JP2001178484A - Method for producing polyhydroxyalkanoic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method for producing a desired polyhydroxyalkanoic acid by a microorganism by utilizing a carbon source other than carbon sources such as sugar, aliphatic hydrocarbon, fatty acid and alcohol which have been utilized hitherto. SOLUTION: This method for producing a polyhydroxyalkanoic acid comprises culturing a microorganism having ability to proliferate by assimilating acetic acid or its salt and produce and accumulate polyhydroxyalkanoic acid in a culture medium containing acetic acid or its salt as a single carbon source.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing polyhydroxyalkanoic acid using a microorganism. In particular, the structural unit of polyhydroxyalkanoic acid is 3
The present invention relates to a method for producing polyhydroxyalkanoic acid containing hydroxyalkanoic acid using a microorganism.

[0002]

2. Description of the Related Art For many years, synthetic polymers derived from petroleum have been used as plastics and the like, but treatment of these plastics and the like discarded after use has become a major social problem. In the past, synthetic polymer materials derived from petroleum have been used as substitutes for metal materials, glass, etc. due to the advantage that they are not easily decomposed, but in recent years they are consumed in large quantities and are discarded in large quantities. Was devastated and accumulated at waste disposal sites. In addition, when incineration is performed, the amount of CO ₂ emission increases, and in some cases, it also causes the generation of harmful substances such as dioxins and environmental hormones. On the other hand, poly 3-hydroxybutyric acid (P
Microbial polyesters represented by HB), unlike synthetic polymers derived from petroleum, have the property of being biodegradable. Therefore, when discarded, the microorganism-produced polyester is biodegraded and taken into the material cycle in the natural world, so that it can be used as a plastic that enables environmental conservation. In addition, application of a microbial polyester as a medical soft member is being studied as a useful possibility that will be useful in the future (Japanese Patent Laid-Open No. 5-159).

[0003] It has been reported that many bacteria produce and accumulate copolymers of PHB and other hydroxyalkanoic acids in the cells ("Biodegradable Plastic Handbook" (edited by Biodegradable Plastics Research Group)). ;
(NTS Corporation), p178-197). In particular, Alcaligenes eutrophus strain H16 (Alcaligenes eutrophus)
s H16, ATCC No. 17699) and variants thereof have been studied in detail for the production of these polymers. This bacterium and its mutant strain are capable of changing the carbon source serving as a substrate, thereby obtaining a copolymer of 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) or a unit containing both units. (JP-B-6-15604, JP-B-7-14352, JP-B-8-19227, etc.).

[0004] In addition to this, utilizing various carbon sources,
The following can be mentioned as an example of causing a microorganism to produce a polyester containing a hydroxyalkanoic acid in a constitutional unit.

[0005] Japanese Patent No. 2,642,937 discloses Pseudomonas oleovorans A.
It is disclosed that a polyester having 3-hydroxyalkanoic acid (3HA) having 6 to 12 carbon atoms as a monomer unit is produced by giving acyclic aliphatic hydrocarbons as a carbon source to strain TCC29347. Japanese Patent Application Laid-Open No. Hei 5-74492 discloses that Methylobacterium
Lactobacillus, Paracoccus, Alcaligenes and Pseudomonas bacteria by contacting them with a primary alcohol having 3 to 7 carbon atoms.
A method for producing a copolyester of HB and 3HV is disclosed.

[0006] JP-A-5-93049 and JP-A-7-265065 disclose that 3HB and 3-hydroxyhexane are obtained by culturing Aeromonas caviae using oleic acid or olive oil as a carbon source. It is disclosed to produce a two-component copolyester of the acid (3HHx).

[0007] Japanese Patent Application Laid-Open No. 9-191893 discloses Comamonas acidovorans IFO13.
It is disclosed that strain 852 produces a polyester having 3HB and 4-hydroxybutyric acid (4HB) as monomer units by culturing using gluconic acid and 1,4-butanediol as carbon sources.

[0008] The above-mentioned example is an example in which a sugar, an aliphatic hydrocarbon, a fatty acid, an alcohol, or the like is used as a carbon source when a microorganism containing a hydroxyalkanoic acid as a constituent unit is produced by a microorganism. On the other hand, when a microorganism containing hydroxyalkanoic acid as a constituent unit, particularly PHA, is produced by a microorganism, there is a case where a culture medium containing acetic acid or a salt thereof is used in culturing, growing, and growing the microorganism. Examples include the following:

Japanese Patent Publication No. 4-012712, Japanese Patent Publication No. 4
-012713, JP-B-8-01927, JP-A-2770378, JP-A-5-00
No. 7492 discloses a method for producing a copolymer of 3HB and 3HV by culturing a microorganism in a medium containing acetate or the like.

JP-B-7-014352, JP-B-7-014
JP-A-014353, JP-B-7-014354, JP-A-5-023189, JP-A-5-064
No. 591, JP-A-6-181784 and JP-A-8-089264 disclose in a method for producing 3HB and 4HB copolymers by microorganisms, in which the microorganisms are grown in advance before production. It is disclosed that acetic acid or a salt thereof can be used in the stage (pre-stage culture or expansion culture).

JP-B-63-032438, JP-B-2-020238 and JP-A-5-093049 disclose that a microorganism is cultured in a medium containing acetate and the like, and the 3HB homogenate is cultured using the cultured microorganism. A method for producing a polymer is disclosed.

JP-B-7-103230, JP-A-5-103230
No. 214081 discloses 3HB, 4HB, and 3H
It is disclosed that in a method for producing a copolymer of V by a microorganism, acetic acid or a salt thereof can be used in the step of growing the microorganism (first-stage culture or growth culture).

JP-B-7-113055, JP-A-6-13055
-009761, 3HB, 4HB, 3HV,
And a method for producing a copolymer of 5-hydroxyvaleric acid (5HV) using a microorganism, in which acetic acid or a salt thereof can be used in the step of growing the microorganism (pre-stage culture or growth culture). It has been disclosed.

JP-A-6-022773, JP-A-6-227773
No. 038739 discloses 3HV, 3-hydroxyoctanoic acid (3HO), and 3-hydroxydecanoic acid (3HO).
It is disclosed that in a method for producing a copolymer of HD) by a microorganism, acetic acid or a salt thereof can be used in the step of growing the microorganism (first-stage culture or growth culture).

JP-A-6-14531 discloses a method for producing a copolymer of HB and 3-hydroxypropionic acid (3HP) by culturing a microorganism in a medium containing acetate or the like and utilizing the microorganism. Is disclosed.

JP-A-6-284892 also discloses a method of culturing microorganisms in a medium containing acetate or the like, and producing 3HB, 3HP and 3HV copolymers using the microorganisms. .

JP-A-6-062875 discloses that acetic acid or a salt thereof can be used for PHA production in a non-aerated state by activated sludge.

[0018]

As described above, conventionally, as a carbon source for producing polyhydroxyalkanoic acid (PHA), which is a microorganism-produced polyester,
Sugars, aliphatic hydrocarbons, fatty acids, alcohols and the like have been used. From the viewpoint of producing PHA under a wider range of conditions, it is important to expand the range of carbon sources that can be used for producing polyhydroxyalkanoic acid (PHA), in addition to these conventionally used carbon sources. come.

The present invention solves the above problems,
An object of the present invention is to utilize a carbon source other than the conventionally used carbon sources such as sugars, aliphatic hydrocarbons, fatty acids, alcohols, and the like to obtain a desired polyhydroxyalkanoic acid (PH) for microorganisms.
An object of the present invention is to provide a novel method for producing A). More specifically, the carbon source is also used for culturing and growing microorganisms, and provides a novel method capable of achieving culturing and growing and producing desired polyhydroxyalkanoic acid (PHA) in the same medium. is there.

[0020]

Means for Solving the Problems The present inventor has made intensive studies and studies to solve the above-mentioned problems. As a result, acetic acid or a salt thereof can be used not only to promote the growth of microorganisms, but also They have found that they can be used as a carbon source when producing hydroxyalkanoic acid (PHA), and have completed the present invention.

That is, the production method of the present invention grows by utilizing acetic acid or a salt thereof in a medium containing acetic acid or a salt thereof as a single carbon source, and produces and accumulates polyhydroxyalkanoic acid. A method for producing polyhydroxyalkanoic acid, comprising culturing a microorganism having an ability.

For example, the polyhydroxyalkanoic acid has the following chemical formula (I):

[0023]

Embedded image (Wherein, R represents CH _3- (CH ₂ ) _n-1- , where n =
A method for producing PHA, which is a polyhydroxyalkanoic acid containing at least one kind of hydroxyalkanoic acid unit described in 3, 5, 7, 9, 11). The microorganism used in the production method of the present invention is a microorganism that can grow using acetic acid or a salt thereof as a single carbon source and has the ability to produce PHA. Preferably, a microorganism belonging to the genus Pseudononas is used. Good to do. More preferably, Pseudomonas chicory eye YN2 strain (Pseudomonas cichorii Y
N2; FERM P-17411), Pseudomonas cichorii H45; FER
MP-17410), Pseudomonas Jessenii P
161 strain (Pseudomonas jessenii P161; FERM P-
17445).

Further, the above-mentioned production method of the present invention can be a production method characterized by comprising a step of recovering polyhydroxyalkanoic acid from microorganisms in the medium.

[0025]

BEST MODE FOR CARRYING OUT THE INVENTION The method for producing polyhydroxyalkanoic acid of the present invention can grow acetic acid or a salt thereof as a single carbon source, and can be used in a medium containing no carbon source other than acetic acid or a salt thereof. A microorganism which produces polyhydroxyalkanoic acid, while growing the microorganism in a medium containing acetic acid or a salt thereof as a single carbon source, and producing a target polyhydroxyalkanoic acid. When a microorganism belonging to the genus Pseudononas having the above characteristics is used as a microorganism to be used, for example, a microorganism capable of producing PHA containing a unit of 3-hydroxyalkanoic acid represented by the above general formula (I) with high efficiency. It is.

In addition, the above three strains, Pseudomonas cichorii YN2;
FERM P-17411), Pseudomonas cichorii H45; FERM P
-17410), Pseudomonas Jesseny P16
1 strain (Pseudomonas jessenii P161; FERM P-17
445) are newly isolated bacteria as preferred strains. The bacteriological properties of each strain are described below.

[0027] The bacteriological properties of each strain are described below.

Mycological properties of strain YN2 Culture temperature: 30 ° C. Cell morphology: Bacillus (0.8 × 1.5-2.0 mm) Gram staining: Negative sporulation: Negative Motility: Positive Colony shape: Round, smooth, low Convex, surface smooth, glossy, translucent Catalase: Positive oxidase: Positive O / F test: Non-fermentative nitrate reduction: Negative Indole production: Positive Glucose acidification: Negative Arginine dihydrolase: Negative urease: Negative Esculin hydrolysis: Negative Gelatin hydrolysis: Negative β-galactosidase: Negative Substrate utilization glucose: Positive L-arabinose: Positive D-mannose: Negative D-mannitol: Negative N-acetyl-D-glucosamine: Negative Maltose: Negative potassium gluconate: Positive n -Capric acid: positive adipic acid: negative dl-malic acid: positive Sodium enate: Positive Phenyl acetate: Positive Fluorescent color production on King's B agar: Positive Growth in 4% NaCl: Positive (weak) Poly-β-hydroxybutyric acid accumulation: Negative (*) Tween 80 hydrolysis: Positive * Judged by staining nutrient agar culture colonies with Sudan Black. Mycological properties of H45 strain Culture temperature: 30 ° C Cell morphology: Bacillus (0.8 × 1.0-1.2 mm) Gram stain: Negative sporulation: Negative Motility: Positive Colony shape: Circular, whole edge smooth, low convex, Surface smooth, glossy, cream Catalase: Positive oxidase: Positive O / F test: Non-fermentative nitrate reduction: Negative Indole production: Negative Glucose acidification: Negative Arginine dihydrolase: Negative urease: Negative esculin hydrolysis: Negative gelatin hydrolysis: Negative β-galactosidase: Negative substrate utilization glucose: Positive L-arabinose: Negative D-mannose: Positive D-mannitol: Positive N-acetyl-D-glucosamine: Positive Maltose: Negative potassium gluconate: Positive n-capric acid: Positive adipic acid: negative dl-malic acid: positive nato citrate Lium: Positive Phenyl acetate: Positive Fluorescent color production on King's B agar: Positive Growth in 4% NaCl: Negative Accumulation of poly-β-hydroxybutyric acid: Negative (*) * Stain nutrient agar culture colonies with Sudan Black Judgment by doing. Mycological properties of strain P161 Culture temperature: 30 ° C Cell morphology: polymorph Spherical (φ0.6 mm) Rod-like (0.6 × 1.5-2.0 mm) Extended gram staining: negative Sporulation: negative Motility: positive colony shape: Circular, whole edge smooth, low convex, surface smooth, light yellow Catalase: Positive oxidase: Positive O / F test: Non-fermentative nitrate reduction: Positive Indole production: Negative Glucose acidification: Negative Arginine dihydrolase: Positive urease: Negative Esculin hydrolysis: Negative Gelatin hydrolysis: Negative β-galactosidase: Negative Substrate utilization glucose: Positive L-arabinose: Positive D-mannose: Positive D-mannitol: Positive N-acetyl-D-glucosamine: Positive Maltose: Negative glucone Potassium acid: Positive n-Capric acid: Positive Adipic acid: Negative dl-malic acid: Positive Sodium citrate: Positive Phenyl acetate: Positive Fluorescent color production on King's B agar: Positive Based on the above microbiological properties, the Burgers Manual of Systematic Bacteriology
Volume 1 (Bergey's Manual of Systematic Bacteriolog
y, Volume 1) (1984) and Bergey's Manual of Determinative Bacteriology
The genus was identified with reference to the ninth edition (1994), and it was determined that it was appropriate to identify both the YN2 strain and the H45 strain as Pseudomonas cichorii.

On the other hand, regarding the P161 strain, only the genus with the highest similarity was found to be Pseudomonas fluorescens (Pseudomonas
fluorescens) was selected, but its mycological properties did not determine its taxonomic position. Therefore, in order to try classification based on genetic characteristics, 16
The base sequence of S rRNA was determined (SEQ ID NO: 1, cDNA
to rRNA), 16S r of known Pseudomonas microorganisms
The homology with the RNA base sequence was examined. As a result, P1
61 strains and Pseudomonas jessenii
jessenii), it was found that the homology of the nucleotide sequence was extremely high. In addition, System. Appl. Microbiol., 2
0, 137-149 (1997), and System.Appl.Microbiol.,
22, 45-58 (1999) also showed high similarity between the mycological properties of Pseudomonas jessenii and the mycological properties of strain P161. From the above results, P16
One strain was deemed appropriate to be assigned to Pseudomonas jessennii.

There has been no report on the production of PHA in any of the strains identified in the above two genera. Therefore, these strains were judged to be new strains, and (YN2 strain: FERM P-17411, H45 strain: F
ERM P-17410, P161 strain: FERM P-1
7445).

In the method for producing a polyhydroxyalkanoic acid according to the present invention, in addition to the YN2 strain, H45 strain and P161 strain, the strain belongs to the genus Pseudomonas and can be grown using acetic acid or a salt thereof as a single carbon source, In addition, microorganisms that produce polyhydroxyalkanoic acid can also be generally used. For example, Pseudomonas oleovorans can also be used. In addition, in addition to microorganisms belonging to the genus Pseudomonas, Aeromonas
s) The genus belongs to the genus, Comamonas or the like, can grow using acetic acid or a salt thereof as a single carbon source, and can also use a microorganism that produces polyhydroxyalkanoic acid.

The medium used in the production method of the present invention includes:
Any medium may be used as long as it has a composition obtained by adding acetic acid or acetate as a carbon source to the composition of an inorganic salt medium containing a phosphate and a nitrogen source such as ammonium salt or nitrate. In addition, it is possible to improve PHA productivity by adjusting the concentration of the nitrogen source contained in the medium. The concentration of acetic acid or acetate added is 0.1%
It is appropriate to select a range of about 1.0% (w / v). When acetic acid is added as free acid,
It is advisable to adjust the pH in the medium to around neutral.

As an example of the inorganic salt medium, the composition of the medium used in the examples showing one embodiment of the present invention is shown below.
(Hereinafter referred to as 1 / 10N-M9 medium). Na ₂ HPO ₄ : 6.3 KH ₂ PO ₄ : 3.0 NH ₄ Cl: 0.1 NaCl: 0.5 g / L, pH = 7.0 Furthermore, for good growth and PHA production, It is preferable to add about 0.3% (v / v) of a minor component solution shown below to a 1 / 10N-M9 medium having the above composition.

Minor component solution nitrilotriacetic acid: 1.5; MgSO ₄ : 3.0; MnSO ₄ : 0.5
_{NaCl: 1.0; FeSO 4: 0.1} ; CaCl 2: 0.1; CoCl 2:
_{0.1; ZnSO 4: 0.1; CuSO} 4: 0.1; AlK (SO 4) 2: 0.1
_{; H 3 BO 3: 0.1;} Na 2 MoO 4: 0.1; NiCl 2: 0.1
g / L In the production method of the present invention, the culture temperature is preferably selected in a temperature range in which the microorganism to be used can grow well. For example, in the above three strains, the temperature range from 20 ° C.
It is appropriate to select a temperature in the range of about 30 ° C. In addition, as a culture method, any method used for ordinary culture of microorganisms such as a batch type, a flow batch type, a continuous culture, a reactor type and the like can be used.

In the production method of the present invention, the target product PHA is recovered from the cells by chloroform extraction, which is usually the simplest method. Other than the extraction method using this organic solvent, without using an organic solvent, for example, treatment with a surfactant such as SDS, treatment with an enzyme such as lysozyme, EDTA,
By removing intracellular components other than PHA by chemical treatment with sodium hypochlorite, ammonia, etc., P
A method of recovering only HA can be adopted.

[0036]

The present invention will be described in more detail with reference to specific examples. These specific examples are examples of the best mode of the present invention, but the present invention is not limited by the following specific examples.

(Example 1) Production of PHA by sodium acetate single carbon source culture using TY2 strain A colony of Pseudomonas chicory eye YN2 strain on a 1/10 N-M9 agar medium containing 0.1% sodium acetate was obtained. The cells were inoculated into 200 mL of a 1 / 10N-M9 liquid medium containing 0.3% of sodium acetate, and cultured at 30 ° C. After 68 hours, the cells were collected by centrifugation.

The cell pellet of the collected YN2 strain was freeze-dried and found to be 90 mg. This lyophilized pellet was suspended in 100 mL of chloroform and extracted with stirring at 60 ° C. for 24 hours. After the extract was filtered, the extract was concentrated with an evaporator, and the concentrate was poured into cold methanol and reprecipitated to obtain a polymer. The polymer was dried under reduced pressure at room temperature and weighed to find that it was 38 mg. The amount ratio of the recovered polymer per dry cell is about 42%.

The composition of the obtained polymer was analyzed by the following procedure.

That is, 10 mg of polymer was added to 25 mL
The mixture was placed in an eggplant-shaped flask and dissolved in 2 mL of chloroform. To this solution was added 2m of a methanol solution containing 3% sulfuric acid.
L was added thereto and reacted at 100 ° C. under reflux for 3.5 hours. After the completion of the reaction, 2 mL of water was added and the mixture was vigorously shaken for 10 minutes, and then the lower chloroform layer separated into two layers was taken out and dehydrated with magnesium sulfate. This sample was subjected to gas chromatography-mass spectrometry (GC-M
S; applied to Shimadzu QP-5050, DB-WAX capillary column (J & W)) to identify peaks of each methyl hydroxyalkanoate contained therein. Table 1 summarizes the analysis results.

(Example 2) Production of PHA by sodium acetate single carbon source culture using strain H45 A colony of Pseudomonas chicoryi strain H45 on a 1/10 N-M9 agar medium containing 0.1% sodium acetate was prepared. The cells were inoculated into 200 mL of a 1 / 10N-M9 liquid medium containing 0.3% of sodium acetate, and cultured at 30 ° C. After 68 hours, the cells were collected by centrifugation.

The cell pellet of the collected H45 strain was freeze-dried to give 50 mg. This lyophilized pellet was suspended in 100 mL of chloroform and extracted with stirring at 60 ° C. for 24 hours. After the extract was filtered, the extract was concentrated with an evaporator, and the concentrate was poured into cold methanol and reprecipitated to obtain a polymer. The polymer was dried under reduced pressure at room temperature and weighed to find 13 mg. The amount ratio of the recovered polymer per dry cell is about 26%.

The composition of the obtained polymer was as described in Example 1 above.
The analysis was performed according to the procedure shown in FIG.

That is, 10 mg of the polymer was added to 25 mL
The mixture was placed in an eggplant-shaped flask and dissolved in 2 mL of chloroform. To this solution was added 2m of methanol solution containing 3% sulfuric acid.
L was added thereto and reacted at 100 ° C. under reflux for 3.5 hours. After the completion of the reaction, 2 mL of water was added and the mixture was vigorously shaken for 10 minutes, and then the lower chloroform layer separated into two layers was taken out and dehydrated with magnesium sulfate. This sample was subjected to gas chromatography-mass spectrometry (GC-M
S; applied to Shimadzu QP-5050, DB-WAX capillary column (J & W)) to identify peaks of each methyl hydroxyalkanoate contained therein. Table 1 summarizes the analysis results.

(Example 3) Production of PHA by sodium acetate single carbon source culture using strain P161 A colony of Pseudomonas jesseny P161 on a 1/10 N-M9 agar medium containing 0.1% of sodium acetate was obtained. 1 / 10N-M containing 0.3% sodium acetate
9 Inoculated in 200 mL of liquid medium and cultured at 30 ° C. 6
Eight hours later, the cells were collected by centrifugation.

The cell pellet of the collected P161 strain was freeze-dried to give 50 mg. This lyophilized pellet was suspended in 100 mL of chloroform, and
For 24 hours. After the extract was filtered, the extract was concentrated with an evaporator, and the concentrate was poured into cold methanol and reprecipitated to obtain a polymer. The polymer was dried under reduced pressure at room temperature and weighed to find that it was 11 mg. The amount ratio of the recovered polymer per dry cell is about 22%.

The composition of the obtained polymer was as described in Example 1 above.
The analysis was performed according to the procedure shown in FIG.

That is, 10 mg of polymer was added to 25 mL
The mixture was placed in an eggplant-shaped flask and dissolved in 2 mL of chloroform. To this solution was added 2m of methanol solution containing 3% sulfuric acid.
L was added thereto and reacted at 100 ° C. under reflux for 3.5 hours. After the completion of the reaction, 2 mL of water was added and the mixture was vigorously shaken for 10 minutes, and then the lower chloroform layer separated into two layers was taken out and dehydrated with magnesium sulfate. This sample was subjected to gas chromatography-mass spectrometry (GC-M
S; applied to Shimadzu QP-5050, DB-WAX capillary column (J & W)) to identify peaks of each methyl hydroxyalkanoate contained therein. Table 1 summarizes the analysis results.

[0049]

[Table 1] The value indicates the peak area ratio (%) of GC-MS. Description of each hydroxyalkanoic acid unit: C4: 3-hydroxybutyric acid, C6: 3-hydroxyhexanoic acid, C8: 3-hydroxyoctanoic acid, C10: 3-hydroxydecanoic acid, C12: 3-hydroxydodecanoic acid, C12 ′: Not identified. It is presumed that the compound contains an unsaturated portion or a branched portion in 3-hydroxydodecanoic acid. C14: 3-hydroxytetradecanoic acid From the analysis results shown in Table 1, the TY2 strain of Example 1 and Example 2 were obtained in an inorganic salt medium to which acetic acid or a salt thereof was added as a single carbon source.
No. H45 strain and the P161 strain of Example 3 both proliferate and produce and accumulate polyhydroxyalkanoic acid in the cells. The polyhydroxyalkanoic acid is at least 3-hydroxyhexanoic acid (3HHx), 3-hydroxyoctanoic acid (3HHx).
O), -hydroxydecanoic acid, 3-hydroxydodecanoic acid, and 3-hydroxytetradecanoic acid. These carbon numbers 6, 8, 10,
The units of 12,14 3-hydroxyalkanoic acids are
As shown in the above general formula (I), it is contained in polyhydroxyalkanoic acid recovered from cells.

[0050]

According to the method for producing polyhydroxyalkanoic acid of the present invention, acetic acid or a salt thereof is used as a single carbon source,
Polyhydroxyalkanoic acids, especially C6, C8, C1
It has become possible to produce polyhydroxyalkanoic acid containing at least one kind of 0, C12, and C14 units using a microorganism.

[0051]

[Sequence List] SEQUENCE LISTING <110> CANON INC. <120> Preparation of Poly-hidroxyalkanoic Acid <130> 4047062 <160> 1 <170> Microsoft Word <210> 1 <211> 1501 <212> DNA <213> Pseudomonas jessenii P161; FERM P-17445 <400> 1 tgaacgctgg cggcaggcct aacacatgca agtcgagcgg 40 atgacgggag cttgctcctg aattcagcgg cggacgggtg 80 agtaatgcct aggaatctgc ctggtagtgg gggacaacgt 120 ctcgaaaggg acgctaatac cgcatacgtc ctacgggaga 160 aagcagggga ccttcgggcc ttgcgctatc agatgagcct 200 aggtcggatt agctagttgg tgaggtaatg gctcaccaag 240 gcgacgatcc gtaactggtc tgagaggatg atcagtcaca 280 ctggaactga gacacggtcc agactcctac gggaggcagc 320 agtggggaat attggacaat gggcgaaagc ctgatccagc 360 catgccgcgt gtgtgaagaa ggtcttcgga ttgtaaagca 400 ctttaagttg ggaggaaggg cattaaccta atacgttagt 440 gttttgacgt taccgacaga ataagcaccg gctaactctg 480 tgccagcagc cgcggtaata cagagggtgc aagcgttaat 520 cggaattact gggcgtaaag cgcgcgtagg tggtttgtta 560 agttggatgt gaaagccccg ggctcaacct gggaactgca 600 ttcaaaactg acaagctaga gtatggtaga gggtggtgga 640 atttcctgtg tagcggtgaa atgcgtagat ataggaagga 680 acaccagtgg cgaaggcgac cacctggact gatactgaca 720 ctgaggtgcg aaagcgtggg gagcaaacag gattagatac 760 cctggtagtc cacgccgtaa acgatgtcaa ctagccgttg 800 ggagccttga gctcttagtg gcgcagctaa cgcattaagt 840 tgaccgcctg gggagtacgg ccgcaaggtt aaaactcaaa 880 tgaattgacg ggggcccgca caagcggtgg agcatgtggt 920 ttaattcgaa gcaacgcgaa gaaccttacc aggccttgac 960 atccaatgaa ctttccagag atggatgggt gccttcggga 1000 acattgagac aggtgctgca tggctgtcgt cagctcgtgt 1040 cgtgagatgt tgggttaagt cccgtaacga gcgcaaccct 1080 tgtccttagt taccagcacg taatggtggg cactctaagg 1120 agactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1160 caagtcatca tggcccttac ggcctgggct acacacgtgc 1200 tacaatggtc ggtacagagg gttgccaagc cgcgaggtgg 1240 agctaatccc acaaaaccga tcgtagtccg gatcgcagtc 1280 tgcaactcga ctgcgtgaag tcggaatcgc tagtaatcgc 1320 gaatcagaat gtcgcggtga atacgttccc gggccttgta 1360 cacaccgccc gtcacaccat gggagtgggt tgcaccagaa 1400 gtagctagtc taaccttcgg gaggacggtt accacggtgt 1440 gattcatgac tggggtgaag tcgtaccaag gtagcc gtag 1480 gggaacctgc ggctggatca c 1501

[Brief description of the drawings]

FIG. 1. Pseudomonas jessennii strain P161 (Ps
eudomonas jessenii P161; FERM P-17445)
1 shows the nucleotide sequence of 16S rRNA.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Sakae Suda 3-30-2 Shimomaruko, Ota-ku, Tokyo Inside Canon Inc. (72) Inventor Tetsuya Yano 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon F term (reference) 4B064 AD83 CA02 CC03 CD07 DA16

Claims (5)

[Claims] 1. A microorganism which has an ability to produce and accumulate polyhydroxyalkanoic acid and grow by assimilating acetic acid or a salt thereof in a medium containing acetic acid or a salt thereof as a single carbon source. A method for producing polyhydroxyalkanoic acid, comprising: 2. The polyhydroxyalkanoic acid has the following formula (I): (Wherein, R represents CH _3- (CH ₂ ) _n-1- , where n =
The method for producing a polyhydroxyalkanoic acid according to claim 1, wherein the method is a polyhydroxyalkanoic acid containing at least one kind of a hydroxyalkanoic acid unit shown in 3, 5, 7, 9, 11). 3. The microorganism which grows using acetic acid or a salt thereof as a single carbon source and has the ability to produce polyhydroxyalkanoic acid is a bacterium belonging to the genus Pseudononas. Or the method for producing a polyhydroxyalkanoic acid according to 2. 4. As a bacterium belonging to the genus Pseudononas, Pseudomonas cichorii YN2; FERM P-1741
1), Pseudomonas chicory eye strain H45 (Pseudomon
as cichoriiH45; FERM P-17410), Pseudomonas jessenii P161 strain (Pseudomonas jessen
ii P161; FERM P-17445), the method for producing a polyhydroxyalkanoic acid according to claim 3, wherein the strain is selected. 5. The method for producing polyhydroxyalkanoic acid according to claim 1, further comprising a step of recovering polyhydroxyalkanoic acid from microorganisms in the medium.