Method for producing polyhydroxyalkanoic acid oligomer

Abstract

To provide a technology for producing polyhydroxyalkanoate oligomers by causing extracellular secretion of polyhydroxyalkanoate oligomers from microbial cells.SOLUTION: Disclosed is a method for producing polyhydroxyalkanoate oligomers as extracellular oligomers of M.W. 200-1000, the method comprising a step of culturing a microorganism in the presence of a carbon source, where the microorganism has a gene encoding a polyhydroxyalkanoate polymerizing enzyme derived from Aeromonas caviae or Bacillus cereus YB4.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing a polyhydroxyalkanoic acid oligomer. [Background technology]

[0002] Polyhydroxyalkanoates (PHAs) are polyesters synthesized intracellularly by microorganisms as an energy storage material. Since polyhydroxyalkanoates are synthesized from renewable biomass, they are expected to be used and spread as so-called biodegradable plastics, as an alternative material to petroleum-derived plastics.

[0003] As a method for producing polyhydroxyalkanoic acid, for example, JP 2015-29484 A discloses a method for producing polyhydroxyalkanoic acid having a weight-average molecular weight of 5,000 to 40,000 and a modified carboxy terminus, which is characterized by culturing a microorganism having a type IV polyhydroxyalkanoic acid synthase gene in the presence of alcohol (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2015-29484 A Summary of the Invention [Problem to be solved by the invention]

[0005] According to the method of Patent Document 1 described above, polyhydroxyalkanoic acid with a reduced molecular weight can be obtained, and it is expected that the crystallization rate of polyhydroxyalkanoic acid resin will be improved if this is used as a crystal nucleating agent. However, from the viewpoint of processability, etc., further improvement of the crystallization rate is required. In the method for producing polyhydroxyalkanoic acid reported so far, the polymer has a weight average molecular weight of 5,000 to 40,000, and is synthesized by microorganisms as an energy storage substance within the cells. Therefore, in order to utilize this, a process of disrupting the cells and separating and purifying the polyhydroxyalkanoic acid is required.

[0006] Therefore, an object of the present invention is to provide a technique for producing polyhydroxyalkanoic acid oligomers by secreting polyhydroxyalkanoic acid oligomers outside of microbial cells, which has the advantages of eliminating the need for the above-mentioned cell disruption step and enabling substance production without being restricted by cell volume. [Means for solving the problem]

[0007] As a result of intensive research conducted by the inventors to solve the above-mentioned problems, they discovered that secretory oligomers can be produced in a culture medium by culturing a microorganism encoding a specific polyhydroxyalkanoate polymerase gene, and thus completed the present invention.

[0008] Specifically, the present invention provides a method for producing polyhydroxyalkanoic acid oligomers, which are extracellular oligomers having a molecular weight of 200 to 1,000, comprising the step of culturing a microorganism encoding a polyhydroxyalkanoic acid polymerase gene derived from Aeromonas caviae or Bacillus cereus YB4 in the presence of a carbon source. Effect of the Invention

[0009] According to the present invention, polyhydroxyalkanoic acid, which has been an endocrine polymer up until now, can be produced as an exocrine oligomer. Polyhydroxyalkanoic acid oligomers are known as physiologically active substances contained in plant growth factors and spider sex pheromones, and diol-terminated polyhydroxyalkanoic acid oligomers in particular are expected to be used as the core substance of polymer materials such as polyurethane, and are high-value-added compounds. [Brief description of the drawings]

[0010] [Figure 1] This is an NMR chart confirming the structure of poly(3-hydroxybutanoic acid)-diethylene glycol oligomer (3HBO). [Diagram 2] 1 is an ESI-TOF-MS chart showing the molecular weight distribution (degree of polymerization) of poly(3-hydroxybutanoic acid-diethylene glycol oligomer) (3HBO). [Diagram 3] FIG. 1 is a diagram for explaining the production mechanisms of 3HBO-EtOH by an ethanol-producing strain and 3HBO by an ethanol non-producing strain. [Figure 4] FIG. 1 shows the results of investigating the effect of adding various diols. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, an embodiment of the present invention will be described in detail. The method for producing a polyhydroxyalkanoic acid oligomer of the present embodiment includes a step of culturing a microorganism encoding a polyhydroxyalkanoic acid polymerase gene derived from Aeromonas caviae or Bacillus cereus YB4.

[0012] It is known that three enzymes, β-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and polyhydroxyalkanoate polymerase (PhaC, PhaEC, or PhaRC), are involved in the synthesis of polyhydroxyalkanoates. Among these, PhaA is an enzyme that catalyzes the reaction in which two molecules of acetyl-CoA are condensed to form acetoacetyl-CoA. In addition, PhaB is an enzyme that catalyzes the reaction in which acetoacetyl-CoA is reduced in the presence of the coenzyme NADPH to form (R)-3-hydroxybutyryl-CoA. In addition, PhaC is an enzyme that has catalytic activity in the reaction that synthesizes poly-3-hydroxybutanoate using 3-hydroxybutyryl-CoA as a substrate. As mentioned above, PhaC can synthesize various polyhydroxyalkanoates using 3-hydroxyacyl-CoA as a substrate, and types I to IV are known based on their substrate specificity, subunit structure, etc.

[0013] In this embodiment, as the polyhydroxyalkanoate synthase gene, a type I polyhydroxyalkanoate synthase gene derived from Aeromonas caviae or a type IV polyhydroxyalkanoate synthase gene derived from Bacillus cereus YB4 is used.

[0014] In this embodiment, the microorganism encoding the gene is not particularly limited as long as it is a microorganism capable of producing polyhydroxyalkanoic acid oligomers when the polyhydroxyalkanoic acid polymerase gene is expressed. Examples of the microorganism include microorganisms belonging to the genus Ralstonia (e.g., Ralstonia eutropha), microorganisms belonging to the genus Pseudomonas, and microorganisms modified to be able to supply a substrate for polyhydroxyalkanoic acid polymerase, such as microorganisms belonging to Escherichia coli (E. coli). Among these, Escherichia coli (E. coli) is preferred from the viewpoints of availability and proliferation ability. In particular, E. coli BW25113ΔadhE, available from the National Institute of Genetics, is preferred. In addition, when wild-type Escherichia coli is used, it is preferred in that it is possible to produce oligomers capped with ethanol on the carboxy terminal side by ethanol produced in the cells.

[0015] Specific examples of the transformant used in this embodiment include E. coli BW25113ΔadhE pGEM-phaCAcAB, which is transformed by introducing a type I polyhydroxyalkanoate synthase gene derived from Aeromonas caviae, and E. coli BW25113ΔadhE pGEM-phaRCYB4AB, which is transformed by introducing a type IV polyhydroxyalkanoate synthase gene derived from Bacillus cereus YB4. These strains have been deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary, and are available under the accession number 1 ... entrustment Number NITE P -02899, NITE P It was given the number -02900.

[0016] Considering the above-mentioned synthetic pathway of polyhydroxybutyric acid, it is preferable to use a microorganism further comprising phaA (β-ketothiolase gene) and phaB (acetoacetyl-CoA reductase gene).

[0017] The basal medium used in this embodiment may be a general medium used for culturing Escherichia coli, for example, LB medium, M9 medium, etc. In this embodiment, the carbon source contains at least one selected from the group consisting of sugars such as glucose, fructose, xylose, maltose, galactose, cellobiose, etc.; fatty acids such as formic acid, acetic acid, lactic acid, propionic acid, hexanoic acid, octanoic acid, decanoic acid, lauric acid, oleic acid, palmitic acid, linoleic acid, linolenic acid, myristic acid, etc.; fatty acid derivatives such as esters and salts of these fatty acids; vegetable oils and fats such as coconut oil, palm oil, palm kernel oil (hereinafter referred to as PKOO); carbon dioxide; and glycerol.

[0018] The concentration of the carbon source is preferably 0.1 to 10% by mass, and more preferably 1 to 5% by mass.

[0019] In this embodiment, the medium preferably further contains an alcohol or a diol, which functions as a chain transfer agent (polymerization terminator) to promote secretion of polyhydroxyalkanoic acid oligomers from the microorganism, thereby enabling a larger amount of polyhydroxyalkanoic acid oligomers to be obtained.

[0020] The alcohol is preferably at least one selected from the group consisting of, for example, methanol, ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, 1-nonanol, 1-decanol, benzyl alcohol, 3-mercapto-1-propanol, 2-propyn-1-ol, polyethylene glycol, polypropylene glycol, 2-propen-1-ol, 3-amino-1-propanol, and 3-butyn-1-ol.

[0021] The alcohol concentration is preferably from 0 to 15% by mass, and more preferably from 1 to 5% by mass.

[0022] The diol is preferably at least one selected from the group consisting of ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, 1,3 propanediol, 1,2 propanediol, 1,4 butanediol, and dipropylene glycol. In particular, diethylene glycol is preferable from the viewpoints of high secretion promoting effect, low cytotoxicity, high efficiency of modification to oligomer termini, and containing a reaction origin for polymerization. Moreover, 1,3 propanediol is preferable from the viewpoints of being able to be synthesized intracellularly by E. coli itself in addition to the above advantages.

[0023] The diol concentration is preferably from 0 to 15% by mass, more preferably from 1 to 10% by mass, and further preferably from 2 to 5% by mass.

[0024] According to the present embodiment, the alcohol or diol used is bonded to the carboxylic acid group at the end of the polyhydroxyalkanoic acid oligomer, thereby modifying the end of the polyhydroxyalkanoic acid oligomer, thereby functioning as a chain transfer agent that increases the amount of the oligomer.

[0025] The culture process will now be described. The microorganisms encoding the polyhydroxyalkanoate polymerase gene derived from Aeromonas caviae or Bacillus cereus YB4 described above are cultured in the presence of a carbon source.

[0026] The timing of adding alcohol or diol to the medium is not particularly limited as long as polyhydroxyalkanoic acid oligomers can be produced, and may be any stage of the early, middle, or late stage of the culture. However, once alcohol or diol is added to the medium, it is preferable to continue the culture in the medium containing the alcohol or diol, and it is preferable not to transfer the medium to a medium not containing alcohol or diol during the culture. This is because if the medium is transferred to a medium not containing alcohol or diol during the culture, the production of polyhydroxyalkanoic acid oligomers may decrease.

[0027] Culture conditions can be set appropriately. For example, a microorganism encoding a polyhydroxyalkanoic acid polymerase gene can be inoculated into a medium at a concentration of 0.1 to 10% by volume and cultured at 20 to 40°C for 12 to 96 hours to secrete polyhydroxyalkanoic acid oligomers into the medium.

[0028] FIG. 3 is a diagram for explaining the production mechanism of 3HBO-EtOH by an ethanol-producing strain and 3HBO by an ethanol-non-producing strain. A transporter present in the cell membrane of E. coli is involved in the secretion of polyhydroxyalkanoic acid oligomers into the medium. When wild-type E. coli is used, ethanol produced within the cells can be used, so that polyhydroxyalkanoic acid oligomers capped with ethanol on the carboxy terminal side can be produced without adding alcohol externally. Here, the inventors have found that the transporter present in the cell membrane of E. coli is different from that of lactate-based oligomers.

[0029] In this embodiment, the method further includes a step of recovering the polyhydroxyalkanoic acid oligomer from the culture medium obtained. A known method can be used to recover the polyhydroxyalkanoic acid oligomer obtained by the above-mentioned production method.

[0030] For example, after the culture is completed, the supernatant and the cells are separated from the culture liquid using a centrifuge or the like, and the supernatant is collected. A good solvent such as chloroform is added to the supernatant fraction, and the mixture is stirred and then allowed to stand, whereby the polyhydroxyalkanoic acid oligomer is fractionated in the chloroform layer. The chloroform layer is then collected to obtain the desired polyhydroxyalkanoic acid oligomer.

[0031] The composition of the polyhydroxyalkanoic acid oligomer thus recovered can be analyzed by, for example, nuclear magnetic resonance (NMR) or other methods, and the molecular weight of the polyhydroxyalkanoic acid oligomer can be measured by, for example, electrospray ionization (ESI)-time-of-flight mass spectrometry (TOF-MS) or other methods. EXAMPLES

[0032] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples, and can be modified within the scope of the above and below-mentioned aims, and all such modifications are included in the technical scope of the present invention.

[0033] 1. Preparation of polyhydroxyalkanoate oligomer-producing candidate strains An expression vector was used which was composed of the target genes phaC, phaA, and phaB, or phaE, phaC, phaA, and phaB, or phaR, phaC, phaA, and phaB for polyhydroxyalkanoic acid synthesis. The polyhydroxyalkanoate synthase gene phaC was derived from Ralstonia eutropha (Re), Aeromonas caviae (Ac), or Delftia acidovorance (Da). phaE and phaC were derived from Allochromatium vinosum (Av). phaR and phaC were derived from Bacillus sp. INT005 (Bs), Bacillus megaterium (Bm), or Bacillus cereus YB4 (YB4). phaA and phaB were derived from Ralstonia eutropha. The seven expression vectors used were: pGEM-phaCReAB, pGEM-phaCAcAB, pGEM-phaCDaAB, pGEM-phaECAvAB, pGEM-phaRCBsAB, pGEM-phaRCBmAB, and pGEM-phaRCYB4AB.

[0034] Next, the seven types of expression vectors were transformed into E. coli BW25113ΔadhE (provided by the National Institute of Genetics) by a standard method to obtain candidate strains for producing the desired polyhydroxyalkanoic acid oligomers (E. coli BW25113ΔadhE pGEM-phaCReAB, E. coli BW25113ΔadhE pGEM-phaCAcAB (provided by the recipient). entrustment Number NITE P-02899), E. coli BW25113ΔadhE pGEM-phaCDaAB, E. coli BW25113ΔadhE pGEM-phaECAvAB, E. coli BW25113ΔadhE pGEM-phaRCBsAB, E. coli BW25113ΔadhE pGEM-phaRCBmAB, E. coli BW25113ΔadhE pGEM-phaRCYB4AB(receiving entrustment Number NITE P -02900) was created.

[0035] 2. Production of polyhydroxyalkanoate oligomers (part 1) Cultivation experiments were carried out to examine whether or not the polyhydroxyalkanoic acid oligomer-producing candidate strains produced oligomers and to examine differences in the amounts produced.

[0036] In detail, first, the production strain candidates obtained in 1 above (E. coli BW25113ΔadhE pGEM-phaCReAB, E. coli BW25113ΔadhE pGEM-phaCAcAB (recipient) entrustment Number NITE P -02899), E. coli BW25113ΔadhE pGEM-phaCDaAB, E. coli BW25113ΔadhE pGEM-phaECAvAB, E. coli BW25113ΔadhE pGEM-phaRCBsAB, E. coli BW25113ΔadhE pGEM-phaRCBmAB, E. coli BW25113ΔadhE pGEM-phaRCYB4AB(receiving entrustment Number NITE P -02900)) was inoculated into LB medium containing 2% by mass glucose and 0.01% by mass ampicillin at a concentration of 1% by volume and cultured at 30°C for 48 hours.

[0037] Each culture solution thus obtained was centrifuged at 6000 rpm for 10 minutes (room temperature) to recover the culture supernatant. The obtained cell pellet was suspended in pure water and centrifuged again under the same conditions as above to obtain a cell pellet. The thus obtained cell pellet was freeze-dried to obtain dry cells. The obtained dry cell weight (g / L) is shown in Table 1.

[0038] Next, the cell mass (g / L) of the above-mentioned dried bacterial cells, the amount of intracellular polymer (polyhydroxyalkanoic acid) (g / L), and the amount of oligomer (polyhydroxyalkanoic acid) (extracellular) in the culture supernatant (g / L) were measured. The cell mass and the amount of intracellular polymer were measured according to the method described in J. Agus et al. / Polymer Degradation and Stability 91 (2006) 1138e1146, and the amount of oligomer was measured according to the method described in ACS Sustainable Chem. Eng. 2017, 5, 3, 2360-2367. These results are summarized in Table 1.

[0039] [Table 1]

[0040] As shown in Table 1, polyhydroxyalkanoate oligomers were produced by all polymerase species, and the production amounts varied greatly depending on the polymerase species, ranging from 0.01 to 1.03 g / L.

[0041] In particular, it was confirmed that the use of Ac-derived and YB4-derived polymerases resulted in a high amount of oligomer secretion.

[0042] 3. Production of polyhydroxyalkanoate oligomers (part 2) As a result of the above 2, the polymerase species Ac and YB4, which produced high oligomers, and Re as a comparison control, were added, and the effect on oligomer production was investigated when culturing was performed in a medium containing a chain transfer agent (polymerization terminator).

[0043] First, E. coli BW25113ΔadhE pGEM-phaCReAB (hereinafter referred to as Re strain) and E. coli BW25113ΔadhE pGEM-phaCAcAB (recipient strain) were entrustment Number NITE P -02899) (hereinafter Ac strain), E. coli BW25113ΔadhE pGEM-phaRCYB4AB (ac strain) entrustment Number NITE P -02900) (hereafter referred to as YB4 strain) was inoculated at a concentration of 1% by volume into LB medium containing 2% by mass glucose, 0.01% by mass ampicillin, and 0 to 15% by volume (0 to 150 mL / L) of diethylene glycol, and cultured at 30°C for 48 hours.

[0044] Each culture solution thus obtained was treated in the same manner as in 2 above to obtain dried cells, and the dry cell weight, cell mass, intracellular polymer amount, and extracellular oligomer amount were then measured in the same manner as in 1 above. The results are shown in Table 2.

[0045] [Table 2]

[0046] As shown in Table 2, the Ac and YB4 strains (recipients) entrustment Number NITE P -02899 and receipt entrustment Number NITE P Unlike the comparative strain Re, the amount of oligomers tended to increase with the amount of diethylene glycol added. entrustment Number NITE P -02899), 1.03-3.14 g / L, YB4 strain (recipient), entrustment Number NITE P -02900), it was 0.21 to 4.98 g / L. In both cases, the highest amount of oligomers was obtained when diethylene glycol was added at a concentration of 50 mL / L.

[0047] In addition to the results of 2 above, a synergistic effect was confirmed by combining the type of polymerization enzyme and the concentration of the chain transfer agent.

[0048] 4. Production of polyhydroxyalkanoate oligomers (part 3) Based on the results of 3 above, the YB4 strain (recipient) had the highest oligomer production. entrustment Number NITE P -02900), the effect of various alcohols and diols that can function as chain transfer agents (polymerization terminators) similar to diethylene glycol on oligomer production was investigated.

[0049] First, the YB4 strain (recipient) obtained in 1 above entrustment Number NITE P -02900) was inoculated at a concentration of 1% by volume into LB medium containing 2% by mass glucose, 0.01% by mass ampicillin, and one of the following chain transfer agent candidates: ethanol, 1-butanol, 1,3-propanediol, 1,2-propanediol, 1,4-butanediol, or dipropylene glycol at 1% by volume (10 mL / L) or 5% by volume (50 mL / L), and cultured at 30°C for 48 hours. We also investigated the effect of using the wild-type strain (E. coli BW25113 pGEM-phaRCYB4AB) in addition to the YB4 strain (E. coli BW25113ΔadhE pGEM-phaRCYB4AB) without the addition of alcohol or diol.

[0050] Each culture solution thus obtained was treated in the same manner as described above in 2 and 3 to obtain dried cells, and the dry cell weight, cell mass, intracellular polymer amount, and extracellular oligomer amount were measured in the same manner as described above in 2 and 3.

[0051] These results are shown in Table 3. For comparison, the results for no addition (quoted from Table 1), 1 volume % (10 mL / L) and 5 volume % (50 mL / L) diethylene glycol addition are also shown.

[0052] [Table 3]

[0053] As shown in Table 3, the use of all alcohols and diols increased the amount of oligomers produced compared to when no addition was made. In particular, for diols (1,3-propanediol, 1,2-propanediol, 1,4-butanediol, and dipropylene glycol), the amount of oligomers increased at 5 vol.% (50 mL / L) compared to 1 vol.% (10 mL / L). The effect of adding 1 vol.% (10 mL / L) of dipropylene glycol was greater than that of adding 1 vol.% (10 mL / L) of diethylene glycol. 1,3-propanediol is a diol that can be produced from sugar by the host E. coli, so it can also be used as an endogenous chain transfer agent.

[0054] From the above results, it was confirmed that the alcohols and diols examined function as chain transfer agents to increase the amount of oligomers. In particular, diethylene glycol, dipropylene glycol, and 1,3-propanediol are considered to be highly useful.

[0055] 5. Production of polyhydroxyalkanoate oligomers (part 4) The YB4 strain obtained in 1 above (recipient entrustment Number NITE P -02900) was inoculated at a concentration of 1% by volume into LB medium containing 4% by mass glucose, 0.01% by mass ampicillin, and any of the following chain transfer agent candidates: ethylene glycol (EG), diethylene glycol (DEG), triethylene glycol (TriEG), tetraethylene glycol (TEG), glycerol, dipropylene glycol (DPG), 1,2-propanediol (1,2-PDO), 1,3-propanediol (1,3-PDO), 1,3-butanediol (1,3-BDO), 1,4-butanediol (1,4-BDO), and 2,3-butanediol (2,3-BDO) at 5% by volume (50 ml / L), and cultured at 30°C for 48 hours. The amount of extracellular oligomers in each culture solution thus obtained was measured by the same method as in 2 and 3 above.

[0056] The results are shown in Table 4 and Figure 4. The alcohols and diols examined were confirmed to function as chain transfer agents that increase the amount of oligomers. In particular, diethylene glycol, triethylene glycol, dipropylene glycol, and 1,3-propanediol are considered to be highly useful.

[0057] [Table 4]

[0058] 6. Structural analysis of polyhydroxyalkanoate oligomers (part 1) The YB4 strain (recipient) mentioned above entrustment Number NITE P The repeating structure and terminal structure of the oligomer obtained by culturing the strain (Citroenzyme-02900) in a medium containing 5% by volume of diethylene glycol (50 mL / L) were analyzed.

[0059] The oligomers were collected by solvent extraction. That is, an equal volume of chloroform was added to the culture solution containing the oligomers, and after stirring for 1 hour, the solution was allowed to stand, and the chloroform layer was collected. The collected solution was washed three times with an equal volume of pure water to remove water-soluble substances. The chloroform layer was collected, dried, and then dissolved in deuterated chloroform containing 0.03% (v / v) TMS (tetramethylsilane), and filtered with a 0.45 μm diameter Millex-HV PVDF filter (manufactured by MILLIPORE).

[0060] Next, regarding the above sample 1 The structure was determined using H-NMR. Specifically, a JEOL NMR instrument was used, and the conditions were 500 MHz at room temperature and 128 scans. Data analysis was performed using ACLICE2 for Windows ver.4.

[0061] These results are shown in Figure 1. As shown in Figure 1, a peak derived from diethylene glycol bound to the end of poly(3-hydroxybutanoic acid) was observed as a triplet at around 4.26 ppm. In addition, other peaks derived from diethylene glycol were observed at around 3.60 and 3.72 ppm, respectively.

[0062] From these results, it was confirmed that, among the oligomers obtained in 3 above, the oligomer obtained by culturing in a medium containing 5 mL / L of diethylene glycol was poly(3-hydroxybutanoic acid) having diethylene glycol attached to the end.

[0063] 7. Structural analysis of polyhydroxyalkanoate oligomers (part 2) The degree of polymerization and terminal structure of the three polyhydroxyalkanoate oligomers mentioned above were analyzed.

[0064] First, the oligomer was recovered by the solvent extraction method in the same manner as in 6 above. An equal amount of chloroform was added to the culture solution containing the oligomer, and after stirring for 1 hour, the solution was allowed to stand, and the chloroform layer was recovered. The recovered solution was washed three times with an equal amount of pure water to remove water-soluble substances. The chloroform layer was recovered, dried, dissolved in methanol, and filtered with a 0.45 μm diameter Millex-HV PVDF filter (manufactured by MILLIPORE Co., Ltd.) to prepare a sample.

[0065] Next, the degree of polymerization and the terminal structure of the above sample were analyzed using ESI-TOF-MS. Specifically, a Bruker ESI-TOF-MS instrument was used, and data collection was performed in positive mode with a full scan between m / z 100 and 1000. The sample was dissolved in methanol and introduced into the ion source at a flow rate of 180 μL / min. The ion spray voltage was set to 250 V. Nitrogen gas was used. Data analysis was performed using the attached software Compass.

[0066] These results are shown in Figure 2. As shown in Figure 2, oligomers (Na adducts) consisting mainly of 2-9-mer 3-hydroxybutanoic acid (chains with mass number 86) with diethylene glycol bonded to the ends were observed, and the molecular weight was 200 to 1000.

[0067] The carboxyl end of the oligomer was bound to diethylene glycol, and the hydroxyl end was confirmed to have a partially dehydrated structure.

Claims

1. A method for producing a polyhydroxyalkanoic acid oligomer having a molecular weight of 200 to 1000 and being an extracellular oligomer, comprising a step of culturing a microorganism encoding a polyhydroxyalkanoic acid polymerase gene derived from Aeromonas caviae or Bacillus cereus YB4 in the presence of a carbon source, The method for producing a polyhydroxyalkanoic acid oligomer, wherein the microorganism is the E. coli BW25113ΔadhE pGEM-phaCAcAB strain deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center under accession number NITE P-02899 or the E. coli BW25113ΔadhE pGEM-phaRCYB4AB strain deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center under accession number NITE P-02900.

2. The method for producing a polyhydroxyalkanoic acid oligomer according to claim 1, wherein the medium further contains at least one chain transfer agent selected from the group consisting of ethanol, diethylene glycol, triethylene glycol, 1,3-propanediol, 1,2-propanediol, 1,4-butanediol, and dipropylene glycol.

3. The method for producing a polyhydroxyalkanoic acid oligomer according to claim 2, wherein the concentration of the chain transfer agent is 1 to 15% by volume.

4. The method for producing a polyhydroxyalkanoic acid oligomer according to any one of claims 1 to 3, wherein the concentration of the carbon source is 0.1 to 10% by mass.

5. The method for producing a polyhydroxyalkanoic acid oligomer according to any one of claims 1 to 3, further comprising a step of recovering the polyhydroxyalkanoic acid oligomer from the obtained culture solution.

6. The method for producing a polyhydroxyalkanoic acid oligomer according to any one of claims 1 to 3, which produces a polyhydroxyalkanoic acid oligomer containing at least 3-hydroxybutanoic acid.

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