KR102177736B1 - Transformed recombinant microorganism producing polyhydroxyalkanoate - Google Patents

Abstract

The present invention relates to a transformed recombinant microorganism producing polyhydroxyalkanoate. The transformed recombinant microorganism according to the present invention can control the composition of 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 4-hydroxyvaleric acid depending on the expression of a fadB gene, an lvaED gene, or an lvaABC gene in a state where the expression of a fadE gene and an atoC gene is suppressed. In addition, depending on the expression of a phaE gene, a phaC gene, or a phaAB gene in the recombinant microorganism, it is possible to be used to produce polyhydroxyalkanoate containing various monomers.

Description

Transformed recombinant microorganism producing polyhydroxyalkanoate {TRANSFORMED RECOMBINANT MICROORGANISM PRODUCING POLYHYDROXYALKANOATE}

The present invention relates to a transformed recombinant microorganism producing polyhydroxyalkanoate.

Plastics are strong, light, and not easily decomposed, so they are used in various ways, including industrial materials and disposable materials, but due to environmental pollution caused by plastic waste, regulations on the use of non-degradable plastics are becoming increasingly severe. In order to solve the problem of environmental pollution caused by plastics, methods of recycling, landfilling, and incineration have been used, but the need for decomposable plastics has emerged since these methods cannot completely solve the environmental problems.

Biodegradable plastic refers to a so-called decaying plastic whose physical and chemical structure is made cotton by organic substances such as bacteria in nature. Types of biodegradable plastics include polyhydroxyalkanoate (PHA), polylactic acid (PLA), and polyglycolic acid (PG), as well as food and chemical additives, and pharmaceuticals. It can be used for materials, degradable packaging (Martin Koller, et al., 2013).

Among them, polyhydroxyalkanoate is a starch that accumulates inside microorganisms for storage of energy and reducing ability when microorganisms lack elements necessary for growth such as nitrogen, oxygen, phosphorus, and magnesium, and carbon sources are abundant. It is a natural polyester material with biological functions like glycogen. Since polyhydroxyalkanoate is biodegradable and biocompatible, it is recognized as a substitute for existing synthetic plastics.

It is known that PHA can be composed of more than 150 types of monomers depending on the type of microorganisms to be produced, the type of chemicals supplied, and the culture conditions. The most representative example is 3-hydroxybutyrate (3HB) homopolyester, which is well decomposed by microorganisms, but has extremely brittle properties, so it has a small practical range as a microbial degradable plastic. Meanwhile, biosynthesis of polyesters such as 3-hydroxybutyrate and 3-hydroxyvaleric acid (3HV) or 4-hydroxyvaleric acid (4HV) copolymer was confirmed. These polyesters exhibit a wide range of physical properties from hard to soft materials depending on the composition ratio of monomers, and thus have the potential as a practical microbial degradable plastic.

However, the monomers 3-hydroxyvaleric acid and 4-hydroxyvaleric acid may be obtained through chemical synthesis or may be obtained through biological methods. However, the chemical synthesis method has a problem that excessive costs are generated due to the use of harsh conditions, expensive substrates, catalysts and organic solvents, and biocompatibility is poor. In addition, biological synthesis using glucose and propionic acid as substrates has a problem in that the production amount and yield of 3-hydroxyvaleric acid and 4-hydroxyvaleric acid are very low.

Martin Koller, et al., POLYHYDROXYALKANOATES: BIODEGRADABLE POLYMERS AND PLASTICS FROM RENEWABLE RESOURCES. ISSN 1580-2949. MTAEC 9, 47(1)5(2013).

It is an object of the present invention to provide a transformed recombinant microorganism that produces polyhydroxyalkanoate.

To achieve the above object, fadE It provides a transformed recombinant microorganism in which the expression of the gene and the atoC gene is suppressed.

In addition, the present invention comprises the steps of culturing the recombinant microorganism according to the present invention; And it provides a method for producing polyhydroxyalkanoate, comprising the step of obtaining polyhydroxyalkanoate produced from the recombinant microorganism.

The recombinant microorganism transformed according to the invention in a fadB expression of fadE gene and atoC gene suppression state Gene, lvaED gene, or lvaABC The composition of 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 4-hydroxyvaleric acid can be adjusted according to the presence or absence of gene expression. In addition, it may be used to produce polyhydroxyalkanoate containing various monomers depending on the expression of the phaE gene, phaC gene, or phaAB gene in the recombinant microorganism.

1 is a schematic diagram of a pathway for producing various polyhydroxyalkanoates from levulinic acid in E. coli.
Figure 2 shows the total amount of PHA produced in the mutant MG1655 strain and the content of 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV), and 4-hydroxyvaleric acid (4HV), which are constituents of PHA ( g/L).
3 is a mass ratio of the total production of PHA produced in the mutant MG1655 strain and of 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV) and 4-hydroxyvaleric acid (4HV), which are components of the PHA. It represents (% wt).
Figure 4 shows a plasmid loaded with lvaABC gene derived from P. putida KT2440 strain.
Figure 5 shows a plasmid loaded with the lvaED gene derived from P. putida KT2440 strain.
Figure 6 shows a plasmid loaded with the phaC gene derived from C. violaceum strain.
7 shows a plasmid loaded with the phaEC gene derived from the T. pfennigii strain.
Figure 8 shows a plasmid loaded with the phaC gene derived from the C. violaceum strain and the phaAB gene derived from the C. necator strain.
9 shows a plasmid loaded with a phaEC gene derived from a T. pfennigii strain and a phaAB gene derived from a C. necator strain.

In one aspect, the present invention provides a transformed recombinant microorganism in which the expression of the fadE gene and the atoC gene is suppressed. At this time, the microorganism may be E. coli .

The term " fadE " used in the present invention may be a polynucleotide having a nucleotide sequence of SEQ ID NO: 22, and the FadE protein encoded by the nucleotide sequence may be a polypeptide having an amino acid sequence of SEQ ID NO: 21. The FadE protein is acyl-coenzyme A dehydrogenase, and 2-enoyl of acyl-Coenzyme A (acyl-CoAs), which is the first step in the beta-oxidation cycle of fatty acid degradation. -Can catalyze the dehydrogenation reaction to CoAs.

In addition, the term " atoC " used in the present invention may be a polynucleotide having a nucleotide sequence of SEQ ID NO: 24, and the AtoC protein encoded by the nucleotide sequence may be a polypeptide having an amino acid sequence of SEQ ID NO: 23. The AtoC protein is an AtoC regulatory protein (AtoC) and is a member of the two-component regulatory system AtoS/AtoC. In the presence of acetoacetate, AtoS/AtoC stimulates the expression of the atoDAEB operon, leading to short chain fatty acid catabolism and activation of the poly-(R)-3-hydroxybutyrate (cPHB) biosynthetic pathway.

In one embodiment, it is possible to additionally inhibit the expression of the fadB gene in the transformed recombinant microorganism.

The term " fadB " used in the present invention may be a polynucleotide having a nucleotide sequence of SEQ ID NO: 26, and the FadB protein encoded by the nucleotide sequence may be a polypeptide having an amino acid sequence of SEQ ID NO: 25. The FadB protein is 3-hydroxyacyl-CoA dehydratase, and is involved in aerobic and anaerobic degradation of long-chain fatty acids through the beta-oxidation cycle. In addition, the FadB protein may catalyze the formation of 3-oxoacyl-CoA from enoyl-CoA through L-3-hydroxyacyl-CoA.

In one embodiment, the lvaED gene may be introduced into the transformed recombinant microorganism. At this time, the lvaED gene may be derived from a P. putida strain, but is not limited thereto.

The term " lvaED " used in the present invention is lvaD Genes and lvaE Means genes. The lvaD Genes and lvaE The gene may be a polynucleotide having a nucleotide sequence of SEQ ID NO: 8 and 10, respectively, and the LvaD enzyme and LvaE enzyme encoded by each nucleotide sequence may be a polypeptide having an amino acid sequence represented by SEQ ID NO: 7 and 9, respectively. . The LvaE enzyme is a short-chain acyl-CoA synthetase, and can be activated by catalyzing levulinic acid with levulinic acid-CoA. The LvaD enzyme is 4-oxopentanoyl-CoA-4-dehydrogenase, which can catalyze the conversion of levulinic acid-CoA to 4-hydroxylvaleric acid-CoA.

In one embodiment, lvaA in the transformed recombinant microorganism, lvaB And lvaC Any one or more genes selected from the group consisting of genes may be introduced. Idae, the lvaA , lvaB And lvaC Any one or more genes selected from the group consisting of genes may be derived from P. putida strain.

The term " lvaABC " used in the present invention means lvaA , lvaB, and lvaC genes. The lvaA gene may be a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 2, and the LvaA enzyme encoded by the nucleotide sequence may be a polypeptide having an amino acid sequence represented by SEQ ID NO: 1. The lvaB gene may be a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 4, and the LvaB enzyme encoded by the nucleotide sequence may be a polypeptide having an amino acid sequence of SEQ ID NO: 3. In addition, the lvaC gene may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 6, and the LvaC enzyme encoded by the nucleotide sequence may be a polypeptide having the amino acid sequence of SEQ ID NO: 5.

Meanwhile, the LvaAB enzyme contributes to the reaction of 4HV-CoA and ATP to produce 4PV-CoA (4-phosphovaleryl-CoA). The LvaC enzyme is a 4PV-CoA (4-phosphovaleryl-CoA) phosphatase/isomerase, which is an enzyme capable of converting 4PV-CoA (4-phosphovaleryl-CoA) into 3-hydroxy pentanoyl-CoA.

In one embodiment, phaC in the transformed recombinant microorganism gene Or phaE Genes can be introduced.

remind phaC The gene may be from a C. violaceum strain. C. violaceum The strain is a Gram-negative strain, and can produce metabolites having anti-viability such as aerocianidin and aerocavin. The phaC gene derived from the C. violaceum strain may be a polynucleotide having a nucleotide sequence of SEQ ID NO: 12, and the PhaC enzyme encoded by the nucleotide sequence may be a polypeptide having an amino acid sequence of SEQ ID NO: 11.

Also, the phaC The gene may be from the T. pfennigii strain. The T. pfennigii strain is a Gram negative strain belonging to the genus Thiococcus . The phaC gene derived from the T. pfennigii strain may be a polynucleotide having a nucleotide sequence of SEQ ID NO: 16, and the PhaC enzyme encoded by the nucleotide sequence may be a polypeptide having an amino acid sequence of SEQ ID NO: 15.

The phaE The gene may be from the T. pfennigii strain. The phaE gene derived from the T. pfennigii strain may be a polynucleotide having a nucleotide sequence of SEQ ID NO: 14, and the PhaE enzyme encoded by the nucleotide sequence may be a polypeptide having an amino acid sequence of SEQ ID NO: 13.

The PhaC enzyme is an enzyme that biosynthesizes polyhydroxyalkanoate by polymerizing monomeric hydroxyalkanoate. In addition, the PhaE enzyme is an enzyme that biosynthesizes poly-(R)-3-hydroxybutyric acid.

In one embodiment, phaA in the transformed recombinant microorganism Or phaB Genes can be introduced.

At this time, the phaA Or phaB The gene may be derived from a C. necator strain. The C. necator strain is a Gram-negative aerobic strain living in the soil. In one embodiment of the present invention, the C. necator strain may be C. necator H16, but is not limited thereto.

The term " phaAB " used in the present invention is phaA and phaB genes. The phaA The gene may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 18, and the PhaA enzyme encoded by the nucleotide sequence may be a polypeptide having the amino acid sequence of SEQ ID NO: 17. Also, the phaB The gene may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 20, and the PhaB enzyme encoded by the nucleotide sequence may be a polypeptide having the amino acid sequence of SEQ ID NO: 19.

The PhaA enzyme is an acetyl-CoA acetyl transferase, which can catalyze the condensation of two acetyl-coA units to form acetoacetyl-CoA. In addition, the PhaB enzyme is an acetoacetyl-CoA reductase, which can catalyze the chiral reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA.

The transformed recombinant microorganism of the present invention can produce polyhydroxyalkanoate.

The term "polyhydroxyalkanoate" used in the present invention is a generic term for a hydroxy acid polyester having various carbon side chains. The polyhydroxyalkanoate is an energy storage material synthesized and accumulated in cells when microorganisms are rich in carbon sources but other growth factors (nitrogen, phosphorus, oxygen, sulfur, etc.). When the factor is provided again, the accumulated polyhydroxyalkanoate is decomposed and used as an energy source. It is known that polyhydroxyalkanoate can contain more than 100 kinds of monomers as constituent elements by changes in the type of microorganisms to be produced, the type of chemical substances supplied, and the culture conditions.

The polyhydroxyalkanoate is poly(3-hydroxybutyric acid), poly(3-hydroxyvaleric acid), poly(4-hydroxyvaleric acid), poly(3-hydroxybutyric acid-co-3-hydric acid) Hydroxyvaleric acid), poly(3-hydroxybutyric acid-co-4-hydroxyvaleric acid), poly(3-hydroxyvaleric acid-co-4-hydroxyvaleric acid) and poly(3-hydroxybutyric acid- It may be any one or more selected from the group consisting of co-4-hydroxyvaleric acid-co-4-hydroxyvaleric acid). Preferably, poly(3-hydroxybutyric acid) (poly(3-Hydroxybutyric acid)), poly(3-hydroxyvaleric acid) (poly(3-Hydroxyvaleric acid)) or poly(4-hydroxyvaleric acid) (poly(4-hydroxyvaleric acid)).

The poly(3-hydroxybutyric acid) may have a structure shown in Formula 1 below.

[Formula 1]

The poly(3-hydroxyvaleric acid) may have a structure shown in Formula 2 below.

[Formula 2]

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

[Formula 3]

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

[Formula 4]

The poly(3-hydroxybutyric acid-co-4-hydroxyvaleric acid) may have a structure shown in Formula 5 below.

[Formula 5]

The poly(3-hydroxyvaleric acid-co-4-hydroxyvaleric acid) may have a structure as shown in Formula 6 below.

[Formula 6]

The poly(3-hydroxybutyric acid-co-4-hydroxyvaleric acid-co-4-hydroxyvaleric acid) may have a structure as shown in Formula 7 below.

[Formula 7]

The term "transformation" used in the present invention means introducing a vector containing a polynucleotide encoding a target protein into a host cell so that the target protein can be expressed in the host cell.

As used herein, the term "transformed recombinant microorganism" refers to levulinic acid as 3-hydroxybutyric acid, 3-hydroxyvaleric acid, 4-hydroxyvaleric acid and/or polyhydroxyalkano for the purposes of the present invention. It refers to a microorganism that has the ability to convert it to A.

Microorganism used in the present invention Xi thigh eggplant (Zymomonas), Escherichia (Escherichia), Pseudomonas (Pseudomonas), alkali jeneseu (Alcaligenes), Salmonella (Salmonella), Shigella (Shigella), Burke holde Liao (Burkholderia), oligo Tropa ( Oligotropha ), Klebsiella ( Klebsiella ), Pichia ( Pichia ), Candida ( Candida ), Hansenula ( Hansenula ), Saccharomyces (S accharomyces ) or Kluyveromyces ( Kluyveromyces ) genus I can. Preferably, the transforming microorganism is Escherichia coli ), more preferably Escherichia coli It may be an MG1655 strain.

In another aspect of the present invention, culturing the transformed recombinant microorganism of the present invention; And it provides a method of producing polyhydroxyalkanoate comprising the step of obtaining polyhydroxyalkanoate from the recombinant microorganism. In this case, the cultivation may be performed in the presence of levulinic acid, and the polyhydroxyalkanoate is as described above.

Any medium and other culture conditions used for culturing the microorganisms of the present invention may be used without any particular limitation as long as it is a medium used for culturing ordinary E. coli. Specifically, the microorganism of the present invention can be cultured in a common medium containing an appropriate carbon source, nitrogen source, personnel, inorganic compounds, amino acids and/or vitamins, etc. under aerobic conditions while controlling temperature and pH.

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

Examples of the nitrogen source may include organic nitrogen sources such as peptone, yeast extract, broth, malt extract, corn steep liquor or soybean meal, and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate. These nitrogen sources may be used alone or in combination.

The medium may contain potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or sodium-containing salt as personnel. In addition, it may contain a metal salt such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and suitable precursors may be included.

The pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the culture in an appropriate manner during 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 without the injection of gas to maintain the anaerobic and microaerobic state.

The temperature of the culture may be generally 20° C. to 40° C., specifically 25° C. to 37° C., more specifically 37° C., but may be changed without limitation depending on the intended purpose. The culture period may be continued until the production amount of the desired useful substance is reached, and specifically, may be 10 to 100 hours, but is not limited thereto.

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

Example One. fadE gene, atoC Genes and fadB Production of transformed E. coli with suppressed gene expression

FadE in E. coli MG1655 strain , It was used as the lambda-red recombinase system to remove the gene, atoC gene and fadB gene. Specifically, the gene fadE, fadB and atoC gene to knock out (knock out) a gene, (Flp recombinase target) frt site and with a homologous nucleotide flip wave dehydratase (flippase) can function in a target region A kanamycin resistance gene having a sequence-cassette (Km-cassette) was constructed through PCR. Thereafter, a recombinase was expressed and replaced with an existing gene. The produced mutations were selected on kanamycin plate medium. Flippase was expressed to remove the inserted kanamycin resistance gene, and colonies without kanamycin resistance gene were selected.

Example 2. lvaABC , lvaED , phaC , phaEC And phaAB Gene introduction

After the lvaABC gene of the P. putida strain was cloned by PCR, it was expressed by IPTG under the PLlacO1 promoter (SEQ ID NO: 27). Later, pBbE6K plasmid (Lee, TS, et al. (2011).BglBrick vectors and datasheets: a synthetic biology platform for gene expression. Journal of biological engineering, 5(), which has kanamycin resistance and ColE1 origin of replication. In 1), 12.), the pBbE6k- lvaABC plasmid was cloned using the Gibson assembly technique.

In addition, the lvaED gene of the P. putida strain was cloned by PCR, and it was expressed by IPTG under the PLlacO1 promoter. Thereafter, the pBbB6a- lvaED plasmid was cloned into the pBbB6a plasmid, which has an ampicillin resistance and an origin of BBR1 replication, using the Gibson assembly technique.

In addition, C. violaceum phaC gene of strain, T. phaEC gene of strain pfennigii, C. necator strains of phaAB After each gene was cloned by PCR, it was expressed by tetracycline antibiotic under the tet promoter. Thereafter, pBbA2c- phaC ( C. violaceum ), pBbA2c- phaEC ( T. pfennigii ), pBbA2c- phaC ( C. pha violaceum )- phaC ( C. violaceum )- phaC C. necator ) or pBbA2c- phaEC ( T. pfennigii ) -phaAB ( C. necator ) plasmids were cloned, respectively.

Each of the prepared plasmids was transformed into each of the transformed E. coli prepared according to Example 1 to prepare recombinant E. coli. This is shown in Table 1 below.

strain name genotype PHA1 E. coli MG1655 △ fadE △ atoC , pBbB6a- lvaED (P. putida ) , pBbA2c- phaEC ( T. pfennigii ) PHA2 E. coli MG1655 △ fadE △ atoC , pBbB6a- lvaED (P. putida ) , pBbA2c- phaC ( C. violaceum ) PHA3 E. coli MG1655 △ fadE △ atoC , pBbB6a- lvaED ( P. putida ) , pBbE6k - lvaABC (P. putida ) , pBbA2c- phaEC ( T. pfennigii ) PHA4 E. coli MG1655 △fadE△atoC, pBbB6a-lvaED(P. putida), pBbE6k - lvaABC (P. putida ), pBbA2c-phaC(C. violaceum) PHA5 E. coli MG1655 △ fadE △ atoC , pBbB6a- lvaED (P. putida ) , pBbA2c- phaEC ( T. pfennigii )- phaAB ( C. necator ) PHA6 E. coli MG1655 △ fadE △ atoC , pBbB6a- lvaED (P. putida ) , pBbA2c- phaC ( C. violaceum ) -phaAB ( C. necator ) PHA7 E. coli MG1655 △ fadE △ atoC , pBbB6a- lvaED ( P. putida ) , pBbE6k - lvaABC (P. putida ) , pBbA2c- phaEC ( T. pfennigii ) -phaAB ( C. necator ) PHA8 E. coli MG1655 △ fadE △ atoC , pBbB6a- lvaED ( P. putida ) , pBbE6k - lvaABC (P. putida ) , pBbA2c- phaC ( C. violaceum ) -phaAB ( C. necator ) PHA9 E. coli MG1655 △ fadE △ atoC △ fadB, pBbB6a- lvaED (P. putida), pBbA2c- phaEC (T. pfennigii) PHA10 E. coli MG1655 △ fadE △ atoC △ fadB, pBbB6a- lvaED (P. putida), pBbA2c- phaC (C. violaceum) PHA11 E. coli MG1655 △ fadE △ atoC △ fadB, pBbB6a- lvaED (P. putida), pBbE6k - lvaABC (P. putida), pBbA2c- phaEC (T. pfennigii) PHA12 E. coli MG1655 △ fadE △ atoC △ fadB, pBbB6a- lvaED (P. putida), pBbE6k - lvaABC (P. putida), pBbA2c- phaC (C. violaceum) PHA13 E. coli MG1655 △ fadE △ atoC △ fadB, pBbB6a- lvaED (P. putida), pBbA2c- phaEC (T. pfennigii) - phaAB (C. necator) PHA14 E. coli MG1655 △ fadE △ atoC △ fadB, pBbB6a- lvaED (P. putida), pBbA2c- phaC (C. violaceum) - phaAB (C. necator) PHA15 E. coli MG1655 △ fadE △ atoC △ fadB, pBbB6a- lvaED (P. putida), pBbE6k - lvaABC (P. putida), pBbA2c- phaEC (T. pfennigii) - phaAB (C. necator) PHA16 E. coli MG1655 △ fadE △ atoC △ fadB, pBbB6a- lvaED (P. putida), pBbE6k - lvaABC (P. putida), pBbA2c- phaC (C. violaceum) - phaAB (C. necator)

The phenotype of the transformed E. coli was selected on each antibiotic-resistant medium, and after extracting the plasmid, the gene sequence was analyzed (macrogen, charge).

Example 3. In transformed E. coli 3- Hydroxybutyric acid 3- Hydroxyvaleric acid , 4- Hydroxyvaleric acid Included Of polyhydroxyalkanoate Production and analysis

After inoculation of the transformed recombinant E. coli produced in Example 2 in 25 ml of Terrific broth (TB) containing 40 mM levulinic acid, 0.1 mM IPTG, and 0.4% glycerol, the OD was around 1 to 5 It was grown for 24 hours. After centrifuging all of the culture solution (25 ml) to obtain a pellet, the supernatant was removed and the remaining cells were washed with distilled water. The washed cells were frozen overnight in a -80°C ultra-low temperature refrigerator, and the frozen cells were freeze-dried overnight. Freeze-dried cells were methanolysis (Methanolysis) in an oven at 100° C. for 3 hours and 30 minutes with 1 g/L benzoate (internal standard) in 2 ml of 85% methanol-15% sulfuric acid solution and 2 ml of chloroform.

After cooling the solution at room temperature and adding 2 ml of distilled water, the solution was sufficiently mixed with a vortex. Thereafter, layer separation was performed at room temperature, and a sample of 1 ml of the chloroform layer was taken, and the monomers of the decomposed PHA were analyzed through the Agilent 7000C GC/MS System (Agilent).

The production amount of 3-hydroxybutyric acid 3-hydroxyvaleric acid or 4-hydroxyvaleric acid of each of the transformed recombinant E. coli and components of the resulting PHA are shown in FIGS. 2 and 3.

<110> UNIST(ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY) <120> TRANSFORMED RECOMBINANT MICROORGANISM PRODUCING POLYHYDROXYALKANOATE <130> FPD/201907-0009 <160> 27 <170> KoPatentIn 3.0 <210> 1 <211> 352 <212> PRT <213> Artificial Sequence <220> <223> P. putida LvaA <400> 1 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> 2 <211> 1059 <212> DNA <213> Artificial Sequence <220> <223> P. putida lvaA <400> 2 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 ttacccgtcc 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> 3 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> P. putida LvaB <400> 3 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> 4 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> P. putida lvaB <400> 4 atgacccaac ccaacgccca cgaattgctc gagatcgccc gcgcgacgct tctggagcag 60 ctgctgccag cgctgcccgg cgagttgcgt tacccggccc tgatgatcgc caacgccatg 120 gccattgcgg cccgcgaaaa ccgcttgggc gctcaggccg aggatcagga gcaggcgcgt 180 ctggccgcct tggtcgatga cgcgccgtcg acattgcccg acctgcgccg ccaactggct 240 cgcgccattc gccagggcag ccatgacgcc ccgcaaaccc ggcgcaccct ggtcgagaca 300 ttacgccaga tcaccgttgc ccgattggcg atcagcaacc ccaaggcctt gccctga 357 <210> 5 <211> 390 <212> PRT <213> Artificial Sequence <220> <223> P. putida LvaC <400> 5 Met Asn Phe Thr Leu Pro Asp Glu Leu Leu Ala Leu Gln Ala Lys Thr 1 5 10 15 Arg Asp Phe Ile Ala Glu Gln Val Ile Pro Phe Glu Asn Asp Pro Arg 20 25 30 Gln Asn Ser His Gly Pro Ser Asp Ala Leu Arg Gln Asp Leu Val Leu 35 40 45 Cys Ala Arg Ala Ala Gly Leu Leu Thr Pro His Ala Ser Arg Glu Met 50 55 60 Gly Gly Leu Glu Leu Ser His Val Ala Lys Ala Ile Val Phe Glu Glu 65 70 75 80 Ala Gly Tyr Ser Pro Leu Gly Pro Val Ala Leu Asn Ile His Ala Pro 85 90 95 Asp Glu Gly Asn Ile His Leu Met Asp Val Val Ala Thr Glu Ala Gln 100 105 110 Lys Asp Arg Trp Leu Arg Pro Leu Val Gln Gly His Ala Arg Ser Cys 115 120 125 Phe Ala Met Thr Glu Pro Ala Pro Gly Ser Gly Ser Asp Pro Ser Met 130 135 140 Leu Arg Thr Thr Ala Thr Arg Asp Gly Asp Asp Tyr Leu Ile Asn Gly 145 150 155 160 Arg Lys Trp Leu Ile Thr Gly Ala Glu Gly Ala Asp Phe Gly Ile Ile 165 170 175 Met Ala Arg Met Glu Asp Gly Thr Ala Thr Met Phe Leu Thr Asp Met 180 185 190 Lys Arg Asp Gly Ile Ile His Glu Arg Gln Leu Asp Ser Leu Asp Ser 195 200 205 Cys Phe Thr Gly Gly His Gly Gln Leu Arg Phe Asp Asn Leu Arg Ile 210 215 220 Pro Ala Ser Asp Val Leu Gly Glu Ile Gly Lys Gly Phe Arg Tyr Ala 225 230 235 240 Gln Val Arg Leu Ala Pro Ala Arg Leu Thr His Cys Met Arg Trp Leu 245 250 255 Gly Ala Ala Arg Arg Ala His Asp Ile Ala Cys Asp Tyr Ala Arg Thr 260 265 270 Arg Asp Ala Phe Gly Lys Pro Leu Gly Glu His Gln Gly Val Gly Phe 275 280 285 Met Leu Ala Asp Asn Met Met Asp Leu His Val Val Arg Leu Ala Val 290 295 300 Trp His Cys Ala Trp Val Leu Asp Gln Gly Arg Arg Ala Asn Val Asp 305 310 315 320 Ser Ser Met Ala Lys Val Ile Ser Ala Glu Ala Leu Trp Arg Val Val 325 330 335 Asp Arg Cys Val Gln Val Leu Gly Gly Arg Gly Val Thr Gly Asp Thr 340 345 350 Val Val Glu Arg Ile Phe Arg Asp Ile Arg Pro Phe Arg Ile Tyr Asp 355 360 365 Gly Pro Ser Glu Val His Arg Met Ser Leu Ala Lys Lys Leu Leu Asp 370 375 380 Gln Arg Leu Glu Ala His 385 390 <210> 6 <211> 1173 <212> DNA <213> Artificial Sequence <220> <223> P. putida lvaC <400> 6 atgaacttca ctctcccgga cgaactgctc gccttgcagg ccaagactcg agacttcatt 60 gccgaacagg tcatcccatt cgagaacgac ccccgccaga acagccacgg ccccagcgac 120 gcactgcgcc aggacctggt gctctgcgcc cgcgccgctg gcttgctgac gcctcacgcc 180 agccgcgaaa tgggcggtct ggaactgagc catgtggcca aggcgatcgt cttcgaagaa 240 gccggctact cgccgctggg cccggtagcg ctgaatatcc atgcgccgga cgaaggcaat 300 atccacctga tggacgtggt cgccaccgaa gcgcagaagg accgctggtt gcgcccgctg 360 gtccagggcc atgcccgttc gtgcttcgcc atgacggagc ctgctccggg ctccggttcg 420 gatccgtcga tgctgcgcac cactgccacc cgcgatggcg acgactacct gatcaatggt 480 cgcaagtggc tgatcaccgg ggccgaaggc gcggacttcg gcatcatcat ggcgcgcatg 540 gaggacggca ccgcgaccat gttcctgacc gacatgaagc gcgacggcat catccatgaa 600 cgtcagctgg actcgctgga cagctgtttt accggcggtc acgggcagct gcgtttcgac 660 aacctgcgta ttccggcgag cgatgtcctc ggcgagatcg gcaagggctt ccggtatgcc 720 caggtgcgcc tggcgcctgc acgcttgact cattgcatgc gctggctcgg tgccgcgcgc 780 cgcgcccacg acatcgcctg cgactatgcg cgcacccggg acgcctttgg caagccgctg 840 ggcgagcacc agggcgtggg tttcatgctg gccgacaaca tgatggacct gcacgtggtg 900 cgtctggcgg tctggcactg cgcctgggtg ctcgaccagg gccggcgcgc caatgtcgat 960 tcgagcatgg ccaaggtgat cagcgccgag gcgctgtggc gggtggtcga tcgttgcgtc 1020 caggtattgg gtggacgcgg ggtgaccggg gacaccgtgg tggagcggat cttccgcgac 1080 attcgcccgt tccgcatcta tgacggcccg agcgaagtgc accgcatgag cctggcgaag 1140 aagctgctcg accagcgcct ggaggcccac tga 1173 <210> 7 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> P. putida LvaD <400> 7 Met Gln Pro Asn Leu Ala Arg Leu Phe Ala Leu Asp Gly Arg Arg Ala 1 5 10 15 Leu Val Thr Gly Ala Ser Ser Gly Leu Gly Arg His Phe Ala Met Thr 20 25 30 Leu Ala Ala Ala Gly Ala Glu Val Val Val Val Thr Ala Arg Arg Gln Ala 35 40 45 Pro Leu Gln Ala Leu Val Glu Ala Ile Glu Val Ala Gly Gly Arg Ala 50 55 60 Gln Ala Phe Ala Leu Asp Val Thr Ser Arg Glu Asp Ile Cys Arg Val 65 70 75 80 Leu Asp Ala Ala Gly Pro Leu Asp Val Leu Val Asn Asn Ala Gly Val 85 90 95 Ser Asp Ser Gln Pro Leu Leu Ala Cys Asp Asp Gln Thr Trp Asp His 100 105 110 Val Leu Asp Thr Asn Leu Lys Gly Ala Trp Ala Val Ala Gln Glu Ser 115 120 125 Ala Arg Arg Met Val Val Ala Gly Lys Gly Gly Ser Leu Ile Asn Val 130 135 140 Thr Ser Ile Leu Ala Ser Arg Val Ala Gly Ala Val Gly Pro Tyr Leu 145 150 155 160 Ala Ala Lys Ala Gly Leu Ala His Leu Thr Arg Ala Met Ala Leu Glu 165 170 175 Leu Ala Arg His Gly Ile Arg Val Asn Ala Leu Ala Pro Gly Tyr Val 180 185 190 Met Thr Asp Leu Asn Glu Ala Phe Leu Ala Ser Glu Ala Gly Asp Lys 195 200 205 Leu Arg Ser Arg Ile Pro Ser Arg Arg Phe Ser Val Pro Ser Asp Leu 210 215 220 Asp Gly Ala Leu Leu Leu Leu Ala Ser Asp Ala Gly Arg Ala Met Ser 225 230 235 240 Gly Ala Glu Ile Val Val Asp Gly Gly His Leu Cys Ser Ser Leu 245 250 255 <210> 8 <211> 768 <212> DNA <213> Artificial Sequence <220> <223> P. putida lvaD <400> 8 atgcagccga accttgcccg actgttcgcc ctcgacgggc gtcgcgccct ggtgaccggg 60 gcctccagcg gcctgggccg tcacttcgcc atgaccctgg ccgccgcagg cgccgaggtg 120 gtggtgaccg ccagacgcca ggcgccgctg caggcgttgg tggaggccat cgaggtggcc 180 ggagggcggg cgcaggcctt tgccctcgat gtgacgagcc gtgaggacat ctgccgggtg 240 ctcgatgccg ccggcccgct ggatgttctg gtcaacaatg cgggggtgag cgacagccag 300 cctttgctag cctgcgatga tcaaacctgg gaccacgtgc tcgacaccaa cctcaagggc 360 gcctgggccg tggcccagga aagcgcccgg cgcatggtgg tggcggggaa ggggggcagc 420 ctgatcaatg tcacctcgat cctcgccagc cgtgtggccg gcgccgtcgg cccttacctg 480 gcggccaagg ccggcctggc ccacctgacc cgcgccatgg cgctggagtt ggcgcgccat 540 ggtatccggg tgaacgccct ggcgcccggc tacgtgatga ctgatttgaa cgaggccttc 600 ctggccagcg aggccggtga caagttgcgc tcgcggatcc ccagccgccg cttcagcgtg 660 ccgtcggacc tggacggcgc cttgctgctg ctcgccagcg atgccgggcg ggcgatgagc 720 ggcgctgaga tcgtggtcga tggcggccac ctgtgcagca gcctgtaa 768 <210> 9 <211> 548 <212> PRT <213> Artificial Sequence <220> <223> P. putida LvaE <400> 9 Met Met Val Pro Thr Leu Glu His Glu Leu Ala Pro Asn Glu Ala Asn 1 5 10 15 His Val Pro Leu Ser Pro Leu Ser Phe Leu Lys Arg Ala Ala Gln Val 20 25 30 Tyr Pro Gln Arg Asp Ala Val Ile Tyr Gly Ala Arg Arg Tyr Ser Tyr 35 40 45 Arg Gln Leu His Glu Arg Ser Arg Ala Leu Ala Ser Ala Leu Glu Arg 50 55 60 Val Gly Val Gln Pro Gly Glu Arg Val Ala Ile Leu Ala Pro Asn Ile 65 70 75 80 Pro Glu Met Leu Glu Ala His Tyr Gly Val Pro Gly Ala Gly Ala Val 85 90 95 Leu Val Cys Ile Asn Ile Arg Leu Glu Gly Arg Ser Ile Ala Phe Ile 100 105 110 Leu Arg His Cys Ala Ala Lys Val Leu Ile Cys Asp Arg Glu Phe Gly 115 120 125 Ala Val Ala Asn Gln Ala Leu Ala Met Leu Asp Ala Pro Pro Leu Leu 130 135 140 Val Gly Ile Asp Asp Asp Gln Ala Glu Arg Ala Asp Leu Ala His Asp 145 150 155 160 Leu Asp Tyr Glu Ala Phe Leu Ala Gln Gly Asp Pro Ala Arg Pro Leu 165 170 175 Ser Ala Pro Gln Asn Glu Trp Gln Ser Ile Ala Ile Asn Tyr Thr Ser 180 185 190 Gly Thr Thr Gly Asp Pro Lys Gly Val Val Leu His His Arg Gly Ala 195 200 205 Tyr Leu Asn Ala Cys Ala Gly Ala Leu Ile Phe Gln Leu Gly Pro Arg 210 215 220 Ser Val Tyr Leu Trp Thr Leu Pro Met Phe His Cys Asn Gly Trp Ser 225 230 235 240 His Thr Trp Ala Val Thr Leu Ser Gly Gly Thr His Val Cys Leu Arg 245 250 255 Lys Val Gln Pro Asp Ala Ile Asn Ala Ala Ile Ala Glu His Ala Val 260 265 270 Thr His Leu Ser Ala Ala Pro Val Val Met Ser Met Leu Ile His Ala 275 280 285 Glu His Ala Ser Ala Pro Pro Val Pro Val Ser Val Ile Thr Gly Gly 290 295 300 Ala Ala Pro Pro Ser Ala Val Ile Ala Ala Met Glu Ala Arg Gly Phe 305 310 315 320 Asn Ile Thr His Ala Tyr Gly Met Thr Glu Ser Tyr Gly Pro Ser Thr 325 330 335 Leu Cys Leu Trp Gln Pro Gly Val Asp Glu Leu Pro Leu Glu Ala Arg 340 345 350 Ala Gln Phe Met Ser Arg Gln Gly Val Ala His Pro Leu Leu Glu Glu 355 360 365 Ala Thr Val Leu Asp Thr Asp Thr Gly Arg Pro Val Pro Ala Asp Gly 370 375 380 Leu Thr Leu Gly Glu Leu Val Val Arg Gly Asn Thr Val Met Lys Gly 385 390 395 400 Tyr Leu His Asn Pro Glu Ala Thr Arg Ala Ala Leu Ala Asn Gly Trp 405 410 415 Leu His Thr Gly Asp Leu Ala Val Leu His Leu Asp Gly Tyr Val Glu 420 425 430 Ile Lys Asp Arg Ala Lys Asp Ile Ile Ile Ser Gly Gly Glu Asn Ile 435 440 445 Ser Ser Leu Glu Ile Glu Glu Val Leu Tyr Gln His Pro Glu Val Val 450 455 460 Glu Ala Ala Val Val Ala Arg Pro Asp Ser Arg Trp Gly Glu Thr Pro 465 470 475 480 His Ala Phe Val Thr Leu Arg Ala Asp Ala Leu Ala Ser Gly Asp Asp 485 490 495 Leu Val Arg Trp Cys Arg Glu Arg Leu Ala His Phe Lys Ala Pro Arg 500 505 510 His Val Ser Leu Val Asp Leu Pro Lys Thr Ala Thr Gly Lys Ile Gln 515 520 525 Lys Phe Val Leu Arg Glu Trp Ala Arg Gln Gln Glu Ala Gln Ile Ala 530 535 540 Asp Ala Glu His 545 <210> 10 <211> 1647 <212> DNA <213> Artificial Sequence <220> <223> P. putida lvaE <400> 10 atgatggttc caaccctcga acacgagctt gctcccaacg aagccaacca tgtcccgctg 60 tcgccgctgt cgttcctcaa gcgtgccgcg caggtgtacc cgcagcgcga tgcggtgatc 120 tatggcgcaa ggcgctacag ctaccgtcag ttgcacgagc gcagccgcgc cctggccagt 180 gccttggagc gggtcggtgt tcagccgggc gagcgggtgg cgatattggc gccgaacatc 240 ccggaaatgc tcgaggccca ctatggcgtg cccggtgccg gggcggtgct ggtgtgcatc 300 aacatccgcc tggaggggcg cagcattgcc ttcatcctgc gtcactgcgc ggccaaggta 360 ttgatctgcg atcgtgagtt cggtgccgtg gccaatcagg cgctggccat gctcgatgcg 420 ccgcccttgc tggtgggcat cgacgatgat caggccgagc gcgccgattt ggcccacgac 480 ctggactacg aagcgttctt ggcccagggc gaccccgcgc ggccgttgag tgcgccacag 540 aacgaatggc agtcgatcgc catcaactac acctccggca ccacggggga ccccaagggc 600 gtggtgctgc atcaccgcgg cgcctacctc aacgcctgcg ccggggcgct gatcttccag 660 ttggggccgc gcagcgtcta cttgtggacc ttgccgatgt tccactgcaa cggctggagc 720 catacctggg cggtgacgtt gtccggtggc acccacgtgt gtctgcgcaa ggtccagcct 780 gatgcgatca acgccgccat cgccgagcat gccgtgactc acctgagcgc cgccccagtg 840 gtgatgtcga tgctgatcca cgccgagcat gccagcgccc ctccggtgcc ggtttcggtg 900 atcactggcg gtgccgcccc gcccagtgcg gtcatcgcgg cgatggaggc gcgtggcttc 960 aacatcaccc atgcctatgg catgaccgaa agctacggtc ccagcacatt gtgcctgtgg 1020 cagccgggtg tcgacgagtt gccgctggag gcccgggccc agttcatgag ccgccagggc 1080 gtcgcccacc cgctgctcga ggaggccacg gtgctggata ccgacaccgg ccgcccggtc 1140 ccggccgacg gccttaccct cggcgagctg gtggtgcggg gcaacactgt gatgaaaggc 1200 tacctgcaca acccagaggc tacccgtgcc gcgttggcca acggctggct gcacacgggc 1260 gacctggccg tgctgcacct ggacggctat gtggaaatca aggaccgagc caaggacatc 1320 atcatttctg gcggcgagaa catcagttcg ctggagatag aagaagtgct ctaccagcac 1380 cccgaggtgg tcgaggctgc ggtggtggcg cgtccggatt cgcgctgggg cgagacacct 1440 cacgctttcg tcacgctgcg cgctgatgca ctggccagcg gggacgacct ggtccgctgg 1500 tgccgtgagc gtctggcgca cttcaaggcg ccgcgccatg tgtcgctcgt ggacctgccc 1560 aagaccgcca ctggaaaaat acagaagttc gtcctgcgtg agtgggcccg gcaacaggag 1620 gcgcagatcg ccgacgccga gcattga 1647 <210> 11 <211> 591 <212> PRT <213> Artificial Sequence <220> <223> C. violaceum PhaC <400> 11 Met Ser Gln Asn Ser Thr Ala Ala Leu Glu Gly Phe Phe Ala Ser Leu 1 5 10 15 Ser Glu Ala Asn Gln Gln Trp Met Gln Gln Phe Val Asn Ser Leu Ser 20 25 30 Gln Pro Pro Ala Pro Asp Gly Ala Ala His Pro Phe Ala Gly Ala Trp 35 40 45 Ala Gln Leu Met Asn Gln Thr Asn Gln Leu Phe Ala Leu Gln Ser Ser 50 55 60 Leu Tyr Gln Gln Gln Leu Asn Leu Trp Ser Gln Phe Leu Gly Gln Ala 65 70 75 80 Ala Gly Gln Glu Ala Ala Ala Glu Ala Gly Ala Lys Pro Ala Asp Arg 85 90 95 Arg Phe Ala Ser Pro Glu Trp Asn Glu His Pro Phe Tyr Asn Phe Leu 100 105 110 Lys Gln Ser Tyr Leu Gln Thr Ser Lys Trp Met Met Glu Leu Val Asp 115 120 125 Lys Thr Gln Leu Asp Glu Asp Ala Lys Asp Lys Leu Ala Phe Ala Thr 130 135 140 Arg Gln Tyr Leu Asp Ala Met Ser Pro Ser Asn Phe Met Leu Thr Asn 145 150 155 160 Pro Asp Val Val Lys Arg Ala Ile Glu Thr Lys Gly Glu Ser Leu Val 165 170 175 Glu Gly Met Lys Asn Met Leu Asp Asp Phe Gln Lys Gly His Ile Ser 180 185 190 Met Ser Asp Glu Ser Lys Phe Glu Ile Gly Lys Asn Leu Val Val Thr 195 200 205 Pro Gly Gln Val Val Phe Arg Asn Glu Leu Ile Glu Leu Ile Gln Tyr 210 215 220 Thr Pro Thr Thr Asp Lys Val Tyr Glu Lys Pro Leu Leu Phe Val Pro 225 230 235 240 Pro Cys Ile Asn Lys Tyr Tyr Leu Met Asp Leu Gln Pro Asp Asn Ser 245 250 255 Met Val Arg His Phe Val Ala Gln Gly Tyr Arg Val Phe Leu Ile Ser 260 265 270 Trp Arg Ser Ala Val Ala Glu Met Lys His Phe Thr Trp Glu Thr Tyr 275 280 285 Ile Glu Lys Gly Val Phe Ala Ala Ala Glu Ala Val Gln Lys Ile Thr 290 295 300 Lys Gln Pro Thr Met Asn Val Leu Gly Phe Cys Val Gly Gly Val Ile 305 310 315 320 Leu Thr Thr Ala Leu Cys Val Ala Gln Ala Lys Gly Leu Lys Tyr Phe 325 330 335 Asp Ser Ala Thr Phe Met Thr Ser Leu Ile Asp His Ala Glu Pro Gly 340 345 350 Glu Ile Ser Phe Phe Ile Asp Glu Ser Val Val Ala Gly Arg Glu Ala 355 360 365 Lys Met Ala Ser Gly Gly Ile Ile Ser Gly Lys Glu Ile Gly Arg Thr 370 375 380 Phe Ala Ser Leu Arg Ala Asn Asp Leu Val Trp Asn Tyr Val Val Asn 385 390 395 400 Asn Tyr Leu Leu Gly Lys Thr Pro Ala Pro Phe Asp Leu Leu Phe Trp 405 410 415 Asn Asn Asp Ala Val Asp Leu Pro Leu Pro Met His Thr Phe Leu Leu 420 425 430 Arg Gln Phe Tyr Met Asn Asn Ala Leu Val Arg Pro Gly Ala Ile Thr 435 440 445 Leu Cys Gly Val Pro Ile Asp Ile Ala Lys Ile Asp Val Pro Val Tyr 450 455 460 Met Phe Ala Ala Arg Asp Asp His Ile Val Leu Trp Ser Ser Ala Phe 465 470 475 480 Ser Gly Leu Lys Tyr Leu Gln Gly Ala Pro Ser Arg Arg Phe Val Leu 485 490 495 Gly Ala Ser Gly His Ile Ala Gly Ser Ile Asn Pro Val Thr Lys Asp 500 505 510 Lys Arg Asn Tyr Trp Ala Asn Asp Thr Leu Pro Leu His Ala Glu Glu 515 520 525 Trp Leu Glu Ser Ala Glu Ser Arg Pro Gly Ser Trp Trp Lys Asp Trp 530 535 540 Asp Ala Trp Leu Ala Pro Gln Ser Gly Lys Gln Val Ala Ala Pro Lys 545 550 555 560 Ser Leu Gly Asn Lys Glu Phe Pro Pro Leu Leu Ala Ala Pro Gly Ser 565 570 575 Tyr Val Leu Ala Lys Ala Met Pro Ser Val Ala Ala Ser Leu Gln 580 585 590 <210> 12 <211> 1776 <212> DNA <213> Artificial Sequence <220> <223> C. violaceum phaC <400> 12 gtgtcccaga atagtaccgc cgcgctggaa ggtttcttcg ccagcctctc cgaggccaac 60 cagcaatgga tgcagcagtt cgtcaactcg ctgtcgcagc ctccagcccc cgacggcgca 120 gcccatccct tcgccggcgc ctgggcgcag ctgatgaacc agaccaacca gctgttcgct 180 ctccagtcct cgctttacca gcagcagctc aatctgtggt cgcaattcct cggccaggcc 240 gccggccagg aagccgccgc cgaagcgggc gccaagccgg ccgaccgccg cttcgcgtcg 300 ccggaatgga acgagcaccc gttctacaac ttcctcaagc agagctatct gcagacctcc 360 aagtggatga tggagctggt ggacaagacc cagctcgacg aggacgccaa ggacaagctg 420 gctttcgcca cccgccagta cctggacgcg atgtccccca gcaacttcat gctgaccaac 480 cccgacgtgg tcaagcgcgc gatcgagacc aagggcgaaa gcctggtcga gggcatgaag 540 aacatgctgg acgacttcca gaagggccac atctcgatgt cggacgagag caagttcgag 600 atcggcaaga acctggtggt caccccgggc caggtggtgt tccgcaacga actgatcgag 660 ctgatccagt acacgccgac caccgacaag gtctacgaga agccgctgct gttcgtgccg 720 ccctgcatca acaagtacta cctgatggac ctgcagccgg acaactccat ggtgcgccac 780 ttcgtcgccc agggttaccg cgtgttcctg atcagctggc gctccgcggt cgccgagatg 840 aagcacttca cctgggaaac ctatatcgag aaaggcgtgt tcgccgcggc ggaagcggtg 900 cagaaaatca ccaagcagcc gacgatgaac gtgctgggct tctgcgtcgg cggcgtcatc 960 ctcaccaccg cgctgtgcgt ggctcaggcc aaggggctga aatacttcga ctccgccacc 1020 ttcatgacct cgctgatcga ccacgccgaa ccgggcgaga tctccttctt catcgacgag 1080 agcgtggtgg ccggccgcga agccaagatg gccagcggcg gcatcatcag cggcaaggaa 1140 atcggccgca ccttcgccag cctgcgcgcc aacgacctgg tgtggaacta cgtggtcaac 1200 aactacctgc tgggcaagac cccggcgccg ttcgacctgc tgttctggaa caacgacgcg 1260 gtggatctgc cgctgccgat gcacaccttc ctgctgcgcc agttctacat gaacaacgcg 1320 ctggtgcgtc cgggcgcgat cacgctgtgc ggcgtgccga tcgacatcgc caagatcgac 1380 gtgccggtct acatgttcgc cgcgcgcgac gaccacatcg tgctgtggag ctccgccttc 1440 tccggcctga aatacctgca gggcgcgccc agccgccgct tcgtgctggg cgcgtccggc 1500 cacatcgccg gctcgatcaa cccggtcacc aaggacaagc gcaactactg ggccaacgac 1560 acgctgccgc tgcacgccga ggaatggctg gaaagcgcgg aaagccgccc cggcagctgg 1620 tggaaggact gggacgcctg gctggcgccg cagtccggca agcaagtggc cgcgcccaaa 1680 agcctgggca acaaggaatt cccgccgctg ctggcggcgc cgggcagtta tgtgctggcc 1740 aaggccatgc cgtccgtcgc cgccagcctg caatga 1776 <210> 13 <211> 367 <212> PRT <213> Artificial Sequence <220> <223> T. pfennigii phaE <400> 13 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 Gln 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> 14 <211> 1104 <212> DNA <213> Artificial Sequence <220> <223> T. pfennigii phaE <400> 14 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> 15 <211> 357 <212> PRT <213> Artificial Sequence <220> <223> T. pfennigii PhaC <400> 15 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> 16 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> T. pfennigii phaC <400> 16 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 acatcccggg 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 ctaa 1074 <210> 17 <211> 393 <212> PRT <213> Artificial Sequence <220> <223> C. necator PhaA <400> 17 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> 18 <211> 1182 <212> DNA <213> Artificial Sequence <220> <223> C. necator phaA <400> 18 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> 19 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> C. necator PhaB <400> 19 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> 20 <211> 741 <212> DNA <213> Artificial Sequence <220> <223> C. necator phaB <400> 20 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> 21 <211> 814 <212> PRT <213> Artificial Sequence <220> <223> E. coli MG1655 FadE <400> 21 Met Met Ile Leu Ser Ile Leu Ala Thr Val Val Leu Leu Gly Ala Leu 1 5 10 15 Phe Tyr His Arg Val Ser Leu Phe Ile Ser Ser Leu Ile Leu Leu Ala 20 25 30 Trp Thr Ala Ala Leu Gly Val Ala Gly Leu Trp Ser Ala Trp Val Leu 35 40 45 Val Pro Leu Ala Ile Ile Leu Val Pro Phe Asn Phe Ala Pro Met Arg 50 55 60 Lys Ser Met Ile Ser Ala Pro Val Phe Arg Gly Phe Arg Lys Val Met 65 70 75 80 Pro Pro Met Ser Arg Thr Glu Lys Glu Ala Ile Asp Ala Gly Thr Thr 85 90 95 Trp Trp Glu Gly Asp Leu Phe Gln Gly Lys Pro Asp Trp Lys Lys Leu 100 105 110 His Asn Tyr Pro Gln Pro Arg Leu Thr Ala Glu Glu Gln Ala Phe Leu 115 120 125 Asp Gly Pro Val Glu Glu Ala Cys Arg Met Ala Asn Asp Phe Gln Ile 130 135 140 Thr His Glu Leu Ala Asp Leu Pro Pro Glu Leu Trp Ala Tyr Leu Lys 145 150 155 160 Glu His Arg Phe Phe Ala Met Ile Ile Lys Lys Glu Tyr Gly Gly Leu 165 170 175 Glu Phe Ser Ala Tyr Ala Gln Ser Arg Val Leu Gln Lys Leu Ser Gly 180 185 190 Val Ser Gly Ile Leu Ala Ile Thr Val Gly Val Pro Asn Ser Leu Gly 195 200 205 Pro Gly Glu Leu Leu Gln His Tyr Gly Thr Asp Glu Gln Lys Asp His 210 215 220 Tyr Leu Pro Arg Leu Ala Arg Gly Gln Glu Ile Pro Cys Phe Ala Leu 225 230 235 240 Thr Ser Pro Glu Ala Gly Ser Asp Ala Gly Ala Ile Pro Asp Thr Gly 245 250 255 Ile Val Cys Met Gly Glu Trp Gln Gly Gln Gln Val Leu Gly Met Arg 260 265 270 Leu Thr Trp Asn Lys Arg Tyr Ile Thr Leu Ala Pro Ile Ala Thr Val 275 280 285 Leu Gly Leu Ala Phe Lys Leu Ser Asp Pro Glu Lys Leu Leu Gly Gly 290 295 300 Ala Glu Asp Leu Gly Ile Thr Cys Ala Leu Ile Pro Thr Thr Thr Pro 305 310 315 320 Gly Val Glu Ile Gly Arg Arg His Phe Pro Leu Asn Val Pro Phe Gln 325 330 335 Asn Gly Pro Thr Arg Gly Lys Asp Val Phe Val Pro Ile Asp Tyr Ile 340 345 350 Ile Gly Gly Pro Lys Met Ala Gly Gln Gly Trp Arg Met Leu Val Glu 355 360 365 Cys Leu Ser Val Gly Arg Gly Ile Thr Leu Pro Ser Asn Ser Thr Gly 370 375 380 Gly Val Lys Ser Val Ala Leu Ala Thr Gly Ala Tyr Ala His Ile Arg 385 390 395 400 Arg Gln Phe Lys Ile Ser Ile Gly Lys Met Glu Gly Ile Glu Glu Pro 405 410 415 Leu Ala Arg Ile Ala Gly Asn Ala Tyr Val Met Asp Ala Ala Ala Ser 420 425 430 Leu Ile Thr Tyr Gly Ile Met Leu Gly Glu Lys Pro Ala Val Leu Ser 435 440 445 Ala Ile Val Lys Tyr His Cys Thr His Arg Gly Gln Gln Ser Ile Ile 450 455 460 Asp Ala Met Asp Ile Thr Gly Gly Lys Gly Ile Met Leu Gly Gln Ser 465 470 475 480 Asn Phe Leu Ala Arg Ala Tyr Gln Gly Ala Pro Ile Ala Ile Thr Val 485 490 495 Glu Gly Ala Asn Ile Leu Thr Arg Ser Met Met Ile Phe Gly Gln Gly 500 505 510 Ala Ile Arg Cys His Pro Tyr Val Leu Glu Glu Met Glu Ala Ala Lys 515 520 525 Asn Asn Asp Val Asn Ala Phe Asp Lys Leu Leu Phe Lys His Ile Gly 530 535 540 His Val Gly Ser Asn Lys Val Arg Ser Phe Trp Leu Gly Leu Thr Arg 545 550 555 560 Gly Leu Thr Ser Ser Thr Pro Thr Gly Asp Ala Thr Lys Arg Tyr Tyr 565 570 575 Gln His Leu Asn Arg Leu Ser Ala Asn Leu Ala Leu Leu Ser Asp Val 580 585 590 Ser Met Ala Val Leu Gly Gly Ser Leu Lys Arg Arg Glu Arg Ile Ser 595 600 605 Ala Arg Leu Gly Asp Ile Leu Ser Gln Leu Tyr Leu Ala Ser Ala Val 610 615 620 Leu Lys Arg Tyr Asp Asp Glu Gly Arg Asn Glu Ala Asp Leu Pro Leu 625 630 635 640 Val His Trp Gly Val Gln Asp Ala Leu Tyr Gln Ala Glu Gln Ala Met 645 650 655 Asp Asp Leu Leu Gln Asn Phe Pro Asn Arg Val Val Ala Gly Leu Leu 660 665 670 Asn Val Val Ile Phe Pro Thr Gly Arg His Tyr Leu Ala Pro Ser Asp 675 680 685 Lys Leu Asp His Lys Val Ala Lys Ile Leu Gln Val Pro Asn Ala Thr 690 695 700 Arg Ser Arg Ile Gly Arg Gly Gln Tyr Leu Thr Pro Ser Glu His Asn 705 710 715 720 Pro Val Gly Leu Leu Glu Glu Ala Leu Val Asp Val Ile Ala Ala Asp 725 730 735 Pro Ile His Gln Arg Ile Cys Lys Glu Leu Gly Lys Asn Leu Pro Phe 740 745 750 Thr Arg Leu Asp Glu Leu Ala His Asn Ala Leu Val Lys Gly Leu Ile 755 760 765 Asp Lys Asp Glu Ala Ala Ile Leu Val Lys Ala Glu Glu Ser Arg Leu 770 775 780 Arg Ser Ile Asn Val Asp Asp Phe Asp Pro Glu Glu Leu Ala Thr Lys 785 790 795 800 Pro Val Lys Leu Pro Glu Lys Val Arg Lys Val Glu Ala Ala 805 810 <210> 22 <211> 2445 <212> DNA <213> Artificial Sequence <220> <223> E. coli MG1655 fadE <400> 22 atgatgattt tgagtattct cgctacggtt gtcctgctcg gcgcgttgtt ctatcaccgc 60 gtgagcttat ttatcagcag tctgattttg ctcgcctgga cagccgccct cggcgttgct 120 ggtctgtggt cggcgtgggt actggtgcct ctggccatta tcctcgtgcc atttaacttt 180 gcgcctatgc gtaagtcgat gatttccgcg ccggtatttc gcggtttccg taaggtgatg 240 ccgccgatgt cgcgcactga gaaagaagcg attgatgcgg gcaccacctg gtgggagggc 300 gacttgttcc agggcaagcc ggactggaaa aagctgcata actatccgca gccgcgcctg 360 accgccgaag agcaagcgtt tctcgacggc ccggtagaag aagcctgccg gatggcgaat 420 gatttccaga tcacccatga gctggcggat ctgccgccgg agttgtgggc gtaccttaaa 480 gagcatcgtt tcttcgcgat gatcatcaaa aaagagtacg gcgggctgga gttctcggct 540 tatgcccagt ctcgcgtgct gcaaaaactc tccggcgtga gcgggatcct ggcgattacc 600 gtcggcgtgc caaactcatt aggcccgggc gaactgttgc aacattacgg cactgacgag 660 cagaaagatc actatctgcc gcgtctggcg cgtggtcagg agatcccctg ctttgcactg 720 accagcccgg aagcgggttc cgatgcgggc gcgattccgg acaccgggat tgtctgcatg 780 ggcgaatggc agggccagca ggtgctgggg atgcgtctga cctggaacaa acgctacatt 840 acgctggcac cgattgcgac cgtgcttggg ctggcgttta aactctccga cccggaaaaa 900 ttactcggcg gtgcagaaga tttaggcatt acctgtgcgc tgatcccaac caccacgccg 960 ggcgtggaaa ttggtcgtcg ccacttcccg ctgaacgtac cgttccagaa cggaccgacg 1020 cgcggtaaag atgtcttcgt gccgatcgat tacatcatcg gcgggccgaa aatggccggg 1080 caaggctggc ggatgctggt ggagtgcctc tcggtaggcc gcggcatcac cctgccttcc 1140 aactcaaccg gcggcgtgaa atcggtagcg ctggcaaccg gcgcgtatgc tcacattcgc 1200 cgtcagttca aaatctctat tggtaagatg gaagggattg aagagccgct ggcgcgtatt 1260 gccggtaatg cctacgtgat ggatgctgcg gcatcgctga ttacctacgg cattatgctc 1320 ggcgaaaaac ctgccgtgct gtcggctatc gttaagtatc actgtaccca ccgcgggcag 1380 cagtcgatta ttgatgcgat ggatattacc ggcggtaaag gcattatgct cgggcaaagc 1440 aacttcctgg cgcgtgctta ccagggcgca ccgattgcca tcaccgttga aggggctaac 1500 attctgaccc gcagcatgat gatcttcgga caaggagcga ttcgttgcca tccgtacgtg 1560 ctggaagaga tggaagcggc gaagaacaat gacgtcaacg cgttcgataa actgttgttc 1620 aaacatatcg gtcacgtcgg tagcaacaaa gttcgcagct tctggctggg cctgacgcgc 1680 ggtttaacca gcagcacgcc aaccggcgat gccactaaac gctactatca gcacctgaac 1740 cgcctgagcg ccaacctcgc cctgctttct gatgtctcga tggcagtgct gggcggcagc 1800 ctgaaacgtc gcgagcgcat ctcggcccgt ctgggggata ttttaagcca gctctacctc 1860 gcctctgccg tgctgaagcg ttatgacgac gaaggccgta atgaagccga cctgccgctg 1920 gtgcactggg gcgtacaaga tgcgctgtat caggctgaac aggcgatgga tgatttactg 1980 caaaacttcc cgaaccgcgt ggttgccggg ctgctgaatg tggtgatctt cccgaccgga 2040 cgtcattatc tggcaccttc tgacaagctg gatcataaag tggcgaagat tttacaagtg 2100 ccgaacgcca cccgttcccg cattggtcgc ggtcagtacc tgacgccgag cgagcataat 2160 ccggttggct tgctggaaga ggcgctggtg gatgtgattg ccgccgaccc aattcatcag 2220 cggatctgta aagagctggg taaaaacctg ccgtttaccc gtctggatga actggcgcac 2280 aacgcgctgg tgaaggggct gattgataaa gatgaagccg ctattctggt gaaagctgaa 2340 gaaagccgtc tgcgcagtat taacgttgat gactttgatc cggaagagct ggcgacgaag 2400 ccggtaaagt tgccggagaa agtgcggaaa gttgaagccg cgtaa 2445 <210> 23 <211> 461 <212> PRT <213> Artificial Sequence <220> <223> E. coli MG1655 AtoC <400> 23 Met Thr Ala Ile Asn Arg Ile Leu Ile Val Asp Asp Glu Asp Asn Val 1 5 10 15 Arg Arg Met Leu Ser Thr Ala Phe Ala Leu Gln Gly Phe Glu Thr His 20 25 30 Cys Ala Asn Asn Gly Arg Thr Ala Leu His Leu Phe Ala Asp Ile His 35 40 45 Pro Asp Val Val Leu Met Asp Ile Arg Met Pro Glu Met Asp Gly Ile 50 55 60 Lys Ala Leu Lys Glu Met Arg Ser His Glu Thr Arg Thr Pro Val Ile 65 70 75 80 Leu Met Thr Ala Tyr Ala Glu Val Glu Thr Ala Val Glu Ala Leu Arg 85 90 95 Cys Gly Ala Phe Asp Tyr Val Ile Lys Pro Phe Asp Leu Asp Glu Leu 100 105 110 Asn Leu Ile Val Gln Arg Ala Leu Gln Leu Gln Ser Met Lys Lys Glu 115 120 125 Ile Arg His Leu His Gln Ala Leu Ser Thr Ser Trp Gln Trp Gly His 130 135 140 Ile Leu Thr Asn Ser Pro Ala Met Met Asp Ile Cys Lys Asp Thr Ala 145 150 155 160 Lys Ile Ala Leu Ser Gln Ala Ser Val Leu Ile Ser Gly Glu Ser Gly 165 170 175 Thr Gly Lys Glu Leu Ile Ala Arg Ala Ile His Tyr Asn Ser Arg Arg 180 185 190 Ala Lys Gly Pro Phe Ile Lys Val Asn Cys Ala Ala Leu Pro Glu Ser 195 200 205 Leu Leu Glu Ser Glu Leu Phe Gly His Glu Lys Gly Ala Phe Thr Gly 210 215 220 Ala Gln Thr Leu Arg Gln Gly Leu Phe Glu Arg Ala Asn Glu Gly Thr 225 230 235 240 Leu Leu Leu Asp Glu Ile Gly Glu Met Pro Leu Val Leu Gln Ala Lys 245 250 255 Leu Leu Arg Ile Leu Gln Glu Arg Glu Phe Glu Arg Ile Gly Gly His 260 265 270 Gln Thr Ile Lys Val Asp Ile Arg Ile Ile Ala Ala Thr Asn Arg Asp 275 280 285 Leu Gln Ala Met Val Lys Glu Gly Thr Phe Arg Glu Asp Leu Phe Tyr 290 295 300 Arg Leu Asn Val Ile His Leu Ile Leu Pro Pro Leu Arg Asp Arg Arg 305 310 315 320 Glu Asp Ile Ser Leu Leu Ala Asn His Phe Leu Gln Lys Phe Ser Ser 325 330 335 Glu Asn Gln Arg Asp Ile Ile Asp Ile Asp Pro Met Ala Met Ser Leu 340 345 350 Leu Thr Ala Trp Ser Trp Pro Gly Asn Ile Arg Glu Leu Ser Asn Val 355 360 365 Ile Glu Arg Ala Val Val Met Asn Ser Gly Pro Ile Ile Phe Ser Glu 370 375 380 Asp Leu Pro Pro Gln Ile Arg Gln Pro Val Cys Asn Ala Gly Glu Val 385 390 395 400 Lys Thr Ala Pro Val Gly Glu Arg Asn Leu Lys Glu Glu Ile Lys Arg 405 410 415 Val Glu Lys Arg Ile Ile Met Glu Val Leu Glu Gln Gln Glu Gly Asn 420 425 430 Arg Thr Arg Thr Ala Leu Met Leu Gly Ile Ser Arg Arg Ala Leu Met 435 440 445 Tyr Lys Leu Gln Glu Tyr Gly Ile Asp Pro Ala Asp Val 450 455 460 <210> 24 <211> 1386 <212> DNA <213> Artificial Sequence <220> <223> E. coli MG1655 AtoC <400> 24 atgactgcta ttaatcgcat ccttattgtg gatgatgaag ataatgttcg ccgtatgctg 60 agcaccgctt ttgcactaca aggattcgaa acacattgtg cgaacaacgg acgcacagca 120 ttacacctgt ttgccgatat tcaccctgat gtggtgttga tggatatccg catgccagag 180 atggacggca tcaaggcact aaaggagatg cgcagccatg agacccggac acccgttatt 240 ctgatgacgg cctatgcgga agtggaaacc gccgtcgaag cgctacgctg cggagccttc 300 gactatgtta ttaaaccgtt tgatctcgat gagttgaatt taatcgttca gcgcgcttta 360 caactccagt caatgaaaaa agagatccgt catctgcacc aggcactgag caccagctgg 420 caatgggggc acattctcac caacagcccg gcgatgatgg acatctgcaa agacaccgcc 480 aaaattgccc tttctcaggc cagcgtcttg attagcggtg aaagcggcac cgggaaagag 540 ttgattgcca gagcgattca ctacaattcg cggcgggcaa aggggccgtt cattaaagtc 600 aactgcgcgg cgctgccgga atcgttgctc gaaagtgaac tgtttggtca tgaaaaaggt 660 gcatttactg gtgcacaaac cttgcgtcag ggattatttg aacgagccaa cgaaggtact 720 ctgctcctcg acgaaattgg cgaaatgccg ctggtactac aagccaaatt actacgcatt 780 ctacaggaac gggaatttga acggattggc ggccatcaga ccataaaagt tgatatccgc 840 atcattgctg ccaccaaccg cgacttgcag gcaatggtaa aagaaggcac cttccgtgaa 900 gatctctttt atcgccttaa cgttattcat ttaatactgc cgcctctgcg cgatcgccgg 960 gaagatattt ccctgttagc taatcacttt ttgcaaaaat tcagtagtga gaatcagcgc 1020 gatattatcg acatcgatcc gatggcaatg tcactgctta ccgcctggtc atggccggga 1080 aatattcgag agctttccaa cgttattgaa cgcgccgtcg tgatgaattc aggcccgatc 1140 attttttctg aggatcttcc gccacagatt cgtcagccag tctgtaatgc tggcgaggta 1200 aaaacagccc ctgtcggtga gcgtaattta aaagaggaaa ttaaacgcgt cgaaaaacgc 1260 atcattatgg aagtgctgga acaacaagaa ggaaaccgaa cccgcactgc tttaatgctg 1320 ggcatcagtc gccgtgcatt gatgtataaa ctccaggaat acggtatcga tccggcggat 1380 gtataa 1386 <210> 25 <211> 729 <212> PRT <213> Artificial Sequence <220> <223> E. coli MG1655 FadB <400> 25 Met Leu Tyr Lys Gly Asp Thr Leu Tyr Leu Asp Trp Leu Glu Asp Gly 1 5 10 15 Ile Ala Glu Leu Val Phe Asp Ala Pro Gly Ser Val Asn Lys Leu Asp 20 25 30 Thr Ala Thr Val Ala Ser Leu Gly Glu Ala Ile Gly Val Leu Glu Gln 35 40 45 Gln Ser Asp Leu Lys Gly Leu Leu Leu Arg Ser Asn Lys Ala Ala Phe 50 55 60 Ile Val Gly Ala Asp Ile Thr Glu Phe Leu Ser Leu Phe Leu Val Pro 65 70 75 80 Glu Glu Gln Leu Ser Gln Trp Leu His Phe Ala Asn Ser Val Phe Asn 85 90 95 Arg Leu Glu Asp Leu Pro Val Pro Thr Ile Ala Ala Val Asn Gly Tyr 100 105 110 Ala Leu Gly Gly Gly Cys Glu Cys Val Leu Ala Thr Asp Tyr Arg Leu 115 120 125 Ala Thr Pro Asp Leu Arg Ile Gly Leu Pro Glu Thr Lys Leu Gly Ile 130 135 140 Met Pro Gly Phe Gly Gly Ser Val Arg Met Pro Arg Met Leu Gly Ala 145 150 155 160 Asp Ser Ala Leu Glu Ile Ile Ala Ala Gly Lys Asp Val Gly Ala Asp 165 170 175 Gln Ala Leu Lys Ile Gly Leu Val Asp Gly Val Val Lys Ala Glu Lys 180 185 190 Leu Val Glu Gly Ala Lys Ala Val Leu Arg Gln Ala Ile Asn Gly Asp 195 200 205 Leu Asp Trp Lys Ala Lys Arg Gln Pro Lys Leu Glu Pro Leu Lys Leu 210 215 220 Ser Lys Ile Glu Ala Thr Met Ser Phe Thr Ile Ala Lys Gly Met Val 225 230 235 240 Ala Gln Thr Ala Gly Lys His Tyr Pro Ala Pro Ile Thr Ala Val Lys 245 250 255 Thr Ile Glu Ala Ala Ala Arg Phe Gly Arg Glu Glu Ala Leu Asn Leu 260 265 270 Glu Asn Lys Ser Phe Val Pro Leu Ala His Thr Asn Glu Ala Arg Ala 275 280 285 Leu Val Gly Ile Phe Leu Asn Asp Gln Tyr Val Lys Gly Lys Ala Lys 290 295 300 Lys Leu Thr Lys Asp Val Glu Thr Pro Lys Gln Ala Ala Val Leu Gly 305 310 315 320 Ala Gly Ile Met Gly Gly Gly Ile Ala Tyr Gln Ser Ala Trp Lys Gly 325 330 335 Val Pro Val Val Met Lys Asp Ile Asn Asp Lys Ser Leu Thr Leu Gly 340 345 350 Met Thr Glu Ala Ala Lys Leu Leu Asn Lys Gln Leu Glu Arg Gly Lys 355 360 365 Ile Asp Gly Leu Lys Leu Ala Gly Val Ile Ser Thr Ile His Pro Thr 370 375 380 Leu Asp Tyr Ala Gly Phe Asp Arg Val Asp Ile Val Val Glu Ala Val 385 390 395 400 Val Glu Asn Pro Lys Val Lys Lys Ala Val Leu Ala Glu Thr Glu Gln 405 410 415 Lys Val Arg Gln Asp Thr Val Leu Ala Ser Asn Thr Ser Thr Ile Pro 420 425 430 Ile Ser Glu Leu Ala Asn Ala Leu Glu Arg Pro Glu Asn Phe Cys Gly 435 440 445 Met His Phe Phe Asn Pro Val His Arg Met Pro Leu Val Glu Ile Ile 450 455 460 Arg Gly Glu Lys Ser Ser Asp Glu Thr Ile Ala Lys Val Val Ala Trp 465 470 475 480 Ala Ser Lys Met Gly Lys Thr Pro Ile Val Val Asn Asp Cys Pro Gly 485 490 495 Phe Phe Val Asn Arg Val Leu Phe Pro Tyr Phe Ala Gly Phe Ser Gln 500 505 510 Leu Leu Arg Asp Gly Ala Asp Phe Arg Lys Ile Asp Lys Val Met Glu 515 520 525 Lys Gln Phe Gly Trp Pro Met Gly Pro Ala Tyr Leu Leu Asp Val Val 530 535 540 Gly Ile Asp Thr Ala His His Ala Gln Ala Val Met Ala Ala Gly Phe 545 550 555 560 Pro Gln Arg Met Gln Lys Asp Tyr Arg Asp Ala Ile Asp Ala Leu Phe 565 570 575 Asp Ala Asn Arg Phe Gly Gln Lys Asn Gly Leu Gly Phe Trp Arg Tyr 580 585 590 Lys Glu Asp Ser Lys Gly Lys Pro Lys Lys Glu Glu Asp Ala Ala Val 595 600 605 Glu Asp Leu Leu Ala Glu Val Ser Gln Pro Lys Arg Asp Phe Ser Glu 610 615 620 Glu Glu Ile Ile Ala Arg Met Met Ile Pro Met Val Asn Glu Val Val 625 630 635 640 Arg Cys Leu Glu Glu Gly Ile Ile Ala Thr Pro Ala Glu Ala Asp Met 645 650 655 Ala Leu Val Tyr Gly Leu Gly Phe Pro Pro Phe His Gly Gly Ala Phe 660 665 670 Arg Trp Leu Asp Thr Leu Gly Ser Ala Lys Tyr Leu Asp Met Ala Gln 675 680 685 Gln Tyr Gln His Leu Gly Pro Leu Tyr Glu Val Pro Glu Gly Leu Arg 690 695 700 Asn Lys Ala Arg His Asn Glu Pro Tyr Tyr Pro Pro Val Glu Pro Ala 705 710 715 720 Arg Pro Val Gly Asp Leu Lys Thr Ala 725 <210> 26 <211> 2190 <212> DNA <213> Artificial Sequence <220> <223> E. coli MG1655 fadB <400> 26 atgctttaca aaggcgacac cctgtacctt gactggctgg aagatggcat tgccgaactg 60 gtatttgatg ccccaggttc agttaataaa ctcgacactg cgaccgtcgc cagcctcggc 120 gaggccatcg gcgtgctgga acagcaatca gatctaaaag ggctgctgct gcgttcgaac 180 aaagcagcct ttatcgtcgg tgctgatatc accgaatttt tgtccctgtt cctcgttcct 240 gaagaacagt taagtcagtg gctgcacttt gccaatagcg tgtttaatcg cctggaagat 300 ctgccggtgc cgaccattgc tgccgtcaat ggctatgcgc tgggcggtgg ctgcgaatgc 360 gtgctggcga ccgattatcg tctggcgacg ccggatctgc gcatcggtct gccggaaacc 420 aaactgggca tcatgcctgg ctttggcggt tctgtacgta tgccacgtat gctgggcgct 480 gacagtgcgc tggaaatcat tgccgccggt aaagatgtcg gcgcggatca ggcgctgaaa 540 atcggtctgg tggatggcgt agtcaaagca gaaaaactgg ttgaaggcgc aaaggcggtt 600 ttacgccagg ccattaacgg cgacctcgac tggaaagcaa aacgtcagcc gaagctggaa 660 ccactaaaac tgagcaagat tgaagccacc atgagcttca ccatcgctaa agggatggtc 720 gcacaaacag cggggaaaca ttatccggcc cccatcaccg cagtaaaaac cattgaagct 780 gcggcccgtt ttggtcgtga agaagcctta aacctggaaa acaaaagttt tgtcccgctg 840 gcgcatacca acgaagcccg cgcactggtc ggcattttcc ttaacgatca atatgtaaaa 900 ggcaaagcga agaaactcac caaagacgtt gaaaccccga aacaggccgc ggtgctgggt 960 gcaggcatta tgggcggcgg catcgcttac cagtctgcgt ggaaaggcgt gccggttgtc 1020 atgaaagata tcaacgacaa gtcgttaacc ctcggcatga ccgaagccgc gaaactgctg 1080 aacaagcagc ttgagcgcgg caagatcgat ggtctgaaac tggctggcgt gatctccaca 1140 atccacccaa cgctcgacta cgccggattt gaccgcgtgg atattgtggt agaagcggtt 1200 gttgaaaacc cgaaagtgaa aaaagccgta ctggcagaaa ccgaacaaaa agtacgccag 1260 gataccgtgc tggcgtctaa cacttcaacc attcctatca gcgaactggc caacgcgctg 1320 gaacgcccgg aaaacttctg cgggatgcac ttctttaacc cggtccaccg aatgccgttg 1380 gtagaaatta ttcgcggcga gaaaagctcc gacgaaacca tcgcgaaagt tgtcgcctgg 1440 gcgagcaaga tgggcaagac gccgattgtg gttaacgact gccccggctt ctttgttaac 1500 cgcgtgctgt tcccgtattt cgccggtttc agccagctgc tgcgcgacgg cgcggatttc 1560 cgcaagatcg acaaagtgat ggaaaaacag tttggctggc cgatgggccc ggcatatctg 1620 ctggacgttg tgggcattga taccgcgcat cacgctcagg ctgtcatggc agcaggcttc 1680 ccgcagcgga tgcagaaaga ttaccgcgat gccatcgacg cgctgtttga tgccaaccgc 1740 tttggtcaga agaacggcct cggtttctgg cgttataaag aagacagcaa aggtaagccg 1800 aagaaagaag aagacgccgc cgttgaagac ctgctggcag aagtgagcca gccgaagcgc 1860 gatttcagcg aagaagagat tatcgcccgc atgatgatcc cgatggtcaa cgaagtggtg 1920 cgctgtctgg aggaaggcat tatcgccact ccggcggaag cggatatggc gctggtctac 1980 ggcctgggct tccctccgtt ccacggcggc gcgttccgct ggctggacac cctcggtagc 2040 gcaaaatacc tcgatatggc acagcaatat cagcacctcg gcccgctgta tgaagtgccg 2100 gaaggtctgc gtaataaagc gcgtcataac gaaccgtact atcctccggt tgagccagcc 2160 cgtccggttg gcgacctgaa aacggcttaa 2190 <210> 27 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> pLacO1 promoter <400> 27 aattgtgagc ggataacaat tgacattgtg agcggataac aagatactga gcacatcagc 60 aggacgcact gacc 74

Claims (28)

As a transformed recombinant microorganism in which expression of fadE gene and atoC gene is suppressed,
In the recombinant microorganism, the lvaED gene is introduced, any one or more genes selected from the group consisting of lvaA , lvaB and lvaC genes are introduced, any one or more genes selected from the group consisting of the phaC gene and the phaE gene are introduced, and the The phaC gene is derived from the C. violaceum strain, and any one or more genes selected from the group consisting of the phaA gene and the phaB gene are introduced, a recombinant microorganism. The method of claim 1,
The fadE will have the nucleotide sequence of SEQ ID NO: 22, the recombinant microorganism. The method of claim 1,
The atoC will have the nucleotide sequence of SEQ ID NO: 24, the recombinant microorganism. The method of claim 1,
The microorganism is Escherichia coli ) that is, a recombinant microorganism. The method of claim 1,
In the recombinant microorganism, the expression of the fadB gene is further suppressed, the recombinant microorganism. The method of claim 5,
The fadB gene will have the nucleotide sequence of SEQ ID NO: 26, the recombinant microorganism. delete The method of claim 1,
The lvaED gene will be derived from a P. putida strain, a recombinant microorganism. The method of claim 1,
The lvaD will have the nucleotide sequence of SEQ ID NO: 8, the recombinant microorganism. The method of claim 1,
The lvaE will have the nucleotide sequence of SEQ ID NO: 10, the recombinant microorganism. delete The method of claim 1,
Any one or more genes selected from the group consisting of the lvaA , lvaB and lvaC genes are derived from the P. putida strain, the recombinant microorganism. The method of claim 1,
The lvaA gene will have the nucleotide sequence of SEQ ID NO: 2, the recombinant microorganism. The method of claim 1,
The lvaB gene will have the nucleotide sequence of SEQ ID NO: 4, the recombinant microorganism. The method of claim 1,
The lvaC gene will have the nucleotide sequence of SEQ ID NO: 6, the recombinant microorganism. delete delete The method of claim 1,
The phaE gene will be derived from the T. pfennigii strain, recombinant microorganism. The method of claim 1,
The phaC gene will have a nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 16, the recombinant microorganism. The method of claim 1,
The phaE gene will have the nucleotide sequence of SEQ ID NO: 14, the recombinant microorganism. delete The method of claim 1,
The phaA or phaB gene is from a C. necator strain, the recombinant microorganism. The method of claim 1,
The phaA gene will have the nucleotide sequence of SEQ ID NO: 18, the recombinant microorganism. The method of claim 1,
The phaB gene will have the nucleotide sequence of SEQ ID NO: 20, the recombinant microorganism. The method of claim 1,
The recombinant microorganism will be capable of producing polyhydroxyalkanoate, the recombinant microorganism. Culturing the microorganism according to claim 1; And
A method for producing polyhydroxyalkanoate comprising the step of obtaining polyhydroxyalkanoate produced from the recombinant microorganism. The method of claim 26,
The culturing is characterized in that culturing the microorganism in the presence of levulinic acid, production method. The method of claim 26,
The polyhydroxyalkanoate is poly(3-hydroxybutyric acid), poly(3-hydroxyvaleric acid), poly(4-hydroxyvaleric acid), poly(3-hydroxybutyric acid-co-3-hydric acid) Hydroxyvaleric acid), poly(3-hydroxybutyric acid-co-4-hydroxyvaleric acid), poly(3-hydroxyvaleric acid-co-4-hydroxyvaleric acid) and poly(3-hydroxybutyric acid- co-4-hydroxyvaleric acid-co-4-hydroxyvaleric acid) is any one or more selected from the group consisting of, the production method.

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