TECHNICAL FIELD

The present invention relates to a microorganism for producing, in a cell thereof, two or more PHAs different in melting point from one another, and a method for producing a PHA high in crystallization/solidification speed, using the microorganism.

BACKGROUND ART

A polyhydroxyalkanoic acid (abbreviated hereinafter to a “PHA”) is a thermoplastic polyester produced and stored as an energy storing substance inside cells of many microorganism species. The PHA, which is produced from various natural carbon sources by microorganisms, is completely biodegraded by a microorganism in the earth or in water to be taken into a carbon cycle process in the natural world. It can be therefore stated that the PHA is an environmentally harmonizing plastic material, which hardly produces any bad effect on the ecological system. In recent years, from the viewpoint of environmental pollution, waste disposal and petroleum resources, synthetic plastics have been becoming serious social problems. Thus, attention has been paid to PHAs as environment-friendly green plastic materials. It has been strongly desired to put PHAs into practical use.

A PHA first discovered in microorganisms is a polyhydroxybutyrate (a PHB), which is a homopolymer made from 3-hydroxybutyric acid (abbreviated hereinafter to 3HB). The PHB is high in crystallinity, and high in crystallization degree to be hard and brittle, and further the PHB is rapidly thermally decomposed at a temperature (180° C.) around the melting point thereof. Accordingly, the PHB has problems that this resin is low in melt workability and a practical use scope thereof is very restrictive.

Thus, in order to lower the PHB in crystallization degree to be improved in brittleness, attempts have been made in which another 3-hydroxyalkanoic acid is introduced into the skeleton of the PHB. In one of the attempts, a copolymer polyester has been discovered which is made from 3HB and 3-hydroxyhexanoic acid (abbreviated hereinafter to 3HH) (this polyester P(3HB-co-3HH) will be abbreviated hereinafter to the PHBH). The PHBH, which contains, as a monomer unit, 3HH having a larger side chain structure (than PHB), is lower in crystallization degree than any PHB to have flexible and soft properties and improved in brittleness. Additionally, the PHBH is low in melting point to be also expected to have improved melt workability. However, the following have been understood: the PHBH is very low in crystallization/solidification speed, and thus, even when cooled to room temperature after heated and melted, the PHBH has a soft property and is viscous for some time; and the PHBH has adhesiveness so that when molded, the PHBH is not immediately released from the mold. For the reason, in cases of putting the PHBH into practical use, some of the cases do not attain actual and continuous production of the PHBH. It has also become evident that working machines used to work existing commodity plastics high in crystallization/solidification speed may not be usable for working the PHBH. In working into a film or sheet, a fiber, a foam, a molded product, or a nonwoven fabric, it is very important when a melt-worked polymer is cooled that the crystallization/solidification speed of this polymer is high since this high speed results in making the producing process of such articles continuous, followed by an improvement of the articles in productivity and a fall in costs thereof.

Thus, attempts have been made for making a PHBH high in crystallization/solidification speed. As an ordinary method therefor, a method of adding, to the PHBH, a nucleating agent has been attempted. According to, for example, Patent Literature 1, boron nitride is used as the nucleating agent for PHBH to produce a crystallization promoting effect. However, this is an expensive material, and further has no biodegradability. Consequently, a less expensive and more biodegradable nucleating agent has been investigated.

Patent Literatures 2 and 3 each disclose a technique of adding a PHB, which is higher in melting point than a PHBH and is further biodegradable, as a nucleating agent to the PHBH to make the resultant high in crystallization/solidification speed. According to these preceding literatures, as a method of blending the PHBH with the PHB, for example, the following has been attempted: a method of dissolving the PHBH and the PHB in a solvent such as hot chloroform, blending these solutions with each other, and then evaporating chloroform to precipitate polymers; a method of pulverizing the two polymers to be blended with each other while the polymers are cooled with dry ice; or blending these polymers in the state that only the PHBH is melted without melting the PHB, or blending these polymers by mixing dry powders of the polymers with each other. However, the method of dissolving the polymers in the solvent to be mixed with each other requires a very large quantity of the solvent for dissolving or crystallizing the PHBH, so as to become high in costs. As the method of blending the PHBH with the PHB, a method is also known in which these polymers are subjected to crystallization with methanol and the resultant mixed polymers are collected. Because of a difference in solubility between the polymer and the nucleating agent at the time of the crystallization, this method has, for example, a probability that the crystallization may not be performed in the state that the nucleating agent is uniformly dispersed. Thus, this method is not practical. In the method of pulverizing the polymers and subsequently blending the polymers with each other, or the method of mixing the dry polymer powders, it is difficult to blend the polymers uniformly with each other. It is therefore anticipated that the effect of the nucleating agent is lowered. As the respective particle diameters of the PHBH and the nucleating agent are smaller, these are more sufficiently blended with each other and further the number of nucleus-forming moieties becomes larger. Thus, a higher advantageous effect is expected. However, in the blending methods described above, the blending effect based on such fine particles is not expectable. Furthermore, in order to disperse the PHB uniformly in the PHBH, working at a temperature not lower than the melting point of the PHB is required. However, ordinary species of the PHB have a high melting point. Additionally, as described above, the species are thermally decomposed at a temperature around the melting point. Thus, when the PHB is dispersed in the PHBH, the PHB and the PHBH are deteriorated by heat, so that a fall in the molecular weight thereof, and other problems are not easily avoidable.

In order to solve these problems, a method has been invented in which a microorganism is caused to produce a PHBH, and a PHA, which is a nucleating agent, in a mixed state by controlling the culture of the microorganism. For example, Patent Literature 4 reports a method of changing a carbon source in the middle of the culture to cause a microorganism to produce a mixture of a PHBH, and a PHB or a PHBH having a low copolymerization proportion of a 3HH monomer. Non-Patent Literature 1 suggests that a culture of a microorganism using a specific plant oil and sodium valerate as carbon sources makes it possible to co-produce, in a cell of the microorganism, a mixture of a PHB, and a copolymer polyester made from 3HB and 3-hydroxyvaleric acid (abbreviated hereinafter to 3HV) (this polyester P(3HB-co-3HV) will be abbreviated hereinafter to the PHBV). These methods do not require independent production of a nucleating agent component such as a PHB to have a large advantage in terms of costs. However, in the method in Patent Literature 4, in which the carbon source is changed in the middle of the culture, two PHAs are non-continuously produced so that the control of the culture is very difficult. Furthermore, the method is low in productivity so that the polymer is not produced stably. Moreover, in the method in Non-Patent Literature 1, a target advantageous effect is obtained only when the specific plant oil is used. Furthermore, it is difficult to control a blend quantity ratio between the two PHAs. Thus, this method is impractical.

Additionally, as an example in which two PHAs are intracellularly co-produced, the following are reported. For example, Non-Patent Literature 2 reports that a wild-type 61-3 strain of the genusPseudomonashas three genes encoding PHA synthases. Two of these three PHA synthases have, as their substrate, a medium-chain-length 3-hydroxyalkanoic acid having a carbon chain length of 6 to 12, and one thereof has, as its substrate, only 3HB. Therefore, when this 61-3 strain is cultured in a culture medium containing a fatty acid such as octanoic acid or dodecanoic acid, a medium-chain-length PHA and a PHB are intracellularly co-produced. Non-Patent Literatures 3 and 4 each report that when a gene encoding a PHB synthase derived from a bacterium that may be of various types is introduced into aPseudomonas oleovoransthat synthesizes a medium-chain-length PHA, the medium-chain-length PHA and a PHB are intracellularly co-produced. Non-Patent Literature 5 reports that when a gene encoding a medium-chain-length PHA synthase derived fromAllochromatium vinosumis introduced intoRalstonia eutrophathat synthesizes a PHB, the PHB and a medium-chain-length PHA are intracellularly co-produced.

CITATION LIST

Patent Literatures

PTL 1: JP H06-157878 A

PTL 2: JP H08-510498 A

PTL 3: WO 2002/50156

PTL 4: JP 2004-250629 A

Non-Patent Literatures

NPLT 1: Wing-Hin Lee, Ching-Yec Loo, Christopher T. Nomura, Kumar Sudesh, Bioresource Technology, vol. 99, pp. 6844-6851, (2008)

NPLT 2: Hiromi Matsusaki, Sumihide Manji, Kazunori Taguchi, Mikiya Kato, Toshiaki Fukui, Yoshiharu Doi, Journal of Bacteriology, vol. 180, pp. 6459-6467, (1998)

NPLT 3: Arnulf Timm, David Byrom, Alexander Steinbuchel, Applied Microbiology and Biotechnology, vol. 33, pp. 296-301, (1990)

NPLT 4: Matthias Liebergesell, Frank Mayer, Alexander Steinbuchel, Applied Microbiology and Biotechnology, vol. 40, pp. 292-300, (1993)

NPLT 5: Kawalpreet K. Aneja, Richard D. Ashby, Daniel K. Y. Solaiman, Biotechnology Letters, vol. 31, pp. 1601-1612, (2009)

SUMMARY OF INVENTION

Technical Problem

An object of the present invention is to improve the crystallization speed of a PHA copolymer which is slowly crystallized to improve the melt workability of the PHA copolymer in working such as injection molding, film molding, blow molding, fiber spinning, extrusion foaming or bead foaming, thereby improving the resultant articles in productivity.

Solution to Problem

In order to solve the above-mentioned problems, the inventors have repeatedly researched to find out that when a microorganism is used which has both of a gene encoding a PHA synthase that synthesizes a copolymer PHA, such as a PHBH, as a main polymer, and a gene encoding a PHA synthase that synthesizes a higher-melting-point PHA serving as a nucleating agent to co-produce the two PHAs in the same cell of the microorganism, the resultant PHA copolymer-containing resin is remarkably improved in crystallization speed. Thus, the present invention has been achieved.

Accordingly, a first aspect of the present invention relates to a method for producing a PHA mixture, including the step of culturing a microorganism having both of a gene encoding a PHA synthase that synthesizes a copolymer PHA (A) and that is derived from the genusAeromonas, and a gene encoding a PHA synthase that synthesizes a PHA (B) different in melting point from the copolymer PHA (A) by 10° C. or more to produce, in a cell of the microorganism, two or more PHAs different in melting point from one another by 10° C. or more simultaneously. The copolymer PHA (A) is preferably a copolymer containing, as monomer units, at least 3HB and 3HH. It is preferred that the resultant PHA mixture shows at least two endothermic peaks between 85° C. and 180° C. in a DSC curve of the mixture and, among these endothermic peaks, an endothermic peak at the highest temperature has a calorie of 0.2 to 20 J/g, or the resultant PHA mixture shows only one endothermic peak between 85° C. and 180° C. in a DSC curve of the mixture, and the endothermic calorie at temperatures higher than 160° C. is from 0.5 to 10 J/g.

A second aspect of the present invention relates to a microorganism including a gene encoding a PHA synthase that synthesizes a copolymer PHA (A) and that is derived from the genusAeromonas, and a gene encoding a PHA synthase that synthesizes a PHA (B) different in melting point from the copolymer PHA (A) by 10° C. or more. The gene encoding the PHA synthase that synthesizes the copolymer PHA (A) and that is derived from the genusAeromonasis preferably a gene encoding a protein having an amino acid sequence shown in SEQ ID NO: 1, or a gene encoding a protein having a sequence homology of 85% or more to the amino acid sequence and having a PHA synthase activity. The gene encoding the PHA synthase that synthesizes the PHA (B) is preferably a gene encoding a PHA synthase derived from a biological species different from the genusAeromonas; is more preferably a gene encoding a PHA synthase derived from the genusCupriavidus; and is even more preferably a gene encoding an amino acid sequence shown in SEQ ID NO: 4 derived fromCupriavidus necator, or a gene encoding a protein having a sequence homology of 85% or more to the amino acid sequence and having a PHA synthase activity.

Furthermore, the microorganism preferably belongs to the genusCupriavidus, and is more preferablyCupriavidus necator. Moreover, the microorganism is preferably a microorganism which further has a gene encoding an (R)-specific enoyl-CoA hydratase, and the expression of the gene encoding the (R)-specific enoyl-CoA hydratase is enhanced; or a microorganism which has a phbA gene and/or a bktB gene, and the expression of the phbA gene is restrained, and/or the expression of the bktB gene is enhanced.

Advantageous Effects of Invention

The present invention makes it possible to remarkably improve the crystallization speed of a PHA copolymer which is slowly crystallized to improve the melt workability or working speed of the PHA copolymer in working such as injection molding, film molding, blow molding, fiber spinning, extrusion foaming or bead foaming.

