JP6990169B2 - Polyhydroxyalkanoic acid having a functional group at the carboxy terminal and its production method - Google Patents

Description

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

Polyhydroxyalkanoates (hereinafter abbreviated as "PHA") are thermoplastic polyesters produced and accumulated as energy storage substances in the cells of many microbial species. PHA produced by microorganisms from various natural carbon sources is completely biodegraded by microorganisms in soil and water and is therefore incorporated into the natural carbon cycle process. Therefore, it can be said that PHA is an environmentally friendly plastic that has almost no adverse effect on the ecosystem. In recent years, as social problems caused by synthetic plastics have become more serious from the viewpoints of environmental pollution, waste treatment, and petroleum resources, PHA has been attracting 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 a problem that it has high crystallinity, is hard and brittle due to its high crystallinity, and is rapidly thermally decomposed at a temperature near the melting point (180 ° C.), so that its melt processability is low and its practical range is extremely limited. is doing.

Therefore, in order to reduce the crystallinity of PHB and improve brittleness, an attempt was made to introduce another 3-hydroxyalkanoic acid into the PHB skeleton. For example, side chains such as 3-hydroxypropionic acid (hereinafter abbreviated as "3HP"), 4-hydroxybutyric acid (hereinafter abbreviated as "4HB"), and 5-hydroxyvaleric acid (hereinafter abbreviated as "5HV") are used. PHB for linear monomers that do not have, and monomers that have side chains such as lactic acid, 3-hydroxyvaleric acid (hereinafter abbreviated as "3HV"), and 3-hydroxyhexanoic acid (hereinafter abbreviated as "3HHx"). 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 thereof, but basically any monomer is introduced, and the crystallinity of PHB is lowered, so that the melt processability is higher than that of PHB. Improve.

In order to further expand the use of PHA, it is necessary to develop a technology for producing a new PHA having physical characteristics significantly different from those of PHA as described above. For that purpose, it is considered effective not only to simply change the length of the PHA main chain and the size of the linear chain, but also to introduce some functional group into the side chain or the carboxy terminal of PHA.

As an example of introducing a functional group into the side chain of PHA, Patent Document 1 has reported that a thioester was introduced into the side chain of a medium-chain PHA having 6 to 14 carbon atoms. Further, in Non-Patent Document 1, a branched chain alkyl group, a cyclohexyl group, an alkyl halide, an acetoxy group, an ester, an alcoholic group, an epoxy group, a thiol group, and a cyano are provided on the side chain of a medium chain PHA having 6 to 14 carbon atoms. Examples of introducing aromatic ring compounds such as a group, a nitro group, a phenyl group and a benzoyl group are summarized.

The introduction of these functional groups is important not only to significantly change the physical properties of PHA, but also to provide a reaction starting point for further chemically modifying the functional group. For example, Non-Patent Document 3 reports that a fluorescent substance or peptide was modified in the double bond of the PHA side chain by using a thiol-enclick reaction. Further, in Non-Patent Document 4, it is reported that a hydroxy group or a carboxy group was introduced into the double bond of the PHA side chain by using a thiol-enclick reaction to change the water repellency of PHA. Further, Non-Patent Document 4 also reports that PHA could be crosslinked by reacting with a multi-branched structure compound. Further, Non-Patent Document 5 reports an example in which a PHA having an azido group in a side chain is produced and the side chain is modified by using an alkyne-azidoclick reaction.

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

On the other hand, Non-Patent Document 6 reports an example in which a functional group is introduced at the carboxy terminal of PHA. Specifically, Non-Patent Document 6 describes Bacillus microorganisms having a type IV PHA synthase gene in the presence of 1.3-propanediol, 2-propin-1-ol, 3-mercapto-1-propanol, and benzyl alcohol. When cultured in, 1-propanol group, 1-propynyl group, 1-propanethiol group, and benzyl group are introduced into the carboxy terminal of PHB, and the molecular weight may be reduced as compared with the case of culturing in the absence of these. It has been reported. Further, it is also reported in Non-Patent Document 6 that no decrease in molecular weight was observed even when cultured in the presence of 2-propen-1-ol or 3-butyne-1-ol.

WO2012 / 038772

Marta Tortajada, Luiziana Ferreira da Silva, Maria Auxiliadora Prioro, International Microbiology, vol. 16, pp. 1-15, 2013 Henry E. Valentin, Pierre A. Berger, Kennes J. et al. Gruys, Maria Filomena de Andrade Rodrigues, Alexander Steinbuchel, Muntian Tran, Jawed Karim, Macromolecules, vol. 32, pp. 7389-7395, 1999 Kenji Tajima, Kosuke Iwamoto, Yoshiharu Sato, Ryosuke Sakai, Toshifumi Satoh, Tohru Dairi, Applied Microbiology. 100, pp. 4375-4383, 2016 Alex C. Levine, Graham W. Heberlig, Christopher T. et al. Nomura, International Journal of Biological Journals, vol. 83, pp. 358-365, 2016 Atahualpa Pinto, Jessesica H. et al. Ciesla, Adriana Paulucci, Bradley P.M. Striff, Christopher T. et al. Nomura, ACS Macro Letters, Vol. 5, pp. 215-219, 2016 Manami Hyakutake, Satoshi Tomizawa, Imai Sugahara, Emi Murata, Kouhei Mizuno, Hideki Abe, Takeharu Tsuge, Polymer. 117, pp. 90-96, 2015

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

As a result of diligent research to solve the above problems, the present inventors have succeeded in producing PHA containing a specific functional group at the carboxy terminal instead of the side chain of PHA, and have completed the present invention. rice field.
The details of the present invention are as follows.
1. 1. The repeating unit is the following general formula (1)
[-C ^* HR ¹ -CH ₂ -CO-O-] ・ ・ ・ (1)
(In the equation, R ¹ is an alkyl group represented by C _n H _{2n + 1} , n is an integer of 1 to 15, and * indicates an asymmetry.)
It is a single or copolymer polymer of R-3-hydroxyalkanoic acid represented by, and the group shown below is bonded to the carboxy terminal depending on whether the polymer is a single polymer or a copolymer polymer. Polyhydroxyalkanoic acid.
(1) A group that binds to the carboxy terminal when it is a single polymer, an alkynyl group having 4 to 8 carbon atoms,
Alkenyl group with 3 to 8 carbon atoms,
A mercaptoalkyl group having 2, 4 to 8 carbon atoms,
Azidated alkyl group having 3 to 8 carbon atoms, or an allyl (poly) oxyalkyl group having 2 to 6 carbon atoms (2) A group having 3 to 8 carbon atoms bonded to the carboxy terminal when it is a copolymer polymer. Alkinyl group,
Alkenyl group with 3 to 8 carbon atoms,
A mercaptoalkyl group with 2 to 8 carbon atoms,
2. Azylated alkyl group having 3 to 8 carbon atoms or allyl (poly) oxyalkyl group having 2 to 6 carbon atoms. A microbially produced polyhydroxyalkanoic acid in which an alkyne group (synonymous with an alkynyl group), an alkene group (synonymous with an alkenyl group), a thiol group (mercapto group) or an azido group is introduced at the carboxy terminal.
The hydroxyalkanoic acid is 3-hydroxybutyric acid, 3-hydrochipropionic acid, 4-hydroxybutyric acid, 3-hydroxyvaleric acid, 5-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 6-hydroxyhexanoic acid, 3-hydroxyheptanoic acid. , 3-Hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxydodecanoic acid.
3. 3. A microbially produced polyhydroxyalkanoic acid having an alkyne group, an alkene group, a thiol group or an azide group introduced at the carboxy terminal.
The hydroxyalkanoic acid is 3-hydroxybutyric acid, 3-hydrochipropionic acid, 4-hydroxybutyric acid, 3-hydroxyvaleric acid, 5-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 6-hydroxyhexanoic acid, 3-hydroxyheptanoic acid. , 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 1 to 3 above, which comprises at least 3-hydroxybutyric acid as a monomer unit.
5. The polyhydroxyalkanoic acid according to 4 above, which further contains 3-hydroxyhexanoic acid as a monomer unit.
6. The method for producing the polyhydroxyalkanoate according to any one of 1 to 5 above, wherein the polyhydroxyalkanoic acid is produced.
An alkyne group, an alkene group, a thiol group at the carboxy terminus, which comprises a step of culturing a microorganism capable of producing polyhydroxyalkanoic acid using an alcohol having an alkyne group, an alkene group, a thiol group, an azido group, or an allyl group. , A method for producing a polyhydroxyalkanoic acid into which an azido group or an allyl group has been introduced.
7. A carboxy terminus comprising the step of culturing a microorganism belonging to the genus Cupriavidus capable of producing polyhydroxyalkanoic acid using an alcohol having an alkyne group, an alkene group, a thiol group, an azido group, or an allyl group and having 2 to 8 carbon atoms. A method for producing a polyhydroxyalkanoic acid into which an alkyne group, an alkene group, a thiol group, an azido group, or an allyl group is introduced.
8. The production method according to 6 or 7 above, wherein the alcohol is a primary alcohol.
9. The production method according to any one of 6 to 8 above, wherein the microorganism is a microorganism having a gene encoding a polyhydroxyalkanoate synthase derived from the genus Aeromonas, Ralstonia or Pseudomonas.
10. 8. The production method according to 8 or 9, wherein the microorganism is a microorganism belonging to the genus Cupriavidus.
11. The production method according to 7 or 10 above, wherein the microorganism is a transformant having Cupriavidus necator as a host.
12. A compound in which the alkyne group, alkene group, thiol group, azido group, or allyl group at the terminal of the polyhydroxyalkanoic acid according to any one of 1 to 5 above is further chemically modified.
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 novel PHA having a specific functional group at the carboxy terminal can be produced. The PHA of the present invention can be expected to improve the physical properties of the conventional PHA by using it as an additive for producing a molded product such as a film or a sheet, and also has various structures by further chemically modifying the functional group. It is thought that guidance to the body is possible.

FIG. 1 is a ¹ H-NMR chart of Example 8. 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.

In the PHA of the present invention, the repeating unit is the following general formula (1).
[-C ^* HR ¹ -CH ₂ -CO-O-] ・ ・ ・ (1)
(In the equation, 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.)
Is a single or copolymer polymer (hereinafter, these polymers are abbreviated as "P3HA") of R-3-hydroxyalkanoic acid (hereinafter, abbreviated as "3HA") represented by, and whether P3HA is a single polymer or co-polymer. The following groups are bonded to the carboxy terminal depending on whether the polymer is a polymer. Preferably, the following group is bonded to the ester bond containing the carboxy group, and the PHA has a triple bond, a double bond, a mercapto group (thiol group), and an allyl group at the terminal.
(1) A group bonded to the carboxy terminal 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, and a mercaptoalkyl group having 3 to 8 carbon atoms. Azylated alkyl group or allyl (poly) oxyalkyl group having 2 to 6 carbon atoms (2) Group bonded to the carboxy terminal when it is a copolymerized polymer Alkinyl group having 3 to 8 carbon atoms, 3 carbon atoms An alkenyl group to 8 to an alkenyl group, a mercaptoalkyl group having 2 to 8 carbon atoms, an azylated alkyl group having 3 to 8 carbon atoms, or an allyl (poly) oxyalkyl group having an alkyl group having 2 to 6 carbon atoms.

In addition, in the above-mentioned non-patent document 6, it is a homopolymer of 3HB having n = 1 in the above general formula (1), and a propynyl group (alkynyl group having 3 carbon atoms) and a mercaptopropyl group (alkynyl group having 3 carbon atoms) at the carboxy terminal thereof ( A thiol group having an alkyl group having 3 carbon atoms) is only disclosed, and the type of functional group is different from that of the present invention. In addition, the types of microorganisms used are different between the two.

That is, the PHA of the present invention is a polyester resin produced from a microorganism called P3HA, which has 3HA as a main monomer unit. Examples of the monomer unit constituting the PHA of the present invention include 3HB (n = ¹ of the alkyl group R1 represented by C _n H _{2n + 1} in the above general formula (1)), 3HV (n = 2). 3HHx (n = 3), 3-hydroxyheptanic 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 PHA of the present invention may be composed of a single species or may be composed of a plurality of species. When a plurality of types of monomer units are contained, two or more types of 3HA may be copolymerized, or one type or two or more types of 3HA may be copolymerized with 4-hydroxyalkanoic acid such as 4HB. It may be a polymer. 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 composed of only the above 3HB as a monomer unit, or may be a copolymer composed of 3HB and other monomer units. Examples of the monomer unit other than 3HB include, in addition to the above-mentioned monomer unit, 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 having 4HV further. (3HB-co-4HV) and the like. Of these, PHBH is preferable.

When the PHA of the present invention is a copolymer, the copolymerization ratio of each monomer unit 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, more preferably 70 mol% or more, and particularly preferably 80 mol% or more. 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%, still more preferably 3 mol%. The upper limit thereof is preferably 20 mol%, more preferably 15 mol%, 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 azido group, or an allyl group is introduced as a specific functional group at the carboxy terminal of the polymer main chain. And. The carboxy terminal of PHA of the present invention has an alkynyl group, an alkenyl group, a thiol group, and an azide group via an alkyl chain, and has an allyl group via an oxyalkyl chain.

Examples of the alkynyl group having 3 to 8 carbon atoms include a linear or branched propynyl group, a butynyl group, a pentynyl group, a hexynyl group, a heptynyl group, and an octynyl group. The one without branching, that is, the straight line is preferable. The number of carbon atoms is preferably 3 to 6, and among them, a propynyl group, a butynyl group, and a hexynyl group are preferable examples.

Examples of the alkenyl group having 3 to 8 carbon atoms include a linear or branched propenyl group, a butenyl group, a pentenyl group, a hexenyl group, a heptenyl group and an octenyl group. The one without branching, that is, the straight line is preferable. The number of carbon atoms is preferably 3 to 6, and among them, a propenyl group, a butenyl group, and a hexenyl group are preferable examples.

Examples of the mercaptoalkyl group having 2 to 8 carbon atoms include a linear or branched mercaptoethyl group, a mercaptopropyl group, a mercaptobutyl group, a mercaptopentyl group, a mercaptohexyl group, a mercaptoheptyl group and a mercaptooctyl group. The one without branching, that is, the straight line is preferable. 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 azized alkyl group having 3 to 8 carbon atoms include a linear or branched propyl azide group, a butyl azide group, a pentyl azide group, a hexyl azide group, and a heptyl azide group. The one without branching, that is, the straight line is preferable. The number of carbon atoms is preferably 3 to 6, and among them, the propyl azide group and the butyl azide group are preferable examples.

Examples of the allyl (poly) oxyalkyl group having 2 to 6 carbon atoms in the alkyl group include those having 1 to 3 oxyalkyls, preferably 1. Examples of the oxyalkyl group include an oxyethyl group, an oxypropyl group, an oxybutyl group, an oxypentyl group and an oxyhexyl group, and among them, an oxyethyl group, an oxypropyl group and an oxybutyl group are preferable examples. The one without branching, that is, the straight line is preferable. The total number of carbon atoms 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 terminal of the carboxyl group as described above, but the polymer side chain may also have a further functional group. However, when the PHA of the present invention is further chemically modified to synthesize a new derivative, it is preferable to have a functional group only at the terminal of the carboxyl group from the viewpoint of reaction control.

The molecular weight of PHA of the present invention is not limited, but when the following production method of the present invention 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, 8,000 to 3,000, or even 10,000 to 100,000 depending on the purpose of use. The number average molecular weight (M _n ) may be about 3000 to 150,000, about 5000 to 1,000,000 or even about 7,000 to 800,000 depending on the purpose of use.

As the above-mentioned method for producing PHA of the present invention, PHA is prepared by using an alcohol having an alkynyl group, an alkenyl group, a thiol group, an azido group, or an allylic group so that the specific functional group can be introduced at the carboxy terminal. Examples thereof include a method of culturing a synthesizable microorganism (hereinafter referred to as “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-propin-1-ol, 3-butyne-1-ol, 4-pentin-1-ol, 5-hexin-1-ol; 2-propen-1-ol, 3-. Butene-1-ol, 4-pentene-1-ol, 5-hexen-1-ol; 2-mercaptoethanol, 3-mercaptopropanol, 4-mercaptobutanol, 5-mercaptopentanol, 6-mercaptohexanol; 4- Azidobutane-1-ol, 5-azidpentane-1-ol, 6-azidohexane-1-ol; ethylene glycol monoallyl ether, propylene glycol monoallyl ether, tetramethylene glycol monoallyl ether, pentamethylene glycol monoallyl ether, etc. Can be mentioned. Among them, 2-propen-1-ol, 3-butene-1-ol, 5-hexene-1-ol, 2-propin-1-ol, 3-butyne-1-ol, 5-hexin-1-ol , 2-Mercaptoethanol, 3-Mercaptopropanol, ethylene glycol monoallyl ether and the like are preferred examples. 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, azido groups, or allyl groups may be used. In addition, 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 stop agent in the chain transfer reaction in PHA synthesis in the microbial cells. In that case, what kind of alcohol functions as a stop agent depends on the substrate specificity of the PHA synthase possessed by the microorganism of the present invention for alcohol. In the production method of the present invention, the above-mentioned microorganism of the present invention is a gene encoding a PHA synthase derived from Aeromonas (Aeromonas), Ralstonia (Ralstonia) or Pseudomonas (Pseudomonas) (hereinafter, "PHA synthase gene"). It is preferable that the microorganism has (abbreviated as), and more specifically, as the PHA synthase gene, the 149th asparagine derived from Aeromonas caviae (Aeromonas caviae) having the amino acid sequence set forth in SEQ ID NO: 1 is used. PHA synthase gene derived from Ralstonia europha (Ralstonia eutropha), which consists of the PHA synthase gene in which serine is artificially replaced with glisin at the 171st aspartic acid, and the amino acid sequence set forth in SEQ ID NO: 2, PHA synthase gene, SEQ ID NO: 3. Pseudomonas Sp. Consisting of the amino acid sequence described in 1. A more preferable example is a PHA synthase gene derived from 61-3 in which serine at position 325 is artificially replaced with threonine, serine at position 477 is replaced with arginine, and glutamine at position 481 is artificially replaced with arginine. Not limited to these. Since the PHA synthase derived from Aeromonas is known to use CoA hydroxyalkanoic acid having 3 to 6 carbon atoms as a substrate, by using a microorganism having a PHA synthase gene derived from Aeromonas, 3HB, 3HP, Homopolymers consisting of 4HB, 3HV, 5HV, and 3HHx, or copolymerized PHA consisting of these monomer units can be produced. Further, since the Ralstonia-derived PHA synthase is known to use hydroxyalkanoates CoA having 3 to 5 carbon atoms as a substrate, 3HB and 3HP can be used by using a microorganism having a Ralstonia-derived PHA synthase gene. Homopolymers consisting of 4, 4HB, 3HV, and 5HV, or copolymerized PHA consisting of these monomer units can be produced. Further, since it is known that the PHA synthase derived from the genus Pseudomonas uses CoA hydroxyalkanoate having 3 to 12 carbon atoms as a substrate, 3HB and 3HP can be used by using a microorganism having a PHA synthase gene derived from the genus Pseudomonas. , 4HB, 3HV, 5HV, 3HHx, 6HHx, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxydodecanoic acid. , Or a copolymerized PHA composed of these monomer units can be produced.

The microbial species of the present invention is not particularly limited and may be either a bacterium or a fungus. For example, the genus Acinetobacter, the genus Aeromonas, the genus Alcaligenes, the genus Allochromatium, the genus Azorhizobium, the genus AzotoBacter, the genus Azotobacter. Genus Delia, genus Candida, genus Caulobacter, genus Chromobacterium, genus Comamonas, genus Cupriavidus, genus Ectothiorhoda, genus Ectothyorhodospira Genus Klebsiella, Genus Metylobacterium, Genus Nocardia, Genus Paracoccus, Genus Pseudomonas, Genus Ralstonia, Genus Rhizobo, Rhizobium Rhodococcus (Rhodococcus) genus, Rhodospirillum (Rhodospirillum) species, rickettsia (rickettsia) genus, Saccharomyces (Saccharomyces) genus, Sinorhizobium (Sinorhizobium) genus, Sphingomonas (Sphingomonas) genus, Synechocystis (Synechocystis) genus, Thiococcus (Chiokokkasu) genus, Thiocystis Examples include microorganisms belonging to the genus (Chiokitis), Vibrio, Watersia, or Zoog / Loea. Among them, microorganisms belonging to the genus Aeromonas, Alcaligenes, Cupriavidus, Escherichia, Pseudomonas, Ralstonia and the like are preferable, and microorganisms belonging to the genus Cupriavidus, Escherycia, Ralstonia and the like are more preferable, and microorganisms belonging to the genus Cupriia are more preferable than those belonging to the genus Cupria. Particularly preferred as the microorganism of the present invention is Cupriavidus necator.

When the microorganism of the present invention does not originally have the PHA synthase gene, or when the PHA synthase gene originally possessed by the microorganism is not the desired PHA synthase gene, for example, the above-mentioned preferable PHA synthase gene is hosted by a gene recombination method. It is also possible to use a transformant introduced into the above-mentioned microorganism. As a method for introducing the PHA synthase gene into the host, it may be carried in a plasmid or introduced at an arbitrary position on the chromosome. At this time, it is preferable that the PHA synthase gene originally possessed by the host loses its function. As a method for deleting the function of the PHA synthase gene, for example, a method for deleting the PHA synthase gene over its entire length or partially, or adding or deleting a base to the PHA synthase gene. Alternatively, a method in which the function of the PHA synthase produced is lost by substitution may be mentioned. Specific methods for inserting or replacing DNA are described, for example, in Green, M. et al. R. and Sambrook, J. Mol. , 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, but if a large amount of the alcohol is used for culturing, there is a high possibility that the growth of microorganisms will be affected. Therefore, it is preferable to use the above alcohol in combination with other carbon sources. As the carbon source other than such 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 carbon sources are not particularly limited, but are preferably sugars such as glucose, fructose and sucrose, palm oil, palm kernel oil (hereinafter abbreviated as "PKO"), corn oil and palms. Fats and oils such as oils, olive oils, soybean oils, rapeseed oils and yatrofa oils, fractionated oils thereof or refined by-products thereof, fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid and myristic acid, and derivatives thereof are preferable. .. In addition, yeast extract, polypeptone, and the like can also be used. More preferably, vegetable oils and fats such as palm oil and palm kernel oil, or palm olein and palm double olein, which are low melting point fractions obtained by separating palm oil and palm kernel oil, or palm kernel oil olein and PFAD (palm oil fatty acid distillation). Products), PKFAD (palm kernel oil fatty acid distillate), refined by-products of fats and oils such as fatty acid distillates of rapeseed oil, and the refined by-products of fats and oils are particularly preferable from the viewpoint of avoiding competition with food.

In the production method of the present invention, the microorganism is cultured using a medium containing the above-mentioned alcohol, a carbon source other than alcohol, a nitrogen source which is a nutrient source other than the carbon source, inorganic salts, and other organic nutrient sources. Is preferable. Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, urea, ammonium sulfate and ammonium phosphate, as well as peptone, meat extract and yeast extract. Examples of the 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 thereof is preferably 0.01 g / L, preferably 0.05 g. / L is more preferable, and 0.1 g / L is more preferable. As the upper limit, as described above, from the viewpoint of suppressing the influence on the growth of microorganisms, about 5 g / L is preferable, 3 g / L is more preferable, and 2 g / L is more preferable. 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, pH at the time of culture, medium and the like may be the culture conditions usually used in the microorganism of the present invention.

In the production method of the present invention, the method for recovering PHA from microbial cells is not particularly limited, but can be carried out by, for example, the following method. After completion of the culture, the cells are separated from the culture solution by a centrifuge or the like, 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. The bacterial cell component is 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. In addition, the supernatant is removed by filtration or centrifugation and dried to recover PHA.

The PHA of the present invention is further transferred to an arbitrary compound by a chemical reaction via a specific alkenyl group, alkynyl group, thiol group, azido group, or allyl group introduced at the carboxy terminal, that is, chemically modified. You can also. For example, when the PHA of the present invention has an alkenyl group or an alkynyl group at the carboxy terminal, the compound containing a thiol group can be rearranged by a thiol-enclick reaction or a thiol-inclick reaction. Conversely, when the PHA of the present invention has a thiol group at the carboxy terminal, the compound containing an alkenyl group or an alkynyl group can be rearranged by a thiol-enclick reaction or a thiol-inclick reaction. Alternatively, when the PHA of the present invention has an alkynyl group at the carboxy terminal, the compound containing an azido group can be rearranged by an alkyne-azidoclick reaction. Conversely, when the PHA of the present invention has an azido group at the carboxy terminal, the compound containing an alkynyl group can be rearranged by an alkyne-azidoclick reaction. Derivatives obtained by such chemical modification are also within the scope of the present invention. Further, the PHA of the present invention and the resin molded body containing the compound which is the derivative thereof are also within the scope of the present invention. In the resin molded product, the PHA of the present invention or a compound which is a derivative thereof may be used alone, or may be mixed with a conventionally known PHA and used.

When the PHA of the present invention has a functional group only at the end of the polymer backbone, 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 terminal can be reacted to induce a graft polymer. Further, by rearranging a PHA having an alkene group at the carboxy terminal to a thiol compound having a multi-branched structure such as pentaerythritol tetrakis (3-mercaptopropionate), it can be induced to be a multi-branched PHA. .. Alternatively, the surface of the resin molded product can be modified by processing a PHA-containing resin composition having an alkene group introduced at the carboxy terminal into a film, sheet, non-woven fabric, or the like, and then rearranging the thiol compound. can.

Further, if the dendrimer is configured by using the PHA of the present invention, it is possible to provide a medical material having high biocompatibility and an agricultural material having an appropriate sustained release property.

This application claims the benefit of priority under Japanese Patent Application No. 2016-062278 filed on March 25, 2016. The entire contents of the specification of Japanese Patent Application No. 2016-062278 filed on March 25, 2016 are incorporated herein by reference.

Examples are shown below and the present invention will be described in more detail, but the present invention is not limited to these examples. The method for breeding the strain, the method for analyzing the monomer unit copolymerization ratio contained in PHA, and the method for analyzing 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. Mol. It can be done by the method described in (2012). In addition, enzymes used for genetic manipulation, cloning hosts, etc. can be purchased from market suppliers and used according to their instruction manuals. The enzyme used in Examples and the like is not particularly limited as long as it can be used for genetic manipulation.

(Analysis method of monomer unit copolymerization ratio 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 unit was calculated.

(Analysis method of molecular weight of PHA)
The molecular weight of PHA was analyzed by gel permission chromatography. The filtrate obtained by dissolving PHA in chloroform at a concentration of 1.5 g / L and filtering with a Φ = 0.2 μm filter was used as an analysis sample. The measurement system used was the GPC system of Shimadzu Corporation. The column was used with two Shodex GPC K-806L (Showa Denko) connected in series, and the column oven was set to 40 ° C. Chloroform was used as the mobile phase, and the flow rate was 1.0 mL / min. As the molecular weight specimen, 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 specimens, and the molecular weight of PHA (weight average molecular weight M _w and number average molecular weight M _n ) was calculated based on the calibration curve.

(Confirmation of carboxy terminal structure of PHA)
¹ It was confirmed by 1 H-NMR whether a predetermined group was introduced at the carboxy terminal. ^{1 1} H-NMR was measured using an NMR instrument manufactured by JEOL Ltd. at room temperature under the conditions of 500 MHz and 256 SCANs. Data analysis is performed by ALICE2 for windows ver. 4 was used. Specific results of Examples 8 and 16 below are shown in FIGS. 1 and 2. For comparison, the results of Comparative Example 1 are also shown in FIG.

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

(culture)
The above microorganisms were cultured under the following conditions.

The composition of the seed medium was 10 g / L meat extract, 10 g / L bactotrypton, 2 g / L yeast extract, 9 g / L sodium dihydrogen phosphate dodecahydrate, and 1.5 g / L dipotassium hydrogen phosphate. 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 made into a solution. The magnesium solution was prepared by dissolving 200 g / L magnesium sulfate heptahydrate in water. The trace metal salt solution is 0.1N hydrochloric acid, 0.218 g / L cobalt chloride hexahydrate, 16.2 g / L iron (III) chloride hexahydrate, 10.3 g / L calcium chloride dihydrate. , 0.118 g / L nickel chloride hexahydrate, 0.156 g / L copper sulfate pentahydrate was dissolved and prepared.

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

PHA production cultures were performed in flasks. 50 mL of PHA production medium was placed in a 500 mL volumetric shaker flask. Immediately before inoculation, 250 μL of magnesium solution, 50 μL of trace metal solution, 1 g of PKO was added, and 2-propen-1-ol was further added at 0.2 g / L. After preparing the medium, 500 μL of the preculture solution was inoculated into the shaking flask, and shaking 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 then sodium lauryl sulfate was added to a final concentration of 3% (w / v). The prepared bacterial cell solution was treated with ultrasonic waves while being ice-cooled to crush the bacterial cells. PHA was recovered as a precipitate from the disrupted bacterial cell solution by centrifugation, washed once with water and ethanol, and then vacuum dried at 60 ° C. for 2 hours to obtain purified PHA.

The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. 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 2-propen-1-ol KNK-005 trc-phaJ4b / ΔphaZ1 as in Example 1. , 2, 6 strains 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. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strain in a medium containing 0.2 g / L of 3-butene-1-ol KNK-005 trc-phaJ4b / ΔphaZ1 as in Example 1. , 2, 6 strains were cultured and purified to obtain purified PHA. However, in the PHA production culture, 3-butene-1-ol was added to the medium at a final concentration of 0.2 g / L instead of 2-propen-1-ol. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. 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-butene-1-ol KNK-005 trc-phaJ4b / ΔphaZ1 as in Example 3. , 2, 6 strains were cultured and purified to obtain purified PHA. However, the final concentration of 3-butene-1-ol added to the medium in the PHA production culture was 1.0 g / L. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strain in a medium containing 0.2 g / L of 5-hexene-1-ol KNK-005 trc-phaJ4b / ΔphaZ1 as in Example 1. , 2, 6 strains were cultured and purified to obtain purified PHA. However, in the PHA production culture, 5-hexene-1-ol was added to the medium at a final concentration of 0.2 g / L instead of 2-propen-1-ol. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. 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 5-hexene-1-ol KNK-005 trc-phaJ4b / ΔphaZ1 as in Example 5. , 2, 6 strains were cultured and purified to obtain purified PHA. However, the final concentration of 5-hexene-1-ol added to the medium was 1.0 g / L. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strain in a medium containing 0.2 g / L of 2-propyne-1-ol KNK-005 trc-phaJ4b / ΔphaZ1 as in Example 1. , 2, 6 strains were cultured and purified to obtain purified PHA. However, in the PHA production culture, 2-propyne-1-ol was added to the medium at a final concentration of 0.2 g / L instead of 2-propen-1-ol. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. 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 2-propyne-1-ol KNK-005 trc-phaJ4b / ΔphaZ1 as in Example 7. , 2, 6 strains were cultured and purified to obtain purified PHA. However, the final concentration of 2-propyne-1-ol added to the medium was 1.0 g / L. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strain in a medium containing 0.2 g / L of 3-butin-1-ol KNK-005 trc-phaJ4b / ΔphaZ1 as in Example 1. , 2, 6 strains were cultured and purified to obtain purified PHA. However, in the PHA production culture, 3-butyne-1-ol was added to the medium at a final concentration of 0.2 g / L instead of 2-propen-1-ol. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. 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-butin-1-ol KNK-005 trc-phaJ4b / ΔphaZ1 as in Example 9. , 2, 6 strains were cultured and purified to obtain purified PHA. However, the final concentration of 3-butyne-1-ol added to the medium was 1.0 g / L. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strain in a medium containing 0.2 g / L of 5-hexin-1-ol KNK-005 trc-phaJ4b / ΔphaZ1 as in Example 1. , 2, 6 strains were cultured and purified to obtain purified PHA. However, in the PHA production culture, 5-hexyne-1-ol was added to the medium at a final concentration of 0.2 g / L instead of 2-propen-1-ol. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. 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 5-hexyne-1-ol KNK-005 trc-phaJ4b / ΔphaZ1 as in Example 11. , 2, 6 strains were cultured and purified to obtain purified PHA. However, the final concentration of 5-hexyne-1-ol added to the medium was 1.0 g / L. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. The results are shown in Table 1.

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

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strain 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. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. The results are shown in Table 1.

Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strain in a medium containing 0.2 g / L of ethylene glycol monoallyl ether KNK-005 trc-phaJ4b / ΔphaZ1,2 as in Example 1. , 6 strains were cultured and purified to obtain purified PHA. However, in PHA production culture, ethylene glycol monoallyl ether was added to the medium so as to have a final concentration of 0.2 g / L instead of 2-propen-1-ol. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. 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 ethylene glycol monoallyl ether KNK-005 trc-phaJ4b / ΔphaZ1,2 as in Example 15. , 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. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. The results are shown in Table 1.

(Comparative Example 1) Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strain in a medium containing no specific alcohol, etc. In PHA production culture, 2-propen-1-ol is not added to the medium. In the same manner as in Example 1, KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strains were cultured and purified to obtain purified PHA. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. 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 KNK-005 trc as in Example 1. -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 so as to have a final concentration of 0.2 g / L instead of 2-propen-1-ol. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. The results are shown in Table 1.

(Comparative Example 3) Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strain in a medium containing 1.0 g / L of 2-aminoethanol hydrochloride KNK-005 trc as in Comparative Example 2. -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. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. The results are shown in Table 1.

(Comparative Example 4) Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strain in a medium containing 0.2 g / L of 3-aminopropanol hydrochloride KNK-005 trc as in Example 1. -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 so as to have a final concentration of 0.2 g / L instead of 2-propen-1-ol. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. The results are shown in Table 1.

(Comparative Example 5) Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strain in a medium containing 1.0 g / L of 2-aminoethanol hydrochloride KNK-005 trc as in Comparative Example 4. -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. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. 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 KNK-005 trc as in Example 1. -PhaJ4b / ΔphaZ1,2,6 strains were cultured and purified to obtain purified PHA. However, in the PHA production culture, 1,2-ethanedithiol was added to the medium at a final concentration of 0.2 g / L instead of 2-propen-1-ol. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. The results are shown in Table 1.

(Comparative Example 7) Production of PHA by KNK-005 trc-phaJ4b / ΔphaZ1,2,6 strain in a medium containing 0.2 g / L of 1,3-propanedithiol KNK-005 trc as in Example 1. -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 instead of 2-propen-1-ol so as to have a final concentration of 0.2 g / L. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. The results are shown in Table 1.

(Results and discussion)
From the results in Table 1, in Examples 1 to 16 in which alcohols having an alkynyl group, an alkenyl group, a thiol group or an allyl group were added to the medium to culture PHA-producing microorganisms, Comparative Example 1 to which these alcohols were not added was used. As compared with the above, the molecular weight of the obtained PHA was lowered in both weight average molecular weight (M _w ) and number average molecular weight (M _n ), so that the added alcohol functioned as a stop agent, and as a result, alkenyl was added to the carboxy terminal. It can be inferred that a PHA into which a group, an alkynyl group, a thiol group or an allyl group has been introduced is produced. However, when 5-hexene-1-ol was added, a significant decrease in the molecular weight of PHA was observed only when the concentration added to the medium was 1 g / L.

1 and 2 are ^{1 1} H-NMR charts of Examples 8 and 16, respectively. For comparison, FIG. 3 shows a ¹ H-NMR chart of Comparative Example 1. Compared with FIG. 3, the peak attributed to the methylene proton in the propynyl group appears at around 4.7 ppm in FIG. 1, and the peak attributed to the allyl group appears at around 4.5 ppm and 5.9 ppm in FIG. 2. It has appeared and it can be seen 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 to be due to the metabolism of these amino alcohols by microorganisms. Further, also in Comparative Examples 6 and 7, no decrease in the molecular weight of PHA was observed even when 1,2-ethanedithiol and 1,3-propanedithiol were added to the medium, and it is considered that dithiol does not function as a terminator. ..

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

(Production Example 1) Preparation of H16 ΔphaZ1,2,6 strain In preparing the H16ΔphaZ1,2,6 strain, first, the PHA synthase is based on the KNK-005 ΔphaZ1,2,6 strain described in WO2014 / 065253. 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 termination codon of the phaZ2 gene, and is described in SEQ ID NO: 5 on the chromosome. It is a strain having a PHA synthase gene.

First, a plasmid for full-length deletion of the PHA synthase gene of the KNK-005 ΔphaZ1,2,6 strain 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 NO: 6 and SEQ ID NO: 7 as a primer pair. KOD-plus (Toyobo) was used as the polymerase. 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 obtained DNA fragments were digested with the restriction enzyme SmiI. did. This DNA fragment is 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) and upstream from the phaC1 gene. A plasmid pNS2X-sacB-ΔphaC1UL for disrupting the PHA synthase gene, which has the base sequence of PHA and the base sequence downstream of the phaC1 gene, was prepared.

This PHA synthase gene disruption plasmid pNS2X-sacB-ΔphaC1UL was introduced into Escherichia coli S17-1 strain (ATCC47055), mixed and cultured on KNK-005 ΔphaZ1, 2, 6 strain and Nutrient Agar medium (DIFCO) for conjugation transfer. Was done.

Simmons agar medium containing 250 mg / L canamycin sulfate (sodium citrate 2 g / L, sodium chloride 5 g / L, magnesium sulfate heptahydrate 0.2 g / L, dihydrogen phosphate) from the above-mentioned strain after transferation. Strains that grow on ammonium 1 g / L, dipotassium hydrogen phosphate 1 g / L, agar 15 g / L, pH 6.8) were selected, and the plasmid was integrated onto the chromosomes of the KNK-005 ΔphaZ1,2,6 strain. Acquired the stock. After culturing this strain in Nutrient Broth medium (DIFCO) for 2 generations, a strain that grows in Nutrient Agar medium containing 15% sucrose was selected. From the obtained strains, strains in which the PHA synthase gene described in SEQ ID NO: 5 on the chromosome was deleted in full length were selected by PCR, and one of them was named H16 ΔphaC1 ΔphaZ1,2,6 strain. H16 ΔphaC1 ΔphaZ1,2,6 strains are C.I. It is a strain in which the necator H16 strain is used as a parent strain, the phaZ1 gene and the phaZ6 gene on the chromosome are deleted in full length, the 16th codon to the termination codon of the phaZ2 gene is deleted, and the phaC1 gene is deleted in full length.

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

First, C.I. A plasmid for inserting the phaC1 gene of the 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 NO: 6 and SEQ ID NO: 9 as a primer pair. KOD-plus was used as the polymerase. 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 of the phaC1 gene, a phaC1 gene, and a base sequence downstream of the phaC1 gene. A plasmid pNS2X-sacB-phaC _Re + UL for disrupting the PHA synthase gene was prepared.

By the same procedure as the above-mentioned PHA synthase gene disruption, a strain in which H16 ΔphaC1 ΔphaZ1,2,6 strain was used as a parent strain and pNS2X-sacB-phaC _Re + UL was integrated on the chromosome was obtained. After culturing this strain in Nutrient Broth medium for 2 generations, a strain that grows in Nutrient Agar medium containing 15% sucrose was selected. From the obtained strains, the strains into which the phaC1 gene on the chromosome was inserted were selected by PCR, and one of them was named H16 ΔphaZ1,2,6 strain. H16 ΔphaZ1, 2, 6 strains are C.I. A gene encoding wild-type PHA synthase derived from Ralstonia europha, with the necator H16 strain as the parent strain, with full-length deletion of the phaZ1 and phaZ6 genes on the chromosome, and deletion of the 16th codon to the termination codon of the phaZ2 gene. Is a strain having a gene on the chromosome.

(Production Example 2) Preparation 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 nucleotide sequence set forth in SEQ ID NO: 11 is inserted on the chromosome is inserted. Was produced. The PHA synthase gene consisting of the nucleotide sequence set forth in SEQ ID NO: 11 is described in Pseudomonas Sp. A PHA synthase consisting of the amino acid sequence shown in SEQ ID NO: 3, which is derived from 61-3 and has artificially replaced serine at position 325 with threonine, serine at position 477 with arginine, and glutamine at position 481 with arginine. It is a gene that encodes.

First, under the expression regulatory sequence set forth in SEQ ID NO: 12, Pseudomonas Sp. A plasmid pCUP2-REP-phaC1 _Ps expressing the PHA synthase gene derived from the 61-3 strain was prepared. The expression regulatory sequence set forth in SEQ ID NO: 11 is described in C.I. It is a promoter of the phaCAB operon derived from the necator H16 strain. C. PCR was performed using the genomic DNA of the necator H16 strain as a template and the DNAs shown in SEQ ID NO: 14 and SEQ ID NO: 15 as a primer pair. KOD-plus was used as the polymerase. 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 NO: 16 and SEQ ID NO: 17 as a primer pair. 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: 14 and SEQ ID NO: 17 as primer pairs, and the obtained DNA fragments were used as 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 to prepare the above-mentioned plasmid pCUP2-REP-phaC1 _Ps . ..

Next, PCR was performed using pCUP2-REP-phaC1 _Ps as a template and the DNAs shown in SEQ ID NO: 14 and SEQ ID NO: 18 as a primer pair. Similarly, PCR was performed using the DNAs shown in SEQ ID NO: 19 and SEQ ID NO: 20 as a primer pair. Similarly, PCR was performed using the DNAs shown in SEQ ID NO: 21 and SEQ ID NO: 17 as primer pairs. PCR was performed 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 used as restriction enzymes MunI and SpeI. Digested with. This DNA fragment was ligated with the DNA fragment obtained by digesting the vector pCUP2 described in JP-A-2007-259708 with MunI and SpeiI using DNA ligase, and the above plasmid pCUP2-REP-phaC1 _Ps ^{S325T, S477R was ligated. , Q481R} was prepared.

Next, a plasmid for inserting the PHA synthase gene set forth in SEQ ID NO: 11 onto the chromosome was prepared. PCR was performed using pCUP2-REP-phaC1 _Ps ^{S325T, S477R, Q481R} as a template and the DNAs shown in SEQ ID NO: 6 and SEQ ID NO: 22 as a primer pair. KOD-plus was used as the polymerase. Similarly, PCR was performed using the DNAs shown in 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 obtained DNA fragments were digested with the restriction enzyme SmiI. did. 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, the PHA synthase gene set forth in SEQ ID NO: 11, and the phaC1 gene. A plasmid pNS2X-sacB-ΔphaC1UL :: STSRQR for disrupting the PHA synthase gene having a downstream base sequence was prepared.

Using the same procedure as for inserting the PHA synthase gene described in Production Example 1, the H16 ΔphaC1 ΔphaZ1,2,6 strain is used as the parent strain, and pNS2X-sacB-ΔphaC1UL :: STSRQR is used to obtain the PHA synthase gene set forth in SEQ ID NO: 11. It was inserted on the chromosome. The obtained strain was named ReSK003 strain. The ReSK003 strain is a strain in which the H16 ΔphaC1 ΔphaZ1,2,6 strain is used as a parent strain and the mutant PHA synthase gene derived from the genus Pseudomonas described in SEQ ID NO: 11 is inserted on the chromosome.

Production of PHA by H16 ΔphaZ1,2,6 strain in a medium containing 1.0 g / L of 2-propen-1-ol H16 ΔphaZ1,2,6 produced in Production Example 1 by the same method 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. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. The results are shown in Table 2.

Production of PHA by H16 ΔphaZ1,2,6 strain in a medium containing 1.0 g / L of 2-propin-1-ol H16 ΔphaZ1,2,6 produced in Production Example 1 by the same method 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 in place of 2-propen-1-ol so as to have a final concentration of 1.0 g / L. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. The results are shown in Table 2.

(Comparative Example 8) Production of PHA by H16 ΔphaZ1, 2, 6 strain in a medium containing no 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 H16 ΔphaZ1,2,6 strains prepared in Production Example 1 were cultured and purified to obtain purified PHA. The obtained PHA was analyzed for the monomer unit copolymerization ratio and the molecular weight. The results are shown in Table 2.

(Results and discussion)
From the results in 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 higher than that in Comparative Example 8 in which these alcohols were not added. However, it can be inferred that the added alcohol functions as a stop agent, and as a result, PHA having a _propenyl group or a _propynyl group introduced at the carboxy terminal is produced. From the above results, PHA in which an alkenyl group or an alkynyl group was introduced into the carboxy terminal by culturing a microorganism having a PHA synthase gene derived from the genus Ralstonia by adding an alcohol having an alkynyl group or an alkenyl group to the medium. Was suggested to be able to produce.

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

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

(Comparative Example 9) Production of PHA by ReSK003 strain in a special alcohol-free medium Production was carried out in the same manner 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 the molecular weight. The results are shown in Table 3.

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

Claims (12)

The repeating unit is the following general formula (1)
[-C ^* HR ¹ -CH ₂ -CO-O-] ・ ・ ・ (1)
(In the equation, R ¹ is an alkyl group represented by C _n H _{2n + 1} , n is an integer of 1 to 9 , and * represents an asymmetric carbon.)
It is a single or copolymer polymer of R-3-hydroxyalkanoic acid shown by, and the group shown below is bonded to the carboxy terminal depending on whether the polymer is a single polymer or a copolymer polymer. , Polyhydroxyalkanoic acid, characterized by having a number average molecular weight of 7,000 to 1500,000 .
(1) A group that binds to the carboxy terminal when it is a single polymer, an alkynyl group having 4 to 8 carbon atoms,
Alkenyl group with 3 to 8 carbon atoms,
A mercaptoalkyl group having 2, 4 to 8 carbon atoms , or an allyl (poly) oxyalkyl group having an alkyl group having 2 to 6 carbon atoms.
(2) A group bonded to the carboxy terminal when it is a copolymerized polymer An alkynyl group having 3 to 8 carbon atoms,
Alkenyl group with 3 to 8 carbon atoms,
A mercaptoalkyl group having 2 to 8 carbon atoms , or an allyl (poly) oxyalkyl group having 2 to 6 carbon atoms. A microbially produced polyhydroxyalkanoic acid having an alkyne group, an alkene group, a thiol group or an allyl group introduced at the carboxy terminal.
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-hydroxyheptane. It is composed of a plurality of types selected from acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, and 3-hydroxydodecanoic acid, and has a number average molecular weight of 7,000 to 1500,000. Characterized polyhydroxyalkanoic acid. A microbially produced polyhydroxyalkanoic acid having an alkyne group, an alkene group, a thiol group or an allyl group introduced at the carboxy terminal.
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-hydroxyheptane. It is composed of a single species selected from acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, and 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.
The thiol group is a mercaptoethyl group, a mercaptobutyl group, a mercaptopentyl group, or a mercaptohexyl group .
The allyl group is an allyloxyethyl group, an allyloxypropyl group, or an allyloxybutyl group.
A polyhydroxyalkanoic acid having a number average molecular weight of 7,000 to 1500,000 . The polyhydroxyalkanoic acid according to claim 1 or 2 , which is a copolymerized polymer of R-3-hydroxyalkanoic acid and contains at least 3-hydroxybutyric acid as a monomer unit. The polyhydroxyalkanoic acid according to claim 1 or 3, which is a homopolymer of R-3-hydroxyalkanoic acid and the monomer unit is 3-hydroxybutyric acid. The polyhydroxyalkanoic acid according to claim 4, further comprising 3-hydroxyhexanoic acid as a monomer unit. The method for producing polyhydroxyalkanoate according to any one of claims 1 to 6 .
Including the step of culturing a microorganism capable of producing polyhydroxyalkanoic acid using an alcohol having an alkyne group, an alkene group, a thiol group , or an allylic group.
The microorganism is a microorganism having a gene encoding a polyhydroxyalkanoic acid synthase derived from the genus Aeromonas, Ralstonia or Pseudomonas, and an alkyne group, an alkene group, a thiol group , or an allyl group is introduced at the carboxy terminal. A method for producing polyhydroxyalkanoic acid. The method for producing the polyhydroxyalkanoic acid according to any one of claims 1 to 6.
At the carboxy terminus, including the step of 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 , or an allyl group. , A method for producing a polyhydroxyalkanoic acid into which an alkyne group, an alkene group, a thiol group , or an allyl group has been introduced. The production method according to claim 7 or 8 , wherein the alcohol is a primary alcohol. The production method according to claim 8 , wherein the microorganism is a transformant having Cupriavidus necator as a host. The compound in which the alkynyl group, the alkenyl group, or the thiol group at the carboxy terminal of the polyhydroxyalkanoic acid according to any one of claims 1 to 6 is further chemically modified, and is the following (1) or (2). A compound that meets the requirements .
(1) A compound obtained by rearranging a compound containing a thiol group with an alkenyl group or an alkynyl group at the carboxy terminal of polyhydroxyalkanoic acid by a thiol-enclick reaction or a thiol-inclick reaction.
(2) A compound obtained by rearranging a compound containing an alkenyl group or an alkynyl group to a thiol group at the carboxy terminal of polyhydroxyalkanoic acid by a thiol-enclick reaction or a thiol-inclick reaction. A molded product containing the polyhydroxyalkanoic acid according to any one of claims 1 to 6 or the compound according to claim 11 .

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