WO2017170423A1

WO2017170423A1 - Alcohol-terminated polyester and polyester production method

Info

Publication number: WO2017170423A1
Application number: PCT/JP2017/012431
Authority: WO
Inventors: 精一田口
Original assignee: 国立大学法人北海道大学; 株式会社Adeka
Priority date: 2016-03-29
Filing date: 2017-03-27
Publication date: 2017-10-05
Also published as: JP7149473B2; JPWO2017170423A1

Abstract

The objective of the present invention is to provide a polyester production method causing polyester to be secreted outside of a microorganism cell. This problem can be solved by this alcohol-terminated polyester having an alcohol residue of 300 molecular weight or lower at a terminal thereof. In addition, the problem can be solved by a polyester production method characterized by the culture of a microorganism capable of producing this polyester in a culture medium containing alcohol of 300 molecular weight or lower.

Description

Alcohol-terminated polyester and method for producing polyester

The present invention relates to an alcohol-terminated polyester and a method for producing the polyester. According to the present invention, polyester produced by microorganisms can be efficiently secreted into the medium.

In recent years, warming due to mass consumption of fossil fuels and future depletion of fossil fuels have been actively discussed, and materials that use renewable biomass to replace these have been developed. Above all, the development of polyesters using microorganisms has attracted attention because it has biodegradability that is easily decomposed and utilized by microorganisms in the environment in addition to its usefulness as a material.
It has long been known that certain microorganisms synthesize polyesters, and the first reported example is polyhydroxybutyrate (PHB) discovered in 1926 at the Pasteur Institute. Since then, over 150 types of constituent units of microbial polyester have been reported, including 3-hydroxybutyrate (3HB), and are collectively called PHA (polyhydroxyalkanoate).

However, many of the microorganisms that can produce polyesters accumulate product in the cells, and when recovering polyesters, it is necessary to crush the cells and to accumulate in the cells. Along with this, there have been cases where growth is hindered or there is an upper limit in the production amount of polyester, which has been an obstacle to mass production.
Various studies have been conducted so far to transfer useful substances produced in the cells outside the cells. When the target substance is a protein or the like, for example, Patent Document 1 discloses a method for producing a target protein outside the cell by using a microorganism transformed with an expression vector containing a gene encoding the OmpF protein of E. coli. Patent Document 2 discloses that the secretion of the target protein is enhanced in low-temperature culture by modifying the Gram-negative bacterium Type I secretion system used for secretion of hemolytic toxin α-hemolysin (HlyA) produced by uropathogenic E. coli. An efficient system is disclosed. However, the method of adding a signal sequence as in

Patent Documents

1 and 2 is difficult to apply to other than proteins.

On the other hand, as a method for transferring products other than proteins to the outside of the cell, a method for producing shikimic acid using a microorganism belonging to a specific genus Citrobacter (Patent Document 3), a microorganism subjected to artificial treatment, or the natural world Microorganism screening method for selecting a strain that grows a microorganism collected from a solid medium and forms a vesicle around the colony and / or a strain that causes turbidity in culture using a transparent liquid medium And a method for efficiently producing lipids containing unsaturated fatty acids using the microorganism (Patent Document 4). Furthermore, for polyesters, a gene encoding a protein having an activity of converting lactic acid into lactic acid CoA and a protein having an activity of synthesizing polyhydroxyalkanoic acid using hydroxyacyl CoA as a substrate for a host microorganism A method for producing an aliphatic polyester has been disclosed in which a recombinant microorganism obtained by introducing and is cultured and the aliphatic polyester is recovered from the medium (Patent Document 5).

However, the methods of

Patent Documents

3 and 4 utilize specific strains that are secreted outside the cells, and the products are greatly limited. Further, in the method of Patent Document 5, the amount of the lactic acid polymer obtained is about 200 mg per liter, and the production amount was not sufficient.

WO2003 / 016538 pamphlet WO2005 / 049823 pamphlet JP 2002-281993 A WO01 / 012780 pamphlet JP 2011-2000153 A

Therefore, an object of the present invention is to provide a method for producing polyester capable of secreting polyester outside the cells of microorganisms.

As a result of diligent research on a method for producing a polyester capable of secreting polyester outside the cells of a microorganism, the present inventor surprisingly added an alcohol having a molecular weight of 300 or less to a culture medium for culturing a microorganism. It has been found that the polyester produced in the cells of the microorganism is efficiently secreted into the medium.
The present invention is based on these findings.
Therefore, the present invention
[1] An alcohol-terminated polyester having an alcohol residue with a molecular weight of 300 or less at the end,
[2] The alcohol-terminated polyester according to [1], wherein the polyester is a polyester comprising α-hydroxy acid and / or β-hydroxy acid,
[3] The alcohol-terminated polyester according to [2], wherein the α-hydroxy acid and / or β-hydroxy acid is lactic acid and / or 3-hydroxybutanoic acid,
[4] The alcohol-terminated polyester according to any one of [1] to [3], wherein the average number of repeating units of the polyester is 2 to 12,
[5] The alcohol-terminated polyester according to [3] or [4], wherein the average lactic acid content is 70 mol% to 100 mol%,
[6] A method for producing polyester, comprising culturing a microorganism capable of producing polyester in a medium containing an alcohol having a molecular weight of 300 or less,
[7] The method for producing a polyester according to [6], wherein the polyester is a polyester comprising α-hydroxy acid and / or β-hydroxy acid,
[8] The method for producing a polyester according to [7], wherein the α-hydroxy acid and / or β-hydroxy acid is lactic acid and / or 3-hydroxybutanoic acid,
[9] The method for producing a polyester according to any one of [6] to [8], wherein a part or all of the polyester is secreted from a microorganism.
[10] The method for producing a polyester according to any one of [6] to [9], wherein the average number of repeating units of the polyester is 2 to 12, and [11] the polyester has a molecular weight of 300 or less at the end of the polyester. A process for producing a polyester according to any one of [6] to [10], comprising an alcohol-terminated polyester having an alcohol residue;
About.
In addition, this specification
[12] An alcohol-terminated polyester having an alcohol residue having a molecular weight of 300 or less at the end of a polyester composed of lactic acid, or a polyester composed of lactic acid and 3-hydroxybutanoic acid,
[13] The alcohol-terminated polyester according to [12], wherein the average number of repeating units of lactic acid and / or 3-hydroxybutanoic acid is 2 to 12,
[14] The alcohol-terminated polyester according to [12] or [13], wherein the average lactic acid content is 70 mol% to 100 mol%,
[15] A method for producing a polyester comprising culturing a polyester comprising lactic acid or a microorganism capable of producing a polyester comprising lactic acid and 3-hydroxybutanoic acid in a medium containing an alcohol having a molecular weight of 300 or less,
[16] The method for producing a polyester according to [15], wherein a part or all of the polyester is secreted from a microorganism.
[17] The method for producing a polyester according to [15] or [16], wherein the polyester has an average number of repeating units of lactic acid and / or 3-hydroxybutanoic acid of 2 to 12, and [18] the polyester is a polyester A process for producing a polyester according to any one of [15] to [17], comprising an alcohol-terminated polyester having an alcohol residue with a molecular weight of 300 or less at the end of
Is disclosed.

According to the polyester of the present invention, it can be efficiently secreted outside the cells of microorganisms.
Moreover, according to the manufacturing method of the polyester of this invention, the polyester containing lactic acid can be efficiently produced by microorganisms. Naturally, there are many L-form lactic acids, but when a specific polyhydroxyalkanoate synthase is used in the present invention, it is possible to secrete a polyester containing D-form lactic acid outside the cells, The obtained polyester is useful as a raw material for biodegradable materials.
The alcohol-terminated polyester obtained according to the present invention can be used as a raw material for food and cosmetics in addition to the alcohol-terminated polyester itself as a polymer raw material.
Further, when the alcohol-terminated polyester obtained by the present invention has a high lactic acid content, it can be suitably used as a lactide raw material for producing polylactic acid. When industrially producing high molecular weight polylactic acid, it is generally carried out by a ring-opening polymerization reaction with a cyclic dimer (lactide) of lactic acid. In general, in order to produce polylactic acid using D-lactic acid or L-lactic acid as a raw material, it is necessary to synthesize lactide once, so that there are problems that the process becomes complicated and costs increase.
By synthesizing lactide from an alcohol-terminated polyester having a high lactic acid content, it is possible to produce polylactic acid more simply and efficiently than in the past.

It is the figure which showed typically the production by the microorganisms of the alcohol termination | terminus polyester obtained by the manufacturing method of this invention. By culturing two types of E. coli (ST / QK (A) and ST / FS / QK (B)) having different mutant polyhydroxyalkanoic acid synthases in a medium supplemented with polyethylene glycol, lactic acid is contained in the medium. It is a graph which shows that the oligomer of the polyester containing was secreted. The black bar indicates Free LA (monomer lactic acid) in the culture supernatant, and the white bar indicates Total LA (monomer and oligomer lactic acid). By culturing two types of Escherichia coli (ST / QK (A) and ST / FS / QK (B)) having different mutant polyhydroxyalkanoate synthases in a medium supplemented with diethylene glycol, lactic acid is contained in the medium. It is a graph which shows that the oligomer of the polyester containing was secreted. The black bar indicates Free LA (monomer lactic acid) in the culture supernatant, and the white bar indicates Total LA (monomer and oligomer lactic acid). It is the graph which showed the lactic acid content rate of the oligomer of the polyester secreted extracellularly by the manufacturing method of this invention. It is the chart of NMR which showed that diethylene glycol was couple | bonded with the extracellular oligomer (A) obtained by the manufacturing method of this invention, the intracellular oligomer (B), and the intracellular polymer (C). It is the graph which implemented the manufacturing method of this invention using the gene disruption mutant of colon_bacillus | E._coli, and measured the quantity of the polyester inside and outside a cell. By culturing E. coli in a medium supplemented with polyethylene glycol (A) or diethylene glycol (B), the amount of lactic acid contained in the oligomer secreted outside the cell (the amount of lactic acid of the monomer was subtracted from the total amount of lactic acid) It is the graph which showed quantity. 3 is a graph showing the results of analyzing the molecular weight of the molecules in the supernatant of the medium obtained by the production method of the present invention by ESI-MS. It is a diagram showing the ^{^{1 H- 1 H COSY NMR (a}} ) and ^{^{1 H- 1 H DOSY NMR (b}} ) extracellular oligomer obtained in this invention. It is the figure which showed ¹ H NMR (a), ¹³ C NMR (b), and ¹ H- ¹³ C HMQC (c) of the extracellular oligomer obtained by the present invention. Is a diagram showing the ^{1 H} NMR of lactide synthesized from the extracellular oligomer obtained in this invention.

[1] Alcohol-terminated polyester The alcohol-terminated polyester of the present invention has an alcohol residue having a molecular weight of 300 or less at the terminal. That is, when the alcohol is a polyhydric alcohol, the alcohol-terminated polyester of the present invention comprises a polyester unit composed of a repeating unit of A in the following general formula (1) and a hydrogen atom, and an alcohol residue composed of B and a hydroxy group.

In the general formula (1), l is not particularly limited, but is 2 to 1000 in some embodiments, 2 to 100 in some embodiments, 2 to 50 in some embodiments, and 2 in some embodiments. ~ 20. For example, when the polyester part is a polyester composed of lactic acid or 3-hydroxybutanoic acid, or a polyester composed of lactic acid and 3-hydroxybutanoic acid, and diethylene glycol, for example, as an alcohol is bonded to the terminal of the polyester part, The alcohol-terminated polyester of the invention has the general formula (2)

It can be expressed as
In the present specification, “polyester” includes oligomers.

<Polyester part>
The repeating unit of the polyester portion of the present invention is not particularly limited as long as it can constitute the polyester, but is preferably a hydroxy acid, more preferably an α-hydroxy acid and / or a β-hydroxy acid. Examples of the hydroxy acid include 2-hydroxypropionic acid (lactic acid), 2-hydroxybutanoic acid, 2-hydroxypentanoic acid, 2-hydroxyhexanoic acid, 2-hydroxyheptanoic acid, 2-hydroxyoctanoic acid, and 2-hydroxynonanoic acid. Α-hydroxy acids such as 2-hydroxydecanoic acid, 3-hydroxypropionic acid, 3-hydroxybutanoic acid, 3-hydroxypentanoic acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3 Β-hydroxy acids such as hydroxynonanoic acid and 3-hydroxydecanoic acid, 4-hydroxybutanoic acid, 4-hydroxypentanoic acid, 4-hydroxyhexanoic acid, 4-hydroxyheptanoic acid, 4-hydroxyoctanoic acid, 4-hydroxy Nonanoic acid, 4-hydroxydecanoic acid, etc. be able to. The repeating unit of the polyester part may be one type of repeating unit or a combination of two or more types of repeating units. In this specification, the repeating unit is referred to as, for example, a hydroxy acid residue, but for convenience, the hydroxy acid residue may be referred to as a “hydroxy acid”.
The polyester portion of the alcohol-terminated polyester of the present invention comprises a repeating unit and a terminal hydroxy group to which no alcohol is bonded. In the polyester portion, for example, a hydroxy acid is polymerized by a condensation reaction.
As one embodiment of the polyester portion of the alcohol-terminated polyester of the present invention, there can be mentioned a terminal hydroxy group to which a lactic acid residue and an alcohol are not bonded. Further, as another embodiment of the polyester portion, there can be mentioned a hydroxy group at a terminal to which a lactic acid residue, a 3-hydroxybutanoic acid residue and an alcohol are not bonded. Furthermore, as another embodiment of the polyester portion, there can be mentioned a hydroxy group at a terminal to which a 3-hydroxybutanoic acid residue and an alcohol are not bonded. In the polyester portion of such an embodiment, lactic acid and lactic acid, lactic acid and 3-hydroxybutanoic acid, or 3-hydroxybutanoic acid and 3-hydroxybutanoic acid are polymerized by a condensation reaction.

The alcohol-terminated polyester of the present invention has the following formula (3)

The lactic acid represented by these can be included. More specifically, the alcohol-terminated polyester of the present invention is a lactic acid repeating unit (4).

The repeating unit is referred to as a lactic acid residue. However, in the present specification, the lactic acid residue is sometimes referred to as “lactic acid” for convenience.

The alcohol-terminated polyester of the present invention has the following formula (5):

The 3-hydroxybutanoic acid represented by these can be included. More specifically, the alcohol-terminated polyester of the present invention contains 3-hydroxybutanoic acid repeating units (6)

The repeating unit is referred to as a 3-hydroxybutanoic acid residue. However, in the present specification, the 3-hydroxybutanoic acid residue is sometimes referred to as “3-hydroxybutanoic acid” for convenience.

The number of repeating units of lactic acid and / or 3-hydroxybutanoic acid in the alcohol-terminated polyester of the present invention is not particularly limited, but is 2 to 1000 in some embodiments and 2 to 100 in some embodiments. In some embodiments, it is 2-50, in some embodiments 2-20, and in some embodiments 2-10. That is, “n + m” in the general formula (2) is not limited, but is 2 to 1000 in an embodiment, 2 to 100 in an embodiment, and 2 to 50 in an embodiment. Is from 2 to 20, and in some embodiments from 2 to 10. Further, m, which is the number of repeating units of 3-hydroxybutanoic acid, is not limited, but in some embodiments, 0 to 1000, in some embodiments, 0 to 100, and in some embodiments, 0 to 50, In some embodiments, 0 to 20, and in some embodiments 0 to 10, but when the alcohol-terminated polyester consists only of 3-hydroxybutanoic acid, the lower limit of m is 2. Further, n, which is the number of repeating units of lactic acid, is 0 to 1000 in an embodiment, 0 to 100 in an embodiment, 0 to 50 in an embodiment, and 0 to 20 in an embodiment. In this case, the lower limit of n is 2 when the alcohol-terminated polyester consists only of lactic acid.
Accordingly, the molecular weight of the alcohol-terminated polyester of the present invention is not particularly limited, but preferably the lower limit of the molecular weight is about 200 and the upper limit of the molecular weight is 100,000. Further, the upper limit of the more preferable molecular weight is 10,000, more preferably 5000, and most preferably 1000.

Further, the ratio of lactic acid to 3-hydroxybutanoic acid in the alcohol-terminated polyester of the present invention is not particularly limited.
For example, when the alcohol-terminated polyester of the present invention is a polyester composed of lactic acid, the ratio of lactic acid is 100 mol%. When the alcohol-terminated polyester is a polyester composed of 3-hydroxybutanoic acid, the ratio of 3-hydroxybutanoic acid is 100 mol%.
On the other hand, when the alcohol-terminated polyester of the present invention is a polyester comprising lactic acid and 3-hydroxybutanoic acid, the content of lactic acid is not particularly limited, but is preferably 70 mol% or more, more preferably 80 mol. % Or more, more preferably 90 mol% or more. Due to the high content of lactic acid, the alcohol-terminated polyester of the present invention can be suitably used as a raw material for biodegradable materials.
In the above general formula (2), the lactic acid residue and the 3-hydroxybutanoic acid residue are represented as being arranged in a block form. The sequence of these residues is a block form, a random form. Any combination of a block-like part and a random part may be used. That is, a 3-hydroxybutanoic acid residue may be present at the end of the polyester portion.

The lactic acid contained in the alcohol-terminated polyester may be D-form or L-form, but D-form is preferred. Natural lactic acid existing in nature has many L-forms. When a biodegradable material is produced, the use of racemic polylactic acid may make the biodegradable material brittle. Therefore, it is preferable to use L-form polylactic acid and D-form polylactic acid to be co-crystallized and to use a stereocomplex type polymer. As mentioned above, since natural D-form polylactic acid is difficult to obtain, D-form polylactic acid is useful as a raw material for biodegradable materials.

《Alcohol part》
In the alcohol moiety of the alcohol-terminated polyester of the present invention, an alcohol forms an ester bond with lactic acid by a condensation reaction, or an alcohol forms an ester bond with 3-hydroxybutanoic acid by a condensation reaction.
In the present specification, an alcohol-derived group bonded to lactic acid or 3-hydroxybutanoic acid is referred to as an alcohol residue. Specifically, the alcohol residue means a residue obtained by removing a hydroxy group from the alcohol. However, in the present specification, the alcohol residue is sometimes referred to as “alcohol” for convenience.

<< Alcohol with a molecular weight of 300 or less >>
The alcohol capable of generating an alcohol residue contained in the alcohol-terminated polyester of the present invention is an alcohol having a molecular weight of 300 or less. Moreover, as a minimum of molecular weight, Preferably it is 50 or more, More preferably, it is 65 or more. The alcohol having a molecular weight of 300 or less is not particularly limited as long as it has a hydroxy group and has a molecular weight of 300 or less, but is preferably a polyhydric alcohol or an alcohol having an ether bond, and more preferably. Is a polyhydric alcohol having an ether bond, more preferably a diol having an ether bond. Another preferred embodiment is an aliphatic alcohol having a monovalent to trivalent hydroxy group. Specific aliphatic alcohols include methanol, ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, cyclohexanol, 1-heptanol, 1-octanol, 1-nonanol, 1-decanol, glycidol , Methylcyclohexanol, 2-methyl-1-butanol, 3-methyl-2-butanol, 4-methyl-2-pentanol, isopropyl alcohol, 2-ethylbutanol, 2-ethylhexanol, 2-octanol, terpineol, dihydro Terpineol, 2-methoxyethanol, 2-ethoxyethanol, 2-n-butoxyethanol, carbitol, ethyl carbitol, n-butyl carbitol, diacetone alcohol, diethylene glycol, triethyleneglycol , Tetraethylene glycol, propylene glycol, trimethylene glycol, dipropylene glycol, tripropylene glycol, 1,2-butylene glycol, 1,3-butylene glycol, 1,4-butylene glycol, pentamethylene glycol, hexylene glycol, Alternatively, glycerin can be mentioned, and diethylene glycol, propanediol or butanediol is preferable. Furthermore, examples of the alcohol include polyether condensed with alcohol, and specific examples include polyethylene glycol or polypropylene glycol.

When the alcohol-terminated polyester of the present invention contains a monohydric alcohol such as methanol or ethanol, the alcohol-terminated polyester of the present invention is a polyester part composed of a repeating unit of A in the following general formula (7) and a hydrogen atom, and It consists of B alcohol residues.

The alcohol-terminated polyester of the present invention can be used as a polymer raw material, food, cosmetic raw material, etc., but can also be derivatized by further reacting with a terminal hydroxyl group. Examples of the group that reacts with the hydroxyl group of the polyester include an isocyanate group, a carboxyl group, a carboxylic acid halide group, and an epoxy group. Accordingly, examples of the low molecular weight compound having a group that reacts with the hydroxyl group of the polyester include isocyanate compounds, carboxylic acids, carboxylic acid halides, and epoxy compounds.

Examples of the monofunctional isocyanate compound include 2,6-diisopropylphenyl isocyanate. Polyfunctional isocyanate compounds include tolylene diisocyanate (TDI), diphenylmethane diisocyanate (MDI), xylylene diisocyanate (XDI), isophorone diisocyanate (IPDI), xylylene diisocyanate (XDI), isophorone diisocyanate (IPDI), methyl. Cyclohexyl diisocyanate (H6TDI), 4,4′-dicyclohexylmethane diisocyanate (H12MDI), 1,3-bis (isocyanatomethyl) cyclohexane (H6XDI), tetramethylxylylene diisocyanate (TMXDI), 2,2,4-trimethylhexa Methylene diisocyanate (TMHDI), hexamethylene diisocyanate (HDI), norbornene diisocyanate (NBDI) 2,4,6-triisopropylphenyl diisocyanate (TIDI), 1,12-diisocyanate dodecane (DDI), 2,4, -bis- (8-isocyanate octyl) -1,3-dioctylcyclobutane (OCDI), or n -Pentane-1,4-diisocyanate.

Monofunctional carboxylic acids include linear or branched saturated aliphatic monofunctional carboxylic acids such as acetic acid, propionic acid, butyric acid, and lauric acid, and linear or branched unsaturated groups such as acrylic acid and methacrylic acid. Mention may be made of aliphatic monofunctional carboxylic acids and aromatic monofunctional carboxylic acids such as benzoic acid and 2-phenoxybenzoic acid.
Examples of the bifunctional carboxylic acid (dicarboxylic acid) include aliphatic dicarboxylic acids such as succinic acid, adipic acid, azelaic acid, sebacic acid, dodecanedicarboxylic acid, and eicosane diacid, 1,2-cyclohexanedicarboxylic acid, 1,3- Cyclohexanedicarboxylic acid, 1,4-cyclohexanedicarboxylic acid, perhydronaphthalenedicarboxylic acid, alicyclic dicarboxylic acid such as dimer acid, fumaric acid, maleic acid, and unsaturated dicarboxylic acid such as itaconic acid, tetrahydrophthalic acid, and Mention may be made of unsaturated alicyclic dicarboxylic acids such as cyclobutene dicarboxylic acid, p-hydroxyethyloxybenzoic acid, and oxyacids such as ε-caprolactone. Further, trifunctional or higher functional carboxylic acids such as trimellitic acid and pyromellitic acid can be exemplified.

Monofunctional carboxylic acid halides include acetyl chloride, acetyl bromide, propanoyl chloride, propanoyl bromide, butanoyl chloride, butanoyl bromide, pentanoyl chloride, pentanoyl bromide, hexanoyl chloride, hexanoyl bromide, benzoyl chloride, and odor Benzoyl chloride.
Examples of polyfunctional carboxylic acid halides include dicarboxylic acid chlorides, dicarboxylic acid bromides, and dicarboxylic acid iodides. More specifically, polyfunctional carboxylic acid having two or more carboxylic acid halide groups. If it is a halide, there is no restriction in particular, for example, biphenyl dicarboxylic acid dichloride, biphenylene dicarboxylic acid dichloride, azobenzene dicarboxylic acid dichloride, terephthalic acid dichloride, isophthalic acid dichloride, naphthalene dicarboxylic acid dichloride, etc. , Aliphatic bifunctional carboxylic acid halides such as adipoyl dichloride, sebacoyl dichloride, cyclopentane dicarboxylic acid dichloride, cyclohexane dicarboxylic acid dichloride, tetrahydrofuran dicarbonate It can be mentioned alicyclic bifunctional carboxylic acid halides such as carbon acid dichloride. Furthermore, examples of the trifunctional carboxylic acid halide include trimesic acid trichloride, 1,3,5-cyclohexane tricarboxylic acid trichloride, and 1,2,4-cyclobutane tricarboxylic acid trichloride.

By using the isocyanate compound, carboxylic acid, or carboxylic acid halide, an alcohol-terminated polyester derivative can be obtained. In this specification, the isocyanate compound, carboxylic acid, or carboxylic acid halide-terminated polyester is obtained by replacing the alcohol with an isocyanate compound, carboxylic acid, or carboxylic acid halide. It can be carried out by replacing with an isocyanate compound, carboxylic acid, or carboxylic acid halide.
Examples of the epoxy compound include ethylene oxide, propylene oxide, and phenylene oxide.

The alcohol-terminated polyester of the present invention is not limited, but can be produced by, for example, microorganisms as described in the production method described later. As shown in FIG. 1, the alcohol-terminated polyester produced by a microorganism includes an extracellular alcohol-terminated polyester that is secreted outside the cells of the microorganism and an intracellular alcohol-terminated polyester that exists inside the cells of the microorganism.

The extracellular alcohol-terminated polyester can be recovered from the culture medium of the microorganism. Therefore, it is easy to collect and purify as compared with polyester accumulated in cells. The number of repeating units of the extracellular alcohol-terminated polyester is not particularly limited, but is 2 to 12 in some embodiments, 2 to 10 in some embodiments, and 2 to 8 in some embodiments. Therefore, when the extracellular alcohol-terminated polyester comprises 3-hydroxybutanoic acid and lactic acid, “n + m” in the general formula (2) is not limited, but is 2 to 12 in some embodiments, and in some embodiments, 2 to 10, and in some embodiments 2 to 8. That is, the extracellular alcohol-terminated polyester contains a large amount of polyester generally called an oligomer. In addition, when the extracellular alcohol-terminated polyester comprises 3-hydroxybutanoic acid, m in the general formula (2) is not limited, but is 2 to 12 in some embodiments and 2 to 10 in some embodiments In some embodiments, it is 2-8. In addition, when the extracellular alcohol-terminated polyester comprises lactic acid, n in the general formula (2) is not limited, but in some embodiments it is 2 to 12, in some embodiments 2 to 10, and in some embodiments 2-8.

The intracellular alcohol-terminated polyester has a larger number of repeating units than the extracellular alcohol-terminated polyester. Therefore, it is useful as a raw material for high molecular weight biodegradable materials. The number of repeating units of the intracellular alcohol-terminated polyester is not particularly limited, but is 2 to 1000 in some embodiments, 2 to 100 in some embodiments, and 2 to 50 in some embodiments. That is, when the intracellular alcohol-terminated polyester comprises 3-hydroxybutanoic acid and lactic acid, “n + m” in the general formula (2) is not limited, but is 2 to 1000 in some embodiments, and in some embodiments, 2 to 100, and in some embodiments 2 to 50. That is, intracellular alcohol-terminated polyesters include high molecular weight polymers and low and medium molecular weight oligomers. In addition, when the intracellular alcohol-terminated polyester comprises 3-hydroxybutanoic acid, m in the general formula (2) is not limited, but is 2 to 12 in some embodiments and 2 to 10 in some embodiments In some embodiments, it is 2-8. In addition, when the intracellular alcohol-terminated polyester comprises lactic acid, n in the general formula (2) is not limited, but in some embodiments it is 2 to 12, in some embodiments 2 to 10, and in some embodiments 2-8.

An alcohol-terminated polyester produced using Escherichia coli as a microorganism is represented by the following formula (8):

The D-form lactic acid residue represented by these can be included. As described above, since natural D-form polylactic acid is difficult to obtain, D-form polylactic acid produced using Escherichia coli as a host is useful as a raw material for biodegradable materials.

[2] Method for Producing Polyester The method for producing a polyester of the present invention is characterized in that a microorganism capable of producing a polyester is cultured in a medium containing an alcohol having a molecular weight of 300 or less. For example, a microorganism capable of producing a polyester composed of lactic acid, a microorganism capable of producing a polyester composed of 3-hydroxybutanoic acid, or a microorganism capable of producing a polyester composed of lactic acid and 3-hydroxybutanoic acid are mixed with an alcohol having a molecular weight of 300 or less. Polyester can be obtained by culturing in a medium containing the same.

《Microorganism》
The microorganism used in the production method of the present invention is not particularly limited as long as it has the ability to produce polyester. For example, a microorganism having a ability to produce polyester consisting of lactic acid, and a polyester producing ability consisting of 3-hydroxybutanoic acid. And microorganisms having the ability to produce polyester comprising lactic acid and 3-hydroxybutanoic acid. Specific examples include bacteria such as Ralstonia and Eutropha.
Moreover, a recombinant microorganism can also be used as a microorganism used for the production method of the present invention. For example, a microorganism that originally has a polyester-producing ability by transforming a microorganism that does not originally have a polyester-producing ability, or a recombinant microorganism that has a polyester-producing ability further enhanced by transforming a microorganism that has a polyester-producing ability. Can be used. By using these transformed recombinant microorganisms, the convenience of operation can be improved and the productivity can be improved.
Microorganisms that can be used as recombinant microorganisms are not particularly limited, but Escherichia coli is particularly preferable, and using tatB, tatE, and araf (deficient mutant) among E. coli can improve the amount of polyester produced per unit medium. It is further preferable in that it can be performed. In addition, pfla and dld deletion mutants (JWMB1 strain) are preferable for improving the polyester production and increasing the content of lactic acid in the oligomer.
As the recombinant microorganism, for example, at least one gene selected from the group consisting of a polyhydroxyalkanoate synthase gene, a propionyl CoA transferase gene, a β-ketothiolase gene, and an acetoacetyl CoA reductase gene is introduced into the microorganism. By expressing at least one of acid synthase, propionyl CoA transferase, β-ketothiolase, and acetoacetyl CoA reductase, it is possible to improve the ability to produce polyester, and efficiently secrete polyester outside the cell. be able to. In particular, by producing polyhydroxyalkanoic acid synthase, the ability to produce polyester can be imparted or efficiently improved. Moreover, the codon of the said gene may be converted according to the codon usage frequency of the host organism used for transformation.

The polyhydroxyalkanoate synthase, propionyl CoA transferase, β-ketothiolase, and acetoacetyl CoA reductase will be described.
(1) Polyhydroxyalkanoic acid synthase The polyhydroxyalkanoic acid synthase is a protein having catalytic activity for a reaction of synthesizing polyhydroxyalkanoic acid using hydroxyacyl CoA as a monomer. Polyhydroxyalkanoic acid synthase catalyzes a reaction for synthesizing polylactic acid using lactyl CoA as a monomer. Furthermore, polyhydroxyalkanoate synthase catalyzes a reaction for synthesizing a hydroxyalkanoic acid-lactic acid copolymer using hydroxyacyl CoA and lactyl CoA as monomers.
A preferred polyhydroxyalkanoate synthase gene to be introduced into the recombinant microorganism used in the present invention is derived from any of Ralstonia, Pseudomonas, Escherichia, and Megasfera Particularly preferred is derived from Pseudomonas sp. 61-3, the base sequence thereof is shown in SEQ ID NO: 1, and the amino acid sequence encoded by the base sequence is shown in SEQ ID NO: 2.
Furthermore, a gene encoding a mutant polyhydroxyalkanoate synthase can also be used as the polyhydroxyalkanoate synthase gene. As such a mutant, the 130th, 325th, 392rd, 477th and 481st amino acids of the amino acid sequence (SEQ ID NO: 2) encoded by the nucleotide sequence shown in SEQ ID NO: 1 are each independently substituted. Single mutation, double mutation in which any two amino acids are substituted, triple mutation in which any three amino acids are substituted, and quadruple mutation in which four amino acids are substituted. Preferably, a gene encoding a mutant having a double mutation in which the 325th Ser is replaced with Thr and the 481st Gln is replaced with Lys in the amino acid sequence of SEQ ID NO: 2, or 325 in the amino acid sequence of SEQ ID NO: 2 It is a gene encoding a mutant having a triple mutation in which the first Ser is replaced with Thr, the 392rd Phe is replaced with Ser, and the 481st Gln is replaced with Lys.

(2) Propionyl CoA transferase gene Propionyl CoA transferase is a protein having an activity of catalyzing a reaction in which CoA is transferred from an appropriate CoA substrate to propionic acid and / or lactic acid. Hereinafter, in this specification, propionyl CoA transferase is described as “PCT”.
Preferred PCT genes in the present invention are those derived from any of Ralstonia, Pseudomonas, Escherichia, and Megasfera. Particularly preferred is derived from Megaphaphaela elsdenii, the base sequence thereof is shown in SEQ ID NO: 3, and the amino acid sequence encoded by the base sequence is shown in SEQ ID NO: 4.

(3) β-ketothiolase gene β-ketothiolase is a protein that catalyzes a reaction in which two molecules of acetyl CoA are condensed to form acetoacetyl CoA. Hereinafter, β-ketothiolase is referred to as “βKT” in the present specification.
A preferred βKT gene in the present invention is derived from any of Ralstonia, Pseudomonas, Escherichia, and Megasfera. Particularly preferably, it is derived from Ralstonia eutropha, the base sequence thereof is shown in SEQ ID NO: 5, and the amino acid sequence encoded by the base sequence is shown in SEQ ID NO: 6.

(4) Acetoacetyl-CoA reductase gene Acetoacetyl-CoA reductase undergoes a reaction in which D (-)-β-hydroxybutyryl-CoA is formed by a reduction reaction that occurs in the presence of a coenzyme such as NADP of acetoacetyl-CoA. It is a protein that catalyzes. Hereinafter, acetoacetyl CoA reductase is referred to as “AACoA-R” in the present specification.
Preferred AACoA-R genes in the present invention are those derived from any of Ralstonia, Pseudomonas, Escherichia, and Megasfera. Particularly preferably, it is derived from Ralstonia eutropha, the base sequence thereof is shown in SEQ ID NO: 7, and the amino acid sequence encoded by the base sequence is shown in SEQ ID NO: 8.

The polyhydroxyalkanoate synthase gene, propionyl CoA transferase, β-ketothiolase, and acetoacetyl CoA reductase used in the present invention have the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, respectively. It may have 1 to several base deletions, substitutions, additions or insertions. Here, the term “several” means 1 to 40, preferably 1 to 20, more preferably 10 or less. The polyhydroxyalkanoate synthase gene, propionyl CoA transferase, β-ketothiolase, and acetoacetyl CoA reductase are bases complementary to the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, respectively. It may be a base sequence of DNA that can hybridize with DNA comprising a sequence under stringent conditions. However, the polyhydroxyalkanoic acid synthase gene is a gene encoding a protein having a catalytic activity for the reaction of synthesizing polyhydroxyalkanoic acid using hydroxyacyl CoA as a monomer, and propionyl CoA transferase gene is propionic acid and / or A gene encoding a protein having catalytic activity for the reaction of transferring CoA to lactic acid, and the β-ketothiolase gene has an activity of catalyzing a reaction in which two molecules of acetyl CoA are condensed to form acetoacetyl CoA It is a gene encoding a protein, and the acetoacetyl CoA reductase gene is a gene encoding a protein having catalytic activity for the reduction reaction of acetoacetyl CoA. The catalytic activity of the reaction in which CoA is transferred to propionic acid and / or lactic acid is, for example, A. E. It can be measured according to the method described in Hofmeister et al. (Eur. J. Biochem., 206, 547-552). The catalytic activity of the reaction in which acetoacetyl CoA is formed from the two molecules of acetyl CoA is measured, for example, by the method described in Slater et al. (J. Bacteriology, 1998, 180, 1979-1987). Can do. The catalytic activity of the reduction reaction of acetoacetyl CoA is, for example, G.M. W. It can be measured by the method described in Haywood et al. (FEMS Microbiology Letters, 1988, Vol. 52, pp. 259-264).

When a microorganism into which the polyhydroxyalkanoic acid synthase gene, propionyl CoA transferase gene, β-ketothiolase gene, and acetoacetyl CoA reductase gene are introduced is used, a polyester having a particularly high lactic acid content (lactic acid content) is obtained. be able to.

"Culture medium"
The medium used in the production method of the present invention can be appropriately selected depending on the microorganism used, but a medium containing a carbon source is usually used. When E. coli is used, for example, LB medium, M9 medium, NZYM medium, SOB medium, 2 × YT medium, or terrific broth can be used.
The carbon source contained in the medium is not particularly limited as long as the polyester of the present invention can be produced.For example, glucose, xylose, fructose, cellobiose, raffinose, maltose, galactose, starch, starch hydrolysate, molasses, Examples include sugars such as waste molasses, and natural products such as wheat and rice. In particular, xylose is preferable when obtaining a polyester having a high lactic acid content (lactic acid content).

<< Alcohol with a molecular weight of 300 or less >>
In the production method of the present invention, an alcohol having a molecular weight of 300 or less is added to the medium. That is, the microorganism is brought into contact with an alcohol having a molecular weight of 300 or less.
As the alcohol having a molecular weight of 300 or less, the “alcohol having a molecular weight of 300 or less” described in the section “[1] Alcohol-terminated polyester” can be used without limitation. Preferably, a polyhydric alcohol or an ether bond is used. More preferably a polyhydric alcohol having an ether bond, and still more preferably a diol having an ether bond. Another preferred embodiment is an aliphatic alcohol having a monovalent to trivalent hydroxy group.
In particular, diethylene glycol, butanediol, or polyethylene glycol is preferable. When polyethylene glycol is used, polyethylene glycol having a molecular weight of 300 or less is used. The molecular weight of polyethylene glycol is preferably 280 or less, more preferably 260 or less.
The lower limit of the molecular weight is preferably 50 or more, more preferably 65 or more.
The alcohol is presumed to be bonded to the end of the polyester portion by a condensation reaction to stop the polymerization of the polyester.
The alcohol concentration added to the medium is not particularly limited as long as the effects of the present invention can be obtained.For example, the concentration that does not inhibit the growth of microorganisms depends on the type of alcohol and the type of microorganism. It can be determined as appropriate. For example, when Escherichia coli and diethylene glycol are used, the concentration of diethylene glycol is preferably 0.01 to 20% by volume, more preferably 0.1 to 10% by volume. When using E. coli and polyethylene glycol, the concentration of polyethylene glycol is preferably 0.01 to 15% by volume, more preferably 0.1 to 10% by volume.

In the method for producing a polyester of the present invention, part or all of the polyester is secreted from microorganisms. That is, the polyester obtained is characterized by being not only accumulated in the cells of microorganisms but also secreted outside the cells. That is, the amount of extracellular polyester produced when alcohol having a molecular weight of 300 or less is contained in the medium is significantly higher than the amount of extracellular polyester produced when no alcohol having a molecular weight of 300 or less is contained in the medium. It has improved.
The polyester obtained by the method for producing a polyester of the present invention includes an alcohol-terminated polyester in which an alcohol residue having a molecular weight of 300 or less is bonded to the terminal of the polyester. That is, although not limited, the alcohol-terminated polyester described in the section “[1] Alcohol-terminated polyester” can be produced. Since the alcohol is bonded to the terminal of the polyester, the polyester can be efficiently secreted outside the cells of the microorganism.

Low molecular weight to high molecular weight polyesters can be produced by the polyester production method of the present invention. The obtained polyester includes not only a polymer compound having a molecular weight of several thousand to several tens of thousands, but also an oligomer having about a dimer to a 10-mer and a copolymer composed of two or more types of monomer units. That is, in the present invention, it is usually possible to produce a polyester having a low molecular weight to a high molecular weight.
Many polyesters secreted extracellularly have a relatively small molecular weight. For example, the average number of repeating units of lactic acid and 3-hydroxybutanoic acid in a polyester is 2 to 12 in an embodiment, and 2 to 10 in an embodiment. And in some embodiments 2-8.
The molecular weight of the polyester in the cell is not particularly limited, but many of them have a relatively large molecular weight. For example, the average number of repeating units of lactic acid and 3-hydroxybutanoic acid in the polyester is 2 to 1000 in some embodiments. In some embodiments, it is 2 to 100, and in some embodiments, 2 to 50.

The recovered polyester can be recovered by a method known to those skilled in the art for recovering a copolyester from a microorganism. For example, the microorganism is collected from the culture solution by centrifugation, washed, dried, suspended in chloroform, and heated to extract the desired copolyester into the chloroform fraction. Methanol is added to precipitate the polyester, the supernatant is removed by filtration or centrifugation, and then dried to obtain a purified copolyester.
The composition of the recovered polyester may be confirmed by a usual method such as gas chromatography or nuclear magnetic resonance.

<Action>
As described above, at least a part of the polyester obtained by the production method of the present invention has an alcohol residue having a molecular weight of 300 or less bonded to the terminal of the polyester. In particular, polyesters secreted extracellularly have a high proportion of bonded alcohol residues having a molecular weight of 300 or less. In the present invention, the reason why the alcohol is bonded to the obtained polyester is not clear in detail, but can be estimated as follows. The added alcohol having a molecular weight of 300 or less seems to act on the termination of polyester biosynthesis after being taken into the cells. As a result, a polyester in which the alcohol is bonded to the terminal is obtained. Specifically, it is considered that alcohol having a molecular weight of 300 or less acts as a chain transfer agent. The chain transfer agent receives radicals from the polymer chain and stops the extension of the polymer, but the chain transfer agent can also attack the monomer to initiate polymerization. It is also possible that the alcohol bound to the end of the polyester has migrated out of the cell by playing a role like a signal sequence. However, the present invention is not limited by the above description.

Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Example 1
Manufacture of polyesters The method described in Example 1 of WO2009 / 131186 DNA encoding propionyl CoA transferase from M. elsdenii, DNA encoding β-ketothiolase from R. eutropha, R. DNA encoding acetoacetyl-CoA reductase from R. eutropha, and Pseudomonas sp. (Pseudomonas sp.) Mutant polyhydroxyalkanoic acid having a double mutation in which the 325th Ser of the polyhydroxyalkanoic acid synthase derived from the 61-3 strain is replaced with Thr and the 481st Gln is replaced with Lys A recombinant plasmid pTV118NpctphaC1 _PS (ST / QK) AB containing DNA encoding a synthetic enzyme (hereinafter sometimes referred to as ST / QK mutant enzyme) was prepared.
Also, instead of the ST / QK mutant enzyme, Pseudomonas sp. (Pseudomonas sp.) Triple alkanoic acid synthase derived from the 61-3 strain was replaced with Thr, Ser at 325, Phe at 392 was replaced with Ser, and Gln at 481 was replaced with Lys. The same as pTV118NpctphaC1 _PS (ST / QK) AB except that it contains a DNA encoding a mutant polyhydroxyalkanoate synthase having a mutation (hereinafter sometimes referred to as ST / FS / QK mutant enzyme). Recombinant plasmid pTV118NpctphaC1 _PS (ST / FS / QK) AB was prepared.
Subsequently, E. coli BW25113 was transformed by the calcium phosphate method, and a transformant in which propionyl CoA transferase, β-ketothiolase, acetoacetyl CoA reductase, and ST / QK mutant enzyme were expressed (hereinafter sometimes referred to as ST / QK E. coli). , And transformants expressing propionyl CoA transferase, β-ketothiolase, acetoacetyl CoA reductase, and ST / FS / QK mutant enzyme (hereinafter sometimes referred to as ST / FS / QK E. coli) Got the body.

The obtained transformant was cultured at 30 ° C. for 48 hours while stirring at 180 rpm using LB medium containing 2 wt% glucose and ampicillin. In advance, polyethylene glycol (PEG 200, sometimes simply referred to as “polyethylene glycol” or “PEG” in the following examples) or diethylene glycol in an amount of 1 to 5% by volume as a final concentration was added to LB medium in advance (hereinafter, alcohol was added to the medium). The same applies to the case of adding to the above).
The culture supernatant was collected, and lactic acid contained in the culture supernatant was measured with a quantification kit (International Ireland, Megazyme). The amount of lactic acid is determined by measuring lactic acid (Free LA) using the culture supernatant as it is (not treated with hydrochloric acid), or adding 80 μL of 5N hydrochloric acid and 20 μL of water to 100 μL of the culture supernatant, and 100 ° C. The hydrolysis was carried out overnight. Thereafter, 200 μL of 2N NaOH was added for neutralization, and lactic acid (Total LA) in the sample was measured. Since Free LA is not treated with hydrochloric acid, monomeric lactic acid in the culture supernatant is measured. Total LA measures the total amount of monomeric and oligomeric lactic acid because oligomeric lactic acid in the culture supernatant is decomposed by hydrochloric acid to become a monomer. Therefore, it is considered that the amount of lactic acid oligomer in the culture supernatant is obtained by subtracting the lactic acid amount of Free LA from the lactic acid amount of Total LA. The results are shown in FIGS. The black bar indicates Free LA, and the white bar indicates Total LA.

In either E. coli expressing ST / QK mutant enzyme or E. coli expressing ST / FS / QK mutant enzyme, polylactic acid is added to the culture supernatant by adding polyethylene glycol (PEG) or diethylene glycol (DEG). Of oligomers were secreted. In particular, by adding diethylene glycol to E. coli expressing ST / FS / QK mutant enzymes, oligomers of polylactic acid outside the cells were significantly secreted (FIGS. 2 and 3).

Example 2
In this example, the ratio of lactic acid to 3-hydroxybutanoic acid contained in the oligomer of polylactic acid secreted into the culture supernatant was measured. The ST / FS / QK E. coli obtained in Example 1 was cultured in the same manner as in Example 1 in a medium to which diethylene glycol was added to obtain a culture supernatant. In the same manner as in Example 1, a sample not treated with hydrochloric acid and a sample treated with hydrochloric acid were prepared from the obtained culture supernatant. For each sample, the amount of lactic acid was measured with a quantitative kit (International Ireland, Megazyme), and the amount of 3-hydroxybutanoic acid was measured with a quantitative kit (Wako Pure Chemical Industries, Ltd., AUTOKIT 3-HB). The results are shown in FIG. The lower part of each bar shows the amount of 3-hydroxybutanoic acid, and the upper part shows the amount of lactic acid.
When diethylene glycol was added so as to be 1% by volume, the lactic acid content in the supernatant was 91 mol%. When diethylene glycol was added so as to be 3% by volume, the content of lactic acid in the supernatant was 96 mol%. When diethylene glycol was added so as to be 5% by volume, the content of lactic acid in the supernatant was 87 mol%. It was found that the oligomer secreted extracellularly contains a large amount of lactic acid.

Example 3
In this example, alcohol was added to the oligomer of extracellular polylactic acid (FIG. 5A), the oligomer of intracellular polylactic acid (FIG. 5B), and the polymer of intracellular polylactic acid (FIG. 5C) using NMR. It was confirmed that they were bonded.
(Separation and sample preparation of extracellular oligomers, intracellular oligomers, and polymers)
Escherichia coli BW25113 having pTV118NpctphaC1ps (ST / FS / QK) AB was cultured at 30 ° C. for 48 hours under the condition of 3% by volume DEG addition with stirring at 180 rpm using LB medium containing 2% by weight glucose and ampicillin, The supernatant and the cells were separated by centrifugation. Extracellular oligomers were extracted by adding 1 part by mass of chloroform to 1 part by mass of the supernatant and mixing. In order to remove excess DEG, 1 part by mass of water was added to 1 part by mass of the chloroform fraction, mixed and washed with water. This operation was repeated twice. In order to quantify the extracted oligomer, LA and 3HB of the chloroform fraction were analyzed by GC / MS.
The high molecular weight body in the microbial cells was obtained by extracting from freeze-dried cells with chloroform for 2 days at room temperature. Cell debris was removed by passing through a PTFE filter, followed by addition of 10 volumes of methanol to precipitate the polymer. The mixture was further incubated at 4 ° C. for 1 day. The polymer precipitated in methanol and the intracellular oligomers were soluble in methanol. The precipitated polymer and intracellular oligomers were separated by centrifugation. The amounts of LA and 3HB in the polymer and intracellular oligomers were analyzed by GC / MS.

(NMR analysis)
It was confirmed by ¹ H-NMR and two-dimensional NMR ( ¹ H- ¹ H DOSY NMR and ¹ H- ¹³ C HMQC) that DEG was bound to the extracellular oligomers, intracellular oligomers and polymers. The sample was dried and then resuspended in deuterated chloroform and measured with a Bruker AMX500 spectrometer.
^{By 1} H- ¹ H DOSY NMR analysis, it was confirmed that DEG bound to the oligomer / polymer behaved as a polymer compound, and showed diffusion similar to that of the polymer / oligomer chain (FIGS. 5A, B, and C).
Furthermore, from ¹ H- ¹³ C HMQC analysis, DEG bound to free DEG was confirmed as a different chemical shift (FIGS. 5D, E, F, and G). The free DEG is a target molecule having two methylene groups, so only two signals can be confirmed. On the other hand, DEG bonded to the oligomer / polymer is not the target molecule, and therefore, the four methylene groups are considered to have different chemical shifts.
FIG. 9A shows ¹ H- ¹ H COSY NMR. The crossover signal of 3.7 ppm / 4.3 ppm indicates that the 4.3 ppm resonance is derived from the proton of (B) of DEG, and therefore DEG was thought to be bound to the polyester at the carboxy terminus. . Furthermore, the weak crossing signals of 1.5 ppm / 4.3 ppm and 1.3 ppm / 4.2 ppm were considered to indicate resonance of polyester lactic acid and 3HB hydroxy-terminal methine protons. Further, FIG. 9 (b) shows ¹ H- ¹ H DOSY NMR, which shows that the diffusion coefficient of DEG is similar to that of D-lactic acid oligomer and is lower than that of unbound DEG. Therefore, the observed DEG was completely bound to the D-lactic acid oligomer.
FIG. 10 (a, b, c) shows ¹ H NMR, ¹³ C NMR, and ¹ H- ¹³ C HMQC. These data also show that the secretory oligomer obtained by adding DEG is D-lactic acid. It was supported to be an oligomeric DEG conjugate.

Example 4
In this example, the amount of intracellular and extracellular polyester was measured using a gene disruption mutant of E. coli. A culture supernatant and Escherichia coli cells were obtained by repeating the procedure of Example 2 except that the gene disruption mutant Escherichia coli shown in FIG. 6 was used instead of Escherichia coli BW25113. The amount of lactic acid was measured for the culture supernatant and the cells.
As shown in FIG. 6, an increase in the amount of polyester produced per unit medium amount was observed when deletion mutants such as tatB, tatE, and araf were used. On the other hand, depending on the defective mutant, growth inhibition of the bacterial cells was also noticeable.
The upper diagram in FIG. 6 shows the amount of lactic acid oligomer produced per liter of the culture solution, and the lower diagram shows the cell weight (Dry Cell Weight) per liter of the culture solution.

Example 5
The operation of Example 1 was repeated to measure Total LA and Free LA in the culture supernatant. FIG. 7 shows the amount of oligomer produced by subtracting Free LA from Total LA.
In ST / QK E. coli, oligomer secretion increased when 5% by volume of polyethylene glycol (PEG) was used as the alcohol. In addition, when diethylene glycol (DEG) was used as the alcohol, secretion of the oligomer increased depending on the volume of DEG.
In ST / FS / QK E. coli, when polyethylene glycol (PEG) was used as the alcohol, almost the same amount of oligomer was secreted in any of 1 to 5% by volume. On the other hand, when diethylene glycol (DEG) was used as the alcohol, secretion of the oligomer increased up to 5% by volume depending on the volume of DEG.

Example 6
In this example, molecules secreted into the culture supernatant were measured by ESI-MS using ST / FS / QK E. coli and 3% by volume of DEG.
The operation of Example 2 was repeated to obtain a culture supernatant.
The obtained culture supernatant was analyzed using ESI-MS (manufactured by Bruker). The results are shown in FIG.

Example 7
In this example, an extracellular polylactic acid oligomer, an intracellular polylactic acid oligomer produced by the production method of the present invention using ST / FS / QK E. coli and 1 to 5% by volume of DEG, and cells The amount of the polylactic acid polymer was measured.
The operation of Example 3 was repeated to obtain an extracellular polylactic acid oligomer, an intracellular polylactic acid oligomer, and an intracellular polylactic acid polymer. As shown in Table 1, by culturing ST / FS / QK E. coli using DEG, oligomers secreted extracellularly increased remarkably.

Example 8
In this example, using the DEG-bonded polyester obtained by culturing in a medium containing 5% by volume DEG of Example 1, and the DEG non-bonded polyester obtained by culturing in a medium not containing DEG, Lactide was synthesized.
10 mg of dry DEG-bonded polyester or non-DEG-bonded polyester was mixed with 10 mg of zinc oxide (catalyst). The mixture was heated in a vacuum at 180 ° C. for 1 hour using an oven (GTO-350RD glass oven: manufactured by Shibata). The evaporated lactide was liquefied in a round bottom flask on ice and the crude lactide was recovered with chloroform. The crude lactide was analyzed by ¹ H-NMR. The lactide obtained from DEG-bonded polyester has a signal of δ 1.68 (d, J = 5.5 Hz, 1H) consistent with the standard D-lactide and δ 5.03 (q, J = 8.5 Hz, 2H). ) Signal (FIG. 11). It was found that D-lactide was produced from DEG-bonded polyester. The lactide obtained from non-DEG polyester also showed a similar signal.
The yield of lactide (% [LA unit contained in μmol lactide] / [LA unit contained in μmol converted DEG-conjugated polyester]) was determined using the amount of lactide produced and benzoic acid as an internal standard for ¹ H-NMR. Calculations were based on the amount of converted oligomers quantified. A commercially available synthetic oligomer (L-lactic acid) was used as a control. The yields of DEG-bonded polyester or non-DEG-bonded polyester were 18% and 12%, respectively (Table 2).

Example 9
In the same manner as in Example 1, R.I. DNA encoding β-ketothiolase from R. eutropha, R. DNA encoding acetoacetyl-CoA reductase from R. eutropha; A recombinant plasmid pTV118NphaCAB containing DNA encoding a polyhydroxyalkanoate synthase derived from R. eutropha was prepared.
Subsequently, E. coli BW25113 was transformed by the calcium phosphate method to obtain a transformant expressing β-ketothiolase, acetoacetyl CoA reductase, and polyhydroxyalkanoate synthase.
The obtained transformant was cultured at 30 ° C. for 48 hours while stirring at 180 rpm using LB medium containing 2 wt% glucose and ampicillin. Any one of compounds A to C in an amount of 3% by volume as a final concentration was previously added to the LB medium.
The culture supernatant was collected and a sample treated with hydrochloric acid was prepared. The amount of 3-hydroxybutanoic acid in the sample was measured with a quantification kit (Wako Pure Chemical Industries, AUTOKIT 3-HB). The results are shown in Table 3.

Example 10
In this example, the effect of adding a carbon source to the LB medium was examined.
In the same manner as in Example 1, E. coli BW25113 was transformed by the calcium phosphate method, and a transformant (ST / FS / QK) in which propionyl CoA transferase, β-ketothiolase, acetoacetyl CoA reductase, ST / FS / QK mutant enzyme was expressed. E. coli) was obtained. As the medium, LB medium containing 100 mg / L ampicillin was prepared, to which diethylene glycol was added to a final concentration of 5% by volume and glucose or xylose was added to a concentration of 20 g / L.
Subsequently, the obtained ST / FS / QK E. coli was cultured at 30 ° C. for 48 hours while stirring at 180 rpm in the above-mentioned two types of media supplemented with glucose or xylose.
After completion of the culture, the culture supernatant was collected and analyzed in the same manner as in Examples 1 and 2. The results are shown in Table 4.

When xylose was used as a carbon source, lactic acid contained in the oligomer increased to 97 mol%.

Example 11
In this example, culture was performed using two gene-deficient (Δpfla and Δdld) mutants JWMB1 as E. coli.
The procedure of Example 10 was repeated except that JWMB1 strain was used as E. coli. The results are shown in Table 5.

When JWMB1 strain was used as E. coli and xylose was used as the carbon source, the amount of oligomer secreted into the medium increased to 8.1 ± 2.9 g / L, and the ratio of lactic acid in the oligomer was 97 ± 1 mol%. It became high.

The alcohol-terminated polyester of the present invention and the polyester obtained by the method for producing the polyester of the present invention can be used as raw materials for biodegradable materials.
As mentioned above, although this invention was demonstrated along the specific aspect, the modification and improvement obvious to those skilled in the art are contained in the scope of the present invention.

Claims

An alcohol-terminated polyester having an alcohol residue with a molecular weight of 300 or less at the end.
The alcohol-terminated polyester according to claim 1, wherein the polyester is a polyester comprising an α-hydroxy acid and / or a β-hydroxy acid.
The alcohol-terminated polyester according to claim 2, wherein the α-hydroxy acid and / or β-hydroxy acid is lactic acid and / or 3-hydroxybutanoic acid.
The alcohol-terminated polyester according to any one of claims 1 to 3, wherein the average number of repeating units of the polyester is 2 to 12.
The alcohol-terminated polyester according to claim 3 or 4, wherein the average lactic acid content is 70 mol% to 100 mol%.
A method for producing polyester, comprising culturing a microorganism capable of producing polyester in a medium containing an alcohol having a molecular weight of 300 or less.
The method for producing a polyester according to claim 6, wherein the polyester is a polyester comprising an α-hydroxy acid and / or a β-hydroxy acid.
The method for producing a polyester according to claim 7, wherein the α-hydroxy acid and / or β-hydroxy acid is lactic acid and / or 3-hydroxybutanoic acid.
The method for producing a polyester according to any one of claims 6 to 8, wherein a part or all of the polyester is secreted from microorganisms.
The method for producing a polyester according to any one of claims 6 to 9, wherein the average number of repeating units of the polyester is 2 to 12.
The method for producing a polyester according to any one of claims 6 to 10, wherein the polyester comprises an alcohol-terminated polyester having an alcohol residue having a molecular weight of 300 or less at the terminal of the polyester.