Genetically recombinant microorganism producing polyhydroxyalkanoate

Abstract

A genetically recombinant microorganism, according to the idea of the present disclosure, comprises at least one foreign gene, wherein the foreign gene comprises a LvaED gene derived from a strain selected from the genus Pseudomonas, and the LvaED gene is introduced into a microorganism selected from the genus Ralstonia.

Description

Genetically recombinant microorganism producing polyhydroxyalkanoate

[0001] The present invention relates to a genetically recombinant microorganism producing polyhydroxyalkanoate.

[0002] As plastic pollution becomes a global issue, research into the production of biodegradable plastics is actively underway to develop solutions for reducing plastic waste. Biodegradable plastics are plastics that decompose in nature, undergoing physical and chemical changes in response to organic matter such as bacteria. Types of biodegradable plastics include polyhydroxyalkanoate (PHA), polylactic acid (PLA), and polyglycolic acid (PG), and can be used as food and chemical additives, pharmaceutical ingredients, and biodegradable packaging.

[0003] Among these, polyhydroxyalkanoate is a biodegradable plastic derived from microorganisms. Compared to other existing biodegradable plastics, it has a high degree of eco-friendliness and allows for various properties to be controlled, so research on it is actively being conducted.

[0004] One aspect of the present disclosure provides a transformed genetically recombinant microorganism capable of producing polyhydroxyalkanoates having more diverse physical properties by introducing the LvaED gene of a Pseudomonas putida strain into a Ralstonia eutropha strain, a microorganism capable of producing polyhydroxyalkanoates including 4-hydroxyvalerate, thereby controlling the molar fraction of 4-hydroxyvalerate.

[0005] One aspect of the present disclosure provides a method for producing polyhydroxyalkanoate, comprising the steps of culturing a recombinant microorganism according to the present disclosure; and obtaining polyhydroxyalkanoate produced from the recombinant microorganism.

[0006] The technical problems to be achieved in this document are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0007] A genetically recombinant microorganism according to the invention comprises at least one foreign gene, wherein the foreign gene comprises an LvaED gene derived from a strain selected from the genus Pseudomonas, and wherein the LvaED gene is introduced into a microorganism selected from the genus Ralstonia.

[0008] A method for producing polyhydroxyalkanoate according to the invention comprises the steps of culturing a genetically recombinant microorganism; and obtaining polyhydroxyalkanoate produced from the genetically recombinant microorganism.

[0009] According to the invention, a transformed genetically recombinant microorganism capable of producing polyhydroxyalkanoates with more diverse properties can be provided by controlling the molar fraction of 4-hydroxyvalerate.

[0010] The effects that can be obtained from the present disclosure are not limited to the effects mentioned above, and other effects that are not mentioned can be clearly understood by a person having ordinary skill in the art to which the present disclosure belongs from the description below.

[0011] Figure 1 is a diagram showing the metabolic circuit for producing polyhydroxyalkanoate when the LvaED gene of the Ralstonia eutropha strain and the Pseudomonas putida strain are introduced.

[0012] Figure 2 is a diagram showing a plasmid into which the LvaED gene of Pseudomonas putida KT2440 strain has been introduced.

[0013] Figure 3 is a diagram showing the metabolic pathway of Pseudomonas putida strain when levulinic acid is used as a substrate.

[0014] Like reference numerals refer to like elements throughout the specification. This specification does not describe all elements of the embodiments, and any general information within the technical field to which the present invention pertains or any information that overlaps between the embodiments is omitted.

[0015] Additionally, when a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0016] Throughout the specification, when we say that an element is "on" another element, this includes not only cases where the element is in contact with the other element, but also cases where another element exists between the two elements.

[0017] The terms first, second, etc. are used to distinguish one component from another, and the components are not limited by the aforementioned terms.

[0018] Singular expressions include plural expressions unless the context clearly indicates otherwise.

[0019] The identification codes in each step are used for convenience of explanation and do not describe the order of each step, and each step may be performed in a different order than specified unless the context clearly indicates a specific order.

[0020] The genetically recombinant microorganism is described in detail with reference to the attached drawings and sequence list below.

[0021] A genetically recombinant microorganism according to the invention comprises at least one foreign gene, wherein the foreign gene comprises an LvaED gene derived from a strain selected from the genus Pseudomonas, and wherein the LvaED gene is introduced into a microorganism selected from the genus Ralstonia. As a result, the recombinant microorganism can produce polyhydroxyalkanoates, and in particular, can increase the molar fraction of 4-hydroxyvalerate.

[0022] Polyhydroxyalkanoate is a biodegradable plastic derived from microorganisms and has higher environmental friendliness than existing biodegradable plastics. The polyhydroxyalkanoate may be at least one selected from the group consisting of poly(3-hydroxybutyrate), poly(3-hydroxyvalerate), poly(4-hydroxyvalerate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-4-hydroxyvalerate), poly(3-hydroxyvalerate-co-4-hydroxyvalerate), and poly(3-hydroxybutyrate-co-4-hydroxyvalerate-co-4-hydroxyvalerate).

[0023] The above poly(3-hydroxybutyrate) may have a structure as shown in the following chemical formula 1.

[0024] [Chemical Formula 1]

[0025]

[0026] The above poly(3-hydroxyvalerate) may have a structure as shown in the following chemical formula 2.

[0027] [Chemical Formula 2]

[0028]

[0029] The above poly(4-hydroxyvalerate) may have a structure as shown in the following chemical formula 3.

[0030] [Chemical Formula 3]

[0031]

[0032] The above poly(3-hydroxybutyrate-co-3-hydroxyvalerate) may have a structure as shown in the following chemical formula 4.

[0033] [Chemical Formula 4]

[0034]

[0035] The above poly(3-hydroxybutyrate-co-4-hydroxyvalerate) may have a structure as shown in Chemical Formula 5 below.

[0036] [Chemical Formula 5]

[0037]

[0038] The above poly(3-hydroxyvalerate-co-4-hydroxyvalerate) may have a structure as shown in Chemical Formula 6 below.

[0039] [Chemical Formula 6]

[0040]

[0041] The above poly(3-hydroxybutyrate-co-4-hydroxyvalerate-co-4-hydroxyvalerate) may have a structure as shown in the following chemical formula 7.

[0042] [Chemical Formula 7]

[0043]

[0044] Poly(3-hydroxybutyrate) is difficult to utilize due to its rigid properties and high melting point, so polymerization of additional monomers is required. 3-hydroxyvalerate and 4-hydroxyvalerate can be included as monomers. In particular, when 4-hydroxyvalerate is included, polyhydroxyalkanoate can have amorphous and elastic characteristics.

[0045] 3-Hydroxyvalerate and 4-hydroxyvalerate can be synthesized from levulinic acid by microorganisms, and levulinic acid is a carbon source with significant potential as a precursor for several high-value-added substances that can be produced from various biomass. Strains that can produce polyhydroxyalkanoates including 3-hydroxyvalerate and 4-hydroxyvalerate from levulinic acid obtained from renewable resources in the wild type include Pseudomonas putida and Ralstonia eutropha. The levulinic acid metabolic pathway of Pseudomonas putida has been revealed to produce 4-hydroxyvalerate-CoA (4HV-CoA) through the LvaED gene and 3-hydroxyvalerate-CoA (3HV-CoA) through the LvaABC genes, but the mechanism related to 4-hydroxyvalerate synthesis in Ralstonia eutropha has not yet been elucidated. As shown in Fig. 3, the levulinic acid metabolic pathway of the Pseudomonas putida strain has been elucidated, making it possible to produce polyhydroxyalkanoates having various monomers through various metabolic engineering methods, such as removal of metabolic pathways and gene introduction within the strain genome.

[0046] A genetically recombinant microorganism according to the invention can produce polyhydroxyalkanoate with an increased 4-hydroxyvalerate molar fraction by introducing the LvaED gene derived from any strain selected from the genus Pseudomonas into a microorganism selected from the genus Ralstonia.

[0047] The genetically recombinant microorganism according to the invention of the present disclosure may be a strain of Pseudomonas putida. Microorganisms of the genus Pseudomonas include Pseudomonas theae, Pseudomonas tolaasii, Pseudomonas fluorescens, Pseudomonas tremae, Pseudomonas viridiflava, Pseudomonas putida, Pseudomonas caricapapayae, Pseudomonas corrugata, Pseudomonas costantinii, Pseudomonas ficuserectae, Pseudomonas fuscovaginae, Pseudomonas marginalis, Pseudomonas mediterranea, Pseudomonas syringae pv. actinidiaeactinidiae), Pseudomonas meliae, Pseudomonas palleroniana, Pseudomonas salomonii, Pseudomonas agarici, Pseudomonas amygdali, Pseudomonas asplenii, Pseudomonas asiatica, Pseudomonas citronellolis, Pseudomonas taiwanensis, Pseudomonas avellanae, Pseudomonas plecoglossicida, It may be Pseudomonas cannabina, Pseudomonas inefficax, Pseudomonas monteilii, etc., and especially Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae pv. actinidiae, Pseudomonas asiatica, Pseudomonas citronelloris, Pseudomonas taiwanensis, Pseudomonas plecoglossida, Pseudomonas inepicax, Pseudomonas monteilii, etc., and especially Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae pv. It may be Actinidiae, Pseudomonas asiatica, Pseudomonas citronellolis, Pseudomonas plecoglossida, etc., and preferably Pseudomonas putida KT2440 strain.

[0048] According to the invention, a genetically recombinant microorganism can be introduced into a Ralstonia eutropha strain by introducing the LvaED gene. In the present invention, the term "LvaED" refers to the LvaD gene and the LvaE gene. The LvaD gene and the LvaE gene may be polynucleotides having the base sequences of SEQ ID NOs: 4 and 5, respectively, and the LvaD enzyme and the LvaE enzyme encoded by the respective base sequences may be polypeptides. The LvaE enzyme is a short-chain acyl-CoA synthetase and can catalyze and activate levulinic acid into levulinic acid-CoA. The LvaD enzyme is a 4-oxopentanoyl-CoA-4-dehydrogenase and can catalyze the conversion of levulinic acid-CoA into 4-hydroxyvaleric acid-CoA.

[0049] A genetically recombinant microorganism according to the invention may further comprise at least one gene selected from the group consisting of LvaA, LvaB, and LvaC. The term "LvaABC" used in the present invention refers to LvaA, LvaB, and LvaC genes. The LvaA gene may be a polynucleotide having a base sequence represented by SEQ ID NO: 1, and the LvaA enzyme encoded by the base sequence may be a polypeptide. The LvaB gene may be a polynucleotide having a base sequence represented by SEQ ID NO: 2, and the LvaB enzyme encoded by the base sequence may be a polypeptide. In addition, the LvaC gene may be a polynucleotide having a base sequence represented by SEQ ID NO: 3, and the LvaC enzyme may be a polypeptide.

[0050] A genetically recombinant microorganism according to the invention may further comprise at least one gene selected from the group consisting of PhaA, PhaB, and PhaC. The term "PhaABC" used in the present invention refers to PhaA, PhaB, and PhaC genes. The PhaA gene may be a polynucleotide having a base sequence of SEQ ID NO: 6, and the PhaA enzyme encoded by the base sequence may be a polypeptide. In addition, the PhaB gene may be a polynucleotide having a base sequence of SEQ ID NO: 7, and the PhaB enzyme encoded by the base sequence may be a polypeptide. The PhaA enzyme is an acetyl-CoA acetyltransferase and can catalyze the condensation of two acetyl-coA units to form acetoacetyl-CoA. In addition, the PhaB enzyme is an acetoacetyl-CoA reductase and can catalyze the chiral reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA. The PhaC gene may be a polynucleotide having the base sequence of SEQ ID NO: 8, and the PhaC enzyme encoded by the base sequence may be a polypeptide. The PhaC enzyme is an enzyme that biosynthesizes polyhydroxyalkanoate by polymerizing the monomer hydroxyalkanoate.

[0051] The term "transformation" or "genetic recombination" 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.

[0052] A genetically recombinant microorganism according to the invention can produce polyhydroxyalkanoates with an increased mole fraction (%) of 4-hydroxyvalerate. When 4-hydroxyvalerate is included, the polyhydroxyalkanoates can have amorphous, elastomeric properties, allowing them to be used in a wide range of applications.

[0053] The following describes a method for producing polyhydroxyalkanoates.

[0054] A method for producing polyhydroxyalkanoate according to the invention comprises the steps of culturing the genetically recombinant microorganism described above; and obtaining polyhydroxyalkanoate produced from the genetically recombinant microorganism. A plasmid, pKM212LvaE,LvaD, which can be used by a Ralstonia eutropha strain into which the LvaED gene has been inserted, is constructed as shown in Fig. 2. When the plasmid is introduced into a Ralstonia wild-type strain using a cloning (transformation) method and then cultured using levulinic acid and glucose as carbon sources, a polymer having a higher molar fraction of 4-hydroxyvalerate than before can be produced. The metabolic pathway for producing a polymer by introducing the LvaED gene of Pseudomonas putida into a Ralstonia eutropha strain is as shown in Fig. 1.

[0055] Ralstonia eutropha produces 3-hydroxybutyryl-CoA through the existing PhaA and PhaB genes, and by introducing the LvaED gene of a Pseudomonas putida strain in addition to the 4-hydroxyvaleryl-CoA production mechanism and 3-hydroxyvaleryl-CoA production mechanism that are possible but not identified in the wild type Ralstonia eutropha strain, the production of 4-hydroxyvaleryl-CoA can be increased. By polymerizing the 3-hydroxybutyryl-CoA, 4-hydroxyvaleryl-CoA, and 3-hydroxyvaleryl-CoA produced in this way through the PhaC gene encoding polyhydroxyalkanoate polymerase (PHA synthase) that Ralstonia eutropha already possesses, polyhydroxyalkanoate having a higher 4-hydroxyvalerate molar fraction can be produced from glucose and levulinic acid than that which can be produced by the existing wild-type Ralstonia eutropha strain.

[0056] In the method for producing polyhydroxyalkanoate according to the invention, the culturing step may be a subculturing step.

[0057] The method for producing polyhydroxyalkanoate according to the invention may be such that the culturing step is performed in a nitrogen-limited environment.

[0058] Polyhydroxyalkanoates are thought to be produced by microorganisms to conserve energy in nutrient-limited environments. Because microorganisms struggle to grow in such environments, a process is necessary to initially produce microorganisms that have grown to a certain level. Therefore, microorganisms are first cultured in nutrient-rich complex media until they reach a certain size, and then subcultured onto nitrogen-limited defined media to induce polyhydroxyalkanoate polymer production.

[0059] The method for producing polyhydroxyalkanoate according to the invention may be such that the culturing step is to cultivate microorganisms using levulinic acid as a main substrate.

[0060] The medium and other culture conditions used for culturing the microorganism of the present invention can be any medium used for conventional culture without particular limitations. Specifically, the microorganism of the present invention can be cultured under aerobic conditions in a conventional medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while controlling temperature, pH, etc.

[0061] The above-mentioned medium may contain potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or sodium-containing salts as personnel. It may also contain metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and appropriate precursors may be included.

[0062] Additionally, the pH of the culture can be adjusted by appropriately adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture during cultivation. Furthermore, to maintain aerobic conditions in the culture, oxygen or oxygen-containing gas can be injected into the culture. To maintain anaerobic and microaerobic conditions, no gas can be injected, or nitrogen, hydrogen, or carbon dioxide can be injected.

[0063] The temperature of the culture medium may generally be 20°C to 40°C, specifically 25°C to 37°C, and more specifically 30°C, but may be varied without limitation depending on the intended purpose. The culture period may continue until the desired amount of useful material is produced, and may specifically be 10 to 100 hours, but is not limited thereto.

[0064] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and the scope of the present invention is not limited to these examples.

[0065] [Example]

[0066] The primers used for plasmid construction are as follows.

[0067] Primer sequence informationLvaE_F_SacIAAAAAgagctcatgttgcagacacgcatcatcaagLvaE_R_KpnIAAAAAggtaccttacaacgtggaaaggaacgcgcLvaD_F_KpnIAAAAAggtacctttcacacaggaaacaatgcagccgaaccttgcccgacLvaD_R_XbaIAAAAAtctagattacaggctgctgcacaggtggc

[0068] The plasmid used was pKM212, which contains the pBBR1 origin, the tac promoter, and kanamycin resistance. To construct a plasmid for specific use, the pKM212 plasmid was digested with SacI and KpnI. The LvaE gene from the genome of the Pseudomonas putida strain was amplified by PCR using the primers LvaE_F_SacI and LvaE_R_KpnI. The amplified gene was digested with SacI and KpnI and ligated to the digested pKM212 plasmid. The constructed plasmid was then transformed into the E. coli XL1-Blue strain, and colonies were selected on a medium supplemented with kanamycin antibiotic.

[0069] The constructed pKM212 LvaE plasmid was digested with KpnI and XbaI. The LvaD gene was amplified by PCR from the genome of Pseudomonas putida strain using the primers LvaD_F_KpnI and LvaD_R_XbaI. The amplified gene was digested with KpnI and XbaI and ligated to the digested pKM 212 LvaE plasmid. After transformation into E. coli XL1-Blue strain, colonies were selected on a medium supplemented with kanamycin antibiotic, thereby constructing the pKM212 LvaE LvaD plasmid.

[0070] The constructed pKM212 LvaE LvaD plasmid was introduced into the Ralstonia eutropha strain via conjugation. The transformation method was as follows.

[0071] The wild-type strain of Ralstonia eutropha and E. coli S17-1 containing pKM212 LvaE LvaD were cultured in LB at 30℃ and 37℃ for one day, respectively. The Ralstonia eutropha strain and E. coli S17-1 strain were mixed in a volume ratio of 1:3 and added dropwise to LB solid medium. After that, the colony was streaked on LB solid medium containing kanamycin antibiotic, and then re-streaked on LB solid medium containing kanamycin and gentamicin antibiotics to confirm that it was a Ralstonia eutropha strain. The plasmid was confirmed by PCR using genetic primers.

[0072] The Ralstonia eutropha strain containing the pKM212 LvaE LvaD plasmid was inoculated into 10 ml of LB liquid medium containing kanamycin and gentamicin antibiotics and cultured at 30°C for 1 day. Then, 1 ml of the culture was inoculated into a 250 ml flask containing 100 ml of LB liquid medium containing kanamycin and gentamicin antibiotics and cultured at 30°C for 24 hours. After 24 h of incubation, MR-N (per liter: 6.67 g KH2PO4, 4 g Na2HPO4, 0.8 g MgSO4·7 H2O, 0.8 g citric acid, 5 ml trace metal solution, Trace metal solution per liter of 0.5 M HCl: 10 g FeSO4·7H2O, 2 g CaCl2, 2.2 g ZnSO4·7H2O, 0.5 g MnSO4·4H2O, 1 g CuSO4·5H2O, 0.1 g (NH4)6Mo7O) was added to provide nitrogen limitation. 24·4H2O, 0.02 g Na2B4O7·10H2O) medium was used for two-stage culture. 100 mL of the culture was centrifuged to collect the pellet, washed with 100 mL of MR-N medium, and cultured in a 250 mL flask at 30°C for 72 hours. Kanamycin and gentamicin were added as antibiotics, and 20 g / L of glucose and 3.5 g / L of levulinic acid were included as carbon sources. All experiments were performed in triplicate.

[0073] After the incubation was completed, the culture solution was centrifuged at 4,000 rpm for 20 minutes at 4°C to collect the pellet, which was washed twice and then dried at 65°C for one day. The dried pellet was reacted with 2 mL of chloroform and 1 mL of PHA solution at 95°C for one day for PHA analysis, and after cooling to room temperature, 1 mL of tertiary water was added and vortexed. The separated reactant (organic solvent layer) was then analyzed by GC.

[0074] As a comparative example, a wild type strain of Ralstonia eutropha was cultured with levulinic acid, and as an example, a Ralstonia eutropha strain was cultured with levulinic acid after introducing the pKM212 LvaE LvaD plasmid, as shown in Table 2 below.

[0075] PHA content (%)PHA conc. (g / L)3HV mol%4HV mol%Comparative Example 44.420.9011.7317.10Example 57.171.3110.8326.75

[0076] PHA: Polyhydroxyalkanoate 3HV: 3-hydroxyvalerate 4HV: 4-hydroxyvalerate

[0077] According to Table 2 above, it was confirmed that the molar fraction of 4-hydroxyvalerate increased from 17.1 mol% to 26.75 mol% by introducing the LvaED gene of the Pseudomonas putida strain into the Ralstonia eutropha strain. This confirmed that introducing the LvaED gene of the Pseudomonas putida strain into the Ralstonia eutropha strain can control and improve the molar fraction of 4-hydroxyvalerate, which explains that polyhydroxyalkanoates with various physical properties can be produced.

[0078] According to one embodiment, a genetically recombinant microorganism is provided that can control the molar fraction of 4-hydroxyvalerate by introducing the LvaED gene of a Pseudomonas putida strain into a Ralstonia eutropha strain, a microorganism having the ability to produce polyhydroxyalkanoate including 4-hydroxyvalerate, thereby providing a transformed genetically recombinant microorganism capable of producing polyhydroxyalkanoate with more diverse physical properties.

[0079] Additionally, the strain may be a Pseudomonas putida strain.

[0080] Additionally, the LvaED gene can be introduced into a Ralstonia eutropha strain.

[0081] Additionally, the LvaD gene may have a base sequence of sequence number 4.

[0082] Additionally, the LvaE gene may have a base sequence of sequence number 5.

[0083] Additionally, the genetically recombinant microorganism may further comprise at least one gene selected from the group consisting of LvaA, LvaB, and LvaC.

[0084] Additionally, the above LvaA gene may have the base sequence of sequence number 1.

[0085] Additionally, the above LvaB gene may have the base sequence of sequence number 2.

[0086] Additionally, the above LvaC gene may have a base sequence of sequence number 3.

[0087] Additionally, the genetically recombinant microorganism may further comprise at least one gene selected from the group consisting of PhaA, PhaB, and PhaC.

[0088] Additionally, the above PhaA gene may have a base sequence of sequence number 6.

[0089] Additionally, the above PhaB gene may have a base sequence of sequence number 7.

[0090] Additionally, the above PhaC gene may have a base sequence of sequence number 8.

[0091] Additionally, the genetically recombinant microorganism can produce polyhydroxyalkanoate having an increased 4-hydroxyvalerate mole fraction (%).

[0092] In addition, the polyhydroxyalkanoate may be at least one selected from the group consisting of poly(3-hydroxybutyrate), poly(3-hydroxyvalerate), poly(4-hydroxyvalerate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-4-hydroxyvalerate), poly(3-hydroxyvalerate-co-4-hydroxyvalerate), and poly(3-hydroxybutyrate-co-4-hydroxyvalerate-co-4-hydroxyvalerate).

[0093] A method for producing polyhydroxyalkanoate according to one embodiment comprises the steps of culturing a genetically recombinant microorganism; and the step of obtaining polyhydroxyalkanoate produced from the genetically recombinant microorganism.

[0094] Additionally, the above culturing step may be subculturing.

[0095] Additionally, the above culturing step may be cultivating microorganisms using levulinic acid as a main substrate.

[0096] Additionally, the culturing step may be performed in a nitrogen-limited environment.

[0097] Additionally, the polyhydroxyalkanoate obtained above may have an increased 4-hydroxyvalerate mole fraction (%).

[0098] Above, a method for producing a genetically recombinant microorganism and polyhydroxyalkanoate according to one embodiment has been described.

[0099] Although the embodiments of the invention disclosed above have been illustrated and described, the disclosed invention is not limited to the specific embodiments described above, and various modifications may be implemented by a person having ordinary skill in the art to which the disclosed invention pertains without departing from the gist claimed in the claims.

[0100] Attached is an electronic file of the sequence list (D: / Auto Backup / Desktop / PSD23-0100-KR0 / Genetically recombinant microorganism producing polyhydroxyalkanoate.xml).

Claims

1. Contains at least one foreign gene, The above foreign gene comprises a LvaED gene derived from any strain selected from the genus Pseudomonas, The above LvaED gene is a genetically recombinant microorganism introduced into a microorganism selected from the genus Ralstonia.

2. In claim 1, The above strain is a genetically recombinant microorganism, a Pseudomonas putida strain.

3. In claim 1, The above LvaED gene is a genetically recombinant microorganism introduced into a Ralstonia eutropha strain.

4. In claim 1, A genetically recombinant microorganism having the LvaD gene as the base sequence of sequence number 4.

5. In claim 1, The LvaE gene is a genetically recombinant microorganism having the base sequence of sequence number 5.

6. In claim 1, The above genetically recombinant microorganism is a genetically recombinant microorganism further comprising at least one gene selected from the group consisting of LvaA, LvaB, and LvaC.

7. In claim 6, A genetically recombinant microorganism having the above LvaA gene having the base sequence of sequence number 1.

8. In claim 6, A genetically recombinant microorganism having the above LvaB gene having the base sequence of sequence number 2.

9. In claim 6, The above LvaC gene is a genetically recombinant microorganism having the base sequence of sequence number 3.

10. In claim 1, The above genetically recombinant microorganism is a genetically recombinant microorganism further comprising at least one gene selected from the group consisting of PhaA, PhaB, and PhaC.

11. In claim 10, A genetically recombinant microorganism having the above PhaA gene having the base sequence of sequence number 6.

12. In claim 10, A genetically recombinant microorganism having the above PhaB gene having the base sequence of sequence number 7.

13. In claim 10, A genetically recombinant microorganism having the above PhaC gene having the base sequence of sequence number 8.

14. In claim 1, The above genetically recombinant microorganism is a genetically recombinant microorganism that produces polyhydroxyalkanoate having an increased mole fraction (%) of 4-hydroxyvalerate.

15. In claim 14, A genetically recombinant microorganism, wherein the polyhydroxyalkanoate is at least one selected from the group consisting of poly(3-hydroxybutyrate), poly(3-hydroxyvalerate), poly(4-hydroxyvalerate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-4-hydroxyvalerate), poly(3-hydroxyvalerate-co-4-hydroxyvalerate), and poly(3-hydroxybutyrate-co-4-hydroxyvalerate-co-4-hydroxyvalerate).

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