Method for producing copolymerized polyhydroxyalkanoate mixture, and transformed microorganism

Abstract

The present invention provides a method for manufacturing a copolymerized polyhydroxyalkanoate mixture. The method includes a step of culturing a microorganism that produces a copolymerized polyhydroxyalkanoate mixture. The above-mentioned mixture contains fraction (I) and fraction (II), wherein fraction (I) contains copolymerized polyhydroxyalkanoates having 3-hydroxybutyric acid structural units and 3-hydroxyhexanoic acid structural units, and the average composition ratio of 3-hydroxyhexanoic acid is 20 mol% or more, and fraction (II) contains polyhydroxyalkanoates having 3-hydroxybutyric acid structural units, and the average composition ratio of 3-hydroxyhexanoic acid is 0 mol% or more and 15 mol% or less. The average composition ratio of 3-hydroxyhexanoic acid in the above-mentioned copolymerized polyhydroxyalkanoate mixture is 22 mol% or less.

Description

Technical Field

[0001] The present invention relates to a method for producing a copolymerized polyhydroxyalkanoate mixture and a transformation microorganism. Background Art

[0002] Polyhydroxyalkanoate (hereinafter sometimes referred to as "PHA") is a polyester-type organic polymer produced by a wide range of microorganisms. PHA is a biodegradable thermoplastic polymer that can be produced using renewable resources as raw materials. As a result, attempts have been made to use PHA as an environmentally friendly raw material or a biocompatible raw material for industrial production and use in various industries.

[0003] So far, it is known that a large number of microorganisms accumulate PHA as an energy storage substance in the bacteria. As a representative example of PHA, poly-3-hydroxybutyric acid (hereinafter sometimes referred to as "P(3HB)"), which is a homopolymer of 3-hydroxybutyric acid (hereinafter sometimes referred to as "3HB"), can be cited. P(3HB) is a thermoplastic polymer. Since it is biodegradable in the natural environment, it has attracted much attention as an environmentally friendly plastic. However, P(3HB) has high crystallinity and therefore has hard and brittle properties, which limits its application range in actual use. In order to expand the scope of application, it is necessary to give P(3HB) flexibility.

[0004] Therefore, a copolymer PHA (hereinafter referred to as "P(3HB-co-3HV)") formed by 3HB and 3-hydroxyvaleric acid (hereinafter referred to as "3HV") and a method for producing the same have been developed (for example, refer to Patent Document 1 and Patent Document 2). P(3HB-co-3HV) is more flexible than P(3HB) and is therefore considered to be able to be used in a wide range of applications. However, in reality, even if the molar fraction of 3HV in P(3HB-co-3HV) is increased, the accompanying changes in physical properties are not significant. In particular, since the flexibility is not improved to the level required for processing into films, sheets, soft packaging containers, etc., it can only be used in limited areas of hard molded bodies such as shampoo bottles and disposable razor handles.

[0005] In order to improve the flexibility of PHA, copolymerized polyhydroxyalkanoate (hereinafter also referred to as "P(3HB-co-3HH)") formed of 3HB and 3-hydroxyhexanoic acid (hereinafter referred to as "3HH") and its production method have been studied (for example, refer to Patent Documents 3 and 4). In these reports, P(3HB-co-3HH) is produced by fermentation using a wild strain of Aeromonas caviae isolated from soil with fatty acids such as oleic acid and palmitic acid as a carbon source.

[0006] In addition, research has been conducted on the mass production of P(3HB-co-3HH) using Cupriavidus necator as a host and PHA synthase derived from Aeromonas caviae. By introducing an R-specific enoyl-CoA hydratase gene into Cupriavidus necator having PHA synthase derived from Aeromonas caviae, or by increasing the expression level of the R-specific enoyl-CoA hydratase gene on the host chromosome, P(3HB-co-3HH) can be produced using plant oils and fats as raw materials, and the 3HH composition ratio of the P(3HB-co-3HH) can be increased to a maximum of about 14 mol% (see Patent Document 5, Patent Document 6 and Non-Patent Document 1).

[0007] In addition, there are also examples in which the 3HH composition ratio of P(3HB-co-3HH) was increased to more than 20 mol % by inhibiting the expression of a gene encoding β-ketothiolase for Coppercopper Staphylococcus aureus having a PHA synthase derived from Aeromonas caviae (see Patent Document 7), wherein the β-ketothiolase has thiolysis activity for β-ketoacyl-CoA having 6 carbon atoms (i.e., β-ketohexanoyl-CoA).

[0008] Research related to the physical properties of P(3HB-co-3HH) has also been conducted (see non-patent document 2). In this report, a fatty acid with a carbon number of 12 or more was used as the sole carbon source to culture Aeromonas caviae, and P(3HB-co-3HH) with various 3HH composition ratios was produced by fermentation. The crystallinity of P(3HB-co-3HH) decreases with the increase of the 3HH composition ratio, and therefore, the hard and brittle properties of P(3HB) gradually show soft properties. When the 3HH composition ratio is further increased, it shows higher softness than P(3HB-co-3HV). That is, P(3HB-co-3HH) can have a wide range of applicable properties from hard polymers to soft polymers by changing the 3HH composition ratio, and can be expected to be used in a wide range of fields.

[0009] On the other hand, although the crystallinity of P(3HB-co-3HH) decreases and the flexibility increases when the 3HH composition ratio is increased, there is a tendency for the processing characteristics to decrease. For example, although P(3HB-co-3HH) with a 3HH composition ratio increased to about 10 mol% is relatively soft, the crystallization rate is slow in injection molding, film molding, blow molding, fiber spinning, extrusion foaming, bead foaming and other processes, so there is a problem of low productivity. In order to solve this problem, the following research has also been conducted: by producing the above-mentioned relatively soft P(3HB-co-3HH) and a copolymer PHA with a low 3HH composition ratio and a high melting point (i.e., high crystallinity) in the same cell, the melt processability and processing speed are improved (see patent document 8).

[0010] However, in the PHA mixture described in Patent Document 8, since the melting point of the low-melting-point component (the PHA component having the highest 3HH composition ratio) exceeds 100°C, it can be inferred that the PHA mixture does not contain more than 20 mol% of a PHA component having a high 3HH composition ratio, and the mechanical properties such as tear strength are insufficient, and there is still room for improvement.

[0011] Prior art literature

[0012] Patent Literature

[0013] Patent Document 1: Japanese Patent Application Laid-Open No. 57-150393

[0014] Patent Document 2: Japanese Patent Application Laid-Open No. 59-220192

[0015] Patent Document 3: Japanese Patent Application Laid-Open No. 5-93049

[0016] Patent Document 4: Japanese Patent Application Laid-Open No. 7-265065

[0017] Patent Document 5: International Publication No. 2011 / 105379

[0018] Patent Document 6: International Publication No. 2015 / 115619

[0019] Patent Document 7: International Publication No. 2019 / 142845

[0020] Patent Document 8: International Publication No. 2017 / 056442

[0021] Non-patent literature

[0022] Non-patent document 1: H. Arikawa, K. Matsumoto, Microb. Cell. Fact., 15, pp. 184 (2016)

[0023] Non-patent document 2: Y. Doi, S. Kitamura, H. Abe, Macromolecules, 28, pp. 4822-4823 (1995) Summary of the invention

[0024] Problems to be solved by the invention

[0025] As described above, it is difficult to obtain a molded product satisfying both good processability and mechanical properties using P(3HB-co-3HH).

[0026] In view of the above-mentioned current situation, an object of the present invention is to provide a method for producing a copolymerized polyhydroxyalkanoate having both excellent processability and mechanical properties.

[0027] Solution to the problem

[0028] The present inventors have conducted intensive studies to solve the above problems and have found that a copolymerized polyhydroxyalkanoate mixture having excellent processability and mechanical properties can be obtained by culturing microorganisms to produce a copolymerized polyhydroxyalkanoate mixture containing two polyhydroxyalkanoate fractions having a specific composition, thereby completing the present invention.

[0029] That is, the present invention relates to a method for producing a copolymerized polyhydroxyalkanoate mixture, the method comprising: a step of culturing a microorganism that produces the copolymerized polyhydroxyalkanoate mixture, wherein the copolymerized polyhydroxyalkanoate mixture contains a polyhydroxyalkanoate fraction (I) and a polyhydroxyalkanoate fraction (II), wherein the polyhydroxyalkanoate fraction (I) contains copolymerized polyhydroxyalkanoates having 3-hydroxybutyric acid structural units and 3-hydroxyhexanoic acid structural units, and the average composition ratio of 3-hydroxyhexanoic acid is 20 mol% or more, and the polyhydroxyalkanoate fraction (II) contains polyhydroxyalkanoates having 3-hydroxybutyric acid structural units, and the average composition ratio of 3-hydroxyhexanoic acid is 0 mol% or more and 15 mol% or less, and the average composition ratio of 3-hydroxyhexanoic acid in the copolymerized polyhydroxyalkanoate mixture is 22 mol% or less.

[0030] The weight ratio of the polyhydroxyalkanoate fraction (I) in the copolymerized polyhydroxyalkanoate mixture is preferably 10 to 90%.

[0031] It is preferred that the average composition ratio of 3-hydroxycaproic acid in the copolymerized polyhydroxyalkanoate mixture is 10 to 22 mol %.

[0032] The microorganism preferably has a gene encoding two types of polyhydroxyalkanoate synthases having different polymerization activities for 3-hydroxyhexanoyl-CoA.

[0033] It is preferred that the amino acid sequences of the two polyhydroxyalkanoate synthases having different polymerization activities for 3-hydroxyhexanoyl-CoA have a sequence identity of 90% or less.

[0034] Preferably, the genes encoding the two types of polyhydroxyalkanoate synthases having different polymerization activities for 3-hydroxyhexanoyl-CoA are the following gene (A) and gene (B), wherein the gene (A) encodes a polyhydroxyalkanoate synthase having a higher polymerization activity for 3-hydroxyhexanoyl-CoA than that of a wild-type polyhydroxyalkanoate synthase, wherein the wild-type polyhydroxyalkanoate synthase is derived from Aeromonas caviae having the amino acid sequence described in SEQ ID NO: 1, and the gene (B) encodes a polyhydroxyalkanoate synthase having a lower polymerization activity for 3-hydroxyhexanoyl-CoA than that of the wild-type polyhydroxyalkanoate synthase, wherein the wild-type polyhydroxyalkanoate synthase is derived from the above-mentioned Aeromonas caviae.

[0035] Preferably, the gene (A) is a polyhydroxyalkanoate synthase gene derived from an Aeromonas microorganism or a mutant thereof. More preferably, the gene (A) is a gene encoding an amino acid sequence having a sequence identity of 99.5 to 100% with respect to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0036] Preferably, the gene (B) is a combination of a portion of a polyhydroxyalkanoate synthase gene derived from a microorganism of the genus Aeromonas and a portion of a polyhydroxyalkanoate synthase gene derived from a microorganism of the genus Cupria. More preferably, the gene (B) is a gene encoding an amino acid sequence having a sequence identity of 90 to 100% with respect to the amino acid sequence shown in SEQ ID NO: 6.

[0037] Preferably, the gene (B) is a polyhydroxyalkanoate synthase gene derived from a microorganism of the genus Chromobacterium or a mutant thereof. More preferably, the gene (B) is a gene encoding an amino acid sequence having 90 to 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5.

[0038] The gene (B) is preferably a polyhydroxyalkanoate synthase gene derived from a Bacillus microorganism or a mutant thereof. More preferably, the gene (B) is a gene encoding an amino acid sequence having 90 to 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8.

[0039] Preferably, the microorganism is a transformed microorganism, wherein the supply of 3-hydroxyhexanoyl-CoA in the cell to polyhydroxyalkanoate synthase is increased compared to a wild-type strain of the microorganism. More preferably, the transformed microorganism is transformed such that the decomposition of intermediate metabolites with a carbon number of 6 in the β-oxidation of oils or fatty acids is suppressed. Further preferably, the transformed microorganism is transformed such that the expression of a gene encoding a β-ketothiolase is suppressed, wherein the β-ketothiolase has a thiolytic activity on β-ketohexanoyl-CoA, which is a β-ketoacyl-CoA with a carbon number of 6.

[0040] The β-ketothiolase preferably has an amino acid sequence that has 90 to 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 10.

[0041] The microorganism is preferably a microorganism having a gene encoding a protein exhibiting R-form-specific enoyl-CoA hydratase activity.

[0042] Preferably, a carbon source containing fats or fatty acids is added in the above-mentioned culturing step. More preferably, the carbon source containing fats or fatty acids is a carbon source containing medium-chain fatty acids having 6 to 12 carbon atoms or glycerides of the medium-chain fatty acids. Further preferably, the medium-chain fatty acids are caproic acid.

[0043] Preferably, the microorganism belongs to the genus Cupriatus, or is a transformant of a microorganism of the genus Cupriatus. More preferably, the microorganism is Insecticidal Cupriatus, or a transformant of Insecticidal Cupriatus.

[0044] The present invention also relates to a transformed microorganism for producing a copolymerized polyhydroxyalkanoate mixture, the transformed microorganism having genes encoding two types of polyhydroxyalkanoate synthases having different polymerization activities for 3-hydroxyhexanoyl-CoA, the transformed microorganism being transformed so that the supply of 3-hydroxyhexanoyl-CoA to polyhydroxyalkanoate synthase in cells is increased compared to a wild-type strain of the transformed microorganism, the copolymerized polyhydroxyalkanoate mixture comprising a polyhydroxyalkanoate fraction (I) and a polyhydroxyalkanoate fraction (II), the polyhydroxyalkanoate fraction (I) comprising copolymerized polyhydroxyalkanoates having 3-hydroxybutyric acid structural units and 3-hydroxyhexanoic acid structural units, and having an average composition ratio of 3-hydroxyhexanoic acid of 20 mol% or more, the polyhydroxyalkanoate fraction (II) comprising polyhydroxyalkanoates having 3-hydroxybutyric acid structural units, and having an average composition ratio of 3-hydroxyhexanoic acid of 0 mol% or more and 15 mol% or less, and the average composition ratio of 3-hydroxyhexanoic acid in the copolymerized polyhydroxyalkanoate mixture being 22 mol% or less.

[0045] Effects of the Invention

[0046] According to the present invention, a copolymerized polyhydroxyalkanoate mixture having both excellent processability and mechanical properties can be produced. According to a preferred embodiment of the present invention, the produced copolymerized polyhydroxyalkanoate mixture is easy to handle and can be easily separated and purified industrially by microorganisms. DETAILED DESCRIPTION

[0047] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to the following embodiments.

[0048] The present invention is a method for producing a copolymerized PHA mixture, comprising the step of culturing microorganisms that produce the copolymerized PHA mixture.

[0049] (Copolymer PHA mixture)

[0050] The copolymerized PHA mixture is composed of a PHA fraction (I) containing copolymerized PHA having 3HB structural units and 3HH structural units, and the average composition ratio of 3HH is 20 mol% or more, and a PHA fraction (II) containing PHA having 3HB structural units, and the average composition ratio of 3HH is 0 mol% or more and 15 mol% or less. The copolymerized PHA mixture can be fractionated into the PHA fraction (I) and the PHA fraction (II) by the MIBK fractionation method described later.

[0051] The above-mentioned PHA fraction (I) is a fraction containing copolymerized PHA having at least 3HB structural units and 3HH structural units, and may contain PHA containing hydroxyalkanoic acid structural units other than 3HB structural units and 3HH structural units. It is preferably a fraction containing copolymerized PHA that does not contain hydroxyalkanoic acid structural units other than 3HB structural units and 3HH structural units and has only 3HB structural units and 3HH structural units, that is, a fraction containing P(3HB-co-3HH).

[0052] Examples of hydroxyalkanoic acid structural units other than the 3HB structural unit and the 3HH structural unit include structural units of hydroxyalkanoic acids such as 3-hydroxypropionic acid, 3HV, 3-hydroxyalkanoic acid having 7 to 16 carbon atoms, 2-hydroxyalkanoic acid having 4 to 16 carbon atoms, 4-hydroxyalkanoic acid (e.g., 4-hydroxybutyric acid), 5-hydroxyalkanoic acid, 6-hydroxyalkanoic acid (e.g., 6-hydroxyhexanoic acid), and lactic acid, but are not limited thereto.

[0053] The average composition ratio of 3HH in the PHA fraction (I) is 20 mol% or more, preferably 22 mol% or more, and more preferably 24 mol% or more. The upper limit of the average composition ratio of 3HH is not particularly limited, but is preferably 35 mol% or less, more preferably 32 mol% or less, and particularly preferably 30 mol% or less.

[0054] The above-mentioned PHA fraction (II) is a fraction containing PHA having a 3HB structural unit. The PHA contained in the PHA fraction (II) may be a homopolymer having only a 3HB structural unit, or may be a copolymer PHA having a 3HB structural unit and a hydroxyalkanoic acid structural unit other than this. In consideration of mechanical properties, a copolymer PHA having a 3HB structural unit and a hydroxyalkanoic acid structural unit other than this is preferred. As the copolymer PHA, a copolymer PHA having a 3HB structural unit, a 3HV structural unit and / or a 3HH structural unit is preferred, a copolymer PHA having a 3HB structural unit and a 3HH structural unit is more preferred, and a copolymer PHA having only a 3HB structural unit and a 3HH structural unit, i.e., P(3HB-co-3HH) is further preferred.

[0055] The average composition ratio of 3HH in the PHA fraction (II) is 0 mol% or more and 15 mol% or less. The lower limit of the average composition ratio of 3HH is preferably 0.1 mol% or more, more preferably 1 mol% or more, further preferably 2 mol% or more, and further preferably 3 mol% or more. The upper limit of the average composition ratio of 3HH is preferably 12 mol% or less, more preferably 10 mol% or less.

[0056] The weight ratio of the PHA fraction (I) in the copolymerized PHA mixture is preferably 10% to 90%, more preferably 20% to 80%, and particularly preferably 30% to 70%. In addition, the weight ratio of the PHA fraction (II) in the copolymerized PHA mixture is preferably 10% to 90%, more preferably 20% to 80%, and particularly preferably 30% to 70%.

[0057] The average composition ratio of 3HH in the copolymerized PHA mixture as a whole is 22 mol% or less. When the average composition ratio of 3HH exceeds 22 mol%, the cohesiveness of the copolymerized PHA mixture increases, and in the separation and purification process of the copolymerized PHA mixture after fermentation production by microorganisms, problems such as formation of undesirable agglomerates, clogging or adhesion to pipes or pump equipment are likely to occur, and there is a tendency that the industrial separation and purification of the copolymerized PHA mixture becomes difficult.

[0058] Taking into account the balance between the mechanical properties of the molded body formed from the mixture and the processing properties and industrial operability of the mixture, the average composition ratio of 3HH shown in the above-mentioned copolymer PHA mixture as a whole is preferably 10 to 22 mol%, more preferably 11 to 20 mol%, further preferably 12 to 18 mol%, and particularly preferably 13 to 17 mol%.

[0059] (MIBK classification method)

[0060] The copolymerized PHA mixture can be fractionated into a PHA fraction (I) having a high average composition ratio of 3HH and a PHA fraction (II) having a low average composition ratio of 3HH by a solvent fractionation method utilizing the difference in solubility in methyl isobutyl ketone (MIBK). The higher the 3HH composition ratio of PHA, the higher the solubility in MIBK. Therefore, after the copolymerized PHA mixture is completely dissolved in high-temperature MIBK, the temperature is lowered to precipitate the PHA component having a low 3HH composition ratio, thereby being fractionated into the PHA fraction (I) and the PHA fraction (II).

[0061] The specific classification steps are described below. First, weigh about 100 mg of the copolymer PHA mixture into a screw-capped test tube, add 10 ml of MIBK and close the lid. Then, shake and mix at 140°C for about 1 to 3 hours and heat to completely dissolve the copolymer PHA mixture. After complete dissolution, place it at 25°C for 1 minute to lower the temperature below the boiling point, quickly transfer all the dissolved liquid to a centrifuge tube whose weight has been measured in advance, and close the lid. The capped centrifuge tube is further placed at 25°C for 15 minutes to precipitate part of the dissolved material. The precipitate and the dissolved liquid are separated by centrifugation (9000 rpm, 5 minutes), and the entire dissolved liquid is transferred to an aluminum cup whose weight has been measured in advance. Add 10 ml of MIBK to the centrifuge tube with the remaining precipitate, mix with a vortex mixer, centrifuge again (9000 rpm, 5 minutes), and transfer the solution to an aluminum cup containing the above-mentioned dissolved liquid. The aluminum cup was heated at 120°C for 30 minutes to volatilize the MIBK and precipitate the dissolved product. Furthermore, the precipitate remaining in the aluminum cup and the precipitate remaining in the centrifuge tube were vacuum dried at 100°C for 6 hours respectively. The precipitate remaining in the aluminum cup was taken as PHA fraction (I), and the precipitate remaining in the centrifuge tube was taken as PHA fraction (II), and they were weighed separately. Confirm that the difference between the total weight of PHA fraction (I) and PHA fraction (II) and the weight of the copolymerized PHA mixture initially measured is within ±3%.

[0062] (Melting behavior of copolymerized PHA mixture)

[0063] The highest melting peak temperature of the copolymerized PHA mixture measured in differential scanning calorimetry is preferably 130°C or higher. By satisfying this condition, the crystallization and solidification of the copolymerized PHA mixture can be performed in a short time, and the processability of the copolymerized PHA mixture can be improved. The highest melting peak temperature is preferably 130 to 165°C, more preferably 130 to 155°C.

[0064] The highest melting peak temperature shown by the above copolymerized PHA mixture is measured in the following manner: using a differential scanning calorimeter, about 2 mg of the above copolymerized PHA mixture is weighed, and the temperature is measured as the temperature of the melting peak on the highest temperature side in the DSC curve obtained when the temperature is increased from -30°C to 200°C at a heating rate of 10°C / min.

[0065] The copolymerized PHA mixture may have, in addition to the highest temperature side melting peak, another melting peak in a region lower than the peak, for example, a melting peak at 100° C. or lower.

[0066] (Copolymer PHA mixture production microorganisms)

[0067] The microorganism used in the production of the copolymerized PHA mixture (hereinafter also referred to as "copolymerized PHA mixture producing microorganism") is not particularly limited as long as it is a microorganism capable of fermenting and producing the copolymerized PHA mixture. It may be a wild strain that originally accumulates PHA, a mutant strain obtained by artificially mutating such a wild strain, or a strain endowed with the ability to accumulate PHA by introducing a foreign PHA synthase gene using a genetic engineering method.

[0068] The copolymerized PHA mixture producing microorganism, or when the microorganism is a transformant, the host of the transformant is not particularly limited, and for example, bacteria belonging to the genus Ralstonia, the genus Cupriavidus, the genus Wautersia, the genus Aeromonas, the genus Escherichia, the genus Alcaligenes, the genus Pseudomonas, etc. are listed as preferred examples. From the viewpoint of safety and PHA productivity, bacteria belonging to the genus Ralstonia, the genus Cupriavidus, the genus Aeromonas, and the genus Wautersia are more preferred, bacteria belonging to the genus Cupriavidus or the genus Aeromonas are further preferred, bacteria belonging to the genus Cupriavidus are further preferred, and Cupriavidus necator is particularly preferred.

[0069] The copolymerized PHA mixture producing microorganism is preferably a microorganism that can efficiently produce two kinds of PHAs with different average composition ratios of 3HH, and has genes encoding two kinds of PHA synthases with different polymerization activities for 3-hydroxyhexanoyl-CoA. 3-Hydroxyhexanoyl-CoA is a precursor of the 3HH structural unit contained in PHA. By making the above-mentioned microorganism have genes encoding two kinds of PHA synthases with different polymerization activities for 3-hydroxyhexanoyl-CoA, a mixture of two kinds of PHAs with greatly different average composition ratios of 3HH, that is, the copolymerized PHA mixture, can be fermented and produced in the cells of the microorganism. The above-mentioned microorganism only needs to have at least two genes encoding PHA synthases with different polymerization activities for 3-hydroxyhexanoyl-CoA, and can also have three or more of the above genes as long as it can ferment and produce the above-mentioned copolymerized PHA mixture.

[0070] The two PHA synthases having different polymerization activities for 3-hydroxyhexanoyl-CoA are not particularly limited, and the sequence identity of the amino acid sequences between the two PHA synthases is preferably 90% or less, more preferably 80% or less, and further preferably 70% or less. It is generally believed that PHA synthases function by forming multimers such as dimers. When the sequence identity of the amino acid sequences between the two PHA synthases is higher than 90%, it is believed that the two PHA synthases form a heterodimer and the copolymerized PHA mixture may not be produced.

[0071] As a specific example of the combination of the two PHA synthases having different polymerization activities for 3-hydroxyhexanoyl-CoA, for example, a combination of gene (A) encoding a PHA synthase having a higher polymerization activity for 3-hydroxyhexanoyl-CoA than that of a wild-type PHA synthase derived from Aeromonas caviae having the amino acid sequence described in SEQ ID NO: 1 and gene (B), encoding a PHA synthase having a lower polymerization activity for 3-hydroxyhexanoyl-CoA than that of a wild-type polyhydroxyalkanoate synthase derived from the above-mentioned Aeromonas caviae.

[0072] Examples of the gene (A) include, for example, a polyhydroxyalkanoate synthase gene derived from a microorganism of the genus Aeromonas or a mutant thereof, and specifically, a gene encoding an amino acid sequence having a sequence identity of 90 to 100% with respect to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 (an amino acid sequence of a PHA synthase mutant derived from a bacterium of the genus Aeromonas). The sequence identity is preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more, and most preferably 99.5% or more.

[0073] Examples of the gene (B) include a gene obtained by combining a portion of a polyhydroxyalkanoate synthase gene derived from a microorganism of the genus Aeromonas and a portion of a polyhydroxyalkanoate synthase gene derived from a microorganism of the genus Cupria, and specifically, a gene encoding an amino acid sequence having a sequence identity of 90 to 100% with respect to the amino acid sequence described in SEQ ID NO: 6. In addition, examples of the gene (B) include a PHA synthase gene derived from a bacterium of the genus Chromobacterium or a mutant thereof, and specifically, a gene encoding an amino acid sequence having a sequence identity of 90 to 100% with respect to the amino acid sequence described in SEQ ID NO: 4 or SEQ ID NO: 5. Alternatively, examples of the gene (B) include a PHA synthase gene derived from a bacterium of the genus Bacillus or a mutant thereof, and specifically, a gene encoding an amino acid sequence having a sequence identity of 90 to 100% with respect to the amino acid sequence described in SEQ ID NO: 7 and SEQ ID NO: 8. For the gene (B), the identities of the above sequences are preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more.

[0074] In order to efficiently produce a copolymerized PHA mixture having a high average composition ratio of 3HH, the copolymerized PHA mixture producing microorganism is preferably a transformed microorganism that has undergone the following transformation: the supply of 3-hydroxyhexanoyl-CoA in the cell relative to the polyhydroxyalkanoate synthase is increased compared to the wild-type strain of the microorganism. Specifically, it is preferably a transformed microorganism that has undergone the following transformation: the decomposition of the intermediate metabolite with a carbon number of 6 in the β-oxidation of oils or fatty acids is suppressed. It can be inferred that as a result of the suppression of the decomposition of the intermediate metabolite with a carbon number of 6 in the β-oxidation, the supply of 3-hydroxyhexanoyl-CoA is increased, and the average composition ratio of 3HH shown in the produced copolymerized PHA mixture is increased.

[0075] Examples of transformed microorganisms transformed in such a manner that the decomposition of intermediate metabolites having 6 carbon atoms in β-oxidation of fats and oils or fatty acids is suppressed include, for example, transformed microorganisms transformed in such a manner that the expression of a gene encoding β-ketothiolase having thiolysis activity for β-ketohexanoyl-CoA, which is β-ketoacyl-CoA having 6 carbon atoms, is suppressed, as described in Patent Document 7. Examples of the gene encoding β-ketothiolase include, for example, a gene encoding an amino acid sequence having a sequence identity of 90 to 100% with respect to the amino acid sequence described in SEQ ID NO: 9 or SEQ ID NO: 10, but are not limited thereto. The sequence identity is preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more.

[0076] In order to inhibit the expression of the gene encoding β-ketothiolase, for example, there can be mentioned a method of completely deleting the enzyme gene in the transformed microorganism, a method of inserting a completely different gene such as a drug resistance gene into the sequence of the enzyme gene, or a method of deleting, replacing, adding or inserting a part of the sequence of the enzyme gene (preferably a region related to enzyme activity), etc. Gene disruption operations include, for example, homologous recombination techniques using vectors containing disruptive genes or disruptive DNA, techniques using transposons, etc. Alternatively, as other methods of destruction, there can be cited known techniques such as the CRISPR / Cas (e.g., Cas9) system for destroying target genes, and TALEN-based genome editing technology (Y. Wang et al., ACS Synth Biol. 2016, 5(7): 721-732; Bogdanove and Voytas, Science, 333: 1843-1846, 2011; Jinek, et al., Science, 337: 816-821, 2012; Shalem, et al., Science, 343: 84-87, 2014; Wang, et al., Science, 343: 80-84, 2014). For example, in the CRISPR / Cas9 system, the guide RNA (gRNA) has a sequence that can bind to a portion of the base sequence of the β-ketothiolase gene to be destroyed, and has the function of targeted transport of Cas9. In addition, the enzyme activity can be eliminated or reduced by mutations such as deletion, substitution, addition, or insertion of the base sequence around the gene, thereby reducing the gene transfer / translation efficiency and mRNA stability.

[0077] In order to efficiently produce a copolymerized PHA mixture having a high average composition ratio of 3HH, the copolymerized PHA mixture producing microorganism is preferably a microorganism having a gene encoding a protein exhibiting R-specific enoyl-CoA hydratase activity. R-specific enoyl-CoA hydratase has the function of converting hexanoyl-CoA to 3-hydroxyhexanoyl-CoA in microbial cells. Therefore, it can be inferred that by making the above-mentioned microorganism have a gene encoding a protein exhibiting R-specific enoyl-CoA hydratase activity, the amount of conversion to 3-hydroxyhexanoyl-CoA will increase, resulting in an increase in the average composition ratio of 3HH exhibited by the copolymerized PHA mixture produced.

[0078] The microorganism having a gene encoding a protein exhibiting the above-mentioned R-specific enoyl-CoA hydratase activity may be a microorganism originally having the gene or a microorganism into which an exogenous gene has been introduced by a genetic engineering method.

[0079] Examples of genes encoding proteins having the above-mentioned exogenous R-specific enoyl-CoA hydratase activity include: a gene derived from Aeromonas caviae encoding an R-specific enoyl-CoA hydratase having the amino acid sequence described in SEQ ID NO: 11; a gene derived from Cupricinus spp. encoding an R-specific enoyl-CoA hydratase having the amino acid sequence described in SEQ ID NO: 12 or SEQ ID NO: 13; a Multifunctional enzyme type 2 (MFE2) gene derived from Yarrowia lipolytica encoding an enzyme having the amino acid sequence described in SEQ ID NO: 14; a gene derived from Drosophila melanogaster encoding an enzyme having the amino acid sequence described in SEQ ID NO: 15; and a gene derived from Drosophila melanogaster encoding an enzyme having the amino acid sequence described in SEQ ID NO: 16. melanogaster), or a gene encoding a protein having a sequence identity of 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more to each of the amino acid sequences described in SEQ ID NOs: 11 to 15 and having R-specific enoyl-CoA hydratase activity, etc., but is not limited thereto.

[0080] In addition, in order to enhance the expression of a gene encoding a protein having R-specific enoyl-CoA hydratase activity, the expression regulatory sequence (promoter sequence and / or SD sequence) for enhancing the expression of the gene can be modified as described in, for example, International Publication No. 2015 / 115619.

[0081] In the case where an exogenous gene is introduced into the copolymerized PHA mixture producing microorganism, the introduced gene may be present on the chromosome possessed by the microorganism serving as the host, or on the DNA of a plasmid, a megaplasmid, etc. From the viewpoint of maintaining the introduced gene, it is preferably present on the chromosome or megaplasmid possessed by the microorganism, and more preferably on the chromosome possessed by the microorganism. In addition, in the case of increasing the expression level of a gene originally maintained by the microorganism serving as the host, the expression level of the gene may also be increased by replacing, deleting or adding the base sequence upstream of the gene.

[0082] Methods for site-specifically replacing or inserting arbitrary DNA into DNA possessed by a microorganism, or methods for deleting arbitrary sites in DNA possessed by a microorganism are well known to those skilled in the art and can be used when producing the transformed microorganism of this embodiment. Although not particularly limited, representative methods include: a method utilizing a transposon and homologous recombination mechanism (Ohman et al., J. Bacteriol., vol. 162: p. 1068 (1985)), a method based on the principle of site-specific introduction using a homologous recombination mechanism and shedding based on a second-stage homologous recombination (Noti et al., Methods Enzymol., vol. 154, p. 197 (1987)), and a method in which a microbial strain in which the sacB gene derived from Bacillus subtilis coexists and the gene is shedding through a second-stage homologous recombination is easily separated into a strain resistant to a sucrose-supplemented medium (Schweizer, Mol. Microbiol., vol. 6, p. 1195 (1992); Lenz et al., J. Bacteriol., vol. 176, p. 4385 (1994)). The method for introducing the vector into cells is not particularly limited, and examples thereof include the calcium chloride method, the electroporation method, the polyethylene glycol method, and the spheroplast method.

[0083] For gene cloning and gene recombination techniques, techniques described in Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989 or 2001) and the like can be used.

[0084] The promoter used to express the introduced gene is not particularly limited. The promoter of the phaC1 gene of Cupricobacterium insecticidalis, the promoter of the phaP1 gene, the lac promoter, lacUV5 promoter, trc promoter, tic promoter, tac promoter derived from Escherichia coli, or the artificially prepared lacN17 promoter having the modified base sequence derived from Escherichia coli shown in SEQ ID NO: 16, the artificially prepared lacN19 promoter having the modified base sequence derived from Escherichia coli shown in SEQ ID NO: 17, etc. can be used.

[0085] (Cultivation of Microorganisms)

[0086] By culturing the copolymerized PHA mixture producing microorganism, the copolymerized PHA mixture can be accumulated in the microbial cells. As a method for culturing the copolymerized PHA mixture producing microorganism, it can be based on a common microbial culture method, as long as it is cultured in a culture medium with an appropriate carbon source. There are no particular limitations on 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. The carbon source is preferably added to the culture medium continuously or intermittently.

[0087] As a carbon source during the culture, any carbon source can be used as long as the copolymer PHA mixture producing microorganism can assimilate it. Although not particularly limited, examples thereof include: sugars such as glucose, fructose, sucrose, and xylose; palm oil, palm kernel oil (including palm olein, palm double olein, and palm kernel olein, which are low melting point fractions obtained by separating them), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil, their fractionated oils, or their purified byproducts; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, and myristic acid, their derivatives, or glycerol, etc. In addition, in the case where the copolymer PHA mixture producing microorganism can utilize gases such as carbon dioxide, carbon monoxide, methane, methanol, and ethanol, and alcohols, these can also be used as carbon sources.

[0088] Among them, the carbon source preferably includes oils or fatty acids. As the above-mentioned oils and fats, vegetable oils or fractionated oils thereof are preferred. The chain length of the above-mentioned oils and fats or fatty acids is preferably shorter, and the above-mentioned carbon source more preferably includes medium-chain fatty acids with 6 to 12 carbon atoms, or glycerides of the medium-chain fatty acids, and the above-mentioned carbon source further preferably includes caproic acid. When the carbon number of the fatty acid contained in the carbon source is 6 to 12, the amount of intermediate metabolites with 6 carbon atoms in β-oxidation increases, and it can be considered that a PHA fraction with a high average composition ratio of 3HH can be obtained efficiently.

[0089] In the manufacture of the copolymerized PHA mixture, it is preferred to culture the microorganisms using a culture medium containing the carbon source, a nitrogen source as a nutrient source other than the carbon source, inorganic salts, and other organic nutrient sources. Although not limited to the following, examples of nitrogen sources include: ammonia; ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate; peptone, meat extract, yeast extract, and the like. Examples of inorganic salts include: potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, and the like. Examples of other organic nutrient sources include: amino acids such as glycine, alanine, serine, threonine, and proline, vitamins such as vitamin B1, vitamin B12, and vitamin C, and the like.

[0090] After the copolymer PHA mixture producing microorganism is cultured for an appropriate period of time so that the copolymer PHA mixture is accumulated in the microbial cells, the copolymer PHA mixture is recovered using a known method. There is no particular limitation on the recovery method, but industrially, recovery based on separation / purification in a water system with low environmental burden is preferred. For example, after the culture is completed, the cells are broken by applying a mechanical shear force or using a surfactant, alkali, enzyme, etc., thereby obtaining a cell lysate in which the cell components other than PHA are dissolved in water. The copolymer PHA mixture is separated from the aqueous phase by filtering and centrifuging the cell lysate and then dried, and the copolymer PHA mixture can be recovered.

[0091] Example

[0092] Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited to these examples. It should be noted that all gene manipulations can be carried out, for example, by the method described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). In addition, the enzymes, cloning hosts, etc. 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.

[0093] (Microbial strain preparation example 1) Preparation of microbial strain (1) for producing copolymerized PHA mixture

[0094] First, a plasmid for PHA synthase gene disruption was prepared as follows.

[0095] By using PCR with synthetic oligo DNA, a DNA fragment (SEQ ID NO: 18) having the base sequences upstream and downstream of the phaC1 structural gene (PHA synthase gene) of the H16 strain of Cupricobacterium spp. was obtained. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Application Laid-Open No. 2007-259708 which had also been digested with SwaI using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare a plasmid vector pNS2X-sacB+phaC1UD for PHA synthase gene disruption.

[0096] Next, a PHA synthase gene-disrupted strain was prepared as follows using the PHA synthase gene-disrupted plasmid vector pNS2X-sacB+phaC1UD.

[0097] Escherichia coli S17-1 strain (ATCC47055) was transformed with the plasmid vector pNS2X-sacB+phaC1UD for PHA synthase gene disruption, and the transformed microorganism thus obtained and KNK005dZ / trc-J4b / dbktB / dA1528 strain were co-cultured on Nutrient Agar medium (manufactured by Difco) for conjugative transfer.

[0098] It should be noted that the KNK005dZ / trc-J4b / dbktB / dA1528 strain is a strain in which the phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the PHA synthase gene on the chromosome is replaced with a modified version of the PHA synthase gene derived from Aeromonas caviae (a gene encoding a PHA synthase having the amino acid sequence recorded in sequence number 2, i.e., the N149S / D171G mutant gene), the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB structural gene is deleted, and the A1528 structural gene is further deleted. The strain can be prepared according to the method described in PCT International Publication No. 2019 / 142845.

[0099] The obtained culture solution was sown 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 growing on the agar medium were selected to obtain strains in which plasmids were introduced into the chromosome of KNK005dZ / trc-J4b / dbktB / dA1528 strain. The strain was cultured for 2 generations using Nutrient Broth medium (manufactured by Difco), diluted and applied on Nutrient Agar medium containing 15% sucrose, and the growing strains were obtained as strains in which plasmids had fallen off. Furthermore, by analysis based on PCR and DNA sequencing, a strain in which the PHA synthase gene on the chromosome was deleted was isolated. The gene-disrupted strain was named KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain.

[0100] In addition, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0101] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO. 19) was obtained, which has the base sequences upstream and downstream of the bktB structural gene (β-ketothiolase gene) of the H16 strain of Cupricinus entomopathogenis, the lacN19 promoter which is a modified form of the lac promoter of Escherichia coli, and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO. 2. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Application Laid-Open No. 2007-259708 which had also been digested with SwaI using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare a plasmid vector pNS2X-sacB+bktbU-lacN19-NSDG-bktbD for introducing the PHA synthase gene.

[0102] Next, using the PHA synthase gene-transferred plasmid vector pNS2X-sacB+bktbU-lacN19-NSDG-bktbD, a PHA synthase gene-transferred strain was prepared as follows.

[0103] The plasmid vector pNS2X-sacB+bktbU-lacN19-NSDG-bktb for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain by using the same conjugation transfer method as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the lacN19 promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 2 were introduced into the position where the bktB gene originally existed on the chromosome. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB::lacN19-NSDG / dA1528 strain.

[0104] In addition, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0105] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO. 20) was obtained, which has the upstream and downstream base sequences of the A1528 structural gene (β-ketothiolase gene) of the H16 strain of Cupricinus entomopathogenis, the lacN17 promoter which is a modified form of the lac promoter of Escherichia coli, and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO. 6. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Application Laid-Open No. 2007-259708 which had also been digested with SwaI using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare a plasmid vector pNS2X-sacB+A1528U-lacN17-AcNSRe12-A1528D for introducing the PHA synthase gene.

[0106] Next, the plasmid vector pNS2X-sacB+A1528U-lacN17-AcNSRe12-A1528D for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB::lacN19-NSDG / dA1528 strain by the same conjugation transfer method as described above. Furthermore, by the same culture as described above and screening based on NutrientAgar medium containing 15% sucrose, a strain was isolated in which the lacN17 promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 6 were introduced into the position where the A1528 gene originally existed on the chromosome. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB::lacN19-NSDG / dA1528::lacN17-AcNSRe12 strain (hereinafter, sometimes referred to as copolymer PHA mixture-producing microbial strain (1)).

[0107] It should be noted that the copolymer PHA mixture producing microbial strain (1) is a strain in which the phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal Cupric bacteria H16 strain are deleted, and a gene encoding a PHA synthase mutant having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase having the amino acid sequence recorded in sequence number 6 (i.e., a gene encoding a PHA synthase formed by combining a part of a polyhydroxyalkanoate synthase gene derived from an Aeromonas microorganism and a part of a polyhydroxyalkanoate synthase gene derived from a Cupric bacteria microorganism) are introduced, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB structural gene (β-ketothiolase gene) is deleted, and the A1528 structural gene (β-ketothiolase gene) is further deleted.

[0108] (Microbial strain preparation example 2) Preparation of microbial strain (2) for producing copolymerized PHA mixture

[0109] First, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0110] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO. 21) was obtained, which has the upstream and downstream base sequences of the bktB structural gene (β-ketothiolase gene) of the H16 strain of Cupricinus entomopathogenis, the lac promoter of Escherichia coli, and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO. 2. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Application Laid-Open No. 2007-259708 which had also been digested with SwaI using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare a plasmid vector pNS2X-sacB+bktbU-lac-NSDG-bktbD for introducing the PHA synthase gene.

[0111] Next, using the PHA synthase gene-transferred plasmid vector pNS2X-sacB+bktbU-lac-NSDG-bktbD, a PHA synthase gene-transferred strain was prepared as follows.

[0112] The plasmid vector pNS2X-sacB+bktbU-lac-NSDG-bktb for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain by the same conjugation transfer method as described above. Furthermore, by the same culture as described above and selection based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the lac promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 2 were introduced into the position where the bktB gene originally existed on the chromosome. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB::lac-NSDG / dA1528 strain.

[0113] Next, the PHA synthase gene introduction plasmid vector pNS2X-sacB+A1528U-lacN17-AcNSRe12-A1528D was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB::lac-NSDG / dA1528 strain by the same conjugation transfer method as described above. Furthermore, by the same culture as described above and screening based on NutrientAgar medium containing 15% sucrose, a strain was isolated in which the lacN17 promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 6 were introduced into the position where the A1528 gene originally existed on the chromosome. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB::lac-NSDG / dA1528::lacN17-AcNSRe12 strain (hereinafter, sometimes referred to as copolymer PHA mixture producing microbial strain (2)).

[0114] It should be noted that the copolymer PHA mixture producing microbial strain (2) is a strain in which the phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal Cupric bacteria H16 strain are deleted, and a gene encoding a PHA synthase mutant having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase having the amino acid sequence recorded in sequence number 6 (i.e., a gene encoding a PHA synthase formed by combining a part of a polyhydroxyalkanoate synthase gene derived from an Aeromonas microorganism and a part of a polyhydroxyalkanoate synthase gene derived from a Cupric bacteria microorganism) are introduced, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB structural gene (β-ketothiolase gene) is deleted, and the A1528 structural gene (β-ketothiolase gene) is further deleted.

[0115] (Microbial strain preparation example 3) Preparation of microbial strain (3) for producing copolymerized PHA mixture

[0116] First, a plasmid for expression of the PHA synthase gene was prepared. The preparation was carried out as described below. By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 22) having a lacN17 promoter as a modified body of the lac promoter of Escherichia coli was obtained. The DNA fragment was digested with restriction enzymes EcoRI and MunI, and the product obtained by cutting the plasmid vector pCUP2 recorded in International Publication No. 2007 / 049716 with MunI was connected to the obtained DNA fragment, and the product connected in the direction in which the restriction enzyme SpeI recognition sequence of pCUP2 was located downstream of the lacN17 promoter was screened out to obtain pCUP2-lacN17. Next, by using PCR with synthetic oligo DNA, a DNA fragment (sequence number 23) was obtained, which had a base sequence of a gene encoding a PHA synthase having an amino acid sequence recorded in sequence number 6 and a gene encoding a PHA synthase having an amino acid sequence recorded in sequence number 2. The DNA fragment was digested with restriction enzymes MunI and SpeI, and the obtained DNA fragment was ligated with a product obtained by cleaving pCUP2-lacN17 with MunI and SpeI to obtain a plasmid pCUP2-lacN17-AcNSRe12-NSDG for expressing the PHA synthase gene.

[0117] Next, the PHA synthase gene expression plasmid pCUP2-lacN17-AcNSRe12-NSDG was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain to obtain the pCUP2-lacN17-AcNSRe12-NSDG / KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain (hereinafter sometimes referred to as the copolymer PHA mixture producing microbial strain (3)).

[0118] As described below, the plasmid vector was introduced into the cells by electroporation. The gene introduction device used was a Gene Pulser manufactured by Biorad, and the sample pool used a gap 0.2 cm manufactured by Biorad. 400 μl of competent cells and 20 μl of expression vector were injected into the sample pool and placed in a pulse device, and electric pulses were applied under the conditions of electrostatic capacitance 25 μF, voltage 1.5 kV, and resistance value 800 Ω. After the pulse, the bacterial liquid in the sample pool was shaken and cultured for 3 hours at 30°C using Nutrient Broth medium (manufactured by DIFCO), and cultured at 30°C for 2 days using a selection plate (Nutrient Agar medium (manufactured by DIFCO), kanamycin 100 mg / L), to obtain a growing copolymer PHA mixture production microbial strain (3).

[0119] It should be noted that the copolymer PHA mixture producing microbial strain (3) is a strain in which the phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal Cupric bacteria H16 strain are deleted, and a gene encoding a PHA synthase mutant having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase having the amino acid sequence recorded in sequence number 6 (i.e., a gene encoding a PHA synthase formed by combining a part of a polyhydroxyalkanoate synthase gene derived from an Aeromonas microorganism and a part of a polyhydroxyalkanoate synthase gene derived from a Cupric bacteria microorganism) are introduced, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB structural gene (β-ketothiolase gene) is deleted, and the A1528 structural gene (β-ketothiolase gene) is further deleted.

[0120] (Microbial strain preparation example 4) Preparation of microbial strain (4) for producing copolymerized PHA mixture

[0121] First, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0122] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO. 24) was obtained, which has the base sequences of the upstream and downstream of the bktB structural gene (β-ketothiolase gene) of the H16 strain of Cupricinus spp., the lac promoter of Escherichia coli, the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO. 6, and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO. 2. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Application Laid-Open No. 2007-259708 which had been similarly digested with SwaI using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare a plasmid vector pNS2X-sacB+bktbU-lac-AcNSRe12-NSDG-bktbD for introducing the PHA synthase gene.

[0123] Next, using the PHA synthase gene-transferred plasmid vector pNS2X-sacB+bktbU-lac-AcNSRe12-NSDG-bktbD, a PHA synthase gene-transferred strain was prepared as follows.

[0124] The plasmid vector pNS2X-sacB+bktbU-lac-AcNSRe12-NSDG-bktbD for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain by the same conjugation transfer method as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the lac promoter, the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 6, and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 2 were introduced into the position where the bktB gene originally existed on the chromosome. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB::lac-AcNSRe12-NSDG / dA1528 strain (hereinafter, sometimes referred to as copolymer PHA mixture producing microbial strain (4)).

[0125] It should be noted that the copolymer PHA mixture producing microbial strain (4) is a strain in which the phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal Cupric bacteria H16 strain are deleted, and a gene encoding a PHA synthase mutant having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase having the amino acid sequence recorded in sequence number 6 (i.e., a gene encoding a PHA synthase formed by combining a part of a polyhydroxyalkanoate synthase gene derived from an Aeromonas microorganism and a part of a polyhydroxyalkanoate synthase gene derived from a Cupric bacteria microorganism) are introduced, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB structural gene (β-ketothiolase gene) is deleted, and the A1528 structural gene (β-ketothiolase gene) is further deleted.

[0126] (Microbial strain preparation example 5) Preparation of microbial strain (5) for producing copolymerized PHA mixture

[0127] First, a plasmid for expressing the PHA synthase gene was prepared. The preparation was performed as described below. By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 26) was obtained, which has a base sequence of a gene encoding a PHA synthase having an amino acid sequence recorded in sequence number 7, a gene encoding a PHA synthase having an amino acid sequence recorded in sequence number 8, and a gene encoding a PHA synthase having an amino acid sequence recorded in sequence number 3. The DNA fragment was digested with restriction enzymes MunI and SpeI, and the obtained DNA fragment was connected to the product obtained by cutting pCUP2-lacN17 with MunI and SpeI to obtain a plasmid pCUP2-lacN17-RCYB4-NSDGST for expressing the PHA synthase gene.

[0128] Next, the PHA synthase gene expression plasmid pCUP2-lacN17-RCYB4-NSDGST was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain by the electroporation method described in Microbial Strain Preparation Example 3, thereby obtaining the pCUP2-lacN17-RCYB4-NSDGST / KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain (hereinafter sometimes referred to as the copolymer PHA mixture production microbial strain (5)).

[0129] It should be noted that the copolymer PHA mixture producing microbial strain (5) is a strain in which the phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 3 and a gene encoding a PHA synthase derived from the genus Bacillus having the amino acid sequence recorded in sequence number 7 and sequence number 8 are introduced, the expression of the R-body specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB structural gene (β-ketothiolase gene) is deleted, and the A1528 structural gene (β-ketothiolase gene) is further deleted.

[0130] (Microbial strain preparation example 6) Preparation of microbial strain (6) for producing copolymerized PHA mixture

[0131] First, a plasmid for introducing the PHA synthase gene was prepared. The preparation was performed as follows. By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 27) was obtained, which has the upstream and downstream base sequences of the A1528 structural gene (β-ketothiolase gene) of the H16 strain of Copperworm, the lacN17 promoter as a modified body of the lac promoter of Escherichia coli, and the base sequence of the gene encoding the PHA synthase having the amino acid sequence recorded in sequence number 5. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained DNA fragment was ligated with the vector pNS2X-sacB recorded in Japanese Patent Publication No. 2007-259708, which was also digested with SwaI, using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-sacB+A1528U-lacN17-CsAG-A1528D for introducing the PHA synthase gene.

[0132] Next, the plasmid vector pNS2X-sacB+A1528U-lacN17-CsAG-A1528D for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB::lac-NSDG / dA1528 strain by the same conjugation transfer method as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the lacN17 promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 5 were introduced into the position where the A1528 gene originally existed on the chromosome. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB::lac-NSDG / dA1528::lacN17-CsAG strain (hereinafter, sometimes also referred to as copolymer PHA mixture producing microbial strain (6)).

[0133] It should be noted that the copolymer PHA mixture producing microbial strain (6) is a strain in which the phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase derived from the genus Chromobacterium having the amino acid sequence recorded in sequence number 5 are introduced, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB structural gene (β-ketothiolase gene) is deleted, and the A1528 structural gene (β-ketothiolase gene) is further deleted.

[0134] (Microbial strain preparation example 7) Preparation of P(3HB-co-3HH) producing microbial strain (1)

[0135] The KNK005dZ strain (hereinafter, sometimes referred to as the P(3HB-co-3HH)-producing microbial strain (1)) is a transformed microorganism in which a PHA synthase gene (a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 2) derived from Aeromonas is introduced into the chromosome of the Cupricinophila spp. H16 strain, and the phaZ1, 2, and 6 genes on the chromosome, which are PHA degrading enzyme genes, are deleted. The transformed microorganism can be prepared according to the method described in PCT International Publication No. 2014 / 065253.

[0136] (Microbial strain preparation example 8) Preparation of P(3HB-co-3HH) producing microbial strain (2)

[0137] First, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0138] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO. 28) was obtained, which has the base sequences upstream and downstream of the phaZ6 structural gene of the Cupricinus entomopathogenis H16 strain, the lacN17 promoter which is a modified form of the lac promoter of Escherichia coli, and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO. 2. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Application Laid-Open No. 2007-259708 which had also been digested with SwaI using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare a plasmid vector pNS2X-sacB+phaZ6U-lacN17-NSDG-phaZ6D for introducing the PHA synthase gene.

[0139] Next, the plasmid vector pNS2X-sacB+phaZ6U-lacN17-NSDG-phaZ6D for introducing the PHA synthase gene was introduced into the KNK005dZ / trc-J4b / dbktB strain by the same conjugation transfer method as described above.

[0140] It should be noted that the KNK005dZ / trc-J4b / dbktB strain is a strain in which the phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-grabbing bacteria H16 strain are deleted, the PHA synthase gene on the chromosome is replaced with a modified version of the PHA synthase gene from Aeromonas caviae (a gene encoding a PHA synthase having the amino acid sequence recorded in sequence number 2, i.e., the N149S / D171G mutant gene), the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, and the bktB structural gene is deleted. The strain can be prepared according to the method described in PCT International Publication No. 2019 / 142845.

[0141] Furthermore, by the same culture as above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the lacN17 promoter and a gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 2 were introduced at the position on the chromosome where the phaZ6 gene originally existed. The obtained strain was named KNK005dZ / trc-J4b / Z6::lacN17-NSDG / dbktB strain (hereinafter, sometimes also referred to as P(3HB-co-3HH) producing microbial strain (2)).

[0142] (Microbial strain preparation example 9) Preparation of P(3HB-co-3HH) producing microbial strain (3)

[0143] First, the plasmid vector pNS2X-sacB+phaC1UD for PHA synthase gene disruption was introduced into the KNK005dZ / trc-J4b / dbktB strain by the same conjugation transfer method as described above.

[0144] Furthermore, a strain lacking the PHA synthase gene on the chromosome was isolated by the same culture and selection using a Nutrient Agar medium containing 15% sucrose as described above. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB strain.

[0145] Next, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0146] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO. 29) was obtained, which has the base sequences upstream and downstream of the phaC1 structural gene of the H16 strain of Cupricinus entomopathogenis and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO. 3. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Application Laid-Open No. 2007-259708 which had also been digested with SwaI using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare a plasmid vector pNS2X-sacB+phaC1U-NSDGST-phaC1D for introducing the PHA synthase gene.

[0147] Next, the plasmid vector pNS2X-sacB+phaC1U-NSDGST-phaC1D for introducing the PHA synthase gene was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB strain by the same conjugation transfer method as described above.

[0148] Furthermore, by the same culture as above and screening using a Nutrient Agar medium containing 15% sucrose, a strain was isolated in which a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 3 was introduced into the position on the chromosome where the phaC1 gene originally existed. The obtained strain was named KNK005dZ / NSDGST / trc-J4b / dbktB strain.

[0149] Next, a plasmid for expression of the PHA synthase gene was prepared. It was prepared as described below. By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 30) was obtained, which has a base sequence of a gene encoding a PHA synthase having an amino acid sequence recorded in sequence number 3. The DNA fragment was digested with restriction enzymes MunI and SpeI, and the obtained DNA fragment was connected to the product obtained by cutting pCUP2 recorded in International Publication No. 2007 / 049716 with MunI and SpeI, and pCUP2-NSDGST was obtained. Next, by using PCR with synthetic oligo DNA, a DNA fragment (sequence number 31) having a trp promoter was obtained. The DNA fragment was digested with restriction enzyme MunI, and the obtained DNA fragment was connected to the product obtained by cutting pCUP2-NSDGST with MunI in a direction in which the gene encoding the PHA synthase was located downstream of the trp promoter, and the plasmid pCUP2-trp-NSDGST for expression of the PHA synthase gene was obtained.

[0150] Next, the PHA synthase gene expression plasmid pCUP2-trp-NSDGST was introduced into the KNK005dZ / NSDGST / trc-J4b / dbktB strain by the electroporation method described in Microbial Strain Preparation Example 3, thereby obtaining the pCUP2-trp-NSDGST / KNK005dZ / NSDGST / trc-J4b / dbktB strain (hereinafter sometimes referred to as the P(3HB-co-3HH) production microbial strain (3)).

[0151] It should be noted that the P(3HB-co-3HH) producing microbial strain (3) is a strain in which the phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, and a gene encoding a PHA synthase mutant from the genus Aeromonas having the amino acid sequence recorded in sequence number 3 is introduced, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, and the bktB structural gene (β-ketothiolase gene) is deleted.

[0152] (Microbial strain preparation example 10) Preparation of P(3HB-co-3HH) producing microbial strain (4)

[0153] First, the plasmid vector pNS2X-sacB+phaC1U-NSDGST-phaC1D for introducing the PHA synthase gene was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain by the same conjugation transfer method as described above.

[0154] Furthermore, by the same culture as above and screening using a Nutrient Agar medium containing 15% sucrose, a strain was isolated in which a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 3 was introduced into the position on the chromosome where the phaC1 gene originally existed. The obtained strain was named KNK005dZ / NSDGST / trc-J4b / dbktB / dA1528 strain.

[0155] Next, the PHA synthase gene expression plasmid pCUP2-trp-NSDGST was introduced into the KNK005dZ / NSDGST / trc-J4b / dbktB / dA1528 strain by the electroporation method described in Microbial Strain Preparation Example 3, thereby obtaining the pCUP2-trp-NSDGST / KNK005dZ / NSDGST / trc-J4b / dbktB / dA1528 strain (hereinafter sometimes referred to as the P(3HB-co-3HH) production microbial strain (4)).

[0156] It should be noted that the P(3HB-co-3HH) producing microbial strain (4) is a strain in which the phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, and a gene encoding a PHA synthase mutant from the genus Aeromonas having the amino acid sequence recorded in sequence number 3 is introduced, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB structural gene (β-ketothiolase gene) is deleted, and the A1528 structural gene (β-ketothiolase gene) is further deleted.

[0157] (Example 1) PHA production based on copolymerized PHA mixture producing microbial strain (1)

[0158] Cultivation studies of the production microbial strain (1) using the copolymerized PHA mixture were conducted under the following conditions.

[0159] (Culture medium)

[0160] The composition of the mother 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).

[0161] The composition of the pre-culture medium is 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, and 0.5v / v% trace metal salt solution (a solution prepared by dissolving 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 in 0.1N hydrochloric acid).

[0162] 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 (a solution prepared by dissolving 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 in 0.1N hydrochloric acid).

[0163] (Measurement method of PHA accumulation ratio)

[0164] 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, freeze-dried, and dried bacterial cells were obtained and the weight was measured. 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 (copolymer PHA mixture) in the bacterial cells. After filtering out the bacterial residue, the mixture was concentrated with an evaporator to a total volume of 30 ml, and then 90 ml of hexanol was gradually added, slowly stirred and allowed to stand for 1 hour. After filtering out the precipitated PHA, it was vacuum dried at 50°C for 3 hours. The weight of the dry PHA was measured to calculate the ratio of the PHA accumulation to the amount of dry bacterial cells.

[0165] (Method for determining the weight ratio of PHA fractions (I) and (II))

[0166] The weight ratio of the PHA fractions (I) and (II) in the copolymerized PHA mixture was determined as follows. First, the dry PHA was fractionated into the PHA fraction (I) and the PHA fraction (II) by the MIBK fractionation method described above, and each fraction was weighed. Next, the weight ratio of each fraction relative to the total weight of the PHA fraction (I) and the PHA fraction (II) was calculated.

[0167] (Method for determining the average composition ratio of 3HH)

[0168] The average composition ratio of 3HH in each of the copolymerized PHA mixture, PHA fraction (I), or PHA fraction (II) was determined as follows. 1 ml of a sulfuric acid-methanol mixture (15:85) and 1 ml of chloroform were added to about 20 mg of the dried copolymerized PHA mixture, PHA fraction (I), or PHA fraction (II), and the mixture was sealed and heated at 100°C for 140 minutes, thereby obtaining the methyl ester of the PHA decomposition product. After cooling, 0.5 ml of deionized water was added thereto and mixed thoroughly, and then allowed to stand until the aqueous layer and the organic layer separated. Then, the monomer unit composition of the PHA decomposition product in the separated organic layer was analyzed by capillary gas chromatography. The capillary gas chromatograph used was Shimadzu Corporation GC-17A, and the capillary chromatographic column used was GL Sciences Co., Ltd. NEUTRA BOND-1 (column length 25 m, column inner diameter 0.25 mm, liquid film thickness 0.4 μm). He was used as the carrier gas, the column inlet pressure was set to 100 kPa, and 1 μl of the sample was injected. The temperature conditions were: the temperature was raised to an initial temperature of 50 to 200°C at a rate of 8°C / min, and further raised to 200 to 290°C at a rate of 30°C / min. Based on the peaks obtained by the analysis under the above conditions, the average composition ratio of 3HH in the copolymerized PHA mixture, PHA fraction (I), or PHA fraction (II) was calculated.

[0169] (Method for measuring melting peak temperature of copolymer PHA mixture)

[0170] Using a differential scanning calorimeter (DSC8500 manufactured by PerkinElmer), about 2 mg of the copolymerized PHA mixture was weighed and the temperature of the melting peak with a melting enthalpy of 0.5 J / g or more detected in the DSC curve obtained when the temperature was increased from -30°C to 200°C at a heating rate of 10°C / min was determined.

[0171] (Processability Evaluation and Pellet Preparation of Copolymer PHA Mixtures)

[0172] 4.5 g of the copolymerized PHA mixture, 0.045 g of pentaerythritol (Mitsubishi Chemical Corporation: Noiraizer P) as an additive, 0.0225 g of behenic acid amide (Nippon Seika Co., Ltd.: BNT-22H), and 0.0225 g of erucic acid amide (Nippon Seika Co., Ltd.: NEUTRON-S) were placed in a small mixer (DSM: DSM Xplore 5 model 2005) and kneaded for 5 minutes at a drum temperature of 170°C and a screw speed of 100 rpm. After the kneading was completed, the linear resin composition in a molten state was discharged through a die head and immediately placed in a water bath heated to 60°C, and the time for crystallization and solidification to occur was measured. The case where solidification occurred within 100 seconds was evaluated as good processability (○).

[0173] Then, the strands crystallized and solidified in the water bath were cut with pliers to prepare resin composition pellets.

[0174] (Evaluation of tear strength)

[0175] The demoulding surface of a PET film (thickness 50 μm) subjected to single-sided demoulding treatment was placed on a 2 mm thick SUS plate (30 cm × 35 cm) in the opposite direction to the SUS plate, and 1.3 g of resin composition particles were placed on the above PET film. Furthermore, a 70 μm pad was set as a spacer to surround the above resin composition particles. Then, a plate identical to the above SUS plate was covered in a manner to clamp the above resin composition particles, and was set on a heated pressing plate of a press machine (manufactured by Shinto Metal Industries, Ltd.: compression molding machine NSF-50) heated to 160°C, and preheated for 5 minutes. After preheating, the pressure was gradually increased to 5 MPa over 2 minutes, and then the pressure was maintained for 2 minutes. After the pressing was completed, it was cooled to room temperature on a cooling plate cooled to about 20°C to obtain a film of about 50 μm thickness. The film was aged for 1 week in an environment of room temperature 23°C and humidity 50%, and used as a film sample.

[0176] The Elmendorf tear strength of the film sample was obtained by dividing the value measured by a light load tear tester (manufactured by Kumagai Riki Kogyo Co., Ltd.: No. 2037 special specification machine) having the function and structure of a standard Elmendorf tear tester defined in JIS P-8116 by the thickness of the film.

[0177] (PHA production and cultivation)

[0178] PHA production culture was carried out as described below. First, a glycerol stock solution (50 μl) of the copolymerized PHA mixture producing microbial strain (1) was inoculated into a mother culture medium (10 ml) and cultured for 24 hours to carry out mother culture. Next, the mother culture solution was inoculated at 1.0 v / v% into a 3L fermenter (MDL-300 manufactured by BEMARUBISHI) to which 1.8L of pre-culture medium was added. The operating conditions were set to a culture temperature of 30°C, a stirring speed of 500 rpm, and an aeration volume of 1.8 L / min. The pH was controlled between 6.7 and 6.8, and cultured for 28 hours to carry out pre-culture. A 14% ammonium hydroxide aqueous solution was used for pH control.

[0179] Next, the pre-culture solution was inoculated at 5.0 v / v% into a 5 L fermenter (MDS-U50 manufactured by BEMARUBISHI) to which 2.5 L of PHA production medium was added. The operating conditions were set to a culture temperature of 33°C, a stirring speed of 420 rpm, and an aeration volume of 2.1 L / min, and the pH was controlled between 6.7 and 6.8. A 25% aqueous solution of ammonium hydroxide 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 ratio of PHA accumulation to the amount of dry bacteria reached more than 80%. The ratio of PHA accumulation to dry bacteria, the average composition ratio of 3HH in the copolymer PHA mixture, PHA fraction (I), or PHA fraction (II), the weight ratio of PHA fractions (I) and (II), the melting peak temperature and melting enthalpy, processability, and Elmendorf tear strength were measured as described above. The results are shown in Table 1.

[0180] (Example 2) PHA production based on copolymerized PHA mixture producing microbial strain (2)

[0181] Cultivation studies of the microbial strain (2) produced using the copolymerized PHA mixture were conducted under the same conditions as in Example 1. Table 1 shows the ratio of the PHA accumulation to the dry microbial cells, the average composition ratio of 3HH in the copolymerized PHA mixture, PHA fraction (I), or PHA fraction (II), the weight ratio of PHA fractions (I) and (II), the melting peak temperature and melting enthalpy, processability, and Elmendorf tear strength.

[0182] (Example 3) PHA production based on copolymerized PHA mixture producing microbial strain (3)

[0183] Cultivation studies of the microbial strain (3) produced using the copolymerized PHA mixture were conducted under the same conditions as in Example 1. Table 1 shows the ratio of the PHA accumulation to the dry microbial cells, the average composition ratio of 3HH in the copolymerized PHA mixture, PHA fraction (I), or PHA fraction (II), the weight ratio of PHA fractions (I) and (II), the melting peak temperature and melting enthalpy, processability, and Elmendorf tear strength.

[0184] (Example 4) PHA production based on copolymerized PHA mixture producing microbial strain (4)

[0185] Cultivation studies of the microbial strain (4) produced using the copolymerized PHA mixture were conducted under the same conditions as in Example 1. Table 1 shows the ratio of the PHA accumulation to the dry microbial cells, the average composition ratio of 3HH in the copolymerized PHA mixture, PHA fraction (I), or PHA fraction (II), the weight ratio of PHA fractions (I) and (II), the melting peak temperature and melting enthalpy, processability, and Elmendorf tear strength.

[0186] (Example 5) PHA production based on copolymerized PHA mixture producing microbial strain (5)

[0187] Cultivation studies of the microbial strain (5) produced using the copolymerized PHA mixture were conducted under the same conditions as in Example 1. Table 1 shows the ratio of the PHA accumulation to the dry cells, the average composition ratio of 3HH in the copolymerized PHA mixture, PHA fraction (I), or PHA fraction (II), the weight ratio of PHA fractions (I) and (II), the melting peak temperature and melting enthalpy, processability, and Elmendorf tear strength.

[0188] (Example 6) PHA production based on copolymerized PHA mixture producing microbial strain (6)

[0189] Cultivation studies of the microbial strain (6) produced using the copolymerized PHA mixture were conducted under the same conditions as in Example 1. The ratio of the PHA accumulation to the dry microbial cells, the average composition ratio of 3HH in the copolymerized PHA mixture, PHA fraction (I), or PHA fraction (II), the weight ratio of PHA fractions (I) and (II), the melting peak temperature and melting enthalpy, processability, and Elmendorf tear strength are shown in Table 1.

[0190] (Comparative Example 1) PHA production based on P(3HB-co-3HH) producing microbial strain (1)

[0191] Cultivation studies using the P(3HB-co-3HH) producing microbial strain (1) were conducted under the same conditions as in Example 1. The ratio of the PHA accumulation amount to the dry bacterial cells, the average composition ratio of 3HH in the P(3HB-co-3HH), MIBK soluble fraction, or MIBK insoluble fraction, the weight ratio of the MIBK soluble fraction and the MIBK insoluble fraction, the melting peak temperature and melting enthalpy, processability, and Elmendorf tear strength are shown in Table 1. It should be noted that in each analysis, PHA accumulated by the P(3HB-co-3HH) producing microbial strain (1) was used instead of the copolymerized PHA mixture. The PHA accumulated by the P(3HB-co-3HH) producing microbial strain (1) is P(3HB-co-3HH). The MIBK soluble fraction and the MIBK insoluble fraction refer to fractions obtained by the same method as the PHA fraction (I) or the PHA fraction (II), respectively.

[0192] (Comparative Example 2) PHA production based on P(3HB-co-3HH) producing microbial strain (2)

[0193] Cultivation studies using the P(3HB-co-3HH) producing microbial strain (2) were conducted under the same conditions as in Example 1. The ratio of the PHA accumulation amount to the dry bacterial cells, the average composition ratio of 3HH in the P(3HB-co-3HH), MIBK soluble fraction, or MIBK insoluble fraction, the weight ratio of the MIBK soluble fraction and the MIBK insoluble fraction, the melting peak temperature and melting enthalpy, processability, and Elmendorf tear strength are shown in Table 1. It should be noted that in each analysis, PHA accumulated by the P(3HB-co-3HH) producing microbial strain (2) was used instead of the copolymerized PHA mixture. The PHA accumulated by the P(3HB-co-3HH) producing microbial strain (2) is P(3HB-co-3HH). The MIBK soluble fraction and the MIBK insoluble fraction refer to fractions obtained by the same method as the PHA fraction (I) or the PHA fraction (II), respectively.

[0194] (Comparative Example 3) PHA production based on P(3HB-co-3HH) producing microbial strain (3)

[0195] Cultivation studies using the P(3HB-co-3HH) producing microbial strain (3) were conducted under the same conditions as in Example 1. The ratio of the PHA accumulation amount to the dry bacterial cells, the average composition ratio of 3HH in the P(3HB-co-3HH), MIBK soluble fraction, or MIBK insoluble fraction, the weight ratio of the MIBK soluble fraction and the MIBK insoluble fraction, the melting peak temperature and melting enthalpy, processability, and Elmendorf tear strength are shown in Table 1. It should be noted that in each analysis, PHA accumulated by the P(3HB-co-3HH) producing microbial strain (3) was used instead of the copolymerized PHA mixture. The PHA accumulated by the P(3HB-co-3HH) producing microbial strain (3) is P(3HB-co-3HH). The MIBK soluble fraction and the MIBK insoluble fraction refer to fractions obtained by the same method as the PHA fraction (I) or the PHA fraction (II), respectively.

[0196] (Comparative Example 4) PHA production based on P(3HB-co-3HH) producing microbial strain (4)

[0197] Cultivation studies using the P(3HB-co-3HH) producing microbial strain (4) were conducted under the same conditions as in Example 1. The ratio of the PHA accumulation amount to the dry bacterial cells, the average composition ratio of 3HH in the P(3HB-co-3HH), MIBK soluble fraction, or MIBK insoluble fraction, the weight ratio of the MIBK soluble fraction and the MIBK insoluble fraction, the melting peak temperature and melting enthalpy, processability, and Elmendorf tear strength are shown in Table 1. It should be noted that in each analysis, PHA accumulated by the P(3HB-co-3HH) producing microbial strain (4) was used instead of the copolymerized PHA mixture. The PHA accumulated by the P(3HB-co-3HH) producing microbial strain (4) is P(3HB-co-3HH). The MIBK soluble fraction and the MIBK insoluble fraction refer to fractions obtained by the same method as the PHA fraction (I) or the PHA fraction (II), respectively.

[0198]

[0199] According to Table 1, the copolymerized PHA mixtures obtained in Examples 1 to 6 have good processability, high Elmendorf tear strength, and excellent mechanical properties. On the other hand, in Comparative Example 1 in which the obtained copolymerized PHA mixture does not contain PHA fraction (I), the Elmendorf tear strength is a very low value. In Comparative Example 2 in which the average composition ratio of 3HH in PHA fraction (I) is not more than 20 mol%, the processability is poor, and the Elmendorf tear strength is also a very low value. In Comparative Examples 3 and 4 in which the average composition ratio of 3HH in the copolymerized PHA mixture is not less than 22 mol% and does not contain PHA fraction (II), the processability is poor. Sequence Listing <110> Kaneka Corporation <120> Method for producing copolymerized polyhydroxyalkanoate mixture, and transformed microorganism <130> B200181 <160> 31 <170> PatentIn version 3.5 <210> 1 <211> 594 <212> PRT <213> Aeromonas caviae <400> 1 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> 2 <211> 594 <212> PRT <213> Aeromonas caviae <400> 2 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> 3 <211> 594 <212> PRT <213> Aeromonas caviae <400> 3 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 Thr 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> 4 <211> 567 <212> PRT <213> Chromobacterium sp. <400> 4 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 Met 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> 5 <211> 567 <212> PRT <213> Chromobacterium sp. <400> 5 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 Gly 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> 6 <211> 585 <212> PRT <213> artificial <220> <223> artificial <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 Ser Ala Met Ala Pro Ala Asn Phe Leu Ala Thr Asn 145 150 155 160 Pro Glu Ala Gln Arg Leu Leu Ile Glu Ser Gly Gly Glu Ser Leu Arg 165 170 175 Ala Gly Val Arg Asn Met Met Glu Asp Leu Thr Arg Gly Lys Ile Ser 180 185 190 Gln Thr Asp Glu Ser Ala Phe Glu Val Gly Arg Asn Val Ala Val Thr 195 200 205 Glu Gly Ala Val Val Phe Glu Asn Glu Tyr Phe Gln Leu Leu Gln Tyr 210 215 220 Lys Pro Leu Thr Asp Lys Val His Ala Arg Pro Leu Leu Met Val Pro 225 230 235 240 Pro Cys Ile Asn Lys Tyr Tyr Ile Leu Asp Leu Gln Pro Glu Ser Ser 245 250 255 Leu Val Arg His Val Val Glu Gln Gly His Thr Val Phe Leu Val Ser 260 265 270 Trp Arg Asn Pro Asp Ala Ser Met Ala Gly Ser Thr Trp Asp Asp Tyr 275 280 285 Ile Glu His Ala Ala Ile Arg Ala Ile Glu Val Ala Arg Asp Ile Ser 290 295 300 Gly Gln Asp Lys Ile Asn Val Leu Gly Phe Cys Val Gly Gly Thr Ile 305 310 315 320 Val Ser Thr Ala Leu Ala Val Leu Ala Ala Arg Gly Glu His Pro Ala 325 330 335 Ala Ser Val Thr Leu Leu Thr Thr Leu Leu Asp Phe Ala Asp Thr Gly 340 345 350 Ile Leu Asp Val Phe Val Asp Glu Gly His Val Gln Leu Arg Glu Ala 355 360 365 Thr Leu Gly Gly Gly Ala Gly Ala Pro Cys Ala Leu Leu Arg Gly Leu 370 375 380 Glu Leu Ala Asn Thr Phe Ser Phe Leu Arg Pro Asn Asp Leu Val Trp 385 390 395 400 Asn Tyr Val Val Asp Asn Tyr Leu Lys Gly Asn Thr Pro Val Pro Phe 405 410 415 Asp Leu Leu Phe Trp Asn Gly Asp Ala Thr Asn Leu Pro Gly Pro Trp 420 425 430 Tyr Cys Trp Tyr Leu Arg His Thr Tyr Leu Gln Asn Glu Leu Lys Val 435 440 445 Pro Gly Lys Leu Thr Val Cys Gly Val Pro Val Asp Leu Ala Ser Ile 450 455 460 Asp Val Pro Thr Tyr Ile Tyr Gly Ser Arg Glu Asp His Ile Val Pro 465 470 475 480 Trp Thr Ala Ala Tyr Ala Ser Thr Ala Leu Leu Ala Asn Lys Leu Arg 485 490 495 Phe Val Leu Gly Ala Ser Gly His Ile Ala Gly Val Ile Asn Pro Pro 500 505 510 Ala Lys Asn Lys Arg Ser His Trp Thr Asn Asp Ala Leu Pro Glu Ser 515 520 525 Pro Gln Gln Trp Leu Ala Gly Ala Ile Glu His His Gly Ser Trp Trp 530 535 540 Pro Asp Trp Thr Ala Trp Leu Ala Gly Gln Ala Gly Ala Lys Arg Ala 545 550 555 560 Ala Pro Ala Asn Tyr Gly Asn Ala Arg Tyr Arg Ala Ile Glu Pro Ala 565 570 575 Pro Gly Arg Tyr Val Lys Ala Lys Ala 580 585 <210> 7 <211> 160 <212> PRT <213> Bacillus cereus <400> 7 Met Ile Asp Gln Lys Phe Asp Pro Leu Gln Ala Trp Lys Asn Ala Tyr 1 5 10 15 Glu Gln Thr Glu Thr Phe Trp Gly Lys Ala Leu Asn Glu Thr Ile Lys 20 25 30 Thr Glu Glu Tyr Ser Ala Trp Met Gly Ser Val Leu Asp Leu Asn Leu 35 40 45 Phe Tyr Gln Lys Ala Leu Asn Asp Thr Thr Lys Asn Tyr Leu Glu Gln 50 55 60 Val Asn Val Pro Thr Lys Glu Asp Ile Ala Arg Val Ala Thr Leu Val 65 70 75 80 Ile Asn Leu Glu Asn Lys Val Asp Asn Ile Glu Glu Phe Leu Glu Glu 85 90 95 Lys Val Glu Ser Val Gly Gln Ala Pro Thr Leu Lys Arg Asp Val Thr 100 105 110 Lys Val Lys Gln Asp Ile Arg Thr Leu Glu Thr Lys Val Asp Gln Ile 115 120 125 Leu Glu Leu Leu Glu Lys Gln Asn Ala Val Leu Ala Lys Leu Gln Glu 130 135 140 Pro Val Lys Glu Glu Val Lys Pro Thr Asn Lys Pro Glu Asn Lys Lys 145 150 155 160 <210> 8 <211> 361 <212> PRT <213> Bacillus cereus <400> 8 Met Thr Thr Phe Ala Thr Glu Trp Glu Lys Gln Leu Glu Leu Tyr Pro 1 5 10 15 Glu Glu Tyr Arg Lys Ala Tyr Arg Arg Val Lys Arg Ala Ser Glu Ile 20 25 30 Leu Leu Arg Glu Pro Glu Pro Gln Val Gly Leu Thr Pro Lys Glu Val 35 40 45 Ile Trp Thr Lys Asn Lys Thr Lys Leu Tyr Arg Tyr Ile Pro Lys Gln 50 55 60 Glu Lys Thr Gln Arg Val Pro Ile Leu Leu Ile Tyr Ala Leu Ile Asn 65 70 75 80 Lys Pro Tyr Ile Met Asp Leu Thr Pro Gly Asn Ser Leu Val Glu Tyr 85 90 95 Leu Val Asp Arg Gly Phe Asp Val Tyr Met Leu Asp Trp Gly Thr Phe 100 105 110 Gly Leu Glu Asp Ser His Leu Lys Phe Asp Asp Phe Val Phe Asp Tyr 115 120 125 Ile Ala Lys Ala Val Lys Lys Val Met Arg Thr Ala Lys Ser Asp Glu 130 135 140 Ile Ser Leu Leu Gly Tyr Cys Met Gly Gly Thr Leu Thr Ser Ile Tyr 145 150 155 160 Ala Ala Leu His Pro His Met Pro Ile Arg Asn Leu Ile Phe Met Thr 165 170 175 Ser Pro Phe Asp Phe Ser Glu Thr Gly Leu Tyr Gly Pro Leu Leu Asp 180 185 190 Glu Lys Tyr Phe Asn Leu Asp Lys Ala Val Asp Thr Phe Gly Asn Ile 195 200 205 Pro Pro Glu Met Ile Asp Phe Gly Asn Lys Met Leu Lys Pro Ile Thr 210 215 220 Asn Phe Val Gly Pro Tyr Val Ala Leu Val Asp Arg Ser Glu Asn Glu 225 230 235 240 Arg Phe Val Glu Ser Trp Arg Leu Val Gln Lys Trp Val Gly Asp Gly 245 250 255 Ile Pro Phe Pro Gly Glu Ser Tyr Arg Gln Trp Ile Arg Asp Phe Tyr 260 265 270 Gln Asn Asn Lys Leu Val Lys Gly Glu Leu Val Ile Arg Gly Gln Lys 275 280 285 Val Asp Leu Ala Asn Ile Lys Ala Asn Val Leu Asn Ile Ser Gly Lys 290 295 300 Arg Asp His Ile Ala Leu Pro Cys Gln Val Glu Ala Leu Leu Asp His 305 310 315 320 Ile Ser Ser Thr Asp Lys Gln Tyr Val Cys Leu Pro Thr Gly His Met 325 330 335 Ser Ile Val Tyr Gly Gly Thr Ala Val Lys Gln Thr Tyr Pro Thr Ile 340 345 350 Gly Asn Trp Leu Glu Glu Arg Ser Asn 355 360 <210> 9 <211> 394 <212> PRT <213> Cupriavidus necator <400> 9 Met Thr Arg Glu Val Val Val Val Ser Gly Val Arg Thr Ala Ile Gly 1 5 10 15 Thr Phe Gly Gly Ser Leu Lys Asp Val Ala Pro Ala Glu Leu Gly Ala 20 25 30 Leu Val Val Arg Glu Ala Leu Ala Arg Ala Gln Val Ser Gly Asp Asp 35 40 45 Val Gly His Val Val Phe Gly Asn Val Ile Gln Thr Glu Pro Arg Asp 50 55 60 Met Tyr Leu Gly Arg Val Ala Ala Val Asn Gly Gly Val Thr Ile Asn 65 70 75 80 Ala Pro Ala Leu Thr Val Asn Arg Leu Cys Gly Ser Gly Leu Gln Ala 85 90 95 Ile Val Ser Ala Ala Gln Thr Ile Leu Leu Gly Asp Thr Asp Val Ala 100 105 110 Ile Gly Gly Gly Ala Glu Ser Met Ser Arg Ala Pro Tyr Leu Ala Pro 115 120 125 Ala Ala Arg Trp Gly Ala Arg Met Gly Asp Ala Gly Leu Val Asp Met 130 135 140 Met Leu Gly Ala Leu His Asp Pro Phe His Arg Ile His Met Gly Val 145 150 155 160 Thr Ala Glu Asn Val Ala Lys Glu Tyr Asp Ile Ser Arg Ala Gln Gln 165 170 175 Asp Glu Ala Ala Leu Glu Ser His Arg Arg Ala Ser Ala Ala Ile Lys 180 185 190 Ala Gly Tyr Phe Lys Asp Gln Ile Val Pro Val Val Ser Lys Gly Arg 195 200 205 Lys Gly Asp Val Thr Phe Asp Thr Asp Glu His Val Arg His Asp Ala 210 215 220 Thr Ile Asp Asp Met Thr Lys Leu Arg Pro Val Phe Val Lys Glu Asn 225 230 235 240 Gly Thr Val Thr Ala Gly Asn Ala Ser Gly Leu Asn Asp Ala Ala Ala 245 250 255 Ala Val Val Met Met Glu Arg Ala Glu Ala Glu Arg Arg Gly Leu Lys 260 265 270 Pro Leu Ala Arg Leu Val Ser Tyr Gly His Ala Gly Val Asp Pro Lys 275 280 285 Ala Met Gly Ile Gly Pro Val Pro Ala Thr Lys Ile Ala Leu Glu Arg 290 295 300 Ala Gly Leu Gln Val Ser Asp Leu Asp Val Ile Glu Ala Asn Glu Ala 305 310 315 320 Phe Ala Ala Gln Ala Cys Ala Val Thr Lys Ala Leu Gly Leu Asp Pro 325 330 335 Ala Lys Val Asn Pro Asn Gly Ser Gly Ile Ser Leu Gly His Pro Ile 340 345 350 Gly Ala Thr Gly Ala Leu Ile Thr Val Lys Ala Leu His Glu Leu Asn 355 360 365 Arg Val Gln Gly Arg Tyr Ala Leu Val Thr Met Cys Ile Gly Gly Gly 370 375 380 Gln Gly Ile Ala Ala Ile Phe Glu Arg Ile 385 390 <210> 10 <211> 392 <212> PRT <213> Cupriavidus necator <400> 10 Met Asn Glu Ala Val Ile Val Ser Thr Ala Arg Thr Pro Leu Ala Lys 1 5 10 15 Ser Trp Lys Gly Ala Phe Asn Met Thr His Gly Ala Thr Leu Gly Gly 20 25 30 His Ala Val Gln His Ala Ile Ala Arg Ala Lys Ile Glu Ala Ala Glu 35 40 45 Val Glu Asp Val Leu Met Gly Cys Ala Asn Pro Glu Gly Ala Thr Gly 50 55 60 Ala Asn Ile Ala Arg Gln Ile Ala Leu Arg Ala Gly Cys Pro Val Thr 65 70 75 80 Val Pro Gly Ala Thr Val Asn Arg Phe Cys Ser Ser Gly Leu Gln Thr 85 90 95 Ile Ala Met Ala Ala Gln Arg Val Ile Ala Asp Glu Gly Asp Ile Phe 100 105 110 Val Ala Gly Gly Val Glu Ser Ile Ser Cys Val Gln Gln Glu Met Asn 115 120 125 Arg His Met Val Gln Glu Ser Trp Leu Leu Lys Asn Lys Pro Glu Ile 130 135 140 Tyr Trp Asn Met Leu Gln Thr Ala Glu Asn Val Ala Lys Arg Tyr Asn 145 150 155 160 Ile Ser Lys Glu Arg Gln Asp Glu Tyr Gly Val Arg Ser Gln Gln Arg 165 170 175 Ala Ala Ala Gly Gln Glu Ala Gly Lys Phe Lys Asp Glu Ile Val Pro 180 185 190 Met Thr Val Leu Ala Gly Val Ala Asp Lys Ser Thr Gly Gln Leu Val 195 200 205 Thr Lys Glu Val Thr Val Ser Ala Asp Glu Gly Ile Arg Ala Asp Thr 210 215 220 Thr Leu Glu Gly Val Ser Lys Ile Arg Ser Ala Val Pro Gly Gly Val 225 230 235 240 Ile Thr Ala Gly Asn Ala Ser Gln Phe Ser Asp Gly Ala Ser Ala Ala 245 250 255 Val Val Met Asn Ala Arg Val Ala Glu Ala Arg Gly Leu Gln Pro Leu 260 265 270 Gly Val Phe Arg Gly Phe Ala Val Ala Gly Cys Glu Pro Asp Glu Met 275 280 285 Gly Ile Gly Pro Val Phe Ala Val Pro Lys Leu Leu Lys Lys Ala Gly 290 295 300 Leu Lys Val Asp Asp Ile Gly Leu Trp Glu Leu Asn Glu Ala Phe Ala 305 310 315 320 Val Gln Val Leu Tyr Cys Ala Asp Thr Leu Gly Ile Pro Met Asp Arg 325 330 335 Leu Asn Val Asn Gly Gly Ala Ile Ala Val Gly His Pro Tyr Gly Val 340 345 350 Ser Gly Ala Arg Leu Val Gly His Ala Leu Ile Glu Gly Lys Arg Arg 355 360 365 Gly Val Lys Tyr Val Val Val Thr Met Cys Ile Gly Gly Gly Gln Gly 370 375 380 Ala Ala Gly Leu Phe Glu Val Leu 385 390 <210> 11 <211> 134 <212> PRT <213> Aeromonas caviae <400> 11 Met Ser Ala Gln Ser Leu Glu Val Gly Gln Lys Ala Arg Leu Ser Lys 1 5 10 15 Arg Phe Gly Ala Ala Glu Val Ala Ala Phe Ala Ala Leu Ser Glu Asp 20 25 30 Phe Asn Pro Leu His Leu Asp Pro Ala Phe Ala Ala Thr Thr Ala Phe 35 40 45 Glu Arg Pro Ile Val His Gly Met Leu Leu Ala Ser Leu Phe Ser Gly 50 55 60 Leu Leu Gly Gln Gln Leu Pro Gly Lys Gly Ser Ile Tyr Leu Gly Gln 65 70 75 80 Ser Leu Ser Phe Lys Leu Pro Val Phe Val Gly Asp Glu Val Thr Ala 85 90 95 Glu Val Glu Val Thr Ala Leu Arg Glu Asp Lys Pro Ile Ala Thr Leu 100 105 110 Thr Thr Arg Ile Phe Thr Gln Gly Gly Ala Leu Ala Val Thr Gly Glu 115 120 125 Ala Val Val Lys Leu Pro 130 <210> 12 <211> 158 <212> PRT <213> Cupriavidus necator <400> 12 Met Arg Thr Ile Ala Ser Leu Glu Glu Leu Glu Gly Leu Gln Gly Gln 1 5 10 15 Glu Val Ala Val Ser Asp Trp Ile Glu Val Thr Gln Gln Gln Val Asn 20 25 30 Gln Phe Ala Asp Ala Thr Gly Asp His Gln Trp Ile His Ile Asp Val 35 40 45 Glu Arg Ala Lys Lys Glu Ser Pro Tyr Gly Gly Pro Ile Ala His Gly 50 55 60 Phe Leu Thr Leu Ser Leu Leu Pro Lys Phe Met His Asn Ala Leu His 65 70 75 80 Met Pro Ser Lys Ile Gly Val Asn Tyr Gly Leu Asn Arg Val Arg Phe 85 90 95 Thr Ala Pro Val Pro Val Gly Ser Lys Leu Arg Ala Arg Ile Lys Leu 100 105 110 Leu Lys Val Glu Arg Leu Asp Pro Leu Pro Lys Ser Pro Glu Leu Val 115 120 125 Gly Ala Gln Ser Thr Trp Glu Val Thr Val Glu Arg Glu Gly Ser Asp 130 135 140 Arg Pro Val Cys Val Ala Glu Ser Ile Thr Arg Arg Tyr Gly 145 150 155 <210> 13 <211> 151 <212> PRT <213> Cupriavidus necator <400> 13 Met Lys Thr Tyr Glu Asn Ile Ala Asp Leu Gln Pro Leu Val Gly Glu 1 5 10 15 Val Ile Gly Thr Ser Glu Trp Leu Ala Leu Asp Gln Ala Arg Ile Asn 20 25 30 Thr Phe Ala Asp Ala Thr Gly Asp His Gln Trp Ile His Val Asp Val 35 40 45 Glu Arg Ala Lys Asn Gly Pro Phe Gly Ala Pro Ile Ala His Gly Phe 50 55 60 Leu Thr Leu Ser Leu Leu Pro Ala Phe Thr His Ser Ala Tyr Arg Ile 65 70 75 80 Arg Asn Ser Ser Thr Gly Val Asn Tyr Gly Leu Asp Lys Val Arg Phe 85 90 95 Pro Ala Pro Val Pro Val Asp Ser Leu Leu Arg Ala Gln Phe Lys Leu 100 105 110 Met Ser Tyr Glu Ala Leu Glu Asn Gly Gly Ala Gln Phe Lys Val Glu 115 120 125 Met Met Val Glu Arg Gln Gly Gly Ser Lys Pro Val Cys Ile Ala Glu 130 135 140 Ser Ile Leu Arg Arg Phe Pro 145 150 <210> 14 <211> 901 <212> PRT <213> Yarrowia lipolytica <400> 14 Met Ser Gly Glu Leu Arg Tyr Asp Gly Lys Val Val Ile Val Thr Gly 1 5 10 15 Ala Gly Gly Gly Leu Gly Lys Ala Tyr Ala Leu Phe Tyr Gly Ser Arg 20 25 30 Gly Ala Ser Val Val Val Asn Asp Leu Gly Gly Asp Phe Lys Gly Asp 35 40 45 Gly Ala Gln Ala Gly Ser Gly Lys Arg Val Ala Asp Val Val Val Asp 50 55 60 Glu Ile Val Ser Lys Gly Gly Lys Ala Val Ala Asn Tyr Asp Ser Val 65 70 75 80 Glu Asn Gly Asp Lys Ile Val Glu Thr Ala Val Lys Ala Phe Gly Ser 85 90 95 Val His Ile Val Ile Asn Asn Ala Gly Ile Leu Arg Asp Ile Ser Phe 100 105 110 Lys Lys Met Thr Asp Lys Asp Trp Asp Leu Val Tyr Lys Val His Val 115 120 125 Phe Gly Ala Tyr Lys Val Thr Arg Ala Ala Trp Pro Tyr Phe Arg Lys 130 135 140 Gln Lys Tyr Gly Arg Val Ile Ser Thr Ser Ser Ala Ala Gly Leu Tyr 145 150 155 160 Gly Asn Phe Gly Gln Thr Asn Tyr Ser Ala Ala Lys Leu Ala Leu Val 165 170 175 Gly Phe Gly Glu Thr Leu Ala Lys Glu Gly Ala Lys Tyr Asn Ile Thr 180 185 190 Ser Asn Val Ile Ala Pro Leu Ala Ala Ser Arg Met Thr Glu Thr Val 195 200 205 Met Pro Glu Asp Ile Leu Lys Leu Leu Lys Pro Glu Tyr Val Val Pro 210 215 220 Leu Val Gly Tyr Leu Thr His Asp Ser Val Thr Glu Ser Tyr Gly Ile 225 230 235 240 Tyr Glu Val Gly Ala Gly Tyr Met Ala Lys Ile Arg Trp Glu Arg Gly 245 250 255 Asn Gly Ala Val Phe Lys Gly Asp Asp Thr Phe Thr Pro Ser Ala Ile 260 265 270 Leu Lys Arg Trp Asp Glu Val Thr Ser Phe Glu Ser Pro Thr Tyr Pro 275 280 285 Asn Gly Pro Ala Asp Phe Phe Lys Tyr Ala Glu Glu Ser Val Lys Arg 290 295 300 Pro Glu Asn Pro Gln Gly Pro Thr Val Ser Phe Lys Asp Gln Val Val 305 310 315 320 Ile Val Thr Gly Ala Gly Ala Gly Ile Gly Arg Ala Tyr Ser His Leu 325 330 335 Leu Ala Lys Leu Gly Ala Lys Val Val Val Asn Asp Phe Gly Asn Pro 340 345 350 Gln Lys Val Val Asp Glu Ile Lys Ala Leu Gly Gly Ile Ala Val Ala 355 360 365 Asp Lys Asn Asn Val Ile His Gly Glu Lys Val Val Gln Thr Ala Ile 370 375 380 Asp Ala Phe Gly Ala Val His Ala Val Val Asn Asn Ala Gly Ile Leu 385 390 395 400 Arg Asp Lys Ser Phe Ala Asn Met Asp Asp Glu Met Trp Gln Leu Ile 405 410 415 Phe Asp Val His Leu Asn Gly Thr Tyr Ser Val Thr Lys Ala Ala Trp 420 425 430 Pro His Phe Leu Lys Gln Lys Tyr Gly Arg Val Ile Asn Thr Thr Ser 435 440 445 Thr Ser Gly Ile Tyr Gly Asn Phe Gly Gln Ala Asn Tyr Ser Ala Ala 450 455 460 Lys Ala Gly Ile Leu Gly Phe Ser Arg Ala Leu Ala Arg Glu Gly Glu 465 470 475 480 Lys Tyr Asn Ile Leu Val Asn Thr Ile Ala Pro Asn Ala Gly Thr Ala 485 490 495 Met Thr Ala Ser Val Phe Thr Glu Glu Met Leu Glu Leu Phe Lys Pro 500 505 510 Asp Phe Ile Ala Pro Ile Thr Val Leu Leu Ala Ser Asp Gln Ala Pro 515 520 525 Val Thr Gly Asp Leu Phe Glu Thr Gly Ser Ala Trp Ile Gly Gln Thr 530 535 540 Arg Trp Gln Arg Ala Gly Gly Lys Ala Phe Asn Thr Lys Lys Gly Val 545 550 555 560 Thr Pro Glu Met Val Arg Asp Ser Trp Ala Lys Ile Val Asp Phe Asp 565 570 575 Asp Gly Asn Ser Thr His Pro Thr Thr Pro Ser Glu Ser Thr Thr Gln 580 585 590 Ile Leu Glu Asn Ile Phe Asn Val Pro Asp Glu Glu Val Glu Glu Thr 595 600 605 Ala Leu Val Ala Gly Pro Gly Gly Pro Gly Ile Leu Asn Lys Glu Gly 610 615 620 Glu Pro Phe Asp Tyr Thr Tyr Thr Tyr Arg Asp Leu Ile Leu Tyr Asn 625 630 635 640 Leu Gly Leu Gly Ala Lys Ala Asn Glu Leu Lys Tyr Val Phe Glu Gly 645 650 655 Asp Asp Asp Phe Gln Thr Val Pro Thr Phe Gly Val Ile Pro Tyr Met 660 665 670 Gly Gly Leu Ile Thr Thr Asn Tyr Gly Asp Phe Val Pro Asn Phe Asn 675 680 685 Pro Met Met Leu Leu His Gly Glu Gln Tyr Leu Glu Ile Arg Gln Trp 690 695 700 Pro Ile Pro Thr Asn Ala Thr Leu Glu Asn Lys Ala Lys Val Ile Asp 705 710 715 720 Val Val Asp Lys Gly Lys Ala Ala Leu Leu Val Thr Ala Thr Thr Thr 725 730 735 Thr Asn Lys Glu Thr Gly Glu Glu Val Phe Tyr Asn Glu Ser Ser Leu 740 745 750 Phe Ile Arg Gly Ser Gly Gly Phe Gly Gly Lys Ser Thr Gly Thr Asp 755 760 765 Arg Gly Ala Ala Thr Ala Ala Asn Lys Pro Pro Ala Arg Ala Pro Asp 770 775 780 Phe Val Lys Glu Ile Lys Ile Gln Glu Asp Gln Ala Ala Ile Tyr Arg 785 790 795 800 Leu Ser Gly Asp Tyr Asn Pro Leu His Ile Asp Pro Ala Phe Ala Ala 805 810 815 Val Gly Asn Phe Asp Arg Pro Ile Leu His Gly Leu Cys Ser Phe Gly 820 825 830 Val Ser Gly Lys Ala Leu Tyr Asp Gln Phe Gly Pro Phe Lys Asn Ala 835 840 845 Lys Val Arg Phe Ala Gly His Val Phe Pro Gly Glu Thr Leu Lys Val 850 855 860 Glu Gly Trp Lys Glu Gly Asn Lys Val Ile Phe Gln Thr Lys Val Val 865 870 875 880 Glu Arg Gly Thr Thr Ala Ile Ser Asn Ala Ala Ile Glu Leu Phe Pro 885 890 895 Lys Asp Ala Lys Leu 900 <210> 15 <211> 598 <212> PRT <213> Drosophila melanogaster <400> 15 Met Ser Ser Ser Asp Gly Lys Leu Arg Tyr Asp Gly Arg Val Ala Val 1 5 10 15 Val Thr Gly Ala Gly Ala Gly Leu Gly Arg Glu Tyr Ala Leu Leu Phe 20 25 30 Ala Glu Arg Gly Ala Lys Val Val Val Asn Asp Leu Gly Gly Thr His 35 40 45 Ser Gly Asp Gly Ala Ser Gln Arg Ala Ala Asp Ile Val Val Asp Glu 50 55 60 Ile Arg Lys Ala Gly Gly Glu Ala Val Ala Asp Tyr Asn Ser Val Ile 65 70 75 80 Asp Gly Ala Lys Val Ile Glu Thr Ala Ile Lys Ala Phe Gly Arg Val 85 90 95 Asp Ile Leu Val Asn Asn Ala Gly Ile Leu Arg Asp Arg Ser Leu Val 100 105 110 Lys Thr Ser Glu Gln Asp Trp Asn Leu Val Asn Asp Val His Leu Lys 115 120 125 Gly Ser Phe Lys Cys Thr Gln Ala Ala Phe Pro Tyr Met Lys Lys Gln 130 135 140 Asn Tyr Gly Arg Ile Ile Met Thr Ser Ser Asn Ser Gly Ile Tyr Gly 145 150 155 160 Asn Phe Gly Gln Val Asn Tyr Thr Ala Ala Lys Met Gly Leu Ile Gly 165 170 175 Leu Ala Asn Thr Val Ala Ile Glu Gly Ala Arg Asn Asn Val Leu Cys 180 185 190 Asn Val Ile Val Pro Thr Ala Ala Ser Arg Met Thr Glu Gly Ile Leu 195 200 205 Pro Asp Ile Leu Phe Asn Glu Leu Lys Pro Lys Leu Ile Ala Pro Val 210 215 220 Val Ala Tyr Leu Cys His Glu Ser Cys Glu Asp Asn Gly Ser Tyr Ile 225 230 235 240 Glu Ser Ala Ala Gly Trp Ala Thr Lys Leu His Met Val Arg Gly Lys 245 250 255 Gly Ala Val Leu Arg Pro Ser Leu Asp Asp Pro Val Thr Ile Glu Tyr 260 265 270 Val Lys Asp Val Trp Ser Asn Val Thr Asp Met Ser Lys Ala Lys His 275 280 285 Leu Gly Ala Ile Ala Glu Ala Ser Gly Thr Leu Leu Glu Val Leu Glu 290 295 300 Lys Leu Lys Glu Gly Gly Gly Asp Ala Ile Glu Asp Ala Phe Glu Phe 305 310 315 320 Asn Ser Lys Glu Leu Ile Thr Tyr Ala Leu Gly Ile Gly Ala Ser Val 325 330 335 Lys Asn Ala Lys Asp Met Arg Phe Leu Tyr Glu Asn Asp Ala Asp Phe 340 345 350 Ala Ala Ile Pro Thr Phe Phe Val Leu Pro Gly Leu Leu Leu Gln Met 355 360 365 Ser Thr Asp Lys Leu Leu Ser Lys Ala Leu Pro Asn Ser Gln Val Asp 370 375 380 Phe Ser Asn Ile Leu His Gly Glu Gln Tyr Leu Glu Ile Val Asp Asp 385 390 395 400 Leu Pro Thr Ser Gly Thr Leu Leu Thr Asn Gly Lys Val Phe Asp Val 405 410 415 Met Asp Lys Gly Ser Gly Ala Val Val Val Thr Asn Ser Glu Ser Phe 420 425 430 Asp Glu Ser Gly Arg Leu Leu Val Arg Asn Gln Ser Thr Thr Phe Ile 435 440 445 Val Gly Ala Gly Lys Phe Gly Gly Lys Lys Asp Pro Ile Ala Gly Val 450 455 460 Val Pro Leu Gln Pro Ala Pro Asn Arg Gln Pro Asp Ala Thr Val Gln 465 470 475 480 Tyr Thr Thr Ser Glu Asp Gln Ala Ala Leu Tyr Arg Leu Ser Gly Asp 485 490 495 Lys Asn Pro Leu His Ile Asp Pro Gln Met Ala Leu Leu Ala Gly Phe 500 505 510 Lys Thr Pro Ile Leu His Gly Leu Cys Thr Leu Gly Phe Ser Val Arg 515 520 525 Ala Val Leu Ala Gln Phe Ala Asp Asn Asn Pro Ala Leu Phe Lys Ala 530 535 540 Val Lys Val Arg Phe Ser Gly Pro Val Ile Pro Gly Gln Thr Leu Arg 545 550 555 560 Val Asp Leu Trp Lys Gln Gly Thr Arg Ile Asn Phe Arg Thr Val Val 565 570 575 Val Glu Thr Gly Lys Glu Val Ile Ser Gly Ala Tyr Val Asp Leu Lys 580 585 590 Ser Ser Gln Ala Lys Leu 595 <210> 16 <211> 29 <212> DNA <213> Escherichia coli <400> 16 tttacactta tgcttccggc tcgtatgtt 29 <210> 17 <211> 31 <212> DNA <213> Escherichia coli <400> 17 tttacacctt tatgcttccg gctcgtatgt t 31 <210> 18 <211> 1056 <212> DNA <213> Artificial <220> <223> Artificial <400> 18 gcgcgcattt aaatctgcca ccacgttgtg ctcggtgatc gccatcatca gcgccacgta 60 gagccagcca atggccacga tgtacatcaa aaattcatcc ttctcgccta tgctctgggg 120 cctcggcaga tgcgagcgct gcataccgtc cggtaggtcg ggaagcgtgc agtgccgagg 180 cggattcccg cattgacagc gcgtgcgttg caaggcaaca atggactcaa atgtctcgga 240 atcgctgacg attcccaggt ttctccggca agcatagcgc atggcgtctc catgcgagaa 300 tgtcgcgctt gccggataaa aggggagccg ctatcggaat ggacgcaagc cacggccgca 360 gcaggtgcgg tcgagggctt ccagccagtt ccagggcaga tgtgccggca gaccctcccg 420 ctttggggga ggcgcaagcc gggtccattc ggatagcatc tccccatgca aagtgccggc 480 cagggcaatg cccggagccg gttcgaatag tgacggcaga gagacaatca aatccgcttg 540 catgagtgcc ggcgtgcgtc atgcacagcg ccggcaggcc tgcaggttcc ctcccgtttc 600 cattgaaagg actacacaat gactgacgtt gtcatcgtat ccgccgcccg caccgcggtc 660 ggcaagtttg gcggctcgct ggccaagatc ccggcaccgg aactgggtgc cgtggtcatc 720 aaggccgcgc tggagcgcgc cggcgtcaag ccggagcagg tgagcgaagt catcatgggc 780 caggtgctga ccgccggttc gggccagaac cccgcacgcc aggccgcgat caaggccggc 840 ctgccggcga tggtgccggc catgaccatc aacaaggtgt gcggctcggg cctgaaggcc 900 gtgatgctgg ccgccaacgc gatcatggcg ggcgacgccg agatcgtggt ggccggcggc 960 caggaaaaca tgagcgccgc cccgcacgtg ctgccgggct cgcgcgatgg tttccgcatg 1020 ggcgatgcca agctggtcga caatttaaat gcgcgc 1056 <210> 19 <211> 3034 <212> DNA <213> Artificial <220> <223> Artificial <400> 19 tacgctagcg taccgagcta tccatttaaa tggagttcca gaccctgctc gactttatcg 60 ccgaagccga actggaccgc gtcggctgct tcgcctactc gccggtggag ggcgccaccg 120 ccaatgacct gccgggcgcg ctgcccgacg aggtgcgcga ggaacgccgc gcccgcttca 180 tggaagtggc cgaagaggtc tcggcgcgcc gcctgcagcg caaggtcggc cagaccctgc 240 gcgtgctggt ggacgaggtc aaccaggatg gcggcatcgg ccgttcgtcc gcggatgcgc 300 cggaaatcga cggcctcgtc tatatcgcgc cgccggaacg ccacgcccag cgctatcgcg 360 ccggcgagtt cgtcgacgtg aagatcaccg gcgccgatgg ccacgacctg tggggcgcgg 420 tctgaaacgg gttgattagg taaaagtacg ctcgttcgat ttcgtccgcg tcccgctata 480 ctgtgcggtg caacataacc tcatggagac aaagtccaat tggcgcaacg caattaatgt 540 gagttagctc actcattagg caccccaggc tttacacctt tatgcttccg gctcgtatgt 600 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa caattgcacg tgcagagaga 660 caatcaaatc atgagccaac catcttatgg cccgctgttc gaggccctgg cccactacaa 720 tgacaagctg ctggccatgg ccaaggccca gacagagcgc accgcccagg cgctgctgca 780 gaccaatctg gacgatctgg gccaggtgct ggagcagggc agccagcaac cctggcaact 840 gatccaggcc cagatgaact ggtggcagga tcagctcaag ctgatgcagc acaccctgct 900 caaaagcgca ggccagccga gcgagccggt gatcaccccg gagcgcagcg atcgccgctt 960 caaggccgag gcctggagcg aacaacccat ctatgactac ctcaagcagt cctacctgct 1020 caccgccagg cacctgctgg cctcggtgga tgccctggag ggcgtccccc agaagagccg 1080 ggagcggctg cgtttcttca cccgccagta cgtcagcgcc atggccccca gcaacttcct 1140 ggccaccaac cccgagctgc tcaagctgac cctggagtcc ggcggccaga acctggtgcg 1200 cgggctggcc ctcttggccg aggatctgga gcgcagcgcc gatcagctca acatccgcct 1260 gaccgacgaa tccgccttcg agctcgggcg ggatctggcg ctcaccccgg gccgggtggt 1320 gcagcgcacc gagctctatg agctgatcca gtacagcccg actaccgaga cggtgggcaa 1380 gacacctgtg ctgatagtgc cccccttcat caacaagtac tacatcatgg acatgcggcc 1440 ccagaactcc ctggtcgcct ggctggtcgc ccagggccag acggtgttca tgatctcctg 1500 gcgcaacccg ggcgtggccc aggcccaaat cgatctcgac gactacgtgg tggacggggt 1560 catcgccgcc ctggacgggg tggaggcggc caccggcgag cgggaggtgc acggcatcgg 1620 ctactgcatc ggcggcactg cactgtcgct cgccatgggc tggctggcgg cgcggcgcca 1680 gaagcagcga gtgcgcaccg ccaccctgtt cactaccctg ctggacttct cccagccagg 1740 ggagctcggc atcttcatcc acgagcccat catagcggcg ctcgaggcgc aaatgaggc 1800 caagggcatc atggacgggc gccagctggc tgtctccttc agcctgctgc gggagaacag 1860 cctctactgg aactactaca tcgacagcta cctcaagggc cagagcccgg tggccttcga 1920 tctgctgcac tggaacagcg acagcaccaa tgtggcgggc aagacccaca acagcctgct 1980 gcgccgtctc tacctggaga accagctggt gaagggggag ctcaagatcc gcaacacccg 2040 catcgatctc ggcaaggtga aaaccccggt actgctggtg tcggccgtgg acgatcacat 2100 cgccctctgg cagggcacct ggcagggcat gaagctgttt ggcggggagc agcgcttcct 2160 cctggcggag tccggccaca tcgccggcat catcaacccg ccggccgcca acaagtacgg 2220 cttctggcac aacggggccg aggccgagag cccggagagc tggctggcag gggcgacgca 2280 ccagggcggc tcctggtggc ccgagatgat gggctttatc cagaaccgtg acgaagggtc 2340 ggagcccgtc cccgcgcggg tgcccgagga ggggctggcc cccgcccccg gccactatgt 2400 caaggtgcgg ctcaaccccg tgtttgcctg cccaacagag gaggacgccg catgacctgc 2460 cggcctggtt caaccagtcg gcagccgact agtggttagc cttgcgcctt gcttcgctga 2520 cgaagaacct ctgcgcgctc ggcctggccg ccgtgctcgc gctggcgggc acggcgcagg 2580 cagcgccggc gaccgagctg tcgaccggcc cggttaacac cgggccagcc ggcggcgaag 2640 gcctgggcat caaccaggcc attcgcgacg gcgaagcccg gcgcggcggc acctcgcttt 2700 ccactggcgc gccaaaaccc ctggcgccgc ggccggaata tgcgtcgctg ccggtctacg 2760 tcggcaaggt cggcgaccag ccggtgcggc tgcgcctggg ccccaagcct gacgagcgcg 2820 acagcgtgcg tggcgaatac gccggccgtg gcgccggtgt gcgcctgctg gcaggcgagt 2880 gggaggacgg cgccttcctg atggaggagt ccgacgacgg cacccgcgta tcgggcaact 2940 gggaaggcag catcgacgcc agcggcgccg tgcgcggcac ctggaccgaa tttaaatccg 3000 aattcactgg ccgtcgtttt acaacgtcgt gact 3034 <210> 20 <211> 2825 <212> DNA <213> Artificial <220> <223> Artificial <400> 20 tacgctagcg taccgagcta tccatttaaa ttgcatgcgg gcttgcacgt gccgcatgcg 60 cggctggtga tccgcaaggg ccaccatgcc gacgtggaca gctactccgc cttcctggag 120 gcggaccgca ccacgcgcac cgggctggcc ggctacctgc gcgagcatgg cgtcaggcgc 180 gtgttctgcg cgggtctggc gacggactac tgcgtggcct ggagcgcgct cgatgcacgc 240 gccgcgggct tcgcggccgc ggtgatcgag gatgcctgcc gcgccatcga cctggaaggg 300 tcgctggcca aagcatggca ggacctcggc gccgccggcg ttgcgcgcgt cacgtccgct 360 gaactgctca agggccaggg ctgaaccgaa ccaccgaact gaacgacaca agaatccg 420 aggagcaaga ccaattggcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 480 caggctttac acttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat 540 ttcacacagg aaacaattgc acgtgcagag agaatcaa atcatgagcc aaccatctta 600 tggcccgctg ttcgaggccc tggcccacta caatgacaag ctgctggcca tggccaaggc 660 ccagacagag cgcaccgcc aggcgctgct gcagaccaat ctggacgatc tgggccaggt 720 gctggagcag ggcagccagc aaccctggca gctgatccag gcccagatga actggtggca 780 ggatcagctc aagctgatgc agcacaccct gctcaaaagc gcaggccagc cgagcgagcc 840 ggtgatcacc ccggagcgca gcgatcgccg cttcaaggcc gaggcctgga gcgaacaacc catctatgac tacctcaagc agtcctacct gctcaccgcc aggcacctgc tggcctcggt 960 ggatgccctg gagggcgtcc cccagaagag ccggggagcgg ctgcgtttct tcacccgcca 1020. gtacgtcagc gccatggccc ccgccaactt ccttgccacc aatcccgagg cgcagcgcct 1080 gctgatcgag tcggggcggcg aatcgctgcg tgccggcgtg cgcaacatga tggaagacct 1140 gacacgcggc aagatctcgc agaccgacga gagcgcgttt gaggtcggcc gcaatgtcgc ggtgaccgaa ggcgccgtgg tcttcgagaa cgagtacttc cagctgttgc agtacaagcc gctgaccgac aaggtgcacg cgcgcccgct gctgatggtg ccgccgtgca tcaacaagta ctacatcctg gacctgcagc cggagagctc gctggtgcgc catgtggtgg agcaggaca 1380 tacggtgttt ctggtgtcgt ggcgcaatcc ggacgccagc atggccggca gcacctggga 1440 cgactacatc gagcacgcgg ccatccgcgc catcgaagtc gcgcgcgaca tcagcggcca ggacaagatc aacgtgctcg gcttctgcgt gggcggcacc attgtctcga ccgcgctggc 1560 ggtgctggcc gcgcgcggcg agcacccggc cgccagcgtc acgctgctga ccacgctgct 1620 ggactttgcc gacacgggca tcctcgacgt ctttgtcgac gagggccatg tgcagttgcg 1680 cgaggccacg ctgggcggcg gcgccggcgc gccgtgcgcg ctgctgcgcg gccttgagct 1740 ggccaatacc ttctcgttct tgcgcccgaa cgacctggtg tggaactacg tggtcgacaa 1800 ctacctgaag ggcaacacgc cggtgccgtt cgacctgctg ttctggaacg gcgacgccac 1860 caacctgccg gggccgtggt actgctggta cctgcgccac acctacctgc agaacgagct 1920 caaggtaccg ggcaagctga ccgtgtgcgg cgtgccggtg gacctggcca gcatcgacgt 1980 gccgacctat atctacggct cgcgcgaaga ccatatcgtg ccgtggaccg cggcctatgc 2040 ctcgaccgcg ctgctggcga acaagctgcg cttcgtgctg ggtgcgtcgg gccatatcgc 2100 cggtgtgatc aacccgccgg ccaagaacaa gcgcagccac tggactaacg atgcgctgcc 2160 ggagtcgccg cagcaatggc tggccggcgc catcgagcat cacggcagct ggtggccgga 2220 ctggaccgca tggctggccg ggcaggccgg cgcgaaacgc gccgcgcccg ccaactatgg 2280 caatgcgcgc tatcgcgcaa tcgaacccgc gcctgggcga tacgtcaaag ccaaggcatg 2340 acctgccggc ctggttcaac cagtcggcag ccgactagta tgtcggctgc gggcaccgcg 2400 cgggccacgc ccgccgacat gctcgcctgg ggccgggagg tgctggccag ccagcccttc 2460 tcggtcctgg tcggcaccga actggcggcg ttgtcgccgg gcaaggctga gttgcgcctg 2520 ccgatccgtc aggacctgcg gcagcagcac ggcttcctgc acggcggcgt ggtcagctac 2580 ctggccgaca atgcgctgac ctacgcgggc ggcgcggcca tggcggtgcc ggtcgtgaca 2640 tccgagtaca agatcaacta cgtgcgcccg gcgatcggcg agctgctggt cgcccgcgcc 2700 gaatgcgtca gcgccggccg ccagcaggcg gtggtgcgct gcgacgtgta cgtcgtcagg 2760 gatggcgagg agaggctctg atttaaatcc gaattcactg gccgtcgttt tacaacgtcg 2820 tgact 2825 <210> 21 <211> 3033 <212> DNA <213> Artificial <220> <223> Artificial <400> 21 tacgctagcg taccgagcta tccatttaaa tggagttcca gaccctgctc gactttatcg 60 ccgaagccga actggaccgc gtcggctgct tcgcctactc gccggtggag ggcgccaccg 120 ccaatgacct gccgggcgcg ctgcccgacg aggtgcgcga ggaacgccgc gcccgcttca 180 tggaagtggc cgaagaggtc tcggcgcgcc gcctgcagcg caaggtcggc cagaccctgc 240 gcgtgctggt ggacgaggtc aaccaggatg gcggcatcgg ccgttcgtcc gcggatgcgc 300 cggaaatcga cggcctcgtc tatatcgcgc cgccggaacg ccacgcccag cgctatcgcg 360 ccggcgagtt cgtcgacgtg aagatcaccg gcgccgatgg ccacgacctg tggggcgcgg 420 tctgaaacgg gttgattagg taaaagtacg ctcgttcgat ttcgtccgcg tcccgctata 480 ctgtgcggtg caacataacc tcatggagac aaagtccaat tggcgcaacg caattaatgt 540 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt 600 gtgtggaatt gtgagcggat aacaatttca cacaggaaac aattgcacgt gcagagagac 660 aatcaaatca tgagccaacc atcttatggc ccgctgttcg aggccctggc ccactacaat 720 gacaagctgc tggccatggc caaggcccag acagagcgca ccgcccaggc gctgctgcag 780 accaatctgg acgatctggg ccaggtgctg gagcagggca gccagcaacc ctggcaactg 840 atccaggccc agatgaactg gtggcaggat cagctcaagc tgatgcagca caccctgctc 900 aaaagcgcag gccagccgag cgagccggtg atcaccccgg agcgcagcga tcgccgcttc 960 aaggccgagg cctggagcga acaacccatc tatgactacc tcaagcagtc ctacctgctc 1020 accgccaggc acctgctggc ctcggtggat gccctggagg gcgtccccca gaagagccgg 1080 gagcggctgc gtttcttcac ccgccagtac gtcagcgcca tggcccccag caacttcctg 1140 gccaccaacc ccgagctgct caagctgacc ctggagtccg gcggccagaa cctggtgcgc 1200 gggctggccc tcttggccga ggatctggag cgcagcgccg atcagctcaa catccgcctg 1260 accgacgaat ccgccttcga gctcgggcgg gatctggcgc tcaccccggg ccgggtggtg 1320 cagcgcaccg agctctatga gctgatccag tacagcccga ctaccgagac ggtgggcaag 1380 acacctgtgc tgatagtgcc ccccttcatc aacaagtact acatcatgga catgcggccc 1440 cagaactccc tggtcgcctg gctggtcgcc cagggccaga cggtgttcat gatctcctgg 1500 cgcaacccgg gcgtggccca ggcccaaatc gatctcgacg actacgtggt ggacggggtc 1560 atcgccgccc tggacggggt ggaggcggcc accggcgagc gggaggtgca cggcatcggc 1620 tactgcatcg gcggcactgc actgtcgctc gccatgggct ggctggcggc gcggcgccag 1680 aagcagcgag tgcgcaccgc caccctgttc actaccctgc tggacttctc ccagccaggg 1740 gagctcggca tcttcatcca cgagcccatc atagcggcgc tcgaggcgca aaatgaggcc 1800 aagggcatca tggacgggcg ccagctggct gtctccttca gcctgctgcg ggagaacagc 1860 ctctactgga actactacat cgacagctac ctcaagggcc agagcccggt ggccttcgat 1920 ctgctgcact ggaacagcga cagcaccaat gtggcgggca agacccacaa cagcctgctg 1980 cgccgtctct acctggagaa ccagctggtg aagggggagc tcaagatccg caacacccgc 2040 atcgatctcg gcaaggtgaa aaccccggta ctgctggtgt cggccgtgga cgatcacatc 2100 gccctctggc agggcacctg gcagggcatg aagctgtttg gcggggagca gcgcttcctc 2160 ctggcggagt ccggccacat cgccggcatc atcaacccgc cggccgccaa caagtacggc 2220 ttctggcaca acggggccga ggccgagagc ccggagagct ggctggcagg ggcgacgcac 2280 cagggcggct cctggtggcc cgagatgatg ggctttatcc agaaccgtga cgaagggtcg 2340 gagcccgtcc ccgcgcgggt gcccgaggag gggctggccc ccgcccccgg ccactatgtc 2400 aaggtgcggc tcaaccccgt gtttgcctgc ccaacagagg aggacgccgc atgacctgcc 2460 ggcctggttc aaccagtcgg cagccgacta gtggtagcc ttgcgccttg cttcgctgac 2520 gaagaacctc tgcgcgctcg gcctggccgc cgtgctcgcg ctggcgggca cggcgcaggcgc 2580 agcgccggcg accgagctgt cgaccggccc ggttaacacc gggccagccg gcggcgaagg 2640 cctgggcatc aaccaggcca ttcgcgacgg cgaagcccgg cgcggcggca cctcgctttc 2700 cactggcgcg ccaaaacccc tggcgccgcg gccggaatat gcgtcgctgc cggtctacgt 2760 cggcaaggtc ggcgaccagc cggtgcggct gcgcctgggc cccaagcctg acgagcgcga 2820 cagcgtgcgt ggcgaatacg ccggccgtgg cgccggtgtg cgcctgctgg caggcgagtg 2880 ggaggacggc gccttcctga tggaggagtc cgacgacggc acccgcgtat cgggcaactg 2940 ggaaggcagc atcgacgcca gcggcgccgt gcgcggcacc tggaccgaat ttaaatccga 3000 attcactggc cgtcgtttta caacgtcgtg act 3033 <210> 22 <211> 140 <212> DNA <213> artificial <220> <223> artificial <400> 22 gcgcgcgaat tcgcgcaacg caattaatgt gagttagctc actcattagg caccccaggc 60 tttacactta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac 120 acaggaaaca attggcgcgc 140 <210> 23 <211> 3706 <212> DNA <213> artificial <220> <223> artificial <400> 23 ggaaacaatt gcacgtgcag agagacaatc aaatcatgag ccaaccatct tatggcccgc 60 tgttcgaggc cctggcccac tacaatgaca agctgctggc catggccaag gcccagacag 120 agcgcaccgc ccaggcgctg ctgcagacca atctggacga tctgggccag gtgctggagc 180 agggcagcca gcaaccctgg cagctgatcc aggcccagat gaactggtgg caggatcagc tcaagctgat gcagcacacc ctgctcaaaa gcgcaggcca gccgagcgag ccggtgatca 300 ccccggagcg cagcgatcgc cgcttcaagg ccgaggcctg gagcgaacaa cccatctatg actacctcaa gcagtcctac ctgctcaccg ccaggcacct gctggcctcg gtggatgccc 420 tggagggcgt cccccagaag agccgggagc ggctgcgttt cttcacccgc cagtacgtca 480 gcgccatggc ccccgccaac ttccttgcca ccaatcccga ggcgcagcgc ctgctgatcg 540 agtcgggcgg cgaatcgctg cgtgccggcg tgcgcaacat gatggaagac ctgacacgcg gcaagatctc gcagaccgac gagagcgcgt ttgaggtcgg ccgcaatgtc gcggtgaccg 660 aaggcgccgt ggtcttcgag aacgagtact tccagctgtt gcagtacaag ccgctgaccg 720 acaaggtgca cgcgcgcccg ctgctgatgg tgccgccgtg catcaacaag tactacatcc tggacctgca gccggagagc tcgctggtgc gccatgtggt ggagcaggga catacggtgt 840 ttctggtgtc gtggcgcaat ccggacgcca gcatggccgg cagcacctgg gacgactaca 900 tcgagcacgc ggccatccgc gccatcgaag tcgcgcgcga catcagcggc caggacaaga 960 tcaacgtgct cggcttctgc gtgggcggca ccattgtctc gaccgcgctg gcggtgctgg 1020 ccgcgcgcgg cgagcacccg gccgccagcg tcacgctgct gaccacgctg ctggactttg 1080 ccgacacggg catcctcgac gtctttgtcg acgagggcca tgtgcagttg cgcgaggcca 1140 cgctgggcgg cggcgccggc gcgccgtgcg cgctgctgcg cggccttgag ctggccaata 1200 ccttctcgtt cttgcgcccg aacgacctgg tgtggaacta cgtggtcgac aactacctga 1260 agggcaacac gccggtgccg ttcgacctgc tgttctggaa cggcgacgcc accaacctgc 1320 cggggccgtg gtactgctgg tacctgcgcc acacctacct gcagaacgag ctcaaggtac 1380 cgggcaagct gaccgtgtgc ggcgtgccgg tggacctggc cagcatcgac gtgccgacct 1440 atatctacgg ctcgcgcgaa gaccatatcg tgccgtggac cgcggcctat gcctcgaccg 1500 cgctgctggc gaacaagctg cgcttcgtgc tgggtgcgtc gggccatatc gccggtgtga 1560 tcaacccgcc ggccaagaac aagcgcagcc actggactaa cgatgcgctg ccggagtcgc 1620 cgcagcaatg gctggccggc gccatcgagc atcacggcag ctggtggccg gactggaccg 1680 catggctggc cgggcaggcc ggcgcgaaac gcgccgcgcc cgccaactat ggcaatgcgc 1740 gctatcgcgc aatcgaaccc gcgcctgggc gatacgtcaa agccaaggca tgacgcttta 1800 aagagtgccg gcgtgcgtca tgcacggcgc cggcaggcct gcaggttccc tcccgtttcc 1860 attgaaagga ctacacaatg agccaaccat cttatggccc gctgttcgag gccctggccc 1920 actacaatga caagctgctg gccatggcca aggcccagac agagcgcacc gcccaggcgc 1980 tgctgcagac caatctggac gatctgggcc aggtgctgga gcagggcagc cagcaaccct 2040 ggcagctgat ccaggcccag atgaactggt ggcaggatca gctcaagctg atgcagcaca 2100 ccctgctcaa aagcgcaggc cagccgagcg agccggtgat caccccggag cgcagcgatc 2160 gccgcttcaa ggccgaggcc tggagcgaac aacccatcta tgactacctc aagcagtcct 2220 acctgctcac cgccaggcac ctgctggcct cggtggatgc cctggagggc gtcccccaga 2280 agagccggga gcggctgcgt ttcttcaccc gccagtacgt cagcgccatg gcccccagca 2340 acttcctggc caccaacccc gagctgctca agctgaccct ggagtccggc ggccagaacc 2400 tggtgcgcgg actggccctc ttggccgagg atctggagcg cagcgccgat cagctcaaca 2460 tccgcctgac cgacgaatcc gccttcgagc tcgggcggga tctggccctg accccgggcc 2520 gggtggtgca gcgcaccgag ctctatgagc tcattcagta cagcccgact accgagacgg 2580 tgggcaagac acctgtgctg atagtgccgc ccttcatcaa caagtactac atcatggaca 2640 tgcggcccca gaactccctg gtcgcctggc tggtcgccca gggccagacg gtattcatga 2700 tctcctggcg caacccgggc gtggcccagg cccaaatcga tctcgacgac tacgtggtgg 2760 atggcgtcat cgccgccctg gacggcgtgg aggcggccac cggcgagcgg gaggtgcacg 2820 gcatcggcta ctgcatcggc ggcaccgccc tgtcgctcgc catgggctgg ctggcggcgc 2880 ggcgccagaa gcagcgggtg cgcaccgcca ccctgttcac taccctgctg gacttctccc 2940 agcccgggga gcttggcatc ttcatccacg agcccatcat agcggcgctc gaggcgcaaa 3000 atgaggccaa gggcatcatg gacgggcgcc agctggcggt ctccttcagc ctgctgcggg 3060 agaacagcct ctactggaac tactacatcg acagctacct caagggtcag agcccggtgg 3120 ccttcgatct gctgcactgg aacagcgaca gcaccaatgt ggcgggcaag acccacaaca 3180 gcctgctgcg ccgtctctac ctggagaacc agctggtgaa gggggagctc aagatccgca 3240 acacccgcat cgatctcggc aaggtgaaga cccctgtgct gctggtgtcg gcggtggacg 3300 atcacatcgc cctctggcag ggcacctggc agggcatgaa gctgtttggc ggggagcagc 3360 gcttcctcct ggcggagtcc ggccacatcg ccggcatcat caacccgccg gccgccaaca 3420 agtacggctt ctggcacaac ggggccgagg ccgagagccc ggagagctgg ctggcagggg 3480 cgacgcacca gggcggctcc tggtggcccg agatgatggg ctttatccag aaccgtgacg 3540 aagggtcaga gcccgtcccc gcgcgggtcc cggaggaagg gctggccccc gcccccggcc 3600 actatgtcaa ggtgcggctc aaccccgtgt ttgcctgccc aacagaggag gacgccgcat 3660 gacctgccgg cctggttcaa ccagtcggca gccgactagt ggatcc 3706 <210> 24 <211> 4875 <212> DNA <213> Artificial <220> <223> Artificial <400> 24 tacgctagcg taccgagcta tccatttaaa tggagttcca gaccctgctc gactttatcg 60 ccgaagccga actggaccgc gtcggctgct tcgcctactc gccggtggag ggcgccaccg 120 ccaatgacct gccgggcgcg ctgcccgacg aggtgcgcga ggaacgccgc gcccgcttca 180 tggaagtggc cgaagaggtc tcggcgcgcc gcctgcagcg caaggtcggc cagaccctgc 240 gcgtgctggt ggacgaggtc aaccaggatg gcggcatcgg ccgttcgtcc gcggatgcgc 300 cggaaatcga cggcctcgtc tatatcgcgc cgccggaacg ccacgcccag cgctatcgcg 360 ccggcgagtt cgtcgacgtg aagatcaccg gcgccgatgg ccacgacctg tggggcgcgg 420 tctgaaacgg gttgattagg taaaagtacg ctcgttcgat ttcgtccgcg tcccgctata 480 ctgtgcggtg caacataacc tcatggagac aaagtccaat tggcgcaacg caattaatgt 540 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt 600 gtgtggaatt gtgagcggat aacaatttca cacaggaaac aattgcacgt gcagagagac 660 aatcaaatca tgagccaacc atcttatggc ccgctgttcg aggccctggc ccactacaat 720 gacaagctgc tggccatggc caaggcccag acagagcgca ccgcccaggc gctgctgcag 780 accaatctgg acgatctggg ccaggtgctg gagcagggca gccagcaacc ctggcagctg 840 atccaggccc agatgaactg gtggcaggat cagctcaagc tgatgcagca caccctgctc 900 aaaagcgcag gccagccgag cgagccggtg atcaccccgg agcgcagcga tcgccgcttc 960 aaggccgagg cctggagcga acaacccatc tatgactacc tcaagcagtc ctacctgctc 1020 accgccaggc acctgctggc ctcggtggat gccctggagg gcgtccccca gaagagccgg 1080 gagcggctgc gtttcttcac ccgccagtac gtcagcgcca tggcccccgc caacttcctt 1140 gccaccaatc ccgaggcgca gcgcctgctg atcgagtcgg gcggcgaatc gctgcgtgcc 1200 ggcgtgcgca acatgatgga agacctgaca cgcggcaaga tctcgcagac cgacgagagc 1260 gcgtttgagg tcggccgcaa tgtcgcggtg accgaaggcg ccgtggtctt cgagaacgag 1320 tacttccagc tgttgcagta caagccgctg accgacaagg tgcacgcgcg cccgctgctg 1380 atggtgccgc cgtgcatcaa caagtactac atcctggacc tgcagccgga gagctcgctg 1440 gtgcgccatg tggtggagca gggacatacg gtgtttctgg tgtcgtggcg caatccggac 1500 gccagcatgg ccggcagcac ctgggacgac tacatcgagc acgcggccat ccgcgccatc 1560 gaagtcgcgc gcgacatcag cggccaggac aagatcaacg tgctcggctt ctgcgtgggc 1620 ggcaccattg tctcgaccgc gctggcggtg ctggccgcgc gcggcgagca cccggccgcc 1680 agcgtcacgc tgctgaccac gctgctggac tttgccgaca cgggcatcct cgacgtcttt 1740 gtcgacgagg gccatgtgca gttgcgcgag gccacgctgg gcggcggcgc cggcgcgccg 1800 tgcgcgctgc tgcgcggcct tgagctggcc aataccttct cgttcttgcg cccgaacgac 1860 ctggtgtgga actacgtggt cgacaactac ctgaagggca acacgccggt gccgttcgac 1920 ctgctgttct ggaacggcga cgccaccaac ctgccggggc cgtggtactg ctggtacctg 1980 cgccacacct acctgcagaa cgagctcaag gtaccgggca agctgaccgt gtgcggcgtg 2040 ccggtggacc tggccagcat cgacgtgccg acctatatct acggctcgcg cgaagaccat 2100 atcgtgccgt ggaccgcggc ctatgcctcg accgcgctgc tggcgaacaa gctgcgcttc 2160 gtgctgggtg cgtcgggcca tatcgccggt gtgatcaacc cgccggccaa gaacaagcgc 2220 agccactgga ctaacgatgc gctgccggag tcgccgcagc aatggctggc cggcgccatc 2280 gagcatcacg gcagctggtg gccggactgg accgcatggc tggccgggca ggccggcgcg 2340 aaacgcgccg cgcccgccaa ctatggcaat gcgcgctatc gcgcaatcga acccgcgcct 2400 gggcgatacg tcaaagccaa ggcatgacgc tttaaagagt gccggcgtgc gtcatgcacg 2460 gcgccggcag gcctgcaggt tccctcccgt ttccattgaa aggactacac aatgagccaa 2520 ccatcttatg gcccgctgtt cgaggccctg gcccactaca atgacaagct gctggccatg 2580 gccaaggccc agacagagcg caccgcccag gcgctgctgc agaccaatct ggacgatctg 2640 ggccaggtgc tggagcaggg cagccagcaa ccctggcagc tgatccaggc ccagatgaac 2700 tggtggcagg atcagctcaa gctgatgcag cacaccctgc tcaaaagcgc aggccagccg 2760 agcgagccgg tgatcacccc ggagcgcagc gatcgccgct tcaaggccga ggcctggagc 2820 gaacaaccca tctatgacta cctcaagcag tcctacctgc tcaccgccag gcacctgctg 2880 gcctcggtgg atgccctgga gggcgtcccc cagaagagcc gggagcggct gcgtttcttc 2940 acccgccagt acgtcagcgc catggccccc agcaacttcc tggccaccaa ccccgagctg 3000 ctcaagctga ccctggagtc cggcggccag aacctggtgc gcggactggc cctcttggcc 3060 gaggatctgg agcgcagcgc cgatcagctc aacatccgcc tgaccgacga atccgccttc 3120 gagctcgggc gggatctggc cctgaccccg ggccgggtgg tgcagcgcac cgagctctat 3180 gagctcattc agtacagccc gactaccgag acggtgggca agacacctgt gctgatagtg 3240 ccgcccttca tcaacaagta ctacatcatg gacatgcggc cccagaactc cctggtcgcc 3300 tggctggtcg cccagggcca gacggtattc atgatctcct ggcgcaaccc gggcgtggcc 3360 caggcccaaa tcgatctcga cgactacgtg gtggatggcg tcatcgccgc cctggacggc 3420 gtggaggcgg ccaccggcga gcgggaggtg cacggcatcg gctactgcat cggcggcacc 3480 gccctgtcgc tcgccatggg ctggctggcg gcgcggcgcc agaagcagcg ggtgcgcacc 3540 gccaccctgt tcactaccct gctggacttc tcccagcccg gggagcttgg catcttcatc 3600 cacgagccca tcatagcggc gctcgaggcg caaaatgagg ccaagggcat catggacggg 3660 cgccagctgg cggtctcctt cagcctgctg cgggagaaca gcctctactg gaactactac 3720 atcgacagct acctcaaggg tcagagcccg gtggccttcg atctgctgca ctggaacagc 3780 gacagcacca atgtggcggg caagacccac aacagcctgc tgcgccgtct ctacctggag 3840 aaccagctgg tgaaggggga gctcaagatc cgcaacaccc gcatcgatct cggcaaggtg 3900 aagacccctg tgctgctggt gtcggcggtg gacgatcaca tcgccctctg gcagggcacc 3960 tggcagggca tgaagctgtt tggcggggag cagcgcttcc tcctggcgga gtccggccac 4020 atcgccggca tcatcaaccc gccggccgcc aacaagtacg gcttctggca caacggggcc 4080 gaggccgaga gcccggagag ctggctggca ggggcgacgc accagggcgg ctcctggtgg 4140 cccgagatga tgggctttat ccagaaccgt gacgaagggt cagagcccgt ccccgcgcgg 4200 gtcccggagg aagggctggc ccccgccccc ggccactatg tcaaggtgcg gctcaacccc 4260 gtgtttgcct gcccaacaga ggaggacgcc gcatgacctg ccggcctggt tcaaccagtc 4320 ggcagccgac tagtggttag ccttgcgcct tgcttcgctg acgaagaacc tctgcgcgct 4380 cggcctggcc gccgtgctcg cgctggcggg cacggcgcag gcagcgccgg cgaccgagct 4440 gtcgaccggc ccggttaaca ccgggccagc cggcggcgaa ggcctgggca tcaaccaggc 4500 cattcgcgac ggcgaagccc ggcgcggcgg cacctcgctt tccactggcg cgccaaaacc 4560 cctggcgccg cggccggaat atgcgtcgct gccggtctac gtcggcaagg tcggcgacca 4620 gccggtgcgg ctgcgcctgg gccccaagcc tgacgagcgc gacagcgtgc gtggcgaata 4680 cgccggccgt ggcgccggtg tgcgcctgct ggcaggcgag tgggaggacg gcgccttcct 4740 gatggaggag tccgacgacg gcacccgcgt atcgggcaac tgggaaggca gcatcgacgc 4800 cagcggcgcc gtgcgcggca cctggaccga atttaaatcc gaattcactg gccgtcgttt 4860 tacaacgtcg tgact 4875 <210> 25 <211> 4874 <212> DNA <213> Artificial <220> <223> Artificial <400> 25 tacgctagcg taccgagcta tccatttaaa tggagttcca gaccctgctc gactttatcg 60 ccgaagccga actggaccgc gtcggctgct tcgcctactc gccggtggag ggcgccaccg 120 ccaatgacct gccgggcgcg ctgcccgacg aggtgcgcga ggaacgccgc gcccgcttca 180 tggaagtggc cgaagaggtc tcggcgcgcc gcctgcagcg caaggtcggc cagaccctgc 240 gcgtgctggt ggacgaggtc aaccaggatg gcggcatcgg ccgttcgtcc gcggatgcgc 300 cggaaatcga cggcctcgtc tatatcgcgc cgccggaacg ccacgcccag cgctatcgcg 360 ccggcgagtt cgtcgacgtg aagatcaccg gcgccgatgg ccacgacctg tggggcgcgg 420 tctgaaacgg gttgattagg taaaagtacg ctcgttcgat ttcgtccgcg tcccgctata 480 ctgtgcggtg caacataacc tcatggagac aaagtccaat tcgcgcaacg caattaatgt 540 gagttagctc actcattagg caccccaggc tttacactta tgcttccggc tcgtatgttg 600 tgtggaattg tgagcggata acaatttcac acaggaaaca attgcacgtg cagagagaca 660 atcaaatcat gagccaacca tcttatggcc cgctgttcga ggccctggcc cactacaatg 720 acaagctgct ggccatggcc aaggcccaga cagagcgcac cgcccaggcg ctgctgcaga 780 ccaatctgga cgatctgggc caggtgctgg agcagggcag ccagcaaccc tggcagctga 840 tccaggccca gatgaactgg tggcaggatc agctcaagct gatgcagcac accctgctca 900 aaagcgcagg ccagccgagc gagccggtga tcaccccgga gcgcagcgat cgccgcttca 960 aggccgaggc ctggagcgaa caacccatct atgactacct caagcagtcc tacctgctca 1020 ccgccaggca cctgctggcc tcggtggatg ccctggaggg cgtcccccag aagagccggg 1080 agcggctgcg tttcttcacc cgccagtacg tcagcgccat ggcccccgcc aacttccttg 1140 ccaccaatcc cgaggcgcag cgcctgctga tcgagtcggg cggcgaatcg ctgcgtgccg 1200 gcgtgcgcaa catgatggaa gacctgacac gcggcaagat ctcgcagacc gacgagagcg 1260 cgtttgaggt cggccgcaat gtcgcggtga ccgaaggcgc cgtggtcttc gagaacgagt 1320 acttccagct gttgcagtac aagccgctga ccgacaaggt gcacgcgcgc ccgctgctga 1380 tggtgccgcc gtgcatcaac aagtactaca tcctggacct gcagccggag agctcgctgg 1440 tgcgccatgt ggtggagcag ggacatacgg tgtttctggt gtcgtggcgc aatccggacg 1500 ccagcatggc cggcagcacc tgggacgact acatcgagca cgcggccatc cgcgccatcg 1560 aagtcgcgcg cgacatcagc ggccaggaca agatcaacgt gctcggcttc tgcgtgggcg 1620 gcaccattgt ctcgaccgcg ctggcggtgc tggccgcgcg cggcgagcac ccggccgcca 1680 gcgtcacgct gctgaccacg ctgctggact ttgccgacac gggcatcctc gacgtctttg 1740 tcgacgaggg ccatgtgcag ttgcgcgagg ccacgctggg cggcggcgcc ggcgcgccgt 1800 gcgcgctgct gcgcggcctt gagctggcca ataccttctc gttcttgcgc ccgaacgacc 1860 tggtgtggaa ctacgtggtc gacaactacc tgaagggcaa cacgccggtg ccgttcgacc 1920 tgctgttctg gaacggcgac gccaccaacc tgccggggcc gtggtactgc tggtacctgc 1980 gccacaccta cctgcagaac gagctcaagg taccgggcaa gctgaccgtg tgcggcgtgc 2040 cggtggacct ggccagcatc gacgtgccga cctatatcta cggctcgcgc gaagaccata 2100 tcgtgccgtg gaccgcggcc tatgcctcga ccgcgctgct ggcgaacaag ctgcgcttcg 2160 tgctgggtgc gtcgggccat atcgccggtg tgatcaaccc gccggccaag aacaagcgca 2220 gccactggac taacgatgcg ctgccggagt cgccgcagca atggctggcc ggcgccatcg 2280 agcatcacgg cagctggtgg ccggactgga ccgcatggct ggccgggcag gccggcgcga 2340 aacgcgccgc gcccgccaac tatggcaatg cgcgctatcg cgcaatcgaa cccgcgcctg 2400 ggcgatacgt caaagccaag gcatgacgct ttaaagagtg ccggcgtgcg tcatgcacgg 2460 cgccggcagg cctgcaggtt ccctcccgtt tccattgaaa ggactacaca atgagccaac 2520 catcttatgg cccgctgttc gaggccctgg cccactacaa tgacaagctg ctggccatgg 2580 ccaaggccca gacagagcgc accgcccagg cgctgctgca gaccaatctg gacgatctgg 2640 gccaggtgct ggagcagggc agccagcaac cctggcagct gatccaggcc cagatgaact 2700 ggtggcagga tcagctcaag ctgatgcagc acaccctgct caaaagcgca ggccagccga 2760 gcgagccggt gatcaccccg gagcgcagcg atcgccgctt caaggccgag gcctggagcg 2820 aacaacccat ctatgactac ctcaagcagt cctacctgct caccgccagg cacctgctgg 2880 cctcggtgga tgccctggag ggcgtccccc agaagagccg ggagcggctg cgtttcttca 2940 cccgccagta cgtcagcgcc atggccccca gcaacttcct ggccaccaac cccgagctgc 3000 tcaagctgac cctggagtcc ggcggccaga acctggtgcg cggactggcc ctcttggccg 3060 aggatctgga gcgcagcgcc gatcagctca acatccgcct gaccgacgaa tccgccttcg 3120 agctcgggcg ggatctggcc ctgaccccgg gccgggtggt gcagcgcacc gagctctatg 3180 agctcattca gtacagcccg actaccgaga cggtgggcaa gacacctgtg ctgatagtgc 3240 cgcccttcat caacaagtac tacatcatgg acatgcggcc ccagaactcc ctggtcgcct 3300 ggctggtcgc ccagggccag acggtattca tgatctcctg gcgcaacccg ggcgtggccc 3360 aggcccaaat cgatctcgac gactacgtgg tggatggcgt catcgccgcc ctggacggcg 3420 tggaggcggc caccggcgag cgggggtgc acggcatcgg ctactgcatc ggcggcaccg 3480 ccctgcgct cgccatgggc tggctggcgg cgcggcgcca gaagcagcgg gtgcgcaccg 3540 ccaccctgtt cactaccctg ctggacttct cccagcccgg ggagcttggc atcttcatcc 3600 acgagcccat catagcggcg ctcgaggcgc aaaatgaggc caagggcatc atggacgggc 3660 gccagctggc ggtctccttc agcctgctgc gggagaacag cctctactgg aactactaca 3720 tcgacagcta cctcaagggt cagagcccgg tggccttcga tctgctgcac tggaacagcg 3780 acagcaccaa tgtggcgggc aagacccaca acagcctgct gcgccgtctc tacctggaga 3840 accagctggt gaagggggag ctcaagatcc gcaacacccg catcgatctc ggcaaggtga 3900 agacccctgt gctgctggtg tcggcggtgg acgatcacat cgccctctgg cagggcacct 3960 ggcagggcat gaagctgttt ggcggggagc agcgcttcct cctggcggag tccggccaca 4020 tcgccggcat catcaacccg ccggccgcca acaagtacgg cttctggcac aacggggccg 4080 aggccgagag cccggagagc tggctggcag gggcgacgca ccagggcggc tcctggtggc 4140 ccgagatgat gggctttatc cagaaccgtg acgaagggtc agagcccgtc cccgcgcggg 4200 tcccggagga agggctggcc cccgcccccg gccactatgt caaggtgcgg ctcaaccccg 4260 tgtttgcctg cccaacagag gaggacgccg catgacctgc cggcctggtt caaccagtcg 4320 gcagccgact agtggttagc cttgcgcctt gcttcgctga cgaagaacct ctgcgcgctc 4380 ggcctggccg ccgtgctcgc gctggcgggc acggcgcagg cagcgccggc gaccgagctg 4440 tcgaccggcc cggttaacac cgggccagcc ggcggcgaag gcctgggcat caaccaggcc 4500 attcgcgacg gcgaagcccg gcgcggcggc acctcgcttt ccactggcgc gccaaaaccc 4560 ctggcgccgc ggccggaata tgcgtcgctg ccggtctacg tcggcaaggt cggcgaccag 4620 ccggtgcggc tgcgcctggg ccccaagcct gacgagcgcg acagcgtgcg tggcgaatac 4680 gccggccgtg gcgccggtgt gcgcctgctg gcaggcgagt gggaggacgg cgccttcctg 4740 atggaggagt ccgacgacgg cacccgcgta tcgggcaact gggaaggcag catcgacgcc 4800 agcggcgccg tgcgcggcac ctggaccgaa tttaaatccg aattcactgg ccgtcgtttt 4860 acaacgtcgt gact 4874 <210> 26 <211> 3558 <212> DNA <213> artificial <220> <223> artificial <400> 26 ggaaacaatt gagaaggaga tatacatatg attgatcaaa aattcgatcc actacaagca 60 tggaaaaatg cttatgaaca aaccgaaaca ttttgggggaa aagcgctcaa tgaaacaatt 120 aaaacagaag aatattctgc ttggatgggc agcgttctag atttgaattt gttttatcaa 180 aaagcattaa atgatacgac aaaaaattat ttagagcagg tgaatgtgcc tacgaaagag 240 gatatcgcta gagtggctac gcttgttat aacttagaaa ataaagtgga taacattgag 300 gagtttctag aagagaaggt agagtcagta ggacaagctc ctacattaaa gcgcgatgtt 360 acgaaagtaa aacaagatat tcgcacatta gaaacgaaag ttgatcaaat tttagaattg 420 ctagaaaagc aaaatgcagt actagcaaaa ctacaagaac ctgtaaaaga agaagtaaaa 480 cctacgaata agccagaaaa taaaaagtga taatagcgct tgtttataac ttatgaaaga 540 cctgcaggag aaggagatat acatatgact acattcgcaa cagaatggga aaagcaatta 600 gagctatacc cagaagagta ccgaaaagca tatcgccgag tgaaaagggc gagtgaaatt 660 ttattacgtg aaccagagcc acaagtagga ttaacgccga aagaggttat ttggacgaag 720 aataagacga agctttatcg ctacattcca aaacaagaaa aaacacaaag agttccaatt 780 ctattaatat atgctcttat tataaacca tatattatgg atttaactcc tggaaatagt 840 ttagtggaat atctagtgga ccgtggtttt gatgtgtata tgcttgattg gggcacattt 900 ggtttagaag atagtcattt gaaatttgat gattttgtgt ttgattatat tgcaaaagca 960 gtgaaaaaag taatgcgaac tgcaaaatcg gacgagattt cttacttgg ttattgcatg 1020 gggggaacgc taacttctat tttgcggca cttcatccac acatgccaat tcgtaaccta 1080 atctttatga caagtccttt tgatttctct gaaacaggat tatatggtcc tttattagac 1140 gagaaatact tcaatctaga taaagcggtt gatacatttg gaaacattcc gccagaaatg 1200 attgattcg gaaacaaaat gttaaaacca attacaaact ttgttggtcc atatgttgct 1260 ttagtagatc gttcagagaa tgagcgcttc gtcgaaagct ggagattggt tcaaaagtgg 1320 gttggtgatg gtattccgtt cccaggtgaa tcatacagac agtggattcg tgatttttat 1380 caaaataata aattggttaa gggtgaactt gtgattcgcg gacaaaaagt agaccttgca 1440 aatattaagg cgaatgtctt aaatttcc gggaaacgtg atcatatcgc tttgccatgt 1500 caagtagaag cattactaga ccatatttct to catch aacaatatgt atgtttaccg acagggcata tgtctatcgt ttacggtgga acagctgtaa aacaaacata tccgacgatt ggaattggc ttgaagagcg ttctaattaa tttaaagagt gccggcgtgc gtcatgcacg gcgccggcag gcctgcaggt tccctcccgt ttccattgaa aggactacac aatgagccaa 1740. ccatcttatg gcccgctgtt cgaggccctg gcccactaca atgacaagct gctggccatg gccaaggccc agacagagcg caccgcccag gcgctgctgc agaccaatct ggacgatctg ggccaggtgc tggagcaggg cagccagcaa ccctggcagc tgatccaggc ccagatgaac tggtggcagg atcagctcaa gctgatgcag cacaccctgc tcaaaagcgc aggccagccg agcgagccgg tgatcacccc ggagcgcagc gatcgccgct tcaaggccga ggcctggagc gaacaaccca tctatgacta cctcaagcag tcctacctgc tcaccgccag gcacctgctg gcctcggtgg atgccctgga gggcgtcccc cagaagagcc gggagcggct gcgtttcttc acccgccagt acgtcagcgc catggcccc agcaacttcc tggccaccaa ccccgagctg ctcaagctga ccctggagtc cggcggccag aacctggtgc gcggactggc cctcttggcc 2280 gaggatctgg agcgcagcgc cgatcagctc aacatccgcc tgaccgacga atccgccttc 2340 gagctcgggc gggatctggc cctgaccccg ggccgggtgg tgcagcgcac cgagctctat 2400 gagctcattc agtacagccc gactaccgag acggtgggca agacacctgt gctgatagtg 2460 ccgcccttca tcaacaagta ctacatcatg gacatgcggc cccagaactc cctggtcgcc 2520 tggctggtcg cccagggcca gacggtattc atgatctcct ggcgcaaccc gggcgtggcc 2580 caggcccaaa tcgatctcga cgactacgtg gtggatggcg tcatcgccgc cctggacggc 2640 gtggaggcgg ccaccggcga gcgggaggtg cacggcatcg gctactgcat cggcggcacc 2700 gccctgtcgc tcgccatggg ctggctggcg gcgcggcgcc agaagcagcg ggtgcgcacc 2760 gccaccctgt tcactaccct gctggacttc tcccagcccg gggagcttgg catcttcatc 2820 cacgagccca tcatagcggc gctcgaggcg caaaatgagg ccaagggcat catggacggg 2880 cgccagctgg cggtcacctt cagcctgctg cgggagaaca gcctctactg gaactactac 2940 atcgacagct acctcaaggg tcagagcccg gtggccttcg atctgctgca ctggaacagc 3000 gacagcacca atgtggcggg caagacccac aacagcctgc tgcgccgtct ctacctggag 3060 aaccagctgg tgaaggggga gctcaagatc cgcaacaccc gcatcgatct cggcaaggtg 3120 aagacccctg tgctgctggt gtcggcggtg gacgatcaca tcgccctctg gcagggcacc 3180 tggcagggca tgaagctgtt tggcggggag cagcgcttcc tcctggcgga gtccggccac 3240 atcgccggca tcatcaaccc gccggccgcc aacaagtacg gcttctggca caacggggcc 3300 gaggccgaga gcccggagag ctggctggca ggggcgacgc accagggcgg ctcctggtgg 3360 cccgagatga tgggctttat ccagaaccgt gacgaagggt cagagcccgt ccccgcgcgg 3420 gtcccggagg aagggctggc ccccgccccc ggccactatg tcaaggtgcg gctcaacccc 3480 gtgtttgcct gcccaacaga ggaggacgcc gcatgacctg ccggcctggt tcaaccagtc 3540 ggcagccgac tagtggat 3558 <210> 27 <211> 2771 <212> DNA <213> Artificial <220> <223> Artificial <400> 27 tacgctagcg taccgagcta tccatttaaa ttgcatgcgg gcttgcacgt gccgcatgcg 60 cggctggtga tccgcaaggg ccaccatgcc gacgtggaca gctactccgc cttcctggag 120 gcggaccgca ccacgcgcac cgggctggcc ggctacctgc gcgagcatgg cgtcaggcgc 180 gtgttctgcg cgggtctggc gacggactac tgcgtggcct ggagcgcgct cgatgcacgc 240 gccgcgggct tcgcggccgc ggtgatcgag gatgcctgcc gcgccatcga cctggaaggg 300 tcgctggcca aagcatggca ggacctcggc gccgccggcg ttgcgcgcgt cacgtccgct 360 gaactgctca agggccaggg ctgaaccgaa ccaccgaact gaacgacaca agaatccg 420 aggagcaaga ccaattcgcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 480 caggctttac acttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat 540 ttcacacagg aaacaattcc acgtgcagag agacaatcaa atcatgcagc agtttgtcaa 600 ttccctgtcg ctcggccagg accagtccga tgcccccat ccgctgaccg gcgcctggtc 660 gcagctgatg agccagacca atcagctctt gcagctgcaa tccagcctct accaacagca 720 attgggcttg tggacgcaat tcctcggcca aaccgccggc aacgacgctt ccgcgccgtc 780 ggccaagccg agcgaccgcc gcttcgcctc gccggagtgg gatgagcatc cgttctacag 840 cttcctcaag cagagctatc tgcaaacctc caagtggatg atggagctgg tggacaagac 900 ccagatcgac gaaagcgcta aggacaagct gtccttcgcc actcgccagt acctggatgc 960 catggcgcc agcaacttca tgctgaccaa ccccgacgtg gtcaaacgcg ccatcgaaac 1020 ccagggcgaa agcctggtgg aaggcatgaa gaacatgatg gaggacatcc agaagggcca 1080 tatctcgatg tccgacgaga gcaagttcca aatcggcaaa aacctggtgg tgacgccggg 1140 cgaggtggtg ttccgcaatg aactgatcga gctgatccag tacacgccga ccaccgaaaa 1200 agtccacgaa aagcccttgc tgtttgtccc gccgtgcatc aacaagtact acctgatgga 1260 tctgcagccg gacaactcca tggtgcgcca cttcgtcggc cagggttacc gcgttttcct 1320 ggtcagctgg cgttccgccg tgcccgagat gaagaacttc acttgggaga cctacatcga 1380 gaaaggcgtg ttcgccgcgg ccgaagcggt tcagaagatc accaagcagc cgaccatgaa 1440 cgcgctgggc ttctgcgtgg gcggcgtgat cctcactacc gcgctgtgcg tggcccaggc 1500 caagggtctg aaatacttcg actccgccac cttcatgacg tcgctgatcg accacgccga 1560 accgggcgag atctccttct tcatcgacga ggcgctggta gccagccgcg aagccaaaat 1620 ggcggccggc ggcatcatca gcggcaagga aatcggacgc actttcgcca gcctgcgcgc 1680 caacgacctg gtgtggaact acgtcgtcaa caactacctg ctgggcaaga ccccggcgcc 1740 gttcgacctg ctgtactgga acaacgacgc ggtggacctg ccgctgccga tgcacacctt 1800 catgctgcgg cagttctaca tcaacaacgc gctgatcacc ccgggcgcca ttacgctgtg 1860 cggcgtcccg atcgacatct ccaagatcga catcccggtg tatatgttcg ccgcgcgcga 1920 agaccacatc gtgctgtgga gttccgccta ctccggcctg aaatatctga gcggcacgcc 1980 aagccgccgc tttgtcctgg gcgcatccgg ccacatcggc ggctcgatca accccgtcac 2040 caaggataag cgcaactact ggaccaacga gcagctgccg gtgaatccgg aagaatggct 2100 ggagggcgcg caaagccatc caggcagctg gtggaaggac tgggacgcct ggctggcccc 2160 gcaatccgga aaacaggttc cggcgcccaa gatgctgggc agcaaggagt tccccccgct 2220 gcagcctgcg ccgggcagct atgtgctcgc caaggcgatg cctcccgtcg ccgccgcctt 2280 gaactgacct gccggcctgg ttcaaccagt cggcagccga ctagtatgtc ggctgcgggc 2340 accgcgcggg ccacgcccgc cgacatgctc gcctggggcc gggaggtgct ggccagccag 2400 cccttctcgg tcctggtcgg caccgaactg gcggcgttgt cgccgggcaa ggctgagttg 2460 cgcctgccga tccgtcagga cctgcggcag cagcacggct tcctgcacgg cggcgtggtc 2520 agctacctgg ccgacaatgc gctgacctac gcgggcggcg cggccatggc ggtgccggtc 2580 gtgacatccg agtacaagat caactacgtg cgcccggcga tcggcgagct gctggtcgcc 2640 cgcgccgaat gcgtcagcgc cggccgccag caggcggtgg tgcgctgcga cgtgtacgtc 2700 gtcagggatg gcgaggagag gctctgattt aaatccgaat tcactggccg tcgttttaca 2760 acgtcgtgac t 2771 <210> 28 <211> 3034 <212> DNA <213> Artificial <220> <223> Artificial <400> 28 gcgcatttaa atccggacct tcgtgcggct caagccccag cacgtcgccg ggcagcgaaa aacccgttac ctgccgccag cccagcggcg attcatagac ctgcttggcg ctgccatggc 120 gcagcgggta gaccgtcagc gcagggcggc cctggcgaaa cacatgctcg ccggtgcgca cctctaccag ctccggctgc agcgccatcg gcggcgtgcgg caggggggct tgtggcccgg 240 cattgtggca aacgtggcga aagaggcaga gcaggcaggc tggggcgccg ctgtccggca tggtgtcatt gtcctccggt gacgatggcc aagtataaaa cgccggcaac gcaagccatc 360 tcgctgcgat ctgcattctt tcgtatggct ggtttaaaaa tttcgcatta cggggcgag 420 gctcgttgcg tttgtgccat aagcgcggga gcacgccggc gggcgtaatg cggattgtga 480 tatgctgcaa cgcaacaata aaggcatagg aggagatcgc gtcacacgat caggagtcct ccaattggcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac acttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacaattgc acgtgcagag aacaatcaa atcatgagcc aaccatctta tggcccgctg ttcgaggccc tggcccacta caatgacaag ctgctggcca tggccaaggc ccagacagag 780 cgcaccgccc aggcgctgct gcagaccaat ctggacgatc tgggccaggt gctggagcag 840 ggcagccagc aaccctggca gctgatccag gcccagatga actggtggca ggatcagctc aagctgatgc agcacaccct gctcaaaagc gcaggccagc cgagcgagcc ggtgatcacc ccggagcgca gcgatcgccg cttcaaggcc gaggcctgga gcgaacaacc catctatgac tacctcaagc agtcctacct gctcaccgcc aggcacctgc tggcctcggt ggatgccctg 1080 1140. gaggcgtcc cccagaagag ccggggagcgg ctgcgtttct tcacccgcca gtacgtcagc gccatggccc ccagcaactt cctggccacc aaccccgagc tgctcaagct gaccctggag tccggcggcc agaacctggt gcgcggactg gccctcttgg ccgaggatct ggagcgcagc 1260 gccgatcagc tcaacatccg cctgaccgac gaatccgcct tcgagctcgg gcgggatctg gccctgaccc cggggccgggt ggtgcagcgc accgagctct atgagctcat tcagtacagc 1380 ccgactaccg agacggtggg caagacacct gtgctgatag tgccgccctt catcaacaag tactacatca tggacatgcg gccccagaac tccctggtcg cctggctggt cgcccagggc 1500. cagacggtat tcatgatctc ctggcgcaac ccggggcgtgg cccaggccca aatcgatctc 1560. gacgactacg tggtggatgg cgtcatcgcc gccctggacg gcgtggaggc ggccaccggc 1620 gagcgggagg tgcacggcat cggctactgc atcggcggca ccgccctgtc gctcgccatg 1680. ggctggctgg cggcgcggcg ccagaagcag cgggtgcgca ccgccaccct gttcactacc 1740 ctgctggact tctcccagcc cggggagctt ggcatcttca tccacgagcc catcatagcg gcgctcgagg cgcaaaatga ggccaagggc atcatggacg ggcgccagct ggcggtctcc ttcagcctgc tgcgggagaa cagcctctac tggaactact acatcgacag ctacctcaag ggtcagagcc cggtggcctt cgatctgctg cactggaaca gcgacagcac caatgtggcg ggcaagaccc acaacagcct gctgcgccgt ctctacctgg agaaccagct ggtgaagggg gagctcaaga tccgcaacac ccgcatcgat ctcggcaagg tgaagacccc tgtgctgctg gtgtcggcgg tggacgatca catcgccctc tggcagggca cctggcaggg catgaagctg tttggcgggg agcagcgctt cctcctggcg gagtccggcc acatcgccgg catcatcaac 2220 ccgccggccg ccaacaagta cggcttctgg cacaacgggg ccgaggccga gagcccggag 2280 agctggctgg caggggcgac gcaccagggc ggctcctggt ggcccgagat gatgggcttt 2340 atccagaacc gtgacgaagg gtcagagccc gtccccgcgc gggtcccgga ggaagggctg 2400 gcccccgccc ccggccacta tgtcaaggtg cggctcaacc ccgtgtttgc ctgcccaaca 2460 gaggaggacg ccgcatgagc ctgacctgcc ggcctggttc aaccagtcgg cagccgacta 2520 gtagtcgggc agcaccaatg cgcatcaagc gcgcacaagt aaagggaggg cgcctgccct 2580 ccctttttcc ttgcagcagc cgcgtcagcc gcgcgagcgg tccttgacga acagcgcagt 2640 caccatgccc agcacgcaca gcgcgaccac atagtgggcc ggtgccagcg gatcctgctt 2700 cagcatcagc gtgacgacca tcggcgtcag cccgccgaag atcgcatacg acacgttgta 2760 cgagaatgac aggcccgaga agcgcacctg cgccgggaac gcattgacca gcacgaacgg 2820 caccgcgccg atggtgccga ccaggaagcc ggtcagcgca tagagcggca gcagcaggtc 2880 ggggcgggtg aagatcgtcg tgtagaacat ataggcgcag atggccagca gcaggccgcc 2940 gacgaacagc gtgcggcgcg caccgatgcg gtcggctaat gcgccggaga cgacacagcc 3000 gatcgtcagg cacagcgtgg cgatttaaat gcgc 3034 <210> 29 <211> 2841 <212> DNA <213> artificial <220> <223> artificial <400> 29 gcgcgcattt aaatctgcca ccacgttgtg ctcggtgatc gccatcatca gcgccacgta 60 gagccagcca atggccacga tgtacatcaa aaattcatcc ttctcgccta tgctctgggg 120 cctcggcaga tgcgagcgct gcataccgtc cggtaggtcg ggaagcgtgc agtgccgagg 180 cggattcccg cattgacagc gcgtgcgttg caaggcaaca atggactcaa atgtctcgga 240 atcgctgacg attcccaggt ttctccggca agcatagcgc atggcgtctc catgcgagaa 300 tgtcgcgctt gccggataaa aggggagccg ctatcggaat ggacgcaagc cacggccgca 360 gcaggtgcgg tcgagggctt ccagccagtt ccagggcaga tgtgccggca gaccctcccg 420 ctttggggga ggcgcaagcc gggtccattc ggatagcatc tccccatgca aagtgccggc 480 cagggcaatg cccggagccg gttcgaatag tgacggcaga gagacaatca aatcatgagc 540 caaccatctt atggcccgct gttcgaggcc ctggcccact acaatgacaa gctgctggcc 600 atggccaagg cccagacaga gcgcaccgcc caggcgctgc tgcagaccaa tctggacgat 660 ctgggccagg tgctggagca gggcagccag caaccctggc agctgatcca ggcccagatg 720 aactggtggc aggatcagct caagctgatg cagcacaccc tgctcaaaag cgcaggccag 780 ccgagcgagc cggtgatcac cccggagcgc agcgatcgcc gcttcaaggc cgaggcctgg 840 agcgaacaac ccatctatga ctacctcaag cagtcctacc tgctcaccgc caggcacctg 900 ctggcctcgg tggatgccct ggagggcgtc ccccagaaga gccgggagcg gctgcgtttc 960 ttcacccgcc agtacgtcag cgccatggcc cccagcaact tcctggccac caaccccgag 1020 ctgctcaagc tgaccctgga gtccggcggc cagaacctgg tgcgcggact ggccctcttg 1080 gccgaggatc tggagcgcag cgccgatcag ctcaacatcc gcctgaccga cgaatccgcc 1140 ttcgagctcg ggcgggatct ggccctgacc ccgggccggg tggtgcagcg caccgagctc 1200 tatgagctca ttcagtacag cccgactacc gagacggtgg gcaagacacc tgtgctgata 1260 gtgccgccct tcatcaacaa gtactacatc atggacatgc ggccccagaa ctccctggtc 1320 gcctggctgg tcgcccaggg ccagacggta ttcatgatct cctggcgcaa cccgggcgtg 1380 gcccaggccc aaatcgatct cgacgactac gtggtggatg gcgtcatcgc cgccctggac 1440 ggcgtggagg cggccaccgg cgagcgggag gtgcacggca tcggctactg catcggcggc 1500 accgccctgt cgctcgccat gggctggctg gcggcgcggc gccagaagca gcgggtgcgc 1560 accgccaccc tgttcactac cctgctggac ttctcccagc ccggggagct tggcatcttc 1620 atccacgagc ccatcatagc ggcgctcgag gcgcaaaatg aggccaaggg catcatggac 1680 gggcgccagc tggcggtcac cttcagcctg ctgcgggaga acagcctcta ctggaactac 1740 tacatcgaca gctacctcaa gggtcagagc ccggtggcct tcgatctgct gcactggaac 1800 agcgacagca ccaatgtggc gggcaagacc cacaacagcc tgctgcgccg tctctacctg 1860 gagaaccagc tggtgaaggg ggagctcaag atccgcaaca cccgcatcga tctcggcaag 1920 gtgaagaccc ctgtgctgct ggtgtcggcg gtggacgatc acatcgccct ctggcagggc 1980 acctggcagg gcatgaagct gtttggcggg gagcagcgct tcctcctggc ggagtccggc 2040 cacatcgccg gcatcatcaa cccgccggcc gccaacaagt acggcttctg gcacaacggg 2100 gccgaggccg agagcccgga gagctggctg gcaggggcga cgcaccaggg cggctcctgg 2160 tggcccgaga tgatgggctt tatccagaac cgtgacgaag ggtcagagcc cgtccccgcg 2220 cgggtcccgg aggaagggct ggcccccgcc cccggccact atgtcaaggt gcggctcaac 2280 cccgtgtttg cctgcccaac agaggaggac gccgcatgac gcttgcatga gtgccggcgt 2340 gcgtcatgca cagcgccggc aggcctgcag gttccctccc gtttccattg aaaggactac 2400 acaatgactg acgttgtcat cgtatccgcc gcccgcaccg cggtcggcaa gtttggcggc 2460 tcgctggcca agatcccggc accggaactg ggtgccgtgg tcatcaaggc cgcgctggag 2520 cgcgccggcg tcaagccgga gcaggtgagc gaagtcatca tgggccaggt gctgaccgcc 2580 ggttcgggcc agaaccccgc acgccaggcc gcgatcaagg ccggcctgcc ggcgatggtg 2640 ccggccatga ccatcaacaa ggtgtgcggc tcgggcctga aggccgtgat gctggccgcc 2700 aacgcgatca tggcgggcga cgccgagatc gtggtggccg gcggccagga aaacatgagc 2760 gccgccccgc acgtgctgcc gggctcgcgc gatggtttcc gcatgggcga tgccaagctg 2820 gtcgacaatt taaatgcgcg c 2841 <210> 30 <211> 1864 <212> DNA <213> Artificial <220> <223> Artificial <400> 30 ggaaacaatt gcacgtgcag agagacaatc aaatcatgag ccaaccatct tatggcccgc 60 tgttcgaggc cctggcccac tacaatgaca agctgctggc catggccaag gcccagacag 120 agcgcaccgc ccaggcgctg ctgcagacca atctggacga tctgggccag gtgctggagc 180 agggcagcca gcaaccctgg cagctgatcc aggcccagat gaactggtgg caggatcagc 240 tcaagctgat gcagcacacc ctgctcaaaa gcgcaggcca gccgagcgag ccggtgatca 300 ccccggagcg cagcgatcgc cgcttcaagg ccgaggcctg gagcgaacaa cccatctatg 360 actacctcaa gcagtcctac ctgctcaccg ccaggcacct gctggcctcg gtggatgccc 420 tggagggcgt cccccagaag agccgggagc ggctgcgttt cttcacccgc cagtacgtca 480 gcgccatggc ccccagcaac ttcctggcca ccaaccccga gctgctcaag ctgaccctgg 540 agtccggcgg ccagaacctg gtgcgcggac tggccctctt ggccgaggat ctggagcgca 600 gcgccgatca gctcaacatc cgcctgaccg acgaatccgc cttcgagctc gggcgggatc 660 tggccctgac cccgggccgg gtggtgcagc gcaccgagct ctatgagctc attcagtaca 720 gcccgactac cgagacggtg ggcaagacac ctgtgctgat agtgccgccc ttcatcaaca 780 agtactacat catggacatg cggccccaga actccctggt cgcctggctg gtcgcccagg 840 gccagacggt attcatgatc tcctggcgca acccgggcgt ggcccaggcc caaatcgatc 900 tcgacgacta cgtggtggat ggcgtcatcg ccgccctgga cggcgtggag gcggccaccg 960 gcgagcggga ggtgcacggc atcggctact gcatcggcgg caccgccctg tcgctcgcca 1020 tgggctggct ggcggcgcgg cgccagaagc agcgggtgcg caccgccacc ctgttcacta 1080 ccctgctgga cttctcccag cccggggagc ttggcatctt catccacgag cccatcatag 1140 cggcgctcga ggcgcaaaat gaggccaagg gcatcatgga cgggcgccag ctggcggtca 1200 ccttcagcct gctgcgggag aacagcctct actggaacta ctacatcgac agctacctca 1260 agggtcagag cccggtggcc ttcgatctgc tgcactggaa cagcgacagc accaatgtgg 1320 cgggcaagac ccacaacagc ctgctgcgcc gtctctacct ggagaaccag ctggtgaagg 1380 gggagctcaa gatccgcaac acccgcatcg atctcggcaa ggtgaagacc cctgtgctgc 1440 tggtgtcggc ggtggacgat cacatcgccc tctggcaggg cacctggcag ggcatgaagc 1500 tgtttggcgg ggagcagcgc ttcctcctgg cggagtccgg ccacatcgcc ggcatcatca 1560 acccgccggc cgccaacaag tacggcttct ggcacaacgg ggccgaggcc gagagcccgg 1620 agagctggct ggcaggggcg acgcaccagg gcggctcctg gtggcccgag atgatgggct 1680 ttatccagaa ccgtgacgaa gggtcagagc ccgtccccgc gcgggtcccg gaggaagggc 1740 tggcccccgc ccccggccac tatgtcaagg tgcggctcaa ccccgtgttt gcctgcccaa 1800 cagaggagga cgccgcatga cctgccggcc tggttcaacc agtcggcagc cgactagtgc 1860 gcgc 1864 <210> 31 <211> 246 <212> DNA <213> Artificial <220> <223> Artificial <400> 31 gcgccaattg tgcttctggc gtcaggcagc catcggaagc tgtggtatgg ctgtgcaggt 60 cgtaaatcac tgcataattc gtgtcgctca aggcgcactc ccgttctgga taatgttttt 120 tgcgccgaca tcataacggt tctggcaaat attctgaaat gagctgttga caattaatca 180 tcgaactagt taactagtac gcaagttcac agcggataac aatttcacac aggaaacaat 240 tggcgc 246

Claims

1. A method for producing a copolymerized polyhydroxyalkanoate mixture, the method comprising: a step of culturing a microorganism that produces the copolymerized polyhydroxyalkanoate mixture, The microorganism is a transformant of the insecticidal copper-greedy bacteria. The microorganism has: A gene encoding the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 (A), and A gene encoding the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 (B), The microorganism is transformed such that the expression of a gene encoding β-ketothiolase having thiolysis activity on β-ketohexanoyl-CoA, which is β-ketoacyl-CoA having 6 carbon atoms, is suppressed, and The microorganism has a gene encoding a protein exhibiting R-body-specific enoyl-CoA hydratase activity, The copolymerized polyhydroxyalkanoate mixture contains a polyhydroxyalkanoate fraction (I) and a polyhydroxyalkanoate fraction (II), The polyhydroxyalkanoate fraction (I) comprises a copolymerized polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit and a 3-hydroxyhexanoic acid structural unit, and the average composition ratio of 3-hydroxyhexanoic acid is 20 mol% or more, The polyhydroxyalkanoate fraction (II) contains polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit, and the average composition ratio of 3-hydroxyhexanoic acid is 0 mol% or more and 15 mol% or less, The average composition ratio of 3-hydroxyhexanoic acid in the copolymerized polyhydroxyalkanoate mixture is 22 mol % or less.

2. The method according to claim 1, wherein: The weight ratio of the polyhydroxyalkanoate fraction (I) in the copolymerized polyhydroxyalkanoate mixture is 10 to 90%.

3. The method according to claim 1 or 2, wherein: The average composition ratio of 3-hydroxyhexanoic acid in the copolymerized polyhydroxyalkanoate mixture is 10 to 22 mol %.

4. The method according to claim 1 or 2, wherein: The amino acid sequence of the β-ketothiolase is shown in sequence number 9 or sequence number 10.

5. The method according to claim 1 or 2, wherein: In the culturing step, a carbon source including oil or fatty acid is added.

6. The method according to claim 5, wherein: The carbon source containing oil or fatty acid is a carbon source containing medium-chain fatty acids having 6 to 12 carbon atoms or glycerides of the medium-chain fatty acids.

7. The method according to claim 6, wherein: The medium chain fatty acid is caproic acid.

8. A transformed microorganism, which is a transformed microorganism of an insecticidal copper-greedy bacterium that produces a copolymerized polyhydroxyalkanoate mixture, The microorganism has: A gene encoding the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 (A), and A gene encoding the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 (B), The microorganism is transformed such that the expression of a gene encoding β-ketothiolase having thiolysis activity on β-ketohexanoyl-CoA, which is β-ketoacyl-CoA having 6 carbon atoms, is suppressed, and The microorganism has a gene encoding a protein exhibiting R-body-specific enoyl-CoA hydratase activity, The copolymerized polyhydroxyalkanoate mixture contains a polyhydroxyalkanoate fraction (I) and a polyhydroxyalkanoate fraction (II), The polyhydroxyalkanoate fraction (I) comprises a copolymerized polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit and a 3-hydroxyhexanoic acid structural unit, and the average composition ratio of 3-hydroxyhexanoic acid is 20 mol% or more, The polyhydroxyalkanoate fraction (II) contains polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit, and the average composition ratio of 3-hydroxyhexanoic acid is 0 mol% or more and 15 mol% or less, The average composition ratio of 3-hydroxyhexanoic acid in the copolymerized polyhydroxyalkanoate mixture is 22 mol % or less.

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