Block copolymer and method for producing the same

Abstract

A copolymer comprising a hydroxycarboxylic acid (A) having a hydroxyl group only at the 2-position and a hydroxycarboxylic acid (B) having a hydroxyl group at a position other than the 2-position, and comprising a homopolymer segment and a copolymer segment, wherein the homopolymer segment is composed of one hydroxycarboxylic acid selected from the hydroxycarboxylic acid (A) and the hydroxycarboxylic acid (B), and the copolymer segment is composed of at least two hydroxycarboxylic acids selected from the hydroxycarboxylic acid (A) and the hydroxycarboxylic acid (B).

Description

Technical Field

[0001] The present invention relates to a block copolymer and a method for producing the same, and more particularly, to a block copolymer having a homopolymer segment and a copolymer segment, and a method for producing the same. Background Art

[0002] Copolymers are polymer compounds synthesized by polymerizing two or more monomers. Compared with other materials such as metals, they have excellent properties such as elasticity, transparency, processability, and lightness. Therefore, they are widely used in various industrial fields such as medicine. Among them, block copolymers composed of chains formed by covalently connecting two or more polymers (segments) with different properties are expected to become materials that have the characteristics of each segment that constitutes them.

[0003] In addition, the polymer compound is usually synthesized using petroleum as a raw material. However, as a countermeasure to the depletion of fossil resources and global warming, which have become problems in recent years, polymer compounds (so-called biopolymers) produced using renewable resources (biomass-derived raw materials) as carbon sources have attracted attention.

[0004] For example, polyhydroxyalkanoic acid (PHA) produced by microorganisms has attracted attention as a biomatrix / biodegradable material. Currently, commercially produced PHA is a copolymer of 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HHx). Furthermore, after the polymerization of non-natural lactic acid units was reported in 2008, PHA containing various non-natural monomers was reported (Non-Patent Document 1).

[0005] In addition, in microorganisms, two or more monomers are usually arranged randomly, and PHA is biosynthesized as a random copolymer. However, the present inventors reported that a block copolymer (P(2HB-b-3HB)) consisting of a homopolymer segment composed of 2-hydroxybutyrate (2HB) and a homopolymer segment composed of 3HB can be biosynthesized by culturing a microorganism expressing CoA transferase and polymerase in a medium containing 2-hydroxybutyrate (2HB) and 3HB (Non-Patent Document 2).

[0006] As described above, although biosynthesis of block copolymers is possible even for biopolymers, further development of block copolymers is required for improvement of physical properties and the like.

[0007] Prior art literature

[0008] Non-patent literature

[0009] Non-patent document 1: Matsumoto et al., 2013, Appl. Microbiol. Biotechnol. 97, 8011

[0010] Non-patent literature 2: Matsumoto K et al., Biomacromolecules, 19(2), 662, 2018 Summary of the invention

[0011] Problems to be solved by the invention

[0012] The present invention has been made in view of the above-mentioned problems in the prior art, and an object of the present invention is to provide a block copolymer having a homopolymer segment and a copolymer segment.

[0013] Means for solving problems

[0014] In order to further improve the block copolymer (P(2HB-b-3HB)) reported in Non-Patent Document 2, the present inventors cultured the microorganism using a culture medium to which 3HHx was added in addition to the conventional 2HB and 3HB. As a result, a block copolymer (P(2HB-b-(3HB-co-3HHx))) consisting of a homopolymer segment composed of 2HB and a random polymer segment composed of 3HB and 3HHx was obtained. That is, unlike conventional polymers having only homopolymer segments, a block copolymer having a random copolymer segment can be biosynthesized.

[0015] It was further confirmed that, when 3-hydroxypropionic acid (3HP), 4-hydroxy-2-methylbutyric acid (4H2MB), 2,4-dihydroxybutyric acid (24DHB), 5-hydroxyvaleric acid (5HV), or 6-hydroxyhexanoic acid (6HHx) is added to a culture medium containing 2HB and 3HB instead of 3HHx, and the microorganism is cultured, block copolymers (P(2HB)-bP(3HB-ran-3HP), P(2HB)-bP(3HB-ran-4H2MB), P(2HB)-bP(3HB-ran-24DHB), P(2HB)-bP(3HB-ran-5HV), P(2HB)-bP(3HB-ran-6HHx)) can be synthesized in the same manner as when 3HHx is used.

[0016] In addition, as a result of culturing the microorganism using a culture medium supplemented with 2-hydroxyacetic acid (glycolic acid, GL) and 3HB, a block copolymer (P(3HB-b-(GL-co-3HB))) consisting of a homopolymer segment composed of 3HB and a copolymer segment composed of GL and 3HB can be obtained.

[0017] Furthermore, as a result of culturing the microorganism using a culture medium supplemented with GL, 3HB and 3HHx, a block copolymer (P(3HB-b-(GL-co-3HB-co-3HHx))) consisting of a homopolymer segment composed of 3HB and a random copolymer segment composed of GL, 3HB and 3HHx can be obtained.

[0018] In addition, it was shown that ldhA (lactate dehydrogenase gene), hadA (CoA transferase gene) and PhaC AR When a glucose assimilating mutant strain of Ralstonia eutropha (polymerase gene) is cultured in a medium containing glucose and 2HB, the microorganism biosynthesizes 3HB and 3HV (3-hydroxyvaleric acid), and further, a block copolymer (P(2HB)-bP(3HB-co-3HV)) can be biosynthesized from these hydroxycarboxylic acids. That is, even if hydroxycarboxylic acids having a hydroxyl group at a position other than the 2-position, such as 3HB and 3HV, are not added to the medium, the block copolymer can be obtained by culturing the microorganism.

[0019] The present invention provides the following inventions more specifically based on the above synthesis examples.

[0020] <1> A copolymer comprising a hydroxycarboxylic acid (A) having a hydroxyl group only at the 2-position and a hydroxycarboxylic acid (B) having a hydroxyl group at a position other than the 2-position, and having a homopolymer segment and a copolymer segment,

[0021] The homopolymer segment is composed of one hydroxycarboxylic acid selected from the group consisting of hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B),

[0022] The copolymer segment comprises at least two hydroxycarboxylic acids selected from the group consisting of hydroxycarboxylic acids (A) and hydroxycarboxylic acids (B).

[0023] <2> The copolymer according to <1>, wherein the hydroxycarboxylic acid (A) is at least one hydroxycarboxylic acid selected from 2-hydroxybutyric acid, 2-hydroxyacetic acid, 2-hydroxypropionic acid, 2-hydroxyvaleric acid, 2-hydroxyhexanoic acid and medium-chain 2-hydroxyalkanoic acid,

[0024] The hydroxycarboxylic acid (B) is at least one hydroxycarboxylic acid selected from 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxypropionic acid, 3-hydroxyvaleric acid, medium-chain 3-hydroxyalkanoic acid, 4-hydroxy-2-methylbutyric acid, 4-hydroxybutyric acid, 2,4-dihydroxybutyric acid, 5-hydroxyvaleric acid and 6-hydroxyhexanoic acid.

[0025] <3> The copolymer according to <1>, wherein the hydroxycarboxylic acid (A) is 2-hydroxybutyric acid, the hydroxycarboxylic acid (B) is 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, and

[0026] The copolymer has a homopolymer segment composed of 2-hydroxybutyric acid and a copolymer segment containing 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

[0027] <4> The copolymer according to <1>, wherein the hydroxycarboxylic acid (A) is 2-hydroxyacetic acid, the hydroxycarboxylic acid (B) is 3-hydroxybutyric acid, and

[0028] The copolymer has a homopolymer segment consisting of 3-hydroxybutyric acid and a copolymer segment containing 2-hydroxyacetic acid and 3-hydroxybutyric acid.

[0029] <5> The copolymer according to <1>, wherein the hydroxycarboxylic acid (A) is 2-hydroxyacetic acid, the hydroxycarboxylic acid (B) is 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, and

[0030] The copolymer has a homopolymer segment consisting of 3-hydroxybutyric acid and a copolymer segment containing 2-hydroxyacetic acid, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

[0031] <6> A method for producing a copolymer according to any one of <1> to <5>, comprising:

[0032] a step of culturing a microorganism expressing CoA transferase and polymerase in a medium containing a hydroxycarboxylic acid (A) and / or a metabolic precursor thereof, and a hydroxycarboxylic acid (B) and / or a metabolic precursor thereof, and

[0033] a step of recovering the copolymer from the culture obtained in the step.

[0034] Effects of the Invention

[0035] According to the present invention, a block copolymer having a homopolymer segment and a copolymer segment, and a method for producing the block copolymer can be provided.

[0036] According to the present invention, for example, a block copolymer (P(2HB-b-(3HB-co-3HHx))) composed of a homopolymer composed of 2HB and a random polymer composed of 3HB and 3HHx can be provided. As shown in the examples described below, the block copolymer has higher stretchability than the copolymer P(2HB-b-3HB) disclosed in Non-Patent Document 2. In particular, P(2HB-b-(3HB-co-3HHx)) has a very high stretchability exceeding 1200%.

[0037] In addition, a homopolymer consisting of 3HB and a block copolymer consisting of a copolymer consisting of GL and 3HB (P(3HB-b-(GL-co-3HB))) can be provided. Furthermore, a homopolymer consisting of 3HB and a block copolymer consisting of a copolymer consisting of GL, 3HB and 3HHx (P(3HB-b-(GL-co-3HB-co-3HHx))) can be provided. As shown in the embodiments described later, these block copolymers are introduced into GL, thereby improving non-enzymatic hydrolysis, thereby exerting high decomposability in vivo. In addition, the copolymer segment of P(3HB-b-(GL-co-3HB-co-3HHx)) adopts a gradient structure. Furthermore, the flexibility of the copolymer is greatly improved by introducing 3HHx. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1A It shows the addition of 2HB, 3HB and 3HHx, using polymerase PhaC AR The copolymer (P(2HB-b-(3HB-co-3HHx))) obtained by 1 A graph showing the results of H NMR spectroscopy analysis.

[0039] Figure 1B It shows the addition of 2HB, 3HB and 3HHx, using polymerase PhaC AR The copolymer (P(2HB-b-(3HB-co-3HHx))) obtained by 13 A graph showing the results of C NMR spectroscopy analysis.

[0040] Figure 1C This is a diagram showing the stress-strain relationship for P(2HB-b-(3HB-co-3HHx)) and the like.

[0041] Figure 2A It shows the addition of 3HB and GL, using polymerase PhaC1 Ps The copolymer obtained by STQK, 1 A graph showing the results of H NMR spectroscopy analysis.

[0042] Figure 2B It shows the addition of 3HB and GL, using polymerase PhaC AR The copolymer (P(3HB-b-(GL-co-3HB))) obtained by 1 A graph showing the results of H NMR spectroscopy analysis.

[0043] Figure 2C This is a molecular weight distribution curve showing the results of gel filtration chromatography analysis of P(3HB-b-(GL-co-3HB)).

[0044] Figure 3AIt shows P(3HB-b-(GL-co-3HB-co-3HHx)), 1 The results of H NMR spectral analysis are shown in FIG. 2. The numbers in the figure correspond to those in Table 2.

[0045] Figure 3B It is a graph showing the results of differential scanning calorimetry of P(3HB-b-(GL-co-3HB-co-3HHx)).

[0046] Figure 3C It is a graph showing the stress-strain relationship for P(3HB-b-(GL-co-3HB-co-3HHx)).

[0047] Figure 4A The results show that the block copolymer composed of P(2HB) homopolymer segments and random copolymer segments containing 3HB is 1 A graph showing the results of H NMR spectroscopy analysis.

[0048] Figure 4B It is a graph showing the results of differential scanning calorimetry of P(2HB)-bP(3HB-ran-5HV).

[0049] Figure 5A is a schematic diagram showing the metabolic pathway for the biosynthesis of P(2HB)-bP(3HB-co-3HV).

[0050] Figure 5B It is a schematic diagram showing that phaCNSDG (modified PHA synthase gene derived from Aeromonas caviae) is replaced with phaCAR (chimeric PHA synthase gene derived from A. caviae and R. eutropha) in the pha operon on the chromosome of R. eutropha H16 NSDG-GG-ΔB1 strain by homologous recombination.

[0051] Figure 5C It is a schematic diagram showing that ldhA (lactate dehydrogenase gene) and hadA (CoA transferase gene) derived from Clostridium difficile are inserted downstream of phaP1 (PHA granule-binding protein gene).

[0052] Figure 5D It is shown that for P(2HB)-bP(3HB-co-3HV) 1 A graph showing the results of H NMR spectroscopy analysis. DETAILED DESCRIPTION

[0053] <Block copolymer>

[0054] As shown in the examples described below, the present invention provides a block copolymer having a homopolymer segment and a copolymer segment, and more specifically provides a copolymer comprising a hydroxycarboxylic acid (A) having a hydroxyl group only at the 2-position, and a hydroxycarboxylic acid (B) having a hydroxyl group at a position other than the 2-position, and having a homopolymer segment and a copolymer segment.

[0055] The homopolymer segment is composed of one hydroxycarboxylic acid selected from the group consisting of hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B),

[0056] The copolymer segment comprises at least two hydroxycarboxylic acids selected from the group consisting of hydroxycarboxylic acids (A) and hydroxycarboxylic acids (B).

[0057] The copolymer of the present invention is obtained by polymerizing hydroxycarboxylic acid through ester bonds and has at least two types of segments.

[0058] In the present invention, the so-called "segment" refers to the constituent unit constituting each block in the block copolymer, which is a unit (homopolymer segment) consisting of one monomer (hydroxycarboxylic acid described later) or a unit (copolymer segment) consisting of multiple monomers. As a "copolymer segment", for example, a random polymerization segment formed by random polymerization of multiple monomers, a gradient polymerization segment in which the monomer composition of the constituent continuously changes, and an alternating polymerization segment formed by alternating polymerization of multiple monomers can be cited. In addition, the number of monomers constituting each segment in the copolymer of the present invention is not particularly limited, but in the case of being used as a material, it is more than 100, usually more than 500, and more preferably more than 4000.

[0059] The "hydroxycarboxylic acid" involved in the present invention is divided into two types: a monohydroxycarboxylic acid having a hydroxyl group only at position 2, and a hydroxycarboxylic acid having a hydroxyl group at a position other than position 2. The latter may also be a hydroxycarboxylic acid having a plurality of hydroxyl groups, in which case, the hydroxyl group may be at position 2 in addition to a position other than position 2.

[0060] As hydroxycarboxylic acids having only a hydroxyl group at the 2nd position (hereinafter referred to as "hydroxycarboxylic acids (A)"), for example, 2-hydroxyacetic acid (glycolic acid, GL), 2-hydroxybutyric acid (2HB), 2-hydroxypropionic acid (lactic acid, 2HP), 2-hydroxyvaleric acid (2-hydroxypentanoic acid), 2-hydroxyhexanoic acid, medium-chain 2-hydroxyalkanoic acid. Among them, medium-chain 2-hydroxyalkanoic acid can be more specifically exemplified by 2-hydroxyalkanoic acids derived from amino acids (derived from the side chain structure of amino acids) (see Sudo M. et al., J Biosci Bioeng. 2019 Oct 18. pii: S1389-1723 (19) 30785-6). In addition, the hydroxycarboxylic acid (A) involved in the present invention refers not only to one monohydroxycarboxylic acid having only a hydroxyl group at the 2nd position, but also to a plurality of monohydroxycarboxylic acids having only a hydroxyl group at the 2nd position. Among them, from the viewpoint of being introduced into a copolymer and improving the non-enzymatic hydrolyzability, and thus easily exerting a high decomposability in the body, 2-hydroxyacetic acid is preferred. 2-Hydroxybutyric acid is preferred from the viewpoints that an isotactic polymer having a stereostructure similar to poly-D-lactic acid (PDLA) is obtained, which imparts transparency to the material, and that the polymer has a lower glass transition temperature than other 2-hydroxycarboxylic acids and is therefore easy to biosynthesize.

[0061] Examples of hydroxycarboxylic acids having a hydroxyl group at a position other than the 2-position (hereinafter also referred to as "hydroxycarboxylic acids (B)") include 3-hydroxypropionic acid (3HP), 3-hydroxybutyric acid (3HB), 3-hydroxyhexanoic acid (3HHx), 3-hydroxyvaleric acid (3-hydroxy-n-valeric acid, 3HV), medium-chain 3-hydroxyalkanoic acid, 4-hydroxy-2-methylbutyric acid (4H2MB), 4-hydroxybutyric acid, 2,4-dihydroxybutyric acid (24DHB), 5-hydroxyvaleric acid (5-hydroxy-n-valeric acid, 5HV), and 6-hydroxyhexanoic acid (6HHx). The hydroxycarboxylic acid (B) involved in the present invention refers to not only one hydroxycarboxylic acid having a hydroxyl group at a position other than the 2-position, but also a plurality of hydroxycarboxylic acids having a hydroxyl group at a position other than the 2-position. Among them, 3-hydroxybutyric acid is preferred from the viewpoint of being able to be most efficiently synthesized from various carbon sources and imparting strength to the material; and 3-hydroxyhexanoic acid is preferred from the viewpoint of improving flexibility by being introduced into a copolymer.

[0062] In the present invention, the combination of these hydroxycarboxylic acids (A) and (B) is not particularly limited as long as the above-mentioned two segments can be formed, and examples thereof include 2-hydroxybutyric acid, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid; 2-hydroxyacetic acid and 3-hydroxybutyric acid; 2-hydroxyacetic acid, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid; 2-hydroxybutyric acid, 3-hydroxybutyric acid and 3-hydroxypropionic acid; 2-hydroxybutyric acid, 3-hydroxybutyric acid and 4-hydroxy-2-methylbutyric acid; 2-hydroxybutyric acid, 3-hydroxybutyric acid and 2,4-dihydroxybutyric acid. 2-Hydroxybutyric acid, 3-hydroxybutyric acid and 5-hydroxyvaleric acid; 2-Hydroxybutyric acid, 3-hydroxybutyric acid and 6-hydroxyhexanoic acid; 2-Hydroxybutyric acid, 3-hydroxypropionic acid and 4-hydroxy-2-methylbutyric acid; 2-Hydroxybutyric acid, 3-hydroxypropionic acid and 2,4-dihydroxybutyric acid; 2-Hydroxybutyric acid, 3-hydroxypropionic acid and 5-hydroxyvaleric acid; 2-Hydroxybutyric acid, 3-hydroxypropionic acid and 6-hydroxyhexanoic acid; 2-Hydroxybutyric acid, 3-hydroxybutyric acid and 3-hydroxyvaleric acid, but 2-Hydroxybutyric acid and 3-Hydroxybutyric acid are excluded.

[0063] In addition, the molecular weight of the copolymer of the present invention composed of such a combination is not particularly limited, but from the viewpoint of obtaining sufficient mechanical strength and maintaining sufficient molecular weight after the molecular weight reduction caused by melt molding, the number average molecular weight is preferably 10,000 or more, more preferably 100,000 or more, and further preferably 1,000,000 or more. In addition, the upper limit is not particularly limited, but from the viewpoint of manufacturing difficulty, it is preferably usually 10,000,000 or less.

[0064] Hereinafter, the copolymer of the present invention will be further described with reference to specific combination examples.

[0065] In the copolymer of the present invention, the copolymer comprising 2-hydroxybutyric acid, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid has a homopolymer segment consisting of 2-hydroxybutyric acid and a copolymer segment comprising 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, wherein the copolymer segment in the copolymer adopts a random copolymer structure.

[0066] The molecular weight (number average molecular weight) of the copolymer is not particularly limited, but is preferably 100,000 or more, more preferably 1,000,000 or more, from the viewpoint of obtaining sufficient mechanical strength and maintaining a sufficient molecular weight even after the molecular weight is reduced by melt molding.

[0067] In addition, from the viewpoint of exerting sufficient flexibility and also exerting the physical properties brought by the block structure, the random copolymer segment of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid is preferably 50 mol% or more of the entire polymer, and more preferably 70 mol% or more. From the viewpoint of exerting sufficient flexibility, the composition of the random segment is preferably 5 mol% or more of 3-hydroxyhexanoic acid, and more preferably 20 mol% or more. The composition of 2-hydroxybutyric acid is uniquely determined by determining the amount ratio of the homopolymer segment to the copolymer segment.

[0068] In the copolymer of the present invention, the copolymer containing 2-hydroxyacetic acid and 3-hydroxybutyric acid has a homopolymer segment composed of 3-hydroxybutyric acid and a copolymer segment containing 2-hydroxyacetic acid and 3-hydroxybutyric acid, wherein the copolymer segment in the copolymer adopts a random copolymer structure.

[0069] The molecular weight (number average molecular weight) of the copolymer is not particularly limited, but is preferably 10,000 or more, more preferably 1,000,000 or more, from the viewpoint of exerting mechanical strength when used as a material and maintaining a sufficient molecular weight even after the molecular weight is reduced by melt molding.

[0070] In addition, from the viewpoint of controlling the mechanical properties of the material, the content ratio of 2-hydroxyacetic acid and 3-hydroxybutyric acid is not particularly limited. When used as a hard segment, 2-hydroxyacetic acid is preferably 50 mol% or more, and more preferably 70 mol% or more. On the other hand, when used as a soft segment, 2-hydroxyacetic acid is preferably 50 mol% or less, and more preferably 10 to 30 mol%.

[0071] In the copolymer of the present invention, the copolymer comprising 2-hydroxyacetic acid, 3-hydroxybutyric acid and 3-hydroxycaproic acid has a homopolymer segment consisting of 3-hydroxybutyric acid and a copolymer segment comprising 2-hydroxyacetic acid, 3-hydroxybutyric acid and 3-hydroxycaproic acid, wherein the copolymer segment in the copolymer adopts a gradient polymerization structure.

[0072] The lower limit of the molecular weight (number average molecular weight) of the copolymer is not particularly limited, but is preferably 10,000 or more, more preferably 1,000,000 or more, from the viewpoint of exerting mechanical strength when used as a material and maintaining a sufficient molecular weight even after the molecular weight is reduced by melt molding.

[0073] In addition, from the viewpoint of the material exhibiting flexibility, the content of 3-hydroxycaproic acid is preferably 10 mol% or more, more preferably 30 mol% or more of the total. In this case, the content of 2-hydroxyacetic acid and 3-hydroxybutyric acid is preferably both 70 mol% or less, and more preferably both 50 mol% or less.

[0074] Similarly, from the viewpoint of flexibility of the material, the hard homopolymer segment is preferably 50 mol% or less, more preferably 30 mol% or less.

[0075] In the copolymer of the present invention, the copolymer comprising 2-hydroxybutyric acid, 3-hydroxybutyric acid, 3-hydroxypropionic acid, 4-hydroxy-2-methylbutyric acid, 2,4-dihydroxybutyric acid, 5-hydroxypentanoic acid or 6-hydroxyhexanoic acid has a homopolymer segment composed of 2-hydroxybutyric acid and a copolymer segment comprising 3-hydroxybutyric acid and 3-hydroxypropionic acid, etc. The copolymer segments in the copolymer adopt a random copolymer structure.

[0076] The molecular weight of the copolymer is preferably 100,000 or more, more preferably about 1,000,000 (eg, 500,000 to 2,000,000, 800,000 to 1,500,000) in order to exhibit strength sufficient for processing into a material.

[0077] The monomer composition of the random segment can be appropriately adjusted by a person skilled in the art to obtain the desired physical properties. When synthesizing a random segment containing 3-hydroxybutyric acid as the main component and 3-hydroxypropionic acid, 4-hydroxy-2-methylbutyric acid, 5-hydroxyvaleric acid or 6-hydroxycaproic acid, the composition other than 3-hydroxybutyric acid is preferably 0 to 50 mol% for the purpose of softening the poly-3-hydroxybutyric acid skeleton. In order to exhibit appropriate flexibility and strength, it is more preferably about 5 to 20 mol% (for example, 1 to 40 mol%, 3 to 30 mol%). When synthesizing a segment containing 2,4-dihydroxybutyric acid for the purpose of hydrophilizing the polymer, the composition of 2,4-dihydroxybutyric acid is preferably about 5 to 20 mol% (for example, 1 to 40 mol%, 3 to 30 mol%). When the purpose is to produce a soft segment using units other than 3-hydroxybutyric acid (3-hydroxypropionic acid, 4-hydroxy-2-methylbutyric acid, 5-hydroxyvaleric acid or 6-hydroxycaproic acid) as the main component, it is preferred to adjust the components other than 3-hydroxybutyric acid to 50 to 100 mol%. In order to exert soft physical properties, it is preferred that the proportion of components other than 3-hydroxybutyric acid is high, and in order to achieve both synthetic efficiency of the polymer, it is preferably about 80 to 95 mol% (for example, 60 to 99 mol%, 70 to 97 mol%).

[0078] In the copolymer of the present invention, the copolymer comprising 2-hydroxybutyric acid, 3-hydroxypropionic acid, 4-hydroxy-2-methylbutyric acid, 2,4-dihydroxybutyric acid, 5-hydroxypentanoic acid or 6-hydroxyhexanoic acid has a homopolymer segment composed of 2-hydroxybutyric acid and a copolymer segment comprising 3-hydroxypropionic acid and 4-hydroxy-2-methylbutyric acid, etc. The copolymer segments in the copolymer adopt a random copolymer structure.

[0079] In the copolymer, the ratio of 3-hydroxypropionic acid to other units in the segment can be any composition, and examples thereof include 100 mol% or 90 mol% of 3-hydroxypropionic acid. The molecular weight is preferably a weight average molecular weight of 100,000 or more.

[0080] In the copolymer of the present invention, the copolymer comprising 2-hydroxybutyric acid, 3-hydroxybutyric acid and 3-hydroxyvaleric acid has a homopolymer segment consisting of 2-hydroxybutyric acid and a copolymer segment comprising 3-hydroxybutyric acid and 3-hydroxyvaleric acid, wherein the copolymer segment in the copolymer adopts a random copolymer structure.

[0081] In the copolymer, the copolymer segment preferably contains 10 to 30 mol % of 3-hydroxyvaleric acid. The molecular weight is preferably a weight average molecular weight of 100,000 or more.

[0082] <Method for producing block copolymer>

[0083] As shown in the examples described below, in microorganisms such as Escherichia coli, CoA is added to the above-mentioned hydroxycarboxylic acid by CoA transferase, and these hydroxycarboxylic acids are polymerized by polymerase, thereby obtaining the above-mentioned copolymer of the present invention. Therefore, the present invention provides a method comprising

[0084] Step 1: a step of culturing a microorganism expressing CoA transferase and polymerase in a medium containing hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B), and

[0085] Step 2: A step of recovering the copolymer from the culture obtained in the above step.

[0086] (CoA transferase)

[0087] In the present invention, "CoA transferase" refers to an enzyme that catalyzes the reaction of transferring CoA (coenzyme A) to the above-mentioned hydroxycarboxylic acid. For example, when the target is a hydroxycarboxylic acid having 5 or less carbon atoms, propionate CoA transferase can be mentioned.

[0088] Propionate CoA transferase (PCT) refers to an enzyme classified as enzyme number (EC): 2.8.3.1. As the PCT involved in the present invention, as long as its catalytic activity is maintained, its source is not particularly limited. From the viewpoint of easy heterohost expression in Escherichia coli, PCT derived from bacteria belonging to the genus Megasphera, Ralstonia, Pseudomonas, or Escherichia is preferred, PCT derived from bacteria belonging to the genus Megasphera is more preferred, and PCT derived from Megasphaera elsdenii (protein consisting of the amino acid sequence described in SEQ ID NO: 1) is further preferred.

[0089] In addition, nucleotide sequence mutations may also occur in nature. And accompanying this, the encoded amino acids may also change. Therefore, as long as the PCT involved in the present invention has the catalytic activity, it also includes a protein (variant) consisting of an amino acid sequence in which one or more amino acids are replaced, deleted, added, and / or inserted in the wild-type amino acid sequence of the bacterial origin (for example, the amino acid sequence recorded in sequence number 1).

[0090] The so-called "plurality" in PCT involved in the present invention means within 100 amino acids, preferably within 80 amino acids, more preferably within 50 amino acids, further preferably within 30 amino acids, more preferably within 20 amino acids, further preferably within 10 amino acids, and particularly preferably within several amino acids (for example, within 5 amino acids, within 3 amino acids, within 2 amino acids).

[0091] Furthermore, under the current technical level, as long as a person skilled in the art can use its gene (nucleotide sequence) information to determine its homologous gene from the same species or other bacteria. As a method for identifying homologous genes, for example, hybridization technology (Southern, EM, J. Mol. Biol., 98: 503, 1975) and polymerase chain reaction (PCR) technology (Saiki, RK, et al. Science, 230: 1350-1354, 1985, Saiki, RK et al. Science, 239: 487-491, 1988) can be cited. In order to identify homologous genes, hybridization reactions are usually carried out under stringent conditions. As stringent hybridization conditions, 6M urea, 0.4% SDS, 0.5×SSC conditions or hybridization conditions with equivalent stringency can be exemplified. If more stringent conditions are used, such as 6M urea, 0.4% SDS, 0.1×SSC conditions, it can be expected that genes with higher homology will be separated.

[0092] Therefore, the PCT involved in the present invention also includes a protein encoded by a DNA that hybridizes with a DNA consisting of a wild-type nucleotide sequence derived from the bacteria under stringent conditions, as long as it has the catalytic activity.

[0093] In addition, the protein encoded by the identified homologous gene usually has a high homology (high similarity) and preferably a high identity with the protein derived from the bacteria. Here, "high" means at least 50% or more, preferably 60% or more, more preferably 70% or more, further preferably 80% or more, more preferably 85% or more (for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more).

[0094] Therefore, the PCT involved in the present invention also includes a protein composed of an amino acid sequence having at least 50% homology (similarity) or identity with the wild-type amino acid sequence derived from the bacteria, as long as it has the above-mentioned catalytic activity.

[0095] In addition, the homology of the sequence can be determined using the BLAST program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). This program is based on the algorithm BLAST of Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). For example, when analyzing an amino acid sequence by BLAST, the parameters are, for example, score=50 and wordlength=3. In addition, when analyzing an amino acid sequence using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. The specific techniques of these analytical methods are well known.

[0096] Whether the above-mentioned variants or homologs of PCT have the above-mentioned catalytic activity can be determined by those skilled in the art by measuring their catalytic activity using the method described in, for example, AE Hofmeister et al. (Eur. J. Biochem., Vol. 206, pp. 547-552).

[0097] In addition to the above-mentioned PCT, when a hydroxycarboxylic acid having a hydroxyl group at the 3-position such as 3HHx is targeted, acyl-CoA synthetase can be used as a suitable example of the CoA transferase involved in the present invention.

[0098] Acyl-CoA synthetase (AlkK) refers to an enzyme classified in enzyme number (EC): 6.2.1.3. As AlkK involved in the present invention, as long as its catalytic activity is maintained, its source is not particularly limited, but from the viewpoint of having sufficient activity on medium-chain 3-hydroxycarboxylic acids, AlkK derived from bacteria belonging to the genus Pseudomonas is preferred, AlkK derived from Pseudomonas oleovorans or Pseudomonas putida is more preferred, AlkK derived from Pseudomonas putida is further preferred, and AlkK derived from Pseudomonas putida KT2440 (protein consisting of the amino acid sequence described in SEQ ID NO: 2) is more preferred (see Wang et al. Appl Environ Microbiol. 2012 Jan; 78(2): 519-527).

[0099] In addition, similar to PCT, the AlkK involved in the present invention also includes a protein (variant) composed of an amino acid sequence in which one or more amino acids are replaced, deleted, added, and / or inserted in the wild-type amino acid sequence derived from the bacteria (e.g., the amino acid sequence recorded in SEQ ID NO: 2) as long as it has the catalytic activity. The so-called "multiple" in the AlkK involved in the present invention refers to within 100 amino acids, preferably within 80 amino acids, more preferably within 50 amino acids, further preferably within 30 amino acids, more preferably within 20 amino acids, further preferably within 10 amino acids, and particularly preferably within several amino acids (e.g., within 5 amino acids, within 3 amino acids, within 2 amino acids).

[0100] Furthermore, the AlkK involved in the present invention, as long as it has the above-mentioned catalytic activity, also includes a protein encoded by a DNA that hybridizes under stringent conditions with a DNA composed of a wild-type nucleotide sequence derived from the above-mentioned bacteria, and a protein composed of an amino acid sequence that has at least 50% homology (similarity) or identity with the wild-type amino acid sequence derived from the above-mentioned bacteria.

[0101] In addition, as to whether the variant or homolog of AlkK has the catalytic activity, a person skilled in the art can determine it by, for example, the following method: the acyl-CoA generated by the reaction of the enzyme is specifically oxidized by acyl-CoA oxidase, and the hydrogen peroxide generated is detected by coloring it with 4-aminoantipyrine, 3-methyl-N-ethyl-N-(2-hydroxyethyl)aniline, N,N-dimethylaniline, N,N-diethylaniline or phenol in the presence of peroxidase.

[0102] (Polymerase)

[0103] In the present invention, the "polymerase" may be any enzyme that catalyzes the reaction of polymerizing the hydroxycarboxylic acid to which CoA has been transferred to form the above-mentioned homopolymer segment and copolymer segment, and examples thereof include polymerases that can recognize (1) hydroxycarboxylic acid having a hydroxyl group only at position 2 and (2) hydroxycarboxylic acid having no hydroxyl group at position 2 and control the polymerization reaction. Preferably, type 1 polyhydroxyalkanoate synthase (PhaC) or a variant thereof having the above-mentioned catalytic activity is exemplified, more preferably type 1 PhaC or a variant thereof derived from bacteria belonging to the genus Aeromonas, and more preferably type 1 PhaC or a variant thereof derived from Aeromonascaviae. More preferably, it is a chimeric polymerase in which the N-terminal portion of type 1 PhaC derived from bacteria belonging to the genus Aeromonas is fused to the C-terminal portion of type 1 PhaC derived from bacteria belonging to the genus Ralstonia, more preferably, it is a chimeric polymerase in which the N-terminal portion of type 1 PhaC derived from Aeromonas caviae is fused to the C-terminal portion of type 1 PhaC derived from Ralstonia eutropha, and even more preferably, it is a chimeric polymerase in which 26% of the N-terminal side of type 1 PhaC derived from Aeromonas caviae is fused to 74% of the C-terminal side of type 1 PhaC derived from Ralstonia eutropha. AR , (protein consisting of the amino acid sequence described in SEQ ID NO: 3)) (see Matsumoto K. et al., Biomacromolecules. 2009 Apr 13; 10(4): 682-685).

[0104] In addition, similar to the above-mentioned CoA transferase, the polymerase involved in the present invention also includes a protein (variant) composed of an amino acid sequence in which one or more amino acids are replaced, deleted, added, and / or inserted in the amino acid sequence of the enzyme (for example, the amino acid sequence described in SEQ ID NO: 3), as long as it has the above-mentioned catalytic activity. The so-called "multiple" in the CoA transferase involved in the present invention means within 100 amino acids, preferably within 80 amino acids, more preferably within 50 amino acids, further preferably within 30 amino acids, more preferably within 20 amino acids, further preferably within 10 amino acids, and particularly preferably within several amino acids (for example, within 5 amino acids, within 3 amino acids, within 2 amino acids).

[0105] As a result of this change, for example, in PhaC AR In the above, at least one amino acid substitution selected from F314X, I320X, G423X, V448X and I505X can be mentioned ("X" means an amino acid different from that before substitution). By the amino acid substitution, the polymerization ability of 3HHx is enhanced, so for example, when 3HHx is used as the hydroxycarboxylic acid (B), the 3HHx fraction in the copolymer of the present invention can be increased.

[0106] Furthermore, as long as the polymerase involved in the present invention has the above-mentioned catalytic activity, it also includes a protein encoded by a DNA that hybridizes under strict conditions with a DNA composed of a nucleotide sequence encoding the enzyme, and a protein composed of an amino acid sequence that has at least 50% homology (similarity) or identity with the amino acid sequence of the enzyme.

[0107] In addition, a person skilled in the art can determine whether a variant or homolog of the polymerase has the catalytic activity by using the method described in Non-Patent Document 2, for example.

[0108] (microorganism)

[0109] In the present invention, the copolymer of the present invention can be produced by culturing a microorganism expressing the above two enzymes. As the microorganism, one expressing these enzymes naturally can be used, but usually a transformant prepared by introducing DNA encoding the enzyme into the microorganism is used.

[0110] As the "microorganism" involved in the present invention, as long as it is a microorganism that can functionally express a polyhydroxyalkanoic acid (PHA) synthase group, it is not particularly limited, but for example, Escherichia coli, hydrogen bacteria (hookworm greedy copper bacteria, Cupriavidus necator), rod-shaped bacteria, yeast, Ralstonia bacteria, Pseudomonas bacteria. Among these microorganisms, from the viewpoint of polymer productivity and production stability, preferably Escherichia coli, hydrogen bacteria, more preferably Escherichia coli. The strain of Escherichia coli is not particularly limited, and from the viewpoint of being able to achieve more stable copolymer production, as a preferred example, E.coli JM109 strain can be cited. In addition, as shown in the examples described below, from the viewpoint of being able to biosynthesize the hydroxycarboxylic acid (B) (3HB, 3HV, 3HHx, etc.) involved in the present invention, preferably, a bacterium of the genus Ralstonia, more preferably Ralstonia eutropha, further preferably a glucose assimilating strain of Ralstonia eutropha (H16 NSDG-GG-ΔB1 strain), more preferably H16CAR-GG-ΔB1 and H16 CAR-GG-ΔB1-ldhAhadA shown in the examples described below.

[0111] In this microorganism, in order to express two kinds of enzymes involved in the present invention, the above-mentioned DNA is usually imported in the form of being integrated into the vector. In addition, homologous recombination can also be utilized to import into the chromosome. In addition, in order to be imported into the DNA (gene) in the chromosome of the microorganism involved in the present invention, it is suitable to be transcribed and then translated into a protein with desired activity, the DNA needs to be integrated in the mode under the control of suitable primers on the chromosome.

[0112] In the present invention, "vector" can basically be constructed as a self-replicating vector, that is, a plasmid that exists as an independent body outside the chromosome and whose replication does not depend on the replication of the chromosome. In addition, when the vector is introduced into the host cell, it can also be integrated into the genome of the host cell and replicated together with the chromosome into which the vector is integrated.

[0113] As such a vector, for example, plasmid DNA and phage DNA can be cited. Examples of vectors for introduction into Escherichia coli include plasmid DNA such as pBLuescript (pBLuescript KS+, pBLuescript SK+, pBLuescriptII KS+, pBLuescriptII KS-, etc.), pBR322, pUC18, and phage DNA such as EMBL3, M13, and λgtII. Examples of vectors for introduction into hydrogen bacteria include pCUP2 (refer to International Publication No. 2007 / 049716), pLA2917 (ATCC37355) having an RK2 replication origin that replicates / maintains in a certain range of hosts, and pJRD215 (ATCC37533) having an RS F1010 replication origin. Examples of vectors for introduction into coryneform bacteria include pAM330, pHM1519, pAJ655, pAJ611, pAJ1844, pCG1, pCG2, pCG4, pCG11, pHK, pPSPTG1, pVC7, and pUC19I. Examples of vectors for introduction into yeast include YEp13 and YCp50. In addition, examples of vectors for introduction into Ralstonia bacteria, Pseudomonas bacteria, and the like include the aforementioned pLA2917 (ATCC37355) and pJRD215 (ATCC37533). In addition, examples of vectors for homologous recombination include pK18mobsacB (Schafer et al., Gene 145, 69-73 (1994)) and pJQ200 (Quandt, J. and Hynes, MP, Gene (1993) 17: 15-21).

[0114] The DNA encoding the above-mentioned enzymes can be inserted into the vector using the known gene recombination techniques conventionally used in the field of genetic engineering. For example, in order to insert the DNA into the vector, the following methods can be used: first, the purified DNA is cut with an appropriate restriction enzyme, and then inserted into the restriction enzyme site or the multiple cloning site of an appropriate vector and connected to the vector.

[0115] During recombination, it is preferred to connect the DNA downstream of a promoter capable of regulating the transcription and translation of each protein of the DNA inserted into the vector. As a promoter, there is no particular limitation as long as it can regulate the transcription of the gene in the host, and it can be obtained as a DNA sequence that controls the expression of a gene encoding any protein of the same or different species as the host cell. For example, when Escherichia coli is used as a host, lac promoter, trp promoter, PL promoter, PR promoter, and T7 promoter can be listed. In addition, the promoter region of the phb operon of hookworm greedy copper bacteria can also be used. When hydrogen bacteria are used as hosts, phaC1 gene promoters derived from the bacteria and phaP1 gene promoters derived from the bacteria can be listed. When rod-shaped bacteria are used as hosts, for example, cell surface protein gene promoters derived from Corynebacterium glutamicum can be listed. When yeast is used as a host, for example, gal1 promoter and gal10 promoter can be listed. When a bacterium of the genus Pseudomonas is used as the microorganism of the present invention, a region containing a promoter upstream of the phaC1Ps gene and upstream of the phbCABRe operon can be used.

[0116] The vector may also contain, as required, a terminator sequence, an enhancer sequence, a splicing signal sequence, a poly A addition signal sequence, a ribosome binding sequence (SD sequence) and other sequences for controlling expression other than the promoter that can be utilized in the microorganism to be introduced into the DNA. In addition, a sequence for inducing expression may also be included in addition to the sequence. As the sequence, the lactose operon that can induce the expression of the gene configured downstream by adding isopropyl-β-D-thiogalactoside (IPTG) may be cited.

[0117] The vector may contain a selection marker gene as required. The selection marker gene may be appropriately selected according to the selection method of the transformed microbial cells, and examples thereof include drug resistance genes such as ampicillin resistance gene, tetracycline resistance gene, neomycin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene, etc.; genes involved in the intracellular biosynthesis of nutrients such as amino acids and nucleic acids (genes that assist in nutritional requirements); genes involved in chemiluminescence such as luciferase; genes encoding fluorescent proteins such as GFP.

[0118] In the vector, DNA encoding the enzyme (CoA transferase, polymerase) involved in the present invention can be inserted one by one, and multiple DNAs can also be inserted into one vector. In the case of inserting multiple DNAs into a vector, if multiple DNAs are inserted into one vector, it is preferred that these DNAs form an operon. Here, the so-called "operon" is a nucleic acid sequence unit consisting of one or more genes transcribed under the control of the same promoter.

[0119] The above DNA or a vector inserted with the DNA can be introduced into a microorganism using a known method. As a method for introducing DNA into a microorganism, for example, a calcium chloride method, a heat shock method, a calcium phosphate method, an electroporation method, a spheroplast method, a lithium acetate method, a conjugation transfection method, and a method using calcium ions can be cited.

[0120] (Cultivation conditions such as culture medium)

[0121] In the present invention, the copolymer of the present invention can be produced by culturing the above-mentioned microorganism in a culture medium containing the hydroxycarboxylic acid (A) and the hydroxycarboxylic acid (B).

[0122] The medium is not particularly limited as long as it can supply a carbon source at a sufficient concentration, and may contain a nitrogen source, inorganic salts, or other organic nutrient sources as nutrient sources other than the carbon source.

[0123] As a carbon source, any carbon source can be used as long as the above-mentioned microorganisms can assimilate it, preferably sugars such as glucose, fructose, and sucrose; oils such as palm oil, palm kernel oil, corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil, and their fractionated oils; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, and myristic acid, or their derivatives, etc.

[0124] Examples of nitrogen sources include ammonia, ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate, peptone, meat extract, and yeast extract. Examples of inorganic salts include potassium dihydrogen phosphate, sodium dihydrogen phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride. Examples of other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, and proline, and vitamins such as vitamin B1, vitamin B12, and vitamin C.

[0125] Examples of the culture medium involved in the present invention include, but are not limited to, LB medium (containing 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L sodium chloride) when culturing Escherichia coli and the like. In addition, when using natural PHA-producing bacteria (e.g., hydrogen bacteria, Pseudomonas putida) as a host, examples include, but are not limited to, nitrogen-limited inorganic salt medium (e.g., containing 9 g / L Na2HPO4, 1.5 g / L KH2PO4, 0.5 g / L NH4Cl, 0.2 mg / L MgSO4, trace elements), and phosphorus-limited inorganic salt medium (e.g., glucose 1 g / L, KNO3 0.5 g / L, MgSO4 (7H20) 0.2 g / L, CaCl2 0.1 g / L, NaCl 0.1 g / L, 8.0 mg K2HPO4 and 2.0 mg KH2PO4 are added thereto to adjust the pH to 7).

[0126] The hydroxycarboxylic acid added to the culture medium is as described above, but may be either an R-isomer or a racemic isomer, but is preferably an R-isomer. In addition, it may be added after adjusting the pH (neutralization, etc.) according to the optimum pH of the culture medium, or it may be added in the form of a salt. Examples of the salt include sodium salts, potassium salts, ammonium salts, and the like. The amount added to the culture medium may be a concentration suitable for copolymerization, and may also be appropriately adjusted to control the monomer composition in the copolymerization synthesized depending on the concentration, for example, 1 to 10 g / L.

[0127] As shown in the examples described below, even if the medium does not contain hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B) themselves, the copolymer of the present invention can be produced by using their metabolic precursors (metabolic starting materials, etc.) as long as the microorganism is capable of biosynthesizing the hydroxycarboxylic acid.

[0128] Therefore, the present invention can also be said to be a method, comprising:

[0129] a step of culturing a microorganism expressing CoA transferase and polymerase in a medium containing hydroxycarboxylic acid (A) and / or its metabolic precursor, and hydroxycarboxylic acid (B) and / or its metabolic precursor; and a step 2 of recovering the copolymer from the culture obtained in the above step.

[0130] The metabolic precursors involved in the present invention are not particularly limited as long as they can be ultimately converted into the hydroxycarboxylic acid involved in the present invention through the metabolic pathway in the microorganism. For example, when the hydroxycarboxylic acid (A) is 2HB, glucose, threonine, 2-ketobutyric acid, etc. can be listed. For example, when the hydroxycarboxylic acid (B) is 3HB, glucose, pyruvic acid, acetyl CoA, acetoacetyl CoA, etc. can be listed. For example, when the hydroxycarboxylic acid (B) is 3HV, 2HB, 2-oxobutyric acid, threonine, propionic acid, propionic acid-IP, propionic acid-CoA, 3-ketovaleryl-CoA, etc. can be listed (please refer to Figure 5A wait).

[0131] As conditions for culturing the above-mentioned microorganisms, in addition to the aforementioned culture medium, the culture temperature is not particularly limited as long as it is within the range in which the microorganisms used can grow and reproduce and the range in which the enzyme activities can be exerted, and is usually 25 to 37°C, preferably 28 to 32°C, and particularly preferably 30°C. In addition, depending on the type of microorganism used, aerobic conditions and anaerobic conditions can be appropriately selected, but from the viewpoint of more easily promoting the synthesis of copolymers, aerobic conditions are preferably used. As for the culture time, it can be appropriately adjusted according to the production amount of the copolymer, etc., but it is usually 12 hours to 1 week, preferably 1 to 3 days.

[0132] (Copolymer recovery, etc.)

[0133] In the present invention, the copolymer of the present invention is recovered from a culture obtained by culturing the above-mentioned microorganisms. The so-called "culture" includes not only the microorganisms obtained by culturing and growing the microorganisms in a culture medium, but also the culture medium containing the secretion products and metabolites of the microorganisms, and their dilutions and concentrates.

[0134] The method for recovering the copolymer from such a culture is not particularly limited, but for example, it can be carried out by the following method. After the culture is completed, the microorganism is separated from the culture medium by centrifugal separation, the microorganism is washed with distilled water and methanol, and dried (for example, freeze-dried). The dried bacterial body is heat-treated with an organic solvent such as chloroform to extract the copolymer. The bacterial body components are removed by filtering the organic solvent solution containing the copolymer, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate the copolymer. Furthermore, the supernatant is removed by filtering and centrifugation, and the copolymer is dried and recovered.

[0135] In addition, as shown in the examples described later, the average molecular weight, monomer composition, structure, etc. of the copolymer obtained in this way can be confirmed by using a known analytical method such as nuclear magnetic resonance (NMR), gel permeation chromatography, gas chromatography, etc.

[0136] Example

[0137] Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to the following examples. In addition, the examples were carried out using the following methods.

[0138] (Example 1)

[0139] <Method for preparing P(2HB-b-(3HB-ran-3HHx))>

[0140] Preparation of pBluescript KS+ plasmid with the promoter region of the phb operon of C.nector, PhaC AR gene, pct gene from Megasphaera elsdenii, and AlkK gene from Pseudomonas putida KT2440 (pBSPreCARpctAlkK). Re In phaC(AR) pct (see Non-Patent Document 2), the AlkK gene was inserted downstream of the pct gene by a conventional method. Then, the plasmid was introduced into Escherichia coli (JM109 strain) by the calcium chloride method to obtain a transformant.

[0141] The transformant was cultured with shaking at 30°C for 48 hours in LB medium containing 1 to 10 g / mL of (R,S)-2HB-Na, 1 to 10 g / mL of (R,S)-3HB-Na, and 1 to 10 g / mL of (R,S)-3HHx-Na.

[0142] The Escherichia coli recovered from the obtained culture solution was freeze-dried and then subjected to a chloroform heating treatment to recover the polymer (see the recovery method described in Taguchi, S. et al., Proc. Natl. Acad. Sci. USA. 2008, 105(45), 17323-17327).

[0143] <Structural analysis of P(2HB-b-(3HB-ran-3HHx))>

[0144] (R,S)-2HB-Na, (R,S)-3HB-Na, and (R,S)-3HHx-Na were added to LB medium at 2.5 g / mL, and the resulting polymers were dissolved in deuterated chloroform. 1 H NMR measurement, calculate the composition and mol fraction. The results are as follows Figure 1A As shown, since only one 5 ppm signal originating from 2HB can be observed, it indicates the presence of P(2HB) segment.

[0145] In addition, the same sample was further provided to 13 C NMR measurement. The results are as follows Figure 1B As shown, it can be seen that since the linkage strength between 3HHx is weak, there is no P(3HHx) homopolymer segment, that is, the structure including 3HHx units is randomly arranged.

[0146] The above results show that the polymer obtained above is a block copolymer composed of a P(2HB) homopolymer segment and a P(3HB-ran-3HHx) random segment.

[0147] <Physical property analysis of P(2HB-b-(3HB-ran-3HHx))>

[0148] About 100 mg of the polymer was dissolved in chloroform, and the solvent was volatilized in a glass petri dish to prepare a solvent cast film. A test piece was cut from the obtained circular film and subjected to a tensile test to calculate parameters such as elongation at break (stretchability) and tensile strength.

[0149] Depend on Figure 1CAs shown in the results, the ternary copolymer P(2HB-b-(3HB-ran-3HHx)) obtained this time showed high stretchability compared to the binary copolymer P(2HB-b-3HB) disclosed in Non-Patent Document 2. In particular, the ternary copolymer showed a very high stretchability exceeding 1200%. These results also suggest that according to the present invention, by controlling the arrangement of monomers and then controlling the high-order structure, a copolymer showing elastomeric properties in which deformation is proportional to stress can be obtained.

[0150] (Example 2)

[0151] <Method for preparing P(3HB-b-(GL-ran-3HB))>

[0152] In the same manner as in Example 1, pBSPreCARpctAlkK was introduced into Escherichia coli (strain JM109) by the calcium chloride method to obtain a transformant.

[0153] Alternatively, a gene encoding PhaC AR Plasmid DNA encoding PhaC1 Ps In addition, the plasmid was replaced by PhaC in pBSPreCARpctAlkK by conventional methods. AR PhaC1 Ps STQK gene, thus made.

[0154] Then, these transformants were cultured with shaking at 30°C for 48 hours in LB medium containing 0 to 12 g / L of (R,S)-GL-Na and 5 g / L of (R,S)-3HB-Na, and the polymer was recovered from the obtained culture solution in the same manner as described in Example 1.

[0155] <Analysis of P(3HB-b-(GL-ran-3HB))>

[0156] The cultured cells were freeze-dried and then extracted with chloroform to extract the polymer in the cells. The chloroform solution after filtering out the cells was concentrated and then an excess of methanol was added to polymerize the cells. The recovered polymer was dissolved in deuterated chloroform and 1 H NMR measurement. The obtained results are shown in Figure 2A and 2B In addition, these figures show the results of analyzing the polymers obtained using LB medium containing (R,S)-GL-Na 12 g / L and (R,S)-3HB-Na 5 g / L. In addition, the monomer composition was calculated based on the peak intensity. The obtained results are shown in Table 1.

[0157] Table 1

[0158]

[0159] <Gel filtration chromatography (GPC) analysis of P(3HB-b-(GL-ran-3HB))>

[0160] The cultured cells were freeze-dried and subjected to chloroform extraction to extract the polymer in the cells. The cells were then filtered using a PTFE membrane and subjected to GPC analysis using chloroform as the mobile phase. The results are shown in Figure 2C In addition, the figure shows the results of analyzing the obtained polymer using LB medium containing 12 g / L of (R,S)-GL-Na and 5 g / L of (R,S)-3HB-Na.

[0161] The results of the above NMR analysis are Figure 2A and 2B In the figure, the four peaks observed at 4.5 to 4.9 ppm are signals attributable to the GL unit, and the peak on the lowest magnetic field side (left side) corresponds to the GL unit sandwiched by 3HB (3HB-GL-3HB). In the case of a random copolymer with a GL fraction of about 20 mol%, it is predicted that the signal of 3HB-GL-3HB is strongly observed.

[0162] Based on the above, PhaC1 Ps The polymer obtained by STQK is a random copolymer (P(GL-ran-3HB)) (refer to Figure 2A ). On the other hand, using PhaC AR The obtained polymer showed strong GL-GL-GL signals, indicating that it contained a structure with high GL density. The GL fraction was estimated to be about 70 mol% from the relative intensity ratio of the four peaks, which was very different from the GL fraction of the entire polymer of about 20 mol%, indicating that the present polymer had a random-block structure (P(3HB-b-(GL-ran-3HB))).

[0163] In addition, in the gel filtration chromatography analysis, when the polymer is a mixture of two or more polymers (for example, a copolymer with P(3HB)), multiple molecular weight distributions are observed. AR The resulting polymers, such as Figure 2C As shown, a single peak was observed, which supports that the polymer has a sequential structure within the same molecule.

[0164] Thus, using PhaC AR The obtained polymer contains P(3HB) homopolymer segments and P(GL-ran-3HB) random copolymer segments, and has a sequence structure with greatly different compositions within the same molecular chain, which has never been synthesized in previous biosynthetic PHAs.

[0165] Such polymers have two advantages. The first is that since a structure is formed by connecting multiple polymers with different physical properties in the same molecule, it is sometimes possible to create new materials derived from its structure. The second is that since a large number of GL units are linked, non-enzymatic hydrolysis is improved (see Matsumoto K. et al., ACS Biomaterials Science & Engineering, 3 (12) 3058, 2017).

[0166] (Example 3)

[0167] <Method for preparing P(3HB-b-(GL-co-3HB-co-3HHx))>

[0168] pBSPreCARpctAlkK was introduced into Escherichia coli (JM109 strain) by the calcium chloride method to obtain a transformant. The transformant was cultured with shaking at 30°C for 48 hours in an LB medium containing (R,S)-GL-Na 2.5-5 g / L, (R,S)-3HB-Na 0-2.5 g / L, and (R,S)-3HHx-Na 1 g / L, and the polymer was recovered from the obtained culture solution by the same method as described in Example 1.

[0169] <Analysis of P(3HB-b-(GL-co-3HB-co-3HHx))>

[0170] The same method as described in the above <Analysis of P(3HB-b-(GL-ran-3HB))> was used to 1 H NMR measurement. The obtained results are shown in Figure 3A . In addition, this figure is the result of analyzing the polymer obtained using LB medium containing (R,S)-GL-Na 5g / L, (R,S)-3HB-Na2.5g / L and (R,S)-3HHx-Na 1g / L, and is obtained by amplifying the signal of the GL unit. In addition, the monomer composition was calculated from the peak area value. The obtained results are shown in Table 2.

[0171] Table 2

[0172]

[0173] <Differential Scanning Calorimetry (DSC) of P(3HB-b-(GL-co-3HB-co-3HHx))>

[0174] The polymer was dissolved in chloroform and the solvent was evaporated in a glass petri dish to prepare a solvent casting film. A test piece of several milligrams was cut from the obtained film and sealed in an aluminum pan to prepare a sample. This sample was subjected to DSC analysis. The DSC analysis was performed by heating from -50°C to 220°C to determine the presence or absence of an endothermic peak associated with crystal melting. The results are shown in Figure 3B .

[0175] As shown in Table 2, according to the present invention, it is possible to synthesize a ternary copolymer containing GL, 3HB and 3HHx. In addition, in the ternary copolymer thus obtained, 3HHx can be increased to 60 mol%, which is a sufficient ratio for imparting flexibility to the polymer properties. In addition, the GL fraction can be increased to about 30 mol%, which can achieve a sufficient GL unit fraction for the purpose of changing the material properties.

[0176] In addition, if Figure 3A As shown, the signal of the GL unit was amplified in the above NMR analysis results, and the relative intensities of the four signals observed showed that the obtained polymer had a gradient structure. In the gradient structure, the two ends and the central part of the molecule have different structures, so it adopts a structure close to a triblock.

[0177] Furthermore, if Figure 3B As shown in FIG. 1 , no crystalline melting peak was observed in the DSC analysis, which shows that the polymer has a structure with a very low degree of crystallinity.

[0178] The above results show that the present polymer, like P(3HB-b-(GL-ran-3HB)) shown in Example 2, contains an internal structure with a high GL fraction. On the other hand, it can be judged that 3HB and 3HHx units are randomly polymerized with each other. Therefore, the present polymer has a special structure (P(3HB-b-(GL-co-3HB-co-3HHx))) in which a GL-rich region is added to the existing 3HB and 3HHx random copolymer.

[0179] <Physical property analysis of P(3HB-b-(GL-co-3HB-co-3HHx))>

[0180] The polymer was dissolved in chloroform and the solvent was evaporated in a glass petri dish to prepare a solvent cast film. A test piece was cut from the obtained film and used for tensile testing. The obtained results are shown in Figure 3C .

[0181] Figure 3CThe tensile test results of P(3HB-b-(GL-co-3HB-co-3HHx)) shown in the figure show that the material has a soft property because the slope is gentle. In addition, it can be slightly stretched. This shows that the softness of the polymer can be greatly improved by introducing the soft 3HHx unit.

[0182] In addition, the non-enzymatic hydrolyzability of the present polymer is increased by introducing the GL unit, and since high degradability in vivo is expected, the present polymer is useful in medical devices (eg, sutures) and the like.

[0183] (Example 4)

[0184] <Method for preparing block copolymer composed of P(2HB) homopolymer segment and random copolymer segment mainly composed of 3-hydroxy acid, and analysis of the block copolymer>

[0185] As described in Example 1, when Escherichia coli JM109 strain introduced with plasmid pBSPreCARpctAlkK is cultured in a medium supplemented with 2HB, 3HB and 3HHx, a block copolymer consisting of a P(2HB) homopolymer segment and a P(3HB-ran-3HHx) random segment is obtained.

[0186] Therefore, in this example, in the culture system described in Example 1, LB medium to which 1.0 g / L of sodium salt of 3-hydroxypropionic acid (3HP), 4-hydroxy-2-methylbutyric acid (4H2MB), 2,4-dihydroxybutyric acid (24DHB), 5-hydroxyvaleric acid (5HV) or 6-hydroxyhexanoic acid (6HHx) was added was used instead of 3HHx for culture (shaking culture at 30°C for 48 hours), and it was confirmed whether copolymers containing each monomer unit could be synthesized in the same manner as in Example 1. The obtained results are shown in Figure 4A and 4B .also, Figure 4A The analysis was performed in the same manner as described in the above <Analysis of P(3HB-b-(GL-ran-3HB)> 1 Results of H NMR measurement. Figure 4B The results obtained by the same analysis as in the above-mentioned <Differential Scanning Calorimetry (DSC) of P(3HB-b-(GL-co-3HB-co-3HHx))> are shown in FIG.

[0187] like Figure 4A As shown, the presence of P(2HB) homopolymer segments is 1 The results were confirmed by H NMR measurement. More specifically, since resonance of methine groups attributable to 2HB was observed at around 5.1 ppm in all polymers, the presence of a P(2HB) segment was indicated.

[0188] In addition, the segment with 3HB as the main component is a random copolymer, which can be confirmed by melting point measurement. Figure 4B In the thermal analysis diagram of differential calorimetry of P(3HB-co-5HV) shown as an example, the peak position indicating the melting point is lower than the melting point (178°C) of P(3HB). This confirms that 5HV is randomly copolymerized. Although not shown in the figure, the same thermal analysis diagram is obtained for other copolymers.

[0189] The above results confirmed that in the obtained polymer, the newly added monomer precursor was incorporated as a random copolymer with the 3HB unit, and the 2HB component was polymerized as a P(2HB) homopolymer segment. More specifically, it was confirmed that through the above-mentioned culture conditions, new ternary block copolymers, P(2HB)-bP(3HB-ran-3HP), P(2HB)-bP(3HB-ran-4H2MB), P(2HB)-bP(3HB-ran-24DHB), P(2HB)-bP(3HB-ran-5HV), and P(2HB)-bP(3HB-ran-6HHx) were obtained.

[0190] Furthermore, in the present invention, even if the precursor 3HB added to the culture medium is replaced with 3HP, a block copolymer consisting of a P(2HB) homopolymer segment and a random copolymer of a segment mainly composed of 3-hydroxy acid can be obtained in the same manner as described above. That is, according to the present invention, P(2HB)-bP(3HP-ran-4H2MB), P(2HB)-bP(3HP-ran-24DHB), P(2HB)-bP(3HP-ran-5HV), P(2HB)-bP(3HP-ran-6HHx), etc. can also be obtained.

[0191] In addition, in the present invention, the synthesis of these polymers depends on the properties of the polymerase, and the monomer-supplying enzyme is not selected as long as it can synthesize the CoA body of the corresponding monomer.

[0192] (Example 5)

[0193] <Method for preparing block copolymer (P(2HB)-bP(3HB-co-3HV)) from glucose and 2HB>

[0194] The inventors consider that Figure 5A As shown, if the microorganism has the biosynthetic pathway shown below, by culturing in the presence of glucose and 2HB, a block copolymer (P(2HB)-bP(3HB-co-3HV)) consisting of a random copolymer of a P(2HB) homopolymer segment and a segment consisting of 3HB and 3HV can be obtained.

[0195] <Biosynthetic pathway>

[0196] 2HB is converted into 2HB-CoA in the presence of HadA (CoA transferase).

[0197] In the glycolysis system, pyruvate is generated from glucose. Acetyl CoA is further generated from pyruvate, which is converted into acetoacetyl CoA in the presence of PhaA, and then biosynthesized into 3HB-CoA in the presence of PhaB (Journal of Biotechnology, 152 (2011), 144-146).

[0198] 2HB is converted into 2-oxobutyrate in the presence of LdhA (lactate dehydrogenase). Propionate and 2-hydroxybutyrate are generated from threonine and 2-oxobutyrate (Letters in Applied Microbiology, 1997, 25, 371-374). Propionate is converted into propionate-CoA via propionate-IP. Propionate-CoA and acetyl-CoA are converted into 3-ketovaleryl-CoA in the presence of PhaA, and further, into 3HV-CoA in the presence of PhaB (Journal of Biotechnology, 152 (2011), 144-146).

[0199] <Construction of a mutant strain of Ralstonia eutropha>

[0200] like Figure 5B As shown, in the pha operon on the chromosome of Ralstonia eutropha H16 NSDG-GG-ΔB1 strain (see Zhang et al., Microbial Cell Factories, volume 18, Article number: 147 (2019)), which is a glucose assimilating mutant strain of Ralstonia eutropha, phaCNSDG (a modified PHA synthase gene derived from Aeromonas caviae) was replaced by phaC AR Gene, construct PhaC AR Expression strain H16CAR-GG-ΔB1. Specifically, a plasmid pK18mobsacB derivative containing the target fragment was introduced into R.eutrpha via the Escherichia coli S17-1 strain with conjugative transfection ability, and strains with the phaCAR gene inserted at the target position on the chromosomal DNA were screened using kanamycin resistance as an indicator, and then the lethality of sacB contained in pK18mobsacB in the presence of sucrose was used to isolate the recombinant strain in which the vector part was removed.

[0201] Furthermore, if Figure 5C As shown, ldhA (lactate dehydrogenase gene) and hadA (CoA transferase gene) derived from Clostridium difficile were inserted downstream of phaP1 (PHA granule-binding protein gene) on the chromosome of the mutant strain, and H16CAR-GG-ΔB1-ldhAhadA capable of expressing these enzymes under the control of the phaP promoter (PphaP, transcriptional activity increased during the PHA synthesis phase) was constructed.

[0202] <Cultivation of mutant strains of R.eutropha and analysis of PHA biosynthesis>

[0203] The above-mentioned modified strain (R.eutrpha H16 CAR-GG-ΔB1-ldhAhadA) was added to the nitrogen source-limited inorganic salt medium with glucose as the single carbon source and 2HB-Na racemate (2HB) was added to try to culture it. The results confirmed that the addition of 2HB caused a strong growth and reproduction hindrance. The avoidance of this growth and reproduction hindrance was studied, and it was found that the addition of valine or pyruvate was effective. Therefore, when 2HB is added to the culture medium, valine or pyruvate, or both, is added for culture. In addition, the composition of the nitrogen source-limited inorganic salt medium is 100ml of water, Na2HPO4·12H2O 0.9g, KH2PO4 0.15g, NH4Cl 0.05g, MgSO4·7H2O 0.02g, and trace element solution 0.1ml. In addition, the final concentrations of other components added to the medium were glucose 2% (w / v), 2HB-Na 0.25% (w / v), pyruvic acid 0.8% (w / v), and valine 0.05%.

[0204] The culture was carried out on a 100 mL scale using a shaking flask, with shaking at 118 strokes / min at 30° C. After 120 hours, bacterial cells were collected from the culture solution by centrifugation and freeze-dried to obtain dry bacterial cells.

[0205] Regarding the amount and composition of PHA accumulated in the bacteria, the PHA contained in the dried bacteria was subjected to methanol decomposition treatment and then analyzed by gas chromatography (GC). This method is the most common method used for the analysis of PHA (refer to PNAS November 11, 2008 vol.105 no.45 17323-17327). By heating the PHA solution in the presence of methanol under acidic conditions, the polymer can be completely decomposed into volatile monomer derivatives, and quantitative / qualitative analysis can be performed by GC. In addition, similar to the methods described in the above-mentioned <Analysis of P(3HB-b-(GL-ran-3HB))> and <Gel filtration chromatography (GPC) analysis of P(3HB-b-(GL-ran-3HB))>, purified PHA is obtained by chloroform extraction and methanol reprecipitation from the dried bacteria, and then used for analysis. 1 Structural analysis by H-NMR and molecular weight determination by GPC.

[0206] The H16 CAR-GG-ΔB1-ldhAhadA strain was cultured in a nitrogen-limited inorganic salt medium containing 2% (w / v) glucose, 0.25% (w / v) 2HB-Na, 0.8% (w / v) pyruvic acid, and 0.05% valine, and the dry bacterial cells were 2.24±0.00 g / L, and the PHA content was 0.42±0.00 g / L (PHA accumulation rate 18.92±0.20 wt%). In the PHA analysis of the dry bacterial cells, it was estimated that it was a copolymer composed of 3HB and 2HB (10.60±0.24 mol%).

[0207] In addition, the 1 The results of H-NMR analysis are as follows Figure 5D As shown in Figure 2, it is shown that the polymer contains a small amount of 3HV units in addition to 3HB and 2HB. Figure 5D As shown in "4.8 to 5.4 ppm", it is strongly suggested that it is a block copolymer having a homopolymer segment of 2HB.

[0208] Furthermore, the detailed composition was determined to be P[(3HB-co-0.5mol%3HV)-block-8.3mol%2HB] by comprehensive analysis of the GPC analysis results. In addition, the number average molecular weight (Mn) = 3.71×10 5 , weight average molecular weight (Mw) = 9.58 × 10 5 , polydispersity index (PDI) = 2.58. Furthermore, the average degree of polymerization can be estimated to be 4,311, of which the degree of polymerization of the 2HB segment is 358.

[0209] Industrial Availability

[0210] As described above, according to the present invention, a block copolymer having a homopolymer segment and a copolymer segment can be provided. The block copolymer has the advantages of each polymer segment as its composition, and can exert various physical properties. Therefore, it is useful in various industrial fields such as medical treatment. In addition, according to the present invention, a block copolymer can be provided by biosynthesis, thereby also contributing to the solution of environmental problems. Sequence Listing <110> Block copolymer and method for producing the same <120> National University Corporation Hokkaido University <130> IBPF21-507WO <150> JP2020-034109 <151> 2020-02-28 <160> 3 <170> PatentIn Version 3.5 <210> 1 <211> 517 <212> PRT <213> Megasphaera elsdenii <400> 1 Met Arg Lys Val Glu Ile Ile Thr Ala Glu Gln Ala Ala Gln Leu Val 1 5 10 15 Lys Asp Asn Asp Thr Ile Thr Ser Ser Ile Gly Phe Val Ser Ser Ala His 20 25 30 Pro Glu Ala Leu Thr Lys Ala Leu Glu Lys Arg Phe Leu Asp Thr Asn 35 40 45 Thr Pro Gln Asn Leu Thr Tyr Ile Tyr Ala Gly Ser Gln Gly Lys Arg 50 55 60 Asp Gly Arg Ala Ala Glu His Leu Ala His Thr Gly Leu Leu Lys Arg 65 70 75 80 Ala Ile Ile Gly His Trp Gln Thr Val Pro Ala Ile Gly Lys Leu Ala 85 90 95 Val Glu Asn Lys Ile Glu Ala Tyr Asn Phe Ser Gln Gly Thr Leu Val 100 105 110 His Trp Phe Arg Ala Leu Ala Gly His Lys Leu Gly Val Phe Thr Asp 115 120 125 Ile Gly Leu Glu Thr Phe Leu Asp Pro Arg Gln Leu Gly Gly Lys Leu 130 135 140 Asn Asp Val Thr Lys Glu Asp Leu Val Lys Leu Ile Glu Val Asp Gly 145 150 155 160 His Glu Gln Leu Phe Tyr Pro Thr Phe Pro Val Asn Val Ala Phe Leu 165 170 175 Arg Gly Thr Tyr Ala Asp Glu Ser Gly Asn Ile Thr Met Asp Glu Glu 180 185 190 Ile Gly Pro Phe Glu Ser Thr Ser Val Ala Gln Ala Val His Asn Cys 195 200 205 Gly Gly Lys Val Val Val Gln Val Lys Asp Val Val Ala His Gly Ser 210 215 220 Leu Asp Pro Arg Met Val Lys Ile Pro Gly Ile Tyr Val Asp Tyr Val 225 230 235 240 Val Val Ala Ala Pro Glu Asp His Gln Gln Thr Tyr Asp Cys Glu Tyr 245 250 255 Asp Pro Ser Leu Ser Gly Glu His Arg Ala Pro Glu Gly Ala Ala Asp 260 265 270 Ala Ala Leu Pro Met Ser Ala Lys Lys Ile Ile Gly Arg Arg Gly Ala 275 280 285 Leu Glu Leu Thr Glu Asn Ala Val Val Asn Leu Gly Val Gly Ala Pro 290 295 300 Glu Tyr Val Ala Ser Val Ala Gly Glu Glu Gly Ile Ala Asp Thr Ile 305 310 315 320 Thr Leu Thr Val Glu Gly Gly Ala Ile Gly Gly Val Pro Gln Gly Gly 325 330 335 Ala Arg Phe Gly Ser Ser Arg Asn Ala Asp Ala Ile Ile Asp His Thr 340 345 350 Tyr Gln Phe Asp Phe Tyr Asp Gly Gly Gly Leu Asp Ile Ala Tyr Leu 355 360 365 Gly Leu Ala Gln Cys Asp Gly Ser Gly Asn Ile Asn Val Ser Lys Phe 370 375 380 Gly Thr Asn Val Ala Gly Cys Gly Gly Phe Pro Asn Ile Ser Gln Gln 385 390 395 400 Thr Pro Asn Val Tyr Phe Cys Gly Thr Phe Thr Ala Gly Gly Leu Lys 405 410 415 Ile Ala Val Glu Asp Gly Lys Val Lys Ile Leu Gln Glu Gly Lys Ala 420 425 430 Lys Lys Phe Ile Lys Ala Val Asp Gln Ile Thr Phe Asn Gly Ser Tyr 435 440 445 Ala Ala Arg Asn Gly Lys His Val Leu Tyr Ile Thr Glu Arg Cys Val 450 455 460 Phe Glu Leu Thr Lys Glu Gly Leu Lys Leu Ile Glu Val Ala Pro Gly 465 470 475 480 Ile Asp Ile Glu Lys Asp Ile Leu Ala His Met Asp Phe Lys Pro Ile 485 490 495 Ile Asp Asn Pro Lys Leu Met Asp Ala Arg Leu Phe Gln Asp Gly Pro 500 505 510 Met Gly Leu Lys Lys 515 <210> 2 <211> 560 <212> PRT <213> Pseudomonas putida <400> 2 Met Leu Gln Thr Arg Ile Ile Lys Pro Ala Glu Gly Ala Tyr Ala Tyr 1 5 10 15 Pro Leu Leu Ile Lys Arg Leu Leu Met Ser Gly Ser Arg Tyr Glu Lys 20 25 30 Thr Arg Glu Ile Val Tyr Arg Asp Gln Met Arg Leu Thr Tyr Pro Gln 35 40 45 Leu Asn Glu Arg Ile Ala Arg Leu Ala Asn Val Leu Thr Glu Ala Gly 50 55 60 Val Lys Ala Gly Asp Thr Val Ala Val Met Asp Trp Asp Ser His Arg 65 70 75 80 Tyr Leu Glu Cys Met Phe Ala Ile Pro Met Ile Gly Ala Val Val His 85 90 95 Thr Ile Asn Val Arg Leu Ser Pro Glu Gln Ile Leu Tyr Thr Met Asn 100 105 110 His Ala Glu Asp Arg Val Val Leu Val Asn Ser Asp Phe Val Gly Leu 115 120 125 Tyr Gln Ala Ile Ala Gly Gln Leu Thr Thr Val Asp Lys Thr Leu Leu 130 135 140 Leu Thr Asp Gly Pro Asp Lys Thr Ala Glu Leu Pro Gly Leu Val Gly 145 150 155 160 Glu Tyr Glu Gln Leu Leu Ala Ala Ala Ser Pro Arg Tyr Asp Phe Pro 165 170 175 Asp Phe Asp Glu Asn Ser Val Ala Thr Thr Phe Tyr Thr Thr Gly Thr 180 185 190 Thr Gly Asn Pro Lys Gly Val Tyr Phe Ser His Arg Gln Leu Val Leu 195 200 205 His Thr Leu Ala Glu Ala Ser Val Thr Gly Ser Ile Asp Ser Val Arg 210 215 220 Leu Leu Gly Ser Asn Asp Val Tyr Met Pro Ile Thr Pro Met Phe His 225 230 235 240 Val His Ala Trp Gly Ile Pro Tyr Ala Ala Thr Met Leu Gly Met Lys 245 250 255 Gln Val Tyr Pro Gly Arg Tyr Glu Pro Asp Met Leu Val Lys Leu Trp 260 265 270 Arg Glu Glu Lys Val Thr Phe Ser His Cys Val Pro Thr Ile Leu Gln 275 280 285 Met Leu Leu Asn Cys Pro Asn Ala Gln Gly Gln Asp Phe Gly Gly Trp 290 295 300 Lys Ile Ile Ile Gly Gly Ser Ser Leu Asn Arg Ser Leu Tyr Gln Ala 305 310 315 320 Ala Leu Ala Arg Gly Ile Gln Leu Thr Ala Ala Tyr Gly Met Ser Glu 325 330 335 Thr Cys Pro Leu Ile Ser Ala Ala His Leu Asn Asp Glu Leu Gln Ala 340 345 350 Gly Ser Glu Asp Glu Arg Val Thr Tyr Arg Ile Lys Ala Gly Val Pro 355 360 365 Val Pro Leu Val Glu Ala Ala Ile Val Asp Gly Glu Gly Asn Phe Leu 370 375 380 Pro Ala Asp Gly Glu Thr Gln Gly Glu Leu Val Leu Arg Ala Pro Trp 385 390 395 400 Leu Thr Met Gly Tyr Phe Lys Glu Pro Glu Lys Ser Glu Glu Leu Trp 405 410 415 Gln Gly Gly Trp Leu His Thr Gly Asp Val Ala Thr Leu Asp Gly Met 420 425 430 Gly Tyr Ile Asp Ile Arg Asp Arg Ile Lys Asp Val Ile Lys Thr Gly 435 440 445 Gly Glu Trp Val Ser Ser Leu Asp Leu Glu Asp Leu Ile Ser Arg His 450 455 460 Pro Ala Val Arg Glu Val Ala Val Val Gly Val Ala Asp Pro Gln Trp 465 470 475 480 Gly Glu Arg Pro Phe Ala Leu Leu Val Ala Arg Asp Gly His Asp Ile 485 490 495 Asp Ala Lys Ala Leu Lys Glu His Leu Lys Pro Phe Val Glu Gln Gly 500 505 510 His Ile Asn Lys Trp Ala Ile Pro Ser Gln Ile Ala Leu Val Thr Glu 515 520 525 Ile Pro Lys Thr Ser Val Gly Lys Leu Asp Lys Lys Arg Ile Arg Gln 530 535 540 Asp Ile Val Gln Trp Gln Ala Ser Asn Ser Ala Phe Leu Ser Thr Leu 545 550 555 560 <210> 3 <211> 585 <212> PRT <213> artificial sequence <220> <223> PhaCAR <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 Asn 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

Claims

1. A copolymer comprising a hydroxycarboxylic acid (A) having a hydroxyl group only at the 2-position and a hydroxycarboxylic acid (B) having a hydroxyl group at a position other than the 2-position, and having a homopolymer segment and a copolymer segment, The homopolymer segment is composed of one hydroxycarboxylic acid selected from the group consisting of hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B), The copolymer segment comprises at least two hydroxycarboxylic acids selected from the group consisting of hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B), The hydroxycarboxylic acid (A) is at least one hydroxycarboxylic acid selected from the group consisting of 2-hydroxybutyric acid, 2-hydroxyacetic acid, 2-hydroxypropionic acid, 2-hydroxyvaleric acid and 2-hydroxycaproic acid, The hydroxycarboxylic acid (B) is at least one hydroxycarboxylic acid selected from 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxypropionic acid, 3-hydroxyvaleric acid, 4-hydroxy-2-methylbutyric acid, 4-hydroxybutyric acid, 2,4-dihydroxybutyric acid, 5-hydroxyvaleric acid and 6-hydroxyhexanoic acid, The number average molecular weight and / or weight average molecular weight of the copolymer is 100,000 or more.

2. The copolymer according to claim 1, wherein the hydroxycarboxylic acid (A) is 2-hydroxybutyric acid, the hydroxycarboxylic acid (B) is 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, and The copolymer has a homopolymer segment composed of 2-hydroxybutyric acid and a copolymer segment containing 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

3. A copolymer comprising a hydroxycarboxylic acid (A) having a hydroxyl group only at the 2-position and a hydroxycarboxylic acid (B) having a hydroxyl group at a position other than the 2-position, and having a homopolymer segment and a copolymer segment, The homopolymer segment is composed of one hydroxycarboxylic acid selected from the group consisting of hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B), The copolymer segment comprises at least two hydroxycarboxylic acids selected from the group consisting of hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B), The combination of hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B) is 2-hydroxyacetic acid and 3-hydroxybutyric acid; 2-hydroxyacetic acid, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid; 2-hydroxybutyric acid, 3-hydroxybutyric acid and 3-hydroxypropionic acid; 2-hydroxybutyric acid, 3-hydroxybutyric acid and 4-hydroxy-2-methylbutyric acid; 2-hydroxybutyric acid, 3-hydroxybutyric acid and 2,4-dihydroxybutyric acid; 2-hydroxybutyric acid, 3-hydroxybutyric acid and 5-hydroxyvaleric acid; 2-hydroxybutyric acid, 3-hydroxybutyric acid and 6-hydroxyhexanoic acid; 2-hydroxybutyric acid, 3-hydroxypropionic acid and 4-hydroxy-2-methylbutyric acid; 2-hydroxybutyric acid, 3-hydroxypropionic acid and 2,4-dihydroxybutyric acid; 2-hydroxybutyric acid, 3-hydroxypropionic acid and 5-hydroxyvaleric acid; 2-hydroxybutyric acid, 3-hydroxypropionic acid and 6-hydroxyhexanoic acid; 2-hydroxybutyric acid, 3-hydroxybutyric acid and 3-hydroxyvaleric acid.

4. A copolymer comprising a hydroxycarboxylic acid (A) having a hydroxyl group only at the 2-position and a hydroxycarboxylic acid (B) having a hydroxyl group at a position other than the 2-position, and having a homopolymer segment and a copolymer segment, The homopolymer segment is composed of one hydroxycarboxylic acid selected from the group consisting of hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B), The copolymer segment comprises at least two hydroxycarboxylic acids selected from the group consisting of hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B), The hydroxycarboxylic acid (A) is 2-hydroxyacetic acid, the hydroxycarboxylic acid (B) is 3-hydroxybutyric acid, and The copolymer has a homopolymer segment consisting of 3-hydroxybutyric acid and a copolymer segment containing 2-hydroxyacetic acid and 3-hydroxybutyric acid.

5. A copolymer comprising a hydroxycarboxylic acid (A) having a hydroxyl group only at the 2-position and a hydroxycarboxylic acid (B) having a hydroxyl group at a position other than the 2-position, and having a homopolymer segment and a copolymer segment, The homopolymer segment is composed of one hydroxycarboxylic acid selected from the group consisting of hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B), The copolymer segment comprises at least two hydroxycarboxylic acids selected from the group consisting of hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B), The hydroxycarboxylic acid (A) is 2-hydroxyacetic acid, the hydroxycarboxylic acid (B) is 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, and, The copolymer has a homopolymer segment consisting of 3-hydroxybutyric acid and a copolymer segment containing 2-hydroxyacetic acid, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

6. A method for producing a copolymer according to any one of claims 1 to 5, comprising: a step of culturing a microorganism expressing CoA transferase and polymerase in a medium containing hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B); and a step of recovering the copolymer from the culture obtained by the step, The CoA transferase is a propionate CoA transferase (PCT) derived from Megasphaera elsdenii and an acyl-CoA synthetase (AlkK) derived from Pseudomonas putida. The polymerase is PhaC AR .

7. A method for producing a copolymer according to any one of claims 1 to 5, comprising: a step of culturing a microorganism expressing CoA transferase and polymerase in a medium containing a metabolic precursor of a hydroxycarboxylic acid (A) and a metabolic precursor of a hydroxycarboxylic acid (B); and a step of recovering the copolymer from the culture obtained by the step, The microorganism expresses ldhA, hadA and PhaC AR The glucose assimilation ability of Ralstonia eutropha was changed. The hydroxycarboxylic acid (A) is 2-hydroxybutyric acid, and the metabolic precursor of the hydroxycarboxylic acid (A) is at least one compound selected from the group consisting of glucose, threonine and 2-ketobutyric acid, The hydroxycarboxylic acid (B) is 3-hydroxyvaleric acid, and the metabolic precursor of the hydroxycarboxylic acid (B) is at least one compound selected from 2-hydroxybutyrate, 2-oxobutyrate, threonine, propionate, propionate-IP, propionate-CoA and 3-ketovaleryl-CoA, or, The hydroxycarboxylic acid (B) is 3-hydroxybutyric acid, and the metabolic precursor of the hydroxycarboxylic acid (B) is at least one compound selected from the group consisting of glucose, pyruvate, acetyl CoA and acetoacetyl CoA.

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