The present invention relates to a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having one or more amino acids deleted, replaced or added. The polypeptide further comprises polyester synthase activity. The invention further relates to a polyester synthase gene comprising DNA coding for a polypeptide, a recombinant vector comprising the gene, and a transformant transformed with the recombinant vector.

Description

18 parts

›This application is a divisional of U.S. patent…

This application is a divisional of U.S. patent application Ser. No. 09/385,742, filed Aug. 30, 1999, now U.S. Pat. No. 6,391,611, which is a continuation-in-part of U.S. patent application Ser. No. 09/052,339, filed Mar. 30, 1998, now U.S. Pat. No. 5,968,805, which claims the benefit under 35 U.S.C. §119 of Japanese Patent Application No. 82965/1997, filed Apr. 1, 1997. The disclosure of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.

›FIELD OF THE INVENTION

The present invention relates to polyester synthase, a gene coding for the enzyme, a recombinant vector containing the gene, a transformant transformed with the vector, and a process for producing polyester synthase by use of the transformant.

›BACKGROUND OF THE INVENTION

Polyesters (e.g., poly-3-hydroxyalkanoic acid) biosynthesized by microorganisms are biodegradable plastics with thermoplasticity ranging widely from rigid matter to viscoelastic rubber.

Poly-3-hydroxybutanoic acid (P(3HB)) is a typical polyester consisting of C4 monomer units, but it is a rigid and brittle polymeric material, so its application is limited. Accordingly, various polyesters such as P(3HB-co-3HV) having (P(3HB)) copolymerized with a C5 monomer unit (3HV) by adding propionic acid etc. to the medium have been prepared and examined to alter the physical properties of the polyester. On the other hand, polyesters consisting of at least C6 monomer units are soft polymeric materials having plasticity.

Polyester-synthesizing microorganisms are roughly divided into 2 groups, that is, those synthesizing polyesters with C3-5 monomer units and those synthesizing polyesters with C6-14 monomer units. The former microorganisms possess a polyester synthase using C3-5 monomer units as the substrate, while the latter microorganisms possess a polyester synthase using C6-14 monomer units as the substrate. Therefore, polyesters with different properties are synthesized by the respective microorganisms.

However, the respective polyesters from such known microorganisms are different in substrate specificity, so with one kind of enzyme given, polyesters (copolymers) having various monomer unit compositions adapted to the object of use are difficult to synthesize.

›SUMMARY OF THE INVENTION

The object of the present invention is to provide a polyester synthase with monomer units having a wide range of carbon atoms as the substrate, a gene coding for the enzyme, a recombinant vector containing the gene, a transformant transformed with the vector, and a process for producing the polyester synthase by use of the transformant.

As a result of their eager research, the present inventors succeeded in cloning a polyester synthase gene from a microorganism belonging to the genus Pseudomonas isolated from soil, to arrive at the completion of the present invention.

That is, the present invention is a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a sequence where in said amino acid sequence, one or more amino acids are deleted, replaced or added, said polypeptide having polyester synthase activity.

Further, the present invention is a polyester synthase gene comprising DNA coding for said polypeptide. The DNA coding for the protein with polyester synthase activity includes, e.g., that of SEQ ID NO:2.

Further, the present invention is a polyester synthase gene comprising the nucleotide sequence of SEQ ID NO:3.

Further, the present invention is a recombinant vector comprising the polyester synthase gene.

Further, the present invention is a transformant transformed with said recombinant vector.

Further, the present invention is a process for producing polyester synthase wherein said transformant is cultured in a medium and polyester synthase is recovered from the resulting culture.

›DETAILED DESCRIPTION OF THE INVENTION · 1 of 2

Hereinafter, the present invention is described in detail.

(1) Cloning of the Polyester Synthase Gene

The polyester synthase gene of the present invention is separated from a microorganism belonging to the genus Pseudomonas.

First, genomic DNA is isolated from a strain having the polyester synthase gene. Such a strain includes, e.g., Pseudomonas sp. Any known methods can be used for preparation of genomic DNA. For example, Pseudomonas sp. is cultured in a bouillon medium and then its genomic DNA is prepared by the hexadecyl trimethyl ammonium bromide method (Current Protocols in Molecular Biology, vol. 1, page 2.4.3., John Wiley & Sons Inc., 1994).

The DNA obtained in this manner is partially digested with a suitable restriction enzyme (e.g., Sau3AI, BamHI, BglII etc.). It is then ligated into a vector dephosphorylated by treatment with alkaline phosphatase after cleavage with a restriction enzyme (e.g., BamHI, BglII etc.) to prepare a library.

Phage or plasmid capable of autonomously replicating in host microorganisms is used as the vector. The phage vector includes, e.g., EMBL3, M13, gt11 etc., and the plasmid vector includes, e.g., pBR322, pUC18, and pBluescript II (Stratagene). Vectors capable of autonomously replicating in 2 or more host cells such as E. coli and Bacillus brevis, as well as various shuttle vectors, can also be used. Such vectors are also cleaved with said restriction enzymes so that their fragment can be obtained.

Conventional DNA ligase is used to ligate the resulting DNA fragment into the vector fragment. The DNA fragment and the vector fragment are annealed and then ligated to produce a recombinant vector.

To introduce the recombinant vector into a host microorganism, any known methods can be used. For example, if the host microorganism is E. coli, the calcium chloride method (Lederberg, E. M. et al., J. Bacteriol. 119, 1072 (1974)) and the electroporation method (Current Protocols in Molecular Biology, vol. 1, page 1.8.4 (1994)) can be used. If phage DNA is used, the in vitro packaging method (Current Protocols in Molecular Biology, vol. 1, page 5.7.1 (1994)) etc. can be adopted. In the present invention, an in vitro packaging kit (Gigapack II, produced by Stratagene etc.) may be used.

To obtain a DNA fragment containing the polyester synthase gene derived from Pseudomonas sp., a probe is then prepared. The amino acid sequences of some polyester synthases have already been known (Peoples, O. P. and Sinskey, A. J., J. Biol. Chem., 264, 15293 (1989); Huisman, G. W. et al., J. Biol. Chem., 266, 2191 (1991); Pieper, U. et al., FEMS Microbiol. Lett., 96, 73 (1992); Timm, A. and Steinbuchel, A., Eur. J. Biochem., 209, 15 (1992), etc.). Well-conserved regions are selected from these amino acid sequences, and nucleotide sequences coding for them are estimated to design oligonucleotides. Examples of such oligonucleotides include, but are not limited to, the sequence 5′-CC(G/C)CAGATCAACAAGTT(C/T)TA(C/G)GAC-3′ (SEQ ID NO:4) reported by Timm, A. and Steinbuchel, A., Eur. J. Biochem., 209, 15 (1992).

Then, this synthetic oligonucleotide is labeled with a suitable reagent and used for colony hybridization of the above genomic DNA library (Current Protocols in Molecular Biology, vol. 1, page 6.0.3 (1994)).

The E. coli is screened by colony hybridization, and a plasmid is recovered from it using the alkaline method (Current Protocols in Molecular Biology, vol. 1, page 1.6.1 (1994)), whereby a DNA fragment containing the polyester synthase gene is obtained. The nucleotide sequence of this DNA fragment can be determined in, e.g., an automatic nucleotide sequence analyzer such as 373A DNA sequencer (Applied Biosystems) using a known method such as the Sanger method (Molecular Cloning, vol. 2, page 13.3 (1989)).

After the nucleotide sequence was determined by the means described above, the gene of the present invention can be obtained by chemical synthesis or the PCR technique using genomic DNA as a template, or by hybridization using a DNA fragment having said nucleotide sequence as a probe.

(2) Preparation of Transformant

The transformant of the present invention is obtained by introducing the recombinant vector of the present invention into a host compatible with the expression vector used in constructing said recombinant vector.

The host is not particularly limited insofar as it can express the target gene. Examples are bacteria such as microorganisms belonging to the genus Alcaligenes, microorganisms belonging to the genus Bacillus, bacteria such as E. coli, yeasts such as the genera Saccharomyces, Candida etc., and animal cells such as COS cells, CHO cells etc.

If microorganisms belonging to the genus Alcaligenes or bacteria such as E. coli are used as the host, the recombinant DNA of the present invention is preferably constituted such that it contains a promoter, the DNA of the present invention, and a transcription termination sequence so as to be capable of autonomous replication in the host. The expression vector includes pLA2917 (ATCC 37355) containing replication origin RK2 and pJRD215 (ATCC 37533) containing replication origin RSF1010, which are replicated and maintained in a broad range of hosts.

The promoter may be any one if it can be expressed in the host. Examples are promoters derived from E. coli, phage etc., such as trp promoter, lac promoter, PL promoter, PR promoter and T7 promoter. The method of introducing the recombinant DNA into bacteria includes, e.g., a method using calcium ions (Current Protocols in Molecular Biology, vol. 1, page 1.8.1 (1994)) and the electroporation method (Current Protocols in Molecular Biology, vol. 1, page 1.8.4 (1994)).

If yeast is used as the host, expression vectors such as YEp13, YCp50 etc. are used. The promoter includes, e.g., gal 1 promoter, gal 10 promoter etc. To method of introducing the recombinant DNA into yeast includes, e.g., the electroporation method (Methods. Enzymol., 194, 182-187 (1990)), the spheroplast method (Proc. Natl. Acad. Sci. USA, 84, 1929-1933 (1978)), the lithium acetate method (J. Bacteriol., 153, 163-168 (1983)) etc.

›DETAILED DESCRIPTION OF THE INVENTION · 2 of 2

If animal cells are used as the host, expression vectors such as pcDNAI, pcDNAI/Amp (produced by Invitrogene) etc. are used. The method of introducing the recombinant DNA into animal cells includes, e.g., the electroporation method, potassium phosphate method etc.

(3) Production of Polyester Synthase

Production of the polyester synthase of the present invention is carried out by culturing the transformant of the present invention in a medium, forming and accumulating the polyester synthase of the present invention in the culture (the cultured microorganism or the culture supernatant) and recovering the polyester synthase from the culture.

A conventional method used for culturing the host is also used to culture the transformant of the present invention.

The medium for the transformant prepared from bacteria such as E. coli etc. as the host includes complete medium or synthetic medium, e.g., LB medium, M9 medium etc. The transformant is aerobically cultured at a temperature ranging from 25 to 37 degrees C. for 12 to 48 hours so that the polyester synthase is accumulated in the microorganism and then recovered.

The carbon source is essential for the growth of the microorganism and includes, e.g., carbohydrates such as glucose, fructose, sucrose, maltose etc.

The nitrogen source includes, e.g., ammonia, ammonium salts such as ammonium chloride, ammonium sulfate, ammonium phosphate etc., peptone, meat extract, yeast extract, corn steep liquor etc. The inorganic matter includes, e.g., monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride etc.

Culture is carried out usually under aerobic conditions with shaking at 25 to 37° C. for more than 2 hours after expression is induced. During culture, antibiotics such as ampicillin, kanamycin, ampicillin, tetracycline etc. may be added to the culture.

To culture the microorganism transformed with the expression vector using an inducible promoter, its inducer can also be added to the medium. For example, isopropyl-D-thiogalactopyranoside (IPTG), indoleacrylic acid (IAA) etc. can be added to the medium.

To culture the transformant from animal cells as the host, use is made of a medium such as RPMI-1640 or DMEM which may be supplemented with fetal bovine serum. Culture is carried out usually in 5% CO2 at 30 to 37° C. for 1 to 7 days. During culture, antibiotics such as kanamycin, penicillin etc. may be added to the medium.

Purification of the polyester synthase can be performed by recovering the resulting culture by centrifugation (after disruption in the case of cells) and subjecting it to affinity chromatography, cation or anion exchange chromatography or gel filtration or to a suitable combination thereof.

Whether the resulting purified substance is the desired enzyme is confirmed by conventional methods such as SDS polyacrylamide gel electrophoresis, Western blotting etc.

›EXAMPLES

Hereinafter, the present invention is described in more detail with reference to the Examples, which, however are not intended to limit the scope of the present invention.

›Examples3

›Example 1

(1) Cloning of the Polyester Synthase Gene From Pseudomonas sp.

First, a genomic DNA library of Pseudomonas sp. was prepared.

Pseudomonas sp. JCM 10015 was cultured overnight in 100 ml bouillon medium (1% meat extract, 1% peptone, 0.5% sodium chloride, pH 7.2) at 30° C. and then genomic DNA was obtained from the microorganism using the hexadecyl trimethyl ammonium bromide method (Current Protocols in Molecular Biology, vol. 1, page 2.4.3 (1994), John Wiley & Sons Inc.).

The resulting genomic DNA was partially digested with restriction enzyme Sau3AI. The vector plasmid used was cosmid vector pLA2917 (ATCC 37355). This plasmid was cleaved with restriction enzyme BglII and dephosphorylated (Molecular Cloning, vol. 1, page 5.7.2 (1989), Cold Spring Harbor Laboratory) and then ligated into the partially digested genomic DNA fragment by use of DNA ligase.

E. coli S17-1 was transformed with this ligated DNA fragment by the in vitro packaging method (Current Protocols in Molecular Biology, vol. 1, page 5.7.2 (1994)) whereby a genomic DNA library from Pseudomonas sp. was obtained.

To obtain a DNA fragment containing the polyester synthase gene from Pseudomonas sp., a probe was then prepared. An oligonucleotide consisting of the sequence 5′-CC(G/C)CAGATCAACAAGTT(C/T)TA(C/G)GAC-3′ (SEQ ID NO:4) reported by Timm, A. and Steinbuchel, A., Eur. J. Biochem., 209, 15 (1992) was synthesized. This oligonucleotide was labeled with digoxigenin using a DIG DNA labeling kit (Boehringer Mannheim) and used as a probe.

Using the probe thus obtained, E. coli carrying a plasmid containing the polyester synthase gene was isolated by colony hybridization from the genomic DNA library from Pseudomonas sp.

When Alcaligenes eutrophus PHB-4 (DSM541) and Pseudomonasputida GPp104 (both of which are strains deficient in an ability to produce polyester) were transformed by the conjugation transfer method with the plasmid containing the polyester synthase gene, both the strains had a reverse ability to produce polyester and showed complementarity.

By recovering the plasmid from the E. coli, a DNA fragment containing the polyester synthase gene was obtained.

The nucleotide sequence of a PstI-XbaI fragment from this fragment was determined by the Sanger method.

As a result, the nucleotide sequence of the 1.8 kbp fragment shown in SEQ ID NO:3 was determined.

By further examining homology to this nucleotide sequence, the polyester synthase gene containing the nucleotide sequence (1680 bp) of SEQ ID NO:2 could be identified in this 1.8 kbp nucleotide sequence. The amino acid sequence encoded by SEQ ID NO:2 is shown in SEQ ID NO:1.

It should be understood that insofar as a protein containing the amino acid sequence of SEQ ID NO:1 or a sequence where in said amino acid sequence, one or more amino acids are deleted, replaced or added has polyester synthase activity, the gene (SEQ ID NO:2 or 3) containing DNA coding for said protein falls under the scope of the polyester synthase gene of the present invention.

Mutations such as deletion, replacement, addition etc. can be induced in the amino acid sequence or nucleotide sequence by the known site-direct mutagenesis method (e.g., Transfomer™ Site-Directed Mutagenesis Kit available from Toyobo).

(2) Preparation of E. coli Transformant

The 1.8 kb PstI-Xbal fragment containing the polyester synthase gene was ligated into the XbaI, PstI site of plasmid vector pBluescript II KS+. The resulting recombinant vector was transformed by the calcium chloride method into Escherichia coli DH5. The resulting transformant was designated Escherichia coli PX18. By extracting the plasmid from this transformant, the 1.8 kb PstI-Xbal fragment containing the polyester synthase gene can be easily obtained. Escherichia coli PX1 8 has been deposited as FERM BP-6297 with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan.

According to the present invention, there are provided a gene coding for polyester synthase, a recombinant vector containing the gene, and a transformant transformed with the vector. The gene of the present invention codes for a polyester synthase using monomers having a wide range of carbon atoms as the substrate, so it is useful in preparing copolymer polyesters having various physical properties.

›Example 2 · 1 of 2

Introduction

Two types of polyhydroxyalkanoate biosynthesis gene loci (phb and pha) of Pseudomonas sp. 61-3 (JCM 10015), which produces a blend of poly(3-hydroxybutyrate) homopolymer [P(3HB)] and a random copolymer (poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) [P(3HB-co-3HA]) consisting of 3HA units from 4 to 12 carbon numbers, were cloned and analyzed at the molecular level. In the present invention, the substrate specificities of PHA synthases were also evaluated by heterologous expression in PHA-negative mutant of R. eutropha.

Materials and Methods

Bacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in this study are listed in Table 1, and the DNA fragments on vectors are illustrated in FIG. 1 . Pseudomonas sp. 61-3 and R. eutropha strains were cultivated at 30° C. in a nutrient-rich (NR) medium containing 10 g of meat extract, 10 g of bactopeptone (Difco), and 2 g of yeast extract, (Difco) in 1 liter of distilled water. Escherichia coli strains were grown at 37° C. on Luria-Bertani (LB) medium (9). When needed, kanamycin (50 mg/liter), tetracycline (12.5 mg/liter), or ampicillin (50 mg/liter) was added to the medium.

DNA manipulations. Isolation of total genomic DNA and plasmids, digestion of DNA with restriction endonucleases, agarose gel electrophoresis, and transformation of E. coli were carried out according to standard procedures (11) or the manufacturers. DNA restriction fragments were isolated from agarose gels by using QIAEX II Gel Extraction Kit (QIAGEN). All other DNA-manipulating enzymes were used as recommended by the manufacturers. Genomic DNA libraries of Pseudomonas sp. 61-3 were constructed with Charomid 9-28 (Nippon gene) and pLA2917 (2) by in vitro packaging using Gigapack II (Stratagene). Conjugation of R. eutropha with E. coli S17-1 harboring broad-host-range plasmids was performed as described by Friedrich et al. (4).

Plasmid construction. Plasmids pJASc22, pJASc60, and pJASc60dC1Z, were constructed as follows: The 2.2-kbp EcoRI-XbaI region containing a gene of the present invention (hereinafter referred to as phaC1 Ps ), and the 6.0-kbp EcoRI-PstI region containing phaC1ZC2D Ps were introduced into pJRD215 as 2.2-kbp and 6.0-kbp ApaI-SacI fragments, to form pJASc22 and pJASc60, respectively (FIG. 1 ). A plasmid pJASc60dC1Z containing phaC2D Ps was constructed by eliminating a BglII-SphI region from a pBluescript II KS+ derivative plasmid carrying the 6.0-kbp EcoRI-PstI region, and introducing the deleted fragment into pJRD215 at the ApaI and SacI sites (FIG. 1 ).

Plasmids pJASc50 and pJASc50dC1Z were constructed as follows: The 5.1-kbp EcoRI-BamHI region containing phaC1ZC2 Ps was introduced into pJRD215 as a 5.1-kbp ApaI-SacI fragment to form pJASc50 (FIG. 1 ). A plasmid pJASc50dC1Z containing a gene of the present invention (hereinafter referred to as phaC2 Ps ) was constructed by eliminating a BglII-SphI region from a pBluescript II KS+ derivative plasmid carrying the 5.1-kbp EcoRI-BamHI region, and introducing the deleted fragment into pJRD215 at the ApaI and SacI sites (FIG. 1 ).

Hybridization experiments. Hybridization was carried out as described by Southern (17). The DNA probes used were a 24-mer synthetic oligonucleotide, 5′-CC(G/C)CAGATCAACAAGTT(C/T)TA(C/G)GAC-3′(SEQ ID NO 7), whose sequence was based on that of a highly conserved region of the polyester synthases of R. eutropha and P. oleovorans as described by Timm and Steinbühel (16). Preparation of digoxigenin-labeled probes and the detection of hybridization signals on membranes were carried out with DIG DNA Labeling and Detection Kit (Boehringer Mannheim) and DIG Oligonucleotide Tailing Kit (Boehringer Mannheim).

Nucleotide sequence analysis. DNA fragments to be sequenced were subcloned into pBluescript II KS+. DNA was sequenced by the modified dideoxy chain-termination method basically as described by Sanger et al. (18) with a 310 Genetic Analyzer (Perkin Elmer). The sequencing reaction was performed according to the manual supplied with the dye terminator cycle sequencing kit (Perkin Elmer). The resulting nucleotide sequence was analyzed with SDC-GENETYX genetic information processing software (Software Development Co., Tokyo, Japan).

Production and analysis of pha. Cells were cultivated on a reciprocal shaker (130 strokes/min) at 30° C. for 72 h in 500-ml flasks containing 100 ml of a nitrogen-limited mineral salt (MS) medium, which consisted of 0.9 g of Na 2 HPO 4 12H 2 O, 0.15 g of KH 2 PO 4 , 0.05 g of NH 4 Cl, 0.02 g of MgSO 4 7H 2 O, and 0.1 ml of trace element solution (8). Filter-sterilized carbon sources were added to the medium as indicated in the text. Determination of cellular PHA content and composition by gas chromatography, isolation of the accumulated PHA, fractionation of the isolated polyesters with acetone, and nuclear magnetic resonance (NMR) analysis of polyesters, were carried out in a manner as described by Kato et al. (8, 9).

Results

Cloning and identification of phb and pha loci of Pseudomonas sp. 61-3. To identify the two possible types of polyester synthase genes in Pseudomonas sp. 61-3, genomic DNA fragments from digestion with several restriction enzymes were hybridized with two different gene probes. One probe is a 1.8-kbp fragment carrying phb synthase gene of R. eutropha (phbC Re ), and the other is a 24-mer synthetic oligonucleotide previously used for identification of pha synthase genes from pseudomonads (16). Southern hybridization analysis using each probe showed different patterns of strong signals (14-kbp HindIII-, 20-kbp EcoRI-, 30-kbp BamHI-, 3.5-kbp PstI-, and 6.3-kbp SacI-fragments with the phbC Re probe, and 17-kbp HindIII-, 1.9-kbp EcoRI-, 16-kbp BamHI-, 3.2-kbp PstI-, and 19-kbp SacI-fragments with the 24-mer oligonucleotide probe). This suggested that the two types of polyester synthase genes are located on different DNA loci in Pseudomonas sp. 61-3.

For cloning of the polyester synthase gene hybridized with the phbC Re and the oligonucleotide probes, a genomic sublibrary of 14-kbp HindIII fragments with a cosmid vector Charomid 9-28, and a total genomic DNA library with a cosmid vector pLA2917 (2) from partially digested genomic DNA using Sau3AI were constructed by in vitro packaging. Positive clones isolated by each hybridization screening were further analyzed by Southern hybridization, and a 6.0-kbp HindIII-ApaI and a 6.0-kbp EcoRI-PstI regions were mapped, respectively.

›Example 2 · 2 of 2

Organization of phb and pha loci. The complete nucleotide sequences of the cloned fragments were determined in both strands. In the 6.0-kbp HindIII-ApaI region (phb locus), four potential open reading frames (ORFs) were identified by computer analysis for protein-coding regions. The nucleotide sequence revealed homologies to genes encoding phb synthase (PhbC Ps ), β-ketothiolase (PhbA Ps ), and NADPH-dependent acetoacetyl-CoA reductase (PhbB Ps ) in R. eutropha (Table 2). The phb locus of Pseudomonas sp. 61-3 was constituted of phbBAC Ps operon, which is a different organization from the corresponding operon in R. eutropha (phbCAB Re ).

In the region upstream of phbB Ps , another ORF (1,137 bp) was oriented in the opposite direction to the other three genes. The ORF was referred to as phbR Ps .

In the 6.0-kbp EcoRI-PstI region (pha locus; SEQ ID NO:8), there are several genes with a similar organization to pha loci of P. oleovorans (6) and P. aeruginosa (16). Two polyester synthase genes, referred to as phaC1 Ps (SEQ ID NO:2) and phaC2 Ps (SEQ ID NO:5), are represented as two large open reading frames in this region (FIG. 1 ). Amino acid sequences encoded by phaC1 Ps and phaC2 Ps are shown in SEQ ID NOS: 1 and 6, respectively. The two PHA synthases of Pseudomonas sp. 61-3 exhibited 53.2% identity each other, which is similar to the homology between the two synthases of P. oleovorans (6). Putative PHA depolymerase is encoded by phaZ Ps located between phaC1 Ps and phaC2 Ps in Pseudomonas sp. 61-3. An ORF was also identified downstream of phaC2 Ps of which deduced amino acid sequence was similar to that of ORF3 of P. aeruginosa (Table 2) (16), then it was designated as phaD Ps . ORF1 upstream of phaC1 Ps was similar to the 3′-terminal region of ORF2 of P. aeruginosa (81.7% identity for the C-terminal 93 amino acids) (16). Two nucleotide sequences resembling the E. coli −35 to −10 consensus sequence of σ 70 -dependent promoter and the E. coli −24 to −12 consensus sequence of σ 54 -dependent promoter were found upstream of phaC1 Ps , although their relevance has not been yet explored.

Complementation studies and heterologous expression. To confirm whether the cloned fragments have functionally active PHA biosynthesis genes, heterologous expression of the genes was investigated in the PHA-negative mutants, R. eutropha PHB 4 (12). Plasmids pJASc60, pJASc22, pJASc60dC1Z, pJASc50 and pJASc50dC1Z harboring the PHA synthase gene were constructed as described Materials and Methods section. These plasmids were mobilized from E. coli S17-1 to R. eutropha PHB 4. The transconjugants were cultivated under nitrogen-limiting conditions in MS medium to promote the PHA biosynthesis from gluconate, octanoate, dodecanoate, or tetradecanoate as a sole carbon source, and were analyzed by gas chromatography to determine the content and composition of the accumulated PHA.

Table 3 shows the results of PHA accumulation in the recombinant strains of R. eutropha PHB 4. The plasmids, pJASc22, pJASc60, pJASc60dC1Z, pJASc50 and pJASc50dC1Z could complement the deficiency of polyester synthases in both the mutant strains, and conferred the ability to accumulate PHA on the hosts.

The recombinant strains of PHB 4 harboring pJASc60, pJASc22, pJASc60dC1Z, pJASc50 and pJASc50dC1Z produced P(3HB) homopolymer from gluconate, while the strains produced P(3HB-co-3HA) copolymer consisting of 3HA of C 4 - to C 12 -monomer units from octanoate, dodecanoate, or tetradecanoate with relatively high 3HB contents (Table 3). 3HB compositions of 30 to 70 mol % were incorporated into the copolymers synthesized by the strains harboring pJASc22, pJASc60dC1Z, pJASc50 and pJASc50dC1Z from the alkanoates (Table 3). Interestingly, PHB 4/pJASc60 produced copolymers composed of much higher 3HB fraction (about 90 mol % 3HB) from octanoate and tetradecanoate. In order to determine whether the polyesters synthesized by PHB 4 strains carrying PHA synthase genes from alkanoates are random copolymers or not, the parameter D values were calculated based on the sequence distribution of 3HB and 3HA units by 13 C-NMR analysis as described by Kamiya et al. (7), which suggested that these polyesters are random copolymers of 3HB and 3HA units (1.4 to 1.6 of D values). As a consequence, both PhaC1 Ps and PhaC2 Ps of Pseudomonas sp. 61-3 were found to be able to incorporate a wide compositional range of 3HA units from C 4 to C 12 into the polyester.

Discussion

R. eutropha PHB 4 strain harboring phaC1 Ps and/or phaC2 Ps produced P(3HB-co-3HA) copolymers consisting of 3HA units of 4 to 12 carbon numbers from alkanoates. These results indicate that both PHA synthases of Pseudomonas sp. 61-3 are able to incorporate 3HB unit into the polyester as well as medium-chain-length 3HA units.

The results described here demonstrate that the polyester synthase gene of the present invention makes it be possible to synthesize P(3HB-co-3HA) random copolymer with a novel composition having a wide range of rigidity or placticity.

FIGURE LEGENDS

FIG. 1 . Organization of pha loci in Pseudomonas sp. 61-3 and DNA fragments including pha locus on broad-host-range vector used in this study.

›REFERENCES

1. Abe, H. et al., 1994. Int. J. Biol. Macromol. 16: 115-119

2. Allen, L. N., and R. S. Hanson. 1985. J. Bacteriol. 161: 955-962.

3. Davison, J., M. et al., 1987. Gene 51: 275-280.

4. Friedrich, B., C. et al., 1981. J. Bacteriol. 147: 198-205.

5. Hebert, M. D., and J. E. Houghton. 1997. J. Bacteriol. 179: 7834-7842.

6. Huismen, G. W. et al., 1991. J. Biol. Chem. 266: 2191-2198.

7. Kamiya, N., Y. et al., 1989. Macromolecules 22: 1676-1682.

8. Kato, M. et al., 1996. Appl. Microbiol. Biotechnol. 45: 363-370.

9. Kato, M. et al., 1996. Bull. Chem. Soc. Jpn. 69: 515-520.

10. Peoples, O. P., and A. J. Sinskey. 1989. J. Biol. Chem. 264: 15298-15303.

11. Sambrook, J. et al., 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

12. Schlegel, H. G. et al., 1970. Microbiol. 71: 283-294.

13. Schubert, P. et al., 1988. J. Bacteriol. 170: 5837-5847.

14. Simon, R. et al., 1983. Bio/Technology 1: 784-791.

15. Slater, S. C. et al., 1988. J. Bacteriol. 170: 4431-4436.

16. Timm, A., and A. Steinbühel. 1992. Eur. J. Biochem. 209: 15-30.

17. Southem, E. M., 1975. J. Mol. Biol. 98: 503-517.

18. Sanger, F., S. et al., 1977. Proc. Natl. Acad. Sci. USA 74: 5463-5467.

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 Asn 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 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

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 Lys 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 His Glu Arg

545 550 555

1680

›DNA

Pseudomonas

atgagtaaca agaatagcga tgacttgaat cgtcaagcct cggaaaacac cttggggctt 60

aaccctgtca tcggcctgcg tggaaaagat ctgctgactt ctgcccgaat ggttttaacc 120

caagccatca aacaacccat tcacagcgtc aagcacgtcg cgcattttgg catcgagctg 180

aagaacgtga tgtttggcaa atcgaagctg caaccggaaa gcgatgaccg tcgtttcaac 240

gaccccgcct ggagtcagaa cccactctac aaacgttatc tacaaaccta cctggcgtgg 300

cgcaaggaac tccacgactg gatcggcaac agcaaactgt ccgaacagga catcaatcgc 360

gctcacttcg tgatcaccct gatgaccgaa gccatggccc cgaccaacag tgcggccaat 420

ccggcggcgg tcaaacgctt cttcgaaacc ggcggtaaaa gcctgctcga cggcctcaca 480

catctggcca aggacctggt aaacaacggc ggcatgccga gccaggtgga catgggcgct 540

ttcgaagtcg gcaagagtct ggggacgact gaaggtgcag tggttttccg caacgacgtc 600

ctcgaattga tccagtaccg gccgaccacc gaacaggtgc atgagcgacc gctgctggtg 660

gtcccaccgc agatcaacaa gttttatgtg tttgacctga gcccggataa aagcctggcg 720

cgcttctgcc tgagcaacaa ccagcaaacc tttatcgtca gctggcgcaa cccgaccaag 780

gcccagcgtg agtggggtct gtcgacttac atcgatgcgc tcaaagaagc cgtcgacgta 840

gtttccgcca tcaccggcag caaagacatc aacatgctcg gcgcctgctc cggtggcatt 900

acctgcaccg cgctgctggg tcactacgcc gctctcggcg agaagaaggt caatgccctg 960

acccttttgg tcagcgtgct cgacaccacc ctcgactccc aggttgcact gttcgtcgat 1020

gagaaaaccc tggaagctgc caagcgtcac tcgtatcagg ccggcgtgct ggaaggccgc 1080

gacatggcca aagtcttcgc ctggatgcgc cctaacgacc tgatctggaa ctactgggtc 1140

aacaactacc tgctgggtaa cgagccaccg gtcttcgaca ttcttttctg gaacaacgac 1200

accacccggt tgcctgctgc gttccacggc gatctgatcg aaatgttcaa aaataaccca 1260

ctggtgcgcg ccaatgcact cgaagtgagc ggcacgccga tcgacctcaa acaggtcact 1320

gccgacatct actccctggc cggcaccaac gatcacatca cgccctggaa gtcttgctac 1380

aagtcggcgc aactgttcgg tggcaaggtc gaattcgtgc tgtccagcag tgggcatatc 1440

cagagcattc tgaacccgcc gggcaatccg aaatcacgtt acatgaccag caccgacatg 1500

ccagccaccg ccaacgagtg gcaagaaaac tcaaccaagc acaccgactc ctggtggctg 1560

cactggcagg cctggcaggc cgagcgctcg ggcaaactga aaaagtcccc gaccagcctg 1620

ggcaacaagg cctatccgtc aggagaagcc gcgccgggca cgtatgtgca tgaacgttaa 1680

1826

›DNA

Pseudomonas

ctgcagtgct ctctgaacta gaaagcaacg ttgtgcaatt aacggtcacc cgagcagtag 60

tacctggcgg ttgctgtgtg actacacagc tggtcccggt actcgtctca ggacaatgga 120

gcgtcgtaga tgagtaacaa gaatagcgat gacttgaatc gtcaagcctc ggaaaacacc 180

ttggggctta accctgtcat cggcctgcgt ggaaaagatc tgctgacttc tgcccgaatg 240

gttttaaccc aagccatcaa acaacccatt cacagcgtca agcacgtcgc gcattttggc 300

atcgagctga agaacgtgat gtttggcaaa tcgaagctgc aaccggaaag cgatgaccgt 360

cgtttcaacg accccgcctg gagtcagaac ccactctaca aacgttatct acaaacctac 420

ctggcgtggc gcaaggaact ccacgactgg atcggcaaca gcaaactgtc cgaacaggac 480

atcaatcgcg ctcacttcgt gatcaccctg atgaccgaag ccatggcccc gaccaacagt 540

gcggccaatc cggcggcggt caaacgcttc ttcgaaaccg gcggtaaaag cctgctcgac 600

ggcctcacac atctggccaa ggacctggta aacaacggcg gcatgccgag ccaggtggac 660

atgggcgctt tcgaagtcgg caagagtctg gggacgactg aaggtgcagt ggttttccgc 720

aacgacgtcc tcgaattgat ccagtaccgg ccgaccaccg aacaggtgca tgagcgaccg 780

ctgctggtgg tcccaccgca gatcaacaag ttttatgtgt ttgacctgag cccggataaa 840

agcctggcgc gcttctgcct gagcaacaac cagcaaacct ttatcgtcag ctggcgcaac 900

ccgaccaagg cccagcgtga gtggggtctg tcgacttaca tcgatgcgct caaagaagcc 960

gtcgacgtag tttccgccat caccggcagc aaagacatca acatgctcgg cgcctgctcc 1020

ggtggcatta cctgcaccgc gctgctgggt cactacgccg ctctcggcga gaagaaggtc 1080

aatgccctga cccttttggt cagcgtgctc gacaccaccc tcgactccca ggttgcactg 1140

ttcgtcgatg agaaaaccct ggaagctgcc aagcgtcact cgtatcaggc cggcgtgctg 1200

gaaggccgcg acatggccaa agtcttcgcc tggatgcgcc ctaacgacct gatctggaac 1260

tactgggtca acaactacct gctgggtaac gagccaccgg tcttcgacat tcttttctgg 1320

aacaacgaca ccacccggtt gcctgctgcg ttccacggcg atctgatcga aatgttcaaa 1380

aataacccac tggtgcgcgc caatgcactc gaagtgagcg gcacgccgat cgacctcaaa 1440

caggtcactg ccgacatcta ctccctggcc ggcaccaacg atcacatcac gccctggaag 1500

tcttgctaca agtcggcgca actgttcggt ggcaaggtcg aattcgtgct gtccagcagt 1560

gggcatatcc agagcattct gaacccgccg ggcaatccga aatcacgtta catgaccagc 1620

accgacatgc cagccaccgc caacgagtgg caagaaaact caaccaagca caccgactcc 1680

tggtggctgc actggcaggc ctggcaggcc gagcgctcgg gcaaactgaa aaagtccccg 1740

accagcctgg gcaacaaggc ctatccgtca ggagaagccg cgccgggcac gtatgtgcat 1800

gaacgttaag ttgtaggcag tctaga 1826

›DNA

Artificial Sequence

Synthetic DNA

ccscagatca acaagttyta sgac 24

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 Gln Leu Gly Arg Val Leu

50 55 60

ctg ggc gaa acg ctg cac acg ccg aac ccg aaa gac aat cgc ttt gcg 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 ctc 384

Met Ser Asp Asp 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 cac aac aac ggg ctg 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 tac 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 ttc gtc 720

Ile Asn Lys 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 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

gca ctg gaa gaa gca ctg aat gtt tgc cgc gct 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 Leu Met Gly Ala Cys 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 agc 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 gaa gcc gcc aag cgc cat tcc 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 Met Ala Lys 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 aaa gaa ccg ccg gcc 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 ttc 1248

Asp Asn Thr 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

ggc atc aac gac cac atc act ccg tgg gac gcg 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 Arg Arg Phe Val Leu Ser Asn Ser Gly His

465 470 475 480

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 ctg agc tgg att 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

545 550 555 560

tga 1683

560

›PRT

Pseudomonas

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 Ser 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

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 Asn 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 Arg 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

›DNA

Artificial Sequence

Synthetic DNA

ccscagatca acaagttyta sgac 24

6029

›DNA · 1 of 2

Pseudomonas

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

acccacaggg 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

atgcatccgc 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 cccgggtaac 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 tgtcgaaggt 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

›DNA · 2 of 2

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 tttcctcgct 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

›Tables in the description — 4

TABLE 1 — BACTERIAL STRAINS AND PLASMIDS USED IN THIS STUDY. a JCM, Japan Collection of Microorganisms; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH.

Strain or Plasmid	Relevant Characteristics	Source Or Reference(s) a
Strains
Pseudomonas sp. strain 61-3	Wild type	JCM 10015 1, 8, 9
R. eutropha PHB − 4	PHA-negative mutant of H16	DSM 541, 12
E. coli DH5α	deoR endA1 gyr A96 hsdR17 (r κ -m κ + ) relA1 supE thi-1	Clontech
	Δ (lacZYA-argFV169) Δ80ΔlacZΔM 15F-λ-
E. coli S17-1	recA and tra genes of plasmid RP4 integrated into	14
	chromosome; auxotrophic for proline and thiamine
Plasmids
pLA29i7	Cosmid; Km r Tc r RK2 replicon; Mob +	2
pJRD215	Cosmid; Km r Sm r RSF1010 replicon; Mob +	3
pBluescript II KS +	Ap r lacPOZ T7 and T3 promoter	Stratagene
pJASc22	pJRD215 derivative; phaC1 Ps	This study
pJASc60	pJRD215 derivative; phaC1 Ps phaZ Ps phaC2 Ps phaD Ps	This study
pJASc60dC1Z	pJRD215 derivative; phaC2 Ps phaD Ps	This study
pJASc50	pJRD215 derivative; phaC1 Ps phaZ Ps phaC2 Ps	This study
pJASc50dC1Z	pJRD215 derivative; phaC2 Ps	This study

TABLE 2 — HOMOLOGY OF THE PRODUCTS OF THE PHB AND PHA LOCI OF PSEUDOMONAS sp. STRAIN 61-3 TO PROTEINS OF OTHER BACTERIA Homology To Other Gene Products

Gene Product	Size of Putative		Amino acid
Designation	Gene Product	Designation a	identity (%)
PhbR Ps	42.3	OruR	25.7
PhbB Ps	26.7	PhbB Re	66.1
PhbA Ps	40.6	PhbA Re	65.8
PhbC Ps	64.3	PhbC Re	53.0
PhaC1 Ps	62.3	PhaC1 Po	83.7
PhaZ Ps	31.7	PhaZ Po	89.0
PhaC2 Ps	62.8	PhaC2 Po	74.8
PhaD Ps	23.5	PhaD Pa	77.2
a OruR, transcriptional regulator for ornithine matabolism of P. aeruginosa(5); PhbB Re , PhbA Re , and PhbC Re , NADPH-dependent acetoacetyl-CoA reductase, β-ketothiolase, and PHB synthase of R. eutropha, respectively (10, 13, 15); PhaC1 Po , PhaZ Po , and PhaC2 Po , PHA synthase 1, PHA depolymerase, and PHA

TABLE 3 — ACCUMULATION OF PHA BY RECOMBINANT R. EUTROPHA PHB − 4 STRAINS HARBORING PHA BIOSYNTHESIS GENES OF PSEUDOMONAS sp. STRAIN 61-3 a

	Dry cell	PHA	PHA composition (mol %) b
Plasmid		Weight	Content	3HB	3HHx	3HO	3HD	3HDD	3H5DD
(Relevant Markers)	Substrate	(g/l)	(wt %)	(C4)	(C6)	(C8)	(C10)	(C12)	(C12′)
pJASc60	Gluconate	0.85	12	100	0	0	0	0	0
(phaC1, phaZ, phaC2, phaD)	Octanoate	0.84	6	92	0	8	0	0	0
	Dodecanoate	0.30	6	100	0	0	0	0	0
	Tetradecanoate	0.69	4	91	0	3	3	3	0
pJASc22	Gluconate	0.74	2	100	0	0	0	0	0
(phaC1)	Octanoate	0.86	13	31	10	59	0	0	0
	Dodecanoate	0.61	5	31	4	23	23	19	0
	Tetradecanoate	0.97	14	46	4	21	18	11	0
pJASc60dC1Z	Gluconate	0.92	20	100	0	0	0	0	0
(phaC2, phaD)	Octanoate	0.74	4	50	7	43	0	0	0
	Dodecanoate	0.34	1	51	0	9	13	27	0
	Tetradecanoate	0.67	5	44	1	16	15	24	0
pJASc50	Gluconate	0.52	2	100	0	0	0	0	0
(phaC1, phaZ, phaC2)	Tetradecanoate	0.78	6	62	2	16	13	7	0
pJASc50dC1Z	Gluconate	0.98	11	100	0	0	0	0	0
(phaC2)	Tetradecanoate	0.75	3	69	0	11	10	10	0
a Cells were cultivated at 30° C. for 72 h in MS medium containing the sodium salt of gluconate (2% wt/vol), octanoate (0.1% wt/vol × 5), dodecanoate, or tetradecanoate (0.5% wt/vol) as a sole carbon source.
b 3HB, 3-hydroxybutyrate; 3HHx, 3-hydroxyhexanoate; 3HO, 3-hydroxyoctanoate; 3HD, 3-hydroxy-decanoate; 3HDD, 3-hydroxydodecanoate; 3H5DD, 3-hydroxy-cis-5-dodecenoate.
8
1	559	PRT	Pseudomonas

5	1683	DNA	Pseudomonas
CDS	(1)...(1683)

1 of 18 part labels are ours — the grant heads the rest

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Section C — Chemistry; metallurgy

C12N9/88

USPC · US Patent Classification

435/232435/141530/350530/300536/23.2435/320.1435/135435/142536/23.7435/252.3

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