WO2017163518A1 - Polyhydroxyalkanoic acid having functional group at terminal carboxyl group and method for producing same - Google Patents

Abstract

Provided are: a novel polyhydroxyalkanoic acid which has a functional group at a terminal carboxy group, and which is able to be chemically modified, while being easily controlled in terms of reaction; and a method for producing this polyhydroxyalkanoic acid. A polyhydroxyalkanoic acid according to the present invention is obtained by introducing a specific alkyne group, alkene group, thiol group, azide group or allyl group into a terminal carboxy group. A production method according to the present invention comprises a step wherein a microorganism that is capable of producing a polyhydroxyalkanoic acid is cultured with use of an alcohol that has an alkyne group, an alkene group, a thiol group, an azide group or an allyl group.

Description

POLYHYDROXYALKANOIC ACID HAVING FUNCTIONAL GROUP AT CARBOXY TERMINAL AND PROCESS FOR PRODUCING THE SAME

The present invention relates to a novel polyhydroxyalkanoic acid having a functional group at the carboxy terminus and a method for producing the same. In particular, the present invention relates to a microbially produced R-3-hydroxyalkanoic acid homopolymer or copolymer [poly (R-3-hydroxyalkanoic acid)] having a hydroxy group at the 3-position and a method for producing the same.

Polyhydroxyalkanoic acid (hereinafter abbreviated as “PHA”) is a thermoplastic polyester that is produced and accumulated as an energy storage substance in the cells of many microbial species. PHA produced from various natural carbon sources by microorganisms is completely biodegraded by microorganisms in soil and water, and is thus incorporated into the natural carbon cycle process. Therefore, it can be said that PHA is an environmentally friendly plastic that has almost no adverse effects on the ecosystem. In recent years, from the viewpoints of environmental pollution, waste disposal, and petroleum resources, as social problems due to synthetic plastics become serious, PHA has attracted attention as an environmentally friendly green plastic, and its practical application is eagerly desired.

The first PHA discovered in microorganisms is polyhydroxybutyrate (hereinafter abbreviated as “PHB”), which is a homopolymer of 3-hydroxybutyric acid (hereinafter abbreviated as “3HB”). PHB has high crystallinity, is hard and brittle because of its high degree of crystallinity, and rapidly decomposes at a temperature near the melting point (180 ° C.), so that melt workability is low and the practical range is extremely limited. is doing.

Therefore, an attempt was made to introduce another 3-hydroxyalkanoic acid into the PHB skeleton in order to improve the brittleness by lowering the crystallinity of PHB. For example, side chains such as 3-hydroxypropionic acid (hereinafter abbreviated as “3HP”), 4-hydroxybutyric acid (hereinafter abbreviated as “4HB”), 5-hydroxyvaleric acid (hereinafter abbreviated as “5HV”), etc. PHB for monomers having a side chain such as linear monomers not having lactic acid, 3-hydroxyvaleric acid (hereinafter abbreviated as “3HV”), 3-hydroxyhexanoic acid (hereinafter abbreviated as “3HHx”), etc. Introduction to the skeleton has been reported so far. The physical properties of PHA obtained vary greatly depending on the type of monomer to be introduced and the copolymerization ratio, but basically any monomer is introduced, so that the crystallinity of PHB is lowered, so that the melt processability is higher than that of PHB. Improve.

In order to further expand the applications of PHA, it is necessary to develop technology for producing new PHA having physical properties greatly different from those described above. For this purpose, it is considered effective not only to simply change the length of the PHA main chain and the size of the straight chain, but also to introduce some functional group into the side chain or carboxy terminus of the PHA.

So far, as an example of introducing a functional group into the side chain of PHA, Patent Document 1 reports that a thioester has been introduced into the side chain of a medium chain PHA having 6 to 14 carbon atoms. Non-Patent Document 1 discloses that a side chain of a medium chain PHA having 6 to 14 carbon atoms has a branched alkyl group, a cyclohexyl group, an alkyl halide, an acetoxy group, an ester, an alkoxy group, an epoxy group, a thiol group, a cyano group. Examples of introducing aromatic ring compounds such as nitro group, nitro group, phenyl group and benzoyl group are summarized.

The introduction of these functional groups is important not only for greatly changing the physical properties of PHA, but also for providing a reaction starting point for further chemically modifying the functional groups. For example, Non-Patent Document 3 reports that a fluorescent substance or a peptide is modified to a double bond of a PHA side chain using a thiol-enclick reaction. Non-Patent Document 4 reports that the water repellency of PHA was changed by introducing a hydroxy group or a carboxy group into a double bond of a PHA side chain by using a thiol-ene click reaction. Furthermore, Non-Patent Document 4 reports that PHA can be crosslinked by reacting with a multi-branched structure compound. Further, Non-Patent Document 5 reports an example in which a PHA having an azide group in the side chain is produced and the side chain is modified using an alkyne-azide click reaction.

Such a method of introducing a functional group into PHA and chemically modifying it into a target is useful for changing the physical properties of PHA. However, when the PHA side chain functional group is used as described above, reaction control is very difficult. For example, when cross-linking is performed using the above multi-branched structure compound, PHA gels if there are many cross-linking points, but if the cross-linking points are small, the effect of modifying physical properties by cross-linking cannot be obtained. In addition, the presence of a plurality of reactive groups on one molecule of PHA chain has caused a problem that the product after the reaction becomes a mixture of various compounds.

On the other hand, Non-Patent Document 6 reports an example in which a functional group is introduced at the carboxy terminus of PHA. Specifically, Non-Patent Document 6 describes a Bacillus genus microorganism having a type IV PHA synthase gene in the presence of 1.3-propanediol, 2-propyn-1-ol, 3-mercapto-1-propanol, and benzyl alcohol. Incubation in 1-propanol group, 1-propynyl group, 1-propanethiol group, and benzyl group is introduced at the carboxy terminus of PHB, and the molecular weight may be lower than that in the absence of these. It has been reported. Furthermore, Non-Patent Document 6 reports that no decrease in molecular weight was observed even when cultured in the presence of 2-propen-1-ol or 3-butyn-1-ol.

WO2012 / 038572

As described above, Non-Patent Document 6 describes a compound in which a functional group is introduced at the carboxy terminus of PHA. However, only four compounds can be actually produced. For example, a propynyl group can be introduced for an alkynyl group. However, it is described that the butynyl group could not be introduced. An object of the present invention is to provide a method capable of producing various novel PHA having a functional group at the carboxy terminus that can be chemically modified and whose reaction control is easy, and a PHA that has not been conventionally produced.

As a result of intensive studies to solve the above problems, the present inventors have succeeded in producing a PHA containing a specific functional group at the carboxy terminus instead of the side chain of PHA, and completed the present invention. It was.
The details of the present invention are as follows.
1. The repeating unit is represented by the following general formula (1)
[—C ^* HR ¹ —CH ₂ —CO—O—] (1)
(In the formula, R ¹ is an alkyl group represented by C _n H _{2n + 1} , n is an integer of 1 to 15, and * is asymmetric.)
R-3-hydroxyalkanoic acid homopolymer or copolymer represented by the following, wherein the following groups are bonded to the carboxy terminus depending on whether the polymer is a homopolymer or a copolymer Polyhydroxyalkanoic acid.
(1) a group bonded to the carboxy terminus when it is a single polymer, an alkynyl group having 4 to 8 carbon atoms,
An alkenyl group having 3 to 8 carbon atoms,
A mercaptoalkyl group having 2 or 4 to 8 carbon atoms,
An azido alkyl group having 3 to 8 carbon atoms, or an allyl (poly) oxyalkyl group having 2 to 6 carbon atoms in the alkyl group (2) a group bonded to the carboxy terminus when the copolymer is a polymer having 3 to 8 carbon atoms An alkynyl group,
An alkenyl group having 3 to 8 carbon atoms,
A mercaptoalkyl group having 2 to 8 carbon atoms,
1. an azido alkyl group having 3 to 8 carbon atoms or an allyl (poly) oxyalkyl group having 2 to 6 carbon atoms in the alkyl group; A microbially produced polyhydroxyalkanoic acid having an alkyne group (synonymous with alkynyl group), alkene group (synonymous with alkenyl group), thiol group (mercapto group) or azide group introduced at the carboxy terminus,
The hydroxyalkanoic acid is 3-hydroxybutyric acid, 3-hydroxypropionic acid, 4-hydroxybutyric acid, 3-hydroxyvaleric acid, 5-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 6-hydroxyhexanoic acid, 3-hydroxyheptanoic acid , Polyhydroxyalkanoic acid composed of a plurality of kinds selected from 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid and 3-hydroxydodecanoic acid.
3. A microbially produced polyhydroxyalkanoic acid having an alkyne group, alkene group, thiol group or azide group introduced at the carboxy terminus;
The hydroxyalkanoic acid is 3-hydroxybutyric acid, 3-hydroxypropionic acid, 4-hydroxybutyric acid, 3-hydroxyvaleric acid, 5-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 6-hydroxyhexanoic acid, 3-hydroxyheptanoic acid , Composed of a single species selected from 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxydodecanoic acid,
The alkyne group is a butynyl group, a pentynyl group, or a hexynyl group;
The alkene group is a propenyl group, a butenyl group, a pentenyl group, or a hexenyl group;
A polyhydroxyalkanoic acid in which the thiol group is a mercaptoethyl group, a mercaptobutyl group, a mercaptopentyl group, or a mercaptohexyl group.
4). 4. The polyhydroxyalkanoic acid according to any one of 1 to 3 above, which contains at least 3-hydroxybutyric acid as a monomer unit.
5. 5. The polyhydroxyalkanoic acid as described in 4 above, further containing 3-hydroxyhexanoic acid as a monomer unit.
6). A method for producing the polyhydroxyalkanoic acid according to any one of 1 to 5, wherein
Including culturing a microorganism capable of producing polyhydroxyalkanoic acid using an alcohol having an alkyne group, an alkene group, a thiol group, an azide group, or an allyl group; , An azido group, or a polyhydroxyalkanoic acid having an allyl group introduced therein.
7). Including culturing a microorganism belonging to the genus Cupriavidus capable of producing polyhydroxyalkanoic acid using an alcohol having 2 to 8 carbon atoms having an alkyne group, an alkene group, a thiol group, an azide group, or an allyl group, A method for producing a polyhydroxyalkanoic acid into which an alkyne group, an alkene group, a thiol group, an azide group, or an allyl group is introduced.
8). 8. The production method according to 6 or 7 above, wherein the alcohol is a primary alcohol.
9. 9. The production method according to any one of the above 6 to 8, wherein the microorganism is a microorganism having a gene encoding a polyhydroxyalkanoate synthase derived from the genus Aeromonas, Ralstonia or Pseudomonas.
10. 10. The production method according to 8 or 9 above, wherein the microorganism is a microorganism belonging to the genus Cupriavidus.
11. 11. The production method according to 7 or 10 above, wherein the microorganism is a transformant using Cupriavidus necator as a host.
12 6. A compound obtained by further chemically modifying the terminal alkyne group, alkene group, thiol group, azide group, or allyl group of the polyhydroxyalkanoic acid according to any one of 1 to 5 above.
13. A molded product containing the polyhydroxyalkanoic acid according to any one of 1 to 5 above or the compound according to 12 above.

According to the present invention, a completely new PHA having a specific functional group at the carboxy terminus can be produced. The PHA of the present invention can be expected not only to improve the physical properties of conventional PHA by using it as an additive for producing molded articles such as films and sheets, but also to have various structures by further modifying the above functional groups. It is thought that guidance to the body is possible.

1 is a ¹ H-NMR chart of Example 8. FIG. FIG. 2 is a ¹ H-NMR chart of Example 16. FIG. 3 is a ¹ H-NMR chart of Comparative Example 1.

Hereinafter, the present invention will be described in more detail.

The PHA of the present invention has a repeating unit represented by the following general formula (1)
[—C ^* HR ¹ —CH ₂ —CO—O—] (1)
(In the formula, R ¹ is an alkyl group represented by C _n H _{2n + 1} , n is an integer of 1 to 15, and * represents an asymmetric carbon.)
Or a copolymer of R-3-hydroxyalkanoic acid (hereinafter abbreviated as “3HA”) (hereinafter, these polymers are abbreviated as “P3HA”), wherein P3HA is a homopolymer or a copolymer. Depending on whether the polymer is a polymer, the following groups are bonded to the carboxy terminus. Preferably, the following groups are bonded to an ester bond containing the carboxy group, and have a triple bond, a double bond, a mercapto group (thiol group), or an allyl group at the terminal of PHA.
(1) A group bonded to the carboxy terminus when it is a single polymer C4-C8 alkynyl group, C3-C8 alkenyl group, C2-C4-8 mercaptoalkyl group, C3-C8 An azido alkyl group or an allyl (poly) oxyalkyl group having 2 to 6 carbon atoms in the alkyl group (2) a group bonded to the carboxy terminus when the copolymer is a polymer 3 to 8 alkynyl group or 3 carbon atoms An alkenyl group having 8 to 8 carbon atoms, a mercaptoalkyl group having 2 to 8 carbon atoms, an azido alkyl group having 3 to 8 carbon atoms, or an allyl (poly) oxyalkyl group having 2 to 6 carbon atoms in the alkyl group

In Non-Patent Document 6 described above, 3HB homopolymer in which n = 1 in the above general formula (1), a propynyl group (alkynyl group having 3 carbon atoms) and a mercaptopropyl group ( Only a thiol group having an alkyl group having 3 carbon atoms is disclosed, and the type of functional group is different from the present invention. In addition, both use different microbial species.

That is, the PHA of the present invention is a polyester resin produced from microorganisms, which is called P3HA with 3HA as a main monomer unit. Examples of the monomer unit constituting the PHA of the present invention include 3HB (in the general formula (1), n = 1 of the alkyl group R ¹ represented by C _n H _{2n +} 1), 3HV (n = 2), 3HHx (n = 3), 3-hydroxyheptanoic acid (n = 4), 3-hydroxyoctanoic acid (n = 5), 3-hydroxynonanoic acid (n = 6), 3-hydroxydecanoic acid (n = 7) , 3-hydroxyundecanoic acid (n = 8), 3-hydroxydodecanoic acid (n = 9), and the like.

The monomer unit in the PHA of the present invention may be composed of a single species or a plurality of species. When plural kinds of monomer units are included, two or more kinds of 3HA may be copolymerized, or 4-hydroxyalkanoic acid such as 4HB may be copolymerized with one or more kinds of 3HA. It may be. The PHA of the present invention preferably contains at least 3HB as a monomer unit. The PHA of the present invention may be a PHB consisting only of the above 3HB as a monomer unit, or may be a copolymer consisting of 3HB and another monomer unit. Examples of monomer units other than 3HB include the above-described monomer units, 3HP, 4HB, 5HV, 6-hydroxyhexanoate (hereinafter abbreviated as “6HHx”), and the like. As an example of such a copolymer, Poly (3HB-co-3HHx) further having 3HHx (hereinafter abbreviated as “PHBH”), Poly having further 3HV (3HB-co-3HV), and Poly further having 4HV (3HB-co-4HV) and the like. Of these, PHBH is preferred.

The copolymerization ratio of each monomer unit when the PHA of the present invention is a copolymer is not particularly limited, but when 3HB is contained as a monomer unit, the copolymerization ratio is more preferably 50 mol% or more. 60 mol% or more is more preferable, 70 mol% or more is more preferable, and 80 mol% or more is particularly preferable. When the PHA of the present invention is PHBH, the lower limit of the 3HHx copolymerization ratio is preferably 1 mol%, more preferably 2 mol%, and even more preferably 3 mol%. Further, the upper limit is preferably 20 mol%, more preferably 15 mol%, and still more preferably 12 mol%.

As described above, the PHA of the present invention is characterized in that an alkynyl group, an alkenyl group, a thiol group (mercapto group), an azide group, or an allyl group is introduced as a specific functional group at the carboxy terminal of the polymer main chain. And The carboxy terminus of the PHA of the present invention has an alkynyl group, an alkenyl group, a thiol group, and an azide group via an alkyl chain, and an allyl group via an oxyalkyl chain.

Examples of the alkynyl group having 3 to 8 carbon atoms include linear or branched propynyl group, butynyl group, pentynyl group, hexynyl group, heptynyl group, and octynyl group. One having no branching, that is, a straight chain is preferred. The number of carbon atoms is preferably 3 to 6, and among them, propynyl group, butynyl group, and hexynyl group are preferable examples.

Examples of the alkenyl group having 3 to 8 carbon atoms include linear or branched propenyl group, butenyl group, pentenyl group, hexenyl group, heptenyl group, and octenyl group. One having no branching, that is, a straight chain is preferred. The number of carbon atoms is preferably 3 to 6, and among them, propenyl group, butenyl group and hexenyl group are preferable examples.

Examples of the mercaptoalkyl group having 2 to 8 carbon atoms include linear or branched mercaptoethyl group, mercaptopropyl group, mercaptobutyl group, mercaptopentyl group, mercaptohexyl group, mercaptoheptyl group, and mercaptooctyl group. One having no branching, that is, a straight chain is preferred. The number of carbon atoms is preferably 2 to 6, and more preferably 3 to 6. For example, a mercaptoethyl group and a mercaptopropyl group are preferable examples.

Examples of the azido alkyl group having 3 to 8 carbon atoms include linear or branched azidated propyl group, azido butyl group, azido pentyl group, azido hexyl group, and azido heptyl group. One having no branching, that is, a straight chain is preferred. The number of carbon atoms is preferably 3 to 6, and among them, preferred examples include azidopropyl group and azidobutyl group.

Examples of the allyl (poly) oxyalkyl group having 2 to 6 carbon atoms in the alkyl group include those having 1 to 3 oxyalkyl, preferably 1. Examples of the oxyalkyl group include an oxyethyl group, an oxypropyl group, an oxybutyl group, an oxypentyl group, and an oxyhexyl group. Among them, an oxyethyl group, an oxypropyl group, and an oxybutyl group are preferable examples. One having no branching, that is, a straight chain is preferred. The total carbon number of the allyl (poly) oxyalkyl group is preferably 2 to 6, and examples thereof include an allyloxyethyl group, an allyloxypropyl group, and an allyloxybutyl group.

The PHA of the present invention is characterized by having a specific functional group at the carboxyl group terminal as described above, but it may further have a functional group in the polymer side chain. However, when a new derivative is synthesized by further chemically modifying the PHA of the present invention, it is preferable that the functional group is present only at the carboxyl group terminal from the viewpoint of reaction control.

The molecular weight of the PHA of the present invention is not limited. However, when the production method of the present invention described below is used, a relatively low molecular weight PHA tends to be obtained. For example, the weight average molecular weight (M _w ) may be about 5,000 to 20,000,000, and may be about 8,000 to 300,000 or even about 10,000 to 100,000 depending on the purpose of use. The number average molecular weight (M _n ) may be about 3000 to 1500,000, and may be about 5000 to 1000000 or even about 7000 to 800000 depending on the purpose of use.

As a method for producing the above-described PHA of the present invention, PHA is produced using an alcohol having the alkynyl group, alkenyl group, thiol group, azide group, or allyl group so that the specific functional group can be introduced at the carboxy terminus. And a method of culturing a synthesizeable microorganism (hereinafter referred to as “the microorganism of the present invention”) (hereinafter referred to as “the production method of the present invention”). The alcohol is preferably a primary alcohol.

Examples of such alcohols include 2-propyn-1-ol, 3-butyn-1-ol, 4-pentyn-1-ol, 5-hexyn-1-ol; 2-propen-1-ol, 3- Buten-1-ol, 4-penten-1-ol, 5-hexen-1-ol; 2-mercaptoethanol, 3-mercaptopropanol, 4-mercaptobutanol, 5-mercaptopentanol, 6-mercaptohexanol; Azidobutan-1-ol, 5-azidopentan-1-ol, 6-azidohexane-1-ol; ethylene glycol monoallyl ether, propylene glycol monoallyl ether, tetramethylene glycol monoallyl ether, pentamethylene glycol monoallyl ether, etc. Is mentioned. Among them, 2-propen-1-ol, 3-buten-1-ol, 5-hexen-1-ol, 2-propyn-1-ol, 3-butyn-1-ol, 5-hexyn-1-ol Preferred examples include 2-mercaptoethanol, 3-mercaptopropanol, and ethylene glycol monoallyl ether. In addition to this, an alcohol having a branched structure in the alkyl chain portion, an alcohol having a plurality of hydroxy groups, or an alcohol having a plurality of alkynyl groups, alkenyl groups, thiol groups, azide groups, or allyl groups may be used. These alcohols may be used alone or in combination of two or more.

In the production method of the present invention, it is considered that the alcohol functions as a terminator in the chain transfer reaction in PHA synthesis in microbial cells. In that case, what alcohol functions as a terminator depends on the substrate specificity of the PHA synthase of the microorganism of the present invention for alcohol. In the production method of the present invention, the microorganism of the present invention is a gene encoding a PHA synthase derived from the genus Aeromonas (Aeromonas), Ralstonia, or Pseudomonas (hereinafter referred to as “PHA synthase gene”). And more specifically, the PHA synthase gene is derived from Aeromonas caviae, which consists of the amino acid sequence shown in SEQ ID NO: 1, and the 149th asparagine is Ralstonia eutropha consisting of the amino acid sequence described in SEQ ID NO: 2, a PHA synthase gene in which 171th aspartic acid is artificially replaced with glycine in serine Rofa) PHA synthase gene derived from, the amino acid sequence set forth in SEQ ID NO: 3, Pseudomonas Sp. PHA synthase genes derived from 61-3, in which the 325th serine is artificially replaced with threonine, the 477th serine with arginine, and the 481st glutamine with arginine, are more preferred examples. It is not limited to these. Since PHA synthase derived from Aeromonas genus is known to use a hydroxyalkanoic acid CoA having 3 to 6 carbon atoms as a substrate, by using a microorganism having a PHA synthase gene derived from Aeromonas genus, 3HB, 3HP, A homopolymer composed of 4HB, 3HV, 5HV, and 3HHx, or a copolymerized PHA composed of these monomer units can be produced. Moreover, since it is known that Ralstonia genus-derived PHA synthase uses a hydroxyalkanoic acid CoA having 3 to 5 carbon atoms as a substrate, 3HB, 3HP can be obtained by using a microorganism having Ralstonia genus-derived PHA synthase gene. Homopolymers composed of 4HB, 3HV, and 5HV, or copolymerized PHA composed of these monomer units can be produced. In addition, since Pseudomonas genus-derived PHA synthase is known to use a hydroxyalkanoic acid CoA having 3 to 12 carbon atoms as a substrate, 3HB, 3HP can be obtained by using a microorganism having a Pseudomonas genus-derived PHA synthase gene. Homopolymer comprising 4HB, 3HV, 5HV, 3HHx, 6HHx, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxydodecanoic acid Alternatively, a copolymerized PHA composed of these monomer units can be produced.

The microorganism species of the present invention is not particularly limited, and may be either bacteria or fungi. For example, Acinetobacter (Acinetobacter), Aeromonas (Aeromonas), Alcaligenes (Alkalinegenes), Allochromatium (Arozobium), Azotobacter (Azotobacter) Deria) genus, Candida genus, Caurobacter genus, Chromobacterium genus, Comamonas genus, Cupriavidus genus, Ectothiorhodospira genus bsiella (Klebsiella), Methylobacterium (Methylobacterium), Nocardia (Nocardia), Paracoccus, Pseudomonas, Ralstonia, Rhizobium, Rhizobium, Rhizobium, Rhizobium Genus, Rhodococcus genus, Rhodospirillum genus, Rickettsia genus, Saccharomyces genus, Sinorhobium genus, S cus (Chiokokkasu) genus, Thiocystis (Chiokisuchisu) genus Vibrio (Vibrio) genus, Wautersia (Woterushia) genus, or Zoog / Loea (Zoogu / Roea) include microorganisms belonging to the genus. Of these, microorganisms belonging to the genus Aeromonas, Alcaligenes, Cupriavidus, Escherichia, Pseudomonas, Ralstonia, etc. are preferred, and microorganisms belonging to the genus Cupriavidus, Escherichia, Ralstonia are more preferred, and the microorganism belonging to the genus Cu belonging to the genus Cu is more preferred than the microorganism belonging to the genus Cu. Particularly preferred as the microorganism of the present invention is Cupriavidus necator.

When the microorganism of the present invention does not originally have a PHA synthase gene, or when the PHA synthase gene originally possessed by the microorganism is not the desired PHA synthase gene, the above-described preferred PHA synthase gene is transformed into a host by gene recombination, for example. It is also possible to use a transformant introduced into the microorganism. The method for introducing the PHA synthase gene into the host may be a format in which the gene is held in a plasmid or a format in which it is introduced at an arbitrary position on the chromosome. At this time, the PHA synthase gene originally possessed by the host is preferably lost its function. Examples of a method of deleting the function of the PHA synthase gene include, as an example, a method of deleting the PHA synthase gene over its entire length or partially, or addition or deletion of a base to the PHA synthase gene. Alternatively, a method in which the function of the produced PHA synthase is lost by substitution, and the like can be mentioned. For specific methods of inserting or replacing DNA, see, for example, Green, M. et al. R. And Sambrook, J.A. , 2012, Molecular Cloning: A Laboratory Manual Fourth Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York may be referred to.

In the production method of the present invention, the alcohol may be used alone as a carbon source for producing PHA. However, if a large amount of the alcohol is used for culturing, the growth of microorganisms is likely to be affected. Therefore, it is preferable to use the alcohol and another carbon source in combination. As such a carbon source other than alcohol, any raw material can be used as long as it is a carbon source that can be assimilated by the microorganism of the present invention. Such a carbon source is not particularly limited, but is preferably sugars such as glucose, fructose, sucrose, palm oil, palm kernel oil (hereinafter abbreviated as “PKO”), corn oil, palm Preferred are oils such as oil, olive oil, soybean oil, rapeseed oil and jatropha oil, fractionated oils or refined by-products thereof, or fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid and myristic acid, and derivatives thereof. . In addition, yeast extract and polypeptone can also be used. More preferably, palm olein, palm double olein, or palm kernel oil olein, PFAD (palm oil fatty acid distillation), which is a low melting point fraction obtained by fractionating palm oil or palm kernel oil, or vegetable oils such as palm oil and palm kernel oil Product), PKFAD (palm kernel oil fatty acid distillate), or rapeseed oil fatty acid distillate, and the like. From the viewpoint of avoiding competition with food, the oil and fat refinement by-product is particularly preferable.

In the production method of the present invention, the microorganism is cultured using a medium containing the alcohol, a carbon source other than alcohol, a nitrogen source that is a nutrient source other than the carbon source, inorganic salts, and other organic nutrient sources. Is preferred. Examples of the nitrogen source include ammonia, ammonium chloride, urea, ammonium sulfate, ammonium phosphate and other ammonium salts, as well as peptone, meat extract, yeast extract and the like. Examples of inorganic salts include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride and the like. Examples of other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, and proline, and vitamins such as vitamin B1, vitamin B12, and vitamin C.

In the production method of the present invention, the concentration of the alcohol in the medium is not particularly limited, but in order to efficiently produce the PHA of the present invention, the lower limit is preferably 0.01 g / L, and 0.05 g / L is more preferable, and 0.1 g / L is more preferable. The upper limit is preferably about 5 g / L, more preferably 3 g / L, and more preferably 2 g / L from the viewpoint of suppressing the influence on the growth of microorganisms as described above. Further, when mercapto alcohol is used as the alcohol, it is particularly preferable to use it at a concentration of 0.8 g / L or less.

Other conditions such as culture temperature, culture time, culture pH, medium, etc. may be those normally used in the microorganism of the present invention.

In the production method of the present invention, a method for recovering PHA from microbial cells is not particularly limited, and for example, the following method can be used. After completion of the culture, the cells are separated from the culture solution with a centrifuge, and the cells are washed with distilled water, methanol, or the like and dried. PHA is extracted from the dried cells using an organic solvent such as chloroform. Cellular components are removed from the organic solvent solution containing PHA by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA. Further, the supernatant is removed by filtration or centrifugation, and dried to recover PHA.

The PHA of the present invention is further rearranged, that is, chemically modified, by a chemical reaction via a specific alkenyl group, alkynyl group, thiol group, azide group, or allyl group introduced at the carboxy terminus. You can also. For example, when the PHA of the present invention has an alkenyl group or an alkynyl group at the carboxy terminus, a compound containing a thiol group can be rearranged by a thiol-ene click reaction or a thiol-in click reaction. Conversely, when the PHA of the present invention has a thiol group at the carboxy terminus, a compound containing an alkenyl group or an alkynyl group can be rearranged by a thiol-ene click reaction or a thiol-in click reaction. Alternatively, when the PHA of the present invention has an alkynyl group at the carboxy terminus, a compound containing an azide group can be rearranged by an alkyne-azido click reaction. Conversely, when the PHA of the present invention has an azide group at the carboxy terminus, a compound containing an alkynyl group can be rearranged by an alkyne-azido click reaction. Derivatives obtained by such chemical modification are also within the scope of the present invention. Furthermore, the PHA of this invention and the resin molding containing the compound which is the derivative of the said are also the category of this invention. In the said resin molding, the compound which is PHA of this invention and its derivative (s) may be used independently, and can also be mixed with a conventionally well-known PHA and used.

When the PHA of the present invention has a functional group only at the terminal of the polymer main chain, the product after the reaction can be limited, which is more preferable. For example, a PHA having a double bond in the side chain and a PHA having a thiol group at the carboxy terminus can be reacted to induce a graft polymer. Further, PHA having an alkene group at the carboxy terminus can be converted to a multibranched PHA by rearranging it to a thiol compound having a multibranched structure such as pentaerythritol tetrakis (3-mercaptopropionate). . Alternatively, a resin composition containing PHA having an alkene group introduced at the carboxy terminus may be processed into a film, sheet, nonwoven fabric, etc., and then the thiol compound is rearranged to modify the surface of the resin molded product. it can.

Furthermore, if a dendrimer is constituted using the PHA of the present invention, it is possible to provide medical materials with high biocompatibility and agricultural materials with moderate sustained release properties.

This application claims the benefit of priority based on Japanese Patent Application No. 2016-062278 filed on Mar. 25, 2016. The entire content of the specification of Japanese Patent Application No. 2016-062278 filed on March 25, 2016 is incorporated herein by reference.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples. In addition, the breeding of a strain, the analysis method of the monomer unit copolymerization ratio contained in PHA, and the analysis method of the molecular weight of PHA are as follows.

(Breeding of strains)
The genetic manipulations in the examples herein are described in Green, M. et al. R. and Sambrook, J. et al. (2012). In addition, enzymes, cloning hosts, etc. used for gene manipulation can be purchased from market suppliers and used in accordance with the instruction manual. In addition, the enzyme used for an Example etc. will not be specifically limited if it can be used for gene manipulation.

(Analyzing method of copolymerization ratio of monomer units contained in PHA)
The monomer unit copolymerization ratio contained in PHA was analyzed using NMR. Specifically, 2 mg of the obtained PHA was dissolved in 2 mL of deuterated chloroform and transferred to a sample tube for measurement. From the area of each detected peak, the copolymerization ratio of the monomer units was calculated.

(Method for analyzing molecular weight of PHA)
The molecular weight of PHA was analyzed by gel permeation chromatography. The filtrate obtained by dissolving PHA in chloroform at a concentration of 1.5 g / L and filtering through a Φ = 0.2 μm filter was used as an analysis sample. The measurement system used was a Shimadzu GPC system. The column was used with two Shodex GPC K-806L (Showa Denko) connected in series, and the column oven was set to 40 ° C. The mobile phase was chloroform and the flow rate was 1.0 mL / min. As the molecular weight standard, about 7 million, about 1.9 million, about 350,000, about 190,000, about 30,000, and about 2,000 polystyrene were used. A calibration curve was prepared from the analysis results of 6 samples, and the molecular weight of PHA (weight average molecular weight M _w and number average molecular weight M _n ) was calculated based on this.

(Confirmation of carboxy terminal structure of PHA)
^{Using 1} H-NMR, it was confirmed whether a predetermined group was introduced at the carboxy terminus. The measurement of ¹ H-NMR was carried out using a JEOL NMR instrument at room temperature under conditions of 500 MHz and about 256 SCAN times. Data analysis was performed using ALICE2 for windows ver. 4 was used. For the following Examples 8 and 16, specific results are shown in FIGS. For comparison, the result of Comparative Example 1 is also shown in FIG.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in a medium containing 0.2 g / L of 2-propen-1-ol As a microorganism, KNK-005 trc- described in WO2015 / 115619 is used. The phaJ4b / ΔphaZ1,2,6 strain was used. KNK-005 trcphaJ4b / ΔphaZ1,2,6 has a full-length deletion of the phaZ1 and phaZ6 genes on the chromosome, a deletion from the 16th codon to the stop codon of the phaZ2 gene, and the sequence number 4 on the chromosome. It is a strain having a mutant PHA synthase gene derived from the described Aeromonas caviae and having an expression regulatory sequence described in SEQ ID NO: 5 inserted upstream of the phaJ4b gene.

(culture)
The microorganism was cultured under the following conditions.

The composition of the seed medium is 10 g / L meat extract, 10 g / L bactotryptone, 2 g / L yeast extract, 9 g / L sodium dihydrogen phosphate dodecahydrate, 1.5 g / L dipotassium hydrogen phosphate and did.

The composition of the PHA production medium is 11 g / L disodium hydrogen phosphate 12 hydrate, 1.9 g / L dipotassium hydrogen phosphate, 1.3 g / L ammonium sulfate, 5 mL / L magnesium solution, 1 mL / L trace metal salt. It was set as the solution. The magnesium solution was prepared by dissolving 200 g / L magnesium sulfate heptahydrate in water. Trace metal salt solution is 0.218 g / L cobalt chloride hexahydrate, 16.2 g / L iron (III) chloride hexahydrate, 10.3 g / L calcium chloride dihydrate in 0.1N hydrochloric acid. 0.118 g / L nickel chloride hexahydrate and 0.156 g / L copper sulfate pentahydrate were prepared.

50 μL of the glycerol stock solution of the strain was inoculated into 10 mL of the seed mother medium, and cultured with shaking at 30 ° C. for 24 hours. The obtained culture broth was used as a pre-culture broth.

PHA production culture was performed in a flask. 50 mL of PHA production medium was placed in a 500 mL shake flask. Immediately before inoculation, 250 μL of magnesium solution, 50 μL of trace metal solution, 1 g of PKO were added, and 2-propen-1-ol was further added to 0.2 g / L. After the medium was prepared, 500 μL of the preculture was inoculated into the shake flask, and shake culture was performed at 30 ° C. for 72 hours.

(Purification)
After completion of the culture, the cells were collected by centrifugation, suspended in water, and sodium lauryl sulfate was added to a final concentration of 3% (w / v). The prepared microbial cell solution was treated with ultrasonic waves while cooling with ice to disrupt the microbial cells. PHA was collected as a precipitate from the disrupted cell solution by centrifugation, washed once with water and ethanol, and then dried in vacuo at 60 ° C. for 2 hours to obtain purified PHA.

The obtained PHA was analyzed for monomer unit copolymerization ratio and molecular weight. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in a medium containing 2-propen-1-ol at 1.0 g / L As in Example 1, KNK-005 trc-phaJ4b / ΔphaZ1 , 2 and 6 were cultured and purified to obtain purified PHA. However, the final concentration of 2-propen-1-ol added to the medium in the PHA production culture was 1.0 g / L. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in medium containing 0.2 g / L of 3-buten-1-ol As in Example 1, KNK-005 trc-phaJ4b / ΔphaZ1 , 2 and 6 were cultured and purified to obtain purified PHA. However, instead of 2-propen-1-ol in PHA production culture, 3-buten-1-ol was added to the medium to a final concentration of 0.2 g / L. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strain in a medium containing 1.0 g / L of 3-buten-1-ol As in Example 3, KNK-005 trc-phaJ4b / ΔphaZ1 , 2 and 6 were cultured and purified to obtain purified PHA. However, the final concentration of 3-buten-1-ol added to the medium in the PHA production culture was 1.0 g / L. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in a medium containing 0.2 g / L of 5-hexen-1-ol As in Example 1, KNK-005 trc-phaJ4b / ΔphaZ1 , 2 and 6 were cultured and purified to obtain purified PHA. However, instead of 2-propen-1-ol in PHA production culture, 5-hexen-1-ol was added to the medium to a final concentration of 0.2 g / L. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in a medium containing 1.0 g / L of 5-hexen-1-ol As in Example 5, KNK-005 trc-phaJ4b / ΔphaZ1 , 2 and 6 were cultured and purified to obtain purified PHA. However, the final concentration of 5-hexen-1-ol added to the medium was 1.0 g / L. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in medium containing 0.2 g / L of 2-propyn-1-ol Similar to Example 1, KNK-005 trc-phaJ4b / ΔphaZ1 , 2 and 6 were cultured and purified to obtain purified PHA. However, in the PHA production culture, 2-propyn-1-ol was added to the medium to a final concentration of 0.2 g / L instead of 2-propen-1-ol. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in a medium containing 2-propyn-1-ol 1.0 g / L As in Example 7, KNK-005 trc-phaJ4b / ΔphaZ1 , 2 and 6 were cultured and purified to obtain purified PHA. However, the final concentration of 2-propyn-1-ol added to the medium was 1.0 g / L. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in medium containing 0.2 g / L of 3-butyn-1-ol As in Example 1, KNK-005 trc-phaJ4b / ΔphaZ1 , 2 and 6 were cultured and purified to obtain purified PHA. However, instead of 2-propen-1-ol in the PHA production culture, 3-butyn-1-ol was added to the medium to a final concentration of 0.2 g / L. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in medium containing 1.0 g / L of 3-butyn-1-ol As in Example 9, KNK-005 trc-phaJ4b / ΔphaZ1 , 2 and 6 were cultured and purified to obtain purified PHA. However, the final concentration of 3-butyn-1-ol added to the medium was 1.0 g / L. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

Production of PHA by strain KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in a medium containing 0.2 g / L of 5-hexyn-1-ol As in Example 1, KNK-005 trc-phaJ4b / ΔphaZ1 , 2 and 6 were cultured and purified to obtain purified PHA. However, instead of 2-propen-1-ol in PHA production culture, 5-hexyn-1-ol was added to the medium to a final concentration of 0.2 g / L. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in medium containing 1.0 g / L of 5-hexyn-1-ol Similar to Example 11, KNK-005 trc-phaJ4b / ΔphaZ1 , 2 and 6 were cultured and purified to obtain purified PHA. However, the final concentration of 5-hexyn-1-ol added to the medium was 1.0 g / L. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in medium containing 0.2 g / L of 2-mercaptoethanol As in Example 1, KNK-005 trc-phaJ4b / ΔphaZ1,2, Six strains were cultured and purified to obtain purified PHA. However, in the PHA production culture, 2-mercaptoethanol was added to the medium to a final concentration of 0.2 g / L instead of 2-propen-1-ol. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in a medium containing 0.2 g / L of 3-mercaptopropanol Similar to Example 1, KNK-005 trc-phaJ4b / ΔphaZ1,2, Six strains were cultured and purified to obtain purified PHA. However, in the PHA production culture, 3-mercaptopropanol was added to the medium to a final concentration of 0.2 g / L instead of 2-propen-1-ol. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in a medium containing 0.2 g / L of ethylene glycol monoallyl ether As in Example 1, KNK-005 trc-phaJ4b / ΔphaZ1,2 6 strains were cultured and purified to obtain purified PHA. However, in the PHA production culture, ethylene glycol monoallyl ether was added to the medium to a final concentration of 0.2 g / L instead of 2-propen-1-ol. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in a medium containing 1.0 g / L of ethylene glycol monoallyl ether As in Example 15, KNK-005 trc-phaJ4b / ΔphaZ1,2 6 strains were cultured and purified to obtain purified PHA. However, the final concentration of ethylene glycol monoallyl ether added to the medium was 1.0 g / L. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

(Comparative Example 1) Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in a medium not containing a specific alcohol or the like, except that 2-propen-1-ol is not added to the medium in the PHA production culture In the same manner as in Example 1, KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strains were cultured and purified to obtain purified PHA. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

(Comparative Example 2) Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strain in a medium containing 0.2 g / L of 2-aminoethanol hydrochloride As in Example 1, KNK-005 trc -PhaJ4b / ΔphaZ1,2,6 strains were cultured and purified to obtain purified PHA. However, in the PHA production culture, 2-aminoethanol hydrochloride was added to the medium to a final concentration of 0.2 g / L instead of 2-propen-1-ol. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

Comparative Example 3 Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in a medium containing 2-aminoethanol hydrochloride 1.0 g / L As in Comparative Example 2, KNK-005 trc -PhaJ4b / ΔphaZ1,2,6 strains were cultured and purified to obtain purified PHA. However, the final concentration of 2-aminoethanol hydrochloride added to the medium was 1.0 g / L. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

(Comparative Example 4) Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in a medium containing 0.2 g / L of 3-aminopropanol hydrochloride As in Example 1, KNK-005 trc -PhaJ4b / ΔphaZ1,2,6 strains were cultured and purified to obtain purified PHA. However, in the PHA production culture, 3-aminopropanol hydrochloride was added to the medium to a final concentration of 0.2 g / L instead of 2-propen-1-ol. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

(Comparative Example 5) Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in a medium containing 2-aminoethanol hydrochloride 1.0 g / L As in Comparative Example 4, KNK-005 trc -PhaJ4b / ΔphaZ1,2,6 strains were cultured and purified to obtain purified PHA. However, the final concentration of 3-aminopropanol hydrochloride added to the medium was 1.0 g / L. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

(Comparative Example 6) Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strain in a medium containing 0.2 g / L of 1,2-ethanedithiol As in Example 1, KNK-005 trc -PhaJ4b / ΔphaZ1,2,6 strains were cultured and purified to obtain purified PHA. However, instead of 2-propen-1-ol in the PHA production culture, 1,2-ethanedithiol was added to the medium to a final concentration of 0.2 g / L. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

(Comparative Example 7) Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 in a medium containing 0.2 g / L of 1,3-propanedithiol As in Example 1, KNK-005 trc -PhaJ4b / ΔphaZ1,2,6 strains were cultured and purified to obtain purified PHA. However, in the PHA production culture, 1,3-propanedithiol was added to the medium to a final concentration of 0.2 g / L instead of 2-propen-1-ol. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 1.

(Results and discussion)
From the results of Table 1, in Examples 1 to 16 in which PHA-producing microorganisms were cultured by adding an alcohol having an alkynyl group, alkenyl group, thiol group or allyl group to the medium, Comparative Example 1 in which these alcohols were not added The molecular weight of the obtained PHA was decreased both in the weight average molecular weight (M _w ) and the number average molecular weight (M _n ), so that the added alcohol functions as a terminator. It can be inferred that PHA into which a group, an alkynyl group, a thiol group or an allyl group is introduced is produced. However, when 5-hexen-1-ol was added, a significant decrease in molecular weight of PHA was observed only when the concentration in the medium was 1 g / L.

1 and 2 are ¹ H-NMR charts of Examples 8 and 16, respectively. For comparison, the ¹ H-NMR chart of Comparative Example 1 is shown in FIG. Compared to FIG. 3, in FIG. 1, a peak attributed to methylene protons in the propynyl group appears around 4.7 ppm, and in FIG. 2, a peak attributed to the allyl group appears around 4.5 ppm and 5.9 ppm. It appears that these groups have been introduced.

On the other hand, in Comparative Examples 2 to 5, no decrease in the molecular weight of PHA was observed even when 2-aminoethanol or 3-amino-1-propanol was added to the medium. This result is considered that these amino alcohols were metabolized by microorganisms. Also in Comparative Examples 6 and 7, even when 1,2-ethanedithiol or 1,3-propanedithiol was added to the medium, no decrease in the molecular weight of PHA was observed, and dithiol would not function as a terminator. .

From the above results, the microorganism having the PHA synthase gene derived from the genus Aeromonas is cultured by adding an alcohol having an alkynyl group, an alkenyl group, a thiol group or an allyl group to the carboxy terminus, It was suggested that PHA introduced with an alkynyl group, a thiol group or an allyl group can be produced.

(Production Example 1) Production of H16 ΔphaZ1,2,6 strain In producing H16ΔphaZ1,2,6 strain, first, based on KNK-005 ΔphaZ1,2,6 strain described in WO2014 / 065253, PHA synthase H16 ΔphaC1 ΔphaZ1,2,6 strains in which the gene was disrupted were prepared by the following procedure. The KNK-005 ΔphaZ1,2,6 strain has a full-length deletion of the phaZ1 and phaZ6 genes on the chromosome, a deletion from the 16th codon to the stop codon of the phaZ2 gene, and the sequence described in SEQ ID NO: 5 on the chromosome. A strain having a PHA synthase gene.

First, a plasmid for full-length deletion of the PHA synthase gene of KNK-005 ΔphaZ1, 2, 6 strain was prepared. C. PCR was performed using the genomic DNA of neator H16 strain as a template and the DNAs shown in SEQ ID NOs: 6 and 7 as primer pairs. As the polymerase, KOD-plus (Toyobo) was used. Similarly, PCR was performed using the DNAs shown in SEQ ID NO: 8 and SEQ ID NO: 9 as primer pairs. PCR was performed under the same conditions using the two types of DNA fragments obtained by the above PCR as templates and the DNAs shown in SEQ ID NO: 6 and SEQ ID NO: 9 as primer pairs, and the resulting DNA fragments were digested with the restriction enzyme SmiI. did. This DNA fragment was ligated with a DNA fragment obtained by digesting the vector pNS2X-sacB described in JP-A-2007-259708 with SmiI using DNA ligase (Ligation High, Toyobo), upstream of the phaC1 gene. And a PHA synthase gene-disrupting plasmid pNS2X-sacB-ΔphaC1UL having the nucleotide sequence of and the downstream of the phaC1 gene.

This plasmid for disrupting the PHA synthase gene pNS2X-sacB-ΔphaC1UL was introduced into E. coli S17-1 strain (ATCC47055), mixed and cultured on KNK-005 ΔphaZ1,2,6 strain on Nutrient Agar medium (DIFCO). Went.

Simmons agar medium containing 2 mg / L of kanamycin sulfate (sodium citrate 2 g / L, sodium chloride 5 g / L, magnesium sulfate heptahydrate 0.2 g / L, dihydrogen phosphate) A strain that grows on ammonium 1 g / L, dipotassium hydrogen phosphate 1 g / L, agar 15 g / L, pH 6.8) is selected, and the plasmid is integrated into the chromosome of KNK-005 ΔphaZ1,2,6. Acquired shares. This strain was cultured for 2 generations in Nutrient Broth medium (DIFCO), and then a strain that grew on Nutrient Agar medium containing 15% sucrose was selected. From the obtained strain, a strain in which the full-length PHA synthase gene described in SEQ ID NO: 5 on the chromosome was deleted was selected by PCR, and one strain was named H16 ΔphaC1, ΔphaZ1, 2, 6 strain. H16 ΔphaC1 ΔphaZ1,2,6 shares are C.I. The strain necator H16 is a parent strain, the phaZ1 gene and phaZ6 gene on the chromosome are deleted in full length, the 16th codon to the stop codon of the phaZ2 gene are deleted, and the phaC1 gene is deleted in full length.

Next, based on the obtained H16 ΔphaC1 ΔphaZ1, 2, 6 strain, C.I. H16ΔphaC1,2,6 strains in which the phaC1 gene derived from necator H16 strain was inserted were prepared by the following procedure.

First, C.I. A plasmid for inserting the phaC1 gene of necator H16 strain on the chromosome was prepared. C. PCR was performed using the genomic DNA of the necator H16 strain as a template and the DNAs shown in SEQ ID NOs: 6 and 9 as primer pairs. As the polymerase, KOD-plus was used. The obtained DNA fragment was digested with the restriction enzyme SmiI. This DNA fragment is ligated with a DNA fragment obtained by digesting pNS2X-sacB with SmiI using DNA ligase, and has a base sequence upstream from the phaC1 gene, a phaC1 gene, and a base sequence downstream from the phaC1 gene A plasmid pNS2X-sacB-phaC _Re + UL for disrupting the PHA synthase gene was prepared.

In the same procedure as in the above PHA synthase gene disruption, a strain in which pNS2X-sacB-phaC _Re + UL was integrated on the chromosome was obtained with the H16 ΔphaC1 ΔphaZ1,2,6 strain as the parent strain. This strain was cultured for 2 generations in Nutrient Broth medium, and then a strain that grew on Nutrient Agar medium containing 15% sucrose was selected. From the obtained strain, a strain into which the phaC1 gene on the chromosome was inserted was selected by PCR, and one strain was named H16 ΔphaZ1,2,6 strain. H16 ΔphaZ1,2,6 strains are C.I. Necator H16 strain as parent strain, phaZ1 gene and phaZ6 gene on the chromosome are deleted in full length, the 16th codon to stop codon of phaZ2 gene is deleted, and a gene encoding a wild-type PHA synthase derived from Ralstonia eutropha On the chromosome.

(Production Example 2) Production of ReSK003 strain Based on the H16 ΔphaC1 ΔphaZ1, 2, 6 strain described in Production Example 1, the ReSK003 strain in which the PHA synthase gene consisting of the base sequence described in SEQ ID NO: 11 is inserted on the chromosome Was made. The PHA synthase gene consisting of the base sequence set forth in SEQ ID NO: 11 is Pseudomonas Sp. A PHA synthase consisting of the amino acid sequence of SEQ ID NO: 3 derived from 61-3, wherein the 325th serine is artificially replaced with threonine, the 477th serine with arginine, and the 481st glutamine with arginine. It is a gene that encodes.

First, under the expression control sequence described in SEQ ID NO: 12, Pseudomonas Sp. Plasmid pCUP2-REP-phaC1 _Ps expressing the PHA synthase gene derived from the 61-3 strain was prepared. The expression control sequence described in SEQ ID NO: 11 is C.I. This is the promoter of the phaCAB operon derived from the necator H16 strain. C. PCR was carried out using the genomic DNA of necator H16 strain as a template and the DNAs shown in SEQ ID NOs: 14 and 15 as primer pairs. As the polymerase, KOD-plus was used. Similarly, Pseudomonas Sp. PCR was performed using the genomic DNA of the 61-3 strain as a template and the DNAs shown in SEQ ID NOs: 16 and 17 as primer pairs. PCR was carried out under the same conditions using the two types of DNA fragments obtained by the above PCR as templates and the DNAs shown in SEQ ID NO: 14 and SEQ ID NO: 17 as primer pairs, and the resulting DNA fragments were converted into restriction enzymes MunI and SpeI. Digested with. This DNA fragment, a DNA fragment obtained by digesting the vector PCUP2 described in JP 2007-259708 by MunI and SpeI, ligated with DNA ligase to prepare the plasmid pCUP2-REP-phaC1 _Ps .

Next, PCR was performed using pCUP2-REP-phaC1 _Ps as a template and the DNAs shown in SEQ ID NOs: 14 and 18 as primer pairs. Similarly, PCR was performed using the DNAs shown in SEQ ID NO: 19 and SEQ ID NO: 20 as primer pairs. Similarly, PCR was performed using the DNAs shown in SEQ ID NO: 21 and SEQ ID NO: 17 as primer pairs. PCR was carried out under the same conditions using the three types of DNA fragments obtained by the above PCR as templates and the DNAs shown in SEQ ID NO: 14 and SEQ ID NO: 17 as primer pairs, and the obtained DNA fragments were subjected to restriction enzymes MunI and SpeI. Digested with. This DNA fragment was ligated with a DNA fragment obtained by digesting the vector pCUP2 described in JP-A-2007-259708 with MunI and SpeI using DNA ligase, and the plasmid pCUP2-REP-phaC1 _Ps ^{S325T, S477R Thus, Q481R} was fabricated.

Next, a plasmid for inserting the PHA synthase gene described in SEQ ID NO: 11 onto the chromosome was prepared. PCR was performed using pCUP2-REP-phaC1 _Ps ^{S325T, S477R, and Q481R} as templates and the DNAs shown in SEQ ID NOs: 6 and 22 as primer pairs. As the polymerase, KOD-plus was used. Similarly, PCR was performed using the DNAs represented by SEQ ID NO: 23 and SEQ ID NO: 9 as primer pairs. PCR was performed under the same conditions using the two types of DNA fragments obtained by the above PCR as templates and the DNAs shown in SEQ ID NO: 6 and SEQ ID NO: 9 as primer pairs, and the resulting DNA fragments were digested with the restriction enzyme SmiI. did. This DNA fragment was ligated with a DNA fragment obtained by digesting pNS2X-sacB with SmiI using DNA ligase, a base sequence upstream from the phaC1 gene, the PHA synthase gene described in SEQ ID NO: 11, and the phaC1 gene A plasmid for destroying PHA synthase gene pNS2X-sacB-ΔphaC1UL :: STSRQR having a downstream base sequence was prepared.

In the same manner as the PHA synthase gene insertion described in Production Example 1, the P16 synthase gene described in SEQ ID NO: 11 was obtained using pNS2X-sacB-ΔphaC1UL :: STSRQR with the H16 ΔphaC1 ΔphaZ1,2,6 strain as the parent strain. Inserted on the chromosome. The obtained strain was named ReSK003 strain. The ReSK003 strain is a strain in which the mutant PHA synthase gene derived from the genus Pseudomonas described in SEQ ID NO: 11 is inserted on the chromosome, with the H16 ΔphaC1 ΔphaZ1,2,6 strain as the parent strain.

Production of PHA by H16 ΔphaZ1,2,6 strain in medium containing 1.0 g / L of 2-propen-1-ol H16 ΔphaZ1,2,6 prepared in Production Example 1 in the same manner as in Example 1. The strain was cultured and purified to obtain purified PHA. However, the final concentration of 2-propen-1-ol added to the medium was 1.0 g / L. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 2.

Production of PHA by H16 ΔphaZ1,2,6 strain in a medium containing 2-propyn-1-ol 1.0 g / L H16 ΔphaZ1,2,6 prepared in Production Example 1 in the same manner as in Example 15. The strain was cultured and purified to obtain purified PHA. However, in the PHA production culture, 2-propin-1-ol was added to the medium so that the final concentration was 1.0 g / L instead of 2-propen-1-ol. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 2.

(Comparative Example 8) Production of PHA by H16 ΔphaZ1,2,6 in a medium not containing a specific alcohol Example 15 except that 2-propen-1-ol was not added to the medium in the PHA production culture. In the same manner, the H16ΔphaZ1,2,6 strain prepared in Production Example 1 was cultured and purified to obtain purified PHA. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 2.

(Results and discussion)
From the results of Table 2, in Examples 17 and 18 in which 2-propen-1-ol or 2-propin-1-ol was added to the medium, the molecular weight of PHA was compared to Comparative Example 8 in which these alcohols were not added. Since both M _w and M _n decreased, it can be inferred that the added alcohol functions as a terminator, and as a result, a PHA having a propenyl group or a propynyl group introduced at the carboxy terminus is produced. From the above results, PHA in which an alkenyl group or alkynyl group is introduced at the carboxy terminus by culturing a microorganism having a PHA synthase gene derived from Ralstonia genus by adding an alcohol having an alkynyl group or alkenyl group to the medium. It was suggested that can be produced.

Production of PHA by ReSK003 strain in a medium containing 2-propen-1-ol at 1.0 g / L In the same manner as in Example 1, the ReSK003 strain prepared in Production Example 2 is cultured and purified to obtain purified PHA did. However, the final concentration of 2-propen-1-ol added to the medium was 1.0 g / L. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 3.

Production of PHA by ReSK003 strain in a medium containing 1.0 g / L of 2-propyn-1-ol In the same manner as in Example 19, the ReSK003 strain prepared in Production Example 1 is cultured and purified to obtain purified PHA did. However, in the PHA production culture, 2-propyn-1-ol was added to the medium to a final concentration of 1.0 g / L instead of 2-propen-1-ol. About the obtained PHA, the monomer unit copolymerization ratio and the molecular weight were analyzed. The results are shown in Table 3.

(Comparative Example 9) Production of PHA by ReSK003 strain in a medium not containing special alcohol Production by the same method as in Example 17 except that 2-propen-1-ol was not added to the medium in the PHA production culture. The ReSK003 strain prepared in Example 1 was cultured and purified to obtain purified PHA. The obtained PHA was analyzed for the monomer unit copolymerization ratio and molecular weight. The results are shown in Table 3.

(Results and discussion)
From the results of Table 3, in Examples 19 and 20 to which 2-propen-1-ol or 2-propyn-1-ol was added, the molecular weight of PHA was M _w compared to Comparative Example 9 to which these alcohols were not added. Since both _{Mn and Mn} decreased, it can be inferred that the added alcohol functions as a terminator, and as a result, PHA having a propenyl group or a propynyl group introduced at the carboxy terminus is produced. From the above results, an alkenyl group or alkynyl group was introduced at the carboxy terminus by culturing a microorganism having a PHA synthase gene derived from the genus Pseudomonas by adding an alcohol having an alkynyl group or alkenyl group to the medium. It was suggested that PHA can be produced.

Claims (13)

1. The repeating unit is represented by the following general formula (1)
   [—C ^* HR ¹ —CH ₂ —CO—O—] (1)
   (In the formula, R ¹ is an alkyl group represented by C _n H _{2n + 1} , n is an integer of 1 to 15, and * represents an asymmetric carbon.)
   R-3-hydroxyalkanoic acid homopolymer or copolymer represented by the following, wherein the following groups are bonded to the carboxy terminus depending on whether the polymer is a homopolymer or a copolymer Polyhydroxyalkanoic acid.
   (1) a group bonded to the carboxy terminus when it is a single polymer, an alkynyl group having 4 to 8 carbon atoms,
   An alkenyl group having 3 to 8 carbon atoms,
   A mercaptoalkyl group having 2 or 4 to 8 carbon atoms,
   An azido alkyl group having 3 to 8 carbon atoms, or an allyl (poly) oxyalkyl group having 2 to 6 carbon atoms in the alkyl group,
   (2) a group bonded to the carboxy terminus when being a copolymer, an alkynyl group having 3 to 8 carbon atoms,
   An alkenyl group having 3 to 8 carbon atoms,
   A mercaptoalkyl group having 2 to 8 carbon atoms,
   An azido alkyl group having 3 to 8 carbon atoms, or an allyl (poly) oxyalkyl group having an alkyl group having 2 to 6 carbon atoms
2. A microbially produced polyhydroxyalkanoic acid having an alkyne group, alkene group, thiol group or azide group introduced at the carboxy terminus;
   The hydroxyalkanoic acid is 3-hydroxybutyric acid, 3-hydroxypropionic acid, 4-hydroxybutyric acid, 3-hydroxyvaleric acid, 5-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 6-hydroxyhexanoic acid, 3-hydroxyheptanoic acid , Polyhydroxyalkanoic acid composed of a plurality of kinds selected from 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid and 3-hydroxydodecanoic acid.
3. A microbially produced polyhydroxyalkanoic acid having an alkyne group, alkene group, thiol group or azide group introduced at the carboxy terminus;
   The hydroxyalkanoic acid is 3-hydroxybutyric acid, 3-hydroxypropionic acid, 4-hydroxybutyric acid, 3-hydroxyvaleric acid, 5-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 6-hydroxyhexanoic acid, 3-hydroxyheptanoic acid , Composed of a single species selected from 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxydodecanoic acid,
   The alkyne group is a butynyl group, a pentynyl group, or a hexynyl group;
   The alkene group is a propenyl group, a butenyl group, a pentenyl group, or a hexenyl group;
   A polyhydroxyalkanoic acid in which the thiol group is a mercaptoethyl group, a mercaptobutyl group, a mercaptopentyl group, or a mercaptohexyl group.
4. The polyhydroxyalkanoic acid according to any one of claims 1 to 3, which contains at least 3-hydroxybutyric acid as a monomer unit.
5. The polyhydroxyalkanoic acid according to claim 4, further comprising 3-hydroxyhexanoic acid as a monomer unit.
6. A process for producing the polyhydroxyalkanoic acid according to any one of claims 1 to 5,
   Including culturing a microorganism capable of producing polyhydroxyalkanoic acid using an alcohol having an alkyne group, an alkene group, a thiol group, an azide group, or an allyl group; , An azido group, or a polyhydroxyalkanoic acid having an allyl group introduced therein.
7. Including culturing a microorganism belonging to the genus Cupriavidus capable of producing polyhydroxyalkanoic acid using an alcohol having 2 to 8 carbon atoms having an alkyne group, an alkene group, a thiol group, an azide group, or an allyl group, A method for producing a polyhydroxyalkanoic acid into which an alkyne group, an alkene group, a thiol group, an azide group, or an allyl group is introduced.
8. The method according to claim 6 or 7, wherein the alcohol is a primary alcohol.
9. The production method according to any one of claims 6 to 8, wherein the microorganism is a microorganism having a gene encoding a polyhydroxyalkanoate synthase derived from the genus Aeromonas, Ralstonia or Pseudomonas.
10. The method according to claim 8 or 9, wherein the microorganism is a microorganism belonging to the genus Cupriavidus.
11. The production method according to claim 7 or 10, wherein the microorganism is a transformant having a Cupriavidus necator as a host.
12. 6. A compound obtained by further chemically modifying the terminal alkyne group, alkene group, thiol group, azide group, or allyl group of the polyhydroxyalkanoic acid according to any one of claims 1 to 5.
13. A molded article containing the polyhydroxyalkanoic acid according to any one of claims 1 to 5 or the compound according to claim 12.

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