pseudomonas sp strain 61-3 <220> <221> CDS <222> (1) .. (1677) <400> 3 atg agt aac aag aat agc gat gac ttg aat cgt caa gcc tcg gaa aac 48 Met Ser Asn Lys Asn Ser Asp Asp Leu Asn Arg Gln Ala Ser Glu Asn 1 5 10 15 acc ttg ggg ctt aac cct gtc atc ggc ctg cgt gga aaa gat ctg ctg 96 Thr Leu Gly Leu Asn Pro Val Ile Gly Leu Arg Gly Lys Asp Leu Leu 20 25 30 act tct gcc cga atg gtt tta acc caa gcc atc aaa caa ccc att cac 144 Thr Ser Ala Arg Met Val Leu Thr Gln Ala Ile Lys Gln Pro Ile His 35 40 45 agc gtc aag cac gtc gcg cat ttt ggc atc gag ctg aag aac gtg atg 192 Ser Val Lys His Val Ala His Phe Gly Ile Glu Leu Lys Asn Val Met 50 55 60 ttt ggc aaa tcg aag ctg caa ccg gaa agc gat gac cgt cgt ttc aac 240 Phe Gly Lys Ser Lys Leu Gln Pro Glu Ser Asp Asp Arg Arg Phe Asn 65 70 75 80 gac ccc gcc tgg agt cag aac cca ctc tac aaa cgt tat cta ca a acc 288 Asp Pro Ala Trp Ser Gln Asn Pro Leu Tyr Lys Arg Tyr Leu Gln Thr 85 90 95 tac ctg gcg tgg cgc aag gaa ctc cac gac tgg atc ggc aac agc aaa 336 Tyr Leu Ala Trp Arg Lys Glu Leu His Asp Trp Ile Gly Asn Ser Lys 100 105 110 ctg tcc gaa cag gac atc aat cgc gct cac ttc gtg atc acc ctg atg 384 Leu Ser Glu Gln Asp Ile Asn Arg Ala His Phe Val Ile Thror Leu Met 115 120 125 acc gaa gcc atg gcc ccg acc aac agt gcg gcc aat ccg gcg gcg gtc 432 Thr Glu Ala Met Ala Pro Thr Asn Ser Ala Ala Asn Pro Ala Ala Val 130 135 140 aaa cgc ttc ttc gaa acc ggc ggt aaa agc ctg ctc gac ggc ctc aca 480 Phe Glu Thr Gly Gly Lys Ser Leu Leu Asp Gly Leu Thr 145 150 155 160 cat ctg gcc aag gac ctg gta aac aac ggc ggc atg ccg agc cag gtg 528 His Leu Ala Lys Asp Leu Val Asn Asn Gly Gly Met Pro Ser Gln Val 165 170 175 gac atg ggc gct ttc gaa gtc ggc aag agt ctg ggg acg act gaa ggt 576 Asp Met Gly Ala Phe Glu Val Gly Lys Ser Leu Gly Thr Thr Glu Gly 180 185 190 gca gtg gtt ttc cgc aac gac gtc ttc atc c ag tac cgg ccg 624 Ala Val Val Phe Arg Asn Asp Val Leu Glu Leu Ile Gln Tyr Arg Pro 195 200 205 acc acc gaa cag gtg cat gag cga ccg ctg ctg gtg gtc cca ccg cag 672 Thr Thr Glu Gln Val His Glu Arg Pro Leu Leu Val Val Pro Pro Gln 210 215 220 atc aac aag ttt tat gtg ttt gac ctg agc ccg gat aaa agc ctg gcg 720 Ile Asn Lys Phe Tyr Val Phe Asp Leu Ser Pro Asp Lys Ser Leu Ala 225 230 235 240 cgc ttc tg ctg agc aac aac cag caa acc ttt atc gtc agc tgg cgc 768 Arg Phe Cys Leu Ser Asn Asn Gln Gln Thr Phe Ile Val Ser Trp Arg 245 250 255 aac ccg acc aag gcc cag cgt gag tgg ggt ctg tcg act tac atc gat Asn Pro Thr Lys Ala Gln Arg Glu Trp Gly Leu Ser Thr Tyr Ile Asp 260 265 270 gcg ctc aaa gaa gcc gtc gac gta gtt tcc gcc atc acc ggc agc aaa 864 Ala Leu Lys Glu Ala Val Asp Val Val Ser Ala Ile Thr Gly Ser Lys 275 280 285 gac atc aac atg ctc ggc gcc tgc tcc ggt ggc att acc tgc acc gcg 912 Asp Ile Asn Met Leu Gly Ala Cys Ser Gly Gly Ile Thr Cys Thr Ala 290 295 300 ctg ctg ggt cac tac gcc gct g ag aag aag gtc aat gcc ctg 960 Leu Leu Gly His Tyr Ala Ala Leu Gly Glu Lys Lys Val Asn Ala Leu 305 310 315 320 acc ctt ttg gtc agc gtg ctc gac acc acc ctc gac tcc cag gtt gca 1008 Thr Leu Leu Val Val Leu Asp Thr Thr Leu Asp Ser Gln Val Ala 325 330 335 ctg ttc gtc gat gag aaa acc ctg gaa gct gcc aag cgt cac tcg tat 1056 Leu Phe Val Asp Glu Lys Thr Leu Glu Ala Ala Lys Arg His Ser Tyr 340 345 350 cag gcc ggc gtg ctg gaa ggc cgc gac atg gcc aaa gtc ttc gcc tgg 1104 Gln Ala Gly Val Leu Glu Gly Arg Asp Met Ala Lys Val Phe Ala Trp 355 360 365 atg cgc cct aac gac ctg atc tgg aac tac tac ctg 1152 Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr Trp Val Asn Asn Tyr Leu 370 375 380 ctg ggt aac gag cca ccg gtc ttc gac att ctt ttc tgg aac aac gac 1200 Leu Gly Asn Glu Pro Pro Le Phe Asp Ile Phe Trp Asn Asn Asp 385 390 395 400 acc acc cgg ttg cct gct gcg ttc cac ggc gat ctg atc gaa atg ttc 1248 Thr Thr Arg Leu Pro Ala Ala Phe His Gly Asp Leu Ile Glu Met Phe 405 410 415 aaa aat aacca ctg gtg cgc gcc aat gca ctc gaa gtg agc ggc acg 1296 Lys Asn Asn Pro Leu Val Arg Ala Asn Ala Leu Glu Val Ser Gly Thr 420 425 430 ccg atc gac ctc aaa cag gtc act gcc gac atc tac tcc ccg Ile Asp Leu Lys Gln Val Thr Ala Asp Ile Tyr Ser Leu Ala Gly 435 440 445 acc aac gat cac atc acg ccc tgg aag tct tgc tac aag tcg gcg caa 1392 Thr Asn Asp His Ile Thr Pro Trp Lys Ser Cys Tyr Lys Ser Ala Gln 450 455 460 ctg ttc ggt ggc aag gtc gaa ttc gtg ctg tcc agc agt ggg cat atc 1440 Leu Phe Gly Gly Lys Val Glu Phe Val Leu Ser Ser Ser Gly His Ile 465 470 475 480 cag agc att ctg aacg ccg aaa tca cgt tac atg acc 1488 Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Lys Ser Arg Tyr Met Thr 485 490 495 agc acc gac atg cca gcc acc gcc aac gag tgg caa gaa aac tca acc 1536 Ser Thr Asp Met Pro Ala Thr Ala Asn Glu Trp Gln Glu Asn Ser Thr 500 505 510 aag cac acc gac tcc tgg tgg ctg cac tgg cag gcc tgg cag gcc gag 1584 Lys His Thr Asp Ser Trp Trp Leu His Trp Gln Ala Trp Gln Ala Glu 515 520 525 cgc tcg ggc aaa ctg aaa aag tcc ccg acc agc ctg ggc aac aag gcc 1632 Arg Ser Gly Lys Leu Lys Lys Ser Pro Thr Ser Leu Gly Asn Lys Ala 530 535 535 540 tat ccg tca gga gaa gcc gc g gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gtg gaa cgt taa 1680 Tyr Pro Ser Gly Glu Ala Ala Pro Gly Thr Tyr Val His Glu Arg 545 550 555 <210> 4 <211> 559 <212> PRT <213> Pseudomonas sp. strain 61-3 <400> 4 Met Ser Asn Lys Asn Ser Asp Asp Leu Asn Arg Gln Ala Ser Glu Asn 1 5 10 15 Thr Leu Gly Leu Asn Pro Val Ile Gly Leu Arg Gly Lys Asp Leu Leu 20 25 30 Thr Ser Ala Arg Met Val Leu Thr Gln Ala Ile Lys Gln Pro Ile His 35 40 45 Ser Val Lys His Val Ala His Phe Gly Ile Glu Leu Lys Asn Val Met 50 55 60 Phe Gly Lys Ser Lys Leu Gln Pro Glu Ser Asp Asp Arg Arg Phe Asn 65 70 75 80 Asp Pro Ala Trp Ser Gln Asn Pro Leu Tyr Lys Arg Tyr Leu Gln Thr 85 90 95 Tyr Leu Ala Trp Arg Lys Glu Leu His Asp Trp Ile Gly Asn Ser Lys 100 105 110 Leu Ser Glu Gln Asp Ile Asn Arg Ala His Phe Val Ile Thr Leu Met 115 120 125 Thr Glu Ala Met Ala Pro Thr Asn Ser Ala Ala As n Pro Ala Ala Val 130 135 140 Lys Arg Phe Phe Glu Thr Gly Gly Lys Ser Leu Leu Asp Gly Leu Thr 145 150 155 160 His Leu Ala Lys Asp Leu Val Asn Asn Gly Gly Met Pro Ser Gln Val 165 170 175 Asp Met Gly Ala Phe Glu Val Gly Lys Ser Leu Gly Thr Thr Glu Gly 180 185 190 Ala Val Val Phe Arg Asn Asp Val Leu Glu Leu Ile Gln Tyr Arg Pro 195 200 205 Thr Thr Glu Glu Gln Val His Glu Arg Pro Leu Leu Val Val Pro Pro Gln 210 215 220 Ile Asn Lys Phe Tyr Val Phe Asp Leu Ser Pro Asp Lys Ser Leu Ala 225 230 235 240 Arg Phe Cys Leu Ser Asn Asn Gln Gln Thr Phe Ile Val Ser Trp Arg 245 250 255 Asn Pro Thr Lys Ala Gln Arg Glu Trp Gly Leu Ser Thr Tyr Ile Asp 260 265 270 270 Ala Leu Lys Glu Ala Val Asp Val Val Ser Ala Ile Thr Gly Ser Lys 275 280 285 Asp Ile Asn Met Leu Gly Ala Cys Ser Gly Gly Ile Thr Cys Thr Ala 290 295 300 Leu Leu Gly His Tyr Ala Ala Leu Gly Glu Lys Lys Val Asn Ala Leu 305 310 315 320 Thr Leu Leu Val Ser Val Leu Asp Thr Thr Leu Asp Ser Gln Val Ala 325 330 335 Leu Phe Val Asp Glu Lys Thr Leu Glu Ala Ala L ys Arg His Ser Tyr 340 345 350 Gln Ala Gly Val Leu Glu Gly Arg Asp Met Ala Lys Val Phe Ala Trp 355 360 365 Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr Trp Val Asn Asn Tyr Leu 370 375 380 Leu Gly Asn Glu Pro Pro Val Phe Asp Ile Leu Phe Trp Asn Asn Asp 385 390 395 400 Thr Thr Arg Leu Pro Ala Ala Phe His Gly Asp Leu Ile Glu Met Phe 405 410 415 Lys Asn Asn Pro Leu Val Arg Ala Asn Ala Leu Glu Val Ser Gly Thr 420 425 430 Pro Ile Asp Leu Lys Gln Val Thr Ala Asp Ile Tyr Ser Leu Ala Gly 435 440 445 Thr Asn Asp His Ile Thr Pro Trp Lys Ser Cys Tyr Lys Ser Ala Gln 450 455 460 Leu Phe Gly Gly Lys Val Glu Phe Val Leu Ser Ser Ser Gly His Ile 465 470 475 480 Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Lys Ser Arg Tyr Met Thr 485 490 495 Ser Thr Asp Met Pro Ala Thr Ala Asn Glu Trp Gln Glu Asn Ser Thr 500 505 510 Lys His Thr Asp Ser Trp Trp Leu His Trp Gln Ala Trp Gln Ala Glu 515 520 525 Arg Ser Gly Lys Leu Lys Lys Ser Pro Thr Ser Leu Gly Asn Lys Ala 530 535 540 Tyr Pro Ser Gly Glu Ala Ala Pro Gly Thr Tyr Val H is Glu Arg 545 550 555 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 5 ccscagatca acaagttyta sgac 24 <210> 6 <211 > 6029 <212> DNA <213> Pseudomonas sp. strain 61-3 <400> 6 gaattcttgc gcgtgcactc tccttccgcc gaagtccagg gccacggcaa acctatcctg 60 caatttggca agatcggcgt aggcctgaac aaggtagaac cggccggtca gtacgcactg 120 aaattgacct tcgacgacgg ccatgacagc ggcctgttca cctgggatta tctgtaccaa 180 ctggcacaac gtcaggaagc actttgggca gattatcttg cagaactcaa agcggctgga 240 aagtcccgcg acccaagcga atccatcgtc aagctgatgc tctaattcag gcctcttgct 300 ctttagaggg cattttctaa tttcatctgt ttgaatgctc cgctgtgcgg caagcgattg 360 gcctgcttgc gaaaaaaatt aaactcgggt aaccaatgga gctggcaagt tccctgcagt 420 gctctctgaa ctagaaagca acgttgtgca attaacggtc acccgagcag tagtacctgg 480 cggttgctgt gtgactacac agctggtccc ggtactcgtc tcaggacaat ggagcgtcgt 540 agatgagtaa caagaatagc gatgacttga atcgtcaagc ctcggaaaac accttggggc 600 ttaaccctgt catcggcctg cgtggaaaag atctgctgac ttctgcccga atggttttaa 660 cccaagccat caaacaaccc attcacagcg tcaagcacgt cgcgcatttt ggcatcgagc 720 tgaagaacgt gatgtttggc aaatcgaagc tgcaaccgga aagcgatgac cgtcgtttca 780 acgaccccgc ctggagtcag aacccactct acaaacgtta tctacaaacc tacctggcgt 840 ggcgcaagga actccacgac tggatcggca acagcaaact gtccgaacag gacatcaatc 900 gcgctcactt cgtgatcacc ctgatgaccg aagccatggc cccgaccaac agtgcggcca 960 atccggcggc ggtcaaacgc ttcttcgaaa ccggcggtaa aagcctgctc gacggcctca 1020 cacatctggc caaggacctg gtaaacaacg gcggcatgcc gagccaggtg gacatgggcg 1080 ctttcgaagt cggcaagagt ctggggacga ctgaaggtgc agtggttttc cgcaacgacg 1140 tcctcgaatt gatccagtac cggccgacca ccgaacaggt gcatgagcga ccgctgctgg 1200 tggtcccacc gcagatcaac aagttttatg tgtttgacct gagcccggat aaaagcctgg 1260 cgcgcttctg cctgagcaac aaccagcaaa cctttatcgt cagctggcgc aacccgacca 1320 aggcccagcg tgagtggggt ctgtcgactt acatcgatgc gctcaaagaa gccgtcgacg 1380 tagtttccgc catcaccggc agcaaagaca tcaacatgct cggcgcctgc tccggtggca 1440 ttacctgcac cgcgctgctg ggtcactacg ccgctctcgg cgagaagaag gtcaatgccc 1500 tgaccctttt ggtcagcgtg ctcgacacca ccctcgactc ccaggttgca ctgttcgtcg 1560 atgagaaaac cctggaagct gccaagcgtc actcgtatca ggccggcgtg ctggaaggcc 1620 gcgacatggc caaagtcttc gcctggatgc gccctaacga cctgatctgg aactactggg 1680 tcaacaacta cctgctgggt aacgagccac cggtcttcga cattcttttc tggaacaacg 1740 acaccacccg gttgcctgct gcgttccacg gcgatctgat cgaaatgttc aaaaataacc 1800 cactggtgcg cgccaatgca ctcgaagtga gcggcacgcc gatcgacctc aaacaggtca 1860 ctgccgacat ctactccctg gccggcacca acgatcacat cacgccctgg aagtcttgct 1920 acaagtcggc gcaactgttc ggtggcaagg tcgaattcgt gctgtccagc agtgggcata 1980 tccagagcat tctgaacccg ccgggcaatc cgaaatcacg ttacatgacc agcaccgaca 2040 tgccagccac cgccaacgag tggcaagaaa actcaaccaa gcacaccgac tcctggtggc 2100 tgcactggca ggcctggcag gccgagcgct cgggcaaact gaaaaagtcc ccgaccagcc 2160 tgggcaacaa ggcctatccg tcaggagaag ccgcgccggg cacgtatgtg catgaacgtt 2220 aagttgtagg cagtctagaa gtccgcggca ctcggaggtg ccgcgagccc taccccatac 2280 agccgaggcc aggcctcgag taatctggag cacgctcagg acggcgtgtc cggcggttta 2340 a cccacaggg cttctgagat gccgcaaccg ttcatattcc gtactgtcga cctggatggc 2400 caaaccatcc gcaccgcagt acgccccggc aagtctcata tgacgccctt gcttattttc 2460 aatggcatcg gcgccaacct ggagctggcg ttcccgttcg tccaggcgct tgacccggac 2520 ctggaggtga ttgccttcga cgttcccggt gttggcggct catcgacgcc cagcatgcct 2580 taccgctttc ccagtctggc caagctgacc gcgcgcatgc tcgactacct ggactacggg 2640 caagtcaacg tcgtgggcgt ttcctggggt ggagcactgg cccagcagtt tgcttacgac 2700 tatccagagc gctgcaaaaa actggtgctt gcggcaaccg cggcaggctc ctttatggtg 2760 ccgggcaagc cgaaagtgct gtggatgatg gcaagcccca ggcgctatat ccagccctcc 2820 catgtgattc gcattgctcc gctgatctat ggcggatcct tccgtcgcga ccccaatctg 2880 gccgcagaac acgccagcaa agtacgttcg gccggcaagc tgggttacta ctggcagctg 2940 ttcgcgggtc tgggctggac cagcattcat tggctgcaca aaattcatca gcccaccctg 3000 gtgctggccg gtgacgacga cccgctgatc ccgctgatca acatgcgcat gctggcctgg 3060 cgaattccca acgcccagct acacataatc gacgatggtc atttgttcct gattacccgc 3120 gccgaagccg ttgcgccgat catcatgaag tttcttcagg aggagcgtca gcgggcagtg 3180 atgcatc cgc acccgacgcc gctcggcaga acttagagtc tcgcggatgt tgaaaggacc 3240 ttcgcctgcg caagaacggg ctggaccgac tatggtgtct gtcttgaatt gatgtgcttg 3300 ttgatggctt gacgaaggag tgttgactca tgagagagaa accaacgccg ggcttgctgc 3360 ccacacccgc gacgttcatc aacgctcaga gtgcgattac cggtctgcgc ggccgggatc 3420 tgttctcgac cctgcgcagc gtggccgccc acggcctgcg tcacccggtg cgcagcgccc 3480 gtcatgttct ggcactgggc ggccagttgg gccgcgtgct gctgggcgaa acgctgcaca 3540 cgccgaaccc gaaagacaat cgctttgcgg acccgacctg gagactgaat ccgttttacc 3600 ggcgcagcct gcaggcctat ctgagctggc agaaacaggt caaaagctgg atcgatgaaa 3660 gcggcatgag tgacgatgac cgcgcccgcg cgcatttcgt cttcgcactg ctcaatgacg 3720 ccgtgtcccc ctccaatacc ctgctcaacc cgctagcgat caaggagctg ttcaactccg 3780 gtggcaacag cctggtccgc ggtctcagcc atttattcga cgacctgatg cacaacaacg 3840 ggctgcccag tcaggtcacc aaacacgcct tcgagattgg caagaccgtg gcaaccaccg 3900 ccgggtccgt ggtgtttcgc aacgagctgc tcgagctgat gcagtacaag ccgatgagcg 3960 aaaaacagta cgccaagccg ttgctgatcg tcccgccgca gattaacaag tactacattt 4020 tcgacctcag cc cgggtaac agcttcgtcc agtacgcatt gaagaatggt ctgcaggtgt 4080 tcgtggtcag ctggcgtaac ccggatgttc gccaccgcga atggggcctg tccagttacg 4140 ttgaggcact ggaagaagca ctgaatgttt gccgcgctat caccggcgcg cgcgacgtca 4200 atctgatggg cgcctgtgct ggcggcctga ccatcgcggc tctgcaaggt catctgcaag 4260 ccaagcggca actgcggcgg gtctccagcg ccagctacct ggtcagcctg ctggatagcc 4320 agatagacag cccggcgacg ttgttcgccg atgagcagac gctggaagcc gccaagcgcc 4380 attcctatca acgaggtgtg ctcgaggggc gcgacatggc gaaaatcttc gcctggatgc 4440 gccccaatga cctgatctgg aactactggg tcaacaacta cctgctgggc aaagaaccgc 4500 cggccttcga cattctgtat tggaacagtg acaacacgcg cctgccagcg gcattccatg 4560 gcgacctgct ggacttcttc aagcacaatc cgctgactca ccccggcggg ctggaggtct 4620 gtggcacgcc tatcgatttg cagaaggtca acgtagacag cttcagcgtg gccggcatca 4680 acgaccacat cactccgtgg gacgcggtgt accgctcgac cctgctgctg ggtggcgacc 4740 ggcgcttcgt actgtccaac agcgggcata tccagagcat cctcaacccg ccgagcaacc 4800 ccaagtccaa ctacatcgag aaccccaagc tcagtggcga tccacgcgcc tggtattacg 4860 acggcaccca tgtcgaag gt agctggtggc cacgttggct gagctggatt caggagcgct 4920 ccggtaccca acgcgaaacc ctgatggccc ttggtaacca gaactatcca ccgatggagg 4980 cggcgccagg tacctacgtg cgcgtgcgct gaattctctc tgcaccacgg tcgggctatt 5040 ggccgtggca tgactcaata accaagaaga ctggatgaaa acccgcgacc ggatcctcga 5100 atgtgccctg caactgttca accaaaaggg cgaaccgaat gtctccacca tggaagttgc 5160 caatgagatg ggcatcagcc ctggcaacct ctattaccac tttcatggca aggaaccgct 5220 gatcctcggc ttgttcgagc gcttccaggc cgaactggtc ccgctgctcg acccgccggc 5280 ggacgtacaa ctggccgctg gagattattg gctgttcctg cacctgatcg tcgagcgcct 5340 ggcgcactac cgcttcctgt ttcaggacct gtccaacctg gccggacgct taccgaaact 5400 ggccaagggc attcgcaacc tgctcaatgc cttgaagcgt accctggcgt cgttgttggc 5460 gcggttgaaa gcgcaaggac agttggtcag cgacacacag gcgctggggc aactggtcga 5520 gcagatcacc atgacgctgc tgttttcact cgactatcaa aggattcttg atcgcgaggg 5580 agaagtgcgg gtggtggtgt accagatcat gatgctggta gcgccgcacc tgctgccacc 5640 ggtgaaattg gcgacggagc aaatggcgtt gcgatatctg gaggagcatg agtgagagag 5700 ctgagtagga caccagatcg ttt cctcgct gatgatcgtt cccacgcgcc gcaaaggaat 5760 gcagcccgtg acgctccgcg tcacaaaagc ggacgcagag cgtccagtga ggcattccca 5820 cgcgggagcg tgggaacgat caattttccg tcagaaacaa aaatgcccga catttacagg 5880 ccgggcgttt ttgtgagccc cgaaaaatca ggactgattg gttggcgtcg gtgaagtcgg 5940 cgcaacagtc ggggtaaccg caggggtcgg tgcagcagcg gagttcgctg tgctgaccgg 6000 agctgcgggg ttggccgcag caactgcag 6029

[0040]

[Sequence List Free Text] SEQ ID NO: 5: Synthetic DNA

[Brief description of the drawings]

FIG. 1 is a diagram showing the gene constitution of a cloned DNA fragment and the inserted DNA fragment in a plasmid used for breeding a polyester-producing strain in the present invention.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) C12N 9/88 C12P 7/62 C12P 7/62 C12N 5/00 A F term (reference) 4B024 AA03 BA07 BA80 CA03 DA05 DA11 EA04 GA11 HA01 4B050 CC01 CC03 CC04 DD02 EE02 LL05 4B064 AD83 AG01 CA02 CA19 CB30 CC03 CC24 DA16 4B065 AA01X AA01Y AA19X AA26X AA41Y AA44X AA73X AA80X AA90X AB01 AC14 CA02 BB02

Claims (7)

[Claims] 1. A protein of the following (a) or (b): (a) a protein containing the amino acid sequence represented by SEQ ID NO: 2 (b) an amino acid sequence represented by SEQ ID NO: 2 containing an amino acid sequence in which one or several amino acids are deleted, substituted or added, and a polyester polymer Protein with enzymatic activity 2. A polyester synthase gene encoding the following protein (a) or (b): (a) a protein containing the amino acid sequence represented by SEQ ID NO: 2 (b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and a polyester synthase Active protein 3. A gene comprising the following DNA (a) or (b): (a) a DNA containing the nucleotide sequence represented by SEQ ID NO: 1 (b) a protein that hybridizes under stringent conditions to a DNA containing the nucleotide sequence represented by SEQ ID NO: 1 and has a polyester synthase activity DNA to encode 4. A recombinant vector containing the gene according to claim 2 or 3. A transformant comprising the recombinant vector according to claim 4. 6. A method for producing a polyester synthase, which comprises culturing the transformant according to claim 5, and collecting a polyester synthase protein from the resulting culture. 7. A method for producing a polyester, comprising culturing the transformant according to claim 5, and collecting the polyester from the resulting culture.