Transformed microorganism, and method for producing polyhydroxyalkanoate using the same

Abstract

The present invention provides a transformed microorganism having a polyhydroxyalkanoate synthase gene and having reduced expression of the A1386 gene and / or the A2405 gene. The transformed microorganism may also have enhanced expression of the minC gene and the minD gene. The present invention also provides a method for producing PHA, comprising a step of culturing the transformed microorganism in the presence of a carbon source.

Description

Technical Field

[0001] The present invention relates to a transformed microorganism and a method for producing polyhydroxyalkanoate using the microorganism. Background Art

[0002] In the context of increasing awareness of environmental issues, food issues, health and safety, and increasing natural or natural desire, the significance and importance of material production using microorganisms (fermentation production, biotransformation, etc.) are increasing, and the production of protein pharmaceuticals, nucleic acids for gene therapy, etc., also uses material production based on microorganisms. For example, the production of ethanol, acetic acid, and medical proteins using microorganisms such as yeast and bacteria has been actively used in industry.

[0003] As an example, the production of polyhydroxyalkanoates (hereinafter also referred to as PHA) based on microorganisms can be cited, and the polyhydroxyalkanoates are expected to be used industrially as biodegradable plastics (refer to non-patent document 1). PHA is a thermoplastic polyester produced and accumulated as an energy storage substance in the cells of multiple microbial species, and is biodegradable. At present, with the improvement of environmental awareness, plastics derived from non-petroleum sources have attracted much attention, especially PHA produced and accumulated by microorganisms in the fungus will be incorporated into the carbon cycle process in nature, so it is expected to have little adverse impact on the ecosystem, and its practical application is urgently desired. In the production of PHA using microorganisms, it is known that, for example, sugars, vegetable oils and fatty acids are given to Cupriavidus bacteria as a carbon source, so that PHA is accumulated in the cells, thereby producing PHA (refer to non-patent documents 2 and 3).

[0004] However, in the production of substances using microorganisms, there are cases where the separation and recovery process of microbial cells and target products is complicated, and the increase in production costs has become a problem. Therefore, improving the separation and recovery efficiency is a major issue in reducing production costs.

[0005] It is known that, in the enlargement of cells in PHA-producing microorganisms, for example, the cell division-inhibiting protein minCD is overexpressed, or a monofunctional peptidoglycan glycosyltransferase, which is one of the peptidoglycan synthases, is destroyed, thereby enlarging the cells of PHA-producing microorganisms (see Non-Patent Document 4 and Patent Document 1). However, there has been no report on the correlation between peptidoglycan hydrolases and the cell morphology of PHA-producing microorganisms.

[0006] Prior art literature

[0007] Non-patent literature

[0008] Non-patent document 1: Anderson AJ., et al., Int. J. Biol. Macromol., 12, 201-105 (1990)

[0009] Non-patent document 2: Sato S., et al., J. Biosci. Bioeng., 120(3), 246-251 (2015)

[0010] Non-patent document 3: Insomphun C., et al., Metab. Eng., 27, 38-45 (2015)

[0011] Non-patent document 4: Shen R., et al., Metab. Eng., 54, 117-126 (2019)

[0012] Patent Document 1: International Publication No. 2016 / 194771 Summary of the invention

[0013] Problems to be solved by the invention

[0014] PHA accumulates in microbial cells. In order to use the PHA accumulated in microbial cells as a biodegradable plastic, the microbial cells are first separated and recovered from the culture solution. When separating and recovering the microbial cells, a centrifuge, a separation membrane, etc. can be used, and the ease and efficiency of separation and recovery depend on the size of the microbial cells. That is, the larger the microbial cells are, the easier it is to efficiently implement separation and recovery using a centrifuge, a separation membrane, etc., so that the production cost is reduced.

[0015] In view of the above-mentioned current situation, an object of the present invention is to provide a transformed microorganism that can accumulate PHA and can be enlarged, and a method for producing PHA using the transformed microorganism.

[0016] Solution to the problem

[0017] The present inventors have found that by reducing the expression of any of the A0302 gene, A0597 gene, A1386 gene, A2272 gene, and A2405 gene, which are presumed to be hydrolases of peptidoglycan in bacteria of the genus Cupriphae, the expression of the A1386 gene or the A2405 gene is reduced, thereby enabling the microbial cells to be enlarged while maintaining the industrially desired PHA accumulation amount, thereby completing the present invention. Furthermore, the inventors have found that by increasing the expression of the minC gene and the minD gene in addition to reducing the expression of the A1386 gene or the A2405 gene, the microbial cells can be further enlarged, thereby completing the present invention.

[0018] That is, the present invention relates to a transformed microorganism having a polyhydroxyalkanoate synthase gene and having reduced expression of the A1386 gene and / or the A2405 gene. Preferably, the A1386 gene is a gene encoding an amino acid sequence having a sequence homology of 85% or more to the amino acid sequence described in sequence number 1, and the A2405 gene is a gene encoding an amino acid sequence having a sequence homology of 85% or more to the amino acid sequence described in sequence number 2. The transformed microorganism may also enhance the expression of the minC gene and the minD gene. Preferably, the minC gene is a gene encoding an amino acid sequence having a sequence homology of 85% or more to the amino acid sequence described in sequence number 3, and the minD gene is a gene encoding an amino acid sequence having a sequence homology of 85% or more to the amino acid sequence described in sequence number 4. The transformed microorganism preferably belongs to the genus Cupriavidus, and is more preferably a transformed microorganism of Cupriavidus hookworm. In addition, the present invention also relates to a method for producing polyhydroxyalkanoate, which comprises a step of culturing the transformed microorganism in the presence of a carbon source. The polyhydroxyalkanoate is preferably a copolymer of two or more hydroxyalkanoic acids, more preferably a copolymer containing 3-hydroxycaproic acid as a monomer unit, and even more preferably a copolymer of 3-hydroxybutyric acid and 3-hydroxycaproic acid.

[0019] Effects of the Invention

[0020] According to the present invention, a transformed microorganism that can accumulate PHA and can be enlarged, and a method for producing PHA using the transformed microorganism can be provided. According to the present invention, since the microbial cells that accumulate PHA are enlarged, it becomes easy to separate and recover the microbial cells from the culture solution, and the production cost can be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a microscopic photograph of the KNK-005 strain (Comparative Example 1) after culture (the scale bar in the photograph represents 10 μm). Figures 2 to 11 same.)

[0022] Figure 2 This is a microscopic photograph of the A0597 deletion disrupted strain (Comparative Example 2) after culture.

[0023] Figure 3 This is a microscopic photograph of the A0302 deletion disrupted strain (Comparative Example 3) after culture.

[0024] Figure 4 This is a microscopic photograph of the A2272 insertion-disrupted strain (Comparative Example 4) after culture.

[0025] Figure 5 This is a microscopic photograph of the A1386 deletion disrupted strain (Example 1) after culture.

[0026] Figure 6 This is a microscopic photograph of the A2405 insertion-disrupted strain (Example 2) after culture.

[0027] Figure 7 This is a microscopic photograph of the A2405 deletion disrupted strain (Example 3) after culture.

[0028] Figure 8 This is a microscopic photograph of the A1386 / A2405 double disrupted strain (Example 4) after culture.

[0029] Fig. 9 This is a microscopic photograph of the minCD expression A1386 disrupted strain (Example 5) after culture.

[0030] Fig.10 This is a microscopic photograph of the minCD expression A2405 disrupted strain (Example 6) after culture.

[0031] Fig.11 This is a microscopic photograph of the minCD-expressing A2405·A1386 disrupted strain (Example 7) after culture. DETAILED DESCRIPTION

[0032] Hereinafter, embodiments of the present invention will be described in detail.

[0033] The transformed microorganism of this embodiment has a PHA synthase gene and has reduced expression of the A1386 gene and / or the A2405 gene. The transformed microorganism may also have enhanced expression of the minC gene and the minD gene.

[0034] (microorganism)

[0035] The transformed microorganism of this embodiment may be a microorganism transformed in a manner that has a PHA synthase gene and reduces the expression of the A1386 gene. In addition, it may be a microorganism transformed in a manner that has a PHA synthase gene and reduces the expression of the A2405 gene. In addition, it may be a microorganism transformed in a manner that has a PHA synthase gene, reduces the expression of the A1386 gene, and enhances the expression of the minC gene and the minD gene. In addition, it may be a microorganism transformed in a manner that has a PHA synthase gene, reduces the expression of the A2405 gene, and enhances the expression of the minC gene and the minD gene. In addition, it may be a microorganism transformed in a manner that has a PHA synthase gene, reduces the expression of the A1386 gene and the A2405 gene. In addition, it may be a microorganism transformed in a manner that has a PHA synthase gene, reduces the expression of the A1386 gene and the A2405 gene, and enhances the expression of the minC gene and the minD gene.

[0036] The host of the transformation microorganism of the present embodiment is not particularly limited, preferably with A1386 gene, A2405 gene, minC gene or minD gene bacteria. As the bacterium, for example, Ralstonia (Ralstonia) genus, Cupriavidus (Cupriavidus) genus, Wautersia (Wautersia) genus, Burkholderia (Burkholderia) genus and other Burkholderia (Burkholderiaceae) section bacteria can be cited as preferred examples. From the viewpoint of safety and PHA productivity, it is more preferably Ralstonia, Cupriavidus genus bacteria, more preferably Cupriavidus genus bacteria, particularly preferably Cupriavidus necator (Cupriavidus necator).

[0037] The host of the transformed microorganism of the present embodiment can be a wild strain originally having a PHA synthase gene, or a mutant strain obtained by artificially mutating such a wild strain, or a strain into which an external PHA synthase gene is introduced by a genetic engineering method. The method for introducing an external PHA synthase gene is not particularly limited, and a method of directly inserting or replacing the gene on the chromosome of the host, a method of directly inserting or replacing the gene on a giant plasmid held by the host, or a method of configuring the gene on a plasmid, a phage, a phagemid or other vector and introducing the gene, etc. can be selected, and two or more of these methods can also be used in combination. Considering the stability of the introduced gene, it is preferably a method of directly inserting or replacing the gene on the chromosome of the host or on a giant plasmid held by the host, and more preferably a method of directly inserting or replacing the gene on the chromosome of the host.

[0038] (PHA synthase gene)

[0039] As PHA synthase genes, there are no particular limitations, and examples include PHA synthase genes of biological origin belonging to the genera Ralstonia, Cupriavidus, Watersella, Alcaligenes, Aeromonas, Pseudomonas, Nocardia, and Chromobacterium, and variants thereof. As the above variant, a base sequence encoding a PHA synthase in which one or more amino acid residues have been deleted, added, inserted, or substituted can be used. For example, a gene having a base sequence encoding a polypeptide represented by an amino acid sequence described in any one of sequence numbers 5 to 9, and a gene having a base sequence encoding a polypeptide represented by an amino acid sequence having a sequence homology of 85% or more to the amino acid sequence and having PHA synthase activity can be listed. As the above sequence homology, it is preferably 90% or more, more preferably 95% or more, further preferably 97% or more, and particularly preferably 99% or more.

[0040] (PHA)

[0041] The type of PHA produced by the transformed microorganism of the present embodiment is not particularly limited as long as it is a PHA that can be produced by the microorganism, but preferably is a homopolymer of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, a copolymer of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms and other hydroxyalkanoic acids (for example, 2-hydroxyalkanoic acid, 4-hydroxyalkanoic acid, 5-hydroxyalkanoic acid, 6-hydroxyalkanoic acid, etc. having 4 to 16 carbon atoms), and a copolymer of two or more monomers selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms. Examples include, but are not limited to, P(3HB) which is a homopolymer of 3-hydroxybutyric acid (abbreviation: 3HB), a copolymer P(3HB-co-3HV) of 3HB and 3-hydroxyvaleric acid (abbreviation: 3HV), a copolymer P(3HB-co-3HH) (abbreviation: PHBH) of 3HB and 3-hydroxyhexanoic acid (abbreviation: 3HH), a copolymer P(3HB-co-4HB) of 3HB and 4-hydroxybutyric acid (abbreviation: 4HB), and PHA containing lactic acid (abbreviation: LA) as a constituent component, such as a copolymer P(LA-co-3HB) of 3HB and LA. Among them, PHBH is preferred from the viewpoint of a wide range of applications as a polymer. It should be noted that, depending on the purpose, the type of PHA to be produced can be appropriately selected by the type of PHA synthase gene retained by the microorganism to be used or introduced separately, the type of gene of the metabolic system related to its synthesis, the culture conditions, and the like.

[0042] (Peptidoglycan)

[0043] Peptidoglycan is a type of high molecular compound containing peptides and sugars that mainly constitutes the cell wall of bacteria. The structure of peptidoglycan varies depending on the bacterial species. As a representative example, in Escherichia coli, it is composed of a sugar chain with alternating repetitions of two amino sugars, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) as units, and a pentapeptide of L-alanine (L-Ala)-γ-D-glutamic acid (Glu)-m-diaminopimelic acid (m-DAP)-D-alanine (D-Ala)-D-Ala. The L-Ala of the pentapeptide is bonded to the MurNAc of the sugar chain through a peptide bond. After removing D-Ala from the pentapeptide to form a tetrapeptide, the m-DAP of the tetrapeptide is bonded to the D-Ala of the tetrapeptide of the other sugar chain, and the two sugar chains are cross-linked, thereby forming a strong structure.

[0044] (Peptidoglycan hydrolase)

[0045] Most bacteria have a variety of peptidoglycan hydrolases such as N-acetylmuramoyl-L-alanine amidase, D-alanyl-D-alanine-endopeptidase, and D-alanyl-D-alanine-carboxypeptidase. N-acetylmuramoyl-L-alanine amidase cuts the bond between MurNAc and the N-terminus of L-Ala in peptidoglycan. D-alanyl-D-alanine-endopeptidase cuts the bond between m-DAP and D-Ala in the cross-linked portion of the tetrapeptide. D-alanyl-D-alanine-carboxypeptidase cuts the bond between D-Ala and D-Ala in the pentapeptide and removes the terminal D-Ala.

[0046] In the UniProtKB database, the protein encoded by the A0597 gene of Coptis necrotica (UniProtKB ID Q0KEW8) was putatively identified as N-acetylmuramoyl-L-alanine amidase.

[0047] In the UniProtKB database, the proteins encoded by the A0302 gene (UniProtKB ID Q0KE26) and the A1386 gene (UniProtKB ID Q0KBU9) of Copperworm were presumed to be D-alanyl-D-alanine-carboxypeptidase.

[0048] In the UniProtKB database, the A2272 and A2405 genes of Cupriphaga necator were putatively encoded cell wall-related hydrolases, but their detailed functions were not reported.

[0049] The A1386 gene is a gene having a base sequence encoding the following polypeptide, and the polypeptide is a polypeptide represented by the amino acid sequence described in SEQ ID NO: 1, or a polypeptide represented by an amino acid sequence having a sequence homology of 85% or more to the amino acid sequence. The sequence homology is preferably 90% or more, more preferably 95% or more, further preferably 97% or more, and particularly preferably 99% or more. It should be noted that the amino acid sequence described in SEQ ID NO: 1 has a sequence homology of about 30% with the amino acid sequence of the protein encoded by the A0302 gene.

[0050] The A2405 gene is a gene having a base sequence encoding the following polypeptide, and the polypeptide is a polypeptide represented by the amino acid sequence described in sequence number 2, or a polypeptide represented by an amino acid sequence having a sequence homology of 85% or more to the amino acid sequence. The above sequence homology is preferably 90% or more, more preferably 95% or more, further preferably 97% or more, and particularly preferably 99% or more. It should be noted that the amino acid sequence described in sequence number 2 has a sequence homology of about 40% with the amino acid sequence of the protein encoded by the A2272 gene.

[0051] (minC gene, minD gene)

[0052] The proteins MinC, MinD and MinE encoded by the minC gene, minD gene and minE gene are proteins that have the function of coordinating and controlling cell division in bacteria (MinCDE system). For example, it is known that in Escherichia coli cells, MinD forms a polymer in an ATP-dependent manner, further forms a complex with MinC, and rapidly vibrates from one pole to the other of the cell. MinC has a role in diaphragm formation during cell division. In addition, it is known that MinE and MinC competitively bind to MinD and have a role in regulating diaphragm formation to occur only in the center of the cell.

[0053] The minC gene in the present invention is a gene having a base sequence encoding a polypeptide represented by the amino acid sequence described in SEQ ID NO: 3 (UniProtKB ID Q0KFI3), or a polypeptide represented by an amino acid sequence having a sequence identity of 85% or more to the amino acid sequence. The sequence identity is preferably 90% or more, more preferably 95% or more, further preferably 97% or more, and particularly preferably 99% or more.

[0054] The minD gene in the present invention is a gene having a base sequence encoding a polypeptide represented by the amino acid sequence described in SEQ ID NO: 4 (UniProtKB ID Q0KFI4), or a polypeptide represented by an amino acid sequence having a sequence identity of 85% or more to the amino acid sequence. The sequence identity is preferably 90% or more, more preferably 95% or more, further preferably 97% or more, and particularly preferably 99% or more.

[0055] (Reduction in gene expression)

[0056] "Reduction of gene expression" in the present invention refers to a state in which the transcription amount of the target gene or the expression amount of the polypeptide encoded by the target gene is reduced compared to a strain in which the expression of the target gene is not reduced. The reduction amount is not particularly limited, and it only needs to be less than 1 times the expression amount of the strain in which the expression of the target gene is not reduced, preferably 0.8 times or less, more preferably 0.5 times or less, further preferably 0.3 times or less, and further preferably 0.2 times or less. The transcription amount of the target gene or the expression amount of the polypeptide encoded by the target gene may be zero. In addition, the situation in which the polypeptide encoded by the gene does not show the original function by modifying the base sequence of the target gene, etc., can also be regarded as a reduction in the expression of the gene. In addition, the expression of the target gene can also be reduced by genetically modifying a microorganism having a PHA synthase gene so that it produces metabolites or proteins that inhibit the function of the polypeptide.

[0057] In the present embodiment, the method for reducing the expression of a gene is not particularly limited, and examples thereof include: a method for deleting a portion or the entire length of a target gene, a method for modifying a "gene expression regulatory sequence" related to the expression of a target gene, and a method for reducing the stability of a transcribed messenger RNA by modifying the target gene and / or its surrounding base sequences. The method for modifying the base sequence is not particularly limited, and can be implemented by replacing, deleting, inserting and / or adding at least a portion of the target gene and / or its surrounding base sequences, and can be performed by methods known to those skilled in the art. In addition, by using antisense RNA, RNA interference (RNAi), CRISPR interference (CRISPRi), etc., to a transformed microorganism having a PHA synthase gene, the expression of the target gene can also be reduced without modifying the target gene and / or its surrounding base sequences.

[0058] (Enhanced gene expression)

[0059] The enhancement of gene expression in the present invention refers to a state in which the transcription amount of the target gene or the expression amount of the polypeptide encoded by the target gene is increased compared to a strain in which the expression of the target gene is not enhanced. The amount of increase is not particularly limited, and it may be more than 1 times, preferably 1.1 times or more, more preferably 1.2 times or more, further preferably 1.5 times or more, and further preferably 2 times or more, compared to a strain in which the expression of the target gene is not enhanced.

[0060] In this embodiment, the method for enhancing the expression of minC gene and minD gene is not particularly limited, and may be a method of introducing the target gene into the host, a method of enhancing the expression level of the target gene originally contained in the host's genomic DNA, or both.

[0061] There are no particular limitations on the method for introducing the target gene into the host. One may select a method of directly inserting or replacing the target gene on the host's chromosome, a method of directly inserting or replacing the target gene on a giant plasmid possessed by the host, or a method of configuring the target gene on a vector such as a plasmid, phage, or phagemid and introducing it. Two or more of these methods may also be used in combination.

[0062] Considering the stability of the introduced gene, it is preferred to directly insert or replace the target gene on the chromosome of the host or on the giant plasmid held by the host, and it is more preferred to directly insert or replace the target gene on the chromosome of the host. In order to reliably express the introduced gene, it is preferably introduced in a manner that the target gene is located downstream of the "gene expression regulatory sequence" originally possessed by the host, or in a form in which the target gene is located downstream of an external "gene expression regulatory sequence". The "gene expression regulatory sequence" in the present invention refers to a DNA sequence comprising a base sequence (such as a promoter sequence) that controls the transcription amount of the gene, and / or a base sequence (such as a Shine-Dalgarno sequence) that regulates the translation amount of the messenger RNA transcribed by the gene. As a "gene expression regulatory sequence", any base sequence existing in nature can be used, and an artificially constructed or modified base sequence can also be used.

[0063] In addition, the method for enhancing the expression level of the target gene originally possessed by the host on the genomic DNA is not particularly limited, and examples thereof include: a method of modifying the "gene expression regulatory sequence" located upstream of the target gene, a method of introducing a foreign "gene expression regulatory sequence" upstream of the target gene, or a method of improving the stability of the transcribed messenger RNA by modifying the target gene and / or its surrounding base sequence, etc.

[0064] Examples of the promoter sequence and Shine-Dalgarno sequence included in the “gene expression regulatory sequence” include, but are not particularly limited to, the base sequences represented by any one of SEQ ID NOs: 10 to 16, or base sequences containing a portion of these base sequences.

[0065] The displacement, deletion, insertion and / or addition of at least a portion of genomic DNA can be carried out by methods well known to those skilled in the art. As a representative method, there is a method utilizing a mechanism of homologous recombination with a transposon (Ohman et al., J. Bacteriol., 162: 1068-1074 (1985)), a method using the site-specific introduction caused by the mechanism of homologous recombination and the shedding caused by the homologous recombination in the second phase as a principle (Noti et al., Methods Enzymol., 154: 197-217 (1987)) etc. In addition, a method can be used to make the sacB gene from Bacillus subtilis coexist and to easily separate the microbial strain with the gene removed as a sucrose resistant strain by homologous recombination in the second stage (Schweizer, Mol. Microbiol., 6: 1195-1204 (1992), Lenz et al., J. Bacteriol., 176: 4385-4393 (1994)). In addition, as another method, a genome editing technology based on the CRISPR / Cas9 system for modifying the target DNA can also be used (Y. Wang et al., ACS Synth Biol. 2016, 5 (7): 721-732). In the CRISPR / Cas9 system, the guide RNA (gRNA) has a sequence that can bind to a part of the base sequence of the genomic DNA to be modified, and has the function of transporting Cas9 to the target site.

[0066] The method for introducing a vector into cells is not particularly limited, and examples thereof include a calcium chloride method, an electroporation method, a polyethylene glycol method, and a spheroplast method.

[0067] By culturing the transformation microorganism of the present embodiment, PHA can be accumulated in the microorganism. As a method for culturing the transformation microorganism of the present embodiment, it can be carried out in a culture medium with an appropriate carbon source according to a conventional microbial culture method. The composition of the culture medium, the method of adding the carbon source, the culture scale, the aeration and stirring conditions, the culture temperature, the culture time, etc. are not particularly limited. The carbon source is preferably added to the culture medium continuously or intermittently.

[0068] As a carbon source during cultivation, any carbon source can be used as long as the transformation microorganism of this embodiment can assimilate it. Examples include: sugars such as glucose, fructose, and sucrose; palm oil, palm kernel oil (including palm olein, palm diolein, palm kernel olein, etc. as low melting point fractions obtained by separation), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, Jatropha oil, etc., their separated oils, or their purified byproducts; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, myristic acid, their derivatives, or glycerol, etc., without particular limitation. In addition, if the transformation microorganism of this embodiment can utilize gases such as carbon dioxide, carbon monoxide, methane, methanol, ethanol, and alcohols, they can also be used as carbon sources.

[0069] In the production of PHA of the present embodiment, it is preferred to culture the above-mentioned microorganisms using a culture medium containing the above-mentioned carbon source, a nitrogen source as a nutrient source other than the carbon source, inorganic salts, and other organic nutrient sources. As nitrogen sources, for example, ammonia; ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate; peptone, meat extract, yeast extract, etc., but are not limited to these. As inorganic salts, for example, potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, etc. can be listed. As other organic nutrient sources, for example, amino acids such as glycine, alanine, serine, threonine, and proline, vitamins such as vitamin B1, vitamin B12, and vitamin C can be listed.

[0070] After culturing for an appropriate period of time to allow PHA to accumulate in the cells, PHA is recovered from the cells using a known method. The recovery method is not particularly limited. For example, after the culture is completed, the cells can be separated from the culture solution using a centrifuge, a separation membrane, etc., and after drying, PHA can be extracted from the dried cells using an organic solvent such as chloroform, and the cell components can be removed from the organic solvent solution containing the PHA by filtration, etc., and a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA, and the supernatant can be removed by filtration or centrifugation, and the PHA can be dried to recover PHA. Alternatively, the cell components other than PHA can be dissolved in water using a surfactant, an alkali, an enzyme, etc., and the PHA particles can be separated from the water phase by filtration or centrifugation, and then dried and recovered.

[0071] According to the present embodiment, since microbial cells with large particle sizes that have accumulated PHA can be obtained, separation of the microbial cells from the culture solution can be easily and efficiently performed.

[0072] Example

[0073] Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited by these examples. It should be noted that all gene manipulations can be performed as described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). In addition, the enzymes and cloning hosts used in gene manipulations can be purchased from suppliers in the market and used according to their instructions. It should be noted that, as enzymes, as long as they can be used in gene manipulations, there is no particular limitation.

[0074] (Production Example 1) Preparation of A0597 Deletion Disruption Strain

[0075] First, a plasmid for gene deletion was prepared. The preparation was performed as described below. By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 17) having the upstream and downstream base sequences of the A0597 structural gene was obtained. The DNA fragment was digested with the restriction endonuclease SwaI, and the obtained DNA fragment was connected with the vector pNS2X-sacB described in Japanese Patent Gazette No. 2007-259708, which was also digested with SwaI, and DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)), and a plasmid vector pNS2X-sacB+A0597UD for gene deletion having the upstream and downstream base sequences of the A0597 structural gene was prepared.

[0076] Next, using the gene deletion disruption plasmid vector pNS2X-sacB+A0597UD, an A0597 deletion disruption strain was prepared as follows.

[0077] The Escherichia coli S17-1 strain (ATCC47055) was transformed with the plasmid vector pNS2X-sacB+A0597UD for gene insertion and destruction, and the transformed microorganism obtained thereby and the KNK-005 strain were mixed and cultured on Nutrient Agar medium (manufactured by Difco) for conjugation transfer. The KNK-005 strain is a transformant in which a PHA synthase gene (a gene encoding a PHA synthase having an amino acid sequence described in sequence number 7) derived from Aeromonas caviae was introduced into the chromosome of the H16 strain of Copperworm nematodes, and can be prepared based on the method described in the specification of U.S. Patent No. 7,384,766.

[0078] The obtained culture solution was inoculated on Simmons' agar medium (sodium citrate 2g / L, sodium chloride 5g / L, magnesium sulfate heptahydrate 0.2g / L, ammonium dihydrogen phosphate 1g / L, dipotassium hydrogen phosphate 1g / L, agar 15g / L, pH 6.8) containing 250mg / L of kanamycin, and strains that can grow on agar medium were selected to obtain strains into which plasmids were introduced on the chromosome of KNK-005 strain. After the strain was cultured for two generations with Nutrient Broth medium (manufactured by Difco), it was diluted and coated on Nutrient Agar medium containing 15% sucrose, and strains that can grow were obtained as strains after plasmid shedding. Further, through analysis based on PCR and DNA sequencer, a strain was isolated in which the start codon to the stop codon of the A0597 structural gene on the chromosome was deleted. The A0597 gene deletion strain was named A0597 deletion destruction strain.

[0079] (Production Example 2) Preparation of A0302 Deletion Disruption Strain

[0080] First, a plasmid for gene deletion was prepared. The preparation was performed as described below. By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 18) having the upstream and downstream base sequences of the A0302 structural gene was obtained. The DNA fragment was digested with the restriction endonuclease SwaI, and the obtained DNA fragment was connected with the vector pNS2X-sacB described in Japanese Patent Gazette No. 2007-259708 and DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)) which was also digested with SwaI, and a plasmid vector pNS2X-sacB+A0302UD for gene deletion having the upstream and downstream base sequences of the A0302 structural gene was prepared.

[0081] Next, the A0302 gene deletion plasmid vector pNS2X-sacB+A0302UD was introduced into the KNK-005 strain by the same method as in Preparation Example 1. Furthermore, a strain in which the start codon to the stop codon of the A0302 structural gene on the chromosome was deleted was isolated by the same method as in Preparation Example 1. The A0302 gene deletion strain was named A0302 deletion disrupted strain.

[0082] (Production Example 3) Preparation of A1386 Deletion Disruption Strain

[0083] First, a plasmid for gene deletion was prepared. The preparation was carried out as described below. By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 19) having the upstream and downstream base sequences of the A1386 structural gene was obtained. The DNA fragment was digested with the restriction endonuclease SwaI, and the obtained DNA fragment was connected with the vector pNS2X-sacB recorded in Japanese Patent Gazette No. 2007-259708 and DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)) which were also digested with SwaI, and a plasmid vector pNS2X-sacB+A1386UD for gene deletion having the upstream and downstream base sequences of the A1386 structural gene was prepared.

[0084] Next, the A1386 gene deletion plasmid vector pNS2X-sacB+A1386UD was introduced into the KNK-005 strain by the same method as in Preparation Example 1. Furthermore, a strain in which the start codon to the stop codon of the A1386 structural gene on the chromosome was deleted was isolated by the same method as in Preparation Example 1. The A1386 gene deletion strain was named A1386 deletion disrupted strain.

[0085] (Production Example 4) Preparation of A2272 insertion-disrupted strain

[0086] First, a plasmid for insertion and destruction of the A2272 gene was prepared. The preparation was carried out as described below. By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 20) having the base sequence of positions 47 to 231 of the A2272 structural gene was obtained. The obtained DNA fragment was digested with the restriction endonuclease SwaI. The DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Gazette No. 2007-259708, which was also digested with SwaI, and DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)) to prepare a plasmid vector pNS2X-sacB-A2272-indel for gene insertion and destruction having the base sequence of positions 47 to 231 of the A2272 structural gene.

[0087] Next, the gene insertion disruption plasmid vector pNS2X-sacB-A2272-indel was introduced into the KNK-005 strain by the same method as in Preparation Example 1. Furthermore, by analysis based on PCR and DNA sequencer, a strain in which the A2272 gene was disrupted by inserting the plasmid into the A2272 structural gene sequence on the chromosome was isolated. The A2272 gene disruption strain was named the A2272 insertion disruption strain.

[0088] (Production Example 5) Preparation of A2405 insertion-disrupted strain

[0089] First, a plasmid for insertion and destruction of the A2405 gene was prepared. The preparation was carried out as described below. By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 21) having the base sequence of positions 7 to 204 of the A2405 structural gene was obtained. The obtained DNA fragment was digested with the restriction endonuclease SwaI. The DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Gazette No. 2007-259708, which was also digested with SwaI, and DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)) to prepare the plasmid vector pNS2X-sacB-A2405-indel for insertion and destruction of the A2405 gene having the base sequence of positions 7 to 204 of the A2405 structural gene.

[0090] Next, the gene insertion disruption plasmid vector pNS2X-sacB-A2405-indel was introduced into the KNK-005 strain by the same method as in Preparation Example 1. Furthermore, by analysis based on PCR and DNA sequencer, a strain in which the A2405 gene was disrupted by inserting the plasmid into the A2405 structural gene sequence on the chromosome was isolated. This A2405 gene disruption strain was named A2405 insertion disruption strain.

[0091] (Production Example 6) Preparation of A2405 Deletion-Disrupted Strain

[0092] First, a plasmid for gene deletion was prepared. The preparation was carried out as described below. By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 22) having the upstream and downstream base sequences of the A2405 structural gene was obtained. The DNA fragment was digested with the restriction endonuclease SwaI, and the obtained DNA fragment was connected with the vector pNS2X-sacB recorded in Japanese Patent Gazette No. 2007-259708 and DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)) which were also digested with SwaI, and a plasmid vector pNS2X-sacB+A2405UD for gene deletion having the upstream and downstream base sequences of the A2405 structural gene was prepared.

[0093] Next, the A2405 gene deletion plasmid vector pNS2X-sacB+A2405UD was introduced into the KNK-005 strain by the same method as in Preparation Example 1. Furthermore, a strain in which the start codon to the stop codon of the A2405 structural gene on the chromosome was deleted was isolated by the same method as in Preparation Example 1. The A2405 gene deletion strain was named A2405 deletion disrupted strain.

[0094] (Production Example 8) Preparation of A1386 / A2405 double disrupted strain

[0095] The plasmid vector pNS2X-sacB+A2405UD for A2405 gene deletion was introduced into the A1386 deletion disrupted strain by the same method as in Preparation Example 1. Furthermore, a strain in which the start codon to the stop codon of the A2405 structural gene on the chromosome was deleted was isolated by the same method as in Preparation Example 1. The strain in which the A1386 gene and the A2405 gene were deleted was named the A1386 / A2405 double disrupted strain.

[0096] (Production Example 9) Preparation of minCD-expressing A1386 deletion disrupted strain

[0097] The preparation of the plasmid vector pNS2X-sacB-PA-minCD for minCD gene expression was carried out. The preparation was carried out as described below. By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 23) having a promoter sequence, a minCD gene sequence, and a base sequence of an introduction region on the genome was obtained. The DNA fragment was digested with the restriction endonuclease SwaI, and the obtained DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Publication No. 2007-259708, which was also digested with SwaI, and DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-PA-minCD for minCD gene expression.

[0098] Next, the plasmid vector pNS2X-sacB-PA-minCD for minCD gene expression was introduced into the A1386 deletion disrupted strain by the same method as in Preparation Example 1. Furthermore, a strain having a promoter sequence and a minCD gene sequence inserted on the chromosome was isolated by the same method as in Preparation Example 1. The minCD gene expression A1386 deletion strain was named as the minCD expression A1386 disrupted strain.

[0099] (Production Example 10) Preparation of minCD-expressing A2405 disrupted strain

[0100] The plasmid vector pNS2X-sacB-PA-minCD for minCD gene expression was introduced into the A2405 deletion disrupted strain by the same method as in Preparation Example 1. Furthermore, a strain having a promoter sequence and a minCD gene sequence inserted into the chromosome was isolated by the same method as in Preparation Example 1. The minCD gene expression A2405 deletion strain was named the minCD expression A2405 disruption strain.

[0101] (Production Example 11) Preparation of minCD-expressing A2405 / A1386 disrupted strain

[0102] The plasmid vector pNS2X-sacB+A1386UD for A1386 gene deletion was introduced into the minCD expression A2405 disrupted strain by the same method as in Preparation Example 1. Furthermore, a strain in which the start codon to the stop codon of the A1386 structural gene on the chromosome was deleted was isolated by the same method as in Preparation Example 1. The minCD gene expression A2405 / A1386 deletion strain was named the minCD expression A2405 / A1386 disrupted strain.

[0103] (Comparative Example 1) PHA production based on KNK-005 strain

[0104] Cultivation studies using the KNK-005 strain were performed under the following conditions.

[0105] (Culture medium)

[0106] The composition of the seed culture medium was set to 1 w / v% meat extract, 1 w / v% Bacto-Tryptone, 0.2 w / v% yeast extract, 0.9 w / v% Na2HPO4·12H2O, 0.15 w / v% KH2PO4, (pH 6.8).

[0107] The composition of the pre-culture medium was set to 1.1w / v% Na2HPO4·12H2O, 0.19w / v% KH2PO4, 1.29w / v% (NH4)2SO4, 0.1w / v% MgSO4·7H2O, 2.5w / v% palm olein, 0.5v / v% trace metal salt solution (1.6w / v% FeCl3·6H2O, 1w / v% CaCl2·2H2O, 0.02w / v% CoCl2·6H2O, 0.016w / v% CuSO4·5H2O, 0.012w / v% NiCl2·6H2O dissolved in 0.1N hydrochloric acid). Palm olein was added as a carbon source at a concentration of 10g / L.

[0108] The composition of the PHA production medium is set to 0.385w / v% Na2HPO4·12H2O, 0.067w / v% KH2PO4, 0.291w / v% (NH4)2SO4, 0.1w / v% MgSO4·7H2O, and 0.5v / v% trace metal salt solution (1.6w / v% FeCl3·6H2O, 1w / v% CaCl2·2H2O, 0.02w / v% CoCl2·6H2O, 0.016w / v% CuSO4·5H2O, and 0.012w / v% NiCl2·6H2O dissolved in 0.1N hydrochloric acid).

[0109] (Method for measuring the ratio of PHA accumulation to dry bacterial cells)

[0110] The ratio of PHA accumulation to dry bacterial cells was determined as follows. The bacterial cells were recovered from the culture solution by centrifugation, washed with ethanol, and freeze-dried to obtain dry bacterial cells. 100 ml of chloroform was added to 1 g of the obtained dry bacterial cells, and the mixture was stirred at room temperature for a day and a night to extract the PHA in the bacterial cells. After filtering out the bacterial residue, the mixture was concentrated to a total volume of 30 ml using an evaporator, and then 90 ml of hexane was gradually added and allowed to stand for 1 hour while slowly stirring. After filtering out the precipitated PHA, it was vacuum dried at 50°C for 3 hours. The weight of the dry PHA was measured, and the ratio of PHA accumulation in the bacterial cells to the dry bacterial cells was calculated.

[0111] (Method for measuring cell diameter)

[0112] The cell diameter was measured as follows. The culture solution after the culture was treated at 65°C for 60 minutes to inactivate the bacterial cells, and then analyzed by a laser diffraction / scattering particle size distribution measuring device (Microtrac MT3300EXII manufactured by Microtrac BEL) to measure the volume mean diameter (MV) of the cells. The measurement was performed under standard settings (particle permeability: transparent, particle refractive index: 1.81, particle shape: non-spherical, solvent refractive index: 1.333).

[0113] (Microscopic observation of cells)

[0114] Microscopic observation of cells was performed as follows. The culture solution after the culture was appropriately diluted, placed on a slide and dried, and then stained with fuchsin. The stained cells were observed under an optical microscope.

[0115] (PHA production and cultivation)

[0116] PHA production culture was carried out as described below. First, a glycerol stock solution (50 μl) of the KNK-005 strain was inoculated into a seed culture medium (10 ml) and cultured for 24 hours to carry out seed culture. Next, the seed culture solution was inoculated at 1.0 v / v% into a 3L fermenter (Jar fermentor) (MDL-300 manufactured by Marubishi Bioengineering) to which 1.8L of pre-culture medium was added. The operating conditions were set to a culture temperature of 33°C, a stirring speed of 500 rpm, and an aeration volume of 1.8 L / min. The pre-culture was carried out by culturing for 28 hours while controlling the pH to between 6.7 and 6.8. A 14% aqueous solution of ammonium hydroxide was used for pH control.

[0117] Next, the pre-culture solution was inoculated at 5.0 v / v% into a 5 L fermenter (MDS-U50 manufactured by Marubishi Bioengineering Co., Ltd.) to which 2.5 L of PHA production medium was added. The operating conditions were set as a culture temperature of 33°C, a stirring speed of 420 rpm, an aeration volume of 2.1 L / min, and the pH was controlled between 6.7 and 6.8. A 25% aqueous ammonium hydroxide solution was used for pH control. A carbon source was added intermittently. Palm olein was used as a carbon source. The culture was carried out until the PHA accumulation reached about 90%. The ratio of PHA accumulation and the cell diameter were measured as described above. The results are shown in Table 1. In addition, photographs taken during microscopic observation of the cells as described above are shown in Figure 1 .

[0118] (Comparative Example 2) PHA production based on A0597 deletion disrupted strain

[0119] Cultivation studies using the A0597 deletion disrupted strain were conducted under the same conditions as in Comparative Example 1. The results of the measurement of the ratio of PHA accumulation and cell diameter are shown in Table 1. In addition, microscopic photographs of the cells performed as described above are shown in Table 1. Figure 2 .

[0120] As a result of the culture study, the cell diameter of the A0597 deletion disrupted strain measured under the above conditions was reduced by more than 10% compared with the parent strain KNK-005. The productivity of PHA was the same as that of KNK-005.

[0121] (Comparative Example 3) PHA production based on A0302 deletion disrupted strain

[0122] Cultivation studies using the A0302 deletion disrupted strain were conducted under the same conditions as in Comparative Example 1. The results of the measurement of the ratio of PHA accumulation and cell diameter are shown in Table 1. In addition, microscopic photographs of the cells performed as described above are shown in Table 1. Figure 3 .

[0123] As a result of the culture study, the cell diameter of the A0302 deletion disrupted strain measured under the above conditions was reduced by more than 10% compared with the parent strain KNK-005. The productivity of PHA was the same as that of KNK-005.

[0124] (Comparative Example 4) PHA production based on A2272 insertion-disrupted strain

[0125] Cultivation studies using the A2272 insertion-disrupted strain were conducted under the same conditions as in Comparative Example 1. The results of the measurement of the ratio of PHA accumulation and cell diameter are shown in Table 1. In addition, microscopic photographs of the cells performed as described above are shown in Table 1. Figure 4 .

[0126] As a result of the culture study, the cell diameter of the A2272 insertion-disrupted strain measured under the above-mentioned conditions was the same as that of the parent strain, KNK-005 strain, and the productivity of PHA was the same as that of KNK-005 strain.

[0127] (Example 1) PHA production based on A1386 deletion disrupted strain

[0128] Cultivation studies using the A1386 deletion disrupted strain were conducted under the same conditions as in Comparative Example 1. The results of the measurement of the ratio of PHA accumulation and cell diameter are shown in Table 1. In addition, microscopic photographs of the cells performed as described above are shown in Table 1. Figure 5 .

[0129] As a result of the culture study, the cell diameter of the A1386 deletion disrupted strain measured under the above conditions was 20% or more larger than that of the parent strain, KNK-005 strain, and the productivity of PHA was the same as that of KNK-005 strain.

[0130] (Example 2) PHA production based on A2405 insertion disruption strain

[0131] Cultivation studies using the A2405 insertion-disrupted strain were conducted under the same conditions as in Comparative Example 1. The results of the measurement of the ratio of PHA accumulation and cell diameter are shown in Table 1. In addition, microscopic photographs of the cells performed as described above are shown in Table 1. Figure 6 .

[0132] As a result of the culture study, the cell diameter of the A2405 insertion-disrupted strain measured under the above conditions was 20% or more larger than that of the parent strain, KNK-005. The productivity of PHA was the same as that of KNK-005.

[0133] (Example 3) PHA production based on A2405 deletion disrupted strain

[0134] Cultivation studies using the A2405 deletion disrupted strain were conducted under the same conditions as in Comparative Example 1. The results of the measurement of the ratio of PHA accumulation and cell diameter are shown in Table 1. In addition, microscopic photographs of the cells performed as described above are shown in Table 1. Figure 7 .

[0135] As a result of the culture study, the cell diameter of the A2405 deletion disrupted strain measured under the above conditions was 20% or more larger than that of the parent strain, KNK-005 strain, and the productivity of PHA was the same as that of KNK-005 strain.

[0136] (Example 4) PHA production based on A1386 / A2405 double disrupted strain

[0137] Cultivation studies using the A1386 / A2405 double disrupted strain were conducted under the same conditions as in Comparative Example 1. The results of the measurement of the ratio of PHA accumulation and cell diameter are shown in Table 1. In addition, microscopic photographs of the cells performed as described above are shown in Table 1. Figure 8 .

[0138] The results of the culture study showed that the cell diameter of the A1386 / A2405 double disrupted strain measured under the above conditions was 40% larger than that of the parent strain KNK-005. That is, A1386 disruption and A2405 disruption had a synergistic or additive effect on the increase in cell diameter. The productivity of PHA was the same as that of KNK-005.

[0139] (Example 5) PHA production based on minCD expression A1386 disrupted strain

[0140] Cultivation studies using the minCD expression A1386 disrupted strain were conducted under the same conditions as in Comparative Example 1. The results of the measurement of the ratio of PHA accumulation and cell diameter are shown in Table 1. In addition, microscopic photographs of the cells performed as described above are shown in Table 1. Fig. 9 .

[0141] As a result of the culture study, the cell diameter of the minCD expression A1386 disrupted strain measured under the above conditions was 20% or more larger than that of the parent strain KNK-005. The productivity of PHA was the same as that of the KNK-005 strain.

[0142] (Example 6) PHA production based on minCD expression A2405 disrupted strain

[0143] Cultivation studies using the minCD expression A2405 disrupted strain were conducted under the same conditions as in Comparative Example 1. The results of the measurement of the ratio of PHA accumulation and cell diameter are shown in Table 1. In addition, microscopic photographs of the cells performed as described above are shown in Table 1. Fig.10 .

[0144] The results of the culture study showed that the cell diameter of the minCD expression A2405 disrupted strain measured under the above conditions was 40% or more larger than that of the parent strain KNK-005. That is, A2405 disruption and minCD expression have a synergistic or additive effect on the increase in cell diameter. The productivity of PHA is the same as that of the KNK-005 strain.

[0145] (Example 7) PHA production based on minCD expression A2405 / A1386 disrupted strain

[0146] Cultivation studies using the minCD expression A2405 / A1386 disrupted strain were conducted under the same conditions as in Comparative Example 1. The results of the measurement of the ratio of PHA accumulation and cell diameter are shown in Table 1. In addition, microscopic photographs of the cells performed as described above are shown in Table 1. Fig.11 .

[0147] The results of the culture study showed that the cell diameter of the minCD expression A2405 / A1386 disrupted strain measured under the above conditions was 50% or more larger than that of the parent strain KNK-005. That is, A1386 disruption, A2405 disruption, and minCD expression had a synergistic or additive effect on the increase in cell diameter. The productivity of PHA was the same as that of the KNK-005 strain.

[0148] It should be noted that the PHA produced in the culture studies of Comparative Examples and Examples was confirmed to be PHBH by HPLC analysis.

[0149] [Table 1]

[0150] Sequence Listing <110> KANEKA CORPORATION <120> Transformed microorganism, and method for producing polyhydroxyalkanoate using the same <130> B190307WO01 <160> twenty three <170> PatentIn version 3.5 <210> 1 <211> 380 <212> PRT <213> Cupriavidus necator <400> 1 Met Ser Met Val Ala Ala Pro Val Ala Glu Ala Ala Thr Lys Ser Ala 1 5 10 15 Thr Thr Gln Lys Thr Ser Lys Lys Gln Val Lys Ser Val Asn Ala Glu 20 25 30 Lys Lys Gly Ala Ser Lys Ile Val Ala Lys Ser Ser Arg Ser Gly Lys 35 40 45 Val Ala Lys Arg Glu Ala Ser Ser Thr Arg Lys Val Val Val Leu Lys 50 55 60 Asn Gly Lys Arg His Thr Val Val Ala Gln Arg Ala Ala Pro Val Arg 65 70 75 80 Ala Ala Phe Thr Pro Ala Lys Pro Ser Leu Gly Glu Ala Met Gly Leu 85 90 95 Arg Asp Thr Asp Asp Ala Leu Ala Leu Arg Ser Ser Val Ala Leu Val 100 105 110 Met Asp Gln Asn Ser Asn Glu Val Leu Phe Gln Lys Asn Ala Ser Ala 115 120 125 Val Leu Pro Ile Ala Ser Ile Thr Lys Leu Met Thr Ala Leu Val Val 130 135 140 Met Asp Ala Arg Leu Pro Met Asp Glu Val Leu Thr Ile Ser Glu Glu 145 150 155 160 Asp Arg Asp Thr Glu Lys His Ser Gly Ser Arg Leu Arg Phe Gly Thr 165 170 175 Gln Leu Thr Arg Gln Glu Leu Leu Leu Leu Ala Leu Met Ser Ser Glu 180 185 190 Asn Arg Ala Ala Ser Ala Leu Gly Arg Asn Tyr Pro Gly Gly Leu Pro 195 200 205 Ala Phe Val Gln Ala Met Asn Arg Lys Ala Arg Glu Leu Gly Met Asn 210 215 220 Asp Ser His Phe Val Asp Ser Ser Gly Leu Ser Ser Ser Asn Val Ser 225 230 235 240 Ser Ala Thr Asp Leu Val Arg Met Val Asn Ala Ala Tyr Arg Asn Pro 245 250 255 Thr Ile Arg Glu Phe Ser Thr Gln Thr Glu His Glu Val Asn Val Leu 260 265 270 Gly Arg Thr Gln His Tyr Val Ser Thr Asn Arg Leu Val Arg Gly Gly 275 280 285 Asn Trp Glu Ile Gly Leu Gln Lys Thr Gly Phe Ile Ser Glu Ala Gly 290 295 300 Gln Cys Leu Val Met Gln Ala Lys Val Gln Gly Arg Asn Val Val Met 305 310 315 320 Val Phe Leu Asp Ser Ala Gly Lys Leu Ser Arg Phe Ala Asp Ala Asn 325 330 335 Arg Val Lys Asp Trp Leu Glu His Ala Pro Ser Pro Ser Ser Pro Gln 340 345 350 Arg Gly Phe Pro Ser Ser Pro Asn Leu Thr Gln Ala Pro Gly Gly Ala 355 360 365 His Ala Ile Leu Ala Ser Gln Gln Ser Arg Gly Ile 370 375 380 <210> 2 <211> 226 <212> PRT <213> Cupriavidus necator <400> 2 Met Gln Arg Ser Val Leu His Ser Leu Ala Arg Ala Ala Val Gly Ile 1 5 10 15 Ala Ile Val Cys Gly Ala Thr Val Ser Asn Gly Val Leu Ala Asp Thr 20 25 30 Val Phe Lys Asp Ala Asp Ala Arg Ile Asp Ala Thr Ala His Ala Ala 35 40 45 Asp Ser His Ala Glu Gly Lys Arg Gly Leu Leu Ser Ser Val Val Asn 50 55 60 Ser Thr Ser Asn Val Ala Ser Lys Ala Gly Asp Leu Val Met Asn Ala 65 70 75 80 Leu Gly Leu Ile Gly Val Arg Tyr Arg Phe Gly Gly Asn Ser Pro Glu 85 90 95 Ser Gly Leu Asp Cys Ser Gly Phe Val Arg Tyr Val Phe Gln Asp Thr 100 105 110 Phe Gly Phe Met Leu Pro Arg Arg Ser Val Glu Ile Ser Arg Val Gly 115 120 125 Thr Thr Val Ala Ala Thr Asp Leu Arg Pro Gly Asp Leu Val Phe Phe 130 135 140 Asn Thr Met Arg Gln Thr Phe Ser His Val Gly Ile Tyr Ile Gly Asp 145 150 155 160 Asn Lys Phe Val His Ala Pro Ser Thr Gly Ser Lys Ile Arg Val Asp 165 170 175 Asp Met Arg Ala Ser Tyr Trp Val Thr Arg Tyr Asn Gly Ala Arg Arg 180 185 190 Ile Glu Asp Gly Lys Glu Gly Gly Ala Asp Gly Leu Gly Asp Met Val 195 200 205 Glu Thr Leu Lys Arg Tyr Asp Pro Lys Val Val Arg Ala Ser Met Tyr 210 215 220 Gly Gly 225 <210> 3 <211> 270 <212> PRT <213> Cupriavidus necator <400> 3 Met Ser Gln Lys Lys Ser Pro Arg Phe Glu Leu Arg Ser Gly Asn Val 1 5 10 15 Asp Ala Leu Leu Leu Ala Leu Gln Thr Ala Asp Met Ala Ala Leu Arg 20 25 30 Asp Asp Leu Leu Ala Arg Phe Glu Ala Thr Pro Asp Phe Phe Ser Asn 35 40 45 Asp Val Ile Ala Leu Asp Leu Arg Ala Leu Glu Asp Asp Ser Glu Val 50 55 60 Ala Leu Gly Thr Val Ile Glu Thr Leu Ala Thr Leu Arg Ala Arg Ala 65 70 75 80 Ile Gly Val Val Ala Arg Pro Gly Gln Arg Glu Trp Ala Glu Arg Phe 85 90 95 Gly Leu Pro Leu Leu Asp Ser Gln Ala Arg Arg Gly Ser Gly Ala Asp 100 105 110 Arg Ala Thr Asp Arg Ala Ala Glu Ala Arg Ala Ala Ala Ala Ala Glu 115 120 125 Gln Ala Ala Ala Asp Gln Ala Ala Arg Glu Glu Ser Ile Arg Ala Ala 130 135 140 Ala Gln Ala Thr Thr Asp Ala Ala Val Ala Ala Ala Ile Arg Gln Thr 145 150 155 160 Gln Thr Met Leu Ile Asp Lys Pro Leu Arg Ser Gly Gln Gln Val Tyr 165 170 175 Ala Gln Gly Asp Val Val Ile Leu Asp Val Val Ser Tyr Gly Ala Glu 180 185 190 Val Ile Ala Glu Gly Asn Ile His Ile Tyr Ala Pro Leu Arg Gly Arg 195 200 205 Ala Leu Ala Gly Val Lys Gly Asn Thr Gly Ala Arg Ile Phe Ser Thr 210 215 220 Cys Met Glu Pro Glu Leu Ile Ser Ile Ala Gly Ile Tyr Arg Thr Ala 225 230 235 240 Glu Gln Thr Leu Pro Ala Asp Val Leu Gly Lys Thr Ala Gln Val Arg 245 250 255 Leu Ala Asp Glu Lys Leu Ile Leu Glu Ala Leu Arg Leu Lys 260 265 270 <210> 4 <211> 271 <212> PRT <213> Cupriavidus necator (Cupriavidus necator) <400> 4 Met Ala Lys Ile Ile Val Val Thr Ser Gly Lys Gly Gly Val Gly Lys 1 5 10 15 Thr Thr Thr Ser Ala Ser Phe Ala Ala Gly Leu Ala Leu Arg Gly His 20 25 30 Lys Thr Ala Val Ile Asp Phe Asp Val Gly Leu Arg Asn Leu Asp Leu 35 40 45 Ile Met Gly Cys Glu Arg Arg Val Val Tyr Asp Leu Ile Asn Val Val 50 55 60 Gln Gly Glu Ala Asn Leu Arg Gln Ala Leu Ile Lys Asp Lys Lys Cys 65 70 75 80 Glu Asn Leu Phe Ile Leu Pro Ala Ser Gln Thr Arg Asp Lys Asp Ala 85 90 95 Leu Thr Arg Glu Gly Val Glu Lys Val Ile Asn Gly Leu Ile Glu Met 100 105 110 Asp Phe Glu Phe Ile Ile Cys Asp Ser Pro Ala Gly Ile Glu Ser Gly 115 120 125 Ala Leu Met Ala Met Tyr Phe Ala Asp Glu Ala Leu Ile Val Thr Asn 130 135 140 Pro Glu Val Ser Ser Val Arg Asp Ser Asp Arg Ile Leu Gly Ile Leu 145 150 155 160 Ala Ser Lys Thr Lys Arg Ala Ser Glu Gly Gly Asp Pro Ile Lys Glu 165 170 175 His Leu Leu Ile Thr Arg Tyr Asn Pro Lys Arg Val His Gly Gly Glu 180 185 190 Met Leu Ser Leu Thr Asp Ile Gln Glu Ile Leu Arg Ile Lys Leu Ile 195 200 205 Gly Val Val Pro Glu Ser Glu Ala Val Leu His Ala Ser Asn Gln Gly 210 215 220 Thr Pro Ala Ile His Leu Glu Gly Ser Asp Val Ala Asp Ala Tyr Gly 225 230 235 240 Asp Val Val Asp Arg Phe Leu Gly Lys Asp Lys Pro Met Arg Phe Thr 245 250 255 Asp Tyr Gln Lys Pro Gly Leu Leu Ser Arg Ile Phe Gly Asn Lys 260 265 270 <210> 5 <211> 589 <212> PRT <213> Cupriavidus necator <400> 5 Met Ala Thr Gly Lys Gly Ala Ala Ala Ser Thr Gln Glu Gly Lys Ser 1 5 10 15 Gln Pro Phe Lys Val Thr Pro Gly Pro Phe Asp Pro Ala Thr Trp Leu 20 25 30 Glu Trp Ser Arg Gln Trp Gln Gly Thr Glu Gly Asn Gly His Ala Ala 35 40 45 Ala Ser Gly Ile Pro Gly Leu Asp Ala Leu Ala Gly Val Lys Ile Ala 50 55 60 Pro Ala Gln Leu Gly Asp Ile Gln Gln Arg Tyr Met Lys Asp Phe Ser 65 70 75 80 Ala Leu Trp Gln Ala Met Ala Glu Gly Lys Ala Glu Ala Thr Gly Pro 85 90 95 Leu His Asp Arg Arg Phe Ala Gly Asp Ala Trp Arg Thr Asn Leu Pro 100 105 110 Tyr Arg Phe Ala Ala Ala Phe Tyr Leu Leu Asn Ala Arg Ala Leu Thr 115 120 125 Glu Leu Ala Asp Ala Val Glu Ala Asp Ala Lys Thr Arg Gln Arg Ile 130 135 140 Arg Phe Ala Ile Ser Gln Trp Val Asp Ala Met Ser Pro Ala Asn Phe 145 150 155 160 Leu Ala Thr Asn Pro Glu Ala Gln Arg Leu Leu Ile Glu Ser Gly Gly 165 170 175 Glu Ser Leu Arg Ala Gly Val Arg Asn Met Met Glu Asp Leu Thr Arg 180 185 190 Gly Lys Ile Ser Gln Thr Asp Glu Ser Ala Phe Glu Val Gly Arg Asn 195 200 205 Val Ala Val Thr Glu Gly Ala Val Val Phe Glu Asn Glu Tyr Phe Gln 210 215 220 Leu Leu Gln Tyr Lys Pro Leu Thr Asp Lys Val His Ala Arg Pro Leu 225 230 235 240 Leu Met Val Pro Pro Cys Ile Asn Lys Tyr Tyr Ile Leu Asp Leu Gln 245 250 255 Pro Glu Ser Ser Leu Val Arg His Val Val Glu Gln Gly His Thr Val 260 265 270 Phe Leu Val Ser Trp Arg Asn Pro Asp Ala Ser Met Ala Gly Ser Thr 275 280 285 Trp Asp Asp Tyr Ile Glu His Ala Ala Ile Arg Ala Ile Glu Val Ala 290 295 300 Arg Asp Ile Ser Gly Gln Asp Lys Ile Asn Val Leu Gly Phe Cys Val 305 310 315 320 Gly Gly Thr Ile Val Ser Thr Ala Leu Ala Val Leu Ala Ala Arg Gly 325 330 335 Glu His Pro Ala Ala Ser Val Thr Leu Leu Thr Thr Leu Leu Asp Phe 340 345 350 Ala Asp Thr Gly Ile Leu Asp Val Phe Val Asp Glu Gly His Val Gln 355 360 365 Leu Arg Glu Ala Thr Leu Gly Gly Gly Ala Gly Ala Pro Cys Ala Leu 370 375 380 Leu Arg Gly Leu Glu Leu Ala Asn Thr Phe Ser Phe Leu Arg Pro Asn 385 390 395 400 Asp Leu Val Trp Asn Tyr Val Val Asp Asn Tyr Leu Lys Gly Asn Thr 405 410 415 Pro Val Pro Phe Asp Leu Leu Phe Trp Asn Gly Asp Ala Thr Asn Leu 420 425 430 Pro Gly Pro Trp Tyr Cys Trp Tyr Leu Arg His Thr Tyr Leu Gln Asn 435 440 445 Glu Leu Lys Val Pro Gly Lys Leu Thr Val Cys Gly Val Pro Val Asp 450 455 460 Leu Ala Ser Ile Asp Val Pro Thr Tyr Ile Tyr Gly Ser Arg Glu Asp 465 470 475 480 His Ile Val Pro Trp Thr Ala Ala Tyr Ala Ser Thr Ala Leu Leu Ala 485 490 495 Asn Lys Leu Arg Phe Val Leu Gly Ala Ser Gly His Ile Ala Gly Val 500 505 510 Ile Asn Pro Pro Ala Lys Asn Lys Arg Ser His Trp Thr Asn Asp Ala 515 520 525 Leu Pro Glu Ser Pro Gln Gln Trp Leu Ala Gly Ala Ile Glu His His 530 535 540 Gly Ser Trp Trp Pro Asp Trp Thr Ala Trp Leu Ala Gly Gln Ala Gly 545 550 555 560 Ala Lys Arg Ala Ala Pro Ala Asn Tyr Gly Asn Ala Arg Tyr Arg Ala 565 570 575 Ile Glu Pro Ala Pro Gly Arg Tyr Val Lys Ala Lys Ala 580 585 <210> 6 <211> 594 <212> PRT <213> Aeromonas caviae <400> 6 Met Ser Gln Pro Ser Tyr Gly Pro Leu Phe Glu Ala Leu Ala His Tyr 1 5 10 15 Asn Asp Lys Leu Leu Ala Met Ala Lys Ala Gln Thr Glu Arg Thr Ala 20 25 30 Gln Ala Leu Leu Gln Thr Asn Leu Asp Asp Leu Gly Gln Val Leu Glu 35 40 45 Gln Gly Ser Gln Gln Pro Trp Gln Leu Ile Gln Ala Gln Met Asn Trp 50 55 60 Trp Gln Asp Gln Leu Lys Leu Met Gln His Thr Leu Leu Lys Ser Ala 65 70 75 80 Gly Gln Pro Ser Glu Pro Val Ile Thr Pro Glu Arg Ser Asp Arg Arg 85 90 95 Phe Lys Ala Glu Ala Trp Ser Glu Gln Pro Ile Tyr Asp Tyr Leu Lys 100 105 110 Gln Ser Tyr Leu Leu Thr Ala Arg His Leu Leu Ala Ser Val Asp Ala 115 120 125 Leu Glu Gly Val Pro Gln Lys Ser Arg Glu Arg Leu Arg Phe Phe Thr 130 135 140 Arg Gln Tyr Val Asn Ala Met Ala Pro Ser Asn Phe Leu Ala Thr Asn 145 150 155 160 Pro Glu Leu Leu Lys Leu Thr Leu Glu Ser Asp Gly Gln Asn Leu Val 165 170 175 Arg Gly Leu Ala Leu Leu Ala Glu Asp Leu Glu Arg Ser Ala Asp Gln 180 185 190 Leu Asn Ile Arg Leu Thr Asp Glu Ser Ala Phe Glu Leu Gly Arg Asp 195 200 205 Leu Ala Leu Thr Pro Gly Arg Val Val Gln Arg Thr Glu Leu Tyr Glu 210 215 220 Leu Ile Gln Tyr Ser Pro Thr Thr Glu Thr Val Gly Lys Thr Pro Val 225 230 235 240 Leu Ile Val Pro Pro Phe Ile Asn Lys Tyr Tyr Ile Met Asp Met Arg 245 250 255 Pro Gln Asn Ser Leu Val Ala Trp Leu Val Ala Gln Gly Gln Thr Val 260 265 270 Phe Met Ile Ser Trp Arg Asn Pro Gly Val Ala Gln Ala Gln Ile Asp 275 280 285 Leu Asp Asp Tyr Val Val Asp Gly Val Ile Ala Ala Leu Asp Gly Val 290 295 300 Glu Ala Ala Thr Gly Glu Arg Glu Val His Gly Ile Gly Tyr Cys Ile 305 310 315 320 Gly Gly Thr Ala Leu Ser Leu Ala Met Gly Trp Leu Ala Ala Arg Arg 325 330 335 Gln Lys Gln Arg Val Arg Thr Ala Thr Leu Phe Thr Thr Leu Leu Asp 340 345 350 Phe Ser Gln Pro Gly Glu Leu Gly Ile Phe Ile His Glu Pro Ile Ile 355 360 365 Ala Ala Leu Glu Ala Gln Asn Glu Ala Lys Gly Ile Met Asp Gly Arg 370 375 380 Gln Leu Ala Val Ser Phe Ser Leu Leu Arg Glu Asn Ser Leu Tyr Trp 385 390 395 400 Asn Tyr Tyr Ile Asp Ser Tyr Leu Lys Gly Gln Ser Pro Val Ala Phe 405 410 415 Asp Leu Leu His Trp Asn Ser Asp Ser Thr Asn Val Ala Gly Lys Thr 420 425 430 His Asn Ser Leu Leu Arg Arg Leu Tyr Leu Glu Asn Gln Leu Val Lys 435 440 445 Gly Glu Leu Lys Ile Arg Asn Thr Arg Ile Asp Leu Gly Lys Val Lys 450 455 460 Thr Pro Val Leu Leu Val Ser Ala Val Asp Asp His Ile Ala Leu Trp 465 470 475 480 Gln Gly Thr Trp Gln Gly Met Lys Leu Phe Gly Gly Glu Gln Arg Phe 485 490 495 Leu Leu Ala Glu Ser Gly His Ile Ala Gly Ile Ile Asn Pro Pro Ala 500 505 510 Ala Asn Lys Tyr Gly Phe Trp His Asn Gly Ala Glu Ala Glu Ser Pro 515 520 525 Glu Ser Trp Leu Ala Gly Ala Thr His Gln Gly Gly Ser Trp Trp Pro 530 535 540 Glu Met Met Gly Phe Ile Gln Asn Arg Asp Glu Gly Ser Glu Pro Val 545 550 555 560 Pro Ala Arg Val Pro Glu Glu Gly Leu Ala Pro Ala Pro Gly His Tyr 565 570 575 Val Lys Val Arg Leu Asn Pro Val Phe Ala Cys Pro Thr Glu Glu Asp 580 585 590 Ala Ala <210> 7 <211> 594 <212> PRT <213> Aeromonas caviae <400> 7 Met Ser Gln Pro Ser Tyr Gly Pro Leu Phe Glu Ala Leu Ala His Tyr 1 5 10 15 Asn Asp Lys Leu Leu Ala Met Ala Lys Ala Gln Thr Glu Arg Thr Ala 20 25 30 Gln Ala Leu Leu Gln Thr Asn Leu Asp Asp Leu Gly Gln Val Leu Glu 35 40 45 Gln Gly Ser Gln Gln Pro Trp Gln Leu Ile Gln Ala Gln Met Asn Trp 50 55 60 Trp Gln Asp Gln Leu Lys Leu Met Gln His Thr Leu Leu Lys Ser Ala 65 70 75 80 Gly Gln Pro Ser Glu Pro Val Ile Thr Pro Glu Arg Ser Asp Arg Arg 85 90 95 Phe Lys Ala Glu Ala Trp Ser Glu Gln Pro Ile Tyr Asp Tyr Leu Lys 100 105 110 Gln Ser Tyr Leu Leu Thr Ala Arg His Leu Leu Ala Ser Val Asp Ala 115 120 125 Leu Glu Gly Val Pro Gln Lys Ser Arg Glu Arg Leu Arg Phe Phe Thr 130 135 140 Arg Gln Tyr Val Ser Ala Met Ala Pro Ser Asn Phe Leu Ala Thr Asn 145 150 155 160 Pro Glu Leu Leu Lys Leu Thr Leu Glu Ser Gly Gly Gln Asn Leu Val 165 170 175 Arg Gly Leu Ala Leu Leu Ala Glu Asp Leu Glu Arg Ser Ala Asp Gln 180 185 190 Leu Asn Ile Arg Leu Thr Asp Glu Ser Ala Phe Glu Leu Gly Arg Asp 195 200 205 Leu Ala Leu Thr Pro Gly Arg Val Val Gln Arg Thr Glu Leu Tyr Glu 210 215 220 Leu Ile Gln Tyr Ser Pro Thr Thr Glu Thr Val Gly Lys Thr Pro Val 225 230 235 240 Leu Ile Val Pro Pro Phe Ile Asn Lys Tyr Tyr Ile Met Asp Met Arg 245 250 255 Pro Gln Asn Ser Leu Val Ala Trp Leu Val Ala Gln Gly Gln Thr Val 260 265 270 Phe Met Ile Ser Trp Arg Asn Pro Gly Val Ala Gln Ala Gln Ile Asp 275 280 285 Leu Asp Asp Tyr Val Val Asp Gly Val Ile Ala Ala Leu Asp Gly Val 290 295 300 Glu Ala Ala Thr Gly Glu Arg Glu Val His Gly Ile Gly Tyr Cys Ile 305 310 315 320 Gly Gly Thr Ala Leu Ser Leu Ala Met Gly Trp Leu Ala Ala Arg Arg 325 330 335 Gln Lys Gln Arg Val Arg Thr Ala Thr Leu Phe Thr Thr Leu Leu Asp 340 345 350 Phe Ser Gln Pro Gly Glu Leu Gly Ile Phe Ile His Glu Pro Ile Ile 355 360 365 Ala Ala Leu Glu Ala Gln Asn Glu Ala Lys Gly Ile Met Asp Gly Arg 370 375 380 Gln Leu Ala Val Ser Phe Ser Leu Leu Arg Glu Asn Ser Leu Tyr Trp 385 390 395 400 Asn Tyr Tyr Ile Asp Ser Tyr Leu Lys Gly Gln Ser Pro Val Ala Phe 405 410 415 Asp Leu Leu His Trp Asn Ser Asp Ser Thr Asn Val Ala Gly Lys Thr 420 425 430 His Asn Ser Leu Leu Arg Arg Leu Tyr Leu Glu Asn Gln Leu Val Lys 435 440 445 Gly Glu Leu Lys Ile Arg Asn Thr Arg Ile Asp Leu Gly Lys Val Lys 450 455 460 Thr Pro Val Leu Leu Val Ser Ala Val Asp Asp His Ile Ala Leu Trp 465 470 475 480 Gln Gly Thr Trp Gln Gly Met Lys Leu Phe Gly Gly Glu Gln Arg Phe 485 490 495 Leu Leu Ala Glu Ser Gly His Ile Ala Gly Ile Ile Asn Pro Pro Ala 500 505 510 Ala Asn Lys Tyr Gly Phe Trp His Asn Gly Ala Glu Ala Glu Ser Pro 515 520 525 Glu Ser Trp Leu Ala Gly Ala Thr His Gln Gly Gly Ser Trp Trp Pro 530 535 540 Glu Met Met Gly Phe Ile Gln Asn Arg Asp Glu Gly Ser Glu Pro Val 545 550 555 560 Pro Ala Arg Val Pro Glu Glu Gly Leu Ala Pro Ala Pro Gly His Tyr 565 570 575 Val Lys Val Arg Leu Asn Pro Val Phe Ala Cys Pro Thr Glu Glu Asp 580 585 590 Ala Ala <210> 8 <211> 567 <212> PRT <213> Chromobacterium sp. <400> 8 Met Gln Gln Phe Val Asn Ser Leu Ser Leu Gly Gln Asp Gln Ser Asp 1 5 10 15 Ala Pro His Pro Leu Thr Gly Ala Trp Ser Gln Leu Met Ser Gln Thr 20 25 30 Asn Gln Leu Leu Gln Leu Gln Ser Ser Leu Tyr Gln Gln Gln Leu Gly 35 40 45 Leu Trp Thr Gln Phe Leu Gly Gln Thr Ala Gly Asn Asp Ala Ser Ala 50 55 60 Pro Ser Ala Lys Pro Ser Asp Arg Arg Phe Ala Ser Pro Glu Trp Asp 65 70 75 80 Glu His Pro Phe Tyr Ser Phe Leu Lys Gln Ser Tyr Leu Gln Thr Ser 85 90 95 Lys Trp Met Met Glu Leu Val Asp Lys Thr Gln Ile Asp Glu Ser Ala 100 105 110 Lys Asp Lys Leu Ser Phe Ala Thr Arg Gln Tyr Leu Asp Ala Met Ala 115 120 125 Pro Ser Asn Phe Met Leu Thr Asn Pro Asp Val Val Lys Arg Ala Ile 130 135 140 Glu Thr Gln Gly Glu Ser Leu Val Glu Gly Met Lys Asn Met Met Glu 145 150 155 160 Asp Ile Gln Lys Gly His Ile Ser Met Ser Asp Glu Ser Lys Phe Gln 165 170 175 Ile Gly Lys Asn Leu Val Val Thr Pro Gly Glu Val Val Phe Arg Asn 180 185 190 Glu Leu Ile Glu Leu Ile Gln Tyr Thr Pro Thr Thr Glu Lys Val His 195 200 205 Glu Lys Pro Leu Leu Phe Val Pro Pro Cys Ile Asn Lys Tyr Tyr Leu 210 215 220 Met Asp Leu Gln Pro Asp Asn Ser Met Val Arg His Phe Val Gly Gln 225 230 235 240 Gly Tyr Arg Val Phe Leu Val Ser Trp Arg Ser Ala Val Pro Glu Met 245 250 255 Lys Asn Phe Thr Trp Glu Thr Tyr Ile Glu Lys Gly Val Phe Ala Ala 260 265 270 Ala Glu Ala Val Gln Lys Ile Thr Lys Gln Pro Thr Met Asn Ala Leu 275 280 285 Gly Phe Cys Val Gly Gly Val Ile Leu Thr Thr Ala Leu Cys Val Ala 290 295 300 Gln Ala Lys Gly Leu Lys Tyr Phe Asp Ser Ala Thr Phe Met Thr Ser 305 310 315 320 Leu Ile Asp His Ala Glu Pro Gly Glu Ile Ser Phe Phe Ile Asp Glu 325 330 335 Ala Leu Val Ala Ser Arg Glu Ala Lys Met Ala Ala Gly Gly Ile Ile 340 345 350 Ser Gly Lys Glu Ile Gly Arg Thr Phe Ala Ser Leu Arg Ala Asn Asp 355 360 365 Leu Val Trp Asn Tyr Val Val Asn Asn Tyr Leu Leu Gly Lys Thr Pro 370 375 380 Ala Pro Phe Asp Leu Leu Tyr Trp Asn Asn Asp Ala Val Asp Leu Pro 385 390 395 400 Leu Pro Met His Thr Phe Met Leu Arg Gln Phe Tyr Ile Asn Asn Ala 405 410 415 Leu Ile Thr Pro Gly Ala Ile Thr Leu Cys Gly Val Pro Ile Asp Ile 420 425 430 Ser Lys Ile Asp Ile Pro Val Tyr Met Phe Ala Ala Arg Glu Asp His 435 440 445 Ile Val Leu Trp Ser Ser Ala Tyr Ser Gly Leu Lys Tyr Leu Ser Gly 450 455 460 Thr Pro Ser Arg Arg Phe Val Leu Gly Ala Ser Gly His Ile Ala Gly 465 470 475 480 Ser Ile Asn Pro Val Thr Lys Asp Lys Arg Asn Tyr Trp Thr Asn Glu 485 490 495 Gln Leu Pro Val Asn Pro Glu Glu Trp Leu Glu Gly Ala Gln Ser His 500 505 510 Pro Gly Ser Trp Trp Lys Asp Trp Asp Ala Trp Leu Ala Pro Gln Ser 515 520 525 Gly Lys Gln Val Pro Ala Pro Lys Met Leu Gly Ser Lys Glu Phe Pro 530 535 540 Pro Leu Gln Pro Ala Pro Gly Ser Tyr Val Leu Ala Lys Ala Met Pro 545 550 555 560 Pro Val Ala Ala Ala Leu Asn 565 <210> 9 <211> 559 <212> PRT <213> Pseudomonas sp. <400> 9 Met Ser Asn Lys Asn Ser Asp Asp Leu Asn Arg Gln Ala Ser Glu Asn 1 5 10 15 Thr Leu Gly Leu Asn Pro Val Ile Gly Leu Arg Gly Lys Asp Leu Leu 20 25 30 Thr Ser Ala Arg Met Val Leu Thr Gln Ala Ile Lys Gln Pro Ile His 35 40 45 Ser Val Lys His Val Ala His Phe Gly Ile Glu Leu Lys Asn Val Met 50 55 60 Phe Gly Lys Ser Lys Leu Gln Pro Glu Ser Asp Asp Arg Arg Phe Asn 65 70 75 80 Asp Pro Ala Trp Ser Gln Asn Pro Leu Tyr Lys Arg Tyr Leu Gln Thr 85 90 95 Tyr Leu Ala Trp Arg Lys Glu Leu His Asp Trp Ile Gly Asn Ser Lys 100 105 110 Leu Ser Glu Gln Asp Ile Asn Arg Ala His Phe Val Ile Thr Leu Met 115 120 125 Thr Glu Ala Met Ala Pro Thr Asn Ser Ala Ala Asn Pro Ala Ala Val 130 135 140 Lys Arg Phe Phe Glu Thr Gly Gly Lys Ser Leu Leu Asp Gly Leu Thr 145 150 155 160 His Leu Ala Lys Asp Leu Val Asn Asn Gly Gly Met Pro Ser Gln Val 165 170 175 Asp Met Gly Ala Phe Glu Val Gly Lys Ser Leu Gly Thr Thr Glu Gly 180 185 190 Ala Val Val Phe Arg Asn Asp Val Leu Glu Leu Ile Gln Tyr Arg Pro 195 200 205 Thr Thr Glu Gln Val His Glu Arg Pro Leu Leu Val Val Pro Pro Gln 210 215 220 Ile Asn Lys Phe Tyr Val Phe Asp Leu Ser Pro Asp Lys Ser Leu Ala 225 230 235 240 Arg Phe Cys Leu Ser Asn Asn Gln Gln Thr Phe Ile Val Ser Trp Arg 245 250 255 Asn Pro Thr Lys Ala Gln Arg Glu Trp Gly Leu Ser Thr Tyr Ile Asp 260 265 270 Ala Leu Lys Glu Ala Val Asp Val Val Ser Ala Ile Thr Gly Ser Lys 275 280 285 Asp Ile Asn Met Leu Gly Ala Cys Ser Gly Gly Ile Thr Cys Thr Ala 290 295 300 Leu Leu Gly His Tyr Ala Ala Leu Gly Glu Lys Lys Val Asn Ala Leu 305 310 315 320 Thr Leu Leu Val Ser Val Leu Asp Thr Thr Leu Asp Ser Gln Val Ala 325 330 335 Leu Phe Val Asp Glu Lys Thr Leu Glu Ala Ala Lys Arg His Ser Tyr 340 345 350 Gln Ala Gly Val Leu Glu Gly Arg Asp Met Ala Lys Val Phe Ala Trp 355 360 365 Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr Trp Val Asn Asn Tyr Leu 370 375 380 Leu Gly Asn Glu Pro Pro Val Phe Asp Ile Leu Phe Trp Asn Asn Asp 385 390 395 400 Thr Thr Arg Leu Pro Ala Ala Phe His Gly Asp Leu Ile Glu Met Phe 405 410 415 Lys Asn Asn Pro Leu Val Arg Ala Asn Ala Leu Glu Val Ser Gly Thr 420 425 430 Pro Ile Asp Leu Lys Gln Val Thr Ala Asp Ile Tyr Ser Leu Ala Gly 435 440 445 Thr Asn Asp His Ile Thr Pro Trp Lys Ser Cys Tyr Lys Ser Ala Gln 450 455 460 Leu Phe Gly Gly Lys Val Glu Phe Val Leu Ser Ser Ser Gly His Ile 465 470 475 480 Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Lys Ser Arg Tyr Met Thr 485 490 495 Ser Thr Asp Met Pro Ala Thr Ala Asn Glu Trp Gln Glu Asn Ser Thr 500 505 510 Lys His Thr Asp Ser Trp Trp Leu His Trp Gln Ala Trp Gln Ala Glu 515 520 525 Arg Ser Gly Lys Leu Lys Lys Ser Pro Thr Ser Leu Gly Asn Lys Ala 530 535 540 Tyr Pro Ser Gly Glu Ala Ala Pro Gly Thr Tyr Val His Glu Arg 545 550 555 <210> 10 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Artificial DNA <400> 10 ttgacaatta atcatccggc tcgtataat 29 <210> 11 <211> 30 <212> DNA <213> Escherichia coli <400> 11 tttacacttt atgcttccgg ctcgtataat 30 <210> 12 <211> 30 <212> DNA <213> Escherichia coli <400> 12 ttgacaatta atcatcgaac tagttaacta 30 <210> 13 <211> 30 <212> DNA <213> Cupriavidus necator <400> 13 tttacacttt atgcttccgg ctcgtatgtt 30 <210> 14 <211> 30 <212> DNA <213> Cupriavidus necator <400> 14 ttgacagcgc gtgcgttgca aggcaacaat 30 <210> 15 <211> 231 <212> DNA <213> Cupriavidus necator <400> 15 tacgccgccc cctgaccagg aacgccgggc cagtcccggc gtttttttat tctatagcgc 60 aattaaccgc cgtcatattg cgtcaccatg attgccggat ggccgcggcg atcccttgct 120 ggaggccggt tccaagaaga tttaaagatg tcacggaatt gtcatacagg gagcatagag 180 ttcgtcttgt caaaaatttg tcattcccaa ccaatgttct ctggaggaca t 231 <210> 16 <211> 17 <212> DNA <213> Cupriavidus necator <400> 16 agagagacaa tcaaatc 17 <210> 17 <211> 890 <212> DNA <213> Artificial sequence <220> <223> Artificial DNA <400> 17 agtgaattcg gatttaaatt gcatgtgcag ctgtccggcg acctgggcgc cggcaagacc 60 acgctgacgc gcacgatcct gcgcgcgctc ggccatgcgg gcaaggtccg cagccccacc 120 tacacgctgt gcgagcccta cgaggtggcc cgcgccgacg gctcgccgct gaccgtctac 180 cacttcgacc tgtaccgctt tgccgatccc gaagagtgga tcgacgcggg ttttcgcgac 240 tgcttcgccg aaccggcctt caacctggtc gagtggccgg aaaaggccgg ccgcctgctc 300 ggggaaccgg atctccatat gttgctccaa tcggacatgg ccggggcgga tgatgccggc 360 gagcggcgga tcgcgacgat gcgcgcctat actcacactg gacttaccct gctgaacgca 420 tgctgagtct ggcgggcaca aaaaaaccgg ggcatgcccc ggttttttca tggctgcgtg 480 cctcaggccc gcgccgcggc ctggtcctcc tgcaaggcat cgcggtcggc atagcgcgac 540 gcgatcaccg caccaccc cagctgcagc tggtgataga ccatcagcgg caccacgc 600 aggcccagcg ccggatggcc ggcaaacagg atcttggcca tcgggatgcc gttggccagg 660 ctcttcttgg agccgcagaa caccgccgtg atctcgtcct cgacagagaa gccaaggcgg 720 cgcgcggtca gcgtggtaaa gcccagcacc acgaacagca gcaccgcggc gatcagcatc 780 actgcgccga tggtctgcca ctggtactgg tgccacaggc cctgggcggt ggcatcgcag 840 aatgaggaat agacgatcag cacgatcacg catttaaatg gatagctcgg <210> 18 <211> 769 <212> DNA <213> The snowstorm <220> <223> The DNA <400> 18 ccgagctatc catttaaatg gacgaccttc accatcggat gcgaagtcag cgcccgatac aagtcgtcga gctgctcgcg gctggtgacc cacaccgtca cggtcaggcc caggtagttg ccgccgctgg aggggcgcat ttccatcttg ccggcgtgga agtcgggatc gaactcctgc 180 accagcgtga cgatggcctc ggcaaagccg tcctgcatcg cgcccatcac cttgatggga 240 aagtggctgg ggtattcgat cagcgactgc tccggcggga tgtcgcccgg cttgttgctg 300 ccttgcttgt cggttgtcat gtcggattct ccggcacgaa accggcgcgc cgcatggcgc 360 gccggcttac gcgcattcag ggtttctgtt ttccgacagt aagaaggtgg cgcggggatg 420 gtccgggtgg cggcgtgccg gcctcaccgg tcccacgcat tgacgacgag ctgcttgatc 480 aggtgcagct tgcggtgaaa gaaatggtcg gcgccgggga tcacgatcac cggcagctcc 540 tgcgggcggg cccagtcgaa cacgctggcc agcggcacgg tgtcgtcctg ttcgccatgg 600 atcacgatgg tgtcggccgg cacctcggct acctgccagc ggctggcggc ggtgcccacc 660 agcaccaggc gctgcgcggg cgtgccggcc tcggccaggc ggcgcgccac gtgggtggtg 720 acgaagctgc cgaacgagaa cccgcccatg atttaaatcc gaattcact 769 <210> 19 <211> 956 <212> DNA <213> Artificial Sequence <220> <223> Artificial DNA <400> 19 agtgaattcg gatttaaatg tatgcggcat gtcactgggt tttgccgacc cggaagcgat 60 cgagaaccag ctgaccacgg aacgtgagcc ggtcagcggg ttcgcgcgtt tcctctcata 120 gcaaagtttg agaaaagttt gtatcaatgt gtaacgatga gtgccgatat acaagacgac 180 gtccgtgtat tggctgggag tgttccaagg ggcagcaagg tgaacccggg tacgcctggg 240 gcaagccaga ggcggttcgc atgcaaacgt gaccttttgg ttgctttttc cgcaatgtgg 300 aaatgtttgc aaatcgaact ttaaggagcg tctgtaagtc tttaatcttg ctaacaattt 360 ctttctttcc tacactagcg ccattcctat gcgctgaacg aatcatgttc cggtctgatt 420 ccattttttc cagattcttc ggctccgcac ccctgtccgc tgttgccacc gcggtcctgg 480 tatcgtacgg aacgcctgac cgggaaaaaa cgcgccgtgc cacggcgcgt ttttcgttct 540 ggcggccgcg gccgtcagag cagcttgcct gggttcatga tcccggccgg gtcgaacacg 600 gccttgatct cgcgcatcag ccgcagttcc agcgggtcct tcatggtcag gaaggcatgg 660 cgcttgagct ggccgatgcc atgctcggcg ctgatgctgc cgccgtagcg catcacttcg 720 tccagcaccg cgtcggtcac cgcatcgcct tgcgtggccg cccagtcctt gggcgcgccg 780 gccgggcgcg acaggttgta gtgcaggttg ccgtcgccga agtgcccgaa gataaagggc 840 cggatggcgg gatcgagccc acgcagccgc gtttccatcg aggtcatgaa ggccggaatc 900 tgctcgatcg ggagcgagat gtcgtgcttc aggtgcgatt taaatggata gctcgg 956 <210> 20 <211> 223 <212> DNA <213> Artificial sequence <220> <223> Artificial DNA <400> 20 agtgaattcg gatttaaata gggcctgctt gcccaggtcc ggctgccgct gctctgcgcc 60 accgccctgc tgctggcggc ctgcgccagt tccccgtctt cgcgccacgc cggcctgccg 120 cggacgccgg gcaaacccat gatcgacccc agcgccggac tggaagagat ttcgatccag 180 gcgatgtccc tggtcggcac acccatttaa atggatagct cgg 223 <210> 21 <211> 238 <212> DNA <213> Artificial sequence <220> <223> Artificial DNA <400> 21 agtgaattcg gatttaaata gcgatcggta cttcattccc tggcgcgtgc cgccgtcgga 60 attgccattg tctgcggtgc gactgtgtcg aatggagtgc tggcggatac ggtattcaag 120 gatgccgacg cccgtatcga tgccacggcc cacgccgcgg attcgcatgc ggaaggcaag 180 cgcggcttgc tgtcgtcggt ggtgaattcc accagcaaca tttaaatgga tagctcgg 238 <210> 22 <211> 926 <212> DNA <213> Artificial Sequence <220> <223> Artificial DNA <400> 22 agtgaattcg gatttaaata cccctatacg cgcaagctga tggcggcggc gcaggtcggc 60 gctggctgac ccggaggtgt gcggcgcagc aacgccgcgg ccccgcctgg ctagcatccg 120 gttgttcgga tcaatccgat aaacaaggtg cgaattcccg cctatatcct tgattcgcca 180 gtcaaatccg gcgaatttgt aacgaacttt gacatgtgaa tgacaaccct ttaccatccc 240 gcgagaacta gttttggggg tggtccgtcc gacgcattgg atcgtgcata gcacgtttgc 300 ggtgcaagac aggcccggaa agcctggtgc ggatgttgca tagggttcac cccgcaggtt 360 cacatgaatt tctcgcgaag ttcacgcgaa tttcacatat aaccagctgc cccggacttg 420 tgccggggct tgctttggaa cgatcaacgg gagaaccagt ttccggcgcg ctacaagcaa 480 aaaggactgc tgcgacagtc cttttttctt tggcggggcg tgctccccgg gctattgcac 540 tgcgaccgtt ccggccggtg ccaggcgggc ctgttccagc cgctgccgca gcgttgccat 600 caccgccgtg gtggcctgtt cgccggccag tatggctcgg ttgcgggcgt tgaagtcgct 660 gccgcccata tcgggcagct cggggcggat caccacgtcg gcgcgcgcca gtgccatctt 720 gttgatcgac tggcccatga tcgcggtggt ctgcagcagc acgccgctct ggcccgcgtt 780 cttctgcgcc gacgggtcgg ccgagatgtt gaccgcgatg acaaagtccg cgcccatgcc 840 gcgcgcggaa tccaccggca ccggctcgac caggccgccg tcgacatagt cgtgaccctg 900 gatcgacatt taaatggata gctcgg 926 <210> 23 <211> 2478 <212> DNA <213> Artificial sequence <220> <223> Artificial DNA <400> 23 agtgaattcg gatttaaatg ctaatggtga gtgtggtctt ggacatcgcg cctcctttac 60 tgcttgttgc cgctaatggc cgcgcaccta tgcagtgcat ccggcaggca ccagtctgaa 120 gccgctgcgc gcaacgcgc gcgaagcggc gccatgccca tgcgccaggc gcatgcctcg 180 ctacttgcgc ggcattgtcc gcccgctcac agcacaatgc gcaaggcgcg tgccaggcat 240 aaactgatgg ccaattgtac gccgccccct gaccaggaac gccgggccag tcccggcgtt 300 ttttattct atagcgcaat taaccgccgt catattgcgt caccatgatt gccggatggc 360 cgcggcgatc ccttgctgga ggccggttcc aagaagattt aaagatgtca cggaattgtc 420 atacagggag catagagttc gtcttgtcaa aaatttgtca ttcccaacca atgttctctg 480 gaggacatat gtcccagaag aaatcgccac gcttcgagct gcgcagtggc aacgtagacg 540 cccctctct cgccctccag accgccgaca tggctgcgct gcgggatgac ctcctcgccc 600 gctttgaagc cacccccgac ttcttttcca atgacgtgat tgcgctggac ctgcgcgc 660 tggaagatga cagcgaagtc gcgcttggca ccgtgatcga gacgctggcc acgctcaggg 720 cccgcgccat cggcgtggtg gccgccccg gccagcgcga gtgggccgag cgcttcggcc 780 tgccgctgct ggacagccag gcccgccgcg gcagtggcgc cgatcgcgcc accgaccgtg 840 ccgccgaggc cagggccgca gccgcggcgg aacaggccgc agccgaccag gccgcgcgcg 900 aggaatccat ccgcgccgcc gcgcaggcca ccaccgacgc cgccgtggcc gctgccatcc 960 gccagaccca gaccatgctg atcgacaagc cgcttcgctc gggccagcag gtctacgcgc 1020 agggcgacgt ggtcatcctg gacgtggtca gctacggcgc cgaggtgatc gccgaaggca 1080 acatccatat ctatgccccg ctgcgcggcc gtgcgctggc gggcgtcaag ggcaacaccg 1140 gcgcgcgcat tttcagcacg tgcatggagc ctgaactgat ttccatcgcc ggcatctacc 1200 ggaccgcgga gcagacgctt ccggccgacg tgctcggcaa gaccgcccag gtgcgcctgg 1260 ccgatgaaaa actgatcctg gaagcgctgc ggctcaagta accgcggcag cccccgggac 1320 cgaattgcag agagcgcaag cttcaactta ttactggacc aaagagccat ggcaaaaatc 1380 atcgttgtga cctccggcaa gggaggcgtc ggcaagacca ccaccagcgc cagctttgcc 1440 gccggcctgg ccctgcgcgg ccacaagact gccgtgatcg acttcgacgt cggcctgcgc 1500 aaccttgacc tgatcatggg ttgcgagcgc cgcgtggtgt acgacctgat caacgtggtg 1560 1620 atcctgccgg cctcgcagac gcgcgacaag gacgcgctca cgcgcgaagg cgtcgagaag 1680 gtcatcaacg gcctgatcga gatggatttc gaattcatca tctgcgactc gccggccggc 1740 atcgagtcgg gcgcgctgat ggcgatgtac ttcgccgacg aggcgctgat cgtgaccaac 1800 ccggaagtgt cgtcggtgcg cgattcggac cgcatcctgg gcatcctggc ctccaagacc 1860 aagcgcgcca gcgaaggcgg cgacccgatc aaagcaacacc tgctgatcac ccgctacaac 1920 cccaagcgtg tgcatggcgg cgaaatgctg tcgctgaccg acatccagga aatcctgcgc 1980 atcaagctga tcggcgtggt gccggagtct gaagccgtgc tgcacgctc gaaccagggc 2040 acgcccgcca tccacctgga aggcagcgac gtggccgacg cctatggcga cgtggtggac 2100 cgcttctcg gcaagcaa gccgatgcgt ttcaccgact accagaagcc gggtctgctc 2160 tcccgcatct tcggcaacaa gtaacctgcc ggcctggttc aaccagtcgg cagccgacta 2220 gtcccggcag ccgccagcgc gctggcctcg cttatcatgg cagctgcgcc gggcggcacg 2280 cgaacggcgc ggcaccaacg atcaacatgc cattgctacc gacacaagac ttccagggcc 2340 agccgctggt ccggatcggc gatgccgaca cgttcctgct gctcgccccg caacacggcg 2400 ggcggctggt ccgctgggtg caccgcggac aggacatcct ctactggccg gacgctgcca 2460 tttaaatgga tagctcgg 2478

Claims

1. A transformed microorganism of Copperobacterium necrotum, which has a gene encoding a polyhydroxyalkanoate synthase having an amino acid sequence shown in SEQ ID NO: 7, and the A1386 gene and / or the A2405 gene are destroyed by deletion and / or insertion, The A1386 gene is a gene encoding the amino acid sequence shown in SEQ ID NO: 1, The A2405 gene is a gene encoding the amino acid sequence shown in sequence number 2.

2. The transformed microorganism according to claim 1, which further enhances the expression of minC gene and minD gene, The minC gene is a gene encoding the amino acid sequence shown in sequence number 3, The minD gene is a gene encoding the amino acid sequence shown in sequence number 4.

3. A method for producing a polyhydroxyalkanoate, the method comprising: A step of culturing the transformed microorganism according to claim 1 or 2 in the presence of a carbon source.

4. The method for producing a polyhydroxyalkanoate according to claim 3, wherein Polyhydroxyalkanoate is a copolymer of two or more hydroxyalkanoic acids.

5. The method for producing a polyhydroxyalkanoate according to claim 4, wherein The polyhydroxyalkanoate is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit.

6. The method for producing a polyhydroxyalkanoate according to claim 5, wherein The polyhydroxyalkanoate is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

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