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

[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 technology]

[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. For this reason, they are widely used in various industrial fields, including medicine. In particular, block copolymers, which consist of chains in which two or more polymers (segments) with different properties are linked by covalent bonds, are expected to become 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 non-natural lactic acid units was reported in 2008, PHAs containing various non-natural 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 by culturing a microorganism expressing CoA transferase and polymerase in a medium containing 2-hydroxybutyrate (2HB) and 3HB, 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 (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. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Matsumoto et al.,2013,Appl.Microbiol.Biotechnol.97,8011 [Non-patent document 2] Matsumoto K et al., Biomacromolecules, 19(2), 662, 2018 Summary of the Invention [Problem to be solved by the invention]

[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. [Means for solving the problem]

[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 previous block copolymers that only have homopolymerized segments, they were able to biosynthesize a block copolymer that also has randomly polymerized segments.

[0010] Furthermore, 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) were added to a medium containing 2HB and 3HB instead of 3HHx, the microorganisms were cultured. It was also confirmed that 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)) could be synthesized, similar to the case 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))) was obtained, in which a homopolymer segment consisting of 3HB was linked to a copolymer segment consisting of GL and 3HB.

[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))) was obtained, in which a homopolymer segment consisting of 3HB was linked to a random copolymer segment consisting of GL, 3HB, and 3HHx.

[0013] In addition, ldhA (lactate dehydrogenase gene), hadA (CoA transferase gene) and PhaC AR When a glucose-utilizing Ralstonia eutropha strain carrying a polymerase gene was cultured in a medium containing glucose and 2HB, the microorganism biosynthesized 3HB and 3HV (3-hydroxyvaleric acid) and was also able to biosynthesize a block copolymer (P(2HB)-bP(3HB-co-3HV)) from these hydroxycarboxylic acids. Specifically, the block copolymer could be obtained by culturing the microorganism without adding to the medium hydroxycarboxylic acids that have a hydroxy group at a position other than the 2-position, such as 3HB and 3HV.

[0014] The present invention is based on the above synthesis examples, and more specifically provides the following. <1> It 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, a homopolymer segment consisting of one hydroxycarboxylic acid selected from the group consisting of hydroxycarboxylic acids (A) and hydroxycarboxylic acids (B); and a copolymer segment containing at least two hydroxycarboxylic acids selected from the group consisting of hydroxycarboxylic acid (A) and hydroxycarboxylic acid (B). <2> 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 acids; 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; <1> The copolymer described in <3> The hydroxycarboxylic acid (A) is 2-hydroxybutyric acid, the hydroxycarboxylic acid (B) is 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, and It has a homopolymer segment consisting of 2-hydroxybutyric acid and a copolymer segment containing 3-hydroxybutyric acid and 3-hydroxyhexanoic acid. <1> The copolymer described in <4> The hydroxycarboxylic acid (A) is 2-hydroxyacetic acid and 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. <1> The copolymer described in <5> 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. <1> The copolymer described in <6> <1> ~ <5> A method for producing the copolymer according to any one of the above, Cultivating 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 step. [Effects of the Invention]

[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, it is possible to provide 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. 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))) can be provided in which a homopolymer of 3HB is bonded to a copolymer of GL and 3HB. Furthermore, a block copolymer (P(3HB-b-(GL-co-3HB-co-3HHx))) can be provided in which a homopolymer of 3HB is bonded to a copolymer of GL, 3HB, and 3HHx. As shown in the Examples below, these block copolymers exhibit improved non-enzymatic hydrolysis due to the introduction of GL, thereby exhibiting 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. [Brief explanation of the drawings]

[0018] [Figure 1A] FIG. 1 is a chart showing the results of 1H NMR spectrum analysis of a copolymer (P(2HB-b-(3HB-co-3HHx))) obtained by charging 2HB, 3HB, and 3HHx and using the polymerizing enzyme PhaCAR. [Figure 1B] FIG. 1 is a chart showing the results of 13C NMR spectrum analysis of a copolymer (P(2HB-b-(3HB-co-3HHx))) obtained by charging 2HB, 3HB, and 3HHx and using the polymerizing enzyme PhaCAR. [Figure 1C] FIG. 1 is a plot showing stress-strain relationships for P(2HB-b-(3HB-co-3HHx)) and others. [Figure 2A] FIG. 1 is a chart showing the results of 1H NMR spectrum analysis of a copolymer obtained by charging 3HB and GL and using the polymerizing enzyme PhaC1PsSTQK. [Figure 2B] FIG. 1 is a chart showing the results of 1H NMR spectrum analysis of a copolymer (P(3HB-b-(GL-co-3HB))) obtained by charging 3HB and GL and using the polymerizing enzyme PhaCAR. [Figure 2C] 1 is a molecular weight distribution curve showing the results of gel filtration chromatography analysis of P(3HB-b-(GL-co-3HB)). [Figure 3A] 1 is a chart showing the results of 1H NMR spectrum analysis of P(3HB-b-(GL-co-3HB-co-3HHx)). The numbers in the chart correspond to those in Table 2. [Figure 3B] 1 is a graph showing the results of differential scanning calorimetry of P(3HB-b-(GL-co-3HB-co-3HHx)). [Figure 3C] FIG. 1 is a plot showing stress-strain relationships for P(3HB-b-(GL-co-3HB-co-3HHx)). [Figure 4A] FIG. 1 is a chart showing the results of 1H NMR spectrum analysis of a block copolymer composed of a P(2HB) homopolymer segment and a random copolymer segment containing 3HB. [Figure 4B] 1 is a graph showing the results of differential scanning calorimetry of P(2HB)-bP(3HB-ran-5HV). [Figure 5A] FIG. 1 is a schematic diagram showing the metabolic pathway for biosynthesis of P(2HB)-bP(3HB-co-3HV). [Figure 5B] FIG. 1 is a schematic diagram showing that phaCNSDG (a modified PHA synthase gene derived from Aeromonas caviae) was replaced by 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. [Figure 5C] FIG. 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). [Figure 5D] FIG. 1 is a chart showing the results of 1H NMR spectrum analysis of P(2HB)-bP(3HB-co-3HV). DETAILED DESCRIPTION OF THE INVENTION

[0019] <Block copolymer> As will be shown in the examples below, the present invention provides a block copolymer having a homopolymer segment and a copolymer segment, and more specifically, It 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, a homopolymer segment consisting of one hydroxycarboxylic acid selected from the group consisting of hydroxycarboxylic acids (A) and hydroxycarboxylic acids (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 of 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, in which case they 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 amino acids). (See Sudo M. et al., J. Biosci. Bioeng. 2019 Oct. 18, p.i.:S1389-1723(19)30785-6.) Furthermore, the hydroxycarboxylic acid (A) of the present invention refers not only to a single 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 provides transparency to the material, produces an isotactic polymer with a three-dimensional structure similar to poly-D-lactic acid (PDLA), and has a lower glass transition temperature than other 2-hydroxycarboxylic acids, facilitating biosynthesis.

[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 acids, 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 the most efficient synthetic candidate 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, and the copolymer segment in the copolymer has 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 molecular weight reduction due to melt molding, it is preferably 100,000 or more, more preferably 1,000,000 or more.

[0030] In order to achieve sufficient flexibility and to exhibit physical properties attributable 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. In order to achieve sufficient flexibility, the composition of this random segment preferably contains 3-hydroxyhexanoic acid at 5 mol % or more, more preferably 20 mol % or more. 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, and 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 viewpoint 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, 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, 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, and 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, it is preferably 10,000 or more, and more preferably 1,000,000 or more, from the viewpoint of exhibiting mechanical strength and maintaining a sufficient molecular weight even after molecular weight reduction due to melt molding.

[0036] In order to ensure the 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 each 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] Those skilled in the art can adjust the monomer composition of the random segments as needed to achieve the desired physical properties. However, when synthesizing random segments containing 3-hydroxybutyric acid as the main component and 3-hydroxypropionic acid, 4-hydroxy-2-methylbutyric acid, 5-hydroxyvaleric acid, or 6-hydroxyhexanoic acid to soften the poly-3-hydroxybutyric acid backbone, 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 adequate flexibility and strength. When synthesizing a segment containing 2,4-dihydroxybutyric acid to hydrophilize the polymer, the content 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 flexible segments using units 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 %. To achieve flexible physical properties, a high proportion of components other than 3-hydroxybutyric acid is preferred, but 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. Furthermore, the molecular weight is preferably a weight-average molecular weight of 100,000 or more.

[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, and the copolymer segment in the copolymer has 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 of producing block copolymer> As will be 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. 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); Step 2: recovering the copolymer from the culture obtained in the previous step. A method is provided.

[0046] (CoA transferase) In the present invention, the term "CoA transferase" refers to an enzyme that catalyzes the reaction in which CoA (coenzyme A) is transferred 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, bacteria belonging to the genus Megasphera are 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, 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 in the same or other bacteria. Methods for identifying homologous genes include, for example, hybridization techniques (Southern, EM, J. Mol. Biol., 98:503, 1975) and polymerase chain reaction (PCR) techniques (Saiki, RK, et al. Science, 230:1350-1354, 1985; Saiki, RK, et al. Science, 239:487-491, 1988). To identify homologous genes, hybridization reactions are typically 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. If more stringent conditions are used, for example, 6 M urea, 0.4% SDS, and 0.1×SSC, it is expected that genes with higher homology will be isolated.

[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 that has 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, for example, score = 50 and word length = 3. When analyzing an amino acid sequence using the Gapped BLAST program, the procedure can be as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When using 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 origin of the AlkK according to the present invention is not particularly limited as long as it retains its catalytic activity. However, from the viewpoint of having sufficient activity toward 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 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 even 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" refers to 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, within 2 amino acids).

[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 then detecting the resulting hydrogen peroxide by color development with 4-aminoantipyrine and 3-methyl-N-ethyl-N-(2-hydroxyethyl)aniline, N,N-dimethylaniline, N,N-diethylaniline, or phenol in the presence of peroxidase.

[0061] (polymerase) In the present invention, the term "polymerization enzyme" refers to any enzyme that catalyzes the polymerization of the aforementioned hydroxycarboxylic acid to which CoA has been transferred to form the aforementioned homopolymerized segment and copolymerized segment. Examples of such enzymes include polymerases that can recognize (1) a hydroxycarboxylic acid having a hydroxy group only at the 2-position and (2) a hydroxycarboxylic acid having no hydroxy group at the 2-position and control the polymerization reaction. Preferred examples include type 1 polyhydroxyalkane enzymes (PhaCs) or variants thereof that have the catalytic activity. More preferred examples include type 1 PhaCs derived from bacteria belonging to the genus Aeromonas or variants thereof, and even more preferred examples include type 1 PhaCs derived from Aeromonas caviae or variants 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 case of 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, within 2 amino acids).

[0063] Such modifications include, for example, PhaC AR In the above, at least one amino acid substitution selected from the group consisting of F314X, I320X, G423X, V448X, and I505X (where "X" represents 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 increase 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 to 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] (microorganisms) In the present invention, the copolymer of the present invention can be produced by culturing a microorganism that expresses the above-mentioned two enzymes. Although microorganisms that naturally express these enzymes can be used as such microorganisms, transformants prepared by introducing DNA encoding the enzymes into a microorganism are usually 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 viewpoints 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 the 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 acids (B) (3HB, 3HV, 3HHx, etc.) of 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 into a vector. Alternatively, it may be introduced into the chromosome by homologous recombination. 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 integrated 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 plasmid DNAs such as pBLuescript (pBLuescript KS+, pBLuescript SK+, pBLuescriptII KS+, pBLuescriptII KS-, etc.), pBR322, and pUC18, and phage DNAs 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, MP, 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, inserted into a restriction enzyme site or a multicloning site of an appropriate vector, and then ligated to the vector.

[0072] During recombination, it is preferable to ligate the DNA inserted into the vector downstream of a promoter capable of regulating the transcription and translation of each protein from the DNA. 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 genes encoding proteins either homologous or heterologous to the host cell. For example, when Escherichia coli is used as the host, the lac promoter, trp promoter, PL promoter, PR promoter, and T7 promoter are examples. The promoter region of the phb operon of Capriavidus necator can also be used. When hydrogen bacteria are used as the host, the phaC1 gene promoter and the phaP1 gene promoter derived from the bacterium are examples. When coryneform bacteria are used as the host, examples include cell surface protein gene promoters derived from Corynebacterium glutamicum. When yeast is used as the host, examples include the gal1 promoter and the gal10 promoter. When Pseudomonas bacteria are used as the microorganism of the present invention, regions containing promoters upstream of the phaC1Ps gene or the phbCABRe operon can be used.

[0073] The vector may optionally contain expression-regulating 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. Such a sequence includes 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 of the present invention (CoA transferase, polymerase), or multiple types of DNA may be inserted into a single 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 known methods, such as the calcium chloride method, heat shock method, calcium phosphate method, electroporation method, spheroplast method, lithium acetate method, conjugative transfer method, and calcium ion method.

[0077] (Culture conditions such as medium) In the present invention, the copolymer of the present invention can be produced by culturing the above-mentioned microorganism in a 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] An example of a medium according to the present invention for culturing Escherichia coli or the like is LB medium (containing 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L sodium chloride), but is not limited to this. Furthermore, when natural PHA-producing bacteria (e.g., hydrogen bacteria, Pseudomonas putida) are used as hosts, examples of suitable media include, but are not limited to, nitrogen-source-limited inorganic salts media (e.g., containing 9 g / L NaHPO, 1.5 g / L KHPO, 0.5 g / L NHCl, 0.2 mg / L MgSO, and trace elements) and phosphorus-source-limited inorganic salts media (e.g., containing 1 g / L glucose, 0.5 g / L KNO, 0.2 g / L MgSO(7H2O), 0.1 g / L CaCl, 0.1 g / L NaCl, to which 8.0 mg KHPO and 2.0 mg KHPO are added and the pH is adjusted to 7).

[0082] The hydroxycarboxylic acid added to the medium is as described above, and may be either the R-form or racemic, preferably the R-form. 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 microorganism is capable of biosynthesizing such hydroxycarboxylic acids.

[0084] Therefore, the present invention provides Cultivating 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; Step 2: recovering the copolymer from the culture obtained in the previous step. It can also be called a method.

[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, pyruvate, 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, for example, Figure 5A).

[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 copolymers, etc.) In the present invention, the copolymer of the present invention is recovered from a culture obtained by culturing the above-mentioned microorganism. The term "culture" includes not only the grown microorganism obtained by culturing the microorganism in a medium, but also the medium containing secreted 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 for example, it can be carried out by the following method. After completion of the culture, the microorganisms are separated from the culture medium by centrifugation or the like, and the microorganisms are washed with distilled water, methanol or the like and dried (for example, freeze-dried). The dried cells are heat-treated using an organic solvent such as chloroform to extract the copolymer. The 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. Further, the supernatant is removed by filtration or centrifugation and dried to recover the copolymer.

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

Examples

[0090] Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples. The examples were carried out using the following methods.

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

[0092] This transformant was cultured with shaking at 30 °C for 48 hours in an 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 Escherichia coli recovered from the obtained culture broth was freeze-dried and 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 added to an LB medium to a concentration of 2.5 g / mL each. After dissolving the obtained polymer in deuterated chloroform, 1 the composition and molar fraction were calculated by performing 1H NMR measurement. As a result, as shown in Fig. 1A, since only one signal at 5 ppm derived from 2HB was observed, it was revealed that a P(2HB) segment was present.

[0095] Furthermore, the same sample was further 13 subjected to 13C NMR measurement. As a result, as shown in Fig. 1B, since the chain intensity between 3HHx units was weak, it was found that a P(3HHx) homopolymer segment did not exist, that is, the structure containing 3HHx units was a random arrangement.

[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] <Physical property analysis of P(2HB-b-(3HB-ran-3HHx))> 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. Test pieces were cut out from the obtained circular film and subjected to a tensile test to calculate parameters such as elongation at break (stretchability) and tensile strength.

[0098] As is clear from the results shown in Figure 1C, the ternary copolymer P(2HB-b-(3HB-ran-3HHx)) obtained this time showed higher stretchability compared to the binary copolymer P(2HB-b-3HB) disclosed in Non-Patent Document 2. In particular, the ternary copolymer showed very high stretchability exceeding 1200%. From these results, according to the present invention, it is also suggested that by controlling the monomer sequence in this way and thus controlling the higher-order structure, a copolymer showing elastomeric properties in which strain and stress are proportional can be obtained.

[0099] (Example 2) <Preparation method of P(3HB-b-(GL-ran-3HB))> Similar to Example 1, pBSPreCARpctAlkK was introduced into Escherichia coli (JM109 strain) by the calcium chloride method to obtain a transformant.

[0100] Also, instead of the plasmid DNA encoding PhaC AR a transformant was prepared by introducing plasmid DNA encoding PhaC1 Ps STQK. This plasmid was prepared by replacing the PhaC AR gene with the PhaC1 Ps STQK gene in pBSPreCARpctAlkK by a conventional method.

[0101] Then, these transformants were cultured with shaking at 30 °C for 48 hours in an LB medium containing (R,S)-GL-Na 0 to 12 g / L and (R,S)-3HB-Na 5 g / L, and the polymer was recovered from the obtained culture solution in the same manner as the method described in Example 1.

[0102] <Analysis of P(3HB-b-(GL-ran-3HB))> After culturing, the cells were freeze-dried and the intracellular polymer was extracted by chloroform extraction. The chloroform solution from which the cells were filtered off was concentrated, and then excess methanol was added to precipitate the polymer. The recovered polymer was dissolved in deuterated chloroform and 1 1H NMR measurement was performed. The results obtained are shown in Figs. 2A and 2B. In these figures, 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 are shown. Also, the monomer composition was calculated based on the peak intensities. The results obtained are shown in Table 1.

[0103] [Table 1]

[0104] <Gel filtration chromatography (GPC) analysis of P(3HB-b-(GL-ran-3HB))> After culturing, the cells were freeze-dried and the intracellular polymer was extracted by chloroform extraction. Then, for the analysis, it was filtered using a PTFE membrane and subjected to GPC analysis with chloroform as the mobile phase. The results obtained are shown in Fig. 2C. In this figure, 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 are shown.

[0105] As a result of the above NMR analysis, in Figs. 2A and 2B, the four peaks observed at 4.5 to 4.9 ppm are signals attributed to the GL units, and the peak on the lowest magnetic field side (left side) corresponds to the GL unit (3HB-GL-3HB) sandwiched by 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 will be strongly observed.

[0106] From the above, PhaC1 PsThe polymer obtained using STQK was determined to be a random copolymer (P(GL-ran-3HB)) (see Figure 2A). On the other hand, PhaC AR For the polymer obtained using it, a strong signal of GL-GL-GL was observed, indicating that the structure contains a high GL density. From the relative intensity ratio of the four peaks, the GL fraction was estimated to be about 70 mol%, which is significantly different from the overall GL fraction of the polymer of about 20 mol%. Thus, it was revealed that this polymer has a random-block structure (P(3HB-b-(GL-ran-3HB))).

[0107] Also, in the above 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. However, for the polymer obtained using PhaC AR as shown in Figure 2C, a single-peaked peak was observed, supporting that the polymer has an array structure within the same molecule.

[0108] Thus, the polymer obtained using PhaC AR contains a P(3HB) homopolymer segment and a P(GL-ran-3HB) random copolymer segment, and has an array structure with a significantly different composition within the same molecular chain, which has no synthetic example in previous biosynthetic PHAs.

[0109] There are mainly two advantages to such polymers. The first is that a structure in which a plurality of polymers with different physical properties are linked within the same molecule can be created, enabling the creation of new materials due to that structure. The second is that since it contains many chains of GL units, its non-enzymatic hydrolyzability is improved (see Matsumoto K.et al., ACS Biomaterials Science & Engineering, 3(12)3058, 2017).

[0110] (Example 3) <Preparation method of P(3HB-b-(GL-co-3HB-co-3HHx))> pBSPreCARpctAlkK was introduced into Escherichia coli (JM109 strain) by the calcium chloride method to obtain a transformant. This transformant was cultured with shaking at 30 °C for 48 hours in an LB medium containing 2.5 - 5 g / L of (R,S)-GL-Na, 0 - 2.5 g / L of (R,S)-3HB-Na, and 1 g / L of (R,S)-3HHx-Na. From the resulting culture broth, a polymer was recovered 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 the method described in <Analysis of P(3HB-b-(GL-ran-3HB))> above, 1 1H NMR measurement was performed. The obtained results are shown in Fig. 3A. The drawing is the result 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. Also, the monomer composition was calculated from the peak area value. The obtained results are shown in Table 2.

[0112]

Table 2

[0113] <Differential Scanning Calorimetry (DSC) of P(3HB-b-(GL-co-3HB-co-3HHx))> The polymer was dissolved in chloroform, and a solvent cast film was prepared by volatilizing the solvent in a glass petri dish. A test piece of several milligrams was cut out from the obtained film and enclosed in an aluminum pan to obtain a sample. This sample was subjected to DSC analysis. The DSC analysis was performed with a temperature increase from -50 °C to 220 °C to judge the presence or absence of an endothermic peak accompanying the melting of crystals. The obtained results are shown in Fig. 3B.

[0114] As shown in Table 2, according to the present invention, it was revealed that it is possible to synthesize a ternary copolymer containing GL, 3HB, and 3HHx. Further, in the ternary copolymer thus obtained, 3HHx can be increased up to 60 mol%. This is a sufficient ratio for the purpose of imparting flexibility to the polymer physical properties. Also, the GL fraction can be increased up to about 30 mo%, and a sufficient GL unit fraction can be achieved for the purpose of modifying the material physical properties.

[0115] Also, as shown in Fig. 3A, in the above NMR analysis results, when the signal of the GL unit was enlarged, based on the relative intensities of the four observed signals, it became clear that the obtained polymer has a gradient structure. In a gradient structure, since both ends and the central part of the molecule have different structures, it takes a structure close to a triblock.

[0116] Furthermore, as shown in Fig. 3B, in the DSC analysis, no crystal melting peak was observed. From this, it is shown that this polymer takes a structure with a very low crystallinity.

[0117] From the above results, it became clear 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 judged 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] <Physical Property Analysis of <P(3HB-b-(GL-co-3HB-co-3HHx))> The polymer was dissolved in chloroform, and a solvent cast film was prepared by volatilizing 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] As shown in FIG. 3C, the tensile test results of P(3HB-b-(GL-co-3HB-co-3HHx)) show that the slope is gentle, indicating that it has soft physical properties. Also, it can be stretched slightly. Thus, it was revealed that the flexibility of this polymer is significantly improved by introducing the soft 3HHx unit.

[0120] In addition, this polymer has increased non-enzymatic hydrolyzability by introducing the GL unit, and for example, high degradability in vivo is expected, so it is useful in medical devices (such as sutures) and the like.

[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 acids, and analysis of the block copolymer> As described in Example 1, when the Escherichia coli JM109 strain introduced with the plasmid pBSPreCARpctAlkK 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, instead of 3HHx, 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) were each added at 1.0 g / L, and culture (shaking culture at 30 °C for 48 hours) was performed using LB medium. Similar to Example 1, it was confirmed whether copolymers containing each monomer unit were synthesized. The obtained results are shown in FIGS. 4A and 4B. In FIG. 4A, similar to the method described in <Analysis of P(3HB-b-(GL-ran-3HB))> above, 1The results of the \(^1H\) NMR measurement are shown. In Figure 4B, similar to the differential scanning calorimetry (DSC) for the above <P(3HB-b-(GL-co-3HB-co-3HHx))>, the analyzed results are shown.

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

[0124] Also, it was confirmed from the melting point measurement that the segment mainly composed of 3HB is a random copolymer. In the thermogram of the differential calorimetry measurement of P(3HB-co-5HV) illustrated in Figure 4B, the peak position representing the melting point is lower than that near the melting point of P(3HB) (178 °C). From this, it can be confirmed that 5HV is randomly copolymerized. Although not shown in the figure, similar thermograms have been obtained for other copolymers.

[0125] From the above results, it was confirmed that the obtained polymers incorporated the newly added monomer precursor as a random copolymer with 3HB units, and the 2HB component was polymerized as a P(2HB) homopolymer segment. More specifically, under the above culture conditions, it was confirmed that novel ternary 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), P(2HB)-b-P(3HB-ran-6HHx) were obtained.

[0126] Furthermore, in the present invention, even if the precursor 3HB added to the culture 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 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.

[0127] Furthermore, 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)-bP(3HB-co-3HV)) from glucose and 2HB> As shown in Figure 5A, the present inventors hypothesized that a microorganism equipped with the biosynthetic pathway shown below could be cultured in the presence of glucose and 2HB to produce a block copolymer (P(2HB)-bP(3HB-co-3HV)) composed of a P(2HB) homopolymer segment and a random copolymer of 3HB and 3HV segments.

[0129] <Biosynthetic pathway> 2HB is converted to 2HB-CoA in the presence of HadA (CoA transferase). In glycolysis, pyruvate is produced from glucose. Acetyl-CoA is then 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). Propionate and 2-hydroxybutyrate are produced from threonine and 2-oxobutyrate (Letters in Applied Microbiology, 1997, 25, 371-374). Propionate 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 to 3HV-CoA in the presence of PhaB (Journal of Biotechnology, 152 (2011), 144-146).

[0130] <Construction of a modified Ralstonia eutropha strain> As shown in Fig. 5B, in the pha operon on the chromosome of the R. eutropha glucose-assimilating modified strain R. eutrpha H16 NSDG-GG-ΔB1 (see Zhang et al., Microbial Cell Factories, volume 18, Article number: 147 (2019)), phaCNSDG (a modified PHA synthase gene derived from Aeromonas caviae) was replaced by homologous recombination with the phaC AR gene, and the PhaC AR expression strain H16 CAR-GG-ΔB1 was constructed. Specifically, a plasmid pK18mobsacB derivative containing the target fragment was introduced into R. eutropha via the conjugative Escherichia coli S17-1 strain with conjugative transfer ability, and a strain in which the phaCAR gene was inserted at the target position on the chromosomal DNA was selected using kanamycin resistance as an indicator. Subsequently, using the lethality of sacB contained in pK18mobsacB in the presence of sucrose, a recombinant strain with the vector part removed was isolated.

[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-binding protein gene) on the chromosome of this modified strain, and H16 CAR-GG-ΔB1-ldhAhadA was constructed, which enables the expression of these enzymes under the control of the phaP promoter (PphaP, transcriptional activity increases during the PHA synthesis phase).

[0132] <Culture of R. eutropha modified strain and analysis of biosynthetic PHA> When the above-mentioned modified strain (R. eutrpha H16 CAR-GG-ΔB1-ldhAhadA) was cultured by adding the racemic form of 2HB-Na (2HB) to a nitrogen-source-limited inorganic salt medium with glucose as the sole carbon source, strong growth inhibition was confirmed by the addition of 2HB. Examination was carried out on the avoidance of this growth inhibition, and it was found that the addition of valine or pyruvate was effective. Therefore, when 2HB was added to the medium, valine or pyruvate, or both, were added for culturing. The composition of the nitrogen-source-limited inorganic salt medium is 0.9 g of Na2HPO4·12H2O, 0.15 g of KH2PO4, 0.05 g of NH4Cl, 0.02 g of MgSO4·7H2O, and 0.1 ml of trace-element solution in 100 ml of water. The final concentrations of the other components added to the medium were 2% (w / v) glucose, 0.25% (w / v) 2HB-Na, 0.8% (w / v) pyruvate, and 0.05% valine.

[0133] The culture was carried out on a 100 mL scale using a shaking flask and shaken at 30 °C at 118 strokes / min. The cells were recovered from the culture broth after 120 hours by centrifugation and freeze-dried to obtain dry cells. A

[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 method used for the analysis of PHA (see 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 is completely decomposed into volatile monomer derivatives, enabling quantitative and qualitative analysis by GC. Also, similar to the methods described in the above <Analysis of P(3HB-b-(GL-ran-3HB))> and <Gel filtration chromatography (GPC) analysis of P(3HB-b-(GL-ran-3HB))>, purified PHA was obtained by chloroform extraction from dried cells and methanol reprecipitation, 1 structural analysis by 1H-NMR and molecular weight measurement by GPC were performed.

[0135] As a result of culturing the H16 CAR-GG-ΔB1-ldhAhadA strain in a nitrogen-source-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, 2.24 ± 0.00 g / L of dried cells were obtained, and the PHA amount was 0.42 ± 0.00 g / L (PHA accumulation rate 18.92 ± 0.20 wt%). In the PHA analysis of the dried cells, it was推测 to be a copolymer consisting of 3HB and 2HB (10.60 ± 0.24 mol%).

[0136] Also, the above 1 As a result of 1H-NMR analysis, as shown in Fig. 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 - 5.4 ppm" in Fig. 5D, it was strongly suggested that it is a block copolymer having a 2HB homo-segment.

[0137] Furthermore, integrating the analysis results by GPC, its detailed composition was determined to be P[(3HB-co-0.5 mol% 3HV)-block-8.3 mol% 2HB]. Also, the number-average molecular weight (Mn) = 3.71×10 5, weight average molecular weight (Mw)=9.58×10 5 The 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. [Industrial Applicability]

[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. The present invention comprises 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, a homopolymer segment consisting of one hydroxycarboxylic acid selected from the group consisting of hydroxycarboxylic acids (A) and hydroxycarboxylic acids (B); and a copolymer segment containing at least two hydroxycarboxylic acids selected from the group consisting of hydroxycarboxylic acids (A) and hydroxycarboxylic acids (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-hydroxypentanoic acid, 2-hydroxyhexanoic acid, and medium-chain 2-hydroxyalkanoic acids; 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; A copolymer having a number average molecular weight or weight average molecular weight of 100,000 or more and a number average molecular weight of 10,000,000 or less.

2. The combination of the hydroxycarboxylic acid (A) and the hydroxycarboxylic acid (B) is 2-hydroxybutyric 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.

3. The hydroxycarboxylic acid (A) is 2-hydroxybutyric acid, the hydroxycarboxylic acid (B) is 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, and 2. The copolymer according to claim 1, which has a homopolymerized segment consisting of 2-hydroxybutyric acid and a copolymerized segment containing 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

4. The hydroxycarboxylic acid (A) is 2-hydroxyacetic acid, the hydroxycarboxylic acid (B) is 3-hydroxybutyric acid, and 2. The copolymer according to claim 1, which has a homopolymerized segment consisting of 3-hydroxybutyric acid and a copolymerized segment containing 2-hydroxyacetic acid and 3-hydroxybutyric acid.

5. The hydroxycarboxylic acid (A) is 2-hydroxyacetic acid, the hydroxycarboxylic acid (B) is 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, and 2. The copolymer according to claim 1, which has a homopolymerized segment consisting of 3-hydroxybutyric acid and a copolymerized 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: Cultivating 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, 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 method, wherein the metabolic precursor of the hydroxycarboxylic acid (B) is at least one compound selected from the group consisting of 2-hydroxybutyric acid, 2-oxobutyric acid, threonine, propionic acid, propionate-IP, propionate-CoA, 3-ketovaleryl-CoA, glucose, pyruvate, acetyl-CoA, and acetoacetyl-CoA.

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