Block copolymer and method for producing same

Abstract

This copolymer contains a hydroxycarboxylic acid (A) having a hydroxy group only at the 2-position and a hydroxycarboxylic acid (B) having a hydroxy group at a position other than the 2-position, and has a homopolymerized segment composed of one hydroxycarboxylic acid selected from the group consisting of the hydroxycarboxylic acid (A) and the hydroxycarboxylic acid (B) and a copolymerized segment containing at least 2 hydroxycarboxylic acids selected from the group consisting of the hydroxycarboxylic acid (A) and the hydroxycarboxylic acid (B).

Description

Block copolymer and method for producing same

[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.

[0002] Copolymers are polymeric compounds synthesized by polymerizing two or more types of monomers. Compared to other materials such as metals, they have superior physical properties, such as elasticity, transparency, processability, and light weight. Therefore, they are widely used in various industrial fields, including medicine. Block copolymers, which consist of chains in which two or more polymer segments with different properties are covalently linked, are expected to be materials that combine the characteristics of each of the constituent segments.

[0003] Although such polymer compounds are usually synthesized using petroleum as a raw material, polymer compounds (so-called biopolymers) produced using renewable resources (biomass-derived raw materials) as carbon sources have been attracting attention in order to address the recent issues of depletion of fossil fuel resources and global warming.

[0004] For example, polyhydroxyalkanoates (PHAs) produced by microorganisms have attracted attention as biobased and biodegradable materials. Currently, commercially produced PHAs are copolymers of 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HHx). Furthermore, since the polymerization of unnatural lactic acid units was reported in 2008, PHAs containing various unnatural monomers have been reported (Non-Patent Document 1).

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

[0006] Thus, even though biosynthesis of block copolymers has become possible, there is a demand for further development of block copolymers in order to improve their physical properties.

[0007] Matsumoto et al. , 2013, Appl. Microbiol. Biotechnol. 97,8011 Matsumoto K et al. , Biomacromolecules, 19(2), 662, 2018

[0008] The present invention has been made in view of the problems of the prior art, and an object of the present invention is to provide a block copolymer having a homopolymer segment and a copolymer segment.

[0009] In order to further improve the block copolymer (P(2HB-b-3HB)) reported in Non-Patent Document 2, the present inventors cultured the microorganism in a medium supplemented with 3HHx in addition to the conventional 2HB and 3HB. As a result, they were able to obtain a block copolymer (P(2HB-b-(3HB-co-3HHx))) in which a homopolymerized segment consisting of 2HB is linked to a randomly polymerized segment consisting of 3HB and 3HHx. In other words, unlike the conventional block copolymers having only homopolymerized segments, they were able to biosynthesize a block copolymer also having randomly polymerized segments.

[0010] Furthermore, it was 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) was added to a medium containing 2HB and 3HB instead of 3HHx and the microorganism was cultured, block copolymers (P(2HB)-b-P(3HB-ran-3HP), P(2HB)-b-P(3HB-ran-4H2MB), P(2HB)-b-P(3HB-ran-24DHB), P(2HB)-b-P(3HB-ran-5HV), and P(2HB)-b-P(3HB-ran-6HHx)) could be synthesized in the same manner as when 3HHx was used.

[0011] Furthermore, when the microorganism was cultured in a medium containing 2-hydroxyacetic acid (glycolic acid, GL) and 3HB, a block copolymer (P(3HB-b-(GL-co-3HB))) in which a homopolymer segment consisting of 3HB was bonded to a copolymer segment consisting of GL and 3HB was obtained.

[0012] Furthermore, when the microorganism was cultured in a medium containing GL, 3HB, and 3HHx, a block copolymer (P(3HB-b-(GL-co-3HB-co-3HHx))) in which a homopolymer segment consisting of 3HB was linked to a random copolymer segment consisting of GL, 3HB, and 3HHx was obtained.

[0013] In addition, ldhA (lactate dehydrogenase gene), hadA (CoA transferase gene) and PhaC ＡＲ It was revealed that when a glucose-utilizing modified strain of Ralstonia eutropha into which a polymerase gene (a hydroxylase gene) had been introduced was cultured in a medium containing glucose and 2HB, the microorganism biosynthesized 3HB and 3HV (3-hydroxyvaleric acid) and could also biosynthesize a block copolymer (P(2HB)-b-P(3HB-co-3HV)) from these hydroxycarboxylic acids. In other words, the block copolymer could be obtained by culturing the microorganism without adding to the medium a hydroxycarboxylic acid having a hydroxy group at a position other than the 2-position, such as 3HB or 3HV.

[0014] The present invention is based on the above synthesis examples, and more specifically provides the following: <1> A copolymer containing a hydroxycarboxylic acid (A) having a hydroxy group only at the 2-position and a hydroxycarboxylic acid (B) having a hydroxy group at a position other than the 2-position, the copolymer having a homopolymer segment containing one hydroxycarboxylic acid selected from the group consisting of the hydroxycarboxylic acid (A) and the hydroxycarboxylic acid (B), and a copolymer segment containing at least two hydroxycarboxylic acids selected from the group consisting of the hydroxycarboxylic acid (A) and the hydroxycarboxylic acid (B). <2> The copolymer according to <1>, wherein 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-hydroxypentanoic acid, 2-hydroxyhexanoic acid, and a medium-chain 2-hydroxyalkanoic acid, and the hydroxycarboxylic acid (B) is at least one hydroxycarboxylic acid selected from the group consisting of 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxypropionic acid, 3-hydroxypentanoic acid, a medium-chain 3-hydroxyalkanoic acid, 4-hydroxy-2-methylbutyric acid, 4-hydroxybutyric acid, 2,4-dihydroxybutyric acid, 5-hydroxypentanoic acid, and 6-hydroxyhexanoic acid. <3> The copolymer according to <1>, wherein the hydroxycarboxylic acid (A) is 2-hydroxybutyric acid and the hydroxycarboxylic acid (B) is 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, and the copolymer has a homopolymer segment comprising 2-hydroxybutyric acid and a copolymer segment comprising 3-hydroxybutyric acid and 3-hydroxyhexanoic acid. <4> The copolymer according to <1>, wherein the hydroxycarboxylic acid (A) is 2-hydroxyacetic acid, the hydroxycarboxylic acid (B) is 3-hydroxybutyric acid, and the copolymer has a homopolymer segment composed of 3-hydroxybutyric acid and a copolymer segment containing 2-hydroxyacetic acid and 3-hydroxybutyric acid.<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 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 the copolymer according to any of <1> to <5>, the method comprising the steps of culturing a microorganism that expresses a CoA transferase and a polymerase in a medium containing the hydroxycarboxylic acid (A) and / or a metabolic precursor thereof and the hydroxycarboxylic acid (B) and / or a metabolic precursor thereof, and recovering the copolymer from the culture obtained in the step.

[0015] According to the present invention, it is possible to provide a block copolymer having a homopolymer segment and a copolymer segment, and a method for producing the same.

[0016] According to the present invention, for example, a block copolymer (P(2HB-b-(3HB-co-3HHx))) in which a homopolymer consisting of 2HB is bonded to a random polymer consisting of 3HB and 3HHx can be provided. As shown in the examples below, this block copolymer has higher extensibility than the copolymer P(2HB-b-3HB) disclosed in Non-Patent Document 2. In particular, P(2HB-b-(3HB-co-3HHx)) has extremely high extensibility of more than 1200%.

[0017] Furthermore, a block copolymer (P(3HB-b-(GL-co-3HB))) in which a homopolymer of 3HB is bonded to a copolymer of GL and 3HB can be provided. Furthermore, a block copolymer (P(3HB-b-(GL-co-3HB-co-3HHx))) in which a homopolymer of 3HB is bonded to a copolymer of GL, 3HB, and 3HHx can be provided. As shown in the examples below, these block copolymers have improved non-enzymatic hydrolysis properties due to the introduction of GL, and therefore exhibit high biodegradability. Furthermore, the copolymer segment of P(3HB-b-(GL-co-3HB-co-3HHx)) has a gradient structure. Furthermore, the introduction of 3HHx significantly improves the flexibility of the copolymer.

[0018] 2HB, 3HB, and 3HHx were added, and the polymerase PhaC ＡＲ The copolymer (P(2HB-b-(3HB-co-3HHx))) obtained using １ 1 is a chart showing the results of H NMR spectrum analysis. ＡＲ The copolymer (P(2HB-b-(3HB-co-3HHx))) obtained using １３ 1 is a chart showing the results of C NMR spectrum analysis. 2 is a plot showing the stress-strain relationship for P(2HB-b-(3HB-co-3HHx)). 3HB and GL were charged, and the polymerizing enzyme PhaC1 Ｐｓ The copolymer obtained using STQK １ 1 is a chart showing the results of H NMR spectrum analysis. ＡＲ The copolymer (P(3HB-b-(GL-co-3HB))) obtained using １ 1 is a chart showing the results of H NMR spectrum analysis. 2 is a molecular weight distribution curve showing the results of gel filtration chromatography analysis of P(3HB-b-(GL-co-3HB)). 3 is a chart showing the results of H NMR spectrum analysis. 4 is a chart showing the results of H NMR spectrum analysis. １1 is a chart showing the results of H NMR spectrum analysis. The numbers in the figure correspond to those in Table 2. 2 is a graph showing the results of differential scanning calorimetry of P(3HB-b-(GL-co-3HB-co-3HHx)). 3 is a plot showing the stress-strain relationship for P(3HB-b-(GL-co-3HB-co-3HHx)). 4 is a block copolymer composed of a P(2HB) homopolymer segment and a random copolymer segment containing 3HB. １

[0023] Figure 1 is a chart showing the results of H NMR spectroscopy. Figure 2 is a graph showing the results of differential scanning calorimetry of P(2HB)-b-P(3HB-ran-5HV). Figure 3 is a schematic diagram showing the metabolic pathway for biosynthesis of P(2HB)-b-P(3HB-co-3HV). Figure 4 is a schematic diagram showing the replacement of phaCNSDG (a modified PHA synthase gene derived from Aeromonas caviae) with phaCAR (a chimeric PHA synthase gene derived from A. caviae and R. eutropha) by homologous recombination in the pha operon on the chromosome of the R. eutropha H16 NSDG-GG-ΔB1 strain. 1 is a schematic diagram showing that ldhA (lactate dehydrogenase gene) and hadA (CoA transferase gene) derived from Clostridium difficile were inserted downstream of phaP1 (PHA granule-associated protein gene). １ FIG. 1 is a chart showing the results of H NMR spectrum analysis.

[0019] <Block Copolymer> As will be described in the Examples below, the present invention provides a block copolymer having a homopolymer segment and a copolymer segment, and more specifically, the present invention provides a copolymer comprising a hydroxycarboxylic acid (A) having a hydroxy group only at the 2-position and a hydroxycarboxylic acid (B) having a hydroxy group at a position other than the 2-position, and having a homopolymer segment comprising one hydroxycarboxylic acid selected from the group consisting of hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B), and a copolymer segment comprising at least two hydroxycarboxylic acids selected from the group consisting of hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B).

[0020] The copolymer of the present invention is obtained by polymerizing hydroxycarboxylic acids through ester bonds and has at least two types of segments.

[0021] In the present invention, a "segment" refers to a structural unit constituting each block in a block copolymer, and is a unit (homopolymerized segment) composed of one type of monomer (hydroxycarboxylic acid, described below) or a unit (copolymerized segment) composed of multiple types of monomers. Examples of "copolymerized segments" include randomly polymerized segments in which multiple types of monomers are randomly polymerized, gradient polymerized segments in which the constituent monomer composition changes continuously, and alternating polymerized segments in which multiple types of monomers are alternately polymerized. Furthermore, the number of monomers constituting each segment in the copolymer of the present invention is not particularly limited, but when used as a material, it is preferably 100 or more, usually 500 or more, and more preferably 4000 or more.

[0022] The "hydroxycarboxylic acids" according to the present invention are divided into two types: monohydroxycarboxylic acids having a hydroxy group only at the 2-position, and hydroxycarboxylic acids having a hydroxy group at a position other than the 2-position. The latter may be hydroxycarboxylic acids having multiple hydroxy groups, and in such cases, may have a hydroxy group at the 2-position in addition to positions other than the 2-position.

[0023] Examples of hydroxycarboxylic acids having a hydroxy group only at the 2-position (hereinafter also referred to as "hydroxycarboxylic acid (A)") include 2-hydroxyacetic acid (glycolic acid, GL), 2-hydroxybutyric acid (2HB), 2-hydroxypropionic acid (lactic acid, 2HP), 2-hydroxypentanoic acid (2-hydroxyvaleric acid), 2-hydroxyhexanoic acid, and medium-chain 2-hydroxyalkanoic acids. More specifically, medium-chain 2-hydroxyalkanoic acids include 2-hydroxyalkanoic acids derived from amino acids (derived from the side chain structure of an amino acid). (See Sudo M. et al., J. Biosci. Bioeng. 2019 Oct. 18. pii: S1389-1723(19)30785-6.) The hydroxycarboxylic acid (A) according to the present invention refers not only to one monohydroxycarboxylic acid having a hydroxy group only at the 2-position, but also to multiple monohydroxycarboxylic acids having a hydroxy group only at the 2-position. Among these, 2-hydroxyacetic acid is preferred because its incorporation into copolymers improves non-enzymatic hydrolysis and thus facilitates high biodegradability. 2-Hydroxybutyric acid is also preferred because it imparts transparency to the material, provides an isotactic polymer with a three-dimensional structure similar to that of poly-D-lactic acid (PDLA), and facilitates biosynthesis due to the polymer's lower glass transition temperature than other 2-hydroxycarboxylic acids.

[0024] Examples of hydroxycarboxylic acids having a hydroxy group at a position other than the 2-position (hereinafter also referred to as "hydroxycarboxylic acid (B)") include 3-hydroxypropionic acid (3HP), 3-hydroxybutyric acid (3HB), 3-hydroxyhexanoic acid (3HHx), 3-hydroxypentanoic acid (3-hydroxyvaleric acid, 3HV), medium-chain 3-hydroxyalkanoic acid, 4-hydroxy-2-methylbutyric acid (4H2MB), 4-hydroxybutyric acid, 2,4-dihydroxybutyric acid (24DHB), 5-hydroxypentanoic acid (5-hydroxyvaleric acid, 5HV), and 6-hydroxyhexanoic acid (6HHx). Note that the hydroxycarboxylic acid (B) according to the present invention refers not only to one hydroxycarboxylic acid having a hydroxy group at a position other than the 2-position, but also to multiple hydroxycarboxylic acids having a hydroxy group at a position other than the 2-position. Among these, 3-hydroxybutyric acid is preferred from the viewpoint of being able to be synthesized most efficiently from various carbon sources and imparting strength to the material, and 3-hydroxyhexanoic acid is preferred from the viewpoint of improving flexibility when introduced into a copolymer.

[0025] In the present invention, the combination of these hydroxycarboxylic acids (A) and hydroxycarboxylic acids (B) is not particularly limited as long as it can form the above-mentioned two types of segments, 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 excluding 2-hydroxybutyric acid and 3-hydroxybutyric acid.

[0026] The molecular weight of the copolymer of the present invention formed from such a combination is not particularly limited, but from the viewpoint of obtaining sufficient mechanical strength and maintaining a sufficient molecular weight even after molecular weight reduction by melt molding, the number average molecular weight is preferably 10,000 or more, more preferably 100,000 or more, and even more preferably 1,000,000 or more. The upper limit is also not particularly limited, but from the viewpoint of production difficulties, it is usually 10,000,000 or less.

[0027] The copolymer of the present invention will be further described below with reference to specific examples of combinations.

[0028] The copolymer of the present invention containing 2-hydroxybutyric acid, 3-hydroxybutyric acid, and 3-hydroxyhexanoic acid has a homopolymer segment consisting of 2-hydroxybutyric acid and a copolymer segment containing 3-hydroxybutyric acid and 3-hydroxyhexanoic acid. The copolymer segments in the copolymer have a random copolymer structure.

[0029] The molecular weight (number average molecular weight) of such a copolymer is not particularly limited, but from the viewpoint of obtaining sufficient mechanical strength and maintaining a sufficient molecular weight even after the molecular weight is reduced by melt molding, it is preferably 100,000 or more, and more preferably 1,000,000 or more.

[0030] Furthermore, from the viewpoint of exhibiting sufficient flexibility and also exhibiting physical properties due to the block structure, the random copolymer segments of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid preferably account for 50 mol % or more, more preferably 70 mol % or more, of the entire polymer. From the viewpoint of exhibiting sufficient flexibility, the composition of this random segment is preferably 5 mol % or more, more preferably 20 mol % or more, of 3-hydroxyhexanoic acid. The composition of 2-hydroxybutyric acid is uniquely determined by determining the quantitative ratio between the homopolymerized segment and the copolymerized segment.

[0031] The copolymer of the present invention containing 2-hydroxyacetic acid and 3-hydroxybutyric acid has a homopolymer segment consisting of 3-hydroxybutyric acid and a copolymer segment containing 2-hydroxyacetic acid and 3-hydroxybutyric acid. The copolymer segment in the copolymer has a random copolymer structure.

[0032] The molecular weight (number average molecular weight) of such a copolymer is not particularly limited, but when used as a material, it is preferably 10,000 or more, and more preferably 1,000,000 or more, from the viewpoints of exhibiting mechanical strength and maintaining a sufficient molecular weight even after molecular weight reduction due to melt molding.

[0033] 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, but when used as a hard segment, the content of 2-hydroxyacetic acid is preferably 50 mol % or more, more preferably 70 mol % or more. On the other hand, when used as a soft segment, the content of 2-hydroxyacetic acid is preferably 50 mol % or less, more preferably 10 to 30 mol %.

[0034] The copolymer of the present invention containing 2-hydroxyacetic acid, 3-hydroxybutyric acid, and 3-hydroxyhexanoic acid has a homopolymer segment consisting of 3-hydroxybutyric acid and a copolymer segment containing 2-hydroxyacetic acid, 3-hydroxybutyric acid, and 3-hydroxyhexanoic acid. The copolymer segment in the copolymer has a gradient polymerization structure.

[0035] The molecular weight (number average molecular weight) of such a copolymer is not particularly limited to a lower limit, but when used as a material, from the viewpoint of exhibiting mechanical strength and maintaining a sufficient molecular weight even after molecular weight reduction due to melt molding, the molecular weight is preferably 10,000 or more, and more preferably 1,000,000 or more.

[0036] From the viewpoint of ensuring flexibility of the material, the content of 3-hydroxyhexanoic acid is preferably 10 mol% or more, more preferably 30 mol% or more, and the contents of 2-hydroxyacetic acid and 3-hydroxybutyric acid are both preferably 70 mol% or less, more preferably 50 mol% or less.

[0037] Similarly, from the viewpoint of imparting flexibility to the material, the content of the hard homopolymerized segment is preferably 50 mol % or less, and more preferably 30 mol % or less.

[0038] The copolymer of the present invention, which contains 2-hydroxybutyric acid, 3-hydroxybutyric acid, 3-hydroxypropionic acid, 4-hydroxy-2-methylbutyric acid, 2,4-dihydroxybutyric acid, 5-hydroxyvaleric acid, or 6-hydroxyhexanoic acid, has a homopolymer segment consisting of 2-hydroxybutyric acid and a copolymer segment containing 3-hydroxybutyric acid, 3-hydroxypropionic acid, etc. The copolymer segment in the copolymer has a random copolymer structure.

[0039] The molecular weight of such a copolymer is preferably a weight-average molecular weight of 100,000 or more, more preferably about 1,000,000 (for example, 500,000 to 2,000,000, or 800,000 to 1,500,000), in order to develop strength sufficient for processing into a material.

[0040] The monomer composition of the random segment can be adjusted as appropriate by those skilled in the art to obtain the desired physical properties, but when synthesizing a random segment containing 3-hydroxybutyric acid as the main component and including 3-hydroxypropionic acid, 4-hydroxy-2-methylbutyric acid, 5-hydroxyvaleric acid, or 6-hydroxyhexanoic acid, with the aim of softening the poly-3-hydroxybutyric acid skeleton, the content of components other than 3-hydroxybutyric acid is preferably 0 to 50 mol%, and more preferably about 5 to 20 mol% (e.g., 1 to 40 mol%, 3 to 30 mol%) to achieve appropriate flexibility and strength. When synthesizing a segment containing 2,4-dihydroxybutyric acid with the aim of hydrophilizing the polymer, the composition of 2,4-dihydroxybutyric acid is preferably about 5 to 20 mol% (e.g., 1 to 40 mol%, 3 to 30 mol%). When the objective is to produce a flexible segment using a unit other than 3-hydroxybutyric acid (3-hydroxypropionic acid, 4-hydroxy-2-methylbutyric acid, 5-hydroxyvaleric acid, or 6-hydroxyhexanoic acid) as the main component, it is preferable to adjust the proportion of components other than 3-hydroxybutyric acid to 50 to 100 mol %. In order to exhibit flexible physical properties, a high proportion of components other than 3-hydroxybutyric acid is preferred, but in order to achieve compatibility with polymer synthesis efficiency, a proportion of about 80 to 95 mol % (e.g., 60 to 99 mol %, 70 to 97 mol %) is preferred.

[0041] The copolymer of the present invention, which contains 2-hydroxybutyric acid, 3-hydroxypropionic acid, and 4-hydroxy-2-methylbutyric acid, 2,4-dihydroxybutyric acid, 5-hydroxyvaleric acid, or 6-hydroxyhexanoic acid, has a homopolymer segment consisting of 2-hydroxybutyric acid and a copolymer segment containing 3-hydroxypropionic acid, 4-hydroxy-2-methylbutyric acid, etc. The copolymer segment in the copolymer has a random copolymer structure.

[0042] In such copolymers, the ratio of 3-hydroxypropionic acid to other units in the segments can be any ratio, for example, 100% or 90 mol% of 3-hydroxypropionic acid. The molecular weight is preferably 100,000 or more in weight average molecular weight.

[0043] The copolymer of the present invention containing 2-hydroxybutyric acid, 3-hydroxybutyric acid, and 3-hydroxyvaleric acid has a homopolymer segment consisting of 2-hydroxybutyric acid and a copolymer segment containing 3-hydroxybutyric acid and 3-hydroxyvaleric acid. The copolymer segments in the copolymer have a random copolymer structure.

[0044] In such a copolymer, the copolymer segment preferably contains 10 to 30 mol % of 3-hydroxyvaleric acid, and the molecular weight is preferably 100,000 or more in weight average molecular weight.

[0045] <Method for Producing Block Copolymer> As shown in the Examples below, in a microorganism such as Escherichia coli, CoA is added to the above-mentioned hydroxycarboxylic acids by a CoA transferase, and these hydroxycarboxylic acids are further polymerized by a polymerase, thereby obtaining the above-mentioned copolymer of the present invention. Therefore, the present invention provides a method comprising: Step 1: culturing a microorganism expressing a CoA transferase and a polymerase in a medium containing a hydroxycarboxylic acid (A) and a hydroxycarboxylic acid (B), and Step 2: recovering the copolymer from the culture obtained in the above step.

[0046] (CoA transferase) In the present invention, the term "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 is an example.

[0047] Propionate CoA transferase (PCT) refers to an enzyme classified under enzyme code (EC): 2.8.3.1. The origin of the PCT of the present invention is not particularly limited as long as it retains its catalytic activity. However, from the viewpoint of ease of expression in a different host in E. 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 (a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1) is even more preferred.

[0048] Furthermore, mutations in nucleotide sequences can occur in nature, and the encoded amino acids can also change accordingly. Therefore, the PCT of the present invention also includes proteins (variants) consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in the wild-type amino acid sequence derived from the bacterium (e.g., the amino acid sequence set forth in SEQ ID NO: 1), as long as it has the catalytic activity.

[0049] In the PCT of the present invention, "multiple" means within 100 amino acids, preferably within 80 amino acids, more preferably within 50 amino acids, even more preferably within 30 amino acids, more preferably within 20 amino acids, even more preferably within 10 amino acids, and particularly preferably within several amino acids (for example, within 5 amino acids, within 3 amino acids, or within 2 amino acids).

[0050] Furthermore, with the current state of the art, those skilled in the art can use the gene (nucleotide sequence) information to identify homologous genes from the same or other bacteria. Methods for identifying homologous genes include, for example, hybridization techniques (Southern, E.M., J. Mol. Biol., 98:503, 1975) and polymerase chain reaction (PCR) techniques (Saiki, R.K., et al. Science, 230:1350-1354, 1985; Saiki, R.K. et al. Science, 239:487-491, 1988). To identify homologous genes, hybridization reactions are usually performed under stringent conditions. Examples of stringent hybridization conditions include 6 M urea, 0.4% SDS, and 0.5x SSC, or hybridization conditions of equivalent stringency. Higher stringency conditions, such as 6 M urea, 0.4% SDS, and 0.1x SSC, are expected to lead to the isolation of genes with higher homology.

[0051] Therefore, the PCT of the present invention also includes proteins encoded by DNA that hybridizes under stringent conditions with DNA consisting of a wild-type nucleotide acid sequence derived from the bacterium, as long as it has the catalytic activity.

[0052] Furthermore, the protein encoded by the identified homologous gene usually has high homology (high similarity), preferably high identity, with that of the bacterium. Here, "high" means at least 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and more preferably 85% or more (e.g., 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more).

[0053] Therefore, the PCT of the present invention also includes proteins consisting of an amino acid sequence having at least 50% homology (similarity) or identity with the wild-type amino acid sequence derived from the bacterium, as long as it has the catalytic activity.

[0054] Sequence homology can be determined using the BLAST program (Altschul et al. J. Mol. Biol., 215:403-410, 1990). This program is based on the BLAST algorithm by 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 using BLAST, the parameters are set to, for example, score = 50 and wordlength = 3. Furthermore, when analyzing amino acid sequences using the Gapped BLAST program, the method can be performed as described by 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. Specific techniques for these analysis methods are known.

[0055] Those skilled in the art can determine whether the above-mentioned PCT variants or homologues have the catalytic activity by measuring their catalytic activity using, for example, the method described in A. E. Hofmeister et al. (Eur. J. Biochem., Vol. 206, pp. 547-552).

[0056] In addition to the above-mentioned PCT, when a hydroxycarboxylic acid having a hydroxy group at the 3-position, such as 3HHx, is the target, acyl-CoA synthetase is used as a suitable example of the CoA transferase of the present invention.

[0057] Acyl-CoA synthetase (AlkK) refers to an enzyme classified under enzyme code (EC): 6.2.1.3. The AlkK of the present invention is not particularly limited in terms of origin, so long as it retains its catalytic activity. From the viewpoint of having sufficient activity on medium-chain 3-hydroxycarboxylic acids, however, AlkK derived from a bacterium belonging to the genus Pseudomonas is preferred, AlkK derived from Pseudomonas oleovorans or Pseudomonas putida is more preferred, AlkK derived from Pseudomonas putida is even more preferred, and AlkK derived from Pseudomonas putida KT2440 (a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2) is more preferred (see Wang et al. Appl Environ Microbiol. 2012 Jan; 78(2): 519-527).

[0058] Similarly to PCT, the AlkK of the present invention also includes proteins (variants) consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in the wild-type amino acid sequence derived from the bacterium (e.g., the amino acid sequence set forth in SEQ ID NO: 2), so long as the AlkK has the catalytic activity. In the AlkK of the present invention, "multiple" means 100 amino acids or less, preferably 80 amino acids or less, more preferably 50 amino acids or less, even more preferably 30 amino acids or less, more preferably 20 amino acids or less, even more preferably 10 amino acids or less, and particularly preferably several amino acids or less (e.g., 5 amino acids or less, 3 amino acids or less, 2 amino acids or less).

[0059] Furthermore, the AlkK of the present invention also includes proteins encoded by DNA that hybridizes under stringent conditions with DNA consisting of a wild-type nucleotide acid sequence derived from the bacterium, and proteins consisting of an amino acid sequence that has at least 50% homology (similarity) or identity to the wild-type amino acid sequence derived from the bacterium, so long as they have the above-mentioned catalytic activity.

[0060] Furthermore, whether or not such a variant or homologue of AlkK has the catalytic activity can be determined by those skilled in the art, for example, by specifically oxidizing acyl-CoA produced by the reaction of the enzyme with acyl-CoA oxidase, and detecting the hydrogen peroxide produced thereby by color development with 4-aminoantipyrine, 3-methyl-N-ethyl-N-(2-hydroxyethyl)aniline, N,N-dimethylaniline, N,N-diethylaniline, or phenol in the presence of peroxidase.

[0061] (Polymerization Enzyme) In the present invention, the term "polymerization enzyme" refers to any enzyme that catalyzes the reaction of polymerizing the hydroxycarboxylic acid to which CoA has been transferred to form the homopolymerized segment and copolymerized segment described above. Examples of the polymerization enzyme include a polymerization enzyme that can recognize (1) a hydroxycarboxylic acid having a hydroxy group only at the 2-position and (2) a hydroxycarboxylic acid that does not have a hydroxy group at the 2-position and control the polymerization reaction. Preferred examples include type 1 polyhydroxyalkane enzyme (PhaC) or a variant thereof having the catalytic activity, more preferred are type 1 PhaC derived from bacteria belonging to the genus Aeromonas or a variant thereof, and even more preferred are type 1 PhaC derived from Aeromonas caviae or a variant thereof. More preferably, it is a chimeric polymerase obtained by fusing the N-terminal portion of type 1 PhaC derived from a bacterium belonging to the genus Aeromonas with the C-terminal portion of type 1 PhaC derived from a bacterium belonging to the genus Ralstonia, more preferably a chimeric polymerase obtained by fusing the N-terminal portion of type 1 PhaC derived from Aeromonas caviae with the C-terminal portion of type 1 PhaC derived from Ralstonia eutropha, and even more preferably a chimeric polymerase (PhaC) obtained by fusing the N-terminal 26% portion of type 1 PhaC derived from Aeromonas caviae with the C-terminal 74% portion of type 1 PhaC derived from Ralstonia eutropha. ＡＲ, (a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3) (see Matsumoto K. et al., Biomacromolecules. 2009 Apr 13; 10(4): 682-685).

[0062] Similarly to the above-mentioned CoA transferases, the polymerases of the present invention also include proteins (variants) consisting of the amino acid sequence of the enzyme (e.g., the amino acid sequence of SEQ ID NO: 3) in which one or more amino acids have been substituted, deleted, added, and / or inserted, so long as they have the catalytic activity. In the CoA transferases of the present invention, "multiple" means within 100 amino acids, preferably within 80 amino acids, more preferably within 50 amino acids, even more preferably within 30 amino acids, more preferably within 20 amino acids, even more preferably within 10 amino acids, and particularly preferably within several amino acids (e.g., within 5 amino acids, within 3 amino acids, or within 2 amino acids).

[0063] Such modifications include, for example, PhaC ＡＲ In the formula (I), at least one amino acid substitution selected from the group consisting of F314X, I320X, G423X, V448X, and I505X (where "X" means an amino acid different from that before the substitution) is included. Such amino acid substitutions enhance the 3HHx polymerization ability, and therefore, when 3HHx is used as the hydroxycarboxylic acid (B), for example, it becomes possible to improve the 3HHx fraction in the copolymer of the present invention.

[0064] Furthermore, the polymerase of the present invention also includes proteins encoded by DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide acid sequence encoding the enzyme, and proteins consisting of an amino acid sequence that has at least 50% homology (similarity) or identity with the amino acid sequence of the enzyme, so long as they have the above-mentioned catalytic activity.

[0065] Furthermore, whether or not such modified or homologous polymerases have the catalytic activity can be determined by those skilled in the art, for example, by using the method described in Non-Patent Document 2.

[0066] (Microorganism) In the present invention, the copolymer of the present invention can be produced by culturing a microorganism that expresses the above-mentioned two enzymes. As such a microorganism, a microorganism that naturally expresses these enzymes can be used, but usually a transformant prepared by introducing DNA encoding the enzymes into a microorganism is used.

[0067] The "microorganism" according to the present invention is not particularly limited as long as it is a microorganism capable of functionally expressing polyhydroxyalkanoate (PHA) synthases, and examples thereof include Escherichia coli, Cupriavidus necator, coryneform bacteria, yeast, Ralstonia bacteria, and Pseudomonas bacteria. Among these microorganisms, from the viewpoint of polymer productivity and production stability, Escherichia coli and hydrogen bacteria are preferred, with Escherichia coli being more preferred. The strain of Escherichia coli is not particularly limited, but an example is E. coli JM109 strain, which enables more stable copolymer production. Furthermore, as will be shown in the Examples below, from the viewpoint of being able to biosynthesize the hydroxycarboxylic acid (B) (3HB, 3HV, 3HHx, etc.) according to the present invention, bacteria of the genus Ralstonia are preferred, Ralstonia eutropha is more preferred, a glucose-assimilating modified strain of Ralstonia eutropha (H16 NSDG-GG-ΔB1 strain) is even more preferred, and H16 CAR-GG-ΔB1 and H16 CAR-GG-ΔB1-ldhAhadA, which will be shown in the Examples below, are more preferred.

[0068] In order to express the two enzymes of the present invention in such a microorganism, the DNA is usually introduced in the form of being incorporated into a vector. Alternatively, it may be introduced into the chromosome by homologous recombination. Note that in order for the DNA (gene) introduced into the chromosome of the microorganism of the present invention to be properly transcribed and further translated into a protein having the desired activity, the DNA must be incorporated so that it is under the control of an appropriate promoter on the chromosome.

[0069] In the present invention, a "vector" refers to a self-replicating vector, i.e., a vector that exists as an extrachromosomal independent entity and whose replication does not depend on chromosomal replication, and can be constructed, for example, based on a plasmid. Alternatively, a vector may be one that, when introduced into a host cell, is integrated into the genome of the host cell and replicated together with the chromosome into which it has been integrated.

[0070] Examples of such vectors include plasmid DNA and phage DNA. Examples of vectors for introduction into E. coli include pBLuescript (pBLuescript KS+, pBLuescript SK+, pBLuescriptII KS+, pBLuescriptII KS-, etc.), plasmid DNA such as pBR322 and pUC18, and phage DNA such as EMBL3, M13, and λgtII. Examples of vectors for introduction into hydrogen bacteria include pCUP2 (see WO 2007 / 049716), pLA2917 (ATCC 37355) with an RK2 replication origin that can be replicated and maintained in a wide range of hosts, and pJRD215 (ATCC 37533) with 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. Examples of vectors for introduction into Ralstonia bacteria, Pseudomonas bacteria, and the like include the aforementioned pLA2917 (ATCC37355) and pJRD215 (ATCC37533). Furthermore, examples of vectors for homologous recombination include pK18mobsacB (Schafer et al., Gene 145, 69-73 (1994)) and pJQ200 (Quandt, J. and Hynes, M.P., Gene (1993) 17:15-21).

[0071] The DNA encoding each of the above enzymes can be inserted into a vector using known gene recombination techniques commonly used in the field of genetic engineering. For example, to insert the DNA into a vector, a method is employed in which purified DNA is first cleaved with an appropriate restriction enzyme, and then inserted into a restriction enzyme site or a multicloning site of an appropriate vector and ligated to the vector.

[0072] During recombination, it is preferable to link the DNA downstream of a promoter capable of regulating the transcription and translation of each protein from the DNA inserted into the vector. The promoter is not particularly limited as long as it can regulate gene transcription in the host, and can be obtained as a DNA sequence that controls the expression of a gene encoding a protein either homologous or heterologous to the host cell. For example, when Escherichia coli is used as the host, examples of the promoter include the lac promoter, trp promoter, PL promoter, PR promoter, and T7 promoter. The promoter region of the phb operon of Capriavidus necator can also be used. When hydrogen bacteria are used as the host, examples of the promoter include the phaC1 gene promoter and the phaP1 gene promoter derived from the bacterium. When coryneform bacteria are used as the host, examples of the promoter include a cell surface protein gene promoter derived from Corynebacterium glutamicum. When yeast is used as the host, examples of the promoter include the gal1 promoter and the gal10 promoter. When a Pseudomonas bacterium is used as the microorganism of the present invention, a region containing a promoter upstream of the phaC1Ps gene or upstream of the phbCABRe operon can be used.

[0073] The vector may optionally contain expression-controlling sequences other than the promoter, such as terminator sequences, enhancer sequences, splicing signal sequences, poly A addition signal sequences, and ribosome binding sequences (SD sequences), which can be used in the microorganism into which the DNA is to be introduced. The vector may also contain an expression-inducing sequence other than the promoter. Examples of such sequences include the lactose operon, which can induce expression of a downstream gene by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG).

[0074] The vector may contain a selection marker gene, if necessary. The selection marker gene may be appropriately selected depending on the method for selecting transformed microbial cells, and examples thereof include drug resistance genes such as ampicillin resistance gene, tetracycline resistance gene, neomycin resistance gene, kanamycin resistance gene, and chloramphenicol resistance gene, genes involved in the intracellular biosynthesis of nutrients such as amino acids and nucleic acids (genes that complement auxotrophy), genes related to chemiluminescence such as luciferase, and genes encoding fluorescent proteins such as GFP.

[0075] A vector may contain one type of DNA encoding each of the enzymes (CoA transferase, polymerase) of the present invention, or multiple types of DNA may be inserted into one vector. When multiple types of DNA are inserted into a single vector, it is preferable that these DNAs form an operon. Here, an "operon" is a nucleic acid sequence unit consisting of one or more genes transcribed under the control of the same promoter.

[0076] The DNA or a vector into which the DNA has been inserted is introduced into a microorganism using a known method, such as the calcium chloride method, heat shock method, calcium phosphate method, electroporation method, spheroplast method, lithium acetate method, conjugative transfer method, or a method using calcium ions.

[0077] (Culture Conditions for Culture Medium, etc.) 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).

[0078] The medium is not particularly limited as long as it supplies a carbon source at a sufficient concentration, but may also contain nutrient sources other than the carbon source, such as a nitrogen source, inorganic salts, and other organic nutrient sources.

[0079] Any carbon source can be used as long as it can be assimilated by the above-mentioned microorganisms, but preferred carbon sources include sugars such as glucose, fructose, and sucrose; oils and fats such as palm oil, palm kernel oil, corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil, and fractionated oils thereof; and fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, and myrinsic acid, and derivatives thereof.

[0080] 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, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride. 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.

[0081] Examples of the medium according to 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 or the like. Furthermore, when using natural PHA-producing bacteria (e.g., hydrogen bacteria, Pseudomonas putida) as a host, nitrogen source-limited inorganic salt medium (e.g., 9 g / L Na ２ HPO ４ , 1.5g / L KH ２ P.O. ４ , 0.5g / L NH ４ Cl, 0.2mg / L MgSO ４, trace element-containing), phosphorus source-limited inorganic salt medium (e.g., glucose 1 g / L, KNO ３ 0.5g / L, MgSO ４ (7H ２ 0) 0.2g / L, CaCl ２ 0.1g / L, NaCl 0.1g / L, and 8.0mg K ２ HPO ４ and 2.0 mg KH ２ P0 ４ and adjusting the pH to 7).

[0082] The hydroxycarboxylic acid added to the medium is as described above, and may be either the R-form or racemic, with the R-form being preferred. Furthermore, the hydroxycarboxylic acid may be added after adjusting (neutralizing, etc.) the pH to match the optimum pH of the medium, or may be added in the form of a salt. Examples of such salts include sodium salts, potassium salts, and ammonium salts. The amount of hydroxycarboxylic acid added to the medium may be any concentration suitable for copolymer synthesis, and may be adjusted appropriately to control the monomer composition during copolymerization, which is synthesized depending on the concentration; for example, the amount is 1 to 10 g / L.

[0083] Furthermore, as will be shown in the Examples below, the copolymer of the present invention can be produced by using metabolic precursors (e.g., metabolic starting materials) of hydroxycarboxylic acids (A) and (B) themselves in a culture medium, as long as the culture medium is a microorganism capable of biosynthesizing such hydroxycarboxylic acids.

[0084] Therefore, the present invention can also be described as a method comprising the steps of: culturing a microorganism expressing a CoA transferase and a 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 step 2: recovering the copolymer from the culture obtained in the step 1.

[0085] The metabolic precursor according to the present invention is not particularly limited as long as it can be ultimately converted into the hydroxycarboxylic acid according to the present invention through a metabolic pathway in a microorganism, and examples thereof include glucose, threonine, 2-ketobutyric acid, etc. when the hydroxycarboxylic acid (A) is 2HB. For example, examples of the metabolic precursor according to the present invention include glucose, pyruvic acid, acetyl-CoA, acetoacetyl-CoA, etc. when the hydroxycarboxylic acid (B) is 3HB. For example, examples of the metabolic precursor according to the present invention include 2HB, 2-oxobutyric acid, threonine, propionic acid, propionic acid-IP, propionic acid-CoA, 3-ketovaleryl-CoA, etc. when the hydroxycarboxylic acid (B) is 3HV (see FIG. 5A, etc.).

[0086] Conditions for culturing the above-mentioned microorganisms include the aforementioned medium and the culture temperature, which is not particularly limited as long as it is within a range in which the microorganisms used can grow and the enzyme activities can be exhibited, and is usually 25 to 37°C, preferably 28 to 32°C, and particularly preferably 30°C. Depending on the type of microorganism used, aerobic or anaerobic conditions can be appropriately selected, but aerobic conditions are preferred from the viewpoint of facilitating copolymer synthesis. The culture time can be appropriately adjusted depending on the amount of copolymer produced, etc., but is usually 12 hours to 1 week, preferably 1 to 3 days.

[0087] (Recovery of copolymer, etc.) In the present invention, the copolymer of the present invention is recovered from a culture obtained by culturing the above-mentioned microorganism. Note that the "culture" includes not only the grown microorganism obtained by culturing the microorganism in a medium, but also the medium containing secretory products, metabolic products, etc. of the microorganism, as well as dilutions and concentrates thereof.

[0088] The method for recovering the copolymer from such a culture is not particularly limited, and can be, for example, the following method. After the culture is completed, the microorganisms are separated from the medium by centrifugation or the like, washed with distilled water and methanol, and dried (for example, freeze-dried). The dried cells are heated using an organic solvent such as chloroform to extract the copolymer. Cell components are removed from the organic solvent solution containing the copolymer by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate the copolymer. The supernatant is then removed by filtration or centrifugation, and the copolymer is recovered by drying.

[0089] Furthermore, the average molecular weight, monomer composition, structure, etc. of the copolymer thus obtained can be confirmed using known analytical methods such as nuclear magnetic resonance (NMR), gel permeation chromatography, and gas chromatography, as shown in the examples described later.

[0090] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. The examples were carried out using the following methods.

[0091] (Example 1) <Method for preparing P(2HB-b-(3HB-ran-3HHx))> A pBluescript KS+ plasmid was inserted with the promoter region of the phb operon of C. nectar, PhaC ＡＲ A plasmid (pBSPreCARpctAlkK) was prepared by inserting the pBSP gene, the pct gene derived from Megasphaera elsdenii, and the AlkK gene derived from Pseudomonas putida KT2440 in this order. Ｒｅ This plasmid was prepared by inserting the AlkK gene downstream of the pct gene in phaC(AR)pct (see Non-Patent Document 2) using a conventional method. This plasmid was then introduced into Escherichia coli (JM109 strain) by the calcium chloride method to obtain a transformant.

[0092] 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.

[0093] The E. coli cells collected from the resulting culture medium were freeze-dried and then heat-treated with chloroform to recover the polymer (see the recovery method described in Taguchi, S. et al., Proc. Natl. Acad. Sci. U.S.A. 2008, 105(45), 17323-17327).

[0094] <Structural analysis of P(2HB-b-(3HB-ran-3HHx))> (R,S)-2HB-Na, (R,S)-3HB-Na, and (R,S)-3HHx-Na were each added to LB medium at a concentration of 2.5 g / mL, and the resulting polymer was dissolved in deuterated chloroform. １ The composition and molar fraction were calculated by H NMR measurement, and as shown in Figure 1A, only one signal at 5 ppm due to 2HB was observed, revealing the presence of P(2HB) segments.

[0095] In addition, the same sample was further １３ The result was C NMR measurement. As shown in Figure 1B, the chain strength between 3HHx units was weak, indicating that there were no P(3HHx) homopolymer segments, i.e., the structure containing 3HHx units was randomly arranged.

[0096] From the above, it was revealed that the polymer obtained above was a block copolymer composed of a P(2HB) homopolymer segment and a P(3HB-ran-3HHx) random segment.

[0097] <Analysis of Physical Properties of P(2HB-b-(3HB-ran-3HHx))> Approximately 100 mg of the polymer was dissolved in chloroform, and the solvent was evaporated in a glass Petri dish to produce a solvent cast film. Test pieces were cut out from the resulting circular film and subjected to tensile tests to calculate parameters such as breaking elongation (extensibility) and tensile strength.

[0098] As is clear from the results shown in Figure 1C, the terpolymer P(2HB-b-(3HB-ran-3HHx)) obtained this time exhibited higher elongation than the binary copolymer P(2HB-b-3HB) disclosed in Non-Patent Document 2. In particular, the terpolymer exhibited extremely high elongation of over 1200%. These results suggest that, according to the present invention, by controlling the monomer sequence and, in turn, the higher-order structure, it is possible to obtain a copolymer that exhibits elastomeric properties in which strain and stress are proportional.

[0099] Example 2 <Method for preparing P(3HB-b-(GL-ran-3HB))> In the same manner as in Example 1, pBSPreCARpctAlkK was introduced into Escherichia coli (JM109 strain) by the calcium chloride method to obtain a transformant.

[0100] Also, PhaC ＡＲ Instead of the plasmid DNA encoding PhaC1 Ｐｓ A transformant was prepared by introducing a plasmid DNA encoding STQK. This plasmid was prepared by inserting PhaC into pBSPreCARpctAlkK by a conventional method. ＡＲ Instead of the gene, PhaC1 Ｐｓ It was constructed by substituting the STQK gene.

[0101] These transformants were then 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 polymers were recovered from the resulting culture solution in the same manner as in Example 1.

[0102] <Analysis of P(3HB-b-(GL-ran-3HB))> After cultivation, the cells were freeze-dried and extracted with chloroform to extract the intracellular polymer. The chloroform solution from which the cells were filtered was concentrated, and then excess methanol was added to precipitate the polymer. The recovered polymer was dissolved in deuterated chloroform, １ H NMR measurements were performed. The results are shown in Figures 2A and 2B. These figures show the results of analyzing a polymer obtained using an LB medium containing 12 g / L of (R,S)-GL-Na and 5 g / L of (R,S)-3HB-Na. The monomer composition was calculated based on the peak intensities. The results are shown in Table 1.

[0103]

[0104] <Gel Permeation Chromatography (GPC) Analysis of P(3HB-b-(GL-ran-3HB))> After cultivation, the cells were freeze-dried and extracted with chloroform to extract the intracellular polymer. For analysis, the polymer was filtered using a PTFE membrane and subjected to GPC analysis using chloroform as the mobile phase. The results are shown in FIG. 2C. This figure shows the results of analyzing the polymer obtained using an LB medium containing 12 g / L of (R,S)-GL-Na and 5 g / L of (R,S)-3HB-Na.

[0105] 2A and 2B, the four peaks observed between 4.5 and 4.9 ppm are signals assigned to GL units, and the peak furthest downfield (left) corresponds to a GL unit sandwiched between 3HB units (3HB-GL-3HB). In the case of a random copolymer with a GL fraction of about 20 mol%, a strong 3HB-GL-3HB signal is expected to be observed.

[0106] From the above, PhaC1 Ｐｓ The polymer obtained using STQK was determined to be a random copolymer (P(GL-ran-3HB)) (see Figure 2A). ＡＲThe polymer obtained using (3HB-b-(GL-ran-3HB)) exhibited a strong GL-GL-GL signal, indicating that it contained a structure with a high GL density. Based on the relative intensity ratio of the four peaks, the GL fraction was estimated to be approximately 70 mol%, which differed significantly from the GL fraction of the entire polymer, approximately 20 mol%, demonstrating that this polymer possessed a random-block structure (P(3HB-b-(GL-ran-3HB))).

[0107] Furthermore, in the gel filtration chromatography analysis, when the polymer is a mixture of two or more types (for example, a copolymer with P(3HB)), multiple molecular weight distributions are observed. ＡＲ As shown in FIG. 2C, a single peak was observed in the polymer obtained using the method, which supports the idea that the polymer has an intramolecular sequence structure.

[0108] In this way, PhaC ＡＲ The polymer obtained using the method contains P(3HB) homopolymer segments and P(GL-ran-3HB) random copolymer segments, and has a sequence structure in which the composition varies greatly within the same molecular chain, which has never been seen in previous biosynthetic PHAs.

[0109] Such polymers have two main advantages. First, they can create structures in which multiple polymers with different physical properties are linked within the same molecule, making it possible to create new materials based on this structure. Second, they contain many GL unit chains, which improves non-enzymatic hydrolysis (see Matsumoto K. et al., ACS Biomaterials Science & Engineering, 3(12)3058, 2017).

[0110] Example 3 <Method for Preparing P(3HB-b-(GL-co-3HB-co-3HHx))> pBSPreCARpctAlkK was introduced into Escherichia coli (strain JM109) by the calcium chloride method to obtain a transformant. This transformant was cultured with shaking at 30°C for 48 hours in LB medium containing 2.5 to 5 g / L of (R,S)-GL-Na, 0 to 2.5 g / L of (R,S)-3HB-Na, and 1 g / L of (R,S)-3HHx-Na, and the polymer was recovered from the obtained culture solution by the same method as described in Example 1.

[0111] <Analysis of P(3HB-b-(GL-co-3HB-co-3HHx))> In the same manner as in the above <Analysis of P(3HB-b-(GL-ran-3HB))>, １ H NMR measurement was performed. The results are shown in Figure 3A. This figure shows the results of analyzing a polymer obtained using an LB medium containing 5 g / L of (R,S)-GL-Na, 2.5 g / L of (R,S)-3HB-Na, and 1 g / L of (R,S)-3HHx-Na, and the signal of the GL unit is enlarged. The monomer composition was calculated from the peak area. The results are shown in Table 2.

[0112]

[0113] <Differential Scanning Calorimetry (DSC) for P(3HB-b-(GL-co-3HB-co-3HHx))> A solvent cast film was prepared by dissolving the polymer in chloroform and volatilizing the solvent in a glass Petri dish. A test piece of several milligrams was cut out from the obtained film and sealed in an aluminum pan to prepare a sample. This sample was subjected to DSC analysis. In the DSC analysis, the temperature was raised from -50°C to 220°C, and the presence or absence of an endothermic peak associated with the melting of the crystals was determined. The results obtained are shown in Figure 3B.

[0114] As shown in Table 2, it has been demonstrated that the present invention makes it possible to synthesize a terpolymer containing GL, 3HB, and 3HHx. Furthermore, in the terpolymer obtained in this manner, the 3HHx content can be increased to 60 mol %, which is sufficient for the purpose of imparting flexibility to the polymer properties. Furthermore, the GL fraction can be increased to approximately 30 mol %, making it possible to achieve a GL unit fraction sufficient for the purpose of modifying the material properties.

[0115] Furthermore, as shown in Figure 3A, when the signal of the GL unit in the NMR analysis results was enlarged, the relative intensities of the four observed signals revealed that the obtained polymer has a gradient structure. In a gradient structure, the ends and the center of the molecule have different structures, resulting in a structure similar to a triblock.

[0116] Furthermore, as shown in Figure 3B, no crystalline melting peak was observed in the DSC analysis, indicating that the polymer has a structure with very low crystallinity.

[0117] From the above results, it was revealed that this polymer contains an internal structure with a high GL fraction, similar to P(3HB-b-(GL-ran-3HB)) shown in Example 2. On the other hand, it can be determined that the 3HB and 3HHx units are polymerized randomly with each other. Therefore, this polymer has a special structure (P(3HB-b-(GL-co-3HB-co-3HHx))) in which a GL-rich region is added to a conventional 3HB, 3HHx random copolymer.

[0118] <Analysis of Physical Properties of P(3HB-b-(GL-co-3HB-co-3HHx))> A solvent cast film was prepared by dissolving the polymer in chloroform and evaporating the solvent in a glass Petri dish. Test pieces were cut out from the obtained film and subjected to a tensile test. The obtained results are shown in FIG. 3C.

[0119] The tensile test results for P(3HB-b-(GL-co-3HB-co-3HHx)) shown in Figure 3C show a gentle slope, indicating that the polymer has soft properties. It can also be slightly stretched. Thus, it has become clear that the introduction of soft 3HHx units into this polymer significantly improves its flexibility.

[0120] Furthermore, the introduction of the GL unit into this polymer increases its non-enzymatic hydrolysis property, and it is expected to be highly degradable in vivo, making it useful for medical devices (e.g., sutures).

[0121] Example 4 Method for preparing a block copolymer composed of a P(2HB) homopolymer segment and a random copolymer segment mainly composed of 3-hydroxy acid, and analysis of the block copolymer As described in Example 1, when Escherichia coli JM109 strain into which the plasmid pBSPreCARpctAlkK has been introduced is cultured in a medium supplemented with 2HB, 3HB, and 3HHx, a block copolymer composed of a P(2HB) homopolymer segment and a P(3HB-ran-3HHx) random segment is obtained.

[0122] Therefore, in this example, in the culture system described in Example 1, LB medium was used to add 1.0 g / L each of the sodium salts 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), instead of 3HHx. Culture was performed (30°C, 48 hours with shaking) and, similarly to Example 1, it was confirmed whether copolymers containing each monomer unit were synthesized. The results obtained are shown in Figures 4A and 4B. In Figure 4A, similarly to the method described above in <Analysis of P(3HB-b-(GL-ran-3HB)>, １ 4B shows the results of H NMR measurement, and Fig. 4C shows the results of analysis similar to that described above in <Differential Scanning Calorimetry (DSC) for P(3HB-b-(GL-co-3HB-co-3HHx)>.

[0123] As shown in Figure 4A, the presence of P(2HB) homopolymer segments １ This was confirmed by H NMR measurement. More specifically, a resonance attributed to the methine group of 2HB was observed around 5.1 ppm in each polymer, indicating the presence of P(2HB) segments.

[0124] Furthermore, melting point measurements confirmed that the segment primarily composed of 3HB was a random copolymer. In the differential scanning calorimetry thermogram of P(3HB-co-5HV) shown in Figure 4B, the peak representing the melting point is lower than the melting point of P(3HB) (178°C). This confirms that 5HV is randomly copolymerized. Although not shown in the figure, similar thermograms were obtained for other copolymers.

[0125] These results confirmed that 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, the culture conditions described above yielded novel triblock copolymers, P(2HB)-b-P(3HB-ran-3HP), P(2HB)-b-P(3HB-ran-4H2MB), P(2HB)-b-P(3HB-ran-24DHB), P(2HB)-b-P(3HB-ran-5HV), and P(2HB)-b-P(3HB-ran-6HHx).

[0126] Furthermore, in the present invention, even if the precursor 3HB added to the medium is replaced with 3HP, block copolymers composed 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)-b-P(3HP-ran-4H2MB), P(2HB)-b-P(3HP-ran-24DHB), P(2HB)-b-P(3HP-ran-5HV), P(2HB)-b-P(3HP-ran-6HHx), etc. can also be obtained.

[0127] In the present invention, the synthesis of these polymers depends on the properties of the polymerase, and the monomer-donating enzyme is not selected as long as the CoA form of the corresponding monomer can be synthesized.

[0128] Example 5 Method for Preparing Block Copolymer (P(2HB)-b-P(3HB-co-3HV)) from Glucose and 2HB As shown in FIG. 5A, the present inventors suspected that a block copolymer (P(2HB)-b-P(3HB-co-3HV)) composed of a P(2HB) homopolymer segment and a random copolymer of segments composed of 3HB and 3HV could be obtained by culturing a microorganism equipped with the biosynthetic pathway shown below in the presence of glucose and 2HB.

[0129] <Biosynthetic pathway> 2HB is converted to 2HB-CoA in the presence of HadA (CoA transferase). In the glycolysis pathway, pyruvate is produced from glucose. Acetyl-CoA is further produced from pyruvate, which is converted to acetoacetyl-CoA in the presence of PhaA, and then 3HB-CoA is biosynthesized in the presence of PhaB (Journal of Biotechnology, 152 (2011), 144-146). 2HB is converted to 2-oxobutyrate in the presence of LdhA (lactate dehydrogenase). Propionic acid and 2-hydroxybutyrate are produced from threonine and 2-oxobutyrate (Letters in Applied Microbiology, 1997, 25, 371-374). Propionic acid is converted to propionate-CoA via propionate-IP. Propionate-CoA and acetyl-CoA are converted to 3-ketovaleryl-CoA in the presence of PhaA, and further converted to 3HV-CoA in the presence of PhaB (Journal of Biotechnology, 152 (2011), 144-146).

[0130] <Construction of a Ralstonia eutropha modified strain> As shown in Figure 5B, in the pha operon on the chromosome of the R. eutropha H16 NSDG-GG-ΔB1 strain (see Zhang et al., Microbial Cell Factories, volume 18, Article number: 147 (2019)), which is a glucose-utilizing modified strain of Ralstonia eutropha, phaCNSDG (a modified PHA synthase gene derived from Aeromonas caviae) was inserted into phaC. ＡＲ The gene was replaced by homologous recombination to form PhaC ＡＲ An expression strain, H16 CAR-GG-ΔB1, was constructed. Specifically, a plasmid pK18mobsacB derivative containing the target fragment was introduced into R. eutrupha via the conjugatively competent E. coli strain S17-1, and strains in which the phaCAR gene had been inserted at the desired site on the chromosomal DNA were selected using kanamycin resistance as an indicator. Subsequently, recombinant strains in which the vector portion had been removed were isolated by utilizing the lethality of sacB contained in pK18mobsacB in the presence of sucrose.

[0131] Furthermore, as shown in FIG. 5C , ldhA (lactate dehydrogenase gene) and hadA (CoA transferase gene) derived from Clostridium difficile were inserted downstream of phaP1 (PHA granule-associated protein gene) on the chromosome of this modified strain to construct H16 CAR-GG-ΔB1-ldhAhadA, which enables expression of these enzymes under the control of the phaP promoter (PphaP, transcriptional activity of which increases during PHA synthesis).

[0132] <Cultivation of R. eutropha modified strain and analysis of biosynthesized PHA> When an attempt was made to culture the above-mentioned modified strain (R. eutropha H16 CAR-GG-ΔB1-ldhAhadA) in a nitrogen source-limited mineral salt medium containing glucose as the sole carbon source and adding 2HB-Na racemate (2HB), strong growth inhibition was confirmed due to the addition of 2HB. Investigations were conducted to avoid this growth inhibition, and it was found that the addition of valine or pyruvic acid was effective. Therefore, when adding 2HB to the medium, valine, pyruvic acid, or both were added and the culture was carried out. The composition of the nitrogen source-limited mineral salt medium was as follows: ２ HPO ４ ・12H ２ O 0.9g, KH ２ P.O. ４ 0.15g NH ４ Cl 0.05g, MgSO ４ ・7H ２ The final concentrations of other components added to the medium were 2% (w / v) glucose, 0.25% (w / v) 2HB-Na, 0.8% (w / v) pyruvic acid, and 0.05% valine.

[0133] The culture was carried out on a 100 mL scale using a shake flask, and was cultured with shaking at 118 strokes / min at 30° C. After 120 hours, the cells were collected from the culture medium by centrifugation and freeze-dried to obtain dry cells.

[0134] The amount and composition of PHA accumulated in the cells were analyzed by gas chromatography (GC) after methanolysis of the PHA contained in the dried cells. This method is the most common technique used for PHA analysis (see PNAS November 11, 2008 vol. 105 no. 45 17323-17327). By heating a PHA solution under acidic conditions in the presence of methanol, the polymer is completely decomposed into volatile monomer derivatives, allowing quantitative and qualitative analysis by GC. Furthermore, purified PHA was obtained from the dried cells by chloroform extraction and methanol reprecipitation, similar to the methods described above in <Analysis of P(3HB-b-(GL-ran-3HB))> and <Gel Permeation Chromatography (GPC) Analysis of P(3HB-b-(GL-ran-3HB))>. １ The structure was analyzed by H-NMR and the molecular weight was measured by GPC.

[0135] The H16 CAR-GG-ΔB1-ldhAhadA strain was cultured in a nitrogen-limited mineral salt medium containing 2% (w / v) glucose, 0.25% (w / v) 2HB-Na, 0.8% (w / v) pyruvate, and 0.05% valine. The resulting dry cells yielded 2.24±0.00 g / L of PHA, with a PHA content of 0.42±0.00 g / L (PHA accumulation rate: 18.92±0.20 wt%). Analysis of the PHA content of the dry cells indicated that the PHA was a copolymer of 3HB and 2HB (10.60±0.24 mol%).

[0136] In addition, the above １ As a result of H-NMR analysis, as shown in Figure 5D, it was revealed that this polymer contains a small amount of 3HV units in addition to 3HB and 2HB. Furthermore, as shown in "4.8 to 5.4 ppm" in Figure 5D, it was strongly suggested that this is a block copolymer containing a 2HB homosegment.

[0137] Furthermore, by combining the results of GPC analysis, the detailed composition was determined to be P[(3HB-co-0.5 mol% 3HV)-block-8.3 mol% 2HB]. The number average molecular weight (Mn) was 3.71 × 10 ５ , weight average molecular weight (Mw)=9.58×10 ５The probable acidity index (PDI) was 2.58. Furthermore, the average degree of polymerization was estimated to be 4,311, of which the degree of polymerization of the 2HB segment was 358.

[0138] As described above, the present invention makes it possible to provide a block copolymer having a homopolymer segment and a copolymer segment. Such a block copolymer combines the characteristics of each of the constituent polymer segments and can exhibit a variety of physical properties. Therefore, it is useful in various industrial fields such as medicine. Furthermore, since the present invention can provide a block copolymer by biosynthesis, it can also contribute to solving environmental problems.

Claims

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

2. The copolymer according to claim 1, wherein 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-hydroxypentanoic acid, 2-hydroxyhexanoic acid, and medium-chain 2-hydroxyalkanoic acid, and the hydroxycarboxylic acid (B) is at least one hydroxycarboxylic acid selected from the group consisting of 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxypropionic acid, 3-hydroxypentanoic acid, medium-chain 3-hydroxyalkanoic acid, 4-hydroxy-2-methylbutyric acid, 4-hydroxybutyric acid, 2,4-dihydroxybutyric acid, 5-hydroxypentanoic acid, and 6-hydroxyhexanoic acid.

3. 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 consisting of 2-hydroxybutyric acid and a copolymer segment containing 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

4. The copolymer according to claim 1, wherein 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. The copolymer according to claim 1, wherein 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 the copolymer according to any one of claims 1 to 5, comprising the steps of: culturing a microorganism expressing a CoA transferase and a 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 recovering the copolymer from the culture obtained in the above step.