KR102331270B1 - Transformed microorganism producing polyhydroxyalkanoate - Google Patents

Abstract

The present invention relates to a transgenic microorganism in which a gene is modified to produce polyhydroxyalkanoate. More specifically, the present invention relates to a transformed microorganism that produces polyhydroxyalkanoate using 3-hydroxybutyric acid, 3-hydroxyvaleric acid, 4-hydroxyvaleric acid, and the like as monomers. The transformed microorganism of the present invention can control the composition and production of polyhydroxyalkanoate by introducing the phaC gene of a heterogeneous strain, and thus can be utilized to produce various types of polyhydroxyalkanoate.

Description

Transformed microorganism producing polyhydroxyalkanoate {TRANSFORMED MICROORGANISM PRODUCING POLYHYDROXYALKANOATE}

The present invention relates to a transgenic microorganism in which a gene is modified to produce polyhydroxyalkanoate. More specifically, the present invention relates to a transformed microorganism that produces polyhydroxyalkanoate using 3-hydroxybutyric acid, 3-hydroxyvaleric acid, 4-hydroxyvaleric acid, and the like as monomers.

Plastic materials, which have revolutionized the material due to their lightness and excellent physical properties, do not decompose in nature, causing serious environmental problems. Currently, the amount of plastic used worldwide exceeds 100 million tons, and the annual consumption in Korea is approaching 7 million tons, and about 20% of it is used for packaging materials, diapers, and syringes that are used once and is the main culprit of serious pollution. is designated as In order to solve the problem of environmental pollution caused by plastics, a solution through recycling of plastic waste is being sought, but the fundamental problem has not been solved. Accordingly, attention is focused on fundamental solutions through the development of biodegradable plastics.

Polyhydroxyalkanoate (PHA) is a chemical that accumulates inside microorganisms for storage of energy and reducing ability when a carbon source is abundant in a state in which the elements necessary for growth such as nitrogen, oxygen, phosphorus, and magnesium are insufficient. It is a natural polyester material. Polyhydroxyalkanoate has properties similar to synthetic polymers derived from conventional petroleum and exhibits biodegradability and biocompatibility, and thus is recognized as a material to replace conventional synthetic plastics.

Polyhydroxyalkanoate forms a form in which a hydroxy acid monomer having a side chain is polymerized. 3-Hydroxybutyric acid (3-Hydroxybutyric acid), 3-hydroxyvaleric acid (3-Hydroxyvaleric acid, 3HV) and 4-hydroxyvaleric acid (4-Hydroxyvaleric acid, 4HV) by combining monomers such as polyhydroxy It can constitute alkanoate, and it is known that there are more than 150 kinds of the monomers. Polyhydroxyalkanoate may have significantly different properties such as brittleness and rigidity depending on the combination of polymerized monomers (Yoshiharu. et. al . Biopolymers . 2002. Weinheim, Germany: Wiley-VCH). .ISBN 978-3-527-30225-3).

Meanwhile, the monomers such as 3-hydroxybutyric acid, 3-hydroxyvaleric acid and 4-hydroxyvaleric acid may be obtained through chemical synthesis or may be obtained through a biological method. However, the chemical synthesis method has problems such as excessive cost due to harsh conditions, use of catalysts and organic solvents, and poor biocompatibility. In addition, biological synthesis has a problem in that production and yield are low.

Yoshiharu. et. al. Biopolymers. 2002. Weinheim, Germany: Wiley-VCH. ISBN 978-3-527-30225-3.

Accordingly, an object of the present invention is to provide a transformed microorganism capable of effectively producing polyhydroxyalkanoate (PHA).

Another object of the present invention is to provide a method for producing polyhydroxyalkanoate using the microorganism.

In order to achieve the above object, one aspect of the present invention provides a transformed microorganism into which the phaC gene of a heterologous strain is introduced.

Another aspect of the present invention, the step of culturing the transformed microorganism; and obtaining polyhydroxyalkanoate produced from the transformed microorganism.

Another aspect of the present invention provides a culture medium composition comprising the transformed microorganism.

The transformed microorganism of the present invention can control the composition and production of polyhydroxyalkanoate by introducing the phaC gene of a heterogeneous strain, and thus can be utilized to produce various types of polyhydroxyalkanoate.

1 is a schematic diagram of a pathway for producing various polyhydroxyalkanoates from levulinic acid.
Figure 2a is a schematic diagram of the structure of the pPROBE-lvaR_P1vaA_phaBCA plasmid used in one embodiment of the present invention.
Figure 2b schematically shows the structure of the pPROBE-lvaR_P1vaA_phaC_P.d plasmid used in one embodiment of the present invention.
Figure 2c is a schematic diagram of the structure of the pPROBE-lvaR_P1vaA_phaC_C.n plasmid used in one embodiment of the present invention.
Figure 2d is pPROBE-lvaR_P1vaA_phaC_T.p used in one embodiment of the present invention. It is a schematic diagram of the structure of the plasmid.
Figure 2e shows pPROBE-lvaR_P1vaA_phaC_C.v. It is a schematic diagram of the structure of the plasmid.
3 is a graph analyzing the PHA production level of the strain according to the presence or absence of phaC gene deletion and levulinic acid (LA) (purple: culture for 24 hours, green: culture for 48 hours, blue: culture for 72 hours).
4 is a graph analyzing the PHA composition of the strain according to the presence or absence of deletion of the phaC gene by GC-MS (Gas Chromatograph Mass Spectrometer).
Figure 5a is a measurement of the composition of polyhydroxyalkanoate (PHA) according to the phaC and medium composition of the introduced heterogeneous strain.
Figure 5b is a measurement of the production of polyhydroxyalkanoate (PHA) according to the phaC and medium composition of the introduced heterogeneous strain.
6a is a graph confirming the composition and production change of polyhydroxyalkanoate (PHA) according to the additional deletion of the phaC gene.
6b is a graph confirming the composition and production change of polyhydroxyalkanoate (PHA) according to the additional deletion of phaC and lvaAB genes.
Figure 6c is a graph confirming the change in the composition and production of polyhydroxyalkanoate (PHA) according to a further deletion of phaC and fadB gene.

Hereinafter, the present invention will be specifically described.

One aspect of the present invention provides a transformed microorganism into which the phaC gene of a heterologous strain is introduced.

As used herein, the term "phaC" is a synthetic enzyme that biosynthesizes polyhydroxyalkanoate by polymerizing hydroxyalkanoate as a monomer.

In one embodiment of the present invention, the heterologous strain is Paracoccus denitrificans ( Paracoccus denitrificans ), Chromobacterium violaceum ( Chromobacterium violaceum ), Cupriavidus necator ( Cupriavidus necator ), Pseudomonas oleoborans ( Pseudomonas oleovorans ) or Thiocapsa pfennigii ).

The phaC gene of the heterogeneous strain may include the phaC gene of Paracoccus denitrificans. The phaC gene of Paracoccus denitrificans may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1. In addition, the phaC gene may have about 70%, 80%, 90%, or 95% or more homology with the nucleotide sequence of SEQ ID NO: 1. On the other hand, the phaC protein encoded by the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. The phaC protein may have about 70%, 80%, 90%, or 95% or more homology to the amino acid sequence of SEQ ID NO: 2.

The phaC gene of the heterologous strain may include the phaC gene of Chromobacterium violaceum. The phaC gene of Chromobacterium violaceum may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3. In addition, the phaC gene may have about 70%, 80%, 90%, or 95% or more homology with the nucleotide sequence of SEQ ID NO: 3. Meanwhile, the phaC protein encoded by the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4. The phaC protein may have about 70%, 80%, 90%, or 95% or more homology to the amino acid sequence of SEQ ID NO: 4.

PhaC gene in the heterogeneous strains may be one containing the phaC gene of Coop Ria bideoseu Neka Torr (Cupriavidus necator). PhaC gene of the Coop Ria bideoseu Neka Torr (Cupriavidus necator) may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5. In addition, the phaC gene may have about 70%, 80%, 90%, or 95% or more homology with the nucleotide sequence of SEQ ID NO: 5. On the other hand, the phaC protein encoded by the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5 may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6. The phaC protein may have about 70%, 80%, 90%, or 95% or more homology to the amino acid sequence of SEQ ID NO: 6.

PhaC gene in the heterogeneous strains may be those containing genes phaA, phaB, and phaC genes in gene Coop Ria bideoseu Neka Torr (Cupriavidus necator). The Coop Ria bideoseu genes phaA, phaB, and phaC genes in gene Neka Torr (Cupriavidus necator) may be a polynucleotide consisting of the nucleotide sequences of SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 5. In addition, the phaA gene, the phaB gene and the phaC gene may have about 70%, 80%, 90%, or 95% or more homology with the base sequences of SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 5, respectively. On the other hand, the phaA protein, phaB protein, and phaC protein encoded by the polynucleotide consisting of the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 5 are each composed of the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 6 It may be a polypeptide. The phaA protein, the phaB protein and the phaC protein may have about 70%, 80%, 90% or 95% or more homology to the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 6, respectively.

The phaC gene of the heterologous strain may include the phaC1 gene and the phaC2 gene of Pseudomonas oleovorans. The phaC1 gene and the phaC2 gene of Pseudomonas oleovorans may be polynucleotides consisting of the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 9, respectively. In addition, the phaC1 gene and the phaC2 gene may have about 70%, 80%, 90%, or 95% or more homology with the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 9, respectively. On the other hand, the phaC1 protein and the phaC2 protein encoded by the polynucleotide consisting of the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 9 may be polypeptides consisting of the amino acid sequences of SEQ ID NO: 8 and SEQ ID NO: 10, respectively. The phaC1 protein and the phaC2 protein may have about 70%, 80%, 90% or 95% or more homology with the amino acid sequences of SEQ ID NO: 8 and SEQ ID NO: 10, respectively.

PhaC gene in the heterogeneous strains may be one containing the phaC gene and the gene of phaE thio kapsa peupen nigiyi (Thiocapsa pfennigii). The thiocapsa pfennigii ( Thiocapsa pfennigii ) phaE gene and phaC gene may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 13, respectively. In addition, the phaE gene and the phaC gene may have about 70%, 80%, 90%, or 95% or more homology with the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 13, respectively. Meanwhile, the phaE protein and the phaC protein encoded by the polynucleotide consisting of the amino acid sequence of SEQ ID NO: 11 and SEQ ID NO: 13 may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 12 and SEQ ID NO: 14, respectively. The phaE protein and the phaC protein may have about 70%, 80%, 90% or 95% or more homology with the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 14, respectively.

In one embodiment of the invention, the transformed microorganism is Xi thigh eggplant (Zymomonas), Escherichia (Escherichia), Pseudomonas (Pseudomonas), alkali jeneseu (Alcaligenes), Salmonella (Salmonella), Shigella (Shigella), Burg holde Liao (Burkholderia), to raise Trojan wave (Oligotropha), keulrep when Ella (Klebsiella), blood teeth (Pichia), Candia (Candida), Hanse Cronulla (Hansenula), Saccharomyces as MY process (S accharomyces), or inclusive Yves It may be a microorganism belonging to the genus Kluyveromyces. In one embodiment of the present invention, the transformed microorganism may be Pseudomonas putida , specifically Pseudomonas putida EM42 strain.

The transformed microorganism may be one of the expression of any one gene selected from the group consisting of lvaAB gene, fadB gene, phaC gene, tesB gene, and combinations thereof suppressed.

The " lvaAB gene" is a gene encoding an enzyme present in the lva operon in the Pseudomonas putida strain. The lvaAB protein encoded by the lvaAB gene contributes to the production of 4PV-CoA (4-phosphovaleryl-CoA) by reacting 4HV-CoA with ATP. Specifically, the lvaAB protein is an enzyme in which phosphotransferase lvaA, an enzyme catalyzing transfer of a phosphate group, and lvaB, a hypothetical protein, are synthesized.

lvaAB inhibition of gene expression may be the expression of the gene lvaA suppressed, may be the expression of the gene lvaB suppressed, and can be the expression of all the genes and lvaA lvaB gene suppression.

In this case, the " lvaA gene" may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 19. The lvaA gene may have about 70%, 80%, 90%, or 95% or more homology with the nucleotide sequence of SEQ ID NO: 19. On the other hand, the lvaA protein encoded by the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 19 may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 20. The lvaA protein may have about 70%, 80%, 90%, or 95% or more homology to the amino acid sequence of SEQ ID NO: 20.

In addition, the lvaB gene may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 21. The lvaB gene may have about 70%, 80%, 90%, or 95% or more homology with the nucleotide sequence of SEQ ID NO: 21. On the other hand, the lvaB protein encoded by the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 21 may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 22. The lvaB protein may have about 70%, 80%, 90% or 95% or more homology to the amino acid sequence of SEQ ID NO: 22.

The " fadB gene" serves to reduce the acyl-CoA-based intermediate compound used as a precursor of PHA synthesis to its initial form. In one embodiment of the present invention, the fadB gene is a gene of Pseudomonas putita strain, and may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 23. The fadB gene may have about 70%, 80%, 90%, or 95% or more homology with the nucleotide sequence of SEQ ID NO: 23. On the other hand, the fadB protein encoded by the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 23 may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 24. The fadB protein may have about 70%, 80%, 90%, or 95% or more homology to the amino acid sequence of SEQ ID NO: 24.

The "phaC gene" of the Pseudomonas putita strain may consist of a phaC1 gene, a phaZ gene and a phaC2 gene. The phaC1 gene, phaZ gene, and phaC2 gene may be polynucleotides consisting of the nucleotide sequences of SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 29, respectively. In addition, the phaC1 gene, phaZ gene, and phaC2 gene of the Pseudomonas putita strain have about 70%, 80%, 90% or 95% or more homology with the nucleotide sequences of SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 29, respectively. can Meanwhile, the phaC protein of the Pseudomonas putita strain may be composed of a phaC1 protein, a phaZ protein, and a phaC2 protein. The phaC protein may be a polypeptide consisting of the amino acid sequences of SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO: 30, respectively, and the amino acids of SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO: 30, respectively. It may have at least about 70%, 80%, 90%, or 95% homology to the sequence.

The " tesB gene" is a gene encoding the tesB protein, which is a short-chain or broad-chain-specific thioesterase that converts 4HV-CoA (4-hydroxyvaleryl-CoA) to 4HV in a Pseudomonas putida strain. am. In one embodiment of the present invention, the tesB gene may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 31. The tesB gene may have about 70%, 80%, 90%, or 95% or more homology with the nucleotide sequence of SEQ ID NO: 31. Meanwhile, the tesB protein encoded by the polynucleotide of SEQ ID NO: 31 may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 32. The tesB protein may have about 70%, 80%, 90%, or 95% or more homology to the amino acid sequence of SEQ ID NO: 32.

In the present invention, "introduction of a gene" and "suppression of expression of a gene" may be used without limitation as long as it is a method used in the art. In one embodiment of the present invention, according to the Gibson method (Gibson, DG, Young, L., Chuang, R.-Y., Venter, JC, Hutchison III, CA, & Smith, HO (2009)), the "gene Introduction" and "suppression of gene expression" were performed. In one embodiment of the present invention, the transformed microorganism may produce polyhydroxyalkanoate (PHA).

The "polyhydroxyalkanoate" is a generic term for hydroxy acid polyesters having various carbon side chains, and includes 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV) and 4-hydroxyvaleric acid. It can be formed by polymerization of one or more monomers selected from the group consisting of acid (4HV), is a carbon and energy storage material of a wide range of bacteria, and exists as an inclusion body in cells. The production route of the polyhydroxyalkanoate is schematically shown in FIG. 1 .

In the present invention, the "3-hydroxybutyric acid" has _{a chemical formula of CH 3} CH ₂ (OH)CHCOOH, and may have a structure as shown in Formula 1 below.

[Formula 1]

In the present invention, the "3-hydroxyvaleric acid" has _{a chemical formula of CH 3} (CH ₂ ) ₂ (OH)CHCOOH, and may have a structure as shown in Formula 2 below.

[Formula 2]

In the present invention, the "4-hydroxyvaleric acid" has _{a chemical formula of CH 3} CH(OH)(CH ₂ ) ₂ COOH, and may have a structure as shown in Chemical Formula 3 below.

[Formula 3]

In the present invention, the polyhydroxyalkanoate is poly(3-hydroxybutyric acid), poly(3-hydroxyvaleric acid), poly(4-hydroxyvaleric acid), poly(3-hydroxybutyric acid-co- 3-hydroxyvaleric acid), poly(3-hydroxybutyric acid-co-4-hydroxyvaleric acid), poly(3-hydroxyvaleric acid-co-4-hydroxyvaleric acid) and poly(3-hydroxy It may be at least one selected from the group consisting of hydroxybutyric acid-co-4-hydroxyvaleric acid-co-4-hydroxyvaleric acid).

The poly(3-hydroxybutyric acid) may have a structure as shown in Formula 4 below.

[Formula 4]

The poly(3-hydroxyvaleric acid) may have a structure as shown in Chemical Formula 5 below.

[Formula 5]

The poly(4-hydroxyvaleric acid) may have a structure as shown in Formula 6 below.

[Formula 6]

The poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) may have a structure as shown in Formula 7 below.

[Formula 7]

The poly(3-hydroxybutyric acid-co-4-hydroxyvaleric acid) may have a structure as shown in Formula 8 below.

[Formula 8]

The poly(3-hydroxyvaleric acid-co-4-hydroxyvaleric acid) may have a structure as shown in Formula 9 below.

[Formula 9]

The poly(3-hydroxybutyric acid-co-4-hydroxyvaleric acid-co-4-hydroxyvaleric acid) may have a structure as shown in Formula 10 below.

[Formula 10]

Another aspect of the present invention comprises the steps of culturing the transformed microorganism of claim 1; and obtaining polyhydroxyalkanoate produced from the transformed microorganism.

The medium and other culture conditions used for culturing the microorganism of the present invention can be used without any particular limitation as long as it is a medium used for culturing a normal microorganism, particularly, a microorganism of the genus Pseudomonas, specifically, the microorganism of the present invention is a suitable carbon source. , a nitrogen source, phosphorus, inorganic compounds, amino acids and / or vitamins can be cultured under aerobic conditions while controlling the temperature, pH, etc. in a conventional medium.

Examples of the carbon source may include sucrose or glucose, molasses containing a large amount of sucrose may also be used as the carbon source, and other appropriate carbon sources may be variously used.

Examples of nitrogen sources may include organic nitrogen sources such as peptone, yeast extract, broth, malt extract, corn steep liquor, and soybean wheat, and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, anmonium carbonate, and ammonium nitrate. have. These nitrogen sources may be used alone or in combination. The medium may contain potassium dihydrogen phosphate, dipotassium hydrogen phosphate and the corresponding sodium-containing salt as phosphorus. It may also contain a metal salt such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins and suitable precursors may be included.

During the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. In addition, in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas may be injected into the culture, or nitrogen, hydrogen or carbon dioxide gas may be injected with or without gas to maintain anaerobic and microaerobic conditions.

The temperature of the culture may be usually 20° C. to 40° C., specifically, 25° C. to 35° C., more specifically, 30° C., but may be changed without limitation depending on the intended purpose. The incubation period may be continued until the desired amount of useful material is reached, and specifically, it may be 10 hours to 100 hours, but is not limited thereto.

The method for producing polyhydroxyalkanoate of the present invention may include obtaining the polyhydroxyalkanoate produced from the culture medium or the microorganism. Methods for recovering polyhydroxyalkanoate from microorganisms or media include methods known in the art, such as centrifugation, filtration, anion exchange chromatography, crystallization and HPLC, but are not limited thereto.

Another aspect of the present invention provides a culture medium composition comprising the transformed microorganism.

The culture medium composition comprising the transforming microorganism is not particularly limited as long as it can be used to produce polyhydroxyalkanoate in high yield, but as an example, the culture of the transformed microorganism, culture supernatant, and lysate , their fractions, etc. As another example, a culture supernatant obtained by centrifuging a culture of a transformed microorganism, a lysate obtained by physically or sonication of a transformed microorganism, the culture, a culture supernatant, It may be a fraction obtained by subjecting the lysate to a method such as centrifugation or chromatography.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Experimental Example 1. Production of transgenic microorganisms with suppressed expression of specific genes

Pseudomonas putida ( P. putita ) In order to delete a specific gene in the EM42 strain, the homologous region of the gene to be deleted was amplified by PCR using the primers in Table 1 below. This PCR result was linked with the pQSAK plasmid treated with restriction enzymes XhoI and XbaI by the Gibsson method to produce a pQSAK suicide plasmid. In this case, the Gibsson method was used to make a master mix using Phusion DNA polymerase (NEB biolabs), Taq DNA ligase (NEB biolabs) and T5 Exonuclease (NEB biolabs).

Primer name sequence information note lvaA _upstream_fwd
(SEQ ID NO: 33) 5'-gaggccctttcgtcttcacctcgagatggttgtagctgagcacg-3' lvaAB deletion lvaA _upstream_rev (SEQ ID NO: 34) 5'-actgctgcatggttctgtaggccctgcc-3' lvaAB deletion lvaB _downstream_fwd (SEQ ID NO: 35) 5'-cctgcaatgaaaccgcatcgaccacccc-3' lvaAB deletion lvaB _downstream_rev (SEQ ID NO: 36) 5'-cgcaaaagaaaatgccgattctagacatggcgaagcacgaacg-3' lvaAB deletion phaC1 _up_fwd (SEQ ID NO: 37) 5'-gaggccctttcgtcttcacctcgaggaacaactgtgggcag-3' phaC1ZC2 deletion phaC1_ up_rev (SEQ ID NO: 38) 5'-cagcacgtagcccagggtgttttccgag-3' phaC1ZC2 deletion phaC2_ down_fwd (SEQ ID NO: 39) 5'-acaccctgggctacgtgctgacccgatg-3' phaC1ZC2 deletion phaC2 _down_rev (SEQ ID NO: 40) 5'-cgcaaaagaaaatgccgattctagagcgtgcgtttgattgcattg-3' phaC1ZC2 deletion fadB_up_fwd (SEQ ID NO: 41) 5'-gaggccctttcgtcttcacctcgagtgtagtcgccttggctc-3' fadB deletion _up_rev fadB (SEQ ID NO: 42) 5'-catttcacgctgttgacggactcacccttg-3' fadB deletion fadB_down_fwd (SEQ ID NO: 43) 5'-tccgtcaacagcgtgaaatggccaagaac-3' fadB deletion _down_rev fadB (SEQ ID NO: 44) 5'-cgcaaaagaaaatgccgattctagatctgcagtcaggcccatc-3' fadB deletion

The pQSAK suicide plasmid information of the EM42 strain and specific genes is shown in Table 2 below.

designation characteristic where to get it strain Pseudomonas putida ( P. putida ) EM42 KT2440 derivative;△prophage1 △prophage4 △prophage3 △?prophage2 △Tn7 △endA-1△endA-2 △hsdRMS △flagellum △Tn4652 CSIC pQSAK suicide plasmid pQSAK-lvaAB suicide plasmid for deletion of lvaA and lvaB genes in P. putida Prepared in Experimental Example 1 pQSAK-tesB suicide plasmid for deletion of tesB gene in P. putida Prepared in Experimental Example 1 pQSAK-phaC1ZC2 Suicide plasmid for deletion of phaC1, phaZ, and phaC2 genes in P. putida Prepared in Experimental Example 1 pQSAK-fadB suicide plasmid for deletion of fadB gene in P. putida Prepared in Experimental Example 1

The pQSAK-lvaAB, pQSAK-fadB, and pQSAK-phaC1ZC2 plasmids were introduced into the EM42 strain by electroporation, followed by primary selection in a medium to which kanamycin was added. Thereafter, introduction of the pQSAK-lvaAB, pQSAK-fadB and pQSAK-phaC1ZC2 plasmids into the EM42 strain was confirmed through PCR analysis. pQSAK-lvaAB, pQSAK-fadB and pQSAK-phaC1ZC2 plasmid introduction can colonies (colony) of the check EM42 strain of 10% is grown on the doped medium cross (sucrose) with a lvaAB gene, fadB gene and phaC gene deletion EM42 A strain (Example 1) was selected.

The pQSAK- phaC1ZC2 , pQSAK-tesB and pQSAK-fadB After the plasmid was introduced into the EM42 strain by electroporation, the plasmid was first selected in a medium to which kanamycin was added. Then, through PCR analysis, pQSAK-phaC1ZC2, pQSAK- tesB and pQSAK-fadB in the EM42 strain Plasmid introduction was confirmed. pQSAK-phaC1ZC2, pQSAK- tesB and pQSAK-fadB plasmid introduced is 10% of the colonies were identified EM42 strain grown in the medium were selected to cross with a strain EM42 phaC gene, fadB gene deletion and tesB gene (Example 2) . On the other hand, the EM42 strain (Example 3) in which the gene was not deleted was selected.

Furthermore, the pQSAK-phaC1ZC2 plasmid was introduced into the EM42 strain by electroporation, and then, the kanamycin-added medium was used for primary selection. Then, pQSAK-phaC1ZC2 in the EM42 strain through PCR analysis The introduction of the plasmid was confirmed. pQSAK-phaC1ZC2 Colonies of the EM42 strain confirmed for plasmid introduction were grown in 10% sucrose medium to select the EM42 strain (Example 4) in which the phaC gene was deleted.

The pQSAK-lvaAB and pQSAK-phaC1ZC2 plasmids were introduced into the EM42 strain by electroporation, and then, primary selection was performed in a medium to which kanamycin was added. Then, pQSAK-lvaAB in the EM42 strain through PCR analysis and the introduction of the pQSAK-phaC1ZC2 plasmid was confirmed. Colonies of the EM42 strain in which the pQSAK-lvaAB and pQSAK-phaC1ZC2 plasmid introduction were confirmed were grown in 10% sucrose medium to select the EM42 strain (Example 5) in which the lvaAB gene and the phaC gene were deleted.

The pQSAK-fadB and pQSAK-phaC1ZC2 plasmids were introduced into the EM42 strain by electroporation, and then, primary selection was made in a medium to which kanamycin was added. Thereafter, introduction of the pQSAK-fadB and pQSAK-phaC1ZC2 plasmids into the EM42 strain was confirmed through PCR analysis. pQSAK-fadB and pQSAK-phaC1ZC2 plasmid introduction can colonies of the identified EM42 strain of 10% is grown on the medium were selected to cross with a strain EM42 fadB gene and phaC gene deletion (Example 6).

The deletion of the gene in the selected EM42 strain was confirmed through PCR band identification and sequencing of the band.

Experimental Example 2. Preparation of a transgenic microorganism into which the phaC gene of a heterologous strain was introduced

In order to introduce the phaC gene of the heterologous strain into the EM42 strain of Examples 1 to 6, Paracoccus denitrificans ( Paracoccus denitrificans ), Chromobacterium violaceum ( Chromobacterium violaceum ), Cupria vidus necator (Cupriavidus necator), Pseudomonas oleovorans (Pseudomonas oleovorans) or thio kapsa peupen nigiyi (Thiocapsa pfennigii) the phaC gene plasmid each pPROBE (Improved gfp and inaZ Broad-Host-Range-Promoter Probe Vectors of - William G. Miller, Johan A levulinic acid (LA) inducible phaC expression system was constructed by cloning together with the lvaR regulator and lvaA promoter in HJ Leveau, and the author of Steven E. Lindow).

Information on the heterologous strain and the pPROBE plasmid into which the phaC gene of each heterologous strain is inserted is shown in Table 3 and FIGS. 2A to 2E.

designation characteristic where to get it strain Cupriavidus necator H16 wild type KCTC 22469 Chromobacterium violaceum wild type KCTC 2897 Paracoccus denitrificans wild type KCTC No. 2530 Pseudomonas oleovolans Gpo1 wild type ATCC 29347 Thiocapsa pfennigii wild type DSM226 pPROBE plasmid pPROBE-lvaR-P _lvaA- phaC_P.d. Plasmid in which phaC of Paracoccus denitrificans is expressed by the LA inducible system Prepared in Experimental Example 2 pPROBE-lvaR-P _lvaA- phaC_C.v. Plasmid in which phaC of Chromobacterium violaceum is expressed by LA inducible system Prepared in Experimental Example 2 pPROBE-lvaR-P _lvaA- phaC_T.p. Plasmids in which phaE and phaC of Thiocapsa pfennigii are expressed by the LA inducible system Prepared in Experimental Example 2 pPROBE-lvaR-PlvaA-phaBCA_phaBCA. Plasmid in which phaA, phaB, and phaC of Cupriavidus necator H16 are expressed by LA inducible system Prepared in Experimental Example 2 pPROBE-lvaR-P _lvaA- phaC_P.o. Plasmids in which phaC1 and phaC2 of Pseudomonas oleovorans are expressed by the LA inducible system Prepared in Experimental Example 2 pPROBE-lvaR-P _lvaA- phaC_C.n. Plasmid in which phaC of Cupriavidus necator H16 is expressed by LA inducible system Prepared in Experimental Example 2

Specifically, the phaC of Paracoccus denitrificans , Chromobacterium violaceum or Cupriavidus necator is a part of the pPROBE plasmid and to include the lvaA promoter at the end. It was amplified by PCR using the primers in Table 4.

Primer name sequence information note phaC_P.d._fwd
(SEQ ID NO: 45) 5'-ctttataaggaggaaaaacatatggccgcgccccgcgcc-3' pPROBE-lvaR-PlvaA-phaC_Pd phaC_P.d._rev (SEQ ID NO: 46) 5'-agtccaagctcagctaattaagcttttacgcccgctcgtggacgtaaaggccc-3' pPROBE-lvaR-PlvaA-phaC_Pd phaC_C.v._fwd
(SEQ ID NO: 47) 5'-ctttataaggaggaaaaacatatgcagcagttcgtcaactcg-3' pPROBE-lvaR-PlvaA-phaC_Cv phaC_C.v._rev (SEQ ID NO: 48) 5'-agtccaagctcagctaattaagctttcattgcaggctggcggc-3' pPROBE-lvaR-PlvaA-phaC_Cv phaC_C.n_fwd (SEQ ID NO: 49) 5'-ctttataaggaggaaaaacatatggcgaccggcaaaggc-3' pPROBE-lvaR-PlvaA-phaC_Cn phaC_C.n_rev (SEQ ID NO: 50) 5'-agtccaagctcagctaattaagctcatgccttggctttgacgtatcg-3' pPROBE-lvaR-PlvaA-phaC_Cn

The pPROBE-lvaR-P _lvaA plasmid was prepared by digestion with HindIII and NdeI. The prepared phaC PCR fragment was ligated with pPROBE-lvaR-PlvaA plasmid by Gibson method (pPROBE-lvaR-P _lvaA- phaC_C.v.; pPROBE-lvaR-P _lvaA- phaC_C.n.; and pPROBE-lvaR, respectively. -P _lvaA -phaC_P.d.). The plasmid prepared thereafter was transduced into the DH10B strain by electroporation, and then obtained from the colonies selected in the antibiotic-added medium.

In Pseudomonas oleovorans , phaC1 and phaC2 were amplified by PCR using the primers in Table 5, respectively, and linked to the pPROBE-lvaR-PlvaA plasmid cut with HindIII and NdeI by Gibson method (pPROBE-lvaR- P _lvaA -phaC_P.o.).

Primer name sequence information note phaC1_fwd
(SEQ ID NO: 63) 5'-aggtgagagtggaggaggtccatttatgcgagacaagtcgaac-3' pPROBE-lvaR-PlvaA-phaC1C2 P.o phaC1_rev (SEQ ID NO: 64) 5'-agtccaagctcagctaattaagctttcagcggatatgcacgtag-3' pPROBE-lvaR-PlvaA-phaC1C2 P.o phaC2_fwd (SEQ ID NO: 65) 5'-ctttataaggaggaaaaacatatgagtgacaagaacaacgaag-3' pPROBE-lvaR-PlvaA-phaC1C2 P.o phaC2_rev (SEQ ID NO: 66) 5'-aaatggacctcctccactctcaccttcagcgttcgtgcacatag-3' pPROBE-lvaR-PlvaA-phaC1C2 P.o

Thiocapsa pfennigii ( Thiocapsa pfennigii ), phaE and phaC were amplified by PCR with the primers of Table 6 below, and linked to the pPROBE-lvaR-PlvaA plasmid digested with HindIII and NdeI by the Gibson method (pPROBE-lvaR-PlvaA- phaC_T.p.).

Primer name sequence information note phaE_T.p._fwd
(SEQ ID NO: 51) 5'-ctttataaggaggaaaaacatatgaacgatacggccaacaag-3' pPROBE-lvaR-PlvaA-phaEC phaE_T.p._rev
(SEQ ID NO: 52) 5'-atggggacatggcactctctcctggtgg-3' pPROBE-lvaR-PlvaA-phaEC phaC_T.p._fwd (SEQ ID NO: 53) 5'-agagagtgccatgtccccattcccgatc-3' pPROBE-lvaR-PlvaA-phaEC phaC_T.p._rev
(SEQ ID NO: 54) 5'-caagctcagctaattaagctttcagccgcgttcgttcag-3' pPROBE-lvaR-PlvaA-phaEC

The phaBCA of Cupriavidus necator was amplified by PCR using the primers in Table 7 below for phaA, phaB and phaC, respectively, and then pPROBE-lvaR-PlvaA plasmid digested with HindIII and NdeI (Fig. 4a) by Gibson method (pPROBE-lvaR-PlvaA-phaBCA_C.n.).

Primer name sequence information note phaA_C.n_fwd
(SEQ ID NO: 55) 5'-caaggcatgaattgaaaggactacacaatgac-3' pPROBE-lvaR-PlvaA-phaBCA_Cn phaA_C.n_rev (SEQ ID NO: 56) 5'-agtccaagctcagctaattaagcttatttgcgctcgactgc-3' pPROBE-lvaR-PlvaA-phaBCA_Cn phaB_C.n_fwd (SEQ ID NO: 57) 5'-ctttataaggaggaaaaacatatgactcagcgcattgcg-3' pPROBE-lvaR-PlvaA-phaBCA_Cn phaB_C.n_rev_RBS (SEQ ID NO: 58) 5'-ttgtctctcttcagcccatatgcaggcc-3' pPROBE-lvaR-PlvaA-phaBCA_Cn phaC_C.n_fwd_RBS (SEQ ID NO: 59) 5'-tatgggctgaagagagacaatcaaatcatgg-3' pPROBE-lvaR-PlvaA-phaBCA_Cn phaC_C.n_rev_RBS (SEQ ID NO: 60) 5'-tcctttcaattcatgccttggctttgac-3' pPROBE-lvaR-PlvaA-phaBCA_Cn

The prepared pPROBE-lvaR-P _lvaA- phaC_C.v.; pPROBE-lvaR-P _lvaA -phaC_C.n.; and pPROBE-lvaR-P _lvaA- phaC_P.d.; pPROBE- lvaR-P _lvaA -phaC_P.o.; pPROBE-lvaR-PlvaA-phaC_T.p.; and pPROBE-lvaR-PlvaA-phaBCA_C.n. Plasmids were obtained from colonies selected in a medium to which antibiotics were added after transduction of each DH10B strain by electroporation.

The obtained pPROBE-lvaR-P _lvaA- phaC_C.v.; pPROBE-lvaR-P _lvaA -phaC_C.n.; and pPROBE-lvaR-P _lvaA- phaC_P.d.; pPROBE-lvaR-P _lvaA -phaC_P.o.; pPROBE-lvaR-PlvaA-phaC_T.p.; and pPROBE-lvaR-PlvaA-phaBCA_C.n. The strains into which the plasmids were introduced in Examples 1 to 6, respectively, are shown in Table 8 below.

pPROBE plasmid Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 pPROBE-lvaR-P _lvaA phaC_P.d. Example 1-1 Example 2-1 Example 3-1 Example 4-1 Example 5-1 Example 6-1 pPROBE-lvaR-P _lvaA- phaC_C.v Example 1-2 Example 2-2 Example 3-2 Example 4-2 Example 5-2 Example 6-2 pPROBE-lvaR-P _lvaA phaC_T.p. Examples 1-3 Example 2-3 Example 3-3 Example 4-3 Example 5-3 Example 6-3 pPROBE-lvaR-PlvaA-phaBCA_phaBCA. Examples 1-4 Example 2-4 Example 3-4 Example 4-4 Example 5-4 Example 6-4 pPROBE-lvaR-P _lvaA- phaC_P.o. Examples 1-5 Example 2-5 Example 3-5 Example 4-5 Example 5-5 Example 6-5 pPROBE-lvaR-P _lvaA- phaC_C.n. Examples 1-6 Example 2-6 Example 3-6 Example 4-6 Example 5-6 Example 6-6

On the other hand, in the case of the control group, no plasmid was used.

Experimental Example 3. Confirmation of the phenotype of the transformed microorganism

Experimental Example 3-1. Comparison of PHA production with or without phaC gene deletion and levulinic acid treatment

For the strains of Examples 3-4 and 4-4, the production of PHA according to the presence or absence of levulinic acid (LA) was compared ( FIG. 3 ).

Specifically, the strain is a trace element (trace element, per liter 2.4 g FeCl _{3 ·} 6H ₂ O, 0.3 g CoCl _{2 ·} 6H ₂ O, 0.15 g CuCl _{2 ·} 2H ₂ O, 0.3 g ZnCl ₂ , 0.3 g Na ₂ MO _4· 2H ₂ O, 0.075 g H ₃ BO ₃ and 0.495 g MnCl _2· 4H ₂ O) and a carbon source (D-glucose purchased from Bioshop) in LB (purchased from Merck) medium with an OD value of 0.5 to It was cultured at 30°C and 200 RPM conditions to become 1. Thereafter, each strain was treated with levulinic acid, and levulinic acid was additionally administered every 24 hours and cultured for 48 to 72 hours.

The pellet was collected by centrifugation of 1 ml of the culture solution, washed with cold distilled water, 1 ml of distilled water and 80 μg/L of Nile red (sigma) diluted with DMSO were mixed with a solution for 1 hour. reacted while After washing with distilled water once more, diluted with PBS (purchased from Bioworld), the fluorescence intensity of Nile Red was analyzed using BD FACSCalibur Flow Cytomerer (BD Biosciences) to measure PHA production from each strain. .

As a result, as shown in FIG. 3 , when neither of the strains of Examples 3-4 and 4-4 were treated with levulinic acid, it was confirmed that PHA was not produced. On the other hand, when the strains of Examples 3-4 and 4-4 were treated with levulinic acid, it was confirmed that the strain of Example 4-4 produced more PHA than the strain Example 3-4. At this time, the horizontal axis means the fluorescence intensity of Nile Red dyed on the PHA granules produced in the strain, the vertical axis means the number of strains, and each line in FIG. 3 means the result according to the incubation time.

Experimental Example 3-2. Confirmation of change in PHA composition due to phaC gene deletion

In addition, in order to confirm whether native PHA production can be suppressed by deletion of the phaC gene, the composition of PHA produced in the strains of Examples 3-4 and 4-4 was analyzed.

Specifically, the strains of Examples 3-4 and 4-4 were inoculated on an LB-agar-plate and cultured overnight at a temperature of 30°C. Three colonies each were selected from the plate, inoculated into 5 ml of LB medium (per liter: 5 g yeast extract, 10 g peptone, and 10 g NaCl), and cultured for 15 hours under shaking conditions.

Then, i) 7.5 g/L glucose, ii) 7.5 g/L levulinic acid, iii) trace element, per liter: 2.4 g FeCl ₃ 6H ₂ O, 0.3 g CoCl ₂ 6H ₂ O, 0.15 g CuCl _2· 2H ₂ O, 0.3 g ZnCl ₂ , 0.3 g Na ₂ MO _4· 2H ₂ O, 0.075 g H ₃ BO ₃ and 0.495 g MnCl _2· 4H ₂ O) and iv) 4Хphosphate buffer (per liter) : NaH ₂ PO ₄ ·H ₂ O 7.50 g and K ₂ HPO ₄ 15.52 g) containing 20 ㎖ or 40 ㎖ of modified M9 medium (modified M9 medium) containing 1 ㎖ flask containing 1 ㎖ culture inoculated.

It was cultured in a shaking incubator at 30 °C and 200 rpm for 24 hours. After that, the culture was supplemented with glucose and levulinic acid at the same concentration again, and then cultured in a shaking incubator at 30°C and 200 rpm. For PHA analysis, 2 ml or 10 ml of the culture was collected and centrifuged at 2,600×g for 20 min at 4°C. The collected pellet was washed twice with cold distilled water and stored at -80°C for further analysis. All experiments were performed in triplicate.

The frozen pellet was freeze-dried using a 2.5 L bench-top freeze dryer to measure biomass content (CDM: cell dry mass). PHA content and composition were confirmed through GC-MS analysis following methanolysis.

Each of the freeze-dried pellets was mixed with 2 ml of chloroform (purchased from Acros Organic, 99.8%) and 2 ml of a methanol-sulfuric acid mixture (15% v/v 95% sulfuric acid) solution, and at 100° C. for 4 hours. Methanolysis was carried out. After the reaction, the solution was cooled at room temperature, 1 ml of distilled water was added, and the solution was mixed by vortexing. Thereafter, phase separation was performed at room temperature overnight, and 100 μl of a sample was collected from the lower organic solvent layer, diluted in 900 μl of chloroform, and 1 ml of a sample for GC-MS analysis was prepared.

The prepared sample was analyzed under the conditions shown in Table 9 below using a 7890B GC equipment equipped with a 5977B Mass Selective Detector.

One column CycloSil-B column
(30 m × 0.320 mm × 0.25 μm) 2 sample volume 2 μl 3 flow rate 1.5 ml/min 4 temperature initially set at 60 ℃ thereafter increased at a rate of 10 ℃/min for 7 minutes and 30 ℃/min until a maximum temperature of 250 ℃was attained, and subsequently held for 3 min

The PHA content (wt %) was calculated as a percentage of CDM on a mass basis, and the PHA monomer composition (mol %) was expressed as a molar percentage of the total number of monomers.

As a result, as shown in FIG. 4 , in the case of Example 3-4 strain (a), native PHA of peaks 1, 2 and 3 were detected, whereas in Example 4-4 strain (b), 1 and 2 And it was confirmed that the native PHA of peak 3 was not detected.

Experimental Example 3-3. Confirmation of PHA composition change according to phaC gene origin and medium composition

In addition, when the medium composition was changed for the strains of Examples 3-1 to 3-4, 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV) and 4- The ratio and production of hydroxyvaleric acid (4HV) were evaluated ( FIGS. 5A and 5B ).

The strains of Examples 3-1 to 3-4 were inoculated on an LB-agar-plate and cultured overnight at a temperature of 30°C. Three colonies each were selected from the plate, inoculated into 5 ml of LB medium (per liter: 5 g yeast extract, 10 g peptone, and 10 g NaCl), and cultured for 15 hours under shaking conditions.

Then, i) 7.5 g/L glucose, ii) 7.5 g/L levulinic acid, iii) trace element, per liter: 2.4 g FeCl ₃ 6H ₂ O, 0.3 g CoCl ₂ 6H ₂ O, 0.15 g CuCl _2· 2H ₂ O, 0.3 g ZnCl ₂ , 0.3 g Na ₂ MO _4· 2H ₂ O, 0.075 g H ₃ BO ₃ and 0.495 g MnCl _2· 4H ₂ O) and iv) 4Хphosphate buffer (per liter) _{: NaH 2 PO 4 · H 2} O 7.50 g , and _{K 2 HPO 4 15.52 g) 20} ㎖ or 40 ㎖ improved M9 medium (modified M9 medium), TB medium (Alfa Aesa) or LA (Sigma-Aldrich of containing 1 ml of the culture was inoculated into a 250 ml flask containing the medium.

It was cultured in a shaking incubator at 30 °C and 200 rpm for 24 hours. After that, the culture was supplemented with glucose and levulinic acid at the same concentration again, and then cultured in a shaking incubator at 30°C and 200 rpm. For PHA analysis, 2 ml or 10 ml of the culture was collected and centrifuged at 2,600×g for 20 min at 4°C. The collected pellet was washed twice with cold distilled water and stored at -80°C for further analysis. All experiments were performed in triplicate.

The frozen pellet was freeze-dried using a 2.5 L bench-top freeze dryer to measure biomass content (CDM: cell dry mass). PHA content and composition were confirmed through GC-MS analysis following methanolysis.

Each of the freeze-dried pellets was mixed with 2 ml of chloroform (purchased from Acros Organic, 99.8%) and 2 ml of a methanol-sulfuric acid mixture (15% v/v 95% sulfuric acid) solution, and at 100° C. for 4 hours. Methanolysis was carried out. After the reaction, the solution was cooled at room temperature, 1 ml of distilled water was added, and the solution was mixed by vortexing. Thereafter, phase separation was performed at room temperature overnight, and 100 μl of a sample was collected from the lower organic solvent layer, diluted in 900 μl of chloroform, and 1 ml of a sample for GC-MS analysis was prepared.

The prepared sample was analyzed under the conditions of Table 9 using the 7890B GC equipment equipped with the 5977B Mass Selective Detector, and the PHA content (wt %) was calculated as a percentage of CDM based on the mass, and the PHA monomer composition (mol %) is expressed as a molar percentage of the total number of monomers.

division badge 3HB 3HV 4HV PHA% of CDW Example 3-1 TB 7.7 60.0 1.3 1.4 Example 3-2 69.8 1342.9 1669.1 38.7 Example 3-3 9.6 269.9 225.8 8.0 Example 3-4 87.1 98.9 4.1 4.0 control 0.0 0.0 0.0 0.0 Example 3-1 M9 & Glu 26.4 1702.8 1009.7 58.3 Example 3-2 31.7 1205.3 0.0 35.4 Example 3-3 0.6 554.4 167.5 37.1 Example 3-4 808.6 1045.6 5.5 49.0 control 0.0 0.0 0.0 0.0 Example 3-3 M9 0.0 51.9 0.0 3.5 control 0.0 0.0 0.0 0.0

As shown in Table 10 and FIGS. 5A and 5B , it was confirmed that the composition ( FIG. 5A ) and the production amount ( FIG. 5B ) of the PHA produced were different depending on the origin of the phaC gene and the composition of the medium.

Experimental Example 3-4. Confirmation of changes in PHA composition due to additional deletion of lavAB, fadB, and phaC genes and additional introduction of phaAB genes

In order to confirm the change in the PHA composition according to the additional deletion of the lavAB, fadB, and phaC genes, the strains of Example 4, Example 4-3, Example 5, Example 5-3, Example 6 and Example 6-3 The ratio and production amount of 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV) and 4-hydroxyvaleric acid (4HV) produced from to 6c). In addition, Example 4-4, Example 5-4, and Example 6- in which pRK415-lvaR-PlvaA-phaAB plasmids were introduced into the Example 4-3, Example 5-3 and Example 6-3 strains, respectively By preparing 4 strains, the ratio and production of 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV) and 4-hydroxyvaleric acid (4HV) produced from the strains were also evaluated. The pRK415-lvaR-PlvaA-phaAB was obtained by amplifying phaA and phaB of Cupriavidus necator using the pRK415 plasmid (Nova lifetech inc.) and the primers of Table 11 below, followed by the same method as in Experimental Example 2. (pRK415-lvaR-PlvaA-phaAB).

Primer name sequence information note phaB_rev_phaBA
(SEQ ID NO: 61) 5'-tcctttcaattcagcccatatgcaggcc-3' pRK415-lvaR-PlvaA-phaBA phaA_fwd_phaBA (SEQ ID NO: 62) 5'-tatgggctgaattgaaaggactacacaatgac-3' pRK415-lvaR-PlvaA-phaBA

At this time, the medium is 7.5 g/L glucose, ii) 7.5 g/L levulinic acid, iii) trace element (per liter: 2.4 g FeCl ₃ 6H ₂ O, 0.3 g CoCl ₂ 6H ₂ O, 0.15 g CuCl _2· 2H ₂ O, 0.3 g ZnCl ₂ , 0.3 g Na ₂ MO _4· 2H ₂ O, 0.075 g H ₃ BO ₃ and 0.495 g MnCl _2· 4H ₂ O) and iv) 4Хphosphate buffer (per liter: the _{NaH 2 PO 4 · H 2 O} 7.50 g , and _{K 2 HPO 4 15.52 g) 20} ㎖ or 40 ㎖ improved M9 medium (M9 modified medium) for containing used.

division badge 3HB 3HV 4HV CDM PHA content (dry wt%) Example 4
(No plasmid) M9 & Glu 0 0 0 2.376666667 0.023174824 Example 4-3
(pPHA_Tp) 0.680519125 59.91356
39.45262
9.43
37.24471
Example 4-4
(pPHA_Tp pPHA_AB) 8.853452
67.26591
23.88064
7.665
38.33716 Example 5
(No plasmid) 0 0 0 1.533333 0.062615 Example 5-3 (pPHA_Tp) 0 2.059927 97.94007 1.16 2.244671 Example 5-4
(pPHA_Tp pPHA_AB) 0 0 0 1.126667 0.251628 Example 6
(No plasmid) 0 0 0 2.33 0.117577 Example 6-3
(pPHA_Tp) 2.41463
74.32329
23.26208
7.213333
34.04846
Example 6-4
(pPHA_Tp pPHA_AB) 1.643472
77.35955
20.99697
2.75
25.52309

As a result, as shown in Table 12 and FIGS. 6a to 6c, lavAB, fadB, and phaC genes were additionally deleted. And it was confirmed that the composition and production of PHA produced according to the additional introduction of the phaAB gene were different.

Through the above examples, a new polymer production method is presented in addition to the existing production method dependent on the supply of monomers or nutrient imbalance, and the production of polymers of various compositions in one strain is achieved by comparing various heterogeneous phaCs and showing each substrate preference. prove that it is possible In addition, we show a method for producing PHA polymers using levulinic acid as a substrate, inducer and carbon source by utilizing the metabolic process of Pseudomonas putida and the LA-inducible promoter. Therefore, using the microorganism according to the present invention, PHA of various monomer compositions can be produced by utilizing levulinic acid in a single strain of Pseudomonas putida without restrictions such as nutritional imbalance or a separate inducer, and is involved in PHA production This means that polymer productivity can be increased through the combination of the Pseudomonas putida gene and the heterologous PhaC gene.

<110> UNIST (ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY) <120> TRANSFORMED MICROORGANISM PRODUCING POLYHYDROXYALKANOATE <130> FPD/202008-0001 <150> KR 10-2019-0107196 <151> 2019-08-30 <160> 66 <170> KoPatentIn 3.0 <210> 1 <211> 1875 <212> DNA <213> Artificial Sequence <220> <223> Paracoccus denitrificans-phaC-gene <400> 1 atggccgcgc cccgcgccca ggctgccgcc ccggcgggga ccggccagtc tgccggactc 60 gctgccgagc cgcatccagc ccgaacaccg ccgccttcgt cgaggcggcc ttcggtcccg 120 gcagccgcct cccaacagct ggcccagaac atcgagcgca tcgaatcgct gacccagcgc 180 ctgatcagcg cgctggcgca gcgccgtccc tcgaatcccg gcgtcgagat gccgggcccc 240 gaccttttcg ccaccgcgac ctcggcctgg atcaagcttc tggccgagca gcccgagcgg 300 gtgatcggcc agcaggtcag ctattggggc gaaaccttgc gccatttcgc cgaggcccag 360 gccgcctttg cccgcggcac cgtgacgccg ccgcccagcg aagggccgcg ggaccggcgc 420 tttgccaacc cgctgtggga ggcgcatccc ttcttcaact tcatcaagcg gcaataccag 480 atcaacgccc aggccctgca ggaggcggcc agcacactgg acctgcccga gatgaccgac 540 cggcgccgga tcgaatggtt cacccgccag atgatcgaca tgatggcgcc gacgaatttt 600 ctggccacca atcccgacga cagctggaaa aggcgctgga gaccgaggga cgaaagcctg 660 gtcaggggcc ttgagaacct ggtgcgcgac gtcgagcaga acagcggcga gctgatcgtg 720 tcgctggccg accgcgatgc cttccgtgtg ggcgagaaca tcggcaccac cgagggcacg 780 gtggtcgcgc gcaccaagct ttacgagctg atccagtaca agcccaccac cgcgcaggtg 840 catgagatcc cgctggtgat ctttccgccc tggatcaaca aattctacat cctcgacctc 900 aagccgcaga acagcctgat caaatggatc gtggaccagg gccatacgct gttcgtggtg 960 gcctggaaga accccgaccc cagctatggc gacaccggca tggacgatta cgtcagcgcc 1020 tatctggagg tgatggaccg ggttctggat ctgaccgacc agaaaaagct gaatgcggtg 1080 ggctattgca tcgccggcac caccctggcg ctgacccctg tcgtgctgaa gcagcgcggc 1140 gacgaccggg tgaacgcggc caccttcttc accgcgctga ccgatttcgc cgaccagggc 1200 gagttcactg cctatctgca ggaggatttc gtctcaggca tcgaggagga ggcggcgcgg 1260 accggcatcc tgggcgcgca gctgatgacg cgcaccttca gcttcctgcg cgccaacgac 1320 ctggtctggg ggccggcgat ccgcagctac atgctgggcg agacgccgcc ggccttcgac 1380 ctgctgttct ggaacggcga cggcaccaac ctgcccgggc gcatggccgt ggaatacctg 1440 cgcggcctgt gccagcagaa ccgcttcgtc aaggaggggt tcgatctgat gggccaccgc 1500 ctgcatgtcg gcgacgtgac cgtgccgctt tgcgccatcg cctgcgagac cgaccatatc 1560 gcgccctgga aggacagctg gcgcggcatc gcgcagatgg gctccaggga caagaccttc 1620 atcctgtccg aatcgggcca tatcgccggc atcgtcaacc cgcccagcaa gaagaaatac 1680 ggccattata cctcggacgc cggtttcggt cagggcgagc agcactggct ggacaaggcc 1740 agccatcacg agggcagctg gtggggccgc tggggcgaat ggctggcccg gcgggcgggg 1800 ggcatggtcg atgcccgcga cccgggcgag ggcttcggcc ctgcgccggg cctttacgtc 1860 cacgagcggg cgtaa 1875 <210> 2 <211> 624 <212> PRT <213> Artificial Sequence <220> <223> Paracoccus denitrificans-phaC-peptide <400> 2 Met Ala Ala Pro Arg Ala Gln Ala Ala Ala Pro Ala Gly Thr Gly Gln 1 5 10 15 Ser Ala Gly Leu Ala Ala Glu Pro His Pro Ala Arg Thr Pro Pro Pro 20 25 30 Ser Ser Arg Arg Pro Ser Val Pro Ala Ala Ala Ser Gln Gln Leu Ala 35 40 45 Gln Asn Ile Glu Arg Ile Glu Ser Leu Thr Gln Arg Leu Ile Ser Ala 50 55 60 Leu Ala Gln Arg Arg Pro Ser Asn Pro Gly Val Glu Met Pro Gly Pro 65 70 75 80 Asp Leu Phe Ala Thr Ala Thr Ser Ala Trp Ile Lys Leu Leu Ala Glu 85 90 95 Gln Pro Glu Arg Val Ile Gly Gln Gln Val Ser Tyr Trp Gly Glu Thr 100 105 110 Leu Arg His Phe Ala Glu Ala Gln Ala Ala Phe Ala Arg Gly Thr Val 115 120 125 Thr Pro Pro Pro Ser Glu Gly Pro Arg Asp Arg Arg Phe Ala Asn Pro 130 135 140 Leu Trp Glu Ala His Pro Phe Phe Asn Phe Ile Lys Arg Gln Tyr Gln 145 150 155 160 Ile Asn Ala Gln Ala Leu Gln Glu Ala Ala Ser Thr Leu Asp Leu Pro 165 170 175 Glu Met Thr Asp Arg Arg Arg Ile Glu Trp Phe Thr Arg Gln Met Ile 180 185 190 Asp Met Met Ala Pro Thr Asn Phe Leu Ala Thr Asn Pro Asp Asp Ser 195 200 205 Trp Lys Arg Arg Trp Arg Pro Arg Asp Glu Ser Leu Val Arg Gly Leu 210 215 220 Glu Asn Leu Val Arg Asp Val Glu Gln Asn Ser Gly Glu Leu Ile Val 225 230 235 240 Ser Leu Ala Asp Arg Asp Ala Phe Arg Val Gly Glu Asn Ile Gly Thr 245 250 255 Thr Glu Gly Thr Val Val Ala Arg Thr Lys Leu Tyr Glu Leu Ile Gln 260 265 270 Tyr Lys Pro Thr Thr Ala Gln Val His Glu Ile Pro Leu Val Ile Phe 275 280 285 Pro Pro Trp Ile Asn Lys Phe Tyr Ile Leu Asp Leu Lys Pro Gln Asn 290 295 300 Ser Leu Ile Lys Trp Ile Val Asp Gln Gly His Thr Leu Phe Val Val 305 310 315 320 Ala Trp Lys Asn Pro Asp Pro Ser Tyr Gly Asp Thr Gly Met Asp Asp 325 330 335 Tyr Val Ser Ala Tyr Leu Glu Val Met Asp Arg Val Leu Asp Leu Thr 340 345 350 Asp Gln Lys Lys Leu Asn Ala Val Gly Tyr Cys Ile Ala Gly Thr Thr 355 360 365 Leu Ala Leu Thr Pro Val Val Leu Lys Gln Arg Gly Asp Asp Arg Val 370 375 380 Asn Ala Ala Thr Phe Phe Thr Ala Leu Thr Asp Phe Ala Asp Gln Gly 385 390 395 400 Glu Phe Thr Ala Tyr Leu Gln Glu Asp Phe Val Ser Gly Ile Glu Glu 405 410 415 Glu Ala Ala Arg Thr Gly Ile Leu Gly Ala Gln Leu Met Thr Arg Thr 420 425 430 Phe Ser Phe Leu Arg Ala Asn Asp Leu Val Trp Gly Pro Ala Ile Arg 435 440 445 Ser Tyr Met Leu Gly Glu Thr Pro Pro Ala Phe Asp Leu Leu Phe Trp 450 455 460 Asn Gly Asp Gly Thr Asn Leu Pro Gly Arg Met Ala Val Glu Tyr Leu 465 470 475 480 Arg Gly Leu Cys Gln Gln Asn Arg Phe Val Lys Glu Gly Phe Asp Leu 485 490 495 Met Gly His Arg Leu His Val Gly Asp Val Thr Val Pro Leu Cys Ala 500 505 510 Ile Ala Cys Glu Thr Asp His Ile Ala Pro Trp Lys Asp Ser Trp Arg 515 520 525 Gly Ile Ala Gln Met Gly Ser Arg Asp Lys Thr Phe Ile Leu Ser Glu 530 535 540 Ser Gly His Ile Ala Gly Ile Val Asn Pro Ser Lys Lys Lys Tyr 545 550 555 560 Gly His Tyr Thr Ser Asp Ala Gly Phe Gly Gin Gly Glu Gln His Trp 565 570 575 Leu Asp Lys Ala Ser His His Glu Gly Ser Trp Trp Gly Arg Trp Gly 580 585 590 Glu Trp Leu Ala Arg Arg Ala Gly Gly Met Val Asp Ala Arg Asp Pro 595 600 605 Gly Glu Gly Phe Gly Pro Ala Pro Gly Leu Tyr Val His Glu Arg Ala 610 615 620 <210> 3 <211> 1707 <212> DNA <213> Artificial Sequence <220> <223> Chromobacterium violaceum-phaC-gene <400> 3 atgcagcagt tcgtcaactc gctgtcgcag cctccagccc ccgacggcgc agcccatccc 60 ttcgccggcg cctgggcgca gctgatgaac cagaccaacc agctgttcgc tctccagtcc 120 tcgctttacc agcagcagct caatctgtgg tcgcaattcc tcggccaggc cgccggccag 180 gaagccgccg ccgaagcggg cgccaagccg gccgaccgcc gcttcgcgtc gccggaatgg 240 aacgagcacc cgttctacaa cttcctcaag cagagctatc tgcagacctc caagtggatg 300 atggagctgg tggacaagac ccagctcgac gaggacgcca aggacaagct ggctttcgcc 360 acccgccagt acctggacgc gatgtccccc agcaacttca tgctgaccaa ccccgacgtg 420 gtcaagcgcg cgatcgagac caagggcgaa agcctggtcg agggcatgaa gaacatgctg 480 gacgacttcc agaagggcca catctcgatg tcggacgaga gcaagttcga gatcggcaag 540 aacctggtgg tcaccccggg ccaggtggtg ttccgcaacg aactgatcga gctgatccag 600 tacacgccga ccaccgacaa ggtctacgag aagccgctgc tgttcgtgcc gccctgcatc 660 aacaagtact acctgatgga cctgcagccg gacaactcca tggtgcgcca cttcgtcgcc 720 cagggttacc gcgtgttcct gatcagctgg cgctccgcgg tcgccgagat gaagcacttc 780 acctgggaaa cctatatcga gaaaggcgtg ttcgccgcgg cggaagcggt gcagaaaatc 840 accaagcagc cgacgatgaa cgtgctgggc ttctgcgtcg gcggcgtcat cctcaccacc 900 gcgctgtgcg tggctcaggc caaggggctg aaatacttcg actccgccac cttcatgacc 960 tcgctgatcg accacgccga accgggcgag atctccttct tcatcgacga gagcgtggtg 1020 gccggccgcg aagccaagat ggccagcggc ggcatcatca gcggcaagga aatcggccgc 1080 accttcgcca gcctgcgcgc caacgacctg gtgtggaact acgtggtcaa caactacctg 1140 ctgggcaaga ccccggcgcc gttcgacctg ctgttctgga acaacgacgc ggtggatctg 1200 ccgctgccga tgcacacctt cctgctgcgc cagttctaca tgaacaacgc gctggtgcgt 1260 ccgggcgcga tcacgctgtg cggcgtgccg atcgacatcg ccaagatcga cgtgccggtc 1320 tacatgttcg ccgcgcgcga cgaccacatc gtgctgtgga gctccgcctt ctccggcctg 1380 aaatacctgc agggcgcgcc cagccgccgc ttcgtgctgg gcgcgtccgg ccacatcgcc 1440 ggctcgatca acccggtcac caaggacaag cgcaactact gggccaacga cacgctgccg 1500 ctgcacgccg aggaatggct ggaaagcgcg gaaagccgcc ccggcagctg gtggaaggac 1560 tgggacgcct ggctggcgcc gcagtccggc aagcaagtgg ccgcgcccaa aagcctgggc 1620 aacaaggaat tcccgccgct gctggcggcg ccgggcagtt atgtgctggc caaggccatg 1680 ccgtccgtcg ccgccagcct gcaatga 1707 <210> 4 <211> 568 <212> PRT <213> Artificial Sequence <220> <223> Chromobacterium violaceum-phaC-peptide <400> 4 Met Gln Gln Phe Val Asn Ser Leu Ser Gln Pro Pro Ala Pro Asp Gly 1 5 10 15 Ala Ala His Pro Phe Ala Gly Ala Trp Ala Gln Leu Met Asn Gln Thr 20 25 30 Asn Gln Leu Phe Ala Leu Gln Ser Ser Leu Tyr Gln Gln Gln Leu Asn 35 40 45 Leu Trp Ser Gln Phe Leu Gly Gln Ala Ala Gly Gln Glu Ala Ala Ala 50 55 60 Glu Ala Gly Ala Lys Pro Ala Asp Arg Arg Phe Ala Ser Pro Glu Trp 65 70 75 80 Asn Glu His Pro Phe Tyr Asn Phe Leu Lys Gln Ser Tyr Leu Gln Thr 85 90 95 Ser Lys Trp Met Met Glu Leu Val Asp Lys Thr Gln Leu Asp Glu Asp 100 105 110 Ala Lys Asp Lys Leu Ala Phe Ala Thr Arg Gln Tyr Leu Asp Ala Met 115 120 125 Ser Pro Ser Asn Phe Met Leu Thr Asn Pro Asp Val Val Lys Arg Ala 130 135 140 Ile Glu Thr Lys Gly Glu Ser Leu Val Glu Gly Met Lys Asn Met Leu 145 150 155 160 Asp Asp Phe Gln Lys Gly His Ile Ser Met Ser Asp Glu Ser Lys Phe 165 170 175 Glu Ile Gly Lys Asn Leu Val Val Thr Pro Gly Gln Val Val Phe Arg 180 185 190 Asn Glu Leu Ile Glu Leu Ile Gln Tyr Thr Pro Thr Thr Asp Lys Val 195 200 205 Tyr Glu Lys Pro Leu Leu Phe Val Pro Cys Ile Asn Lys Tyr Tyr 210 215 220 Leu Met Asp Leu Gln Pro Asp Asn Ser Met Val Arg His Phe Val Ala 225 230 235 240 Gln Gly Tyr Arg Val Phe Leu Ile Ser Trp Arg Ser Ala Val Ala Glu 245 250 255 Met Lys His Phe Thr Trp Glu Thr Tyr Ile Glu Lys Gly Val Phe Ala 260 265 270 Ala Ala Glu Ala Val Gln Lys Ile Thr Lys Gln Pro Thr Met Asn Val 275 280 285 Leu Gly Phe Cys Val Gly Gly Val Ile Leu Thr Thr Ala Leu Cys Val 290 295 300 Ala Gln Ala Lys Gly Leu Lys Tyr Phe Asp Ser Ala Thr Phe Met Thr 305 310 315 320 Ser Leu Ile Asp His Ala Glu Pro Gly Glu Ile Ser Phe Phe Ile Asp 325 330 335 Glu Ser Val Val Ala Gly Arg Glu Ala Lys Met Ala Ser Gly Gly Ile 340 345 350 Ile Ser Gly Lys Glu Ile Gly Arg Thr Phe Ala Ser Leu Arg Ala Asn 355 360 365 Asp Leu Val Trp Asn Tyr Val Val Asn Asn Tyr Leu Leu Gly Lys Thr 370 375 380 Pro Ala Pro Phe Asp Leu Leu Phe Trp Asn Asn Asp Ala Val Asp Leu 385 390 395 400 Pro Leu Pro Met His Thr Phe Leu Leu Arg Gln Phe Tyr Met Asn Asn 405 410 415 Ala Leu Val Arg Pro Gly Ala Ile Thr Leu Cys Gly Val Pro Ile Asp 420 425 430 Ile Ala Lys Ile Asp Val Pro Val Tyr Met Phe Ala Ala Arg Asp Asp 435 440 445 His Ile Val Leu Trp Ser Ser Ala Phe Ser Gly Leu Lys Tyr Leu Gln 450 455 460 Gly Ala Pro Ser Arg Arg Phe Val Leu Gly Ala Ser Gly His Ile Ala 465 470 475 480 Gly Ser Ile Asn Pro Val Thr Lys Asp Lys Arg Asn Tyr Trp Ala Asn 485 490 495 Asp Thr Leu Pro Leu His Ala Glu Glu Trp Leu Glu Ser Ala Glu Ser 500 505 510 Arg Pro Gly Ser Trp Trp Lys Asp Trp Asp Ala Trp Leu Ala Pro Gln 515 520 525 Ser Gly Lys Gln Val Ala Ala Pro Lys Ser Leu Gly Asn Lys Glu Phe 530 535 540 Pro Pro Leu Leu Ala Ala Pro Gly Ser Tyr Val Leu Ala Lys Ala Met 545 550 555 560 Pro Ser Val Ala Ala Ser Leu Gln 565 <210> 5 <211> 1770 <212> DNA <213> Artificial Sequence <220> <223> Cupriavidus necator-phaC-gene <400> 5 atggcgaccg gcaaaggcgc ggcagcttcc acgcaggaag gcaagtccca accattcaag 60 gtcacgccgg ggccattcga tccagccaca tggctggaat ggtcccgcca gtggcagggc 120 actgaaggca acggccacgc ggccgcgtcc ggcattccgg gcctggatgc gctggcaggc 180 gtcaagatcg cgccggcgca gctgggtgat atccagcagc gctacatgaa ggacttctca 240 gcgctgtggc aggccatggc cgagggcaag gccgaggcca ccggtccgct gcacgaccgg 300 cgcttcgccg gcgacgcatg gcgcaccaac ctcccatatc gcttcgctgc cgcgttctac 360 ctgctcaatg cgcgcgcctt gaccgagctg gccgatgccg tcgaggccga tgccaagacc 420 cgccagcgca tccgcttcgc gatctcgcaa tgggtcgatg cgatgtcgcc cgccaacttc 480 cttgccacca atcccgaggc gcagcgcctg ctgatcgagt cgggcggcga atcgctgcgt 540 gccggcgtgc gcaacatgat ggaagacctg acacgcggca agatctcgca gaccgacgag 600 agcgcgtttg aggtcggccg caatgtcgcg gtgaccgaag gcgccgtggt cttcgagaac 660 gagtacttcc agctgttgca gtacaagccg ctgaccgaca aggtgcacgc gcgcccgctg 720 ctgatggtgc cgccgtgcat caacaagtac tacatcctgg acctgcagcc ggagagctcg 780 ctggtgcgcc atgtggtgga gcagggacat acggtgtttc tggtgtcgtg gcgcaatccg 840 gacgccagca tggccggcag cacctgggac gactacatcg agcacgcggc catccgcgcc 900 atcgaagtcg cgcgcgacat cagcggccag gacaagatca acgtgctcgg cttctgcgtg 960 ggcggcacca ttgtctcgac cgcgctggcg gtgctggccg cgcgcggcga gcacccggcc 1020 gccagcgtca cgctgctgac cacgctgctg gactttgccg acacgggcat cctcgacgtc 1080 tttgtcgacg agggccatgt gcagttgcgc gaggccacgc tgggcggcgg cgccggcgcg 1140 ccgtgcgcgc tgctgcgcgg ccttgagctg gccaatacct tctcgttctt gcgcccgaac 1200 gacctggtgt ggaactacgt ggtcgacaac tacctgaagg gcaacacgcc ggtgccgttc 1260 gacctgctgt tctggaacgg cgacgccacc aacctgccgg ggccgtggta ctgctggtac 1320 ctgcgccaca cctacctgca gaacgagctc aaggtaccgg gcaagctgac cgtgtgcggc 1380 gtgccggtgg acctggccag catcgacgtg ccgacctata tctacggctc gcgcgaagac 1440 catatcgtgc cgtggaccgc ggcctatgcc tcgaccgcgc tgctggcgaa caagctgcgc 1500 ttcgtgctgg gtgcgtcggg ccatatcgcc ggtgtgatca acccgccggc caagaacaag 1560 cgcagccact ggactaacga tgcgctgccg gagtcgccgc agcaatggct ggccggcgcc 1620 atcgagcatc acggcagctg gtggccggac tggaccgcat ggctggccgg gcaggccggc 1680 gcgaaacgcg ccgcgcccgc caactatggc aatgcgcgct atcgcgcaat cgaacccgcg 1740 cctgggcgat acgtcaaagc caaggcatga 1770 <210> 6 <211> 589 <212> PRT <213> Artificial Sequence <220> <223> Cupriavidus necator-phaC-peptide <400> 6 Met Ala Thr Gly Lys Gly Ala Ala Ala Ser Thr Gln Glu Gly Lys Ser 1 5 10 15 Gln Pro Phe Lys Val Thr Pro Gly Pro Phe Asp Pro Ala Thr Trp Leu 20 25 30 Glu Trp Ser Arg Gln Trp Gln Gly Thr Glu Gly Asn Gly His Ala Ala 35 40 45 Ala Ser Gly Ile Pro Gly Leu Asp Ala Leu Ala Gly Val Lys Ile Ala 50 55 60 Pro Ala Gln Leu Gly Asp Ile Gln Gln Arg Tyr Met Lys Asp Phe Ser 65 70 75 80 Ala Leu Trp Gln Ala Met Ala Glu Gly Lys Ala Glu Ala Thr Gly Pro 85 90 95 Leu His Asp Arg Arg Phe Ala Gly Asp Ala Trp Arg Thr Asn Leu Pro 100 105 110 Tyr Arg Phe Ala Ala Ala Phe Tyr Leu Leu Asn Ala Arg Ala Leu Thr 115 120 125 Glu Leu Ala Asp Ala Val Glu Ala Asp Ala Lys Thr Arg Gln Arg Ile 130 135 140 Arg Phe Ala Ile Ser Gln Trp Val Asp Ala Met Ser Pro Ala Asn Phe 145 150 155 160 Leu Ala Thr Asn Pro Glu Ala Gln Arg Leu Leu Ile Glu Ser Gly Gly 165 170 175 Glu Ser Leu Arg Ala Gly Val Arg Asn Met Met Glu Asp Leu Thr Arg 180 185 190 Gly Lys Ile Ser Gln Thr Asp Glu Ser Ala Phe Glu Val Gly Arg Asn 195 200 205 Val Ala Val Thr Glu Gly Ala Val Val Phe Glu Asn Glu Tyr Phe Gln 210 215 220 Leu Leu Gln Tyr Lys Pro Leu Thr Asp Lys Val His Ala Arg Pro Leu 225 230 235 240 Leu Met Val Pro Cys Ile Asn Lys Tyr Tyr Ile Leu Asp Leu Gln 245 250 255 Pro Glu Ser Ser Leu Val Arg His Val Val Glu Gin Gly His Thr Val 260 265 270 Phe Leu Val Ser Trp Arg Asn Pro Asp Ala Ser Met Ala Gly Ser Thr 275 280 285 Trp Asp Asp Tyr Ile Glu His Ala Ala Ile Arg Ala Ile Glu Val Ala 290 295 300 Arg Asp Ile Ser Gly Gln Asp Lys Ile Asn Val Leu Gly Phe Cys Val 305 310 315 320 Gly Gly Thr Ile Val Ser Thr Ala Leu Ala Val Leu Ala Ala Arg Gly 325 330 335 Glu His Pro Ala Ala Ser Val Thr Leu Leu Thr Thr Leu Leu Asp Phe 340 345 350 Ala Asp Thr Gly Ile Leu Asp Val Phe Val Asp Glu Gly His Val Gln 355 360 365 Leu Arg Glu Ala Thr Leu Gly Gly Gly Ala Gly Ala Pro Cys Ala Leu 370 375 380 Leu Arg Gly Leu Glu Leu Ala Asn Thr Phe Ser Phe Leu Arg Pro Asn 385 390 395 400 Asp Leu Val Trp Asn Tyr Val Val Asp Asn Tyr Leu Lys Gly Asn Thr 405 410 415 Pro Val Pro Phe Asp Leu Leu Phe Trp Asn Gly Asp Ala Thr Asn Leu 420 425 430 Pro Gly Pro Trp Tyr Cys Trp Tyr Leu Arg His Thr Tyr Leu Gln Asn 435 440 445 Glu Leu Lys Val Pro Gly Lys Leu Thr Val Cys Gly Val Pro Val Asp 450 455 460 Leu Ala Ser Ile Asp Val Pro Thr Tyr Ile Tyr Gly Ser Arg Glu Asp 465 470 475 480 His Ile Val Pro Trp Thr Ala Ala Tyr Ala Ser Thr Ala Leu Leu Ala 485 490 495 Asn Lys Leu Arg Phe Val Leu Gly Ala Ser Gly His Ile Ala Gly Val 500 505 510 Ile Asn Pro Pro Ala Lys Asn Lys Arg Ser His Trp Thr Asn Asp Ala 515 520 525 Leu Pro Glu Ser Pro Gln Gln Trp Leu Ala Gly Ala Ile Glu His His 530 535 540 Gly Ser Trp Trp Pro Asp Trp Thr Ala Trp Leu Ala Gly Gln Ala Gly 545 550 555 560 Ala Lys Arg Ala Ala Pro Ala Asn Tyr Gly Asn Ala Arg Tyr Arg Ala 565 570 575 Ile Glu Pro Ala Pro Gly Arg Tyr Val Lys Ala Lys Ala 580 585 <210> 7 <211> 1680 <212> DNA <213> Artificial Sequence <220> <223> Pseudomonas oleovorans-phaC1-gene <400> 7 atgagtgaca agaacaacga agacctgaaa cgccaggcct ctgaaaacac cctgggactc 60 aacccggtaa tcggcattcg cggcaaggat ctgctgacct ctgcccgtat ggtgctcgcc 120 caggcactca agcaaccctt ccacagtgcc aagcatgtcg cccacttcgg cctggagctg 180 aagaacgtca tcctcggtca gtccgcgctc aaacccgaag acggtgaccg ccgcttcaat 240 gatccgacct ggagccagaa cccactgtat cgccgttatc tgcagaccta cctggcctgg 300 cgcaaggagc tgcatgactg gatcgaacac agttcgctgt ccgagcagga cgccagtcgc 360 ggccacttcg tcatcaacct gatgaccgaa gccatggcgc cgagcaacag catggccaac 420 ccggcggcag tcaaacgttt cttcgaaacc ggcggcaaga gcctgctcga cggcctttcg 480 cacctggcca aggacatggt gcacaacggc ggcatgccca gcctggtgaa catggaggcc 540 ttcgaggtcg gcaagaacct ggccaccacc gacggcgccg tggtgtttcg caacgacgtg 600 ctggagctga tccagtacaa gccgatcacc gagagcgtgc acgagcgccc gctgctggtg 660 gtgccaccgc agatcaacaa gttctacgta ttcgacctgt caccggaaaa aagcctggcg 720 cgcttcctgt tgcgcagcca ggtgcagacc ttcgtggtca gctggcgcaa cccgaccaag 780 gcgcagcgtg agtggggcct gtccacctac atcgaggcgc tcaaggaagc catcgacgtg 840 atctgcgcca tcaccggcag caaggacgtc aacatgcttg gcgcctgctc cggcggcctg 900 accactgcct cgctgctcgg tcactatgca gcgcttggcc agcagaaggt caacgccctg 960 accctgctgg tcagcgtgct cgacactcag ctagacaccc aggttgccct gttcgccgac 1020 gagaagccc tggaagccgc caaacgccgc tcctaccagg ccggcgtact ggagggtagt 1080 gacatggcca aggtgttcgc ctggatgcgc cccaacgacc tgatctggaa ctactgggtc 1140 aacaactacc tgctcggcaa cgagccaccg gtgttcgaca tcctctactg gaacaacgac 1200 accacgcgcc tgccggccgc gctgcgtggc gagttcatcg agatgttccg gaccaaccca 1260 ctgacccgcc cgggcgcgct ggaagtctgc ggcacaccga tcgacctgaa gcaggtcacc 1320 tgtgatttct tcgtcgtcgc tggcaccacc gaccacatca ccccctggga ttcctgctac 1380 aagtcggcgc acctgttcgg cggcaaatgc gagttcgtgc tgtccaacag cggccatatc 1440 caaagcattc tcaacccgcc gggcaacccc aaggcgcgct acaagaccaa tagcgcgatg 1500 ccgctggacc cgaaagcctg gcaggaaagc tcgaccaagc acgccgactc ctggtggcta 1560 cattggcaga cgtggctgag cgaacgctcg ggcgaaacca agaatgctcc acaggcgctg 1620 ggcaacaaga aattcccggc tggcgaagcc gcaccaggca cctatgtgca cgaacgctga 1680 1680 <210> 8 <211> 559 <212> PRT <213> Artificial Sequence <220> <223> Pseudomonas oleovorans-phaC1-peptide <400> 8 Met Ser Asp Lys Asn Asn Glu Asp Leu Lys Arg Gln Ala Ser Glu Asn 1 5 10 15 Thr Leu Gly Leu Asn Pro Val Ile Gly Ile Arg Gly Lys Asp Leu Leu 20 25 30 Thr Ser Ala Arg Met Val Leu Ala Gln Ala Leu Lys Gln Pro Phe His 35 40 45 Ser Ala Lys His Val Ala His Phe Gly Leu Glu Leu Lys Asn Val Ile 50 55 60 Leu Gly Gln Ser Ala Leu Lys Pro Glu Asp Gly Asp Arg Arg Phe Asn 65 70 75 80 Asp Pro Thr Trp Ser Gln Asn Pro Leu Tyr Arg Arg Tyr Leu Gln Thr 85 90 95 Tyr Leu Ala Trp Arg Lys Glu Leu His Asp Trp Ile Glu His Ser Ser 100 105 110 Leu Ser Glu Gln Asp Ala Ser Arg Gly His Phe Val Ile Asn Leu Met 115 120 125 Thr Glu Ala Met Ala Pro Ser Asn Ser Met Ala Asn Pro Ala Ala Val 130 135 140 Lys Arg Phe Phe Glu Thr Gly Gly Lys Ser Leu Leu Asp Gly Leu Ser 145 150 155 160 His Leu Ala Lys Asp Met Val His Asn Gly Gly Met Pro Ser Leu Val 165 170 175 Asn Met Glu Ala Phe Glu Val Gly Lys Asn Leu Ala Thr Thr Asp Gly 180 185 190 Ala Val Val Phe Arg Asn Asp Val Leu Glu Leu Ile Gln Tyr Lys Pro 195 200 205 Ile Thr Glu Ser Val His Glu Arg Pro Leu Leu Val Val Pro Gln 210 215 220 Ile Asn Lys Phe Tyr Val Phe Asp Leu Ser Pro Glu Lys Ser Leu Ala 225 230 235 240 Arg Phe Leu Leu Arg Ser Gln Val Gln Thr Phe Val Val Ser Trp Arg 245 250 255 Asn Pro Thr Lys Ala Gln Arg Glu Trp Gly Leu Ser Thr Tyr Ile Glu 260 265 270 Ala Leu Lys Glu Ala Ile Asp Val Ile Cys Ala Ile Thr Gly Ser Lys 275 280 285 Asp Val Asn Met Leu Gly Ala Cys Ser Gly Gly Leu Thr Thr Ala Ser 290 295 300 Leu Leu Gly His Tyr Ala Ala Leu Gly Gln Gln Lys Val Asn Ala Leu 305 310 315 320 Thr Leu Leu Val Ser Val Leu Asp Thr Gln Leu Asp Thr Gln Val Ala 325 330 335 Leu Phe Ala Asp Glu Lys Thr Leu Glu Ala Ala Lys Arg Arg Ser Tyr 340 345 350 Gln Ala Gly Val Leu Glu Gly Ser 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 Tyr Trp Asn Asn Asp 385 390 395 400 Thr Thr Arg Leu Pro Ala Ala Leu Arg Gly Glu Phe Ile Glu Met Phe 405 410 415 Arg Thr Asn Pro Leu Thr Arg Pro Gly Ala Leu Glu Val Cys Gly Thr 420 425 430 Pro Ile Asp Leu Lys Gln Val Thr Cys Asp Phe Phe Val Val Ala Gly 435 440 445 Thr Thr Asp His Ile Thr Pro Trp Asp Ser Cys Tyr Lys Ser Ala His 450 455 460 Leu Phe Gly Gly Lys Cys Glu Phe Val Leu Ser Asn Ser Gly His Ile 465 470 475 480 Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Lys Ala Arg Tyr Met Thr 485 490 495 Asn Ser Ala Met Pro Leu Asp Pro Lys Ala Trp Gln Glu Ser Ser Thr 500 505 510 Lys His Ala Asp Ser Trp Trp Leu His Trp Gln Thr Trp Leu Ser Glu 515 520 525 Arg Ser Gly Glu Thr Lys Asn Ala Pro Gln Ala Leu Gly Asn Lys Lys 530 535 540 Phe Pro Ala Gly Glu Ala Ala Pro Gly Thr Tyr Val His Glu Arg 545 550 555 <210> 9 <211> 1683 <212> DNA <213> Artificial Sequence <220> <223> Pseudomonas oleovorans-phaC2-gene <400> 9 atgcgagaca agtcgaaccc ggcttcactg ccggcacccg ccagtttcat gaacgcccag 60 agcgcggtgg tgggcgtgca cggtcgcgat ctgctgtcca ccatgcgcct gctggctgcc 120 cagggcctga agaatccggt gcgcagtgga cgtcatctgc tggccttcgg cggccagctt 180 ggccgggtgc tgctcggcga taccctgcac aaggtcaatc cgcaggatgc gcgcttcgct 240 gatccgacct ggcacctcaa tcccttctac cgccgcagcc tgcaggccta tctggcgtgg 300 cagaaacaac tggccgcgtg gatcgacgac agcgacctgt cggccgatga ccgctcccgc 360 gcgcgctttc tcgcctcact gatgagcgac gccctgtcgc cctccaacag cccgctcaac 420 ccgcaggcgc tcaaggagct gttcaacacc ggtggcagca gtgccttcaa gggcttgcgc 480 catctgctcg acgacctgct gaacaacgac ggcctgccca gccaggtcag caaacacgcc 540 ttcgaagtcg gccgcaatct ggcctgcacg ccaggtgccg tggtgtttcg caacgagctg 600 ctggaactga tccagtacaa accgatgagc gaaaagcagt acctgcgccc gctgctgatc 660 gtgccgccac agatcaataa gtactacata ttcgatctat ccaacgacaa gagcttcgtc 720 cagtacgcac tgaagaacgg cctgcagacc ttcatgatca gctggcgcaa ccccgaccca 780 cggcatcgcg aatggggcct gtcgagctat gtgcaggccg tcgaggaggc cgtcgatgcc 840 tgccgcgcca tctccggcag caaggacgtc aacctgctcg gcgcctgcgc cggcggtctg 900 accatcgccg ccctgcaagg ccacctgcag gcacgccgcc aactgcgcaa gattgccagc 960 gccacctaca tggtcagtct gctcgatagc cagatcgaca gcccggcgat gctctttgcc 1020 gacgaggaaa ccctcgagtc ggcaaagcgc cgctcctatc agcaaggcgt gctggacggt 1080 cgcgacatgg ccagggtatt cgcctggatg cgtcccaacg acctgatctg gaactactgg 1140 gtcaacaact acctgctcgg caggcagccg ccagccttcg acatcctcta ctggaacaac 1200 gacaacacgc gcctgcccgc tgcactgcat ggcgacctga tcgacttctt caagcacaat 1260 ccgcttagcc gcaacggcgg cctcgaagtc tgcggtacgc cggtggatct gaccaaggtc 1320 aacgtcgaca gtttcagcgt ggccggcatc aacgaccaca tcacgccctg ggatgcggtc 1380 tatcgctcga ccctgctgct tggcggcaac cgccgtttca tcctgtccaa cagcgggcat 1440 atccagagca ttctcaaccc gccgggcaac cccaaggcca actactacga gaacaccaag 1500 ctcacctccg accctcgcgc ctggtatcac gatgccacgc accagcaagg tagttggtgg 1560 ccacagtggc tggaatggat gcaggcgcgt tccggcgcgc aacgcgaaac cctgatggcg 1620 ctgggcaacc agaaccatcc accgatggag gcagcgcccg gcacctacgt gcatatccgc 1680 tga 1683 <210> 10 <211> 560 <212> PRT <213> Artificial Sequence <220> <223> Pseudomonas oleovorans-phaC2-peptide <400> 10 Met Arg Asp Lys Ser Asn Pro Ala Ser Leu Pro Ala Pro Ala Ser Phe 1 5 10 15 Met Asn Ala Gln Ser Ala Val Val Gly Val His Gly Arg Asp Leu Leu 20 25 30 Ser Thr Met Arg Leu Leu Ala Ala Gln Gly Leu Lys Asn Pro Val Arg 35 40 45 Ser Gly Arg His Leu Leu Ala Phe Gly Gly Gln Leu Gly Arg Val Leu 50 55 60 Leu Gly Asp Thr Leu His Lys Val Asn Pro Gln Asp Ala Arg Phe Ala 65 70 75 80 Asp Pro Thr Trp His Leu Asn Pro Phe Tyr Arg Arg Ser Leu Gln Ala 85 90 95 Tyr Leu Ala Trp Gln Lys Gln Leu Ala Ala Trp Ile Asp Asp Ser Asp 100 105 110 Leu Ser Ala Asp Asp Arg Ser Arg Ala Arg Phe Leu Ala Ser Leu Met 115 120 125 Ser Asp Ala Leu Ser Pro Ser Asn Ser Pro Leu Asn Pro Gln Ala Leu 130 135 140 Lys Glu Leu Phe Asn Thr Gly Gly Ser Ser Ala Phe Lys Gly Leu Arg 145 150 155 160 His Leu Leu Asp Asp Leu Leu Asn Asn Asp Gly Leu Pro Ser Gln Val 165 170 175 Ser Lys His Ala Phe Glu Val Gly Arg Asn Leu Ala Cys Thr Pro Gly 180 185 190 Ala Val Val Phe Arg Asn Glu Leu Leu Glu Leu Ile Gln Tyr Lys Pro 195 200 205 Met Ser Glu Lys Gln Tyr Leu Arg Pro Leu Leu Ile Val Pro Gln 210 215 220 Ile Asn Lys Tyr Tyr Ile Phe Asp Leu Ser Asn Asp Lys Ser Phe Val 225 230 235 240 Gln Tyr Ala Leu Lys Asn Gly Leu Gln Thr Phe Met Ile Ser Trp Arg 245 250 255 Asn Pro Asp Pro Arg His Arg Glu Trp Gly Leu Ser Ser Tyr Val Gln 260 265 270 Ala Val Glu Glu Ala Val Asp Ala Cys Arg Ala Ile Ser Gly Ser Lys 275 280 285 Asp Val Asn Leu Leu Gly Ala Cys Ala Gly Gly Leu Thr Ile Ala Ala 290 295 300 Leu Gln Gly His Leu Gln Ala Arg Arg Gln Leu Arg Lys Ile Ala Ser 305 310 315 320 Ala Thr Tyr Met Val Ser Leu Leu Asp Ser Gln Ile Asp Ser Pro Ala 325 330 335 Met Leu Phe Ala Asp Glu Glu Thr Leu Glu Ser Ala Lys Arg Arg Ser 340 345 350 Tyr Gln Gln Gly Val Leu Asp Gly Arg Asp Met Ala Arg Val 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 Arg Gln Pro Pro Ala Phe Asp Ile Leu Tyr Trp Asn Asn 385 390 395 400 Asp Asn Thr Arg Leu Pro Ala Ala Leu His Gly Asp Leu Ile Asp Phe 405 410 415 Phe Lys His Asn Pro Leu Ser Arg Asn Gly Gly Leu Glu Val Cys Gly 420 425 430 Thr Pro Val Asp Leu Thr 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 Asn Arg Arg Phe Ile Leu Ser Asn Ser Gly His 465 470 475 480 Ile Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Lys Ala Asn Tyr Tyr 485 490 495 Glu Asn Thr Lys Leu Thr Ser Asp Pro Arg Ala Trp Tyr His Asp Ala 500 505 510 Thr His Gln Gln Gly Ser Trp Trp Pro Gln Trp Leu Glu Trp Met Gln 515 520 525 Ala Arg Ser Gly Ala Gln Arg Glu Thr Leu Met Ala Leu Gly Asn Gln 530 535 540 Asn His Pro Pro Met Glu Ala Ala Pro Gly Thr Tyr Val His Ile Arg 545 550 555 560 <210> 11 <211> 1104 <212> DNA <213> Artificial Sequence <220> <223> Thiocapsa pfennigii-phaE-gene <400> 11 atgaacgata cggccaacaa gaccagcgac tggctggaca tccaacgcaa gtactgggag 60 acctggtcgg agctcggccg caagaccttg ggtctggaga agaccccggc caatccttgg 120 gccggcgccc tcgatcattg gtggcagacg gtctcgcccg ccgcccccaa cgacctggtt 180 cgcgacttca tggagaagct cgccgagcag ggcaaggcct tcttcggcct caccgactac 240 ttcacgaagg gcctcggcgg cagtagcggt acgcagggct gggacaccct ctcgaagacc 300 atcgacgaca tgcaaaaggc cttcgccagc ggccggatcg aaggcgacga gaccttccgc 360 cgcctgatgg ccttctggga gatgccgctc gacaactggc agcgcaccat gtcctcgctg 420 tccccggtgc ccggcgacct gctgcgcaac atgccgcacg accaagtcag ggacagcgtc 480 gaccgcatcc tctcggcacc cgggctcggc tacacgcgcg aggagcaggc ccgctaccag 540 gatctgatcc gccgctcgct ggagtaccag tcggccctga acgaatacaa cggcttcttc 600 ggccagctcg gtgtcaagtc cctcgagcgg atgcgcgcct tcctgcaggg acaggccgag 660 aagggcgtcg ccatcgagtc ggcgcgcacc ctctacgacg cctgggtcgg ctgctgcgaa 720 gaggtctatg ccgaggaggt cagctccgcc gactacgcgc acatccacgg ccgcctcgtc 780 aacgcccaga tggccctcaa gcagcgcatg tcgaccatgg tcgacgaggt cctcggcgcg 840 atgccgctgc cgacccgcag cgagctgcgc acgctccagg atcggctcca ggagtcgcgc 900 ggcgagggca agcgccagcg ccaagagatc gagacgctga agcggcaggt cgcggccttg 960 gccggcggcg cccagcccgc gccccaggcc tccgcccagc ccagcacccg gcccgcgccg 1020 gcgacggccc cggcggcgag cgcggcgccc aagcgcagca ccacgacccg ccgcaagacc 1080 accaagccca ccaccggcca gtga 1104 <210> 12 <211> 367 <212> PRT <213> Artificial Sequence <220> <223> Thiocapsa pfennigii-phaE-peptide <400> 12 Met Asn Asp Thr Ala Asn Lys Thr Ser Asp Trp Leu Asp Ile Gln Arg 1 5 10 15 Lys Tyr Trp Glu Thr Trp Ser Glu Leu Gly Arg Lys Thr Leu Gly Leu 20 25 30 Glu Lys Thr Pro Ala Asn Pro Trp Ala Gly Ala Leu Asp His Trp Trp 35 40 45 Gln Thr Val Ser Pro Ala Ala Pro Asn Asp Leu Val Arg Asp Phe Met 50 55 60 Glu Lys Leu Ala Glu Gln Gly Lys Ala Phe Phe Gly Leu Thr Asp Tyr 65 70 75 80 Phe Thr Lys Gly Leu Gly Gly Ser Ser Gly Thr Gin Gly Trp Asp Thr 85 90 95 Leu Ser Lys Thr Ile Asp Asp Met Gln Lys Ala Phe Ala Ser Gly Arg 100 105 110 Ile Glu Gly Asp Glu Thr Phe Arg Arg Leu Met Ala Phe Trp Glu Met 115 120 125 Pro Leu Asp Asn Trp Gln Arg Thr Met Ser Ser Leu Ser Pro Val Pro 130 135 140 Gly Asp Leu Leu Arg Asn Met Pro His Asp Gln Val Arg Asp Ser Val 145 150 155 160 Asp Arg Ile Leu Ser Ala Pro Gly Leu Gly Tyr Thr Arg Glu Glu Gln 165 170 175 Ala Arg Tyr Gln Asp Leu Ile Arg Arg Ser Leu Glu Tyr Gln Ser Ala 180 185 190 Leu Asn Glu Tyr Asn Gly Phe Phe Gly Gln Leu Gly Val Lys Ser Leu 195 200 205 Glu Arg Met Arg Ala Phe Leu Gln Gly Gln Ala Glu Lys Gly Val Ala 210 215 220 Ile Glu Ser Ala Arg Thr Leu Tyr Asp Ala Trp Val Gly Cys Cys Glu 225 230 235 240 Glu Val Tyr Ala Glu Glu Val Ser Ser Ala Asp Tyr Ala His Ile His 245 250 255 Gly Arg Leu Val Asn Ala Gln Met Ala Leu Lys Gln Arg Met Ser Thr 260 265 270 Met Val Asp Glu Val Leu Gly Ala Met Pro Leu Pro Thr Arg Ser Glu 275 280 285 Leu Arg Thr Leu Gln Asp Arg Leu Gln Glu Ser Arg Gly Glu Gly Lys 290 295 300 Arg Gln Arg Gln Glu Ile Glu Thr Leu Lys Arg Gln Val Ala Ala Leu 305 310 315 320 Ala Gly Gly Ala Gln Pro Ala Pro Gln Ala Ser Ala Gln Pro Ser Thr 325 330 335 Arg Pro Ala Pro Ala Thr Ala Pro Ala Ala Ser Ala Ala Pro Lys Arg 340 345 350 Ser Thr Thr Thr Arg Arg Lys Thr Thr Lys Pro Thr Thr Gly Gln 355 360 365 <210> 13 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> Thiocapsa pfennigii-phaC-gene <400> 13 atgtccccat tcccgatcga catccggccc gacaagctga ccgaggagat gctggagtac 60 agccgcaagc tcggcgaggg tatgcagaac ctgctcaagg ccgaccagat cgacacaggc 120 gtcaccccca aggacgtcgt ccaccgcgag gacaagctgg tcctctaccg ctaccggcgc 180 ccggcgcagg tggcgaccca gacgatcccg ctgctgatcg tctacgccct cgtcaatcgg 240 ccctacatga ccgacatcca ggaggatcgc tcgacgatca agggcctgct cgccaccggt 300 caggacgtct atctgatcga ctggggctac ccggatcagg ccgaccgggc gctgaccctc 360 gatgactaca tcaacggcta catcgaccgc tgcgtcgact acctgcgcga gacccacggc 420 gtcgaccagg tcaacctgct cgggatctgc cagggcgggg ccttcagcct ctgctacacg 480 gccctgcact ccgagaaggt caaaaacctc gtcaccatgg tcacgccggt cgacttccag 540 accccgggca acctgctctc ggcctgggtc cagaacgtcg acgtcgacct ggccgtcgac 600 accatgggca acatccccggg cgaactgctc aactggacct tcctgtcgct caagcccttc 660 agcctgaccg gccagaagta cgtcaacatg gtcgacctgc tcgacgacga ggacaaggtc 720 aagaacttcc tgcggatgga gaagtggatc ttcgacagcc cggaccaggc cggcgagacc 780 ttccgccagt tcatcaagga cttctaccag cgcaacggct tcatcaacgg cggcgtcctg 840 atcggcgatc aggaggtcga cctgcgcaac atccgctgcc cggtcctgaa catctacccg 900 atgcaggacc acctggtgcc gccggatgcc tccaaggccc tcgcgggact gacctccagc 960 gaggactaca cggagctcgc cttccccggc gggcacatcg gcatctacgt cagcggcaag 1020 gcgcaggaag gagtcacccc ggcgatcggc cgctggctga acgaacgcgg ctga 1074 <210> 14 <211> 357 <212> PRT <213> Artificial Sequence <220> <223> Thiocapsa pfennigii-phaC-peptide <400> 14 Met Ser Pro Phe Pro Ile Asp Ile Arg Pro Asp Lys Leu Thr Glu Glu 1 5 10 15 Met Leu Glu Tyr Ser Arg Lys Leu Gly Glu Gly Met Gln Asn Leu Leu 20 25 30 Lys Ala Asp Gln Ile Asp Thr Gly Val Thr Pro Lys Asp Val Val His 35 40 45 Arg Glu Asp Lys Leu Val Leu Tyr Arg Tyr Arg Arg Pro Ala Gln Val 50 55 60 Ala Thr Gln Thr Ile Pro Leu Leu Ile Val Tyr Ala Leu Val Asn Arg 65 70 75 80 Pro Tyr Met Thr Asp Ile Gln Glu Asp Arg Ser Thr Ile Lys Gly Leu 85 90 95 Leu Ala Thr Gly Gln Asp Val Tyr Leu Ile Asp Trp Gly Tyr Pro Asp 100 105 110 Gln Ala Asp Arg Ala Leu Thr Leu Asp Asp Tyr Ile Asn Gly Tyr Ile 115 120 125 Asp Arg Cys Val Asp Tyr Leu Arg Glu Thr His Gly Val Asp Gln Val 130 135 140 Asn Leu Leu Gly Ile Cys Gln Gly Gly Ala Phe Ser Leu Cys Tyr Thr 145 150 155 160 Ala Leu His Ser Glu Lys Val Lys Asn Leu Val Thr Met Val Thr Pro 165 170 175 Val Asp Phe Gln Thr Pro Gly Asn Leu Leu Ser Ala Trp Val Gln Asn 180 185 190 Val Asp Val Asp Leu Ala Val Asp Thr Met Gly Asn Ile Pro Gly Glu 195 200 205 Leu Leu Asn Trp Thr Phe Leu Ser Leu Lys Pro Phe Ser Leu Thr Gly 210 215 220 Gln Lys Tyr Val Asn Met Val Asp Leu Leu Asp Asp Glu Asp Lys Val 225 230 235 240 Lys Asn Phe Leu Arg Met Glu Lys Trp Ile Phe Asp Ser Pro Asp Gln 245 250 255 Ala Gly Glu Thr Phe Arg Gln Phe Ile Lys Asp Phe Tyr Gln Arg Asn 260 265 270 Gly Phe Ile Asn Gly Gly Val Leu Ile Gly Asp Gln Glu Val Asp Leu 275 280 285 Arg Asn Ile Arg Cys Pro Val Leu Asn Ile Tyr Pro Met Gln Asp His 290 295 300 Leu Val Pro Pro Asp Ala Ser Lys Ala Leu Ala Gly Leu Thr Ser Ser 305 310 315 320 Glu Asp Tyr Thr Glu Leu Ala Phe Pro Gly Gly His Ile Gly Ile Tyr 325 330 335 Val Ser Gly Lys Ala Gln Glu Gly Val Thr Pro Ala Ile Gly Arg Trp 340 345 350 Leu Asn Glu Arg Gly 355 <210> 15 <211> 1182 <212> DNA <213> Artificial Sequence <220> <223> Cupriavidus necator-phaA-gene <400> 15 atgactgacg ttgtcatcgt atccgccgcc cgcaccgcgg tcggcaagtt tggcggctcg 60 ctggccaaga tcccggcacc ggaactgggt gccgtggtca tcaaggccgc gctggagcgc 120 gccggcgtca agccggagca ggtgagcgaa gtcatcatgg gccaggtgct gaccgccggt 180 tcgggccaga accccgcacg ccaggccgcg atcaaggccg gcctgccggc gatggtgccg 240 gccatgacca tcaacaaggt gtgcggctcg ggcctgaagg ccgtgatgct ggccgccaac 300 gcgatcatgg cgggcgacgc cgagatcgtg gtggccggcg gccaggaaaa catgagcgcc 360 gccccgcacg tgctgccggg ctcgcgcgat ggtttccgca tgggcgatgc caagctggtc 420 gacaccatga tcgtcgacgg cctgtgggac gtgtacaacc agtaccacat gggcatcacc 480 gccgagaacg tggccaagga atacggcatc acacgcgagg cgcaggatga gttcgccgtc 540 ggctcgcaga acaaggccga agccgcgcag aaggccggca agtttgacga agagatcgtc 600 ccggtgctga tcccgcagcg caagggcgac ccggtggcct tcaagaccga cgagttcgtg 660 cgccagggcg ccacgctgga cagcatgtcc ggcctcaagc ccgccttcga caaggccggc 720 acggtgaccg cggccaacgc ctcgggcctg aacgacggcg ccgccgcggt ggtggtgatg 780 tcggcggcca aggccaagga actgggcctg accccgctgg ccacgatcaa gagctatgcc 840 aacgccggtg tcgatcccaa ggtgatgggc atgggcccgg tgccggcctc caagcgcgcc 900 ctgtcgcgcg ccgagtggac cccgcaagac ctggacctga tggagatcaa cgaggccttt 960 gccgcgcagg cgctggcggt gcaccagcag atgggctggg acacctccaa ggtcaatgtg 1020 aacggcggcg ccatcgccat cggccacccg atcggcgcgt cgggctgccg tatcctggtg 1080 acgctgctgc acgagatgaa gcgccgtgac gcgaagaagg gcctggcctc gctgtgcatc 1140 ggcggcggca tgggcgtggc gctggcagtc gagcgcaaat aa 1182 <210> 16 <211> 393 <212> PRT <213> Artificial Sequence <220> <223> Cupriavidus necator-phaA-peptide <400> 16 Met Thr Asp Val Val Ile Val Ser Ala Ala Arg Thr Ala Val Gly Lys 1 5 10 15 Phe Gly Gly Ser Leu Ala Lys Ile Pro Ala Pro Glu Leu Gly Ala Val 20 25 30 Val Ile Lys Ala Ala Leu Glu Arg Ala Gly Val Lys Pro Glu Gln Val 35 40 45 Ser Glu Val Ile Met Gly Gln Val Leu Thr Ala Gly Ser Gly Gln Asn 50 55 60 Pro Ala Arg Gln Ala Ala Ile Lys Ala Gly Leu Pro Ala Met Val Pro 65 70 75 80 Ala Met Thr Ile Asn Lys Val Cys Gly Ser Gly Leu Lys Ala Val Met 85 90 95 Leu Ala Ala Asn Ala Ile Met Ala Gly Asp Ala Glu Ile Val Val Ala 100 105 110 Gly Gly Gln Glu Asn Met Ser Ala Ala Pro His Val Leu Pro Gly Ser 115 120 125 Arg Asp Gly Phe Arg Met Gly Asp Ala Lys Leu Val Asp Thr Met Ile 130 135 140 Val Asp Gly Leu Trp Asp Val Tyr Asn Gln Tyr His Met Gly Ile Thr 145 150 155 160 Ala Glu Asn Val Ala Lys Glu Tyr Gly Ile Thr Arg Glu Ala Gln Asp 165 170 175 Glu Phe Ala Val Gly Ser Gln Asn Lys Ala Glu Ala Ala Gln Lys Ala 180 185 190 Gly Lys Phe Asp Glu Glu Ile Val Pro Val Leu Ile Pro Gln Arg Lys 195 200 205 Gly Asp Pro Val Ala Phe Lys Thr Asp Glu Phe Val Arg Gln Gly Ala 210 215 220 Thr Leu Asp Ser Met Ser Gly Leu Lys Pro Ala Phe Asp Lys Ala Gly 225 230 235 240 Thr Val Thr Ala Ala Asn Ala Ser Gly Leu Asn Asp Gly Ala Ala Ala 245 250 255 Val Val Val Met Ser Ala Ala Lys Ala Lys Glu Leu Gly Leu Thr Pro 260 265 270 Leu Ala Thr Ile Lys Ser Tyr Ala Asn Ala Gly Val Asp Pro Lys Val 275 280 285 Met Gly Met Gly Pro Val Pro Ala Ser Lys Arg Ala Leu Ser Arg Ala 290 295 300 Glu Trp Thr Pro Gln Asp Leu Asp Leu Met Glu Ile Asn Glu Ala Phe 305 310 315 320 Ala Ala Gln Ala Leu Ala Val His Gln Gln Met Gly Trp Asp Thr Ser 325 330 335 Lys Val Asn Val Asn Gly Gly Ala Ile Ala Ile Gly His Pro Ile Gly 340 345 350 Ala Ser Gly Cys Arg Ile Leu Val Thr Leu Leu His Glu Met Lys Arg 355 360 365 Arg Asp Ala Lys Lys Gly Leu Ala Ser Leu Cys Ile Gly Gly Gly Met 370 375 380 Gly Val Ala Leu Ala Val Glu Arg Lys 385 390 <210> 17 <211> 741 <212> DNA <213> Artificial Sequence <220> <223> Cupriavidus necator-phaB-gene <400> 17 atgactcagc gcattgcgta tgtgaccggc ggcatgggtg gtatcggaac cgccatttgc 60 cagcggctgg ccaaggatgg ctttcgtgtg gtggccggtt gcggccccaa ctcgccgcgc 120 cgcgaaaagt ggctggagca gcagaaggcc ctgggcttcg atttcattgc ctcggaaggc 180 aatgtggctg actgggactc gaccaagacc gcattcgaca aggtcaagtc cgaggtcggc 240 gaggttgatg tgctgatcaa caacgccggt atcacccgcg acgtggtgtt ccgcaagatg 300 acccgcgccg actgggatgc ggtgatcgac accaacctga cctcgctgtt caacgtcacc 360 aagcaggtga tcgacggcat ggccgaccgt ggctggggcc gcatcgtcaa catctcgtcg 420 gtgaacgggc agaagggcca gttcggccag accaactact ccaccgccaa ggccggcctg 480 catggcttca ccatggcact ggcgcaggaa gtggcgacca agggcgtgac cgtcaacacg 540 gtctctccgg gctatatcgc caccgacatg gtcaaggcga tccgccagga cgtgctcgac 600 aagatcgtcg cgacgatccc ggtcaagcgc ctgggcctgc cggaagagat cgcctcgatc 660 tgcgcctggt tgtcgtcgga ggagtccggt ttctcgaccg gcgccgactt ctcgctcaac 720 ggcggcctgc atatgggctg a 741 <210> 18 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> Cupriavidus necator-phaB-peptide <400> 18 Met Thr Gln Arg Ile Ala Tyr Val Thr Gly Gly Met Gly Gly Ile Gly 1 5 10 15 Thr Ala Ile Cys Gln Arg Leu Ala Lys Asp Gly Phe Arg Val Val Ala 20 25 30 Gly Cys Gly Pro Asn Ser Pro Arg Arg Glu Lys Trp Leu Glu Gln Gln 35 40 45 Lys Ala Leu Gly Phe Asp Phe Ile Ala Ser Glu Gly Asn Val Ala Asp 50 55 60 Trp Asp Ser Thr Lys Thr Ala Phe Asp Lys Val Lys Ser Glu Val Gly 65 70 75 80 Glu Val Asp Val Leu Ile Asn Asn Ala Gly Ile Thr Arg Asp Val Val 85 90 95 Phe Arg Lys Met Thr Arg Ala Asp Trp Asp Ala Val Ile Asp Thr Asn 100 105 110 Leu Thr Ser Leu Phe Asn Val Thr Lys Gln Val Ile Asp Gly Met Ala 115 120 125 Asp Arg Gly Trp Gly Arg Ile Val Asn Ile Ser Ser Val Asn Gly Gln 130 135 140 Lys Gly Gln Phe Gly Gln Thr Asn Tyr Ser Thr Ala Lys Ala Gly Leu 145 150 155 160 His Gly Phe Thr Met Ala Leu Ala Gln Glu Val Ala Thr Lys Gly Val 165 170 175 Thr Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Asp Met Val Lys 180 185 190 Ala Ile Arg Gln Asp Val Leu Asp Lys Ile Val Ala Thr Ile Pro Val 195 200 205 Lys Arg Leu Gly Leu Pro Glu Glu Ile Ala Ser Ile Cys Ala Trp Leu 210 215 220 Ser Ser Glu Glu Ser Gly Phe Ser Thr Gly Ala Asp Phe Ser Leu Asn 225 230 235 240 Gly Gly Leu His Met Gly 245 <210> 19 <211> 1059 <212> DNA <213> Artificial Sequence <220> <223> Pseudomonas putida-lvaA-gene <400> 19 atgagcagtt caccaacgat ttccccggcc agcgatacgt tcgcggccat gactgacgat 60 caccgcctgg ccgagttcat ccgcgagcag gcctcggcaa cgcgggtggt catccaggcg 120 cgcaagcgcc tgagcggcgg cgctatccag gaaaactggc tgctggacct gctgatcgaa 180 ggcggcccgt gggccggtgt ccggcgttgg gtactgcgca gcgatgcgct ttcagcgcta 240 cccgccagcc ttgaccgtga acaggagttc gccgtgctgc aggtggttta ccaggccggc 300 gtgaaagtgc cacgcccgct ctggctgtgc cgcgatgtgc gcgtgcatgg gcgggtgttc 360 ttcctgatgg agtatgtgcc gggtagcgct gccggccgcg cgctcagcac cggcgccggt 420 cctcagggcc gggcgcaact ggcgacgcag cttggcgcca acctggcgcg tctgcatcag 480 gtccgcccgc cgtgcgccac gctgtgcttc ctgtccgttc cggacagctc gccggccctg 540 gcgaccatcg acgcctaccg ccgctacctc gacaccctcg ccgatgccta tccggtgctg 600 gaatggggcc tgcgctggtg cgagctgcat gcgccgcgca gcagcaccct gtgcctgttg 660 caccgtgact accgcaccgg caactacctg gccagcgaag aagggctgga ggccgtgctc 720 gactgggagt tcaccggctg gggagatcct tgcgaggacc tcggctggtt caccgcccgt 780 tgctggcgtt ttaccccgtcc agacctcgaa gccggcggca ttggccagct ggaggatttt 840 ctgcgtggtt atcacgaggt gtcttcgctg tgcatcgagc gcagtcggct ccactactgg 900 caagtcatgg ccaccctgcg ctgggcggtg attgccttgc agcaagggca gcgccatctg 960 tccggtgaag aaccgtcgct cgagctagca ctgacagccc ggctgttgcc ggagctcgaa 1020 ctcgacatcc tgcacatgac cggagccgaa gcgccatga 1059 <210> 20 <211> 352 <212> PRT <213> Artificial Sequence <220> <223> Pseudomonas putida-lvaA-peptide <400> 20 Met Ser Ser Ser Pro Thr Ile Ser Pro Ala Ser Asp Thr Phe Ala Ala 1 5 10 15 Met Thr Asp Asp His Arg Leu Ala Glu Phe Ile Arg Glu Gln Ala Ser 20 25 30 Ala Thr Arg Val Val Ile Gln Ala Arg Lys Arg Leu Ser Gly Gly Ala 35 40 45 Ile Gln Glu Asn Trp Leu Leu Asp Leu Leu Ile Glu Gly Gly Pro Trp 50 55 60 Ala Gly Val Arg Arg Trp Val Leu Arg Ser Asp Ala Leu Ser Ala Leu 65 70 75 80 Pro Ala Ser Leu Asp Arg Glu Gln Glu Phe Ala Val Leu Gln Val Val 85 90 95 Tyr Gln Ala Gly Val Lys Val Pro Arg Pro Leu Trp Leu Cys Arg Asp 100 105 110 Val Arg Val His Gly Arg Val Phe Phe Leu Met Glu Tyr Val Pro Gly 115 120 125 Ser Ala Ala Gly Arg Ala Leu Ser Thr Gly Ala Gly Pro Gln Gly Arg 130 135 140 Ala Gln Leu Ala Thr Gln Leu Gly Ala Asn Leu Ala Arg Leu His Gln 145 150 155 160 Val Arg Pro Pro Cys Ala Thr Leu Cys Phe Leu Ser Val Pro Asp Ser 165 170 175 Ser Pro Ala Leu Ala Thr Ile Asp Ala Tyr Arg Arg Tyr Leu Asp Thr 180 185 190 Leu Ala Asp Ala Tyr Pro Val Leu Glu Trp Gly Leu Arg Trp Cys Glu 195 200 205 Leu His Ala Pro Arg Ser Ser Thr Leu Cys Leu Leu His Arg Asp Tyr 210 215 220 Arg Thr Gly Asn Tyr Leu Ala Ser Glu Glu Gly Leu Glu Ala Val Leu 225 230 235 240 Asp Trp Glu Phe Thr Gly Trp Gly Asp Pro Cys Glu Asp Leu Gly Trp 245 250 255 Phe Thr Ala Arg Cys Trp Arg Phe Thr Arg Pro Asp Leu Glu Ala Gly 260 265 270 Gly Ile Gly Gln Leu Glu Asp Phe Leu Arg Gly Tyr His Glu Val Ser 275 280 285 Ser Leu Cys Ile Glu Arg Ser Arg Leu His Tyr Trp Gln Val Met Ala 290 295 300 Thr Leu Arg Trp Ala Val Ile Ala Leu Gln Gln Gly Gln Arg His Leu 305 310 315 320 Ser Gly Glu Glu Pro Ser Leu Glu Leu Ala Leu Thr Ala Arg Leu Leu 325 330 335 Pro Glu Leu Glu Leu Asp Ile Leu His Met Thr Gly Ala Glu Ala Pro 340 345 350 <210> 21 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Pseudomonas putida-lvaB-gene <400> 21 atgacccaac ccaacgccca cgaattgctc gagatcgccc gcgcgacgct tctggagcag 60 ctgctgccag cgctgcccgg cgagttgcgt tacccggccc tgatgatcgc caacgccatg 120 gccattgcgg cccgcgaaaa ccgcttgggc gctcaggccg aggatcagga gcaggcgcgt 180 ctggccgcct tggtcgatga cgcgccgtcg acatgcccg acctgcgccg ccaactggct 240 cgcgccattc gccagggcag ccatgacgcc ccgcaaaccc ggcgcaccct ggtcgagaca 300 ttacgccaga tcaccgttgc ccgattggcg atcagcaacc ccaaggcctt gccctga 357 <210> 22 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Pseudomonas putida-lvaB-peptide <400> 22 Met Thr Gln Pro Asn Ala His Glu Leu Leu Glu Ile Ala Arg Ala Thr 1 5 10 15 Leu Leu Glu Gln Leu Leu Pro Ala Leu Pro Gly Glu Leu Arg Tyr Pro 20 25 30 Ala Leu Met Ile Ala Asn Ala Met Ala Ile Ala Ala Arg Glu Asn Arg 35 40 45 Leu Gly Ala Gln Ala Glu Asp Gln Glu Gln Ala Arg Leu Ala Ala Leu 50 55 60 Val Asp Asp Ala Pro Ser Thr Leu Pro Asp Leu Arg Arg Gln Leu Ala 65 70 75 80 Arg Ala Ile Arg Gln Gly Ser His Asp Ala Pro Gln Thr Arg Arg Thr 85 90 95 Leu Val Glu Thr Leu Arg Gln Ile Thr Val Ala Arg Leu Ala Ile Ser 100 105 110 Asn Pro Lys Ala Leu Pro 115 <210> 23 <211> 2148 <212> DNA <213> Artificial Sequence <220> <223> Pseudomonas putida-fadB-gene <400> 23 atgatttacg aaggtaaagc catcacggtt aaggctcttg aaagtggcat cgtcgagctc 60 aagttcgacc tcaagggtga gtccgtcaac aagttcaacc gccttaccct gaacgagctg 120 cgccaggccg tcgatgccat ccgggccgat gcttcggtca agggcgtgat cgtcaggagt 180 ggcaaggacg tgttcatcgt cggcgccgac atcaccgagt tcgtcgacaa cttcaagctg 240 cctgaggccg aactggtcgc tggcaacctg gaagccaatc gcatcttcaa cgcgttcgaa 300 gacctcgaag tgccgaccgt tgccgccatc aacggcatcg cgctgggcgg cggcctggaa 360 atgtgcctgg cggccgacta ccgggtcatg tccaccagcg ccaggatcgg cctgccggaa 420 gtcaagctgg gtatctaccc gggctttggc ggtaccgtgc gcctgccgcg cctgatcggc 480 tcggacaacg ccatcgagtg gatcgccgcc ggcaaggaaa accgtgccga agatgccctg 540 aaagtggggg ccgtcgacgc cgtggtcgcc cctgagctgc tgctggccgg tgccctcgac 600 ctgatcaagc gtgccatcag tggcgagctg gactacaagg ccaagcgcca gccgaagctg 660 gaaaagctca agctcaatgc catcgagcag atgatggcct tcgagactgc caagggcttc 720 gtcgctggcc aggccggccc gaactacccg gccccggtcg aagcgatcaa gagcatccag 780 aaagccgcca acttcggtcg cgacaaggcc ctggaagtcg aagccgcagg ctttgccaag 840 ctggccaaga cctctgtcgc cgagagcctg atcggcttgt tcctcaacga tcaggaactc 900 aagcgcaagg ccaaggcgca tgacgagatc gcccacgacg tgaagcaggc cgccgtgctc 960 ggcgccggca tcatgggcgg cggtatcgcc taccagtcgg cggtcaaagg tacgccgatc 1020 ctgatgaagg acatccgcga ggaagccatt cagctgggtc tgaacgaggc ctccaagttg 1080 cttggcaacc gcgtcgagaa gggccgcctg accccggcca agatggccga ggccctcaac 1140 gccattcgcc cgaccctgtc ctatggcgat ttcgccaatg tcgacatcgt cgtcgaggct 1200 gtggtcgaga acccgaaggt caagcaagcg gtactggcgg aagtggaagg ccaggtgaag 1260 gacgatgcga tcctcgcttc caacacctct accatctcca tcaacctgct ggccaaggcg 1320 ctcaagcgcc cggaaaactt cgtcggcatg cacttcttca acccggtgca catgatgccg 1380 ctggttgaag tgatccgtgg cgagaagtcc agtgacgtgg cggtcgccac caccgtggcc 1440 tacgccaaga aaatgggcaa gaacccgatc gtggtcaacg actgcccggg ctttttggtc 1500 aaccgcgtgc tgttcccgta ctttggcggt tttgccaagc tggtcagcgc cggtgtcgac 1560 ttcgtgcgca tcgacaaggt catggagaag ttcggctggc cgatgggccc agcctacttg 1620 atggacgtgg tcggcatcga caccggccac cacggccgtg acgtcatggc cgaaggcttc 1680 ccggatcgca tgaaggacga gcgccgctcg gcagtcgacg cgttgtacga ggccaaccgc 1740 ctgggccaga agaacggtaa gggcttctac gcctacgaaa ccgacaagcg cggcaagccg 1800 aagaaggtct tcgatgccac cgtgctcgac gtgctcaaac cgatcgtgtt cgagcagcgt 1860 gaagtcactg acgaagacat catcaactgg atgatggtcc cgctgtgcct tgagaccgtg 1920 cgttgcctgg aagacggcat cgtcgaaacc gctgccgaag ccgacatggg cctggtctac 1980 ggcattggtt tccctccctt ccgcggtggt gcgctgcgtt acatcgactc gatcggtgtg 2040 gccgaattcg tcgccctggc cgatcagtat gccgacctgg ggccgctgta ccacccgacc 2100 gccaagctgc gtgaaatggc caagaacggc cagcgcttct tcaactga 2148 <210> 24 <211> 715 <212> PRT <213> Artificial Sequence <220> <223> Pseudomonas putida-fadB-peptide <400> 24 Met Ile Tyr Glu Gly Lys Ala Ile Thr Val Lys Ala Leu Glu Ser Gly 1 5 10 15 Ile Val Glu Leu Lys Phe Asp Leu Lys Gly Glu Ser Val Asn Lys Phe 20 25 30 Asn Arg Leu Thr Leu Asn Glu Leu Arg Gln Ala Val Asp Ala Ile Arg 35 40 45 Ala Asp Ala Ser Val Lys Gly Val Ile Val Arg Ser Gly Lys Asp Val 50 55 60 Phe Ile Val Gly Ala Asp Ile Thr Glu Phe Val Asp Asn Phe Lys Leu 65 70 75 80 Pro Glu Ala Glu Leu Val Ala Gly Asn Leu Glu Ala Asn Arg Ile Phe 85 90 95 Asn Ala Phe Glu Asp Leu Glu Val Pro Thr Val Ala Ala Ile Asn Gly 100 105 110 Ile Ala Leu Gly Gly Gly Gly Leu Glu Met Cys Leu Ala Ala Asp Tyr Arg 115 120 125 Val Met Ser Thr Ser Ala Arg Ile Gly Leu Pro Glu Val Lys Leu Gly 130 135 140 Ile Tyr Pro Gly Phe Gly Gly Thr Val Arg Leu Pro Arg Leu Ile Gly 145 150 155 160 Ser Asp Asn Ala Ile Glu Trp Ile Ala Ala Gly Lys Glu Asn Arg Ala 165 170 175 Glu Asp Ala Leu Lys Val Gly Ala Val Asp Ala Val Val Ala Pro Glu 180 185 190 Leu Leu Leu Ala Gly Ala Leu Asp Leu Ile Lys Arg Ala Ile Ser Gly 195 200 205 Glu Leu Asp Tyr Lys Ala Lys Arg Gln Pro Lys Leu Glu Lys Leu Lys 210 215 220 Leu Asn Ala Ile Glu Gln Met Met Ala Phe Glu Thr Ala Lys Gly Phe 225 230 235 240 Val Ala Gly Gln Ala Gly Pro Asn Tyr Pro Ala Pro Val Glu Ala Ile 245 250 255 Lys Ser Ile Gln Lys Ala Ala Asn Phe Gly Arg Asp Lys Ala Leu Glu 260 265 270 Val Glu Ala Ala Gly Phe Ala Lys Leu Ala Lys Thr Ser Val Ala Glu 275 280 285 Ser Leu Ile Gly Leu Phe Leu Asn Asp Gln Glu Leu Lys Arg Lys Ala 290 295 300 Lys Ala His Asp Glu Ile Ala His Asp Val Lys Gln Ala Ala Val Leu 305 310 315 320 Gly Ala Gly Ile Met Gly Gly Gly Gly Ile Ala Tyr Gln Ser Ala Val Lys 325 330 335 Gly Thr Pro Ile Leu Met Lys Asp Ile Arg Glu Glu Ala Ile Gln Leu 340 345 350 Gly Leu Asn Glu Ala Ser Lys Leu Leu Gly Asn Arg Val Glu Lys Gly 355 360 365 Arg Leu Thr Pro Ala Lys Met Ala Glu Ala Leu Asn Ala Ile Arg Pro 370 375 380 Thr Leu Ser Tyr Gly Asp Phe Ala Asn Val Asp Ile Val Val Glu Ala 385 390 395 400 Val Val Glu Asn Pro Lys Val Lys Gln Ala Val Leu Ala Glu Val Glu 405 410 415 Gly Gln Val Lys Asp Asp Ala Ile Leu Ala Ser Asn Thr Ser Thr Ile 420 425 430 Ser Ile Asn Leu Leu Ala Lys Ala Leu Lys Arg Pro Glu Asn Phe Val 435 440 445 Gly Met His Phe Phe Asn Pro Val His Met Met Pro Leu Val Glu Val 450 455 460 Ile Arg Gly Glu Lys Ser Ser Asp Val Ala Val Ala Thr Thr Val Ala 465 470 475 480 Tyr Ala Lys Lys Met Gly Lys Asn Pro Ile Val Val Asn Asp Cys Pro 485 490 495 Gly Phe Leu Val Asn Arg Val Leu Phe Pro Tyr Phe Gly Gly Phe Ala 500 505 510 Lys Leu Val Ser Ala Gly Val Asp Phe Val Arg Ile Asp Lys Val Met 515 520 525 Glu Lys Phe Gly Trp Pro Met Gly Pro Ala Tyr Leu Met Asp Val Val 530 535 540 Gly Ile Asp Thr Gly His His Gly Arg Asp Val Met Ala Glu Gly Phe 545 550 555 560 Pro Asp Arg Met Lys Asp Glu Arg Arg Ser Ala Val Asp Ala Leu Tyr 565 570 575 Glu Ala Asn Arg Leu Gly Gln Lys Asn Gly Lys Gly Phe Tyr Ala Tyr 580 585 590 Glu Thr Asp Lys Arg Gly Lys Pro Lys Lys Val Phe Asp Ala Thr Val 595 600 605 Leu Asp Val Leu Lys Pro Ile Val Phe Glu Gln Arg Glu Val Thr Asp 610 615 620 Glu Asp Ile Ile Asn Trp Met Met Val Pro Leu Cys Leu Glu Thr Val 625 630 635 640 Arg Cys Leu Glu Asp Gly Ile Val Glu Thr Ala Ala Glu Ala Asp Met 645 650 655 Gly Leu Val Tyr Gly Ile Gly Phe Pro Pro Phe Arg Gly Gly Ala Leu 660 665 670 Arg Tyr Ile Asp Ser Ile Gly Val Ala Glu Phe Val Ala Leu Ala Asp 675 680 685 Gln Tyr Ala Asp Leu Gly Pro Leu Tyr His Pro Thr Ala Lys Leu Arg 690 695 700 Glu Met Ala Lys Asn Gly Gln Arg Phe Phe Asn 705 710 715 <210> 25 <211> 1680 <212> DNA <213> Artificial Sequence <220> <223> Pseudomonas putida-phaC1-gene <400> 25 atgagtaaca agaacaacga tgagctacag cggcaggcct cggaaaacac cctggggctg 60 aacccggtca tcggcatccg ccgcaaggac ctgttgagct cggcacgcac cgtgctgcgc 120 caggccgtgc gccaaccgct gcacagcgcc aagcatgtgg ctcactttgg cctggagctg 180 aagaacgtgt tgctgggcaa atccagcctg gccccggaca gcgacgaccg tcgcttcaat 240 gacccggcct ggagcaacaa cccgctgtac cgccgctacc tgcaaaccta cctggcctgg 300 cgcaaggagc tgcaggactg ggtgagcagc agcgacctgt ccccccagga catcagccgc 360 ggccagttcg tcatcaacct gatgaccgag gccatggcgc cgaccaatac cctgtccaac 420 ccggctgcgg tcaaacgctt cttcgaaacc ggcggcaaga gcctgctcga tggcctgtcc 480 aacctggcca aggacatggt caacaacggc ggcatgccca gccaggtgaa catggatgcc 540 ttcgaagtgg gcaagaacct gggcaccagc gaaggcgcgg tggtgtaccg caacgatgtg 600 ctggaactga tccagtacag ccccatcacc gagcaggtgc atgcccgtcc gctgctggtg 660 gtgccaccgc agatcaacaa gttctacgtg ttcgacctca gcccggaaaa gagcctggcg 720 cgcttctgcc tgcgctcgca gcagcagacc ttcatcatca gctggcgcaa cccgaccaag 780 gcccagcgtg aatggggcct gtccacctac atcgatgcgc tgaaagaagc cgtcgacgcg 840 gtgctgtcga ttaccggcag caaggacctg aacatgctcg gcgcctgctc cggtggcatc 900 acttgtaccg cactggtggg ccactatgcc gccattggcg agaacaaggt caacgccctg 960 accctgctgg tcagcgtgct ggacaccacc atggacaacc aggttgcttt gtttgtcgac 1020 gagcagacct tggaggccgc caagcgccac tcctatcagg cgggcgtgct ggaaggcagc 1080 gaaatggcca aggtgttcgc ctggatgcgc cccaacgacc tgatctggaa ctactgggta 1140 aacaactacc tgctcggcaa tgagcccccc gtgttcgaca tcctgttctg gaacaacgac 1200 accacgcgcc tgccggccgc cttccacggc gacctgatcg aaatgttcaa gagcaacccg 1260 ctgacccgcc ccgacgccct ggaagtgtgc ggcaccgcga tcgacctgaa acaggtcaaa 1320 tgcgacatct acagcctcgc cggcaccaac gaccacatca ccccctggcc gtcatgctac 1380 cgctcggcac atctgttcgg cggcaagatc gaattcgtac tgtccaacag cgggcatatc 1440 cagagcatcc tcaacccgcc gggcaacccg aaggcacgtt tcatgaccgg tgccgatcgc 1500 ccgggtgacc cggtggcctg gcaggaaaat gccatcaagc atgcagactc ctggtggttg 1560 cactggcaga gttggctggg cgagcgtgcc ggcgcgctga aaaaggcacc gacccgcctg 1620 ggcaaccgta cctatgccgc cggcgaagcc tccccaggca cctacgttca cgagcgttga 1680 1680 <210> 26 <211> 559 <212> PRT <213> Artificial Sequence <220> <223> Pseudomonas putida-phaC1-peptide <400> 26 Met Ser Asn Lys Asn Asn Asp Glu Leu Gln Arg Gln Ala Ser Glu Asn 1 5 10 15 Thr Leu Gly Leu Asn Pro Val Ile Gly Ile Arg Arg Lys Asp Leu Leu 20 25 30 Ser Ser Ala Arg Thr Val Leu Arg Gln Ala Val Arg Gln Pro Leu His 35 40 45 Ser Ala Lys His Val Ala His Phe Gly Leu Glu Leu Lys Asn Val Leu 50 55 60 Leu Gly Lys Ser Ser Leu Ala Pro Asp Ser Asp Asp Arg Arg Phe Asn 65 70 75 80 Asp Pro Ala Trp Ser Asn Asn Pro Leu Tyr Arg Arg Tyr Leu Gln Thr 85 90 95 Tyr Leu Ala Trp Arg Lys Glu Leu Gln Asp Trp Val Ser Ser Ser Asp 100 105 110 Leu Ser Pro Gln Asp Ile Ser Arg Gly Gln Phe Val Ile Asn Leu Met 115 120 125 Thr Glu Ala Met Ala Pro Thr Asn Thr Leu Ser Asn Pro Ala Ala Val 130 135 140 Lys Arg Phe Phe Glu Thr Gly Gly Lys Ser Leu Leu Asp Gly Leu Ser 145 150 155 160 Asn Leu Ala Lys Asp Met Val Asn Asn Gly Gly Met Pro Ser Gln Val 165 170 175 Asn Met Asp Ala Phe Glu Val Gly Lys Asn Leu Gly Thr Ser Glu Gly 180 185 190 Ala Val Val Tyr Arg Asn Asp Val Leu Glu Leu Ile Gln Tyr Ser Pro 195 200 205 Ile Thr Glu Gln Val His Ala Arg Pro Leu Leu Val Val Pro Pro Gln 210 215 220 Ile Asn Lys Phe Tyr Val Phe Asp Leu Ser Pro Glu Lys Ser Leu Ala 225 230 235 240 Arg Phe Cys Leu Arg Ser Gln Gln Gln Thr Phe Ile Ile 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 Ala Val Leu Ser Ile Thr Gly Ser Lys 275 280 285 Asp Leu Asn Met Leu Gly Ala Cys Ser Gly Gly Ile Thr Cys Thr Ala 290 295 300 Leu Val Gly His Tyr Ala Ala Ile Gly Glu Asn Lys Val Asn Ala Leu 305 310 315 320 Thr Leu Leu Val Ser Val Leu Asp Thr Thr Met Asp Asn Gln Val Ala 325 330 335 Leu Phe Val Asp Glu Gln Thr Leu Glu Ala Ala Lys Arg His Ser Tyr 340 345 350 Gln Ala Gly Val Leu Glu Gly Ser Glu 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 Ser Asn Pro Leu Thr Arg Pro Asp Ala Leu Glu Val Cys Gly Thr 420 425 430 Ala Ile Asp Leu Lys Gln Val Lys Cys Asp Ile Tyr Ser Leu Ala Gly 435 440 445 Thr Asn Asp His Ile Thr Pro Trp Pro Ser Cys Tyr Arg Ser Ala His 450 455 460 Leu Phe Gly Gly Lys Ile Glu Phe Val Leu Ser Asn Ser Gly His Ile 465 470 475 480 Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Lys Ala Arg Phe Met Thr 485 490 495 Gly Ala Asp Arg Pro Gly Asp Pro Val Ala Trp Gln Glu Asn Ala Ile 500 505 510 Lys His Ala Asp Ser Trp Trp Leu His Trp Gln Ser Trp Leu Gly Glu 515 520 525 Arg Ala Gly Ala Leu Lys Lys Ala Pro Thr Arg Leu Gly Asn Arg Thr 530 535 540 Tyr Ala Ala Gly Glu Ala Ser Pro Gly Thr Tyr Val His Glu Arg 545 550 555 <210> 27 <211> 852 <212> DNA <213> Artificial Sequence <220> <223> Pseudomonas putida-phaZ-gene <400> 27 atgccgcaac cctatatttt caggaccgtc gagctggaca accagtccat ccgcaccgct 60 gttcgccccg gcaagccgca cctgacgccg ttgctgatct ttaacggcat cggcgccaac 120 ctcgagctgg tgttcccgtt catcgatgca cttgacccgg acctggaagt catcgccttt 180 gatgtgcccg gggtcggcgg ctcgtctacg ccacgcaacc cgtaccgctt ccctgggctg 240 gccaagctga ccgcgcggat gctcgactac ctcgactacg gccaggtcaa cgtcatcggc 300 gtgtcctggg gcggcgccct ggcccagcag tttgctcacg attaccccga gcgctgcaag 360 aagctggtgc tggccgccac cgctgccggt gcggtaatgg tgccaggcaa gcccaaggtg 420 ctgtggatga tggccagccc ccggcgttac gtgcagccat cgcatgtcat ccgcattgcg 480 ccgatgatct atggcggcgg cttccgacgt gaccccgacc tggccatgca ccatgccgcc 540 aaggtgcgct ccggcggcaa gctgggctac tactggcagc tgttcgcagg gctcggctgg 600 accagcatcc actggctgca caagatccgg cagcccaccc tggtactggc tggcgacgac 660 gacccgttga tcccgctgat caacatgcgc ctgctggcct ggcggattcc caatgcccag 720 ctacacatta tcgacgacgg ccatctgttc ctgatcaccc gtgccgaagc cgtcgccccg 780 atcatcatga agttcctgca ggaagaacgt cagcgtgcgg tcatgcatcc ccgtccggcc 840 tcgggggggt ga 852 <210> 28 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> Pseudomonas putida-phaZ-peptide <400> 28 Met Pro Gln Pro Tyr Ile Phe Arg Thr Val Glu Leu Asp Asn Gln Ser 1 5 10 15 Ile Arg Thr Ala Val Arg Pro Gly Lys Pro His Leu Thr Pro Leu Leu 20 25 30 Ile Phe Asn Gly Ile Gly Ala Asn Leu Glu Leu Val Phe Pro Phe Ile 35 40 45 Asp Ala Leu Asp Pro Asp Leu Glu Val Ile Ala Phe Asp Val Pro Gly 50 55 60 Val Gly Gly Ser Ser Thr Pro Arg Asn Pro Tyr Arg Phe Pro Gly Leu 65 70 75 80 Ala Lys Leu Thr Ala Arg Met Leu Asp Tyr Leu Asp Tyr Gly Gln Val 85 90 95 Asn Val Ile Gly Val Ser Trp Gly Gly Ala Leu Ala Gln Gln Phe Ala 100 105 110 His Asp Tyr Pro Glu Arg Cys Lys Lys Leu Val Leu Ala Ala Thr Ala 115 120 125 Ala Gly Ala Val Met Val Pro Gly Lys Pro Lys Val Leu Trp Met Met 130 135 140 Ala Ser Pro Arg Arg Tyr Val Gln Pro Ser His Val Ile Arg Ile Ala 145 150 155 160 Pro Met Ile Tyr Gly Gly Gly Phe Arg Arg Asp Pro Asp Leu Ala Met 165 170 175 His His Ala Ala Lys Val Arg Ser Gly Gly Lys Leu Gly Tyr Tyr Trp 180 185 190 Gln Leu Phe Ala Gly Leu Gly Trp Thr Ser Ile His Trp Leu His Lys 195 200 205 Ile Arg Gln Pro Thr Leu Val Leu Ala Gly Asp Asp Asp Pro Leu Ile 210 215 220 Pro Leu Ile Asn Met Arg Leu Leu Ala Trp Arg Ile Pro Asn Ala Gln 225 230 235 240 Leu His Ile Ile Asp Asp Gly His Leu Phe Leu Ile Thr Arg Ala Glu 245 250 255 Ala Val Ala Pro Ile Ile Met Lys Phe Leu Gln Glu Glu Arg Gln Arg 260 265 270 Ala Val Met His Pro Arg Pro Ala Ser Gly Gly 275 280 <210> 29 <211> 1683 <212> DNA <213> Artificial Sequence <220> <223> Pseudomonas putida-phaC2-gene <400> 29 atgacagaca aaccggccaa aggatcgaca acgctccccg ccacccgcat gaacgtgcag 60 aacgccatcc tcggcctgcg cggccgcgac ctgctttcca cgctgcgcaa cgtcggccgc 120 cacggcctgc gccacccgct gcataccgcg catcatctgc tggcgcttgg cgggcagttg 180 gggcgggtga tgctggggga cacgccctac cagccgaacc cgcgtgacgc acgcttcagt 240 gacccgacct ggagccagaa cccgttctac cgccgcggcc tgcaagccta tctggcctgg 300 cagaagcaga cacgccagtg gatcgatgaa agccatttga acgacgatga tcgagcccgc 360 gcccacttcc tgttcaacct gatcaacgat gcgctggcgc ccagcaactc actgctcaat 420 ccgttggcgg tcaaggagct gttcaacacc ggcggccaga gcctggtgcg cggcgtggct 480 cacctgctcg acgacctgcg tcacaacgat gggctgcctc gtcaggtgga cgagcgcgcc 540 ttcgaagtgg gcgttaacct ggccgcaacc cctggcgcag tggtatttcg caacgagctg 600 ctggagctga tccagtactc gccgatgagc gaaaagcagc acgcacgccc actgctggtc 660 gtgccgcctc agatcaacaa gttctacatc ttcgacctca gcgcgaccaa cagcttcgtc 720 cagtacatgc tcaaaagcgg cttgcaggtg ttcatggtca gctggcgcaa ccccgaccca 780 cgccaccgtg aatggggcct ttccagctat gtgcaagccc tggaggaagc gctcaatgcc 840 tgccgcagta tcagcggcaa ccgcgacccc aacctgatgg gtgcctgtgc cggcggcctg 900 accatggccg cactgcaagg ccatctgcaa gccaagaagc aattgcgccg ggtgcgcagt 960 gccacgtatc tggtcagctt gctggacagc aagttcgaaa gcccggccag cctgttcgcc 1020 gatgagcaga ccatcgaagc ggccaagcga cgctcctatc agcgtggcgt gctggacggt 1080 ggtgaagtgg cgcggatctt cgcctggatg cggcccaacg acctgatctg gaactactgg 1140 gtaaacaact acctgctcgg caagacaccg ccggcgttcg acatcctgta ctggaatgcc 1200 gacagcacgc gcctgcccgc cgcgctgcat ggcgacctgc tggagttttt caagctcaac 1260 cccttgacct acgcgtccgg gctggaggtg tgcggtacgc cgatcgacct gcagcaggtc 1320 aatatcgaca gctttaccgt ggccggcagc aacgaccaca tcacaccatg ggatgcggtg 1380 taccgctcgg ccttgctgct gggtggcgag cggcgcttcg tgctggccaa cagcgggcat 1440 atccagagca tcatcaaccc gccaggcaac cccaaggcct actacctggc caaccccaag 1500 ctgagcagcg acccacgcgc ctggttccac gacgccaagc gcagtgaagg cagctggtgg 1560 ccgttgtggc tggagtggat caccgcacgc tccggcctgc tcaaggcacc gcgtactgaa 1620 ctgggcaacg ccacttaccc accgctaggc cccgcgccag gcacctacgt gctgacccga 1680 tga 1683 <210> 30 <211> 560 <212> PRT <213> Artificial Sequence <220> <223> Pseudomonas putida-phaC2-peptide <400> 30 Met Thr Asp Lys Pro Ala Lys Gly Ser Thr Thr Leu Pro Ala Thr Arg 1 5 10 15 Met Asn Val Gln Asn Ala Ile Leu Gly Leu Arg Gly Arg Asp Leu Leu 20 25 30 Ser Thr Leu Arg Asn Val Gly Arg His Gly Leu Arg His Pro Leu His 35 40 45 Thr Ala His His Leu Leu Ala Leu Gly Gly Gln Leu Gly Arg Val Met 50 55 60 Leu Gly Asp Thr Pro Tyr Gln Pro Asn Pro Arg Asp Ala Arg Phe Ser 65 70 75 80 Asp Pro Thr Trp Ser Gln Asn Pro Phe Tyr Arg Arg Gly Leu Gln Ala 85 90 95 Tyr Leu Ala Trp Gln Lys Gln Thr Arg Gln Trp Ile Asp Glu Ser His 100 105 110 Leu Asn Asp Asp Asp Arg Ala Arg Ala His Phe Leu Phe Asn Leu Ile 115 120 125 Asn Asp Ala Leu Ala Pro Ser Asn Ser Leu Leu Asn Pro Leu Ala Val 130 135 140 Lys Glu Leu Phe Asn Thr Gly Gly Gln Ser Leu Val Arg Gly Val Ala 145 150 155 160 His Leu Leu Asp Asp Leu Arg His Asn Asp Gly Leu Pro Arg Gln Val 165 170 175 Asp Glu Arg Ala Phe Glu Val Gly Val Asn Leu Ala Ala Thr Pro Gly 180 185 190 Ala Val Val Phe Arg Asn Glu Leu Leu Glu Leu Ile Gln Tyr Ser Pro 195 200 205 Met Ser Glu Lys Gln His Ala Arg Pro Leu Leu Val Val Pro Gln 210 215 220 Ile Asn Lys Phe Tyr Ile Phe Asp Leu Ser Ala Thr Asn Ser Phe Val 225 230 235 240 Gln Tyr Met Leu Lys Ser Gly Leu Gln Val Phe Met Val Ser Trp Arg 245 250 255 Asn Pro Asp Pro Arg His Arg Glu Trp Gly Leu Ser Ser Tyr Val Gln 260 265 270 Ala Leu Glu Glu Ala Leu Asn Ala Cys Arg Ser Ile Ser Gly Asn Arg 275 280 285 Asp Pro Asn Leu Met Gly Ala Cys Ala Gly Gly Leu Thr Met Ala Ala 290 295 300 Leu Gln Gly His Leu Gln Ala Lys Lys Gln Leu Arg Arg Val Arg Ser 305 310 315 320 Ala Thr Tyr Leu Val Ser Leu Leu Asp Ser Lys Phe Glu Ser Pro Ala 325 330 335 Ser Leu Phe Ala Asp Glu Gln Thr Ile Glu Ala Ala Lys Arg Arg Ser 340 345 350 Tyr Gln Arg Gly Val Leu Asp Gly Gly Glu Val Ala Arg 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 Thr Pro Pro Ala Phe Asp Ile Leu Tyr Trp Asn Ala 385 390 395 400 Asp Ser Thr Arg Leu Pro Ala Ala Leu His Gly Asp Leu Leu Glu Phe 405 410 415 Phe Lys Leu Asn Pro Leu Thr Tyr Ala Ser Gly Leu Glu Val Cys Gly 420 425 430 Thr Pro Ile Asp Leu Gln Gln Val Asn Ile Asp Ser Phe Thr Val Ala 435 440 445 Gly Ser Asn Asp His Ile Thr Pro Trp Asp Ala Val Tyr Arg Ser Ala 450 455 460 Leu Leu Leu Gly Gly Glu Arg Arg Phe Val Leu Ala Asn Ser Gly His 465 470 475 480 Ile Gln Ser Ile Ile Asn Pro Pro Gly Asn Pro Lys Ala Tyr Tyr Leu 485 490 495 Ala Asn Pro Lys Leu Ser Ser Asp Pro Arg Ala Trp Phe His Asp Ala 500 505 510 Lys Arg Ser Glu Gly Ser Trp Trp Pro Leu Trp Leu Glu Trp Ile Thr 515 520 525 Ala Arg Ser Gly Leu Leu Lys Ala Pro Arg Thr Glu Leu Gly Asn Ala 530 535 540 Thr Tyr Pro Pro Leu Gly Pro Ala Pro Gly Thr Tyr Val Leu Thr Arg 545 550 555 560 <210> 31 <211> 870 <212> DNA <213> Artificial Sequence <220> <223> Pseudomonas putida-tesB-gene <400> 31 atgagtcatg tgttggacga cctggtcgac ctgttgagcc tcgaatccat cgaggagaac 60 ctgttccgtg gacgtagcca ggacctgggc ttccgccagc tttacggtgg ccaagtgctg 120 ggccagtcgt tgtcggccgc cagtcagaca gtcgaggacg cgcgccatgt gcattcgttg 180 cacggctact tcctgcgccc gggcgatgcc agccttccag tggtgtactc ggtagaccgc 240 gtgcgcgatg gcggcagctt cagtacacgg cgggtgacgg cgatccagaa gggccagacc 300 atcttcacct gcagcgcgtc gttccagtac gacgaggaag gcttcgagca ccaggcgcag 360 atgcctgacg tggtcggccc ggaaaacctg cctaccgaag tcgaactggc ccatgccatg 420 gccgaccagt tgcccgagcg catccgcgac aaggtgctgt gcgccaagcc gatcgagatc 480 cgcccggtca ccgagcgcga cccgttcaac cccaagccgg gcgacccggt gaagtacgcc 540 tggttccgcg ccgacggcaa cctgcctgac gtgcctgccc tgcacaagta catgctggcc 600 tacgcctcgg acttcggcct gctgaccacg gcgttgctgc cccatggcaa atcggtgtgg 660 cagcgcgaca tgcagatcgc cagcctcgac cattcgctgt ggtttcacgg caacctgcgc 720 gccgaccagt ggctactgta cgccaccgat agcccctggg ccggcaattc ccgtggcttc 780 tgccgcggca gcattttcaa ccaggccggg caactggtgg catcgtcgag ccaggaaggc 840 ctgattcgcc atcgcaagga ctgggcatga 870 <210> 32 <211> 289 <212> PRT <213> Artificial Sequence <220> <223> Pseudomonas putida-tesB-peptide <400> 32 Met Ser His Val Leu Asp Asp Leu Val Asp Leu Leu Ser Leu Glu Ser 1 5 10 15 Ile Glu Glu Asn Leu Phe Arg Gly Arg Ser Gln Asp Leu Gly Phe Arg 20 25 30 Gln Leu Tyr Gly Gly Gln Val Leu Gly Gln Ser Leu Ser Ala Ala Ser 35 40 45 Gln Thr Val Glu Asp Ala Arg His Val His Ser Leu His Gly Tyr Phe 50 55 60 Leu Arg Pro Gly Asp Ala Ser Leu Pro Val Val Tyr Ser Val Asp Arg 65 70 75 80 Val Arg Asp Gly Gly Ser Phe Ser Thr Arg Arg Val Thr Ala Ile Gln 85 90 95 Lys Gly Gln Thr Ile Phe Thr Cys Ser Ala Ser Phe Gln Tyr Asp Glu 100 105 110 Glu Gly Phe Glu His Gln Ala Gln Met Pro Asp Val Val Gly Pro Glu 115 120 125 Asn Leu Pro Thr Glu Val Glu Leu Ala His Ala Met Ala Asp Gln Leu 130 135 140 Pro Glu Arg Ile Arg Asp Lys Val Leu Cys Ala Lys Pro Ile Glu Ile 145 150 155 160 Arg Pro Val Thr Glu Arg Asp Pro Phe Asn Pro Lys Pro Gly Asp Pro 165 170 175 Val Lys Tyr Ala Trp Phe Arg Ala Asp Gly Asn Leu Pro Asp Val Pro 180 185 190 Ala Leu His Lys Tyr Met Leu Ala Tyr Ala Ser Asp Phe Gly Leu Leu 195 200 205 Thr Thr Ala Leu Leu Pro His Gly Lys Ser Val Trp Gln Arg Asp Met 210 215 220 Gln Ile Ala Ser Leu Asp His Ser Leu Trp Phe His Gly Asn Leu Arg 225 230 235 240 Ala Asp Gln Trp Leu Leu Tyr Ala Thr Asp Ser Pro Trp Ala Gly Asn 245 250 255 Ser Arg Gly Phe Cys Arg Gly Ser Ile Phe Asn Gln Ala Gly Gln Leu 260 265 270 Val Ala Ser Ser Ser Gln Glu Gly Leu Ile Arg His Arg Lys Asp Trp 275 280 285 Ala <210> 33 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> lvaA_upstream_fwd <400> 33 gaggcccttt cgtcttcacc tcgagatggt tgtagctgag cacg 44 <210> 34 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> lvaA_upstream_rev <400> 34 actgctgcat ggttctgtag gccctgcc 28 <210> 35 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> lvaB_downstream_fwd <400> 35 cctgcaatga aaccgcatcg accacccc 28 <210> 36 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> lvaB_downstream_rev <400> 36 cgcaaaagaa aatgccgatt ctagacatgg cgaagcacga acg 43 <210> 37 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> phaC1_up_fwd <400> 37 gaggcccttt cgtcttcacc tcgaggaaca actgtgggca g 41 <210> 38 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> phaC1_up_rev <400> 38 cagcacgtag cccagggtgt tttccgag 28 <210> 39 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> phaC2_down_fwd <400> 39 acaccctggg ctacgtgctg acccgatg 28 <210> 40 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> phaC2_down_rev <400> 40 cgcaaaagaa aatgccgatt ctagagcgtg cgtttgattg cattg 45 <210> 41 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> fadB_up_fwd <400> 41 gaggcccttt cgtcttcacc tcgagtgtag tcgccttggc tc 42 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> fadB_up_rev <400> 42 catttcacgc tgttgacgga ctcacccttg 30 <210> 43 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> fadB_down_fwd <400> 43 tccgtcaaca gcgtgaaatg gccaagaac 29 <210> 44 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> fadB_down_rev <400> 44 cgcaaaagaa aatgccgatt ctagatctgc agtcaggccc atc 43 <210> 45 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> phaC_P.d._fwd <400> 45 ctttataagg aggaaaaaca tatggccgcg ccccgcgcc 39 <210> 46 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> phaC_P.d._rev <400> 46 agtccaagct cagctaatta agcttttacg cccgctcgtg gacgtaaagg ccc 53 <210> 47 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> phaC_C.v._fwd <400> 47 ctttataagg aggaaaaaca tatgcagcag ttcgtcaact cg 42 <210> 48 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> phaC_C.v._rev <400> 48 agtccaagct cagctaatta agctttcatt gcaggctggc ggc 43 <210> 49 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> phaC_C.n_fwd <400> 49 ctttataagg aggaaaaaca tatggcgacc ggcaaaggc 39 <210> 50 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> phaC_C.n_rev <400> 50 agtccaagct cagctaatta agctcatgcc ttggctttga cgtatcg 47 <210> 51 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> phaE_T.p._fwd <400> 51 ctttataagg aggaaaaaca tatgaacgat acggccaaca ag 42 <210> 52 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> phaE_T.p._rev <400> 52 atggggacat ggcactctct cctggtgg 28 <210> 53 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> phaC_T.p._fwd <400> 53 agagagtgcc atgtccccat tcccgatc 28 <210> 54 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> phaC_T.p._rev <400> 54 caagctcagc taattaagct ttcagccgcg ttcgttcag 39 <210> 55 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> phaA_C.n_fwd <400> 55 caaggcatga attgaaagga ctacacaatg ac 32 <210> 56 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> phaA_C.n_rev <400> 56 agtccaagct cagctaatta agcttatttg cgctcgactg c 41 <210> 57 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> phaB_C.n_fwd <400> 57 ctttataagg aggaaaaaca tatgactcag cgcattgcg 39 <210> 58 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> phaB_C.n_rev_RBS <400> 58 ttgtctctct tcagcccata tgcaggcc 28 <210> 59 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> phaC_C.n_fwd_RBS <400> 59 tatgggctga agagagacaa tcaaatcatg g 31 <210> 60 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> phaC_C.n_rev_RBS <400> 60 tcctttcaat tcatgccttg gctttgac 28 <210> 61 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> phaB_rev_phaBA <400> 61 tcctttcaat tcagcccata tgcaggcc 28 <210> 62 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> phaA_fwd_phaBA <400> 62 tatgggctga attgaaagga ctacacaatg ac 32 <210> 63 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> phaC1_fwd <400> 63 aggtgagagt ggaggaggtc catttatgcg agacaagtcg aac 43 <210> 64 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> phaC1_rev <400> 64 agtccaagct cagctaatta agctttcagc ggatatgcac gtag 44 <210> 65 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> phaC2_fwd <400> 65 ctttataagg aggaaaaaca tatgagtgac aagaacaacg aag 43 <210> 66 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> phaC2_rev <400> 66 aaatggacct cctccactct caccttcagc gttcgtgcac atag 44

Claims (14)

As a transgenic microorganism into which the phaC gene of a heterologous strain is introduced,
The microorganism is a Pseudomonas genus ( Pseudomonas sp ) strain,
The transgenic microorganism has suppressed expression of the phaC gene,
The microorganism can produce polyhydroxyalkanoate (PHA),
The polyhydroxyalkanoate is any one monomer selected from the group consisting of 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV), 4-hydroxyvaleric acid (4HV), and combinations thereof. Which is formed by polymerization, transformed microorganisms. According to claim 1,
The heterologous strain is Paracoccus denitrificans ( Paracoccus denitrificans ), Chromobacterium violaceum ( Chromobacterium violaceum ), Cupriavidus necator ( Cupriavidus necator ), Pseudomonas oleovorans ( Pseudomonas oleovorans ) or Thio ( Thiocapsa pfennigii ), a transforming microorganism. According to claim 1,
The phaC gene of the heterologous strain is Pseudomonas oleovorans ( Pseudomonas oleovorans ) The transformed microorganism comprising the phaC1 gene and the phaC2 gene. According to claim 1,
The phaC gene of the heterologous strain is thiocapsa pfennigii ( Thiocapsa pfennigii ) The transformed microorganism comprising the phaE gene and the phaC gene. According to claim 1,
The phaC gene to the two kinds of strain comprises genes phaA, phaB, and phaC genes in the gene Coop Ria bideoseu Neka Torr (Cupriavidus necator), transformed microorganism. According to claim 1,
The phaC gene to the two kinds of strains including phaC gene of Coop Ria bideoseu Neka Torr (Cupriavidus necator), transformed microorganism. According to claim 1,
The phaC gene of the heterogeneous strain is Chromobacterium violaceum ( Chromobacterium violaceum ) The transformed microorganism comprising the phaC gene. According to claim 1,
The phaC gene of the heterologous strain is Paracoccus denitrificans ( Paracoccus denitrificans ) The transformed microorganism comprising the phaC gene. According to claim 1,
The microorganism is Pseudomonas putida ( Pseudomonas putida ) The strain, a transforming microorganism. According to claim 1,
The transformed microorganism is lvaAB gene, fadB gene, tesB gene and that the, transformed microorganism further suppressed the expression of any one gene selected from the group consisting of. delete delete Culturing the transformed microorganism of claim 1; and
A method for producing polyhydroxyalkanoate, comprising the step of obtaining polyhydroxyalkanoate produced from the transformed microorganism. A culture medium comprising the transformed microorganism of claim 1.

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